an essential role of the immune system in …rb593gv5868/kavya rakhra... · an essential role of...
TRANSCRIPT
AN ESSENTIAL ROLE OF THE IMMUNE SYSTEM IN
REMODELING THE TUMOR MICROENVIRONMENT
UPON ONCOGENE INACTIVATION
A DISSERTATION SUBMITTED TO THE PROGRAM IN IMMUNOLOGY AND
THE COMMITTEE ON GRADUATE STUDIES OF STANFORD UNIVERSITY IN
PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY
Kavya Rakhra
August 2011
http://creativecommons.org/licenses/by-nc/3.0/us/
This dissertation is online at: http://purl.stanford.edu/rb593gv5868
© 2011 by Kavya Rakhra. All Rights Reserved.
Re-distributed by Stanford University under license with the author.
This work is licensed under a Creative Commons Attribution-Noncommercial 3.0 United States License.
ii
I certify that I have read this dissertation and that, in my opinion, it is fully adequatein scope and quality as a dissertation for the degree of Doctor of Philosophy.
Dean Felsher, Primary Adviser
I certify that I have read this dissertation and that, in my opinion, it is fully adequatein scope and quality as a dissertation for the degree of Doctor of Philosophy.
Sheri Krams
I certify that I have read this dissertation and that, in my opinion, it is fully adequatein scope and quality as a dissertation for the degree of Doctor of Philosophy.
Calvin Kuo
I certify that I have read this dissertation and that, in my opinion, it is fully adequatein scope and quality as a dissertation for the degree of Doctor of Philosophy.
Ronald Levy
Approved for the Stanford University Committee on Graduate Studies.
Patricia J. Gumport, Vice Provost Graduate Education
This signature page was generated electronically upon submission of this dissertation in electronic format. An original signed hard copy of the signature page is on file inUniversity Archives.
iii
iv
ABSTRACT
The phenomenon of oncogene addiction has been presumed to occur in a cell
autonomous manner independent of the tumor microenvironment, through the
induction of proliferative arrest, apoptosis, differentiation and/or senescence. Immune
effectors have been implicated in both the induction and restraint of tumorigenesis but
their role in tumor regression upon the therapeutic inactivation of an oncogene is
unclear. Here we show that an intact immune system is required to mediate sustained
tumor regression upon oncogene inactivation in conditional mouse models of MYC
induced T-cell acute lymphoblastic leukemia (T-ALL) and BCR-ABL induced pro-B-
cell lymphocytic leukemia (B-ALL). We used these transgenic mouse models of
conditional oncogene inactivation, to show that the absence of an intact immune
system results in a 10-1000-fold reduction in the rate, extent, and duration of tumor
regression upon oncogene inactivation.
We demonstrate that CD4+ T-cells are critical to elicit oncogene addiction
upon inactivation of the MYC oncogene in a mouse model of T-ALL. The absence of
CD4+ T-cells had no effect on the ability of MYC inactivation to induce proliferative
arrest or apoptosis of tumor cells but markedly attenuated cellular senescence of tumor
cells and the shutdown of angiogenesis in the tumors. CD4+ T-cells were required
upon MYC inactivation to elicit inflammatory cytokines that regulate the
microenvironment. Provocatively, immune effectors knocked out for thrombospondins
failed to induce sustained tumor regression. Hence, CD4+ T-cells are critical immune
v
effectors, required for the remodeling of the tumor microenvironment through the
secretion of chemokines like thrombospondins in order to elicit oncogene addiction.
Most strategies to identify therapeutic agents utilize in vitro models or in vivo
xenograft models overlooking the effect of the immune system. Our results argue for
the necessity of models that include an intact host immune system to properly evaluate
the potential efficacy of targeted therapeutics for maximum clinical impact. Our
results also imply that the efficacy of new and existing cancer targeted therapeutics
can be increased by combining them with strategies to inhibit angiogenesis, induce
cellular senescence or increase CD4+ T-cell infiltration in the tumor
microenvironment.
vi
ACKNOWLEDGEMENTS
This Ph.D. required a lot of moral support and various colleagues, friends and
family members have listened to the incessant grumblings and travails of being a
graduate student over the past several years. It's going to be impossible to individually
mention everyone's contribution and I'd like to start by acknowledging everyone who
shared in the joy, excitement, enthusiasm, depression, anger, frustration and
experimental failure that are an inherent part of graduate school.
I did my first rotation in Dean's lab and knew almost immediately that this
would be my lab. I was mentored by Alice and Pavan and was introduced to tumor
immunology for the very first time. I continued to work with them through my
formative years in graduate school and have benefited greatly from this experience.
I've learnt so much from them, not just about scientific experimental design and career
development but also about fitness, music, movies, books and how to be a well
rounded individual.
Dean runs a fantastic high energy lab that has accommodated my constant
stream of questions, disorganized desk and bench and tendency to burst into song for
no apparent reason. This allowed me to enjoy coming into work every morning and
created a „microenvironment‟ for me to succeed. Dean has always been supportive,
encouraging or exacting as the situation warranted and allowed me to realize my
scientific potential. He has also served as an excellent role model as someone who has
it all, a clinical career, a research laboratory pushing the frontiers of cancer biology
and a wonderful family and I hope to have at least some combination of these in the
vii
years to come. I would also like to thank my committee members Sheri Krams, Ron
Levy and Calvin Kuo for their help navigating through my enormous project and
always bringing me back to the fundamentals of immunology.
My family of course, has supported me through all of my life and grad school
was no different. My parents would always make an effort to try and understand what
I've been working on and make me give lab meeting presentations when I went home
for the holidays. I love that they've managed to stay actively involved in my life even
though I've been 9000 miles away from them. It's also been awesome to have my
brother living in NYC, just far enough that we don't get on each others' nerves but
close enough to always have a place to visit for the holidays and have my phone bills
paid. To all of my family who've always called on my birthday and who are always
proud of me, thank you!!
I was also fortunate to have several friends who were always willing to lend an
ear or a beer or provide some good Indian food or catch a late night movie show. In no
particular order, I want to acknowledge Lux, Bharey, Addu, Div, Anu, Yashas,
Mallika, Peter, Cat, Alper, Lavoo, Nammo, Shariq, Anshul, Justine, Kiri, Masumi,
Nalini, Simona, Shreekar, Prasanthi, RSS, Ditch, Cherry, Ranji, Indra and Sanketh.
I'd also like to thank current and previous Felsher lab members Alice, Pavan,
Peter, Alper, Emelyn, Stacey, Tahera, Lowen, Qiwei, Hanan, Vanessa, Ramya, Prajna,
Yulin, Natalie, Ling, Dina, and Shelly for participating in various scientific and non-
scientific activities (happy hours, birthday cakes, dish walks, disney land, manicures,
tattoos, marathons, bay to breakers, etc.) with me and enriching my time in the Felsher
lab. All in all, it was so much more than just a Ph.D.
viii
TABLE OF CONTENTS
Abstract………………………………….………………………………………………....…..iv
Acknowledgements……………………………………………….……………………….…..vi
List of Tables…………………………………………….………………………………..…...xi
List of Figures……..…………………………………………….…………………………….xii
Chapter 1: Introduction
1.1 Overview: ............................................................................................................................. 2
1.2 Oncogenes and cancer: ......................................................................................................... 3
1.3 MYC: .................................................................................................................................... 4
1.3.1 The estrogen receptor-tamoxifen regulatory system: ........................................................ 5
1.3.2 The tetracycline regulatory system: .................................................................................. 6
1.4 Oncogene addiction: ............................................................................................................. 8
1.5 Mechanisms of tumor regression upon oncogene inactivation: ......................................... 11
1.5.1 Apoptosis:........................................................................................................................ 12
1.5.2 Inhibition of angiogenesis: .............................................................................................. 12
1.5.3 Cellular senescence: ........................................................................................................ 14
1.6 Interaction of a tumor with its immune microenvironment:............................................... 16
1.6.1 Role of CD4+ T-cells in the tumor microenvironment: ................................................... 21
1.6.2 Role of tumor associated marcophages (TAMs) in the tumor microenvironment….......23
1.6.2.1 TAMs and tumor progression: ................................................. 25
ix
1.6.2.2 TAMs and tumor inhibition: .................................................... 26
1.6.3 Role of eosinophils in the tumor microenvironment: ...................................................... 27
1.6.3.1 Eosinophils and tumor progression: ......................................... 28
1.6.3.1 Eosinophils and tumor inhibition: ............................................ 29
1.7 References: ......................................................................................................................... 33
Chapter 2: Contribution of the immune system to oncogene inactivation mediated tumor
regression
2.1 Overview: ........................................................................................................................... 44
2.2 Contribution of the adaptive immune system:.................................................................... 44
2.3 Contribution of the innate immune system: ....................................................................... 56
2.4 Contribution of T-cells to regression of primary MYC induced lymphoma: ...................... 60
2.5 Contribution of the adaptive immune system in other models of oncogene induced
hematologic malignancies: ....................................................................................................... 62
2.6 Contribution of an antigen specific immune response: ...................................................... 64
2.7 References: ......................................................................................................................... 96
Chapter 3: Discussion of findings, implications of results and future direction
3.1 Overview: ........................................................................................................................... 99
3.2 The adaptive immune system remodels the tumor microenvironment: ............................. 99
3.3 Potential role of T-regs and an antigen specific immune response: ................................. 105
3.4 Potential role of the innate immune system: .................................................................... 107
3.5 Implications: ..................................................................................................................... 110
x
3.6 References: ....................................................................................................................... 114
Appendix I: Materials and Methods ................................................................................... 119
xi
LIST OF TABLES
Chapter 1: Introduction
Table 1: Mechanisms of tumor regression upon oncogene inactivation……….…...31
Table 2: CD4+ T-cell polarization and cytokine secretion profile…………………..32
LIST OF FIGURES
Chapter 1: Introduction
Figure 1: A bi-transgenic mouse model of conditional MYC expression…………………….30
Chapter 2: Contribution of the immune system to oncogene inactivation mediation
tumor regression
Figure 1: An intact immune system is required for sustained tumor regression.....................66
Figure 2: An intact immune system is required for sustained tumor regression upon MYC
inactivation……………………………………………………………………………..…......69
Figure 3: CD4+ T-cells home to the tumor and are sufficient to induce sustained tumor
regression upon MYC inactivation……………………………………………….....................71
Figure 4: Splenocytes depleted of CD4+ T-cells home to the tumor microenvironment and
verification of RAG1-/-
reconstitution……………………………….……..………………….73
Figure 5: The immune system does not influence apoptosis and cellular arrest upon MYC
inactivation…….……………………………………………………………………………...75
Figure 6: An intact immune system is required for the inhibition of angiogenesis upon MYC
inactivation………..…………………………………………………………………………..77
Figure 7: TSP-1 expression in the tumor microenvironment is required for sustained tumor
regression upon MYC inactivation…………………………………………………………….79
Figure 8: An intact immune system is required for the induction of cellular senescence upon
MYC inactivation…………………………………………………………...............................81
xiii
Figure 9: Cytokines produced by the immune system contribute to sustained tumor regression
upon MYC inactivation ……………………………………….................................................83
Figure 10: Macrophages infiltrate tumors regressing in WT and CD8-/-
hosts upon MYC
inactivation…………………………………………………………………............................85
Figure 11: Increased levels of iNOS, Arg-1 and TSP-1 in macrophages from tumor bearing
WT host…………….……………………………………………………................................87
Figure 12: Cyclosporine A treatment inhibits induction of senescence and inhibition of
angiogenesis in primary MYC induced T-ALL…………………………………...…..............89
Figure 13: An intact immune system is required for sustained regression of tumors in a
conditional mouse model of BCR-ABL-induced B-ALL …………………………..................91
Figure 14: Increased Foxp3 expression in tumors regressing in WT hosts upon MYC
inactivation ……………………………………………………...............................................93
Figure 15: Transplanted tumors are rejected upon re-challenge in WT hosts that have
previously exhibited sustained tumor regression…………………………………...................95
Chapter 3: Discussion of findings, implications of results and future direction
Figure 1: Model of the interaction of the immune system with oncogene addiction……….113
1
CHAPTER 1: Introduction
2
1.1 Overview:
To set the stage for this dissertation thesis, I will discuss two important
discoveries in the field of cancer biology. The first is the discovery of oncogene
addiction, the phenomenon by which highly complex tumor cells that are a
consequence of multiple genetic and epigenetic changes become exquisitely dependent
upon a single oncogene for their continued growth and survival [1-2]. The generation
of conditional mouse models of cancer in which oncogene expression could be
spatially and temporally controlled allowed the study of the mechanisms by which
targeting oncogenes reversed the cancer phenotype. Consequently, inactivating
oncogenes in tumor cells has been therapeutically exploited to cause tumor regression
in a variety of cancer patients and drugs like Imatinib Mesylate or Gleevec, a small
molecule inhibitor of the BCR-ABL oncogene [3], have seen great clinical success.
The second important discovery is the complex interaction of the immune
system with tumor cells [4-5]. The immune system can influence various aspects of
tumor initiation, growth, progression [6-7] and tumor regression in response to various
anti-cancer radiation- and chemo-therapeutics [8-10]. While Gleevec and other
targeted therapies have been effective in causing tumor regression in various cancers
[11-14], the contribution of the immune system to tumor regression mediated by the
targeted inactivation of an oncogene remained unknown and studying this became the
focus of my graduate work.
In this introductory chapter, I describe the role of oncogenes in cancer with
particular emphasis on the MYC oncogene, various conditional mouse models
3
designed to study the role of MYC in tumorigenesis, mechanisms of oncogene
addiction, various targeted therapies based on the concept of oncogene addiction and
how the immune response interacts with cancer focusing on the role of the CD4+ T-
cells of the adaptive immune system and macrophages of the innate immune system.
This will set the stage to understand the experiments described in this thesis (Chapter
2) and the discussion and interpretation of the results of these experiments (Chapter 3).
1.2 Oncogenes and cancer:
Oncogenes are genes that either initiate and/or are involved in progression of
cancer [15]. They often encode altered forms of proteins normally involved in cell
proliferation and apoptosis. These could be proteins like transcription factors, growth
factors, growth factor receptors, signal transducers or apoptosis regulators. In a normal
cell, genes that encode these proteins are known as proto-oncogenes. Proto-oncogenes
can be activated to oncogenes by genetic events such as mutation, gene fusion, gene
translocation, chromosomal rearrangement, gene amplification or juxtaposition of a
gene to enhancer elements [15].
In several mouse models of cancer, it has been demonstrated that tumors arise
after a period of latency upon oncogene activation. This suggests that oncogene
activation alone is not always sufficient for tumorigenesis. Other genetic events must
occur in addition to oncogene activation in order to cause neoplasia [16-18]. In fact,
tumorigenesis is known to occur as a result of sequential genetic aberrations, each of
which confers some growth advantage to a normal cell which eventually renders it
cancerous [19].
4
1.3 MYC:
The MYC protein belongs to a family of basic helix loop helix transcription
factors that include c-MYC, N-MYC and L-MYC. For the purpose of this dissertation,
we will focus on c-MYC. For the remainder of this text, MYC refers to c-MYC unless
otherwise specified. MYC is a proto-oncogene found on chromosome 8 in humans and
on chromosome 15 in mice [20]. It encodes a transcription factor which controls the
expression of genes involved in critical cellular functions like cell cycle regulation,
apoptosis, proliferation, metabolism, angiogenesis, adhesion and differentiation. In
normal cells, the expression of MYC is tightly controlled, however in cancer cells
MYC expression is dysregulated due to genetic abnormalities [21-22]. Dysregulation
of MYC expression can occur due to chromosomal translocation resulting in MYC
being expressed from the immunoglobulin locus which is active in B-cells [23] or
gene amplification that results in increased copies of the MYC gene and hence
increased MYC expression [24]. Abnormal genetic events that lead to increased MYC
transcription or mRNA stability can also cause increased MYC protein expression.
Furthermore, increased MYC activity can often result from mutations in pathways
upstream of MYC such as the RAS and β-catenin pathways [21-22].
This dysregulation of MYC expression leads to tumorigenesis due to the
disruption of cellular functions related to cell cycle progression, metabolism, apoptosis
and genomic instability. Aberrant MYC expression is known to occur in 100% of
Burkitt‟s lymphomas, 90% of gynecological cancers, 80% of breast cancers, 70% of
colon cancers, 50% of hepatocellular cancers, 50% of T-ALLs and 5% of adult acute
5
lymphoblastic leukemia in humans. Thus MYC is the quintessential oncogene since it
causes cellular transformation upon inappropriate expression [21-22].
As an important gene that mediates human cancers, MYC‟s role in
tumorigenesis has been explored through several genetic mouse models in which MYC
overexpression induces tumor formation. In order to recapitulate sporadic human
cancer, mouse models were designed to regulate MYC expression in a time dependent
and tissue specific manner using a number of different genetic strategies [18, 25-27].
This ensures that the tumor initiating mutation occurs in cells surrounded by an
un-mutated tumor microenvironment to accurately model spontaneous tumor initiation
seen in humans [28]. Several conditional models of MYC induced cancer have been
designed and are discussed below:
1.3.1 The estrogen receptor-tamoxifen regulatory system:
In this genetic system, the MYC gene is fused to the hormone binding domain
of the estrogen receptor (ER). In the absence of any ER ligands, the MYC-ER fusion
protein forms a complex with intracellular proteins like HSP-90, thus preventing MYC
from entering the nucleus. However, in the presence of an appropriate ER ligand such
as 17β-estradiol, MYC-ER is released from these intracellular complexes and can be
transported into the nucleus where it can function as a transcription factor. This allows
MYC function to be estrogen dependent [29]. To prevent the effects of endogenous
estrogen on this fusion protein, second generation fusion proteins have been generated
in which MYC is fused to a mutant murine estrogen receptor G525R which can no
longer bind to 17β-estradiol, but can still bind to the synthetic steroid
6
4-hydroxytamoxifen (4OHT). This allows MYC function to be dependent on
exogenous 4OHT. In mouse models, tissue specificity of MYC expression is achieved
by cloning the MYC-ER fusion protein downstream of a tissue specific promoter [30].
The advantage of this model is that MYC function is controlled at the protein
level and not at the level of transcription. This ensures that MYC becomes functional
within minutes of systemic administration of 4-OHT and this can be easily reversed by
withdrawing 4-OHT administration. This strategy has been used to overexpress MYC
in pancreatic β cells [31], primary rodent fibroblasts [30] and thymocytes [32].
1.3.2 The tetracycline regulatory system:
Derived from the bacterial tetracycline resistance operon, this genetic system
has been designed to incorporate two regulatory elements. One element is a
tetracycline transactivator (tTA) and the second element is a tetracycline response
element (TRE) consisting of tet-O sequences of the bacterial operon within a minimal
promoter. The gene of interest (in this case MYC) is cloned downstream of the tet-O
promoter. In order to induce target gene expression, tTA must bind to the tet-O
promoter to activate it. Gene expression from the tet-O promoter can be controlled by
using tetracycline or tetracycline analogs like doxycycline (dox). In the presence of
dox, tTA can no longer bind to the tet-O promoter, thus causing gene expression to be
turned off. This is known as the Tet-Off system (Figure 1) [33-34].
Conditional mouse models of MYC induced cancers are generated by
crossing a transgenic mouse line expressing the tTA element in a tissue specific
manner with a transgenic mouse line that carries the human MYC transgene under the
7
control of the TRE. The progeny of this cross will express human MYC in tissues in
which tTA is expressed. Gene expression can be turned off by administering
doxycycline to these mice in their drinking water and by delivering a doxycycline
injection intra-peritoneally (IP). This model has been used to overexpress MYC in the
bone, liver, mammary glands and hematopoietic system [18, 26, 35].
A variation of this genetic switch is the Tet-On system, in which a modified
tTA element known as the reverse tTA (rtTA) is used. The rtTA element requires dox
to bind to the tet-O promoter and thus transgene expression turned on only in the
presence of doxycycline. In the absence of doxycycline, rtTA cannot bind the tet-O
promoter and transgene expression is off [33-34].
The data described in chapter 2 have been generated using a model of MYC
induced lymphoma, using the Tet-Off system. In this model, tTA expression is
restricted to the lymphoid compartment as it is transcribed downstream of the
Eµ-Immunoglobulin heavy chain enhancer. Once crossed with mice bearing a
tet-O-MYC construct, MYC expression is induced in the lymphoid compartment. When
MYC is turned on from birth, these mice develop lymphoma within 8-10 weeks. The
phenotype of this lymphoma is CD4+CD8
+ in 90% of the mice analyzed [18] (Figure
1).
These models have been helpful in characterizing the role of MYC in tumor
initiation and progression and have also served as excellent means to test novel cancer
therapeutics against MYC induced cancers. Additionally, the conditional regulation of
MYC expression in these models has facilitated the analysis of the consequences of
8
inactivating MYC in tumors initiated by this oncogene. It has been shown that various
tumors initiated by MYC overexpression such as hepatocellular carcinomas [35],
osteosarcomas [26], lymphomas [18] and pappilomas [36] undergo regression upon
MYC inactivation. These tumor cells are dependent on MYC for their growth and
survival and are thus addicted to the MYC oncogene. These models have been used
extensively to study the mechanisms of tumor regression upon oncogene inactivation.
It is important to note that whether or not tumor cells are dependent on MYC is highly
contextual and in certain cases inactivation of MYC fails to cause tumor regression of
a MYC induced tumor [37].
1.4 Oncogene addiction:
Tumor cells are notorious for their genetic and epigenetic complexity and are
known to harbor several mutations. Despite this, they often show dependence on a
single oncogene or pathway for their growth and survival, a phenomenon defined as
oncogene addiction [1, 38]. As a consequence of this addiction, when an oncogene
that tumor cells are dependent on is inactivated, the tumor undergoes regression.
The phenomenon of oncogene addiction has been demonstrated in several
mouse models. In addition to the inducible models of MYC initiated tumorigenesis
described earlier, oncogene addiction has also been shown to occur in conditional
mouse models of H-RAS induced melanoma [39], K-RAS induced lung
adenocarcinomas [40] and BCR-ABL induced leukemia [41] and others [2].
9
With the advent of targeted therapeutics like Imatinib Mesylate or Gleevec,
compelling evidence accrued suggesting that oncogene addiction occurs in tumors
from human patients. Gleevec inhibits the BCR-ABL oncogene that causes chronic
myelogenous leukemia (CML). The mechanism of action of Gleevec involves
preventing access of the ATP molecule to the constitutively active BCR-ABL tyrosine
kinase by competitive inhibition. This prevents tyrosine phosphorylation and
activation of the proteins involved in the BCR-ABL signaling cascade which is
required for CML cells to proliferate [3]. Gleevec can also inhibit other tyrosine
kinases like KIT and has also proved to be effective in the treatment of
Gastrointestinal Stromal tumors (GIST) [42].
