Tumor and Stem Cell Biology
Cancer Stem Cell Vaccination Confers SignificantAntitumor Immunity
Ning Ning1,4, Qin Pan1,5, Fang Zheng1,6, Seagal Teitz-Tennenbaum1, Martin Egenti1, Ji Yet2, Mu Li1,Christophe Ginestier3, Max S. Wicha3, Jeffrey S. Moyer2, Mark E.P. Prince2, Yingxin Xu4,Xiao-Lian Zhang5, Shiang Huang6, Alfred E. Chang1, and Qiao Li1
AbstractMost studies of cancer stem cells (CSC) involve the inoculation of cells from human tumors into immunosup-
pressed mice, preventing an assessment on the immunologic interactions and effects of CSCs. In this study, weexamined the vaccination effects produced by CSC-enriched populations fromhistologically distinctmurine tumorsafter their inoculation into different syngeneic immunocompetent hosts. Enriched CSCs were immunogenic andmore effective as an antigen source than unselected tumor cells in inducing protective antitumor immunity.Immune sera fromCSC-vaccinated hosts contained high levels of IgGwhich bound to CSCs, resulting in CSC lysis inthe presence of complement. CTLs generated from peripheral blood mononuclear cells or splenocytes harvestedfrom CSC-vaccinated hosts were capable of killing CSCs in vitro. Mechanistic investigations established thatCSC-primed antibodies and T cells were capable of selective targeting CSCs and conferring antitumor immunity.Together, these proof-of-concept results provide a rationale for a new type of cancer immunotherapy based onthe development of CSC vaccines that can specifically target CSCs. Cancer Res; 72(7); 1853–64. �2012 AACR.
IntroductionClinical trials to treat patients with cancer using adoptively
transferred T cells (1–3) or dendritic cells (DC; refs. 4–6) haveshown therapeutic efficacy for patients with advanced dis-eases. However, the clinical responses to such immunothera-peutic approaches have been confined to a limited percentageof treated patients. To date, bulk tumor masses with hetero-geneous populations of cancer cells have been used as a sourceof antigen either to generate effector T cells or to prime DCvaccines. Human tumors are composed of heterogeneous
tumor cell clones that differ with respect to proliferation,differentiation, and ability to initiate daughter tumors. Theinability to target cancer stemcells (CSC)with current immuneapproaches may be a significant factor for treatment failures.
The identification of human CSCs (7–17) presents a newparadigm for the development of cancer treatments. Thesestem cells have been shown to be relatively resistant toconventional chemotherapeutic regimens and radiation (18,19) and are postulated to be the cells responsible for the relapseand progression of cancers after such therapies. In an analo-gous fashion, the CSC phenomenon may adversely affect thedevelopment of effective immunotherapies for cancer. Thesetherapies have involved targeting cells that express differen-tiated tumor antigens. However, such antigens may be selec-tively expressed on differentiated tumor cells. CSCs that do notexpress these antigens may thus escape these immunologicinterventions.
While a few studies have evaluated the resistance of CSCs tothe cytotoxic effects of chemotherapy (18, 20–23) and low-doseradiation treatment (19), the immunogenicity of CSCs andtheir susceptibility to immune-based therapy have not beendetermined. So far, the majority of CSC studies have beenconducted using human tumors inoculated into severelyimmunosuppressed hosts [e.g., severe combined immunode-ficient (SCID) mice]. These hosts represent very useful modelsfor the studies of the biology, tumorigenicity, and signalingpathways of human CSCs as well as for the screening of smallmolecules which may lead to the development of new drugsthat target CSCs. A very recent report described the isolation ofcancer-initiating cells (CIC) using ALDEFLUOR/ALDH as amarker from human head and neck, breast, and pancreaticcarcinoma cell lines and the generation of ALDH1A1-specific
Authors' Affiliations: Departments of 1Surgery, 2Otolaryngology, and3Internal Medicine, University of Michigan, Ann Arbor, Michigan; 4Depart-ment of General Surgery, Chinese PLA General Hospital, Beijing; 5StateKey Laboratory of Virology, Department of Immunology and Hubei Prov-ince Key Laboratory of Allergy and Immune-related Diseases, WuhanUniversity School of Medicine; and 6Center for Stem Cell Research andApplication, Institute of Hematology, Union Hospital, Tongji Medical Col-lege, Huazhong University of Science and Technology, Wuhan, China
Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).
N. Ning, Q. Pan, and F. Zheng contributed equally to the work.
Current address for C. Ginestier: Centre de Recherche en Canc�erologiede Marseille, U1068-Inserm-Institut Paoli-Calmettes-CNRS, 27, Bd LeiRoure, BP 30059, 13273 Marseille Cedex 09, France.
Current address for N. Ning: Department of General Surgery, HainanBranch of Chinese PLA General Hospital, Hainan, China.
Corresponding Author: Qiao Li, University of Michigan ComprehensiveCancer Center, 1150 West Medical Center Drive, 1520 MSRB-1, Ann Arbor,MI 48109. Phone: 734-615-1977; Fax734-763-4135; E-mail: [email protected]
doi: 10.1158/0008-5472.CAN-11-1400
�2012 American Association for Cancer Research.
