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Epigenetic silencing of THY1 tracks the acquisition of the Notch1-EGFR signaling in a

xenograft model of CD44+/CD24low/CD90+ myoepithelial cells

Micaela Montanari1,2

, Maria Rita Carbone1, Luigi Coppola

1, Mario Giuliano

2, Grazia Arpino

2,

Rossella Lauria2, Agostina Nardone

1,3, Felicia Leccia

4, Meghana V.Trivedi

3,5, Corrado Garbi

1,

Roberto Bianco2, Enrico V. Avvedimento

1, Sabino De Placido

2,6, and Bianca Maria Veneziani

1,6

1Department of Molecular Medicine and Medical Biotechnology, University of Naples Federico II,

Naples, Italy.

2Department of Clinical Medicine and Surgery, Oncology Division, University of Naples Federico

II, Naples, Italy.

3Lester and Sue Smith Breast Center and Dan L. Duncan Comprehensive Cancer Center, and

Department of Medicine, Baylor College of Medicine, Houston, Texas, USA.

4CEINGE-Biotecnologie Avanzate, Naples, Italy.

5Department of Pharmacy Practice and Translational Research, University of Houston College of

Pharmacy, Houston, Texas, USA.

6Oncotech, School of Medicine and Surgery, University of Naples Federico II, Naples, Italy.

Running title: Epigenetic silencing of Thy1 in CD44+/CD24low/CD90+ myoepithelial cells.

Key words: CD90/thy1, myoepithelial cells, TNBC triple negative breast cancer, basal-like breast

cancer, tumor heterogeneity, epithelial-mesenchymal phenotype, breast cancer tailored therapy

on May 4, 2021. © 2018 American Association for Cancer Research. mcr.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on September 21, 2018; DOI: 10.1158/1541-7786.MCR-17-0324

http://mcr.aacrjournals.org/

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Additional information.

Corresponding author:

Bianca Maria Veneziani, Department of Molecular Medicine and Medical Biotechnology,

University of Naples Federico II, Via S. Pansini 5, 80131 Napoli, Italy. Phone: 39-081-7463758. E-

mail: [email protected]

Funding

This work was supported by Ministero dell’Universita` e Ricerca, PRIN Grant 2015B7M39T (to S.

DePlacido and B.M. Veneziani), Grant MOVIE of the Rete delle Biotecnologie in Campania (to

B.M. Veneziani), PON 03PE_00146_1 BIOBIOFAR (to R. Bianco, S. DePlacido. and B.M.

Veneziani) M. Montanari is supported by a post-Doctoral Fellowship from POR CREME, M.R.

Carbone is supported by a fellowship from Dottorato di Ricerca (PhD) in Medicina Molecolare e

Biotecnologie Mediche, University Federico II of Naples, Italy.

Competing financial interests

The authors declare that they have no competing financial interests.




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Abstract

The surface glycoprotein Thy1 is a marker of myoepithelial precursor cells, which are basal cells

with epithelial-mesenchymal intermediate phenotype originating from the ectoderm. Myoepithelial

precursor cells are lost during progression from in situ to invasive carcinoma. To define the

functional role of Thy1-positive cells within the myoepithelial population we tracked Thy1

expression in human breast cancer samples, isolated Thy-positive myoepithelial progenitor cells

(CD44+/CD24low/CD90+) and established long term cultures (parental cells). Parental cells were

used to generate a xenograft model to examine Thy1 expression during tumor formation. Post-

transplantation cell cultures loss Thy1 expression through methylation at the THY1 locus and this is

associated with an increase in EGFR and Notch1 transcript levels. Thy1-low cells are sensitive to

the EGFR/HER2 dual inhibitor lapatinib. High Thy1 expression is associated with poorer relapse-

free survival in breast cancer patients. Thy1 methylation may track the shift of bipotent progenitors

into differentiated cells. Thy1 is a good candidate biomarker in basal-like breast cancer.

Implications: Our findings provide evidence that Thy1 expression is lost in xenografts due to

promoter methylation. Thy1-low cells with increased EGFR and Notch1 expression are responsive

to target therapy. Because DNA methylation is often altered in early cancer development, candidate

methylation markers may be exploited as biomarkers for basal-like breast cancer.




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INTRODUCTION

The human breast gland is a ductal tree covered with a monolayer of polarized epithelial cells

whose basal surface lies on contractile myoepithelial cells that are confined by the basement

membrane and surrounded by an interstitial stroma. Myoepithelial cells originate from the ectoderm

and are basal cells, namely, cells in the basal position adjacent to the basement membrane (1).