Other successful examples of cancer therapies based on oncogene inactivation
and tyrosine kinase inhibition are:
Traztazumab/Herceptin which blocks HER2 function in breast
cancers [12].
Erlotinib/Tarceva which blocks EGFR function in non-small cell
lung cancers (NSCLC) [14].
Sorafenib/Nexavar to block B-RAF and VEGFR function in
melanoma and renal cell carcinoma [11, 13].
While current therapies based on oncogene addiction are limited to tyrosine
kinase inhibitors, several efforts are underway to target other oncogenes like MYC,
RAS and β-catenin. Strategies being tested to target MYC include using anti-sense
oligonucleotides or small interfering RNA (siRNA) to inhibit MYC mRNA, using
10
phosphorodiamidate morpholino oligomers (PMOs) to prevent mRNA translation, and
targeting the interaction of MYC with its binding partner MAX. However these
strategies have seen limited success and active research is being done to improve the
efficacy, sensitivity, specificity and delivery of these drugs [43-44].
Since oncogene addiction was first characterized in human tumor derived cell
lines in vitro, it was largely thought to be a cancer cell autonomous phenomenon that
occurred independently of tumor-stromal interactions [45]. However, it is becoming
increasingly evident that overexpression of an oncogene can cause changes in the
tumor microenvironment. Activation of the RET oncogene in normal human
thymocytes induces an inflammatory response leading to tumor tissue remodeling,
angiogenesis and metastasis, all of which contribute to the maintenance of the
transformed state of the tumor [46]. Oncogenic RAS upregulates expression of the
cytokines IL-6 [47] and IL-8 [48] which in turn contributes to tumorigenesis. In a
MYC-induced model of lymphoma, MYC overexpression is associated with the
activation of macrophages which can cause tumor suppression [49]. Furthermore,
endogenous MYC levels have also been shown to maintain the angiogenic tumor
microenvironment in certain tumor models [50]. This dynamic cross talk between the
oncogene and the tumor microenvironment suggested that this interplay might be
fundamental to eliciting oncogene addiction.
Additionally, the complex interaction of the immune system with tumor cells
has been investigated in great detail. The immune system can play a significant role in
influencing not only aspects of tumor initiation, growth and progression, but also the
11
outcome of a cancer therapy [6-7, 9-10]. This has been well studied for various
radiation- and chemo-therapies [10]. The kind of tumor cell apoptosis caused by a
specific anti-cancer therapy determines the contribution of the immune system to the
therapy [8-9, 51].
This suggested that there might be a significant contribution of the immune
system to tumor regression mediated by targeted therapeutics as well. My goals were
to ascertain whether or not there is a non-cell autonomous, immune based component
to oncogene addiction and to characterize this contribution by identifying key players
of the immune system that are involved and the mechanisms by which they influence
oncogene addiction.
1.5 Mechanisms of tumor regression upon oncogene inactivation:
Tumor regression upon oncogene inactivation has been demonstrated in
several inducible mouse models of cancer as described above. Analysis of tumor
regression in these mouse models shows that inactivation of the oncogene after tumor
establishment leads to tumor regression through a number of different mechanisms
depending on the oncogene being inactivated and the context of the tumor (Table 1).
For example, even brief oncogene inactivation can induce a permanent loss of a
neoplastic phenotype in osteosarcoma [26] and lymphoma [18] but not in epithelial
tumors such as hepatocellular carcinoma [35] and breast carcinoma [25].
We investigated the influence of the immune system on various mechanisms of
regression known to occur upon inactivation of the MYC oncogene. Experiments were
12
performed to study the mechanisms of apoptosis, inhibition of angiogenesis and
irreversible cellular growth arrest or senescence known to contribute to tumor
regression upon MYC inactivation in the conditionally inducible Eµ-tTA; tet-O-MYC
model of MYC induced murine lymphoma [18, 52-53].
1.5.1 Apoptosis:
Studies have shown that both MYC overexpression [54-55] and loss of MYC
expression [56] can lead to apoptosis. The precise mechanisms governing MYC‟s role
in apoptosis have not been clearly elucidated though several cases of MYC induced
apoptosis appear to involve p53 activity [57-58]. However MYC induced apoptosis can
also occur in the absence of p53 [59]. Apoptosis that occurs upon reduction of MYC
levels can occur through increasing p27kip1
[60] and caspase activation [61].
In the conditional model of MYC induced lymphoma used in our experiments,
after 3-6 days of MYC inactivation, certain areas of the tumor appear to be undergoing
apoptosis in a cell autonomous manner, which contributes to tumor regression upon
MYC inactivation [18].
1.5.2 Inhibition of angiogenesis:
Angiogenesis is the process by which new blood vessels are generated from
pre-existing blood vessels. Angiogenesis is required for tumor cells to grow and
become invasive and targeting angiogenesis in cancer is being actively studied as a
means of controlling tumor growth [62].
13
The angiogenic microenvironment in a tumor is governed by an “angiogenic
switch” that depends on a balance of pro- and anti-angiogenic factors. If there is a
predominance of pro-angiogenic factors (VEGF, bFGF), the angiogenic switch is
considered “on” and this leads to tumor vascularization and thus to tumor growth.
However if there is an abundance of anti-angiogenic factors (TSP-1, endostatin,
angiostatin), then the angiogenic switch is turned “off” and tumor vascularization is
prevented [63].
In a model of conditional MYC induced lymphoma that is also p53-/-
, it has
been shown that MYC inactivation alone could not cause sustained tumor regression.
In order for regression to be sustained upon MYC inactivation, the angiogenic switch
had to be turned off through the production of Thrombospondin-1 (TSP-1), a potent
anti-angiogenic factor [52].
TSP-1 is an extracellular matrix glycoprotein known to be secreted by various
cell types such as platelets, endothelial cells, fibroblasts, vascular smooth muscle cells,
bone marrow stromal cells, monocytes and macrophages [64-65]. TSP-1 was the first
endogenous inhibitor of angiogenesis to be identified. It inhibits angiogenesis by
preventing endothelial cell migration, inducing endothelial cell apoptosis and
antagonizing the effect of pro-angiogenic factor VEGF. TSP-1 is a pleiotropic
molecule and its effects are not restricted to inhibition of angiogenesis. Its other
functions include activation of latent TGF-β, suppression of nitric oxide signaling,
modulation of thrombosis, regulations of T-cell chemotaxis, and regulation of
inflammation by influencing the functioning of several immune cell types [66-68].
14
Interestingly, TSP-1 expression is activated by tumor suppressor genes like
p53 and PTEN [69-70] and repressed by oncogenes like RAS, MYC and c-JUN [71-
72]. Downregulation of TSP-1 expression by MYC occurs due to increased TSP-1
mRNA turnover [73]. Thus tumor regression seen upon MYC inactivation in a
conditional model of MYC induced tumorigenesis can be attributed to the angiogenic
switch being turned off by increased TSP-1 expression. Other mechanisms by which
increased TSP-1 expression causes tumor regression include activation of TGF-β and
recruitment of macrophage infiltration to the tumor site [65].
1.5.3 Cellular senescence:
Several years ago, it was noticed that cells growing in culture enter a state of
permanent proliferative arrest after a fixed number of cell divisions [74]. This
irreversible cellular growth arrest is known as cellular senescence and was first
characterized as a mechanism of cellular aging. More recently there has been evidence
that cellular senescence also functions as a mechanism of tumor suppression and can
prevent cells from undergoing neoplastic transformation. Senescent cells have been
identified in pre-malignant and benign tumors but not in malignant tumors suggesting
that cellular senescence could be a barrier to tumor progression [75-77].
It has also been observed that cells that accumulate oncogenic insults and
induce cellular senescence programs as a mechanism of tumor suppression, can only
become malignant if the senescence program is bypassed due to mutations in genes
like p53 and the p16INK4a locus [77]. Oncogenic insults known to drive cells into
senescence are events like RAS or MYC overexpression or DNA damage [78-80].
15
In addition to being an important restraint to tumorigenesis, cellular senescence
has also been implicated as an important mechanism of tumor regression upon
oncogene inactivation. Suppression of MYC expression even in normal fibroblasts can
induce senescence [81]. Wu et. al. have shown that upon inactivation of MYC in
conditional primary tumor models of MYC induced lymphoma and hepatocellular
carcinoma, tumor cells undergo cellular senescence. This is shown by the upregulation
of senescence-associated acidic β-galactosidase (SA β–gal) and the increased
expression cyclin-dependent kinase inhibitors like p16INK4A and p21CIP1. Senescent
tumor cells are also known to up regulate heterochromatin foci [53]. Thus, cellular
senescence has been documented as an important mechanism of regression upon
oncogene inactivation.
Recent studies have revealed that senescent cells develop a secretory
phenotype that results in the secretion of inflammatory mediators such as interleukins,
chemokines, growth factors and proteins that alter the extra-cellular matrix. These
mediators can significantly alter the tumor microenvironment and cause increased
angiogenesis, increased cell motility and proliferation leading to tumor promotion [82-
86].
Several of the mediators of this senescence associated secretory phenotype
(SASP) like MCP-1, IL-8 and IL-15 can cause infiltration of immune cells including
macrophages, natural killer (NK) cells and neutrophils, to the tumor
microenvironment. Depending on the amount of these factors secreted and the type of
immune cells that infiltrate the tumor microenvironment, this could be beneficial or
16
harmful to the tumor. In a model of p53 mediated senescence in the context of a
murine liver tumor, it was shown that senescence causes infiltration of neutrophils and
macrophages which cause senescent liver tumor cells to be cleared away, thus causing
an anti-tumor response [87]. However, mediators secreted by senescent cells are also
known to attract TH-2 polarized T-cells and M2 polarized macrophages which are
known to be tumor promoting [82, 88].
Thus, on one hand, senescence appears to be a mechanism of tumor
suppression by causing irreversible cellular arrest, but on the other, it can also lead to
tumor promotion depending on the SASP of the senescent cells and the context of the
tumor. The parameters that determine the net effect of cellular senescence on a tumor
are not well understood and are most likely governed by complex interactions of
senescent tumor cells with immune cells of the tumor microenvironment. This
duplicity of character is also seen with respect to infiltrating immune cells. Studies
have shown that depending on the polarization of infiltrating immune cells and the
type of tumor they infiltrate, they can either promote or antagonize tumor growth [6-7,
89-91]. The relationship between immune cells and cancer is discussed in further
detail in the next section.
1.6 Interaction of a tumor with its immune microenvironment:
The immune system can be subdivided into the innate immune system and the
adaptive immune system. The innate immune system comprises cells like the
macrophages, dendritic cells, NK cells, mast cells, neutrophils and basophils which are
17
the first line of defense in the body and can recognize danger signals on invading
pathogens and self cells in a non-specific way.
The adaptive immune system comprises the B-cells and the CD4+ and CD8
+ T-
cells. These cells bear receptors that can recognize specific antigens on pathogens or
tumor cells with the help of antigen presenting cells. Cells of the adaptive immune
system can also develop long lived memory against the antigens that they recognize in
order to make subsequent immune responses to the same pathogen faster and more
efficient.
Since early studies of the functionality of the immune system were done with
respect to invading pathogens that were foreign to the body, the interaction of the
immune system with cancer cells that are known to be self cells was largely ignored.
However recent evidence such as increased incidence of tumor formation in
immunocompromised mice and human patients has led to extensive characterization
of the interaction of the immune system with various aspects of tumor formation,
progression and regression [4-5].
We now know that tumor cells interact intimately with the immune system.
Tumors co-evolve with the immune system and while the immune response can
protect the host from tumors by causing tumor cell death, it can also modify the tumor
cells in such a way that eventually tumors can escape the immune response. The
interaction of tumor cells with immune cells has been extensively studied and
Schreiber et al. have proposed the “Cancer Immunoediting” model to understand this
interaction. It is proposed that this interaction occurs in three distinct but continuous
18
phases: Elimination, Equilibrium and Escape. These phases are briefly described
below [4]:
Elimination: As a tumor grows invasively, it elicits a local inflammatory
response and causes innate immune cells like macrophages and NK cells to home to
the site of the tumor. These cells recognize the transformed cells and secrete
cytokines like IFN-γ which causes further tumor cell death. This eventually leads to a
cascade of immune activation that results in the presentation of tumor antigens to T-
cells in the tumor draining lymph nodes and recruitment of tumor antigen specific
CD4+ and CD8
+ T-cells of the adaptive immune system to the site of the tumor. These
cells can now recognize and eliminate the tumor cells. If this elimination is complete,
the host remains tumor free. However, if the elimination is incomplete, the next phase
of interaction (Equilibrium) between the tumor cells and the infiltrating immune cells
ensues.
Equilibrium: Tumor cells that have not been eliminated enter into a dynamic
equilibrium with the infiltrating immune cells. During this phase, the tumor cells do
not proliferate. The immune cells exert a Darwinian selective pressure on the tumor
cells during this phase.
Escape: At the end of the equilibrium phase, in response to the selection
pressure exerted by the immune cells, tumor cell variants arise that can escape the
immune response. These tumor cell variants can now proliferate and manifest as a
clinical tumor in the host.
19
Clinical studies of breast cancer, colon cancer, neuroblastoma and melanoma
have demonstrated that increased presence of tumor infiltrating lymphocytes (TILs)
correlated with better patient survival [92-95]. Mice lacking various components of
the immune system such as RAG1-/-
mice (lacking the adaptive immune system),
perforin-/-
mice (lacking lymphocyte cytotoxicity), and IFN-γ-/-
mice showed increased
incidence of tumor formation in various assays [4, 96]. This suggested that the
immune system, particularly the T-cells, could play a protective role against tumor
formation.
However, there is also ample evidence that various immune cell populations
that are involved in establishing chronic inflammatory responses such as T-cells,
macrophages and mast cells can promote tumor development in certain tumor models.
This can occur either by directly enhancing tumor cell survival or by inhibiting anti-
tumor immune responses in the tumor microenvironment. For example, when
macrophages are depleted in a mouse model of cervical cancer, tumorigenesis
decreases [97]. Creation of a pro-inflammatory environment by these cells occurs
through secretion of pro-inflammatory cytokines like IL-1, IL-6, VEGF and TNF-α.
This leads to the formation of a pro-angiogenic tumor microenvironment which
promotes tumor growth. Chronic inflammation can contribute to tumor initiation
through inducing genotoxic stresses, to tumor maintenance by causing tumor cell
proliferation and to tumor progression by causing tumor cells to be invasive [7].
Additionally, subsets of innate immune cells known as myeloid derived
suppressor cells (MDSCs) and subsets of adaptive immune cells known as regulatory
20
T-cells (T-regs) have been shown to inhibit anti-tumor T-cell function [98-99].
MDSCs inhibit T-cell proliferation by metabolizing L-arginine in the tumor
microenvironment that is required for T-cell proliferation or by producing reactive
oxygen species (ROS) and peroxynitrite which can cause T-cell suppression [100]. T-
regs can either kill activated T-cells or inhibit them from proliferating through
multiple mechanisms including secretion of immunosuppressive cytokines like IL-10
and TGF-β [101-103].
Thus it is clear that the immune system plays a dual role in the tumor
microenvironment. The anti-tumor immune response is counter-acted by the creation
of a pro-tumor microenvironment through the secretion of pro-inflammatory cytokines
and through recruitment of cells that suppress the anti-tumor immune response. The
contribution of the immune system varies depending on the context of the tumor and
the composition of the tumor microenvironment. Moreover different components of
the immune system can be pro- or anti-tumor in the same microenvironment
depending on their temporal and spatial recruitment to the tumor microenvironment
[5].
We have identified anti-tumor CD4+ T-cells as being important to mediate the
effects of oncogene inactivation. This warrants a discussion of the role of CD4+ T-
cells in the tumor microenvironment. CD4+ T-cells often exert their anti-tumor effect
through innate immune cells like macrophages and eosinophils and their roles in the
tumor microenvironment are also discussed below.
21
1.6.1 Role of CD4+ T-cells in the tumor microenvironment
While studying the anti-tumor function of T-cells, focus has largely been on
the CD8+
T-cell population. This has been for three reasons. The first being, CD8+ T
cells are known to be cytotoxic and their ability to kill infected cells has been well
characterized. Moreover CD4+ T-cells are required to provide help to the CD8
+ T-cells
in order for them to perform their cytotoxic function, thus implicating the CD4+ T-
cells with an indirect role in anti-tumor immunity [104]. Secondly, most tumors that
were studied did not express MHC Class II molecules that are required to enable CD4+
T-cells to recognize tumor antigens. On the other hand, MHC Class I molecules,
expressed in all nucleated cells, were found on the surface of most tumor cells. Since
CD8+ T-cells recognize antigen only when presented by MHC Class I molecules, they
were presumed to be the primary mediators of immunity against tumors. Third, studies
using adoptive transfer of purified populations of T-cells into tumor bearing hosts
often showed that CD8+ T-cells could induce an anti-tumor response comparable to
CD4+ and CD8
+ T-cells together, provided that the CD8
+ T-cells were activated. Once
again, this argued for an indirect role of the CD4+ T-cells in this immune response
[105]. For these reasons, the role of CD8+ T-cells has been extensively characterized
in the context of anti-tumor immunity while that of CD4+ T-cells has been under
appreciated.
The earliest reports providing evidence that CD4+ T-cells could exert an anti-
tumor response independent of CD8+ T-cells date back to 1985 [106]. More recent
reports have shown that CD4+ T-cell anti-tumor immunity can be superior to CD8
+ T-
cell anti-tumor immunity in specific tumors [107]. The mechanism by which CD4+ T-
22
cells exert their anti-tumor effect is known to involve macrophages [108] or NK cells
[107] or eosinophils [109] depending on the kind of tumor and the context of CD4+ T-
cell activation. Some reports have also attributed a cytotoxic function to CD4+ T-cells
enabling them to directly kill tumor cells on which MHC Class II expression has been
upregulated by exposure to IFN-γ [110].
Depending on the context in which CD4+ T-cells are activated, they can
differentiate into a range of polarization states as described below (Table 2) [89]. The
polarization state of the CD4+ T-cells and the context of the tumor they infiltrate
determine their role in the tumor microenvironment. For example, increased TH-17
cell infiltration in human hepatocellular carcinoma correlates with a poor prognosis
[111]. Similar pro-tumor roles for TH-17 cells have been reported in mouse models of
mammary and epithelial cancers [112-113]. However in a model of B16 murine
melanoma, it has been shown that TH-17 cells can eradicate established tumors [114].
TH-1 and TH-2 subsets of CD4+ T-cells are also known to elicit anti-tumor
responses [115-116], but it is generally accepted that TH-1 cells play a more important
role in anti-tumor immunity [117] as TH-2 cells have also been implicated in pro-
tumor immunity in several models of cancer [91]. The different immune infiltrating
cells and the various cytokines, chemokines and other immune mediators secreted in
the tumor microenvironment determine whether the net effect of the immune response
is pro- or anti-tumor [118]. The role of CD4+ T-cells in cancer has not yet been clearly
elucidated due to the complex biology of these cells and the different effects they elicit
depending on their polarization and site of action. The work presented in this thesis
23
uncovers a novel role for CD4+ T-cells in remodeling the tumor microenvironment
upon oncogene inactivation.
1.6.2 Role of tumor associated macrophages (TAMs) in the tumor
microenvironment:
Similar to macrophages found in normal tissues, tumor associated
macrophages (TAMs) also have specialized functions. These functions are dependent
on the particular tumor microenvironment and anatomical location of the tumor [119].
Human clinical data from several different cancers such as breast, prostate, kidney and
bladder cancers, has shown that the presence of TAMs is correlated with poor
prognosis of disease [119]. This agrees with data generated from a mouse model of
breast cancer in which mice lacking macrophages, show slower progression of tumors
and lesser incidence of lung metastasis [120]. However, a small number of clinical
studies of TAMs in stomach cancer [121], colorectal cancer [122] and melanoma
[123] have shown that high levels of TAM infiltration correlate with favorable disease
prognosis. This illustrates the different roles that macrophages can play in different
tumor microenvironments.
Cytokine signals in the tumor microenvironment determine TAM functional
polarization. These signals determine macrophage receptor expression and cytokine
production thus determining macrophage function [124]. At any given time, the state
of macrophage polarization can lie in between a spectrum of states, the ends of which
are defined as M1 or classically activated macrophages and M2 or alternatively
activated macrophages. This nomenclature parallels the TH-1/TH-2 polarization states
24
of the CD4+ helper T-cells described earlier [124-125]. TH-1 cytokines like IFN-γ can
induce M1 macrophage polarization either alone or in conjunction with microbial
products like LPS or cytokines like TNF. They produce IL-12, IL-23, IL-1β, TNF, IL-
6 and reactive oxygen and nitrogen species. Reactive nitrogen species are formed due
to metabolism of arginine through the iNOS pathway, a hallmark of M1 macrophages.
These classically activated M1 macrophages can present antigens and can cause tumor
cell cytotoxicity [125].
On the other hand, TH-2 cytokines like IL-4 and IL-13 induce M2 polarization
of macrophages. These macrophages produce IL-10 and show increased expression of
IL-1 decoy receptor and IL-1RA [124, 126]. Alternatively activated macrophages
function to dampen the inflammatory response and enhance tissue remodeling and
repair. These macrophages can cause cellular proliferation through the generation of
ornithine and polyamines by metabolism of arginine through the arginase pathway, a
hallmark of M2 macrophages. These macrophages are responsible for tumor
progression.
TAMs are derived from circulating monocytes in response to monocyte
chemotactic factors derived from the tumor. One such factor that is frequently
produced by several different tumors is CCL2 or monocyte chemotactic protein-1
(MCP-1) [124, 127]. Other tumor derived macrophage chemoattractants include
colony-stimulating factor-1 (CSF-1), CCL3, CCL4, CCL5, CCL7, CCL8 and VEGF
[119]. TAM polarization is often skewed towards the M2 alternative state of activation
25
[128] and these macrophages can influence several aspects of tumor progression and
carcinogenesis as discussed below.
1.6.2.1 TAMs and tumor progression:
Promoting tumor angiogenesis: As described earlier, angiogenesis is required in order
for tumors to grow and become malignant. TAMs are capable of secreting several pro-
angiogenic factors such as VEGF, TNF-α, IL-8 and bFGF. TAMs can also secrete
enzymes that modulate angiogenesis such as MMP-2, MMP-7, MMP-9 and MMP-12
[119]. Furthermore, hypoxic regions of a tumor can also attract TAMs [129]. TAMs
that accumulate in regions of hypoxia upregulate hypoxia inducible transcription
factors HIF-1 and HIF-2 which can induce the expression of angiogenic proteins in
these TAMs. [130-132].
Suppressing the immune response: M1 macrophages can function as antigen
presenting cells and are robust inducers of an anti-tumor immune response; however
TAMs are often M2 macrophages which cannot perform these functions. In fact, M2
TAMs can suppress a T-cell mediated immune response by inhibiting T-cell
proliferation and activation by secreting immunosuppressive cytokines like IL-10,
TGF-β and prostaglandins [133-134].