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CD8T cells in vitro (24). These T cells eliminated CICs in vivo byadoptive transfer to immunodeficient (SCID) mice bearinghuman tumor xenografts. However, the absence of adaptiveimmune responses in the SCID mouse precludes the ability toinvestigate the host immune response to CSCs. Althoughnormal mouse mammary stem cells have been isolated (25),there is a need to develop model systems where CSCs can beisolated in the immunocompetent host to evaluate the immu-nogenicity of CSCs.
In this study, we isolated and assessed the tumorigenicity ofmurine CSCs in 2 histologically different tumors from 2 genet-ically distinct immunocompetent hosts. From there, we eval-uated the immunogenicity induced by purified CSCs used as asource of antigen to prime DCs as a vaccine. We found thatCSC-based vaccines conferred effective protective antitumorimmunity which was associated with the induction of humoraland cellular responses that directly targeted CSCs via comple-ment-dependent cytotoxicity (CDC) and CTLs, respectively.
Materials and MethodsMice
Female C57BL/6 (B6) and C3H/HeNCrMTV (C3H)mice werefrom Charles River Laboratories. All the animals were main-tained in a pathogen-free environment and used at age 8 weeksor older. The University of Michigan Laboratory of AnimalMedicine (Ann Arbor, MI) approved all the animal protocols.
Murine tumorsD5 is a clone which our laboratory produced (26) from the
B16-BL6 tumor line that is a poorly immunogenicmelanoma ofspontaneous origin syngeneic to B6 mice (27, 28). SCC7 is aspontaneously arising squamous cell cancer syngeneic to C3Hmice also described in our previous report (29).
ALDEFLUOR assayThe ALDEFLUOR Kit (StemCell Technologies) labels the
ALDEFLUORþ/ALDHhigh population including the stem/pro-genitor cells (30–33). TheALDEFLUORassay uses afluorescentsubstrate of the enzyme (BAAA) freely diffusible across cellmembranes. Polar fluorescent products (BAA) accumulatewhen this substrate is oxidized in cells that express aldehydedehydrogenase (ALDH). Consequently, cells with high levels ofALDH enzymatic activity stain more brightly (ALDEFLUORþ
also referred to as ALDHþ or ALDHhigh) than cells with lowerALDH (ALDEFLUOR� also referred to as ALDH� or ALDHlow).The fluorescent product BAA is trapped in the cells, due to itsnegative charges. In each experiment, a sample of cells wasstained under identical conditions with specific ALDH inhib-itor diethylaminobenzaldehyde (DEAB) as negative control.Flow cytometry–based sorting is conducted using a FACStar-PLUS. The sorting gates are established using as negativecontrols the propidium iodide (PI)-stained cells for viabilityand the ALDEFLUOR-stained cells treated with DEAB.
Test of tumorigenicity of ALDEFLUORþ cellsEqual number of ALDEFLUORþ or ALDEFLUOR� tumor
cells mixed with Matrigel (BD Biosciences; 1:1) were injected
into the opposite side of the syngeneic mice. Tumor size wasmeasured every 3 to 4 days.
VaccinationTo examine the protective antitumor immunity induced
by vaccination with DCs pulsed with the lysate ofALDEFLUORþ cells (CSC-TPDC), ALDEFLUORþ/ALDHhigh
and ALDEFLUOR�/ALDHlow cells were isolated as describedabove either from cultured D5 and SCC7 cells or from freshlyharvested growing tumors from initial respectiveALDEFLUORþ D5 or SCC cell injection. ALDEFLUORþ,ALDEFLUOR�, and unsorted cells were frozen and thawed 3times to make cell lysate. Bone marrow–derived DCs werecultured in interleukin (IL)-4 and granulocyte macrophagecolony—stimulating factor (GM-CSF) as previously describedin our laboratory (5, 27) and were pulsed with tumor lysate togenerate tumor lysate–pulsed DCs (TPDC). After 24 hours ofcoculture, normal animals were vaccinated with CSC-TPDC orDC pulsed with lysate from unsorted heterogeneous tumorcells (H-TPDC) or DCs pulsed with sorted ALDEFLUOR� celllysate (ALDHlow-TPDC) at the same DC to tumor cell lysateratio as CSC-TPDC.
Tumor challengeAfter vaccination, the B6 mice were challenged with the
heterogeneous D5 tumor cells intravenously and the lungsharvested 20 days later to enumerate lung metastases. In SCC7model, the C3H mice were challenged with the heterogeneousSCC7 tumor cells subcutaneously on the opposite side of thevaccine and the tumor size was monitored.
Antibody productionTo test systematic immune responses conferred by CSC-
based vaccine, spleens were harvested at the end of experi-ments. Spleen B cells were activated with lipopolysaccharide(LPS) plus anti-CD40 (FGK45) monoclonal antibody (mAb)ascites as previously described (28). After activation, super-natants were collected and analyzed for IgG production.
CSC binding by immune plasmaPlasmawas collected from vaccinated hosts at the end of the
experiments. IgG level was tested using ELISA. ALDEFLUORþ
cells were washed with fluorescence-activated cell-sorting(FACS) buffer, blocked with anti-CD16/CD32 (BD biosciences),and incubated with the plasma with equal quantity of IgG for60minutes on ice. Cells were washed again and incubated withfluorescein isothiocyanate (FITC) anti-mouse IgG (0.5 mg/106
cells) for 30 minutes on ice. Cells were then washed and theirbinding to plasma IgG was detected using flow cytometry.
Antibody and complement-mediated cytotoxicityCSC lysis mediated by antibodies in plasma was assessed by
incubation of 105 viable ALDHþ CSCs or ALDH� non-CSCs(serving as control) with plasma in test tubes on ice for 1 hourfollowed by culture in the presence of rabbit complement(Calbiochem) in a 37�C water bath for another 1 hour. Viablecells were then counted after trypan blue staining to calculateCSC lysis: % of viable cells ¼ viable cells counted after plasma
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and complement incubation/105. Lower percentage of viablecells at the end of incubation indicates more cell lysis.
CTL cytotoxicityCTLs were generated from the peripheral blood mononu-
clear cells (PBMC) or splenocytes harvested from vaccinatedanimals by anti-CD3/CD28 activation and IL-2 expansion,which consistently results in more than 90% of CD3þ T cells(data not shown). CTL-mediated CSC cytotoxicity was testedusing the lactate dehydrogenase (LDH)ReleaseAssay (CytoTox96 Non-Radioactive Cytotoxicity Assay, Promega) according tothe manufacturer's protocol. The following formula was usedto calculate cytotoxicity:
% Cytotoxicity
¼ Experimental�Effector spontaneous�Target spontaneous
Target maximum� Target spontaneous�100
Statistical analysisThe significance of difference in tumorigenicity, metastatic
nodules, tumor size, the concentration of IgG, and CSC lysisby antibodies or CTLs was determined using unpaired t test.
P values<0.05were considered statistically significant betweenthe experiment groups.
ResultsIdentification of CSCs in two syngeneicimmunocompetent animal models
We have previously described the isolation of stem cell–enriched populations using ALDEFLUOR/ALDH as a marker(30, 33, 34). Using this technique, we identified CSC-enrichedpopulations in 2 different immunocompetent murine tumormodels. As indicated in Fig. 1, approximately 4% to 6% of thecultured murine melanoma D5 and squamous cancer SCC7cells are ALDEFLUORþ/ALDHþ/high; with the rest (�95%)being ALDEFLUOR�. The existence of a small percentage ofALDEFLUORþ cells in established murine tumors was con-firmed by analyzing freshly harvested tumor cells. Processedtumor cells from in vivo established D5 and SCC7 murinetumors also revealed approximately 5% of the ALDEFLUORþ
cells (Fig. 1). To determine the purity of the sorted cells, thewhole ALDEFLUORþ/ALDHþ/high cells and an approximatelyequal number (5%–15%) of the ALDEFLUOR� cells used for the
ALDH+/high
91.8%
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With DEAB
With DEAB With DEAB
With DEAB
ALDH-/low
95.6%
Figure 1. Existence of ALDEFLUORþ/ALDHþ/high populations in murine D5 melanoma and SCC7 squamous cell tumors. The ALDEFLOUR Kit labels theALDEFLUOR-positive population including the stem/progenitor cells. The ALDEFLOUR assay isolates the populationwith a high ALDH enzymatic activity. Asnegative control, an aliquot of each sample of cells was treated with 50 mmol/L DEAB, a specific ALDH inhibitor. To test the purity of the sorted cells, thewhole ALDEFLUORþ/ALDHþ/high cells (4%–6%) and an approximately equal number (5%–15%) of the ALDEFLUOR� cells used for the subsequentimmunogenicity analyses (ALDH�/low cells) were collected and restained with ALDEFLOUR using the same staining protocol. The percentages ofALDHþ/high and ALDH�/low cells are listed with the purity of 7 of the 8 restains being higher than 90%.
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subsequent immunogenicity analyses (ALDH�/low cells) werecollected and restained with ALDEFLUOR using the samestaining protocol. High percentages (>90% in 7 of the 8restains) of the ALDH�/low cells (in blue) and ALDHþ/high cells(in red) after restaining confirmed the purity of originallysorted cells (Fig. 1).
The tumorigenicity of sorted D5 melanoma ALDEFLUORþ
cells was evaluated in the syngeneic immunocompetentC57BL/6 host. ALDEFLUORþ D5 cells (5,000 per inoculum)generated large size tumors in 19 days (Fig. 2A), whereas equalnumbers of ALDEFLUOR� cells injected into the opposite sideof the same mouse failed to generate tumors. Separate mice
0 10 20 300
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Figure 2. Testing of tumorigenicity of ALDEFLUORþ populations in D5 and SCC7 tumor models. Equal numbers of D5 (A) or SCC7 (B) ALDEFLUORþ andALDEFLUOR� cells were injected into the opposite side of the same mouse. Tumor growth was then observed. ALDEFLUORþ cells can form tumors moreefficiently than ALDEFLUOR� cells.