Tumor cell growth: In various tumors, the presence of TAMs correlates with increased
tumor cell proliferation [135-137]. TAMs are known to upregulate the expression of
factors that promote tumor cell survival and proliferation such as EGF [138], PDGF,
hepatocytes growth factor, TGF-β [139] and bFGF [140].
26
Tumor invasion: TAMs are capable of secreting proteolytic enzymes like cathepsin-b
and various matrix metalloproteinases (MMPs) [139] that can cause areas of
extracellular matrix and basement membrane to breakdown. This allows the tumor
cells to invade into surrounding normal tissue.
Tumor metastasis: The presence of TAMs has also been correlated with increased
tumor metastasis. TAMs contribute to tumor cells leaving their primary site and
growth of tumor cells at secondary sites of metastasis [141-142]. In fact, De nardo
et.al. have done elegant studies in a mouse model of breast carcinoma showing that
IL-4 produced by TH-2 CD4+ T-cells can induce M2 TAMs to express EGF to promote
lung metastases of breast cancer [143].
It is important to note that the pro-tumor functions of TAMs described above
are highly dependent on the cytokine milieu of the tumor microenvironment and
depend on the context of the tumor. Different factors released in the tumor
microenvironment cause macrophages to express different gene programs that
determine their function. This function can also be anti-tumor in certain cases as
described below.
1.6.2.2 TAMs and tumor inhibition:
Although uncommon, certain tumor microenvironments have been shown to
harbor TAMs that can prevent tumor growth through different mechanisms:
Inhibiting tumor angiogenesis: TAMs in certain tumor microenvironments are known
to produce MMP-12. MMP-12 is an enzyme that is known to be involved in the
27
production of the potent angiogenic inhibitor angiostatin [144]. However, most reports
of TAMs in the tumor microenvironment suggest that they are pro-angiogenic.
Promoting tumor cell cytotoxicity and generating an anti-tumor response: In certain
tumor microenvironments, as the tumor grows, it remodels the surrounding stroma,
thus releasing pro-inflammatory cytokines that serve as danger signals to attract and
activate M1 macrophages [145]. These M1 TAMs metabolize L-arginine using the
iNOS enzyme which results in the production of reactive nitric oxide species which
cause tumor cell death [146]. In addition, they produce IL-12 and TNF-α which causes
further tumor cell death. These M1 macrophages can also act as antigen presenting
cells and present tumor antigens to T-cells in the tumor draining lymph nodes causing
activation of anti-tumor T-cells and their infiltration into the tumor thus contributing
to tumor eradication [147]. Macrophages can also interact with NK cells in some
tumor microenvironments to get activated and generate an anti-tumor response [148].
1.6.3 Role of eosinophils in the tumor microenvironment:
Eosinophils are granulocytic cells of the innate immune system. They are
found in circulation in the peripheral blood as well as in mucosal tissues and within
primary and secondary lymphoid organs [149]. These cells contain specialized
granules carrying cytotoxic cationic proteins like major basic protein (MBP) and
eosinophil peroxidase (EPO) in their cytoplasm [150-152]. Additionally, eosinophils
can secrete cytokines like IL-4, IL-5, IL-6, IL-10, IL-13 and TNF-α and G-MCSF.
Eosinophils are also rich sources of various lipid mediators like leukotrienes [149,
153].
28
Secretion of these mediators allows eosinophils to modulate the immune
microenvironment to cause anti-pathogen responses, recruitment of other immune
cells, tissue remodeling and repair. Eosinophils are recruited to their site of action by
IL-5 and eotaxin-1 often secreted by TH-2 cells. Historically eosinophils function as
effector cells of the TH-2 immune response and have been implicated in the immune
response to parasites like helminthes [154] and in allergic responses like asthma [149].
Eosinophils can also function as phagocytic cells and help in the clearance of bacterial
and viral infections [155].
Eosinophils can also influence the tumor microenvironment to generate both
pro- and anti-tumor responses. Like all the other immune cell populations discussed so
far, the contribution of eosinophils to the tumor microenvironment also depends on the
context of the tumor and the specific tumor microenvironment. However, unlike other
immune cell populations, their role in tumor progression has not been extensively
studied. Described below is the evidence for the role of eosinophils in cancer.
1.6.3.1 Eosinophils and tumor progression:
It has been shown that eosinophil ablation in a rodent model of squamous cell
carcinoma, delayed the onset of tumor development and reduced tumor burden
suggesting that eosinophils might play role in tumor promotion [156]. Eosinophils are
also known to play a role in angiogenesis and can secrete VEGF in hypoxic
microenvironments [157]. Eosinophils can also secrete other pro-angiogenic factors
like PDGF, bFGF and IL-6 [149] and have been shown to promote angiogenesis in
29
various pathological conditions involving tissue eosinophilia. It has been proposed that
this can occur in tumors as well [158].
1.6.3.1 Eosinophils and tumor inhibition:
Eosinophils can accumulate in a tumor microenvironment either as a result of a
TH-2 adaptive immune response [109] or independently [159]. Increased eosinophil
infiltration of tumors correlates with increased survival in patients with esophageal
squamous cell carcinoma, gastric cancer, head and neck cancer and colorectal
carcinoma [160] suggesting that eosinophils could play a role in inhibiting tumor
growth. Furthermore, Eotaxin-1/IL-5-/-
mice showed increased MCA induced
tumorigenesis which also suggests an anti-tumor role for eosinophils [161].
Possible mechanisms by which eosinophils can exert an anti-tumor effect
include tumor cell cytotoxicity by releasing eosinophil granules [162] and recruiting
an anti-tumor immune response dependent on eotaxin-1 and STAT-6. Interestingly
eosinophils have been shown to induce a TH-2 anti-tumor response contrary to
evidence that suggests that most TH-2 responses are pro-tumor [109].
30
Figure 1: A bi-transgenic mouse model of conditional MYC expression
Figure 1: A bi-transgenic mouse model of conditional MYC expression. The
tetracycline regulatory system for conditional oncogene expression. Doxycycline
prevents transcription of the target gene, MYC. In this model, the transactivating
protein (tTA is driven by a lymphocyte specific promoter, SRα and immunoglobulin
heavy chain enhancer, Eμ.
31
Table 1: Mechanisms of tumor regression upon oncogene inactivation
ONCOGENE
CANCER
MECHANISM OF
REGRESSION
BCR-ABL
Lymphoblastic leukemia
Apoptosis
c-MYC
T- and B-cell lymphoma,
Acute myeloid leukemia
Cell cycle arrest, Differentiation,
Apoptosis
Osteosarcoma
Differentiation
Hepatocellular
carcinoma
Apoptosis, Differentiation
Pancreatic islet cell
carcinoma
Growth arrest, Differentiation, Cell
adhesion, Vascular collapse
RAS
Melanoma
Apoptosis
Glioblastoma
Apoptosis
MET
Hepatocellular carcinoma
Decreased proliferation, Apoptosis
32
Table 2: CD4+ T-cell polarization and cytokine secretion profile
POLARIZATION
STATE
TRANSCRIPTION
FACTOR
INDUCTION
SECRETION
TH-1
T-bet
IL-12, IFN-γ
IFN-γ, TNF-α, IL-2, IL-10,
MCP-1, MIP1α
TH-2
GATA3
IL-4
IL-4,IL-5, IL-6, IL-10,
IL-25, IL-33
TH-17
ROR-γ
TGF-β, IL6,
IL-21,IL-1,
IL-23
IL-17A,IL-17F, IL-2,
IL-9, IL- 10, IL-21, TNF-α,
CCL-2
T-reg
FoxP3
TGF-β
TGF-β, IL-10
33
1.7 References:
1. Weinstein, I.B., Cancer. Addiction to oncogenes--the Achilles heal of cancer.
Science, 2002. 297(5578): p. 63-4.
2. Weinstein, I.B. and A. Joe, Oncogene addiction. Cancer Res, 2008. 68(9): p.
3077-80; discussion 3080.
3. Druker, B.J., et al., Efficacy and safety of a specific inhibitor of the BCR-ABL
tyrosine kinase in chronic myeloid leukemia. N Engl J Med, 2001. 344(14): p.
1031-7.
4. Dunn, G.P., et al., Cancer immunoediting: from immunosurveillance to tumor
escape. Nat Immunol, 2002. 3(11): p. 991-8.
5. Schreiber, R.D., L.J. Old, and M.J. Smyth, Cancer immunoediting: integrating
immunity's roles in cancer suppression and promotion. Science, 2011.
331(6024): p. 1565-70.
6. de Visser, K.E., A. Eichten, and L.M. Coussens, Paradoxical roles of the
immune system during cancer development. Nat Rev Cancer, 2006. 6(1): p. 24-
37.
7. Grivennikov, S.I., F.R. Greten, and M. Karin, Immunity, inflammation, and
cancer. Cell, 2010. 140(6): p. 883-99.
8. Apetoh, L., et al., Molecular interactions between dying tumor cells and the
innate immune system determine the efficacy of conventional anticancer
therapies. Cancer Res, 2008. 68(11): p. 4026-30.
9. Zitvogel, L., et al., The anticancer immune response: indispensable for
therapeutic success? J Clin Invest, 2008. 118(6): p. 1991-2001.
10. Zitvogel, L., O. Kepp, and G. Kroemer, Immune parameters affecting the
efficacy of chemotherapeutic regimens. Nat Rev Clin Oncol, 2011. 8(3): p.
151-60.
11. Escudier, B., et al., Sorafenib in advanced clear-cell renal-cell carcinoma. N
Engl J Med, 2007. 356(2): p. 125-34.
12. Hudis, C.A., Trastuzumab--mechanism of action and use in clinical practice.
N Engl J Med, 2007. 357(1): p. 39-51.
13. Karasarides, M., et al., B-RAF is a therapeutic target in melanoma. Oncogene,
2004. 23(37): p. 6292-8.
14. Pao, W., et al., EGF receptor gene mutations are common in lung cancers
from "never smokers" and are associated with sensitivity of tumors to gefitinib
and erlotinib. Proc Natl Acad Sci U S A, 2004. 101(36): p. 13306-11.
15. Croce, C.M., Oncogenes and cancer. N Engl J Med, 2008. 358(5): p. 502-11.
16. Adams, J.M., et al., The c-myc oncogene driven by immunoglobulin enhancers
induces lymphoid malignancy in transgenic mice. Nature, 1985. 318(6046): p.
533-8.
17. Alexander, W.S., J.W. Schrader, and J.M. Adams, Expression of the c-myc
oncogene under control of an immunoglobulin enhancer in E mu-myc
transgenic mice. Mol Cell Biol, 1987. 7(4): p. 1436-44.
18. Felsher, D.W. and J.M. Bishop, Reversible tumorigenesis by MYC in
hematopoietic lineages. Mol Cell, 1999. 4(2): p. 199-207.
34
19. Hanahan, D. and R.A. Weinberg, The hallmarks of cancer. Cell, 2000. 100(1):
p. 57-70.
20. Sakaguchi, A.Y., P.A. Lalley, and S.L. Naylor, Human and mouse cellular myc
protooncogenes reside on chromosomes involved in numerical and structural
aberrations in cancer. Somatic Cell Genet, 1983. 9(3): p. 391-405.
21. Gardner, L., Lee, L. and Dang, C., The c-Myc Oncogenic Transcription
Factor. 2002.
22. Oster, S.K., et al., The myc oncogene: MarvelouslY Complex. Adv Cancer Res,
2002. 84: p. 81-154.
23. Erikson, J., et al., Translocation of an immunoglobulin kappa locus to a region
3' of an unrearranged c-myc oncogene enhances c-myc transcription. Proc
Natl Acad Sci U S A, 1983. 80(24): p. 7581-5.
24. Wong, A.J., et al., Gene amplification of c-myc and N-myc in small cell
carcinoma of the lung. Science, 1986. 233(4762): p. 461-4.
25. Boxer, R.B., et al., Lack of sustained regression of c-MYC-induced mammary
adenocarcinomas following brief or prolonged MYC inactivation. Cancer Cell,
2004. 6(6): p. 577-86.
26. Jain, M., et al., Sustained loss of a neoplastic phenotype by brief inactivation of
MYC. Science, 2002. 297(5578): p. 102-4.
27. Pelengaris, S., et al., Reversible activation of c-Myc in skin: induction of a
complex neoplastic phenotype by a single oncogenic lesion. Mol Cell, 1999.
3(5): p. 565-77.
28. Jonkers, J. and A. Berns, Conditional mouse models of sporadic cancer. Nat
Rev Cancer, 2002. 2(4): p. 251-65.
29. Soucek, L. and G.I. Evan, The ups and downs of Myc biology. Curr Opin Genet
Dev, 2010. 20(1): p. 91-5.
30. Littlewood, T.D., et al., A modified oestrogen receptor ligand-binding domain
as an improved switch for the regulation of heterologous proteins. Nucleic
Acids Res, 1995. 23(10): p. 1686-90.
31. Pelengaris, S., M. Khan, and G.I. Evan, Suppression of Myc-induced apoptosis
in beta cells exposes multiple oncogenic properties of Myc and triggers
carcinogenic progression. Cell, 2002. 109(3): p. 321-34.
32. Rudolph, B., A.O. Hueber, and G.I. Evan, Reversible activation of c-Myc in
thymocytes enhances positive selection and induces proliferation and
apoptosis in vitro. Oncogene, 2000. 19(15): p. 1891-900.
33. Giuriato, S., et al., Conditional animal models: a strategy to define when
oncogenes will be effective targets to treat cancer. Semin Cancer Biol, 2004.
14(1): p. 3-11.
34. Freundlieb, S., The Tet System: Powerful, Inducible Gene Expression. 2007.
35. Shachaf, C.M., et al., MYC inactivation uncovers pluripotent differentiation
and tumour dormancy in hepatocellular cancer. Nature, 2004. 431(7012): p.
1112-7.
36. Flores, I., et al., Defining the temporal requirements for Myc in the
progression and maintenance of skin neoplasia. Oncogene, 2004. 23(35): p.
5923-30.
35
37. Tran, P.T., et al., Combined Inactivation of MYC and K-Ras oncogenes
reverses tumorigenesis in lung adenocarcinomas and lymphomas. PLoS One,
2008. 3(5): p. e2125.
38. Weinstein, I.B. and A.K. Joe, Mechanisms of disease: Oncogene addiction--a
rationale for molecular targeting in cancer therapy. Nat Clin Pract Oncol,
2006. 3(8): p. 448-57.
39. Chin, L., et al., Essential role for oncogenic Ras in tumour maintenance.
Nature, 1999. 400(6743): p. 468-72.
40. Fisher, G.H., et al., Induction and apoptotic regression of lung
adenocarcinomas by regulation of a K-Ras transgene in the presence and
absence of tumor suppressor genes. Genes Dev, 2001. 15(24): p. 3249-62.
41. Huettner, C.S., et al., Reversibility of acute B-cell leukaemia induced by BCR-
ABL1. Nat Genet, 2000. 24(1): p. 57-60.
42. Demetri, G.D., et al., Efficacy and safety of imatinib mesylate in advanced
gastrointestinal stromal tumors. N Engl J Med, 2002. 347(7): p. 472-80.
43. Ponzielli, R., et al., Cancer therapeutics: targeting the dark side of Myc. Eur J
Cancer, 2005. 41(16): p. 2485-501.
44. Vita, M. and M. Henriksson, The Myc oncoprotein as a therapeutic target for
human cancer. Semin Cancer Biol, 2006. 16(4): p. 318-30.
45. Sharma, S.V. and J. Settleman, Oncogene addiction: setting the stage for
molecularly targeted cancer therapy. Genes Dev, 2007. 21(24): p. 3214-31.
46. Borrello, M.G., et al., Induction of a proinflammatory program in normal
human thyrocytes by the RET/PTC1 oncogene. Proc Natl Acad Sci U S A,
2005. 102(41): p. 14825-30.
47. Ancrile, B., K.H. Lim, and C.M. Counter, Oncogenic Ras-induced secretion of
IL6 is required for tumorigenesis. Genes Dev, 2007. 21(14): p. 1714-9.
48. Sparmann, A. and D. Bar-Sagi, Ras-induced interleukin-8 expression plays a
critical role in tumor growth and angiogenesis. Cancer Cell, 2004. 6(5): p.
447-58.
49. Reimann, M., et al., Tumor Stroma-Derived TGF-beta Limits Myc-Driven
Lymphomagenesis via Suv39h1-Dependent Senescence. Cancer Cell, 2010.
17(3): p. 262-272.
50. Sodir, N.M., et al., Endogenous Myc maintains the tumor microenvironment.
Genes Dev, 2011. 25(9): p. 907-16.
51. Panaretakis, T., et al., The co-translocation of ERp57 and calreticulin
determines the immunogenicity of cell death. Cell Death Differ, 2008. 15(9): p.
1499-509.
52. Giuriato, S., et al., Sustained regression of tumors upon MYC inactivation
requires p53 or thrombospondin-1 to reverse the angiogenic switch. Proc Natl
Acad Sci U S A, 2006. 103(44): p. 16266-71.
53. Wu, C.H., et al., Cellular senescence is an important mechanism of tumor
regression upon c-Myc inactivation. Proc Natl Acad Sci U S A, 2007. 104(32):
p. 13028-33.
54. Evan, G.I., et al., Induction of apoptosis in fibroblasts by c-myc protein. Cell,
1992. 69(1): p. 119-28.
36
55. Shi, Y., et al., Role for c-myc in activation-induced apoptotic cell death in T
cell hybridomas. Science, 1992. 257(5067): p. 212-4.
56. Kaptein, J.S., et al., Anti-IgM-mediated regulation of c-myc and its possible
relationship to apoptosis. J Biol Chem, 1996. 271(31): p. 18875-84.
57. Hermeking, H. and D. Eick, Mediation of c-Myc-induced apoptosis by p53.
Science, 1994. 265(5181): p. 2091-3.
58. Prasad, V.S., et al., Upregulation of endogenous p53 and induction of in vivo
apoptosis in B-lineage lymphomas of E(mu)-myc transgenic mice by
deregulated c-myc transgene. Mol Carcinog, 1997. 18(2): p. 66-77.
59. Hsu, B., et al., Evidence that c-myc mediated apoptosis does not require wild-
type p53 during lymphomagenesis. Oncogene, 1995. 11(1): p. 175-9.
60. D'Agnano, I., et al., Myc down-regulation induces apoptosis in M14 melanoma
cells by increasing p27(kip1) levels. Oncogene, 2001. 20(22): p. 2814-25.
61. Russo, P., et al., c-myc down-regulation induces apoptosis in human cancer
cell lines exposed to RPR-115135 (C31H29NO4), a non-peptidomimetic
farnesyltransferase inhibitor. J Pharmacol Exp Ther, 2003. 304(1): p. 37-47.
62. Carmeliet, P. and R.K. Jain, Angiogenesis in cancer and other diseases.
Nature, 2000. 407(6801): p. 249-57.
63. Hanahan, D. and J. Folkman, Patterns and emerging mechanisms of the
angiogenic switch during tumorigenesis. Cell, 1996. 86(3): p. 353-64.
64. Chen, D., et al., Vascular smooth muscle cell growth arrest on blockade of
thrombospondin-1 requires p21(Cip1/WAF1). Am J Physiol, 1999. 277(3 Pt
2): p. H1100-6.
65. Martin-Manso, G., et al., Thrombospondin 1 promotes tumor macrophage
recruitment and enhances tumor cell cytotoxicity of differentiated U937 cells.
Cancer Res, 2008. 68(17): p. 7090-9.
66. Lawler, J., The functions of thrombospondin-1 and-2. Curr Opin Cell Biol,
2000. 12(5): p. 634-40.
67. Lawler, J., Thrombospondin-1 as an endogenous inhibitor of angiogenesis and
tumor growth. J Cell Mol Med, 2002. 6(1): p. 1-12.
68. Lawler, J., Thrombospondin 1. UCSD-Nature Molecule Pages, 2010.
69. Dameron, K.M., et al., Control of angiogenesis in fibroblasts by p53
regulation of thrombospondin-1. Science, 1994. 265(5178): p. 1582-4.
70. Schwarte-Waldhoff, I., et al., Smad4/DPC4-mediated tumor suppression
through suppression of angiogenesis. Proc Natl Acad Sci U S A, 2000. 97(17):
p. 9624-9.
71. Tikhonenko, A.T., D.J. Black, and M.L. Linial, Viral Myc oncoproteins in
infected fibroblasts down-modulate thrombospondin-1, a possible tumor
suppressor gene. J Biol Chem, 1996. 271(48): p. 30741-7.
72. Watnick, R.S., et al., Ras modulates Myc activity to repress thrombospondin-1
expression and increase tumor angiogenesis. Cancer Cell, 2003. 3(3): p. 219-
31.
73. Janz, A., et al., Activation of the myc oncoprotein leads to increased turnover
of thrombospondin-1 mRNA. Nucleic Acids Res, 2000. 28(11): p. 2268-75.
37
74. Hayflick, L., The Limited in Vitro Lifetime of Human Diploid Cell Strains. Exp
Cell Res, 1965. 37: p. 614-36.
75. Collado, M., et al., Tumour biology: senescence in premalignant tumours.
Nature, 2005. 436(7051): p. 642.
76. Michaloglou, C., et al., BRAFE600-associated senescence-like cell cycle arrest
of human naevi. Nature, 2005. 436(7051): p. 720-4.
77. Narita, M. and S.W. Lowe, Senescence comes of age. Nat Med, 2005. 11(9): p.
920-2.
78. Chen, Q., et al., Oxidative DNA damage and senescence of human diploid
fibroblast cells. Proc Natl Acad Sci U S A, 1995. 92(10): p. 4337-41.
79. Grandori, C., et al., Werner syndrome protein limits MYC-induced cellular
senescence. Genes Dev, 2003. 17(13): p. 1569-74.
80. Serrano, M., et al., Oncogenic ras provokes premature cell senescence
associated with accumulation of p53 and p16INK4a. Cell, 1997. 88(5): p. 593-
602.
81. Guney, I., S. Wu, and J.M. Sedivy, Reduced c-Myc signaling triggers
telomere-independent senescence by regulating Bmi-1 and p16(INK4a). Proc
Natl Acad Sci U S A, 2006. 103(10): p. 3645-50.
82. Coppe, J.P., et al., The senescence-associated secretory phenotype: the dark
side of tumor suppression. Annu Rev Pathol, 2010. 5: p. 99-118.
83. Coppe, J.P., et al., Senescence-associated secretory phenotypes reveal cell-
nonautonomous functions of oncogenic RAS and the p53 tumor suppressor.
PLoS Biol, 2008. 6(12): p. 2853-68.
84. Krtolica, A., et al., Senescent fibroblasts promote epithelial cell growth and
tumorigenesis: a link between cancer and aging. Proc Natl Acad Sci U S A,
2001. 98(21): p. 12072-7.