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were injected with much lower numbers of ALDEFLUORþ D5cells. In 4 weeks, 500 injected ALDEFLUORþ cells formedtumors (Fig. 2A). In contrast, the curve in Fig. 2A shows thatas many as 50,000 ALDEFLUOR� D5 cells did not grow. Thetumorigenicity of sorted SCC7 ALDEFLUORþ population wasevaluated in the syngeneic immunocompetent C3H host. Aswas the case for the D5 model, only the ALDEFLUORþ (as fewas 2,000) cells grew into tumorswhereas equal numbers or evenmuch greater numbers (as many as 200,000) of ALDEFLUOR�
cells failed to generate tumor (Fig. 2B). These results indicatethat the ALDEFLUOR� tumor cells are less tumorigenic thanALDEFLUORþ cells.Collectively, these data indicate that ALDEFLUOR/ALDH
can serve as a reliable marker for the enrichment of murine D5and SCC7 CSCs. This has allowed us to characterize CSC-induced antitumor immunity in the immunocompetentmurine host in the subsequent experiments.
CSC vaccination confers significant protectiveantitumor immunityDCs pulsed with whole tumor lysate have been reported to
be an effective vaccine for cancer both in animal studies (35)and in clinical trials (4) including the findings reported by ourown group (5, 27). To examine whether DCs pulsed with thelysate of CSCs are more effective in inducing antitumor immu-nity, we evaluated the protective antitumor immunity inducedby vaccination with DCs pulsed with the lysate ofALDEFLUORþ cells (CSC-TPDC) and used DCs pulsedwith the lysate of whole unsorted heterogeneous tumor cells(H-TPDC) as a positive control. In the D5melanomamodel, weused D5 CSCs as a source of antigen. D5 subcutaneous tumorswere established and used as a source of CSCs by sorting forALDEFLUORþ cells. DCs were pulsed with tumor lysate togenerate TPDC to be used as a vaccine. Normal immunocom-petent B6 mice were immunized with D5 CSC-TPDC or D5H-TPDC (at the same lysate:DC ratio as D5 CSC-TPDC).Control groups received PBS. The TPDCs were inoculatedsubcutaneously for 2 doses (106/each) given 1 week apart.Seven days after the last vaccine, themicewere challengedwiththe heterogeneous D5 tumor cells intravenously and the lungsharvested 20 days later to enumerate lung metastases. Thestudy scheme and results are illustrated in Fig. 3. Comparedwith nonvaccinated, PBS-injected animals (PBS), H-TPDCinduced protective immunity against tumor growth, whichcorroborates with previous observations (27, 35). Of note, thepulsing of tumor cell lysate to DC to generate H-TPDCs wassuboptimal compared with what has been reported in the past(4, 5, 27, 34), which may partially explain why H-TPDCs did notimmunize effectively (Fig. 3). In these experiments, DCs werepulsed with the lysate of ALDEFLUORþ cells to generate CSC-TPDCs at the ratio of DCs to ALDEFLUORþ cells 3:1 (D5) and10:1 (SCC7), respectively. This ratio is much lower than the DC:unsorted tumor cell ratio (1:3) as previously described by ourgroup (4, 5, 27). We used fewer tumor cells to pulse DCs togenerate CSC-TPDC due to the following reasons: (i) thenumber of ALDEFLUORþ cells obtained was limited and (ii)we wanted to see the antitumor potential of DCs pulsed withthis limited number of ALDEFLUORþ cells compared with the
DCs pulsed with the same number of unsorted cells. Impor-tantly, mice that received CSC-TPDC prepared at an identicallow lysate to DC ratio resulted in significantly fewer lungmetastases than the PBS control group as well as the H-TPDCvaccine group. These results suggested that D5 CSCs areimmunogenic and can induce an immune response, evenunder a suboptimal CSC to DC pulsing condition, which ledto decreased lung colonization upon tumor challenge. Signif-icantly, these experiments showed a not yet recognized gainand beneficial effect against tumor growth mediated by CSC-TPDC versus H-TPDC (P¼ 0.018 in experiment 1 and P¼ 0.001in experiment 2).
In the SCC7 model, subcutaneous SCC7 tumors wereestablished and used as a source of CSC by sorting forALDEFLUORþ cells. Normal C3H animals were vaccinatedwith CSC-TPDC (DCs pulsed with ALDEFLUORþ SCC7 cells)or DCs pulsed with lysate from unsorted heterogeneousSCC7 cells (H-TPDC). The TPDCs were inoculated subcuta-neously for 3 doses given 1 week apart on days �14, �7, and0 in the right flank of C3H mice. CSC-TPDC- or H-TPDC–vaccinated mice were challenged with unsorted SCC7 tumorcells subcutaneously into the left flank on day 0 and tumorgrowth monitored. Compared with nonvaccinated controlanimals, H-TPDCs induced protective immunity againsttumor growth to a modest extent (Fig. 4A–C). In contrast,mice that received CSC-TPDC showed significant inhibitionof tumor growth compared with the no-treatment group(Fig. 4A; P ¼ 0.003), and the tumors were much smaller thanthose growing in the H-TPDC–treated hosts (Fig. 4B). Theseresults confirmed our previously observed CSC-inducedprotective immunity against D5 melanoma in a secondtumor model syngeneic to a different immunocompetenthost, as well as in a different tumor setting (subcutaneoustumor growth) that enriched CSCs are immunogenic andmore effective as an antigen source than unsorted hetero-geneous tumor cells in inducing immunity of the host toreject the challenge of tumor cells.
We repeated the experiment as shown in Fig. 4A, but addedone control group: ALDHlowTPDC, for example, DCs pulsedwith sorted ALDH�/low SCC7 cell lysate. As shown in Fig. 4C,vaccination with ALDHhighTPDC (CSC-TPDC) induced signif-icantly higher protective immunity against tumor than H-TPDC as well as ALDHlowTPDC. Furthermore, we have carriedout additional experiments with the D5 tumor model byevaluating the efficacy of CSC-TPDCs in the protection ofhosts against subcutaneous tumor challenge. As shownin Fig. 4D, vaccination of DCs pulsed with ALDHþ/high D5 celllysate (ALDHhighTPDC) induced significantly higher protectiveimmunity against subcutaneous tumor challenge thanH-TPDCs as well as ALDHlowTPDC vaccination. We analyzedthe phenotype of the tumors growing from ALDFLUORþ cells.The ALDEFLUORþ population regenerated the initial hetero-geneity of the tumor by reconstitution of both ALDEFLUORþ
and ALDEFLUOR� cell populations. To verify our findingsusing the ALDELFUORþ/ALDHþ/high cells isolated directlyfrom the cultured cell lines, we separated ALDELFUORþ cellsisolated from freshly harvested growing tumor inmurine hostswhich resulted fromprevious ALDELFUORþ cell injection. The
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stem cell nature of the ALDELFUORþ cells isolated fromfreshly harvested growing tumors was verified both in vitroand in vivo; in both D5 and SCC7 models (Supplementary Figs.S1 and S2 and Table S1). The data shown in Fig. 4D weregenerated by pulsing DCs with lysates from ALDELFUORþ/ALDHþ/high cells isolated freshly from the growing D5 tumorfollowed by D5 subcutaneous challenge. Consistent with ourearly findings using cultured tumor cells as a source of CSCs,these experiments showed that vaccination of DCs pulsed withthe lysate of ALDELFUORþ/ALDHþ/high cells isolated fromgrowing tumor induced significantly higher protective immu-nity against tumor than H-TPDCs as well as DCs pulsed with
the lysate of ALDH�/low cells also isolated from growingtumors (Fig. 4D). It is worth noting that the P values ofthe difference between PBS versus H-TPDCs and PBS vs.ALDHlowTPDC growth curves in Fig. 4C using SCC7 was<0.05 at all time points except for day 11 (P ¼ 0.523 and0.308, respectively) after tumor inoculation. In contract, therewas no significant difference between PBS versus H-TPDC andPBS versus ALDHlowTPDC growth curves in Fig. 4D using D5.However, Fig. 4C and D both showed that vaccination of DCspulsed with the lysate of ALDHþ/high cells induced significantlyhigher antitumor immunity than PBS, H-TPDC, as well as DCspulsed with the lysate of ALDH�/low cells in 2 tumor models.
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Expt. 1 Expt. 2
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Figure 3. Vaccination of DCs pulsedwith ALDELFUORþ D5 cell lysate(CSC-TPDC) induced significantlyhigher protective immunity againsttumor than whole D5 tumor celllysate pulsed DCs (H-TPDC). A,CSC-induced protective antitumorimmunity in the D5 melanomamodel syngeneic to B6 host in apulmonary metastatic model. Themean numbers of lung metastases(SEM) are depicted in the graph.B, images of tumors fromrepresentative animals in (A). Twoof the 2 independently carried outexperiments are shown.
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These data further highlight the advantage of using CSCs invaccination versus the traditional vaccine strategy using bulkunsorted tumor cells (H-TPDC) or using ALDH�/low cells.
Systemic humoral and cellular responses in CSC-TPDCvaccinatedhosts anddirect targeting ofCSCsbyantibodyand CTLsTo define possible mechanisms underlying CSC-induced
protective antitumor immunity, we harvested the splenocytesfrom the animals subjected to H-TPDC and CSC-TPDC vacci-nation. These cells were secondarily activated in vitro and theculture supernatants collected for antibody detection. Wefound significantly higher IgG production by LPS/anti-CD40–activated splenocytes isolated from the animals vacci-natedwithD5CSC-TPDCor SCC7CSC-TPDC thanD5H-TPDC(P ¼ 0.004) or SCC7 H-TPDC (Fig. 5A; P ¼ 0.031). These datashowed systemic humoral responses in CSC-TPDC–vaccinatedimmunocompetent hosts.In the experiments shown in Figs. 3 and 4, the enhanced
CSC-induced protective antitumor immunity is postulated tooccur by inhibiting the growth of CSCs present in the unsortedtumor inoculums. To examine this hypothesis, we harvested
the sera and PBMCs from the animals subjected to vaccinationto determine the specificity of the immune responses to CSCs.Using flow cytometry (Fig. 5B), we observed that immune serafrom D5 CSC-TPDC–vaccinated hosts bound to D5 CSCs(>80%) much more efficiently than the binding of the serafrom D5 H-TPDC–vaccinated hosts or sera from PBS-injectedhosts to D5 CSCs (30.6% and 30.1%, respectively). Similarly,immune sera from SCC7 CSC-TPDC–vaccinated hosts boundto SCC7CSCs (26.4%) significantlymore than the binding of thesera from SCC7 H-TPDC–vaccinated hosts (4.0%) or the back-ground binding by sera from PBS-injected hosts (0.9%) to SCC7CSCs (Fig. 5B).