85. Parrinello, S., et al., Stromal-epithelial interactions in aging and cancer:
senescent fibroblasts alter epithelial cell differentiation. J Cell Sci, 2005.
118(Pt 3): p. 485-96.
86. Coppe, J.P., et al., Secretion of vascular endothelial growth factor by primary
human fibroblasts at senescence. J Biol Chem, 2006. 281(40): p. 29568-74.
87. Xue, W., et al., Senescence and tumour clearance is triggered by p53
restoration in murine liver carcinomas. Nature, 2007. 445(7128): p. 656-60.
88. Allavena, P., et al., The inflammatory micro-environment in tumor
progression: the role of tumor-associated macrophages. Crit Rev Oncol
Hematol, 2008. 66(1): p. 1-9.
89. Muranski, P. and N.P. Restifo, Adoptive immunotherapy of cancer using
CD4(+) T cells. Curr Opin Immunol, 2009. 21(2): p. 200-8.
90. DeNardo, D.G., P. Andreu, and L.M. Coussens, Interactions between
lymphocytes and myeloid cells regulate pro- versus anti-tumor immunity.
Cancer Metastasis Rev, 2010. 29(2): p. 309-16.
91. Johansson, M., D.G. Denardo, and L.M. Coussens, Polarized immune
responses differentially regulate cancer development. Immunol Rev, 2008.
222: p. 145-54.
38
92. Clemente, C.G., et al., Prognostic value of tumor infiltrating lymphocytes in
the vertical growth phase of primary cutaneous melanoma. Cancer, 1996.
77(7): p. 1303-10.
93. Naito, Y., et al., CD8+ T cells infiltrated within cancer cell nests as a
prognostic factor in human colorectal cancer. Cancer Res, 1998. 58(16): p.
3491-4.
94. Rilke, F., et al., Prognostic significance of HER-2/neu expression in breast
cancer and its relationship to other prognostic factors. Int J Cancer, 1991.
49(1): p. 44-9.
95. Palma, L., N. Di Lorenzo, and B. Guidetti, Lymphocytic infiltrates in primary
glioblastomas and recidivous gliomas. Incidence, fate, and relevance to
prognosis in 228 operated cases. J Neurosurg, 1978. 49(6): p. 854-61.
96. Dunn, G.P., C.M. Koebel, and R.D. Schreiber, Interferons, immunity and
cancer immunoediting. Nat Rev Immunol, 2006. 6(11): p. 836-48.
97. Giraudo, E., M. Inoue, and D. Hanahan, An amino-bisphosphonate targets
MMP-9-expressing macrophages and angiogenesis to impair cervical
carcinogenesis. J Clin Invest, 2004. 114(5): p. 623-33.
98. Gabrilovich, D.I., et al., Mechanism of immune dysfunction in cancer mediated
by immature Gr-1+ myeloid cells. J Immunol, 2001. 166(9): p. 5398-406.
99. Shimizu, J., S. Yamazaki, and S. Sakaguchi, Induction of tumor immunity by
removing CD25+CD4+ T cells: a common basis between tumor immunity and
autoimmunity. J Immunol, 1999. 163(10): p. 5211-8.
100. Gabrilovich, D.I. and S. Nagaraj, Myeloid-derived suppressor cells as
regulators of the immune system. Nat Rev Immunol, 2009. 9(3): p. 162-74.
101. Marie, J.C., et al., TGF-beta1 maintains suppressor function and Foxp3
expression in CD4+CD25+ regulatory T cells. J Exp Med, 2005. 201(7): p.
1061-7.
102. Sakaguchi, S., et al., Regulatory T cells: how do they suppress immune
responses? Int Immunol, 2009. 21(10): p. 1105-11.
103. Bopp, T., et al., Cyclic adenosine monophosphate is a key component of
regulatory T cell-mediated suppression. J Exp Med, 2007. 204(6): p. 1303-10.
104. Ossendorp, F., et al., Specific T helper cell requirement for optimal induction
of cytotoxic T lymphocytes against major histocompatibility complex class II
negative tumors. J Exp Med, 1998. 187(5): p. 693-702.
105. Melief, C.J., Tumor eradication by adoptive transfer of cytotoxic T
lymphocytes. Adv Cancer Res, 1992. 58: p. 143-75.
106. Greenberg, P.D., D.E. Kern, and M.A. Cheever, Therapy of disseminated
murine leukemia with cyclophosphamide and immune Lyt-1+,2- T cells. Tumor
eradication does not require participation of cytotoxic T cells. J Exp Med,
1985. 161(5): p. 1122-34.
107. Perez-Diez, A., et al., CD4 cells can be more efficient at tumor rejection than
CD8 cells. Blood, 2007. 109(12): p. 5346-54.
108. Corthay, A., et al., Primary antitumor immune response mediated by CD4+ T
cells. Immunity, 2005. 22(3): p. 371-83.
39
109. Mattes, J., et al., Immunotherapy of cytotoxic T cell-resistant tumors by T
helper 2 cells: an eotaxin and STAT6-dependent process. J Exp Med, 2003.
197(3): p. 387-93.
110. Heller, K.N., C. Gurer, and C. Munz, Virus-specific CD4+ T cells: ready for
direct attack. J Exp Med, 2006. 203(4): p. 805-8.
111. Zhang, J.P., et al., Increased intratumoral IL-17-producing cells correlate with
poor survival in hepatocellular carcinoma patients. J Hepatol, 2009. 50(5): p.
980-9.
112. Nam, J.S., et al., Transforming growth factor beta subverts the immune system
into directly promoting tumor growth through interleukin-17. Cancer Res,
2008. 68(10): p. 3915-23.
113. Xiao, M., et al., IFNgamma promotes papilloma development by up-regulating
Th17-associated inflammation. Cancer Res, 2009. 69(5): p. 2010-7.
114. Muranski, P., et al., Tumor-specific Th17-polarized cells eradicate large
established melanoma. Blood, 2008. 112(2): p. 362-73.
115. Hung, K., et al., The central role of CD4(+) T cells in the antitumor immune
response. J Exp Med, 1998. 188(12): p. 2357-68.
116. Nishimura, T., et al., Distinct role of antigen-specific T helper type 1 (Th1) and
Th2 cells in tumor eradication in vivo. J Exp Med, 1999. 190(5): p. 617-27.
117. Nishimura, T., et al., The critical role of Th1-dominant immunity in tumor
immunology. Cancer Chemother Pharmacol, 2000. 46 Suppl: p. S52-61.
118. Coussens, L.M. and Z. Werb, Inflammation and cancer. Nature, 2002.
420(6917): p. 860-7.
119. Lewis, C.E. and J.W. Pollard, Distinct role of macrophages in different tumor
microenvironments. Cancer Res, 2006. 66(2): p. 605-12.
120. Lin, E.Y., et al., Colony-stimulating factor 1 promotes progression of
mammary tumors to malignancy. J Exp Med, 2001. 193(6): p. 727-40.
121. Ohno, S., et al., The degree of macrophage infiltration into the cancer cell nest
is a significant predictor of survival in gastric cancer patients. Anticancer Res,
2003. 23(6D): p. 5015-22.
122. Funada, Y., et al., Prognostic significance of CD8+ T cell and macrophage
peritumoral infiltration in colorectal cancer. Oncol Rep, 2003. 10(2): p. 309-
13.
123. Piras, F., et al., The predictive value of CD8, CD4, CD68, and human
leukocyte antigen-D-related cells in the prognosis of cutaneous malignant
melanoma with vertical growth phase. Cancer, 2005. 104(6): p. 1246-54.
124. Mantovani, A., et al., Macrophage polarization: tumor-associated
macrophages as a paradigm for polarized M2 mononuclear phagocytes.
Trends Immunol, 2002. 23(11): p. 549-55.
125. Mantovani, A., et al., The chemokine system in diverse forms of macrophage
activation and polarization. Trends Immunol, 2004. 25(12): p. 677-86.
126. Gordon, S. and P.R. Taylor, Monocyte and macrophage heterogeneity. Nat
Rev Immunol, 2005. 5(12): p. 953-64.
127. Conti, I. and B.J. Rollins, CCL2 (monocyte chemoattractant protein-1) and
cancer. Seminars in Cancer Biology, 2004. 14(3): p. 149-154.
40
128. Sica, A., et al., Tumour-associated macrophages are a distinct M2 polarised
population promoting tumour progression: potential targets of anti-cancer
therapy. Eur J Cancer, 2006. 42(6): p. 717-27.
129. Murdoch, C., A. Giannoudis, and C.E. Lewis, Mechanisms regulating the
recruitment of macrophages into hypoxic areas of tumors and other ischemic
tissues. Blood, 2004. 104(8): p. 2224-34.
130. Burke, B., et al., Expression of HIF-1alpha by human macrophages:
implications for the use of macrophages in hypoxia-regulated cancer gene
therapy. J Pathol, 2002. 196(2): p. 204-12.
131. White, J.R., et al., Genetic amplification of the transcriptional response to
hypoxia as a novel means of identifying regulators of angiogenesis. Genomics,
2004. 83(1): p. 1-8.
132. Lewis, J.S., et al., Expression of vascular endothelial growth factor by
macrophages is up-regulated in poorly vascularized areas of breast
carcinomas. J Pathol, 2000. 192(2): p. 150-8.
133. Sica, A., et al., Autocrine production of IL-10 mediates defective IL-12
production and NF-kappa B activation in tumor-associated macrophages. J
Immunol, 2000. 164(2): p. 762-7.
134. Kambayashi, T., et al., Potential involvement of IL-10 in suppressing tumor-
associated macrophages. Colon-26-derived prostaglandin E2 inhibits TNF-
alpha release via a mechanism involving IL-10. J Immunol, 1995. 154(7): p.
3383-90.
135. Tsutsui, S., et al., Macrophage infiltration and its prognostic implications in
breast cancer: the relationship with VEGF expression and microvessel density.
Oncol Rep, 2005. 14(2): p. 425-31.
136. Wang, F.Q., et al., Matrilysin (MMP-7) promotes invasion of ovarian cancer
cells by activation of progelatinase. Int J Cancer, 2005. 114(1): p. 19-31.
137. Hamada, I., et al., Clinical effects of tumor-associated macrophages and
dendritic cells on renal cell carcinoma. Anticancer Res, 2002. 22(6C): p.
4281-4.
138. Goswami, S., et al., Macrophages promote the invasion of breast carcinoma
cells via a colony-stimulating factor-1/epidermal growth factor paracrine
loop. Cancer Res, 2005. 65(12): p. 5278-83.
139. Pollard, J.W., Tumour-educated macrophages promote tumour progression
and metastasis. Nat Rev Cancer, 2004. 4(1): p. 71-8.
140. Lewis, C. and C. Murdoch, Macrophage responses to hypoxia: implications
for tumor progression and anti-cancer therapies. Am J Pathol, 2005. 167(3):
p. 627-35.
141. Leek, R.D., et al., Association of macrophage infiltration with angiogenesis
and prognosis in invasive breast carcinoma. Cancer Res, 1996. 56(20): p.
4625-9.
142. Hanada, T., et al., Prognostic value of tumor-associated macrophage count in
human bladder cancer. Int J Urol, 2000. 7(7): p. 263-9.
41
143. DeNardo, D.G., et al., CD4(+) T cells regulate pulmonary metastasis of
mammary carcinomas by enhancing protumor properties of macrophages.
Cancer Cell, 2009. 16(2): p. 91-102.
144. O'Reilly, M.S., et al., Angiostatin: a novel angiogenesis inhibitor that mediates
the suppression of metastases by a Lewis lung carcinoma. Cell, 1994. 79(2): p.
315-28.
145. Lamagna, C., M. Aurrand-Lions, and B.A. Imhof, Dual role of macrophages
in tumor growth and angiogenesis. J Leukoc Biol, 2006. 80(4): p. 705-13.
146. Keller, R., M. Geiges, and R. Keist, L-arginine-dependent reactive nitrogen
intermediates as mediators of tumor cell killing by activated macrophages.
Cancer Res, 1990. 50(5): p. 1421-5.
147. Dunn, G.P., L.J. Old, and R.D. Schreiber, The immunobiology of cancer
immunosurveillance and immunoediting. Immunity, 2004. 21(2): p. 137-48.
148. Diefenbach, A., et al., Ligands for the murine NKG2D receptor: expression by
tumor cells and activation of NK cells and macrophages. Nat Immunol, 2000.
1(2): p. 119-26.
149. Shamri, R., J.J. Xenakis, and L.A. Spencer, Eosinophils in innate immunity: an
evolving story. Cell Tissue Res, 2011. 343(1): p. 57-83.
150. Gleich, G.J., D.A. Loegering, and J.E. Maldonado, Identification of a major
basic protein in guinea pig eosinophil granules. J Exp Med, 1973. 137(6): p.
1459-71.
151. Carlson, M.G., C.G. Peterson, and P. Venge, Human eosinophil peroxidase:
purification and characterization. J Immunol, 1985. 134(3): p. 1875-9.
152. Larson, K.A., et al., The identification and cloning of a murine major basic
protein gene expressed in eosinophils. J Immunol, 1995. 155(6): p. 3002-12.
153. Bandeira-Melo, C. and P.F. Weller, Eosinophils and cysteinyl leukotrienes.
Prostaglandins Leukot Essent Fatty Acids, 2003. 69(2-3): p. 135-43.
154. Klion, A.D. and T.B. Nutman, The role of eosinophils in host defense against
helminth parasites. J Allergy Clin Immunol, 2004. 113(1): p. 30-7.
155. Cline, M.J., J. Hanifin, and R.I. Lehrer, Phagocytosis by human eosinophils.
Blood, 1968. 32(6): p. 922-34.
156. Wong, D.T., et al., Eosinophil ablation and tumor development. Oral Oncol,
1999. 35(5): p. 496-501.
157. Nissim Ben Efraim, A.H., R. Eliashar, and F. Levi-Schaffer, Hypoxia
modulates human eosinophil function. Clin Mol Allergy, 2010. 8: p. 10.
158. Samoszuk, M., Eosinophils and human cancer. Histol Histopathol, 1997.
12(3): p. 807-12.
159. Cormier, S.A., et al., Pivotal Advance: eosinophil infiltration of solid tumors is
an early and persistent inflammatory host response. J Leukoc Biol, 2006.
79(6): p. 1131-9.
160. Ellyard, J.I., L. Simson, and C.R. Parish, Th2-mediated anti-tumour immunity:
friend or foe? Tissue Antigens, 2007. 70(1): p. 1-11.
161. Simson, L., et al., Regulation of carcinogenesis by IL-5 and CCL11: a
potential role for eosinophils in tumor immune surveillance. J Immunol, 2007.
178(7): p. 4222-9.
42
162. Huland, E. and H. Huland, Tumor-associated eosinophilia in interleukin-2-
treated patients: evidence of toxic eosinophil degranulation on bladder cancer
cells. J Cancer Res Clin Oncol, 1992. 118(6): p. 463-7.
43
CHAPTER 2: CONTRIBUTION OF
THE IMMUNE SYSTEM TO
ONCOGENE INACTIVATION
MEDIATED TUMOR REGRESSION
Portions of this chapter are adapted from "CD4+ T-Cells Contribute
to the Remodeling of the Microenvironment Required for Sustained
Tumor Regression upon Oncogene Inactivation"
Kavya Rakhra*, Pavan Bachireddy*, Tahera Zabuawala, Robert Zeiser,
Liwen Xu,1 Andrew Kopelman, Alice C. Fan, Qiwei Yang, Lior
Braunstein, Erika Crosby, Sandra Ryeom, and Dean W. Felsher
Cancer Cell, 18: 485-498, November 2010
*Authors contributed equally
44
2.1 Overview:
Using the conditionally regulatable model of MYC induced T-cell lymphoma
(Eµ-tTA X tet-O-MYC) described earlier [1], experiments were designed to investigate
the role of the adaptive immune system during tumor regression upon MYC
inactivation. Data were generated using both transplanted and primary tumor models
and a compelling role for CD4+ T-cells in remodeling the tumor microenvironment
upon MYC inactivation was identified. CD4+ T-cells remodel the tumor
microenvironment to cause inhibition of angiogenesis and induction of cellular
senescence programs which is required for sustained tumor regression to occur upon
MYC inactivation. These results were also extended to a conditionally regulatable
model of BCR-ABL induced pro-B-cell lymphocytic leukemia [2].
2.2 Contribution of the adaptive immune system:
The adaptive immune system is required for rapid, complete and sustained tumor
regression upon MYC inactivation:
The Eµ-tTAXtet-O-MYC murine model of lymphoma is an excellent model to
study the addiction of tumor cells to the MYC oncogene since the expression of MYC
can be spatially and temporally regulated. In this model, when MYC is turned on from
birth, mice develop lymphoma within 8-10 weeks. Upon MYC inactivation, the
established lymphoma undergoes sustained regression [1]. Tumor cell lines were
generated from primary tumors derived from this mouse model and transduced with a
luciferase transgene. This allowed transplantation of these MYC dependent tumors into
45
various immunocompetent and immmunodeficient hosts and allowed us to monitor
tumor growth and regression kinetics using bioluminescence imaging [3].
Tumor cell lines were transplanted into wildtype (WT) immunocompetent
hosts and into hosts with different immunodeficiencies such as RAG1-/-
, SCID,
RAG2cγc-/-
, CD4-/-
CD8-/-
, CD4-/-
and CD8-/-
hosts. Tumors were allowed to grow to a
comparable size in all cohorts of mice, as quantified by average radiance, a measure of
bioluminescence signal. At this stage, MYC was inactivated by treating these mice
with doxycycline (dox). Dox was administered both intra-peritoneally (i.p.) and orally
in the drinking water. We found that initial tumor regression upon oncogene
inactivation, occurred both in the presence and in the absence of the immune system as
tumors regressed in all cohorts of mice (Figure 1A, B). However, the quality and
extent of tumor regression varied significantly between WT and immunodeficient
hosts.
Severely immune compromised hosts (SCID and RAG2-/-
cc-/-
mice deficient
in the adaptive immune system and NK cells) demonstrated significantly delayed
kinetics of tumor regression upon MYC inactivation compared to wild-type (WT) hosts
Figure 1 D, SCID versus WT p < 0.001) and failed to execute complete tumor
elimination with up to 1,000-fold more minimal residual disease (MRD) after MYC
inactivation (Figure 1E, SCID versus WT, p < 0.001; RAG2-/-
cc-/-
versus WT p =
0.01 at the nadir of luciferase activity upon MYC inactivation). Similarly, less severely
immune compromised hosts (RAG1-/-
and CD4-/-
CD8-/-
) also exhibited delayed
kinetics (Figure 1B, D, RAG1-/-
versus WT, p = 0.02; CD4-/-
CD8-/-
versus WT, p =
46
0.02) and a significantly increased MRD (Figure 1E, RAG1-/-
versus WT, p = 0.01;
CD4-/-
CD8-/-
versus WT, p < 0.01). Hence, an intact immune system is required for
rapid and complete tumor regression.
Previously, we have described that after MYC inactivation some tumors will
recur within 2 months [4-5]. To determine if host immune status influenced the
frequency of tumor recurrence, we continued to observe mice for 80 days after MYC
inactivation. We observed that tumors recurred at a statistically significant increase in
frequency in SCID, RAG2-/-
cc-/-
, RAG1-/-
, and CD4-/-
CD8-/-
hosts (87.5%, 100%,
100%, and 80% respectively) compared to WT hosts (9%) (Figure 1F, immune
compromised hosts versus WT, p < 0.0001). Surprisingly, CD4-/-
but not CD8-/-
hosts
exhibited a significant influence on tumor recurrence (Figure 1F, 28.5%, 0%
respectively). Correspondingly, CD4+, but not CD8
+ T-cell deficiency alone was
sufficient to impede sustained tumor regression compared to WT mice (Figure 1F, WT
versus CD4-/-
p = 0.02). Similar results could be obtained using non-luciferase labeled
tumors (Figure 2A). By qPCR analysis it was confirmed that doxycycline treatment
resulted in similar suppression of transgenic MYC expression regardless of host
immune status (Figure 2B). Hence, defects in the host immune system prevented
sustained tumor regression upon MYC inactivation. Specifically a deficiency in CD4+
T-cells alone was sufficient to increase tumor recurrence upon oncogene inactivation.
47
CD4+ T-cells home to the tumor and are sufficient to restore sustained tumor
regression:
We further investigated the notion that CD4+ T-cells were playing a critical
role in the mechanism by which MYC inactivation was inducing tumor regression.
First, we examined if CD4+ T-cells were homing to the tumor site upon oncogene
inactivation. Upon adoptive transfer into RAG1-/-
hosts, luciferase+ CD4
+ T-cells
rapidly localized to the tumor site upon MYC inactivation as seen by bioluminescence
imaging of these tumors before and after MYC inactivation (Figure 3A). Inactivating
this oncogene causes CD4+ T-cells to localize at the tumor site as early as 4 days after
oncogene inactivation, peak at day 12 and persist up to 3 weeks after MYC
inactivation. Thus, MYC inactivation is associated with trafficking of CD4+ T-cells to
sites of tumor involvement. Notably, CD4+ T-cell depleted luciferase
+ splenocytes also
localized to the site of the tumor upon MYC inactivation, suggesting the recruitment of
additional host immune effector populations (Figure 4A).
Next, we evaluated if we could restore the ability of MYC inactivation to
induce sustained tumor regression in immune compromised hosts by adoptively
transferring specific lymphocyte populations into RAG1-/-
mice. By FACS analysis,
we confirmed reconstitution of effector cells (Figure 4B). As expected, RAG1-/-
mice
adoptively transferred with splenic lymphocytes exhibited sustained regression (Figure
3B). As described above, RAG1-/-
hosts demonstrated a significant amount of MRD
after MYC inactivation compared to WT hosts (Figure 3B, C, RAG1-/-
versus WT, p =
0.007). Importantly, reconstitution of immunodeficient hosts with naive CD8+ T-cells
48
continued to have a significant burden of MRD (Figure 3C, RAG1-/-
CD8+ versus WT,
p = 0.03) whereas reconstitution of RAG1-/-
hosts with naive CD4+ T-cells completely
eliminated MRD, similar to WT hosts upon MYC inactivation (Figure 3C, RAG1-/-
CD4+ versus WT, p = 0.09). Moreover, RAG1
-/- hosts adoptively transferred with
CD4+ T-cells exhibited statistically significant prolonged tumor-free survival
compared to RAG1-/-
or RAG1-/-
hosts reconstituted with CD8+
T-cells (Figure 3B, D,
RAG1-/-
versus RAG1-/-
CD4+ p = 0.007, RAG1
-/-CD4
+ versus RAG1
-/-CD8
+ p = 0.03).
Hence, restoration of CD4+ T-cells alone was sufficient for the ability of MYC
inactivation to eliminate MRD and induce sustained tumor regression.