To evaluate the immunologic significance of the binding ofCSC-primed antibody toCSCs,we examined antibody andCDCof CSCs. Immune sera from D5 CSC-TPDC–vaccinated hostsmediated significantlymore efficientD5CSC lysis than the seracollected from D5 H-TPDC–vaccinated (P ¼ 0.002) or PBS-treated (P¼ 0.004) hosts (Fig. 6A). Such CDCmediated by CSC-primed antibody was CSC specific because sera from the sameD5 CSC-TPDC–vaccinated hosts resulted in minimal lysis ofALDH� D5 cells (Fig. 6A). Of note, this enhanced D5 CSC lysiscorrelated with the significantly increased protective
H-TPDC vaccinated CSC-TPDC vaccinated
47 days after SCC7 injection
Mouse 1
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vs. ALDHlow
-TPDC or H-TPDC
Figure 4. CSC-induced protective antitumor immunity was confirmed in the second tumor model (SCC7) syngeneic to a different immunocompetent host(C3H) in a subcutaneous tumor setting. The mean tumor sizes (SEM) for the groups are depicted in the graph (A). Data are representative of 3 experimentscarried out. B, images of tumors from representative animals used in (A). C, experiments repeating (A) with one additional control group: DCs pulsed withsorted ALDEFLUOR�SCC7 cell lysate (ALDHlow-TPDC). D, CSC-induced protective antitumor immunity was verified in D5 tumormodel with a subcutaneoustumor challenge using the ALDEFLUORþ and ALDEFLUOR� cells isolated from freshly harvested growing D5 tumor.
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antitumor immunity induced by D5 CSC-TPDC vaccination(Fig. 3). Similarly, immune sera from SCC7 CSC-TPDC–vacci-nated hosts mediated significantly increased SCC7 CSCs lysiscompared with the sera collected from SCC7 H-TPDC–vacci-nated hosts (P ¼ 0.001) or from PBS-treated hosts (P¼ 0.001),but not the ALDH� SCC7 cells (Fig. 6B), indicating again therelative specificity of the CSC-TPDC–induced humoralresponse toward ALDHþ CSCs. This enhanced SCC7 CSCdestruction correlatedwith the increased protective antitumorimmunity induced by SCC7 CSC-TPDC vaccination (Fig. 4).
To provide further evidence that the enhanced CSC-inducedantitumor immunity is due to direct targeting of CSCs, weharvested PBMCs from D5 or SCC7 CSC-TPDC–vaccinated
animals to generate CTLs by activation in vitro with anti-CD3/CD28 mAb in the presence of IL-2. These activated cellswere assessed for cytotoxicity against ALDHþ or ALDH�
tumor cells. D5 CSC-TPDC–primed CTLs killed D5 CSCsefficiently (approximately 60%) and significantly more thanD5 H-TPDC–primed CTLs (�20%; Fig. 7A; P ¼ 0.003). Con-currently, the killing of ALDH� D5 cells by D5 CSC-TPDC–primed CTLs was significantly less effective (�20%; P¼ 0.005).In contrast, D5 H-TPDC–primed CTLs killed ALDH� D5 cells(�40%) more than D5 CSC-TPDC–primed CTLs (P ¼ 0.03).SCC7 CSC-TPDC–primed CTLs killed SCC7 CSCs efficiently(�60%) and significantly more than their killing of unsortedSCC7 cells (�20%; P ¼ 0.001) or ALDH� SCC7 cells
0
10
20
30
40
50
ng
/mL
CD3/28/IL-2No activation LPS/CD40
P = 0.001
P = 0.004
P = 0.002
PBS
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0
50
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ng
/mL
LPS/CD40CD3/28/IL-2No activation
P = 0.031
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II antibody only PBS–treatedII antibody only PBS–treated
CSC-TPDC–treatedH-TPDC–treated
30.1 %0.1 %
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H-TPDC–treated CSC-TPDC–treated
R2 R2
R2 R2
0.1% 0.9%
4.0% 26.4%
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A
B
Figure 5. A, systemic humoral responses in CSC-TPDC vaccinated immunocompetent host. Splenocytes were harvested from the animals subjectedtoH-TPDCorCSC-TPDC vaccination andwere activatedwith anti-CD3/anti-CD28/IL-2 for T cells orwith LPS/anti-CD40 for B cells. The culture supernatantswere then collected for IgG detection using ELISA. Data are representative of 2 experiments carried out. B, plasma harvested from D5CSC-TPDC–treated orSCC7 CSC-TPDC–treated animals binds to ALDHþ D5 CSCs and ALDHþ SCC7 CSCs, respectively. Data were repeated in a second experiment forthe D5 model and are representative of 3 experiments carried out for the SCC7 model. SSC, side scatter.