The adaptive immune system is not required to induce proliferative arrest or apoptosis:
Previously, we have shown that upon MYC inactivation in a transgenic model
of T-ALL, tumor cells undergo proliferative arrest and apoptosis [1]. We determined if
the mechanism by which immune cells were contributing to the process of tumor
regression was through effects on proliferation and apoptosis of tumor cells before and
after MYC inactivation (Figure 5A, B). After 4 days of MYC inactivation, tumors from
both wildtype and immunodeficient hosts exhibited an overall loss of pleomorphic
characteristics as evidenced by a marked reduction in cell size and nuclear to
cytoplasmic ratio, similarly in both cohorts. Importantly, we observed that upon MYC
inactivation there were marked changes in the total number of cells per field and we
carefully controlled for these changes in our quantification of TUNEL and Ki67
staining to measure apoptosis and proliferation respectively.
49
To measure apoptosis, TUNEL staining was performed. Apoptosis occurred
equivalently upon MYC inactivation regardless of host immune status (Figure 5A)
suggesting that initial tumor regression occurs similarly regardless of the presence or
absence of an immune system. This suggests that initial tumor regression might be a
cell autonomous process. Quantification of TUNEL staining revealed a 2-fold increase
in the extent of apoptosis upon MYC inactivation in tumors from WT hosts (Figure
5B, WT MYC On versus Off, p = 0.05). Moreover, the apoptosis in regressing tumors
from WT hosts was not significantly different from that of regressing tumors in either
RAG1-/-
or CD4-/-
hosts (Figure 5B, WT versus RAG1-/-
, CD4-/-
MYC Off, p = 0.3 and
0.3 respectively). Finally there was a small but statistically insignificant increase in the
levels of apoptosis upon MYC inactivation in RAG1-/-
and CD4-/-
hosts (Figure 5B,
RAG1-/-
, CD4-/-
MYC On versus Off, p = 0.07 and 0.09 respectively). Hence, the
absence of the immune system does not seem to impede apoptosis of tumor cells upon
MYC inactivation.
Next, changes in cellular proliferation upon MYC inactivation were measured
by Ki67 staining. MYC inactivation in tumors from both WT and immunodeficient
hosts resulted in a significant reduction in Ki67 staining (Figure 5A, B, WT, RAG1-/-
,
CD4-/-
MYC On versus MYC Off, p < 0.01). Interestingly, in comparison to WT hosts,
RAG1-/-
but not CD4-/-
hosts, underwent a statistically significant further decrease in
Ki67 staining upon MYC inactivation (WT versus RAG1-/-
or CD4-/-
MYC Off p = 0.02
or p < 0.05, respectively). Thus, the absence of the host immune system either has no
effect or modestly enhanced the effect of MYC inactivation in inducing proliferative
arrest.
50
The adaptive immune system is required to inhibit angiogenesis in the tumor
microenvironment:
Since we could not account for the influence of host immune status on tumor
regression and recurrence upon MYC inactivation through effects on apoptosis or
proliferative arrest, we considered that other mechanisms were likely to be
responsible. We have previously reported that sustained regression of tumors upon
MYC inactivation requires the angiogenic switch to be turned off and this is achieved
by the secretion of anti-angiogenic extra-cellular matrix glycoprotein thrombospondin-
1 (TSP-1) [4]. We examined if an intact host immune system was required for MYC
inactivation to induce the shutdown of angiogenesis through the secretion of TSP-1.
We quantified TSP-1 expression in transplanted tumors before and after MYC
inactivation from WT, RAG1-/-
and CD4-/-
hosts, using immunohistochemistry. Upon
MYC inactivation there was a robust 3.5-fold induction of TSP-1 in tumors from WT
hosts but not in RAG1-/-
or CD4-/-
hosts (Figure 6A, WT versus RAG1-/-
, CD4-/-
MYC
Off, p = 0.001). Furthermore, while tumors in WT mice demonstrated very little
change in mean vascular density (MVD) as measured by CD31 staining upon MYC
inactivation (Figure 6B), RAG1-/-
and CD4-/-
mice exhibited a 5- and 12-fold, increase
respectively, in tumor MVD upon MYC inactivation (Figure 6A, B, RAG1-/-
MYC On
versus Off, p < 0.0001; CD4-/-
MYC On versus Off, p = 0.07). We also found that
tumors from CD8-/-
hosts showed induction of TSP-1 expression upon MYC
inactivation (data not shown). Thus, the absence of host immune effectors, in
51
particular CD4+ T-cells, markedly impairs the ability of MYC inactivation to shut
down angiogenesis.
Finally, our results suggested that TSP-1 expression requires host immune
cells and specifically CD4+ T cells. Indeed, we found that TSP-1 protein expression is
markedly decreased in spleens of immune compromised versus wild type hosts (Figure
6C). Upon isolating CD4+ T-cells from wild type spleens, we show that TSP-1
production is induced upon activation of CD4+ T-cells (Figure 6D), thus
demonstrating that CD4+ T-cells are a source of TSP-1.
Thrombospondin expression is required for sustained tumor regression upon MYC
inactivation:
Our results suggested to us the possibility that specific cytokines may be
critical to the remodeling of the tumor and the tumor microenvironment upon MYC
inactivation. We used two approaches to investigate the role of TSP-1. First, we
reconstituted RAG1-/-
mice with splenocytes from either TSP-1,2+/+
(WT) or TSP-1,2-/-
mice. Both TSP-1 and 2 have been implicated in the inhibition of angiogenesis and
have similar structural domains [6-7]. By FACS analysis, we verified equivalent
immune reconstitution (Figure 7A). Indeed, RAG1-/-
mice reconstituted with
TSP-1,2-/-
splenocytes completely failed to protect from sustained tumor regression
upon MYC inactivation compared to RAG1-/-
mice reconstituted with WT splenocytes
(Figure 7B, relapse rate WT versus TSP-1,2-/-
, 10% versus 100%, p = 0.02). We
conclude that TSP expression in immune effectors is important for sustained tumor
regression upon MYC inactivation.
52
Next, we addressed whether we could bypass the requirement for TSP-1
expression from host immune cells, by artificially introducing TSP-1 into tumor cells.
We compared tumor recurrence upon MYC inactivation in RAG1-/-
hosts transplanted
with tumors transduced with an empty vector control versus tumors transduced with a
TSP-1 expression vector. TSP-1 overexpressing tumors exhibited a delay in kinetics
(mean latency 80 versus 102 days) and a decreased frequency of tumor recurrence
(100% versus 40%) resulting in a statistically significant survival advantage (Figure
7C, RAG1-/-
TSP-1+ versus RAG1
-/-, p = 0.02). Thus, TSP-1 overexpression of tumor
cells is sufficient to increase the duration and frequency of sustained tumor regression
upon MYC inactivation in immune compromised hosts.
Encouraged by these results, we used our tumor model to study the effect of
3TSR, a synthetic peptide based drug designed to incorporate the three
thrombospondin repeats (TSR) that are known to mediate the anti-angiogenic activity
of TSP-1 [8]. We transplanted tumor cell lines into 2 cohorts of SCID mice. After
tumors had reached a comparable size, we inactivated MYC in both cohorts. Upon
MYC inactivation, one cohort was treated with a daily injection of 3 TSR (a gift from
Jack Lawler) while the other cohort received a mock injection containing PBS. When
we compared tumor recurrence upon MYC inactivation, we found that median survival
in the control cohort was 18 days compared to 23 days in the cohort treated with 3
TSR. Although there was a slight increase in survival with 3 TSR drug treatment, this
was not found to be statistically significant (Figure 7D).
53
The adaptive immune system is required to induce cellular senescence upon MYC
inactivation:
Another mechanism that contributes to sustained tumor regression upon
oncogene inactivation, in this conditional model of MYC induced lymphoma is the
induction of cellular senescence [9]. Since cellular senescence is known to be
governed by the inflammatory signaling pathway [10-12], we investigated whether
induction of cellular senescence was influenced by the immune system. We
investigated this by assaying for levels of senescence-associated acidic β-gal (SA- β -
Gal) activity and the induction of cyclin dependent kinase inhibitors like p16INK4a
and p21. Tumors from WT hosts expressed a 20-fold increase in (SA-β-Gal) activity
upon MYC inactivation and demonstrated a 26- and 6-fold increase in senescence-
associated markers, p16INK4a and p21, respectively, upon MYC inactivation (Figure
8A, B). In contrast, MYC inactivation in tumors from RAG1-/-
and CD4-/-
mice did not
result in an increase in SA-ß-Gal or in the induction of p16INK4a or p21 (Figure 8A,
B, WT versus RAG1-/-
MYC Off SA-ß-Gal p = 0.01, p16 staining p = 0.002, p21
staining p = 0.01. WT versus CD4-/-
MYC Off SA- ß-Gal, p = 0.009, p16 staining, p =
0.0005, p21 staining, p = 0.004). Thus, in immune deficient mice, MYC inactivation is
impeded from inducing cellular senescence in tumor cells. Notably, CD4+ T-cells
specifically appeared to be required for this induction of cellular senescence.
54
Understanding the cytokine profile in the tumor microenvironment in the presence and
absence of the immune system:
The processes of angiogenesis and cellular senescence are governed by the
secretion of several different cytokines. The angiogenic switch is determined by a
delicate balance of pro-angiogenic factors like VEGF and anti-angiogenic factors like
TSP-1 [13]. Similarly recent findings have suggested that cytokine loops might drive
cellular senescence [14]. We now know that cytokines like IL-6 and IL-8 are required
for both replicative and oncogene induced senescence to occur [10-11].
In our tumor model, we have demonstrated that the presence of the immune
system is required for the shutdown of angiogenesis and induction of cellular
senescence to occur upon MYC inactivation. Moreover, cytokines are the key
mediators of immune cell functions. Thus, we decided to assay levels of various
cytokines in the tumor microenvironment before and after MYC inactivation, in the
presence and absence of an immune system using the luminex assay. The limitation of
this assay is that we were only able to gauge levels of the 21 mouse cytokines that this
assay has been optimized for.
We measured relative fold changes in cytokine levels upon MYC inactivation
in tumors from WT and RAG1-/-
hosts (Figure 9A). MYC inactivation in tumors from
WT compared to RAG1-/-
hosts revealed an up regulation of anti-proliferative and
anti-angiogenic (“anti-tumor”) cytokines that suggest potential involvement by other
immune effectors. Eotaxin-1 and IL-5 (Figure 9A, WT versus RAG1-/-
fold change
upon MYC inactivation p = 0.02 and p = 0.003 respectively) are potent TH2 cytokines
55
that have been implicated in the recruitment of an eosinophil-mediated anti-tumor
inflammatory response [15]. IFN- was observed to increase over 4-fold upon MYC
inactivation in the WT hosts with virtually no change in the absence of the host
immune system (WT versus RAG1-/-
fold change upon MYC inactivation p = 0.03).
On the other hand, TNF- was significantly downregulated in RAG1-/-
hosts (RAG1-/-
MYC On versus Off, p = 0.02), while its upregulation was close to statistical
significance in the WT hosts (WT MYC On versus Off, p = 0.07). Both cytokines have
been established as critical mediators of potent CD4+ anti-tumor activity [16-17].
Interestingly, MCP-1, a potent chemo-attractant of inflammatory tumor-associated
macrophages (TAMs) [18-19], was significantly downregulated in the tumors from
immunodeficient hosts compared to WT hosts (WT versus RAG1-/-
fold change upon
MYC inactivation p = 0.008).
Further, we also measured the downregulation of “pro-tumor” cytokines in
tumors from WT and RAG1-/-
hosts. Upon MYC inactivation, vascular endothelial
growth factor (VEGF) was significantly downregulated almost 4-fold in WT hosts
(WT MYC On versus Off, p = 0.01) whereas no significant change in its expression
could be detected in tumors from immunodeficient hosts. IL-1 levels were
significantly decreased in WT hosts compared to RAG1-/-
hosts upon MYC
inactivation (WT versus RAG1-/-
fold change upon MYC inactivation, p = 0.02);
downregulation of these two cytokines suggests enhanced suppression of angiogenesis
in the presence of an intact host immune system upon MYC inactivation [20-21].
56
Finally, RAG1-/-
hosts that had been reconstituted with CD4+ T-cells exhibited
similar changes in chemokine expression to WT hosts upon MYC inactivation (Figure
9B). The anti-tumor cytokines (eotaxin, IFN- and RANTES) increased, while the pro-
tumor cytokine, VEGF decreased in protein expression (Figure 9B). Overall, we
conclude that changes in the cytokine milieu in the tumor microenvironment upon
MYC inactivation are profoundly influenced by host immune status and that CD4+ T-
cells alone appear to be responsible for the regulation of many of these changes.
2.3 Contribution of the innate immune system:
Chemokines that attract macrophages and eosinophils are upregulated upon MYC
inactivation in tumors regressing in WT hosts:
We observed that there was a 3-fold higher level of MCP-1 in tumors from WT
compared to RAG1-/-
hosts when MYC was on compared to a 7-fold higher expression
of MCP-1 in tumors from WT compared to RAG1-/-
hosts upon MYC inactivation
(Figure 9C, WT versus RAG1-/-
MYC ON, p = 0.001, MYC Off p = 0.02). We
observed similar results for monocyte chemoattractant protein-3 (MCP-3), which can
also attract macrophages to tissues and is known to be produced by various tumor cells
[22] (Figure 9D, WT versus RAG1-/-
MYC ON 2-fold higher expression in WT, p =
0.02, MYC Off 7-fold higher expression in WT p = 0.006).
The most dramatic differences between the WT and RAG1-/-
tumor
microenvironments were observed in the protein levels of eotaxin-1. Eotaxin-1 levels
appear to be significantly higher in the WT microenvironment compared to the
57
RAG1-/-
microenvironment even when MYC is on. (Figure 9E, WT versus RAG1-/-
MYC On, 8-fold higher expression in WT, p = 0.04). Upon MYC inactivation, there is
an increase in eotaxin-1 expression in WT hosts while there is no change in expression
in RAG1-/-
hosts (WT versus RAG1-/-
MYC Off, 20-fold higher expression in WT, p =
0.007).
Macrophages infiltrate the WT and CD8-/-
tumor microenvironment upon MYC
inactivation:
After observing increased levels of expression of macrophage chemoattractants
like MCP-1 and MCP-3 in the WT tumor microenvironment, we investigated whether
there was a difference in macrophage recruitment to the tumor in WT and RAG1-/-
hosts before and after MYC inactivation.
We measured the amount of macrophage infiltration by staining tumor sections
before and after MYC inactivation for F480, a macrophage surface marker. We
performed immunohistochemistry and quantified the amount of macrophages stained
and found that upon MYC inactivation, macrophage recruitment occurred in tumors
from WT hosts but not in tumors from RAG1-/-
hosts (Figure 10A, B, WT versus
RAG1-/-
p = 0.004). This correlated with our observation of increased expression of
macrophage chemoattractants upon MYC inactivation in tumors from WT compared to
RAG1-/-
hosts (Figure 9C, D).
We also investigated macrophage infiltration in tumors transplanted into CD4-/-
and CD8-/-
hosts. As we had identified that CD4+ T-cells played a critical role in
mediating sustained tumor regression and previous literature has implicated CD4+
T-
58
cells of the tumor microenvironment in the recruitment, differentiation and activation
of macrophages [23], we checked whether tumors growing in CD4-/-
hosts had
impaired macrophage recruitment. Upon MYC inactivation, tumors growing in CD4-/-
hosts had significantly less macrophage infiltration compared to tumors form WT and
CD8-/-
hosts (Figure 10A, B, WT versus CD4-/-
MYC Off, p < 0.0001, CD4-/-
versus
CD8-/-
MYC Off, p < 0.0001).
Furthermore, we observed that amongst WT, RAG1-/-
, CD4-/-
and CD8-/-
hosts,
only tumors from CD8-/-
hosts were infiltrated by macrophages when MYC was on.
Interestingly, upon MYC inactivation, tumors from CD8-/-
hosts had the highest
amount of macrophage infiltration (Figure 10A, B, CD8-/-
versus WT, 10-fold higher
in the CD8-/-
, MYC Off, p < 0.0001). These results suggest that CD4+ T-cells are
required for macrophage recruitment to the tumor microenvironment upon MYC
inactivation. At this time, we have not investigated the expression levels of MCP-1
and MCP-3 in tumor lysates from CD4-/-
and CD8-/-
hosts.
Macrophages cultured from WT tumor bearing mice express both iNOS and
arginase-1:
After observing macrophages in the tumor microenvironment of WT mice
upon MYC inactivation, we wanted to characterize these macrophages to study
whether they were pro- or anti-tumor. To do this, we first attempted to isolate
macrophages from the tumor microenvironment using magnetically activated cell
sorting (MACS). However, the number of macrophages obtained using this technique
was insufficient to perform RNA extraction experiments.
59
To circumvent this issue, we isolated splenic macrophages from tumor bearing
WT and SCID mice and cultured them in vitro as described before [24]. We then used
qPCR analysis to investigate which macrophage markers were upregulated before and
after MYC inactivation.
Our results show that splenic macrophages from WT tumor bearing mice
before and after MYC inactivation express equivalent levels of iNOS and arginase-1.
Splenic macrophages from tumor bearing SCID mice did not show appreciable levels
of expression of iNOS and arginase-1 (Figure 11A, B, WT versus SCID, MYC On,
iNOS p = 0.04, arginase-1 p = 0.01 , MYC Off, iNOS p = 0.007 , arginase-1, p <
0.0001). Instead, splenic macrophages from SCID mice showed increased expression
of arginase-2 both before and after MYC inactivation compared to splenic
macrophages isolated from WT mice (Figure 11C, WT versus SCID, MYC Off p <
0.0001).
As described in the introduction to this thesis, M1 macrophages produce iNOS
while M2 macrophages produce arginase-1. Arginase-2 is an isoform of the arginase-1
gene and both these isoforms can metabolize arginine into urea and ornithine [25].
Arginase-1 is usually expressed cytoplasmically in hepatic tissues where as arginase-2
is expressed mitochondrially in non-hepatic tissues [26]. The role of arginase-2
expression in determining macrophage polarity is not clear. There have been reports
suggesting that arginase-2 expressing macrophages might be polarized towards the M1
phenotype in experimental atherosclerosis [27].
60
Interestingly we also found that upon MYC inactivation, splenic macrophages
from WT tumor bearing hosts expressed significantly higher levels of TSP-1
compared to when MYC was on. Macrophages from SCID hosts expressed
significantly lower levels of TSP-1 before and after MYC inactivation compared to
macrophages form WT hosts (Figure 11D, WT MYC On versus MYC Off p = 0.001,
WT versus SCID, MYC Off, p = 0.0001). This suggests that macrophages could be a
potential source of TSP-1 in the tumor microenvironment upon MYC inactivation,
described to occur in section 2.2.
2.4 Contribution of T-cells to regression of primary MYC induced
lymphoma:
Characterizing the contribution of the immune system to tumor regression mediated by
MYC inactivation in a primary model of MYC induced T-cell lymphoma:
All of the experiments described above have been performed by transplanting
tumor cell lines into various WT and immunodeficient hosts. A caveat of this
experimental design is that results obtained from these experiments might be
influenced by the fact that primary tumors were adapted to growth in-vitro while
generating tumor cell lines. Furthermore, the host immune response elicited by a
transplanted tumor may differ from the immune response to a tumor that arises
spontaneously within the host. To address whether the immune system could influence
shut down of angiogenesis and induction of cellular senescence in a spontaneously
61
arising tumor, we performed experiments in primary tumor hosts of the murine Eµ-
tTA X tet-O-MYC lymphoma model [1].
Since 90% of the tumors arising in this mouse model are known to be
CD4+CD8
+, we were unable to use antibody mediated depletion of T-cells to study the
effect of T-cell function in primary transgenic tumor regression upon MYC
inactivation. Instead, we inhibited T-cell function in primary tumor hosts using
Cyclosporine A, a drug obtained from the fungus, Tolypocladium inflatum, which
prevents T-cell activation and function through inhibiting the N-FAT pathway [28].
Notably, we first determined that cyclosporine A did not have any direct effects on the
proliferation of tumor cells in vitro (Figure 12A).
Compared to untreated primary transgenic mice, cyclosporine A treated
primary transgenic mice illustrated a marked inhibition of the ability of MYC
inactivation to induce both cellular senescence as measured by staining for SA β-
galactosidase (Figure 12B, 70% versus 1%, p < 0.01), p16 (Figure 12B, 6% versus
2%, p < 0.05) and p21 (Figure 12B, 0.5% versus 0.1%, p < 0.01) as well as the
suppression of angiogenesis as measured by decrease in staining for CD31 (Figure
12B, 0.2% versus 0.4%, p = 0.05) and the induction of TSP-1 (Figure 12B, 6% versus
1%, p = 0.0006). Thus, cyclosporine A mediated immune suppression in the primary
tumor model blocked the ability of MYC inactivation to induce senescence and shut
down angiogenesis. We observed no effects on apoptosis as measured by TUNEL
staining (data not shown). However, interestingly, cyclosporine A treatment may
suppress the ability of MYC inactivation to induce proliferative arrest, suggesting that
62
in primary tumors, cyclosporine A mediated suppression of the immune system
engenders this additional consequence. Long term experiments to check whether
cyclosporine A treated primary tumor hosts would relapse upon MYC inactivation
were inconclusive due to nephrotoxicity caused by cyclosporine A upon frequent
administration.
2.5 Contribution of the adaptive immune system in other models of
oncogene induced hematologic malignancies:
Characterizing the contribution of the immune system to tumor regression mediated by
oncogenes other than MYC:
After establishing a significant role for the immune system in the regression of
lymphomas upon MYC inactivation, we wanted to investigate whether the immune
response would contribute similarly to the regression of hematopoietic tumors induced
by oncogenes other than MYC. To test this, we used conditionally regulatable models
of RAS induced lymphoma (Eµ-tTA X tet-O-RAS) and BCR-ABL induced pro-B-cell
acute lymphocytic leukemia (Eµ-tTA X tet-O-BCR-ABL).
Tumor cell lines were derived from primary conditionally regulatable RAS
induced tumors and transplanted sub-cutaneously into WT and RAG1-/-
mice. After
allowing tumors to grow to a comparable size, RAS was inactivated by administering
dox to the mice intra-peritoneally and orally in their drinking water. Tumor regression
was seen to occur in both WT and RAG1-/-
hosts. These mice were followed up to 6
63
months post RAS inactivation and no evidence of tumor relapse was seen either in WT
or RAG1-/-
hosts (data not shown).
We then tested the contribution of the immune system to BCR-ABL
inactivation in transplanted tumors derived from cell lines generated from a
conditionally regulatable model of BCR-ABL induced B-ALL. Similar to MYC
inactivation, tumors underwent sustained regression upon BCR-ABL inactivation in
WT hosts while 100% of the immunodeficient hosts relapsed within 14 days of BCR-
ABL inactivation (Figure 13A, WT versus RAG1-/-
, p = 0.006). Hence, BCR-ABL
inactivation also induces sustained tumor regression only in immune intact hosts. We
demonstrate that a host immune system is required for BCR-ABL inactivation in B-
ALL to induce cellular senescence, TSP-1 expression and sustained tumor regression.
We conclude that for both MYC and BCR-ABL inactivation, an intact host immune
system is required to elicit oncogene addiction.
We investigated the effect of host immune status on the ability of BCR-ABL
inactivation to induce changes in the tumor microenvironment (Figure 13B). Upon
BCR-ABL inactivation, there was a non-significant decrease in Ki67 expression in
tumors transplanted into immunocompetent hosts. In tumors from immunodeficient
hosts, Ki67 expression upon BCR-ABL inactivation did not change. Ki67 expression
was higher in tumors transplanted into immunodeficient hosts compared to those
transplanted into immune intact hosts (Figure 13B, WT BCR-ABL off versus
immunodeficient BCR-ABL off p = 0.03). Cellular senescence increased upon BCR-
ABL inactivation in tumors from wild type hosts versus immunodeficient hosts as
64
measured by increased SA-β-gal staining (4% versus 0.4%, p = 0.05), p16 staining
(0.1% versus 0%) and p21 staining (0.3% versus 0%). Finally, there was a 3-fold
increase in TSP-1 upon BCR-ABL inactivation in tumors from immunocompetent
hosts while TSP-1 expression did not change upon BCR-ABL inactivation in
immunodeficient hosts (Figure 13B, TSP-1 panel, WT BCR-ABL on versus BCR-ABL
off, p < 0.0001; WT BCR-ABL Off versus immunodeficient BCR-ABL Off, p =
0.0001). We were unable to, measure any significant CD31 expression in the
BCR-ABL induced tumors.
2.6 Contribution of an antigen specific immune response:
Differential recruitment of regulatory T-cells (T-regs) to the tumor microenvironment
upon MYC inactivation:
T-regs are a population of immune cells that can suppress a T-cell mediated
immune response. This has evolved from a need to inhibit auto-reactive
T-cells and to keep immune responses to foreign antigens in check [29-30]. The best
characterized population of suppressor T-cells are the CD4+CD25
+ Foxp3 expressing
regulatory T-cells. Since we had evidence that CD4+ T-cells home to the tumor
microenvironment upon MYC inactivation, (Figure 3A), we decided to investigate
whether a subset of these cells were T-regs.
We used immunohistochemistry to test for the expression of Foxp3, a
transcription factor responsible for the development of T-regs [31], in tumor sections
before and after MYC inactivation from WT and RAG1-/-
hosts. We observed an 8-fold
65
increase in Foxp3 expression upon MYC inactivation in tumors from WT hosts (Figure
14A, B MYC On versus MYC Off, p = 0.002). As expected, tumors in RAG1-/-
hosts
did not show significant Foxp3 expression as these hosts are T-cell deficient.
Antigen specificity of the anti-tumor response to transplanted MYC induced tumors:
We wanted to investigate whether tumors from the EµtTAXtet-O-MYC model
could elicit an antigen specific immune response upon transplantation into WT hosts.
We tested this indirectly by studying whether anti-tumor immune memory was
generated in WT hosts in which tumors had previously regressed upon MYC
inactivation. The rationale behind this experiment was that immune memory is always
generated in response to specific antigens and if anti-tumor immune memory was
generated against MYC induced lymphomas, this would suggest that a tumor antigen
specific immune response had occurred.
We generated a cohort of WT mice in which tumors were established and
underwent regression by MYC inactivation. After complete tumor regression was
achieved, doxycycline delivery was removed from these mice. We then identified the
minimum number of luciferase labeled tumor cells required to be transplanted into
naïve WT hosts in order to achieve sub-cutaneous tumor growth. This minimum
number of tumor cells was transplanted into naïve WT mice and WT mice in which
tumors had previously undergone sustained regression. Tumor growth was monitored
using bioluminescence imaging. We found that while tumors were able to grow in the
naïve mice, all the mice that were re-challenged with tumor remained tumor free
(Figure 15, Naïve WT versus Re-challenged WT, p < 0.001).
66
Figure 1: An intact immune system is required for sustained tumor regression
67
Figure 1: An intact immune system is required for sustained tumor regression.
1(A,B): Graphical representation and representative data are shown of tumor
regression and relapse kinetics as measured by bioluminescence imaging. Luciferase-
labeled tumor cell lines from our conditional mouse T-ALL model [1] were injected
subcutaneously into different cohorts of mice (WT n = 11, CD8-/-
n = 7, CD4-/-
n = 6,
CD4-/-
CD8-/-
n = 5, SCID n = 8, RAG1-/-
n = 7, RAG2-/-
cc-/-
n = 3) and tumor
regression and relapse kinetics were monitored. MYC was inactivated by administering
doxycycline (dox) to the mice when tumors reached a comparable bioluminescence
signal (108p/s/sr/cm
2). Data is presented as bioluminescence signal (average radiance)
plotted against time after MYC inactivation. 1(C): Representative bioluminescence
images of tumors regressing in the different immunodeficient hosts. Data shown is
representative of 3 different experiments. 1(D): Quantitative analysis of tumor
regression in the indicated hosts 8 days post MYC inactivation. 1(E): Quantification of
minimum residual disease in the indicated hosts at the maximally regressed state of the
tumor. Data is presented as the minimum bioluminescence signal after MYC
inactivation. Each symbol represents an individual animal. Average signal for each
cohort is indicated by solid black lines. 1(F): Kaplan Meier curves of tumor-free
survival in the various immunodeficient genotypes. A mouse was scored as a relapse
when its tumor bioluminescence signal first begins to increase after tumor regression.
The table shows the results of a log-rank test to compare the WT survival curve with
those of the indicated immunodeficient mice. Data shown are representative 3
different experiments repeated with 2 cell lines and 1 primary tumor.
68
For panels 1(D E), statistical significance (p value evaluated by unpaired Student‟s t-
test) is shown. * p < 0.01, ** p < 0.001, *** p < 0.0001
69
Figure 2: An intact immune system is required for sustained tumor regression
upon MYC inactivation
Figure 2: An intact immune system is required for sustained tumor regression
upon MYC inactivation. 2(A): Kaplan-Meier survival curve of WT versus. SCID
70
mice. Mice were transplanted with 10^7 unlabeled lymphoma cells and tumors were
treated with doxycyline when they reached a size of 1000 mm3. Mice were scored as
relapses when they showed signs of morbidity. 2(B): Tumors were harvested 0 and 4
days after MYC inactivation, snap frozen in liquid nitrogen and stored in -80oc. RNA
was extracted from frozen tumor samples and a qPCR was run using primers specific
for human MYC and UBC (housekeeping gene). Relative fold change in MYC
expression normalized to UBC is plotted against tumors from different hosts. Error
bars are represented as +/- SEM.
71
Figure 3: CD4+ T-cells home to the tumor and are sufficient to induce sustained
tumor regression upon MYC inactivation
Figure 3: CD4+ T-cells home to the tumor and are sufficient to induce sustained
tumor regression upon MYC inactivation. 3(A): Representative images of
bioluminescence signal from luciferase+
CD4+ T-cells that home to the tumor
microenvironment. RAG1-/-
mice were reconstituted with luciferase+ CD4
+ T-cells and
unlabeled tumor cell lines were injected s.c. 8 days post reconstitution. When tumors
72
grew to a size of 1000 mm3
MYC was inactivated and bioluminescence imaging was
used to observe and measure the distribution pattern of luciferase+ CD4
+ T-cells. Data
is represented as bioluminescence signal (average radiance) plotted against time after
MYC inactivation (n=3). 3(B): Graphical representation of tumor regression and
relapse kinetics as measured by bioluminescence imaging. RAG1-/-
mice were
reconstituted with CD4+ (n=5) or CD8
+ (n=6) T-cells isolated from spleens and lymph
nodes of WT mice using magnetically activated cell sorting (MACS). 8 days post
reconstitution, luciferase+ tumor cell lines were injected s.c. MYC was inactivated
when tumors in all hosts reached a comparable bioluminescence signal and tumor
regression and relapse kinetics were monitored. Data is presented as bioluminescence
signal (average radiance) plotted against time after MYC inactivation. WT (n=3) and
RAG1-/-
(n=3) mice were used as positive and negative controls. 3(C): Quantification
of minimum residual disease in the indicated hosts. Bioluminescence signals of tumors
at their maximally regressed state are plotted against genotype. Statistical significance
(p value evaluated by unpaired Student‟s t-test) is shown. * p < 0.01, ** p < 0.001,
*** p < 0.0001 3(D): Kaplan Meier curves of tumor-free survival in the reconstituted
RAG1-/-
, RAG1-/-
and WT mice. A mouse was scored as a relapse when the
bioluminescence signal first begins to increase after tumor regression. The table shows
the results of a log-rank test to compare the survival curve of RAG1-/-
reconstituted
with CD4+ T-cells with survival curves of the other indicated genotypes. Data shown
are representative 3 different experiments. reconst. = reconstituted with, rec =
reconstituted with
73
Figure 4: Splenocytes depleted of CD4+ T-cells home to the tumor
microenvironment and verification of RAG1-/-
reconstitution
74
Figure 4: Splenocytes depleted of CD4+ T-cells home to the tumor
microenvironment and verification of RAG1-/-
reconstitution. 4(A): Representative
bioluminescence images of CD4+ T-cell depleted luciferase
+ splenocytes homing to
the tumor microenvironment. Luciferase+
splenocytes were depleted for CD4+ T-cells
were injected i.v into RAG1-/-
mice. 8 days after reconstitution, unlabeled tumors were
injected s.c. MYC was inactivated and bioluminescence imaging was used to observe
the distribution pattern of the CD4+ T-cell depleted splenocytes. 4(B): FACS analysis
of peripheral blood of RAG1-/-
mice reconstituted with CD4+ or CD8
+ T-cells, 8 days
post reconstitution. Data shown is representative of 4 different experiments.
75
Figure 5: The immune system does not influence apoptosis and cellular arrest
upon MYC inactivation
Figure 5: The immune system does not influence apoptosis and cellular arrest
upon MYC inactivation. 5(A): Micrographs of Hematoxylin and Eosin staining (top
panel), TUNEL (middle panel) and Ki67 (bottom panel) immunostaining of tumors
derived from untreated (MYC On) and six-day dox treated mice (MYC Off) from WT
(left panel) RAG1-/-
(middle panel) and CD4-/-
(right panel) hosts. Scale Bar = 100 μm.
76
5(B): Quantitative representation of TUNEL (left panel) and Ki67 (right panel)
immunostaining shown in 3(A). Quantification of TUNEL and Ki67 immunostaining
is presented as the average percentage of TUNEL-positive cells and area of Ki67-
positive regions, respectively, within the tumors. At least five different fields from
three different tumors injected with at least two different tumor cell lines for each
different condition. Statistical significance (p value evaluated by unpaired Student‟s t-
test) is shown. * p < 0.01, ** p < 0.001, *** p < 0.0001
77
Figure 6: An intact immune system is required for the inhibition of angiogenesis
upon MYC inactivation
78
Figure 6: An intact immune system is required for the inhibition of angiogenesis
upon MYC inactivation. 6(A): Micrographs of TSP-1 (top panel) and CD31 (bottom
panel) immunofluorescence staining of tumors derived from untreated (MYC On) and
4 day dox treated (MYC Off) mice of the indicated genotypes. Scale Bar = 100μm.
6(B): Quantification of TSP-1 (left panel) and CD31 (right panel) staining shown in
6(A). Quantification is presented as the average percentage of positively stained
regions within the tumors. At least five different fields from two different tumors were
analyzed for each different condition. Statistical significance (p value evaluated by
unpaired Student‟s t-test) is shown. * p < 0.01, ** p < 0.001, *** p < 0.0001 6(C):
Spleens from WT and RAG1-/-
mice were harvested, lysed and probed for TSP-1 by
western blot analysis. HSP-90 was probed as a loading control. 6(D): Splenocytes
were harvested from wild-type mice and purified by positive selection with either anti-
CD45 or anti-CD4 coated beads. Isolated cells were cultured for 3 days in the presence
of anti-CD3 and anti-CD28, harvested, lysed and probed by western blot analysis for
TSP-1. β-actin was probed as a loading control.
79
Figure 7: TSP-1 expression in the tumor microenvironment is required for
sustained tumor regression upon MYC inactivation
80
Figure 7: TSP-1 expression in the tumor microenvironment is required for
sustained tumor regression upon MYC inactivation. 7(A): FACS analysis of
RAG1-/-
mice reconstituted with splenocytes from either WT or TSP-1,2-/-
mice. 8
days after reconstitution, mice were bled from the tail vein to check for reconstitution
by flow cytometry. Data shown is representative of 3 different experiments 7(B):
Kaplan-Meier curves of tumor free survival of reconstituted RAG1-/-
, RAG1-/-
and WT
mice. RAG1-/-
mice were reconstituted with splenocytes from WT (n=18) or TSP-1,2-/-
(n=16) mice i.v. 8 days post reconstitution mice were transplanted with unlabelled
lymphoma cells s.c. Tumors were allowed to grow to a size of 1000mm3 after which
MYC was inactivated and mice were scored for relapse. WT (n=8) and RAG1-/-
(n=11)
mice were used as positive and negative controls. The table shows the results of a log-
rank test to compare the survival curve of RAG1-/-
reconstituted with TSP-1,2-/-
splenocytes with survival curves of the other indicated genotypes. Data shown are
representative 3 different experiments. 7(C): Kaplan-Meier curves of tumor free
survival of RAG1-/-
mice injected with tumor cell lines that were either transduced
with retrovirally expressed TSP-1 (n=5) or with an empty vector (n=5). Tumors were
allowed to grow to a size of 1000mm3 after which MYC was inactivated and mice
were scored for relapse. 7(D): Kaplan-Meier survival curve of SCID mice treated with
PBS (vehicle control) or 3 TSR (drug). Mice were transplanted with 10^7 unlabeled
lymphoma cells and tumors were treated with either doxycycline alone or in
combination with 3 TSR when they reached a size of 1000 mm3. Mice were scored as
relapses when they showed signs of morbidity.
81
Figure 8: An intact immune system is required for the induction of cellular
senescence upon MYC inactivation
Figure 8: An intact immune system is required for the induction of cellular
senescence upon MYC inactivation. 8(A): Micrographs of Senescence Associated β-
galactosidase (SA β-gal, top panel), p16 (middle panel) and p21 (bottom panel)
immunostaining of tumors derived from untreated (MYC On) and four-day dox treated
(MYC Off) mice of the indicated genotypes. Scale Bar = 100 μm. 8(B): Quantification
of SA-β-gal (left panel), p16 (middle panel) and p21 (right panel) staining shown in
82
8(A). Quantification is presented as the average percentage of positively stained
regions within the tumors. At least five different fields from three different tumors
injected with at least two different tumor cell lines were analyzed for each different
condition. Statistical significance (p value evaluated by unpaired Student‟s t-test) is
shown. * p < 0.01, ** p < 0.001, *** p < 0.0001
83
Figure 9: Cytokines produced by the immune system contribute to sustained
tumor regression upon MYC inactivation
84
Figure 9: Cytokines produced by the immune system contribute to sustained
tumor regression upon MYC inactivation. Graphical representation of fold change
of indicated cytokines upon MYC inactivation in tumors from 9(A): WT and RAG1-/-
hosts and 9(B): RAG1-/-
hosts reconstituted with CD4+ T-cells. Tumors were
harvested at tumor onset and 4 days after MYC inactivation and run on a luminex
platform to check for the expression of 21 different cytokines. The significant fold
changes in the various cytokines upon MYC inactivation were log2 transformed and
plotted for various pro- and anti-tumor cytokines. 9(C,D,E): Quantification of protein
levels represented as concentration (pg/ml) obtained from running the luminex assay
on lysates derived from untreated (MYC On) and four-day dox (MYC Off) treated
tumors from WT and RAG1-/-
mice. 9(C): MCP-1 9(D): MCP-3 9(E): Eotaxin-1. Data
are representative of three different tumors run in duplicate. Statistical significance (p
value evaluated by unpaired Student‟s t-test) is shown. * p < 0.01, ** p < 0.001, *** p
< 0.0001
* on top of the bars represents significance in cytokine expression upon MYC
inactivation in the indicated host.
85
Figure 10: Macrophages infiltrate tumors regressing in WT and CD8-/-
hosts
upon MYC inactivation
86
Figure 10: Macrophages infiltrate tumors regressing in WT and CD8-/-
hosts
upon MYC inactivation. 10(A): Micrographs of F480 immunostaining (top panel) of
tumors derived from untreated (MYC On) and three-day dox treated mice (MYC Off)
from WT, RAG1-/-
, CD4-/-
and CD8-/-
hosts (ordering is from top to bottom). Scale Bar
= 100 μm. 10(B): Quantitative representation of F480 (bottom panel) immunostaining
shown in 10(A). Quantification of F480 immunostaining is presented as the average
percentage of F480-positive regions, respectively, within the tumors. At least five
different fields from two different tumors were analyzed for each different condition.
Statistical significance (p value evaluated by unpaired Student‟s t-test) is shown. ** p
< 0.001, *** p < 0.0001
87
Figure 11: Increased levels of iNOS, Arg-1 and TSP-1 in macrophages from
tumor bearing WT hosts
Figure 11: Increased levels of iNOS, Arg-1 and TSP-1 in macrophages from
tumor bearing WT hosts. Quantification of relative gene expression compared to
housekeeping gene UBC from mRNA derived from in vitro cultured splenic
macrophages from WT and SCID mice bearing untreated (MYC On) and four-day dox
(MYC Off) treated tumors. 11(A): iNOS 11(B): Arginase-1 11(C): Arginase-2 11(D):
Thrombospondin-1 Data are representative of 2 different tumors injected for each
88
condition, run in triplicate. Statistical significance (p value evaluated by unpaired
Student‟s t-test) is shown. ** p < 0.001, *** p < 0.0001
89
Figure 12: Cyclosporine A treatment inhibits induction of senescence and
inhibition of angiogenesis in primary MYC induced T-ALL
90
Figure 12: Cyclosporine A treatment inhibits induction of senescence and
inhibition of angiogenesis in primary MYC induced T-ALL. 12(A): Conditional
MYC overexpressing tumor cell lines were treated in vitro with either 500 ng/ml
cyclosporine A, 1000 ng/ml cyclosporine A or 20 ng/ml doxycycline and their growth
was compared to untreated cells at 24, 48 and 72 hours post drug treatment.12(B):
Micrographs and quantification of Hematoxylin and Eosin, Ki67, SA-β-gal, p16, p21,
CD31 and TSP-1 immunostaining (ordered from top to bottom) of tumors derived
from untreated and cyclosporine A treated primary tumor bearing mice (MYC On and
4 day dox treated MYC Off). Scale Bar = 100μm. Quantification is presented as the
average percentage of positively stained regions within the tumors. At least five
different fields from two different tumors were analyzed for each different condition.
Statistical significance (p value evaluated by unpaired Student‟s t-test) is shown. * p <
0.01, ** p < 0.001, *** p < 0.0001
91
Figure 13: An intact immune system is required for sustained regression of
tumors in a conditional mouse model of BCR-ABL-induced B-ALL
92
Figure 13: An intact immune system is required for sustained regression of
tumors in a conditional mouse model of BCR-ABL-induced B-ALL. 13(A):
Kaplan-Meier curves of tumor free survival of RAG1-/-
(n=9) and WT (n=4) mice
transplanted with unlabelled leukemia cells i.p. When mice were moribund with
tumor, BCR-ABL was inactivated, and mice were scored for relapse. 13(B):
Micrographs and quantification of Ki67, SA-β-gal, p16, p21, and TSP-1
immunostaining (ordered from top to bottom) of tumors derived from untreated (BCR-
ABL On) and doxycycline treated (BCR-ABL Off) wildtype and immunodeficient
tumor bearing mice. Scale Bar = 100μm. Quantification is presented as the average
percentage of positively stained regions within the tumors. At least five different fields
from two different tumors were analyzed for each different condition. Statistical
significance (p value evaluated by unpaired Student‟s t-test) is shown. * p < 0.01, ** p
< 0.001, *** p < 0.0001
93
Figure 14: Increased Foxp3 expression in tumors regressing in WT hosts upon
MYC inactivation
Figure 14: Increased Foxp3 expression in tumors regressing in WT hosts upon
MYC inactivation. 14(A): Micrographs of Foxp3 immunostaining of tumors derived
from untreated (MYC On) and four-day dox treated mice (MYC Off) from WT (top
panel) and RAG1-/-
(bottom panel) hosts. 14(B): Quantitative representation of Foxp3
94
immunostaining shown in 14(A). Quantification of Foxp3 immunostaining is
presented as the average percentage of Foxp3-positive regions, respectively, within the
tumors. At least five different fields from three different tumors were analyzed for
each different condition. Statistical significance (p value evaluated by unpaired
Student‟s t-test) is shown. ** p < 0.001
95
Figure 15: Transplanted tumors are rejected upon re-challenge in WT hosts that
have previously exhibited sustained tumor regression
Figure 15: Transplanted tumors are rejected upon re-challenge in WT hosts that
have previously exhibited sustained tumor regression. Graphical representation and
representative data are shown of tumor growth as measured by bioluminescence
imaging. Luciferase-labeled tumor cell lines from our conditional mouse T-ALL
model [1] were injected subcutaneously into different cohorts of WT mice (Naïve n =
5, WT mice that have previously exhibited tumor regression and were being re-
challenged n = 5). Data is presented as bioluminescence signal (average radiance)
plotted against time after tumor challenge (days).
96
2.7 References:
1. Felsher, D.W. and J.M. Bishop, Reversible tumorigenesis by MYC in
hematopoietic lineages. Mol Cell, 1999. 4(2): p. 199-207.
2. Huettner, C.S., et al., Reversibility of acute B-cell leukaemia induced by BCR-
ABL1. Nat Genet, 2000. 24(1): p. 57-60.
3. Contag, C.H., et al., Visualizing gene expression in living mammals using a
bioluminescent reporter. Photochem Photobiol, 1997. 66(4): p. 523-31.
4. Giuriato, S., et al., Sustained regression of tumors upon MYC inactivation
requires p53 or thrombospondin-1 to reverse the angiogenic switch. Proc Natl
Acad Sci U S A, 2006. 103(44): p. 16266-71.
5. Karlsson, A., et al., Genomically complex lymphomas undergo sustained tumor
regression upon MYC inactivation unless they acquire novel chromosomal
translocations. Blood, 2003. 101(7): p. 2797-803.
6. Kazerounian, S., K.O. Yee, and J. Lawler, Thrombospondins in cancer. Cell
Mol Life Sci, 2008. 65(5): p. 700-12.