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(�25%; Fig. 7A; P ¼ 0.002). We also carried out CTL experi-ments using the effector cells generated from the splenocytesharvested from the vaccination experiments shown in Fig. 3. Asrevealed in Fig. 7B, D5 CSC-TPDC–primed CTLs killed ALDHþ
D5 CSCs significantly higher (P < 0.01 at all effector:targetratios) than ALDH� D5 non-CSCs. In addition, we haveobserved a reduction of residual ALDHhigh CSCs within thetumors growing in the hosts subjected to ALDHhigh-TPDCvaccination compared with ALDHlow-TPDC vaccination (Sup-plementary Table S2).Together, these results provide direct experimental evidence
that CSCs can be destroyed by CSC vaccine–primed antibodiesand T cells. The immunologic targeting of CSCs may provide anovel approach for the development of more effective cancerimmunotherapies.
DiscussionALDEFLUOR/ALDH has been successfully used as a mark-
er to isolate stem cell–enriched populations in humancancers (30–33). Using this marker, we enriched CSCs from2 histologically distinct murine tumors to investigate immu-nologic strategies which may specifically target CSCs in 2genetically different immunocompetent hosts. Schatton andcolleagues recently identified a subpopulation enriched forhuman malignant melanoma–initiating cells (MMIC)defined by expression of the chemoresistance mediatorABCB5 (17). We have reported that ALDH is a CSC markerfor human head and neck squamous cell cancers (36).Controversy exists about the existence of CSCs in human
melanomas (37) as tumorigenicity of non-CSCs is evidentwhen extreme immunocompromised hosts (NOD/SCID/IL-2rgnull) are used. However, Civenni and colleagues recentlyfound that the avoidance of trypsin for isolating CSCs fromhuman melanomas which leaves intact surface epitopes ontumor cells results in the ability to isolate cells with genuineCSC properties (38). In our murine studies, we have avoidedthe use of trypsin to isolate CSCs from freshly harvestedgrowing tumors. These reports that CSCs have been iden-tified in human melanoma and squamous cell cancersprovide a rationale to explore CSCs as a target for immu-notherapies in our murine models.
Studies have shown that CSCs are resistant to chemotherapy(18, 20–23). These studies highlight the limitation of currentcancer chemotherapies that are unable to target CSC popula-tions. Novel therapeutic strategies are needed, particularly bytargeting the CSCs. We have recently reported that CXCR1blockade can selectively target human breast CSCs in vitro andin xenografts (34). CXCR1 is a receptor for IL-8 which stimu-lates the self-renewal of CSCs. In the present study, we haveidentified a different method to selectively target CSCs usingthe host immune system.We have been able to show that CSCscan be selectively targeted by both B- andT-cellmechanisms inan active immunization protocol. This was done in 2 genet-ically distinct immunocompetent hosts using a pulmonarymetastasis model and a subcutaneous tumor model, respec-tively. Cancer immunotherapy using DNA vaccine (39), adop-tively transferred T cells (40) or DCs (27, 41), has shown theinvolvement and modulation of host immune effector cells. T-and B-cell depletion in the recipient mice to show absent
ALDH–
CM CM PBS H-TPDC CSC-TPDC CSC-TPDC0
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100
% o
f v
iab
le c
ell
s
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+complement
P < 0.001 P < 0.001
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P = 0.002
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CM CM PBS H-TPDC CSC-TPDC CSC-TPDC0
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f v
iab
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ell
s
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+complement
P < 0.001
P < 0.001
P = 0.001P = 0.001
Target cells: ALDH–
Figure 6. Targeting of CSCs byCSC-primed antibody andCDC. Plasma antibody and complement-mediated (CM) CSC lysis was assessed by incubating 105
viable ALDHþ CSCs or ALDH� non-CSCs (as control) with plasma harvested from animals subjected to PBS, H-TPDC, or CSC-TPDC treatment intest tubes for 1 hour followed by cell culture in the presence of rabbit complement for another 1 hour. Viable cells were then counted under amicroscope aftertrypan blue staining to calculate cell lysis:%of viable cells¼ viable ALDHþor ALDH� cells after plasma and complement incubation/105. Lower percentage ofviable cells at the end of incubation indicates more cell lysis. Data of CDCs were replicated in a second experiment for both the (A) D5 and (B) SCC7tumor models.
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effects on tumor behavior would confirm that CSC vaccinationinduces cellular and humoral anti-CSC responses and under-score the relative role of host T and B cells in the immuneresponse induced by CSC-based vaccines.
Several reports have described the killing of CSCs via non-specific immune effector cells, such as natural killer (NK) cells(42). Contag and colleagues reported a modified approach tokill CSCs which involved cytokine-induced killer cells (IFN-gand anti-CD3 activated) that were used as cellular vehicles todeliver oncolytic virus to CSCs but no CSC-specific antibodiesor T cells were identified, even though immune componentsnecessary for targeting the virus itself were examined andidentified (43). Interestingly, Todaro and colleagues describedkilling of human colon CSCs by gd T lymphocytes in vitro (44),but no in vivo protective or therapeutic experiments werecarried out to correlate the immunologic significance of thein vitro CSC killing with potential in vivo antitumor immunityagainst tumor growth or development.