7. Lawler, J., The functions of thrombospondin-1 and-2. Curr Opin Cell Biol,
2000. 12(5): p. 634-40.
8. Miao, W.M., et al., Thrombospondin-1 type 1 repeat recombinant proteins
inhibit tumor growth through transforming growth factor-beta-dependent and -
independent mechanisms. Cancer Res, 2001. 61(21): p. 7830-9.
9. Wu, C.H., et al., Cellular senescence is an important mechanism of tumor
regression upon c-Myc inactivation. Proc Natl Acad Sci U S A, 2007. 104(32):
p. 13028-33.
10. Acosta, J.C., et al., Chemokine signaling via the CXCR2 receptor reinforces
senescence. Cell, 2008. 133(6): p. 1006-18.
11. Kuilman, T., et al., Oncogene-induced senescence relayed by an interleukin-
dependent inflammatory network. Cell, 2008. 133(6): p. 1019-31.
12. Kuilman, T. and D.S. Peeper, Senescence-messaging secretome: SMS-ing
cellular stress. Nat Rev Cancer, 2009. 9(2): p. 81-94.
13. Hanahan, D. and J. Folkman, Patterns and emerging mechanisms of the
angiogenic switch during tumorigenesis. Cell, 1996. 86(3): p. 353-64.
14. Bartek, J., Z. Hodny, and J. Lukas, Cytokine loops driving senescence. Nat
Cell Biol, 2008. 10(8): p. 887-9.
15. Simson, L., et al., Regulation of carcinogenesis by IL-5 and CCL11: a
potential role for eosinophils in tumor immune surveillance. J Immunol, 2007.
178(7): p. 4222-9.
16. Qin, Z. and T. Blankenstein, CD4+ T cell--mediated tumor rejection involves
inhibition of angiogenesis that is dependent on IFN gamma receptor
expression by nonhematopoietic cells. Immunity, 2000. 12(6): p. 677-86.
17. Thomas, W.D. and P. Hersey, TNF-related apoptosis-inducing ligand (TRAIL)
induces apoptosis in Fas ligand-resistant melanoma cells and mediates CD4 T
cell killing of target cells. J Immunol, 1998. 161(5): p. 2195-200.
97
18. Allavena, P., et al., The inflammatory micro-environment in tumor
progression: the role of tumor-associated macrophages. Crit Rev Oncol
Hematol, 2008. 66(1): p. 1-9.
19. Hu, H., et al., Tumor cell-microenvironment interaction models coupled with
clinical validation reveal CCL2 and SNCG as two predictors of colorectal
cancer hepatic metastasis. Clin Cancer Res, 2009. 15(17): p. 5485-93.
20. Kowanetz, M. and N. Ferrara, Vascular endothelial growth factor signaling
pathways: therapeutic perspective. Clin Cancer Res, 2006. 12(17): p. 5018-22.
21. Shchors, K., et al., q. Genes Dev, 2006. 20(18): p. 2527-38.
22. Conti, I. and B.J. Rollins, CCL2 (monocyte chemoattractant protein-1) and
cancer. Seminars in Cancer Biology, 2004. 14(3): p. 149-154.
23. Hung, K., et al., The central role of CD4(+) T cells in the antitumor immune
response. J Exp Med, 1998. 188(12): p. 2357-68.
24. Alatery, A. and S. Basta, An efficient culture method for generating large
quantities of mature mouse splenic macrophages. J Immunol Methods, 2008.
338(1-2): p. 47-57.
25. Shi, O., et al., Structure of the murine arginase II gene. Mamm Genome, 1998.
9(10): p. 822-4.
26. Maarsingh, H., T. Pera, and H. Meurs, Arginase and pulmonary diseases.
Naunyn Schmiedebergs Arch Pharmacol, 2008. 378(2): p. 171-84.
27. Khallou-Laschet, J., et al., Macrophage plasticity in experimental
atherosclerosis. PLoS One, 2010. 5(1): p. e8852.
28. Ho, S., et al., The mechanism of action of cyclosporin A and FK506. Clin
Immunol Immunopathol, 1996. 80(3 Pt 2): p. S40-5.
29. Belkaid, Y., Regulatory T cells and infection: a dangerous necessity. Nat Rev
Immunol, 2007. 7(11): p. 875-88.
30. Maloy, K.J. and F. Powrie, Regulatory T cells in the control of immune
pathology. Nat Immunol, 2001. 2(9): p. 816-22.
31. Fontenot, J.D., M.A. Gavin, and A.Y. Rudensky, Foxp3 programs the
development and function of CD4+CD25+ regulatory T cells. Nat Immunol,
2003. 4(4): p. 330-6.
98
CHAPTER 3: DISCUSSION OF
FINDINGS, IMPLICATIONS OF
RESULTS, AND
FUTURE DIRECTION
99
3.1 Overview:
Oncogene addiction is often studied with an emphasis on what happens to the
tumor cells upon oncogene inactivation. My work has shifted this emphasis from the
tumor cells to the immune cells of the tumor microenvironment to study the
contribution of the immune system to tumor regression mediated by oncogene
inactivation. Oncogene addiction had been presumed to be a largely cell autonomous
process [1] but results described in this thesis have shown that interactions between
the tumor microenvironment and the immune system are essential for sustained tumor
regression to occur upon oncogene inactivation.
3.2 The adaptive immune system remodels the tumor
microenvironment:
We have demonstrated that in the absence of an intact adaptive immune
system, we see a 10-1000-fold reduction in the rate, extent, and duration of tumor
regression upon MYC inactivation in the Eµ-tTA X tet-O-MYC mouse model of T-
ALL. Provocatively, we found that the absence of CD4+
T-cells alone was sufficient to
markedly impede sustained tumor regression. Thus, oncogene addiction is not
necessarily cell autonomous. The immune system, specifically CD4+ T-cells, may play
a critical role in enabling MYC inactivation to elicit changes in the microenvironment
and in cytokine expression that appear to be required for cellular senescence and the
shutdown of angiogenesis. TSP-1 is one critical cytokine that must be expressed by
immune effectors to cooperate with MYC inactivation to induce sustained tumor
regression. Importantly, our results generalized to primary tumors from MYC-induced
100
T-ALL bearing hosts that had been treated with the immunosuppressive agent
cyclosporine A and a conditional transgenic model of BCR-ABL induced B-ALL. We
conclude that oncogene inactivation may induce tumor regression through immune
cell dependent mechanisms. Our findings have potentially important implications for
the development of targeted therapeutics, for they suggest that testing potential
therapeutics in vitro or in vivo in immune deficient hosts may significantly
underestimate their potential efficacy. Our findings also imply that the efficacy of
existing targeted therapeutics can be improved by combining them with strategies to
increase CD4+ T-cell infiltration of tumors or strategies to inhibit angiogenesis and
induce cellular senescence in the tumor microenvironment.
Our observations are consistent with a multitude of reports that document the
role of the immune system in neoplasia [2-5]. Tumors co-evolve in the context of an
intact immune system through the process of immune editing, resulting in tumor
elimination, dormancy or evolving to escape the immune system and progress to full
malignancy [4, 6-7]. Hence, MYC-induced tumors may evolve to subvert the immune
elimination response. Then, upon MYC inactivation, a massive recruitment of CD4+ T-
cells occurs that is associated with marked changes in cytokine production in the
tumor microenvironment leading to cellular senescence and the shutdown of
angiogenesis.
Provocatively, CD4+ T-cells emerged as the critical host effector population
for sustained tumor regression upon MYC inactivation. The rationale used to obtain
data described in chapter 2 was to observe how CD4+ T-cells influenced the different
101
mechanisms of tumor regression known to occur upon MYC inactivation. We deviated
from classical immunological approaches which would entail investigating the
interaction of CD4+ T-cells with tumor cells with respect to antigen presentation and
CD4+ T-cell activation through antigen recognition in the context of MHC II on the
surface of antigen presenting cells and uncovered a non-canonical role for CD4+ T-
cells in the tumor microenvironment.
CD4+ T-cells may play a direct role in tumor cytotoxicity as has been
described before [8] but we have not performed experiments to test whether or not this
occurs in our lymphoma model. CD4+
T-cells have been previously implicated in the
restraint of tumor growth through regulation of antigen dependent mechanisms
involving either macrophages or cytotoxic T-cells [9-11]. Some reports also suggest
that the anti-tumor effect of CD4+ T-cells is mediated by eosinophils [12-13] and we
explore these possibilities briefly. Further characterization of CD4+ T-cells in this
model is warranted to understand what cytokines they secrete and whether they are
polarized along the TH-1, TH-2 or TH-17 pathway. Future experiments to interrogate
whether or not the CD4+ T-cells that influence tumor regression in this model system
are antigen specific also need to be performed. Additionally, important parameters like
the kinetics of CD4+ T-cell activation post tumor transplantation and post MYC
inactivation need to be determined.
Notably, hosts deficient in CD4+ T-cells exhibited impaired kinetics, degree
and durability of tumor regression as well as reduced senescence and suppression of
angiogenesis upon MYC inactivation. Moreover, the reconstitution of CD4+ T-cells
102
into RAG1-/-
hosts alone was capable of restoring the ability of MYC inactivation to
induce sustained tumor regression. Indeed, the reconstitution of CD4+ T-cells
into
RAG1-/-
hosts had more potent effects on tumor regression compared with the
depletion of these cells, perhaps reflecting that in hosts that are congenitally defective
in a specific immune compartment there may be compensation from other immune
effectors [14].
Intriguingly, host CD4+ T-cells sculpted the tumor‟s response to MYC
inactivation, likely not by their modest influence upon apoptosis or proliferation, but
by dramatically inducing cellular senescence and the shutdown of angiogenesis,
processes previously shown by us to be integral to the ability of MYC inactivation to
effect sustained tumor regression. Moreover, two of the hallmarks of oncogene
addiction, both the induction of cellular senescence and the suppression of
angiogenesis, have been linked to the expression of cytokines known to be expressed
by CD4+ T-cells [15-18]. We found that CD4
+ T-cells were required to effect changes
in the microenvironment and have identified TSP-1 as one of the critical chemokines
that might mediates these changes. Our results are consistent with other reports that
immune effectors and associated changes in chemokines occur upon restoration of the
tumor suppressor p53 in both liver cancer [19] and upon MYC inactivation in
lymphoma [20].
Thus, CD4+ T-cells are one important component of the mechanism of tumor
regression upon oncogene inactivation. It should be noted, that upon MYC inactivation
tumors recurred in only 28.5% of the CD4-/-
hosts compared to 100% of the RAG1-/-
103
and SCID hosts. This suggests that other host immune effectors are likely to contribute
and we recognize the possibility that other innate and adaptive immune compartments
are also involved including macrophages, eosinophils, NK cells, mast cells, and B-
cells. Recent work suggests that mast cells and macrophages both may be critical [5,
19]. Indeed, it is possible that CD4+ T-cells are mediating part of the effects we have
observed by recruiting these effector populations.
We specifically identified TSP-1 as being critical for the mechanism by which
host immune effectors mediate tumor regression upon MYC inactivation. TSP-1 is a
potent cytokine that has been implicated in the regulation of many cellular processes
including the regulation of angiogenesis [21-25]. Furthermore, TSP-1 has also been
implicated in the regulation of lymphocyte homing and function [26]. Our results
suggest that TSP-1 is required for the ability of CD4+ T-cells to contribute to sustained
regression upon oncogene inactivation and we showed that this could be in part due to
the ability of TSP-1to inhibit angiogenesis upon MYC inactivation.
Moreover, TSP-1 has been shown to activate latent TGF-β [27]. Notably, TGF-
β can play a tumor suppressive role in certain tumor microenvironments [28-29].
TGF-β can also contribute to both the restraint of tumor onset as well as oncogene
addiction through the regulation of cellular senescence upon MYC activation and
inactivation [30]. Thus, it is tempting to speculate that TSP-1 may contribute to
oncogene addiction via its influence on TGF-β.
In addition to TSP-1, we identified several other cytokines including eotaxin-1,
IL-5, IFN- and TNF- as possible candidates for mediating changes in cellular
104
senescence and angiogenesis upon MYC inactivation, consistent with reports that these
chemokines may be involved in these processes [16, 31]. Also the observed
downregulation of other cytokines such as VEGF, IL-1, and MCP-1 could contribute
to these mechanisms [32-34]. IFN- and TNF- have been previously implicated in
the regulation of cellular quiescence and angiogenesis [16-18, 31], and eotaxin-1 and
IL-5 have demonstrated potent anti-tumor activity in numerous mouse models of
cancer [35]. Notably, tumor regression induced by the restoration of p53 expression
was also associated with marked changes in chemokine expression [19].
We also found that in primary transgenic tumor hosts, an immune
compromised state induced via treatment with cyclosporine A greatly impeded the
consequences of oncogene inactivation. Our observations in the primary tumor model
are important because they show that our results generalize in the case when
endogenous tumor-host interactions evolved throughout tumorigenesis. Cyclosporine
A treatment is well known to increase the frequency of hematological malignancies in
patients [36-37]. Our results imply that this agent may impede sensitivity to oncogene
directed therapies. Intriguingly, we observe that treatment with cyclosporine A seems
to affect proliferation of primary tumors when MYC is on suggesting T-cells and/or B-
cells could enhance tumor development and progression as has been reported in
several cases [38-39]. If this were the case, it would imply that MYC influences T-cell
function and in the presence of MYC, T-cells are pro-tumor, but in its absence, T-cells
play an anti-tumor role.
105
We also found that an immune intact host is required for BCR-ABL
inactivation to induce sustained tumor regression in B-ALL. Similar to MYC
inactivation, inactivation of the BCR-ABL oncogene resulted in the induction of
cellular senescence, the shutdown of tumor angiogenesis, and ultimately sustained
tumor regression only in the presence of the host immune system. However, different
from MYC inactivation, BCR-ABL inactivation appeared to be less capable of
suppressing cellular proliferation. Hence, the host immune system appears to be
generally important in mediating the consequences of oncogene inactivation. We
recognize that there are likely to be differences in the specific contribution of the
immune system in different types of tumors and this contribution would also depend
on the oncogene being inactivated.
We are currently in the process of designing experiments to test whether
Gleevec, a small molecule inhibitor of the BCR-ABL tyrosine kinase [40] requires an
intact immune system to cause sustained tumor regression in this model of B-ALL and
whether CD4+ T-cells can improve the quality of Gleevec induced regression. If the
immune system contributes to sustained tumor regression initiated by Gleevec, the
findings of this thesis have immediate clinical relevance and efforts can be made
towards testing a combination of Gleevec and anti-angiogenic therapy or CD4+ T-cell
reconstitution to treat chronic myelogenous leukemia patients.
3.3 Potential role of T-regs and an antigen specific immune response:
We have generated data suggesting a potential role for T-regs in the tumor
microenvironment upon MYC inactivation. As T-regs are known to suppress the
106
immune response, they are thought to help the tumor evade an anti-tumor immune
surveillance response [41]. In fact, in several solid tumor models, it has been shown
that increased T-reg infiltration of tumors correlates with poor prognosis [42-43] and
T-regs are generally thought to be tumor promoting. The role of T-regs in
hematological malignancies is not entirely clear. One study has shown that increased
T-reg infiltration in tumor biopsies of patients with cutaneous T-cell lymphoma
(CTCL) is associated with improved survival [44]. One possible explanation is that the
T-regs can inhibit the proliferation of malignant T-cells and thus inhibit tumor growth.
As tumors in our MYC induced lymphoma model are double positive CD4+CD8
+ T-
cells, T-regs might have a direct anti-tumor effect in this model and this warrants
further investigation.
The major part of the work performed in this thesis characterizes a non-
canonical role of the immune system in the anti-tumor response. However, we have
preliminary evidence indicating that there is an antigen specific anti-tumor response
that develops in our MYC induced transplanted tumor model. We show that WT mice
in which tumors have achieved sustained regression are able to reject tumor re-
challenge. It is not clear whether this anti-tumor immune memory develops after
tumor transplantation when MYC is on or after MYC has been inactivated and the
tumor begins to regress. Furthermore, it is not clear whether the immune memory that
is generated contributes to sustained tumor regression upon MYC inactivation.
107
3.4 Potential role of the innate immune system:
There is extensive literature describing the complex role of tumor associated
macrophages in promoting and/or inhibiting tumor growth and progression [45-47].
However the role of macrophages in a tumor microenvironment upon oncogene
inactivation had not been previously studied. We show that macrophage
chemoattractants MCP-1 and MCP-3 are expressed at higher levels in tumors
regressing in WT hosts compared to RAG1-/-
hosts. Upon comparing macrophage
infiltration after MYC inactivation in these hosts, we observe that tumors from WT
hosts show significantly more macrophage infiltration compared to tumors from
RAG1-/-
hosts.
It should be noted that even when MYC is activated, tumors from WT hosts
show significantly increased levels of MCP1- and MCP-3 compared to RAG1-/-
hosts,
but no significant macrophage infiltration is observed in the tumor microenvironments
of either host when MYC is on.
In another model of MYC induced lymphoma, macrophages have been
described to be recruited to the tumor microenvironment in response to MYC induced
apoptosis [48]. Once recruited to the microenvironment, these macrophages
phagocytose apoptotic tumor cells and secrete TGF-β that can induce cellular
senescence in the non-apoptotic tumor cells [48]. While this process has been
described as a mechanism of tumor suppression upon MYC activation, we show that in
our model of MYC induced lymphoma, macrophages accumulate in the tumor
microenvironment only upon MYC inactivation in WT and CD8-/-
hosts. We have
108
shown that MYC inactivation leads to apoptosis and cellular senescence of tumor cells
(Figure 5, 8) in WT hosts and it would be worthwhile to investigate whether
macrophage infiltration contributes to inducing this cellular senescence response.
We do not observe significant macrophage infiltration in tumors from RAG1-/-
hosts and those from CD4-/-
hosts show reduced macrophage infiltration compared to
WT and CD8-/-
hosts upon MYC inactivation. This suggests that the presence of CD4+
T-cells is required to elicit macrophage infiltration in these regressing tumors. This is
in accordance with other reports that CD4+
T-cells recruit macrophages into the tumor
microenvironment [12, 39].
To fully understand the role played by macrophages in our tumor model
system, it will be essential to characterize the functional polarization of the tumor
infiltrating macrophages. At present, we have not been able to successfully extract
RNA from the infiltrating macrophages as we have been unable to retrieve a
significant number of macrophages from the tumor microenvironment. Instead, we
attempted to characterize the phenotype of splenic macrophages from tumor bearing
mice to get a better idea of macrophage functional polarization. We find that splenic
macrophages from WT mice carrying tumors before and after MYC inactivation
express both iNOS and arginase-1 suggesting that both M1 and M2 macrophages may
be present in the spleen of WT tumor bearing mice.
We did not observe either iNOS or arginase-1 expression in splenic
macrophages cultured from tumor bearing SCID hosts. Interestingly, however, we did
observe arginase-2 expression in these cultured macrophages. Arginase-2 was not
109
expressed by WT splenic macrophages. The role of arginase-2 in macrophage
polarization is not well elucidated and without analyzing several other markers of
macrophage polarization (IL-6, IL-10, IL-1β, CCL-17,CXCL9, MRC-1) we cannot
definitively identify the polarization state of these aringase-2 expressing macrophages.
We also found that splenic macrophages from WT hosts upregulated the
expression of TSP-1 upon MYC inactivation. This indicates that macrophages could be
a potential source of increased TSP-1 expression seen in tumors from WT hosts upon
MYC inactivation (Figure 6). However to confirm this, TSP-1 expression from tumor
infiltrating macrophages will have to be measured.
Another innate immune cell type that might contribute to tumor regression
upon MYC inactivation in our tumor model is the eosinophil. We found that eotaxin-1,
a potent eosinophil chemoattractant, was upregulated several fold in tumor lysates
from WT hosts compared to RAG-1-/-
hosts. Future experiments need to be performed
to investigate whether or not there are significant differences in infiltration of
eosinophils in tumors from WT and immunodeficient hosts before and after MYC
inactivation.
Several confirmatory experiments need be performed to interrogate the role of
macrophages and eosinophils in tumor regression upon MYC inactivation. Current
efforts are underway to knock out macrophages using clodronate liposomes [49] to
study their effect in tumor regression and relapse. Similarly, strategies to deplete
eosinophils can be used if preliminary eosinophil infiltration data suggests that this
cell type is involved. There have been several descriptions of anti-tumor immune
110
responses mediated by macrophages and eosinophils together [50-51]. Since neither
macrophages nor eosinophils have inherent tumor specificity, they require adaptive
immune cells to confer anti-tumor specificity. The most likely candidate cell would be
CD4+ T-cells that are known to recruit both macrophages and eosinophils into various
tumor microenvironments [12]. Once we understand the roles played by various innate
populations in mediating tumor regression to targeted therapeutics, strategies to
combine these therapeutics with immune based therapies can be designed.
3.5 Implications:
We propose a model whereby oncogene addiction is a consequence of both cell
autonomous processes such as proliferative arrest and apoptosis as well as host-
immune dependent mechanisms such as cellular senescence and angiogenesis (Figure
1). Immediately upon oncogene inactivation, tumor cells are eliminated primarily in a
cell autonomous manner, and hence we observe a similar effect of oncogene
inactivation in vitro or in vivo and in immune intact or deficient hosts. However, the
kinetics of tumor cell elimination and the extent of tumor elimination, or minimal
residual disease, as well as the durability of sustained tumor regression are all dictated
by the presence of an immune system and appear to be strongly associated with its
ability to elicit cellular senescence and shut down angiogenesis. These latter processes
have been proposed to contribute to the constraint of minimal residual disease by
others [52], and our data extends this paradigm to oncogene addiction. Thus, host
immune effectors appear to be critical to the mechanism of sustained tumor regression
elicited by oncogene inactivation, and their absence invariably results in tumor
111
recurrence. Specifically, we demonstrate that CD4+
T-cells are one critical component
to this phenomenon and that, moreover, TSP-1 emerges as a possible cytokine
regulating these processes.
Another implication of our work is that other immune effectors and
chemokines/cytokines (including IFN-, eotaxin-1, IL-5, TNF-, and MCP-1) are
likely to be involved. Numerous reports have indicated that immune cells and
inflammation can be important to the pathogenesis of cancer through many effects on
the tumor microenvironment [19,53-54]. The host immune system is critical for the
remodeling of the tumor microenvironment required to elicit oncogene addiction and
induce sustained tumor regression. The precise cues that come from the tumor cells
vary depending on their oncogenic state dictating whether immune effectors are
directed to either support tumor growth or mediate tumor regression.