Our immunemonitoring studies revealed direct targeting ofCSCs by antibody and CTLs. This was evident by the produc-tion of IgG by the splenocytes isolated from the hosts subjectedto CSC-TPDC vaccination and the binding of the antibody tothe CSCs which resulted in the CSC lysis via CDC. In addition,CTLs generated from the PBMCs and splenocytes obtained
from CSC-vaccinated hosts selectively killed CSCs. Pellegattaand colleagues used a murine brain tumor cell line GL261 andreported that neurospheres (NS) from glioblastoma multi-forme are enriched in CSCs and that DC loaded withGL261-NS protected mice (45). However, no evidence wasdescribed for the direct targeting of CSCs, and no immunologicevaluation was conducted to elucidate themechanisms poten-tially involved in the protection. Xu and colleagues reported ahuman glioblastoma-derived stem-like cell study in vitro (46).They used a rat 9L CSC brain tumor model and found thatneurosphere-pulsed DCs prolonged survival in rats bearing 9Ltumors and induced IFN-g mRNA expression in CD8 cells.Nevertheless, no direct and specific targeting and killing of theCSCs, either by antibody-mediated cytotoxicity or by T-cell–mediated cytotoxicity was examined. Our studies provide thefirst direct experimental evidence that CSCs can be selectivelytargeted and destroyed by CSC vaccine–primed antibodies andT cells, and such immunologic targeting of CSCs is associatedwith enhanced in vivo CSC vaccine–conferred antitumorimmunity.
The immunogenic response elicited against bulk tumor cellsmay be skewed toward differentiation antigens expressed onthese cells. This might mask immunologic responses to CSCswhich represent only a minor percentage of tumor cells. Our
H-TPDC CSC-TPDC H-TPDC CSC-TPDC0
20
40
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80
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toxic
ity %
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P = 0.003 P = 0.005
P = 0.03
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toxic
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Effect cell:target cell
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* P < 0.01
(at all ratios)
Unsorted
SCC7 cells
ALDH+
SCC7 cells
ALDH–
SCC7 cells
A
B
Figure 7. Targeting of CSCs byCSC-primed CTLs. Cytotoxicitiesof CSCs mediated by CTLsgenerated from the PBMCs (A)harvested from the CSC-TPDC–vaccinated animals are shown. Thekilling of CSCs mediated by theCTLs was measured by an LDHrelease assay as described in theMaterials and Methods. Higherpercentage of cytotoxicityindicates more cell lysis. Data werereplicated in a second experimentfor both the D5 and SCC7 tumormodels. Cytotoxicity of ALDHþ
versus ALDH� D5 cells by effectorcells generated from thesplenocytes (B) of mice vaccinatedwith D5 CSC-TPDC was alsomeasured by the LDH releaseassay as in (A). Data arerepresentative of 2 experimentscarried out.
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results suggest that CSCs, which may not express immuno-genic differentiation antigens (47), can elicit an anti-CSCresponse when presented as a vaccine. Furthermore, as thisimmune response is specifically directed against the CSCpopulations, it may have a greater biologic effect than onedirected at bulk tumor cell populations. Schatton and collea-gues reported that human ABCB5þ melanoma-initiating cellsmanifested immunomodulatory functions that were immuno-suppressive to T-cell activation in culture (48). Instead of an invitro system, we used an in vivo approach to activate host T-celland antibody responses which resulted in the induction ofspecific anti-CSC immunity. Furthermore, we used CSC lysatefor vaccine preparation instead of using intact CSCs. Identi-fication of the respective murine melanoma D5 and SCC7 CSCantigens responsible for the observed effects in this studywarrants further investigation. We have shown herein prefer-ential induction by ALDHþ cell–based DC vaccines of ahumoral response to ALDHþ CSCs. Use of polyclonal serafrom CSC-vaccinated recipients in immunoprecipitationassays may lead to the identification of differentially expressedcandidate antigens in CSCs that can be validated experimen-tally in vaccination studies.It is important to determine the therapeutic efficacy of
established tumors with CSC-based vaccines. Because estab-lished tumors are composed of a very small percentage of CSCs,we postulate that the use of CSC-based vaccines alonewill have
minimal effect on established tumors and will require otheradjunctive therapies. Furthermore, CSCs isolated from highlypassaged cell lines or from freshly harvested tumors afterinoculation may possess immune activity that is not a featureof CSCs obtained directly from spontaneously arising tumors.The lack of using spontaneous tumors remains one of thelimitations of the findings in this study. Nevertheless, evalu-ation of the immunogenicity of CSCs from tumors derived fromimmunocompetent hosts is novel, and the use of CSCs in suchsyngeneic hosts permits the assessment of immune responsesagainst these cells. Our animal models will allow us to explorecombinatorial approaches for the therapy of more establishedtumors by selectively targeting CSCs.
Disclosure of Potential Conflicts of InterestNo potential conflicts of interests were disclosed.
Grant SupportThis work was supported by TheWill and Jeanne Caldwell Endowed Research
Fund of the University ofMichigan Comprehensive Cancer Center, and in part byNIH grant CA82529, and the Gillson Longenbaugh Foundation.
The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.
Received April 22, 2011; revised December 6, 2011; accepted February 6, 2012;published online April 2, 2012.
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