We infer that oncogene addiction is not solely cell autonomous. CD4+ T-cells
are required for both the tumor intrinsic mechanisms of cellular senescence and the
host-associated mechanism of shutdown of angiogenesis. A deficiency in CD4+ T-
cells may render the treatment of tumors in patients less efficacious and impede the
complete elimination of tumor cells. Indeed, AIDS patients exhibit not only a more
than 100-fold increased frequency of lymphomas often associated with MYC
overexpression but are also much less responsive to therapy [55-56], suggesting that
CD4+ T-cells may contribute to the efficacy of therapeutic agents.
Furthermore, methods used to identify targeted therapies that rely on the in
vitro study of cell lines or in vivo analysis of xenograft models in immune
112
compromised hosts may underestimate the efficacy of a therapy by failing to faithfully
recapitulate tumor-host interactions [57-58]. Our model system can provide an
experimental strategy to further dissect the role of specific immune effectors and
cytokines in the mechanism of tumor regression upon MYC inactivation and their role
in the shutdown of angiogenesis and induction of cellular senescence. Our
experimental approach can be generalized to study other oncogenes and cancers. Our
findings support the notion that modulation of CD4+ T-cell function may enhance the
efficacy of therapeutics for cancer [59-60]. The careful choice of a combination of
targeted and immune therapy may therefore be more efficacious in mediating
sustained tumor regression.
We anticipate that the various components of the innate and adaptive immune
system will contribute differently to different targeted therapeutics. Our work suggests
that the efficacy of targeted therapeutics can be improved by understanding the
intricacies of host-tumor interactions in individual patients and exploiting this
knowledge to mediate sustained tumor regression.
113
Figure 1: Model of the interaction of the immune system with oncogene addiction
Figure 1: Model of the interaction of the immune system with oncogene
addiction. Proposed model depicting the importance of the immune system for
eliciting oncogene addiction through induction of cellular senescence and inhibition of
angiogenesis based upon our observations in two different conditional transgenic
mouse tumor models (MYC induced T-ALL and BCR-ABL induced B-ALL).
114
3.6 References:
1. Sharma, S.V. and J. Settleman, Oncogene addiction: setting the stage for
molecularly targeted cancer therapy. Genes Dev, 2007. 21(24): p. 3214-31.
2. de Visser, K.E., A. Eichten, and L.M. Coussens, Paradoxical roles of the
immune system during cancer development. Nat Rev Cancer, 2006. 6(1): p. 24-
37.
3. de Visser, K.E., L.V. Korets, and L.M. Coussens, De novo carcinogenesis
promoted by chronic inflammation is B lymphocyte dependent. Cancer Cell,
2005. 7(5): p. 411-23.
4. Dunn, G.P., et al., Cancer immunoediting: from immunosurveillance to tumor
escape. Nat Immunol, 2002. 3(11): p. 991-8.
5. Soucek, L., et al., Mast cells are required for angiogenesis and macroscopic
expansion of Myc-induced pancreatic islet tumors. Nat Med, 2007. 13(10): p.
1211-8.
6. Guerra, N., et al., NKG2D-deficient mice are defective in tumor surveillance in
models of spontaneous malignancy. Immunity, 2008. 28(4): p. 571-80.
7. Teng, M.W., et al., Immune-mediated dormancy: an equilibrium with cancer. J
Leukoc Biol, 2008. 84(4): p. 988-93.
8. Perez-Diez, A., et al., CD4 cells can be more efficient at tumor rejection than
CD8 cells. Blood, 2007. 109(12): p. 5346-54.
9. Corthay, A., et al., Primary antitumor immune response mediated by CD4+ T
cells. Immunity, 2005. 22(3): p. 371-83.
10. Dranoff, G., et al., Vaccination with irradiated tumor cells engineered to
secrete murine granulocyte-macrophage colony-stimulating factor stimulates
potent, specific, and long-lasting anti-tumor immunity. Proc Natl Acad Sci U S
A, 1993. 90(8): p. 3539-43.
11. Qin, Z. and T. Blankenstein, CD4+ T cell--mediated tumor rejection involves
inhibition of angiogenesis that is dependent on IFN gamma receptor
expression by nonhematopoietic cells. Immunity, 2000. 12(6): p. 677-86.
12. Hung, K., et al., The central role of CD4(+) T cells in the antitumor immune
response. J Exp Med, 1998. 188(12): p. 2357-68.
13. Mattes, J., et al., Immunotherapy of cytotoxic T cell-resistant tumors by T
helper 2 cells: an eotaxin and STAT6-dependent process. J Exp Med, 2003.
197(3): p. 387-93.
14. Xing, Z., et al., Protection by CD4 or CD8 T cells against pulmonary
Mycobacterium bovis bacillus Calmette-Guerin infection. Infect Immun, 1998.
66(11): p. 5537-42.
15. Acosta, J.C., et al., Chemokine signaling via the CXCR2 receptor reinforces
senescence. Cell, 2008. 133(6): p. 1006-18.
16. Beatty, G. and Y. Paterson, IFN-gamma-dependent inhibition of tumor
angiogenesis by tumor-infiltrating CD4+ T cells requires tumor
responsiveness to IFN-gamma. J Immunol, 2001. 166(4): p. 2276-82.
17. Kuilman, T., et al., Oncogene-induced senescence relayed by an interleukin-
dependent inflammatory network. Cell, 2008. 133(6): p. 1019-31.
115
18. Muller-Hermelink, N., et al., TNFR1 signaling and IFN-gamma signaling
determine whether T cells induce tumor dormancy or promote multistage
carcinogenesis. Cancer Cell, 2008. 13(6): p. 507-18.
19. Xue, W., et al., Senescence and tumour clearance is triggered by p53
restoration in murine liver carcinomas. Nature, 2007. 445(7128): p. 656-60.
20. Giuriato, S., et al., Sustained regression of tumors upon MYC inactivation
requires p53 or thrombospondin-1 to reverse the angiogenic switch. Proc Natl
Acad Sci U S A, 2006. 103(44): p. 16266-71.
21. Jimenez, B., et al., Signals leading to apoptosis-dependent inhibition of
neovascularization by thrombospondin-1. Nat Med, 2000. 6(1): p. 41-8.
22. Lawler, J., The functions of thrombospondin-1 and-2. Curr Opin Cell Biol,
2000. 12(5): p. 634-40.
23. Short, S.M., et al., Inhibition of endothelial cell migration by thrombospondin-
1 type-1 repeats is mediated by beta1 integrins. J Cell Biol, 2005. 168(4): p.
643-53.
24. Zaslavsky, A., et al., Platelet-derived thrombospondin-1 (TSP-1) is a critical
negative regulator and potential biomarker of angiogenesis. Blood, 2010.
25. Kazerounian, S., K.O. Yee, and J. Lawler, Thrombospondins in cancer. Cell
Mol Life Sci, 2008. 65(5): p. 700-12.
26. Li, S.S., et al., Endogenous thrombospondin-1 is a cell-surface ligand for
regulation of integrin-dependent T-lymphocyte adhesion. Blood, 2006. 108(9):
p. 3112-20.
27. Beyne-Rauzy, O., et al., Tumor necrosis factor alpha induces senescence and
chromosomal instability in human leukemic cells. Oncogene, 2004. 23(45): p.
7507-16.
28. Shchors, K., et al., q. Genes Dev, 2006. 20(18): p. 2527-38.
29. Su, X., et al., Tumor microenvironments direct the recruitment and expansion
of human Th17 cells. J Immunol, 2010. 184(3): p. 1630-41.
30. Kowanetz, M. and N. Ferrara, Vascular endothelial growth factor signaling
pathways: therapeutic perspective. Clin Cancer Res, 2006. 12(17): p. 5018-22.
31. Simson, L., et al., Regulation of carcinogenesis by IL-5 and CCL11: a
potential role for eosinophils in tumor immune surveillance. J Immunol, 2007.
178(7): p. 4222-9.
32. Cockburn, I.T. and P. Krupp, The risk of neoplasms in patients treated with
cyclosporine A. J Autoimmun, 1989. 2(5): p. 723-31.
33. Opelz, G. and B. Dohler, Lymphomas after solid organ transplantation: a
collaborative transplant study report. Am J Transplant, 2004. 4(2): p. 222-30.
34. Andreu, P., et al., FcRgamma activation regulates inflammation-associated
squamous carcinogenesis. Cancer Cell, 2010. 17(2): p. 121-34.
35. DeNardo, D.G., et al., CD4(+) T cells regulate pulmonary metastasis of
mammary carcinomas by enhancing protumor properties of macrophages.
Cancer Cell, 2009. 16(2): p. 91-102.
36. Druker, B.J., et al., Efficacy and safety of a specific inhibitor of the BCR-ABL
tyrosine kinase in chronic myeloid leukemia. N Engl J Med, 2001. 344(14): p.
1031-7.
116
37. Shevach, E.M., Fatal attraction: tumors beckon regulatory T cells. Nat Med,
2004. 10(9): p. 900-1.
38. Salama, P., et al., Tumor-infiltrating FOXP3+ T regulatory cells show strong
prognostic significance in colorectal cancer. J Clin Oncol, 2009. 27(2): p. 186-
92.
39. Beyer, M. and J.L. Schultze, Regulatory T cells in cancer. Blood, 2006.
108(3): p. 804-11.
40. Gjerdrum, L.M., et al., FOXP3+ regulatory T cells in cutaneous T-cell
lymphomas: association with disease stage and survival. Leukemia, 2007.
21(12): p. 2512-8.
41. Lewis, C.E. and J.W. Pollard, Distinct role of macrophages in different tumor
microenvironments. Cancer Res, 2006. 66(2): p. 605-12.
42. Mantovani, A., et al., Macrophage polarization: tumor-associated
macrophages as a paradigm for polarized M2 mononuclear phagocytes.
Trends Immunol, 2002. 23(11): p. 549-55.
43. Sica, A., et al., Tumour-associated macrophages are a distinct M2 polarised
population promoting tumour progression: potential targets of anti-cancer
therapy. Eur J Cancer, 2006. 42(6): p. 717-27.
44. Reimann, M., et al., Tumor Stroma-Derived TGF-beta Limits Myc-Driven
Lymphomagenesis via Suv39h1-Dependent Senescence. Cancer Cell, 2010.
17(3): p. 262-272.
45. Zeisberger, S.M., et al., Clodronate-liposome-mediated depletion of tumour-
associated macrophages: a new and highly effective antiangiogenic therapy
approach. British Journal of Cancer, 2006. 95(3): p. 272-281.
46. Nathan, C.F. and S.J. Klebanoff, Augmentation of Spontaneous Macrophage-
Mediated Cytolysis by Eosinophil Peroxidase. Journal of Experimental
Medicine, 1982. 155(5): p. 1291-1308.
47. vanderVliet, A., et al., Formation of reactive nitrogen species during
peroxidase-catalyzed oxidation of nitrite - A potential additional, mechanism
of nitric oxide-dependent toxicity. Journal of Biological Chemistry, 1997.
272(12): p. 7617-7625.
48. Aguirre-Ghiso, J.A., Models, mechanisms and clinical evidence for cancer
dormancy. Nat Rev Cancer, 2007. 7(11): p. 834-46.
49. Coussens, L.M. and Z. Werb, Inflammation and cancer. Nature, 2002.
420(6917): p. 860-7.
50. Greten, F.R. and M. Karin, The IKK/NF-kappaB activation pathway-a target
for prevention and treatment of cancer. Cancer Lett, 2004. 206(2): p. 193-9.
51. Carbone, A., Emerging pathways in the development of AIDS-related
lymphomas. Lancet Oncol, 2003. 4(1): p. 22-9.
52. Boshoff, C. and R. Weiss, AIDS-related malignancies. Nat Rev Cancer, 2002.
2(5): p. 373-82.
53. Ronnov-Jessen, L. and M.J. Bissell, Breast cancer by proxy: can the
microenvironment be both the cause and consequence? Trends Mol Med,
2009. 15(1): p. 5-13.
117
54. Weigelt, B. and M.J. Bissell, Unraveling the microenvironmental influences on
the normal mammary gland and breast cancer. Semin Cancer Biol, 2008.
18(5): p. 311-21.
55. Lake, R.A. and B.W. Robinson, Immunotherapy and chemotherapy--a
practical partnership. Nat Rev Cancer, 2005. 5(5): p. 397-405.
56. Gattinoni, L., et al., Adoptive immunotherapy for cancer: building on success.
Nat Rev Immunol, 2006. 6(5): p. 383-93.
118
APPENDIX I: MATERIALS AND
METHODS
119
Materials and Methods
Transgenic Mice: The generation and characterization of Tet system transgenic lines
for conditional expression of MYC, have been described [1]. CD4-/-
, CD8-/-
, CD4-/-
CD8-/-
and RAG1-/-
in the FVB/N background were generously provided by Lisa
Coussens (University of California, San Francisco). TSP-1,2-/-
mice were generously
provided by Ben Barres (Stanford University). Luciferase+L2G85 mice were
generously provided by Robert Negrin (Stanford University). Tet-o-BCR-ABL mice
were generously provided by Daniel Tenen (Harvard Unviersity). Genotyping was
performed by PCR on genomic DNA from tails. Animals were housed in the Stanford
vivarium as per animal protocols approved by Stanford University.
Tumor Surveillance and Tumorigenicity Assays: Transgenic mice were observed
biweekly for tumor development. When mice were moribund with tumor burden, they
were either humanely euthanized or treated with doxycycline in their drinking water
(100 μg/ml) to follow tumor regression and relapse. To monitor for tumor regression
and relapse, percent survival was measured as the time between doxycycline treatment
(if tumor regression occurred within 1 week) and relapse, which is defined as
recurrence of signs of morbidity. Statistical comparison of Kaplan–Meier curves is
based on the log-rank test. For transplantation experiments, primary tumors were first
adapted to in vitro growth as described [1] and then 107 cells were washed once in
PBS before subcutaneous (s.c.) or intra-peritoneal (i.p) injection into FVB/N and
immunodeficient syngeneic mice. 5 different tumors were adapted to in vitro growth
for this study. For primary tumor experiments, mice were treated with either
120
doxycycline or cyclosporine A (Bedford Labs, Ohio) i.p (20 mg/kg) or both when first
signs of tumor were observed (unkempt fur, distended abdomen and labored
breathing).
Reconstitution of RAG1-/-
mice: RAG1-/-
mice were injected intravenously (i.v.) with
either (i) 20X106 splenocytes from WT or TSP-1,2
-/- mice or (ii) 4X10
6 CD4
+ or CD8
+
T-cells isolated from spleens and lymph nodes of WT mice using Magnetically
Activated Cell Sorting (MACS). 8 days post reconstitution, mice were bled from the
tail vein and CD4+ and CD8
+ T-cell reconstitution was verified using FACS. Mice
were then transplanted with tumor s.c. Tumors were allowed to grow to a size of 1000
mm3 after which MYC was inactivated by administration of doxycyline and mice were
either imaged by bioluminescence or scored as relapses when they showed signs of
morbidity.
Cell Culture: Tumor-derived cell lines were generated by mechanical disruption of
tumor tissue followed by Ficoll–Paque purification of the single cell suspension. 5 cell
lines were generated from 5 different tumors. Cells were then maintained in vitro as
described [1].
Retrovirus Constructs, Virus Production and Tumor Cell Infection: MSCV-Puro-
LUC construct, a modified version of the pDON plasmid vector (Takara Mirus Bio,
Madison, WI), was kindly provided by Mobin Karimi and Robert Negrin (Stanford
University). Retrovirus containing supernatants were prepared by transient
transfection of 293T cells, and viral titers were measured as described [2]. Tumor cells
were incubated with retrovirus containing supernatants for 12 h at 32°C in media
121
containing 4 μg/ml polybrene. Cells were then expanded at 37°C for an additional 48 h
and cells containing MSCV-Puro-LUC were selected with puromycin.
In Vivo Bioluminescence Imaging: Tumor cells, expressing the luciferase enzyme,
were injected s.c. into syngeneic mice. Tumors were allowed to develop until reaching
a similar bioluminescent signal. Tumor regression was then induced by doxycycline
treatment (100 μg/ml). Mice developing transplanted tumors were anesthetized with a
combination of inhaled isoflorane/oxygen delivered by the Xenogen XGI-8 5-port Gas
Anaesthesia System. The substrate d-luciferin (150 mg/kg) was injected into the
animal's peritoneal cavity 10 min. before imaging. Animals were then placed into a
light-tight chamber and imaged with an IVIS-200 cooled CCD camera (Xenogen,
Alameda, CA). First, a grayscale body surface reference image (digital photograph)
was taken under weak illumination. Next, photons emitted from luciferase expressing
cells within the animal and transmitted through the tissues were collected for a period
of 5 sec to 1 min and quantified by the software program Living Image (Xenogen) as
an overlay on the image analysis program “Igor” (Wavemetrics, Seattle, WA). For
anatomical localization, a pseudocolor image representing light intensity (blue, least
intense; red, most intense) was generated in Living Image and superimposed over the
gray scale whole body reference image as described previously [3]. Living Image was
used to collect, archive, and analyze photon fluxes and transform them into
pseudocolor images by using Living Image software (Xenogen). At least 5 mice per
group were injected with tumors expressing luciferase.
122
Western Blotting: Spleens were harvested from naïve WT and RAG1-/-
mice and
were snap frozen in liquid nitrogen and stored at -80oc. Frozen samples were lysed in
RIPA buffer and protein lysates were run on a 5% SDS-PAGE gel to test for TSP-1
protein expression by western blotting. Anti-mouse TSP-1 Ab-11 (Neomarkers,
1:1000) was used. Blots were stripped and re-probed with anti-HSP-90 (BD
Pharmingen, 1:1000) as a loading control.
Quantitative PCR: Tumors were harvested from WT and RAG1-/-
hosts at tumor
onset and 4 days post MYC inactivation and were snap frozen in liquid nitrogen and
stored at -80oc. RNA was extracted from frozen tumor samples using Nucleospin
mRNA extraction kits (Machery-Nagel). c-DNA was synthesized using a reverse
transcriptase reaction performed with Superscript II (Invitrogen) by using 2 μg of total
RNA. Quantitative PCR was done by using an ABI PRISM 7900HT cycler (Applied
Biosystems) using SYBR green as a method of detection.
Immunohistochemistry and Immunofluorescence: Mice were euthanized at tumor
onset and 4 and 6 days after MYC inactivation, and transplanted tumors were
harvested and fixed in neutral buffered formalin for paraffin sections and embedded in
OCT freezing medium (Tissue-tek) for frozen sections. Paraffin embedded tumor
sections were deparaffinized by successive incubations in xylene, 95% ethanol, 90%
ethanol, 70% ethanol followed by PBS. Epitopes were unmasked by steaming in
DAKO antigen retrieval solution for 45 minutes and rinsed twice in PBS. Frozen
Sections were immunostained with mouse anti-TSP1 (clone A6.1, 1:50; Lab Vision,
Fremont, CA), or an isotype matched control (Pharmingen), and paraffin embedded
123
sections were immunostained with p16 (1:100, Santa Cruz F-12 antibody) or p21
(1:100 Santa Cruz M-19 antibody) overnight at 4oc. This was followed by incubation
for 2 h with goat anti-rat Alexa 594 (1:500; Molecular Probes) or for 30 minutes at
room temperature with biotinylated anti-mouse (1:300 Vectastain ABC kits). Sections
were mounted in Vectashield mounting media containing dapi (Vector Labs) to stain
the nuclei or developed using 3,3'-Diaminobenzidine (DAB) and counterstained with
hematoxylin. Images were obtained on a Nikon microscope and analyzed by using
Metamoprh software (Meta Imaging Series). Statistical analysis of quantification of
immunostaining was done using an unpaired Student‟s t-test.
Senescence associated β-galactosidase assay: Mice were euthanized at tumor onset
and 4 and 6 days after MYC inactivation, and transplanted tumors were embedded in
OCT freezing medium (Tissue-tek) and stored at -80oc. 8 μm thick tissue sections
were cut using a cryostat. Sections were fixed in 0.5% glutaraldehyde for 10 minutes
and washed with PBS. Sections were then stained for 5-7 hours in a solution
containing 1 mM potassium ferricyanide, 1 mM potassium ferrocyanide and 1mM
magnesium chloride in PBS (pH=5.5).
Microvessel Density: Transplanted tumors were harvested at tumor onset and 6 days
after MYC inactivation and paraffin embedded. Microvessel density was determined
by immunofluorescence staining of deparaffinized tumor sections with an anti-CD31
mAb (B.D. PharMingen; 1:500) overnight at room temperature followed by a goat
anti-rat Alexa 594 (Molecular Probes; 1:500) for 2 h at room temperature. Regions of
highest vessel density were captured at a X200 magnification and area stained by
124
vessels was calculated. At least five fields were counted in a representative tumor
section, and at least three different transplanted tumors were counted.
Luminex cytokine Assay: The concentration of 21 cytokines was measured from
tumor tissue lysates from WT and RAG1-/-
mice at tumor onset and 4 days post MYC
inactivation. Concentrations were measured using Luminex xMAP technology. Data
were obtained as mean fluorescence intensity based on a standard curve generated for
each cytokine. Preconfigured kits are purchased from Panomics/Affymetrix and assay
is performed according to manufacturer‟s recommendation with the following
modifications. Samples are added in duplicates (25ul) to a 96 well filter plate
containing assay buffer. Following the addition of Standards (7 point dilutions) and
controls, the appropriate mix of antibody linked to polystyrene beads are added. The
plate is covered with foil and incubated for 2 hours at room temperature while shaking
at a constant speed (500 rpm). Incubation is continued overnight at 4oC without
shaking. Following overnight incubation the plate is allowed to warm up for 20
minutes and is then vacuum filtered, washed 2X with 140ul of wash buffer to remove
unbound sample. Biotinylated detection antibody solution (25ul) is then added to the
bead mixture in the plate and incubated for 2 hours with shaking at room temperature
as above. The mixture is vacuum filtered and washed 2X to remove excess detector
antibody. SA-PE (50ul) is added to the wells and incubated for 30 minutes with
shaking at room temperature. The plate is vacuum filtered, washed 2X, and re-
suspended in 120ul reading buffer and incubated for 3 minutes at room temperature
with shaking. The plate is transferred to the Luminex reader for quantitative analysis.
Individual cytokines are identified and classified by the Red laser and cytokine levels
125
are quantified using the Green laser. Digital images of the bead array are captured
following laser excitation and are processed on a computer workstation. Standard
curves and reports of the unknown cytokine levels in the samples are prepared using
BeadView and MiraiBio software.
126
References:
1. Felsher, D.W. and J.M. Bishop, Reversible tumorigenesis by MYC in
hematopoietic lineages. Mol Cell, 1999. 4(2): p. 199-207.
2. Pear, W.S., et al., Production of high-titer helper-free retroviruses by transient
transfection. Proc Natl Acad Sci U S A, 1993. 90(18): p. 8392-6.
3. Contag, C.H., et al., Visualizing gene expression in living mammals using a
bioluminescent reporter. Photochem Photobiol, 1997. 66(4): p. 523-31.