Interest in basal cells was stimulated after molecular gene profiling divided breast cancer into five

intrinsic subtypes, one of which displays basal-like gene expression (2). Basal-like breast cancers

are generally aggressive (3) and most are triple-negative, i.e., they test negative for estrogen

receptor (ER), progesterone receptor (PgR) and HER2 (HER) (4). Treatment of patients with basal-

like triple-negative breast cancer (TNBC) is challenging because of the heterogeneity of the disease

and the absence of well-defined druggable targets (5).

In breast cancer, myoepithelial cells are considered tumor suppressors because they inhibit

epithelial cell growth and invasion (6), and because their oncosuppressive function disappears

during progression from in situ to invasive carcinoma (7). Disappearance of the basement

membrane and of the myoepithelial cell layer distinguishes invasive from in situ carcinomas (8),

and the gene expression profiles of myoepithelial cells associated with in situ cancer are distinct

from those in normal breast (9). The signals that initiate these changes are unknown, although it is

recognized that tumor-associated fibroblasts and myofibroblasts counteract the tumor suppressor

function of myoepithelial cells by promoting tumorigenesis (10, 11) and cancer progression (11).

Thy1 (also known as “CD90”) is a surface glycoprotein of 25–28 kDa (12) that is expressed on

the cytoplasmic membrane of diverse cell types (13). The structural gene for human Thy1 lies on

the long arm of chromosome 11 (11q23.3) (14). Thy1 triggers a variety of cellular functions,

namely, proliferation, differentiation, wound repair and apoptosis. In lung cancer, Thy1

differentiates between malignant pleural mesothelioma and lung carcinoma (15). In melanoma, it

contributes to metastasis seeding by mediating the adhesion of melanoma cells to endothelial cells

(16). Thy1 has been associated with tumor suppression in human ovarian cancer (17). It is also a




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cancer stem cell marker in esophageal cancer (18), high-grade gliomas (19) and hepatocarcinoma

(20). In breast cancer, Thy1-expressing cells are undifferentiated cancer progenitor/stem cells (21).

Thy1 is up-regulated in the epithelial-to-mesenchymal transition (EMT) core signature (22).

Moreover, it mediates the interactions of breast cancer stem cells with tumor-associated

macrophages to maintain and reinforce the cancer stem cell state (23).

We previously demonstrated that sorted CD44+/CD24

low cells display differential expression of

surface markers that identify heterogeneous myoepithelial phenotypes (24). Among these surface

markers, Thy1/CD90 was commonly found highly expressed. In this study, we have investigated

the relevance of Thy1 as a tracer biomarker of myoepithelial precursor cells also in relation to

receptor profile, in our model of sorted breast cancer stem/progenitor cells. We show that

xenotransplantation of CD44+/CD24low/CD90+ myoepithelial cells in mouse reduces Thy1

expression through methylation of THY1 in conjunction with the acquisition of the Notch1-EGFR

signaling.

MATERIALS AND METHODS

Cell lines and materials

Cell lines MCF-7, MDA-MB231 (MDA), BT474 and MCF10A were from ATCC (American Type

Culture Collection). HER2-18 cells (25) were kindly provided by Dr. R. Schiff. Cells were

maintained in standard medium consisting of minimal essential Dulbecco/Ham F12 (1:1)

(DMEM/F12) (Sigma-Aldrich) supplemented with 2 mM glutamine (Sigma-Aldrich), 1%

penicillin/streptomycin (Life Technologies, Carlsbad, CA, USA), 15 mM HEPES (Sigma-Aldrich)

and 5% fetal bovine serum (FBS) and at 37°C in a humidified atmosphere of 5% CO2 air. Cell

cultures were routinely checked for mycoplasma with Hoechst 33258 (Sigma-Aldrich) staining,

mycoplasma-negative were used for experiments. Adherent and non-adherent 24-well ultra-low

binding plates were used (Corning, NY, USA). Fetal bovine serum was purchased from Gibco

(Invitrogen, Milan, Italy). The monoclonal anti-pancytokeratin and anti-vimentin antibodies were




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purchased from Sigma-Aldrich. Multicolor flow cytometry was performed with antihuman

monoclonal antibodies (MoAbs) that were conjugated with phycoerythrin (PE), fluorescein

isothiocyanate (FITC), PE-Cy7 (PE-Cy7) or Alexa Fluor 647. Phycoerythrin-conjugated MoAbs

against CD10, CD29, CD49f, CD61, and FITC-conjugated MoAbs against CD49b, CD90, CD227,

CD324, and CD326 were from BD Biosciences and BD Pharmingen (San Jose, CA ,USA); PE-

conjugated MoAbs against CD133 were from Miltenyi Biotech (Auburn, CA, USA); Alexa

Fluor647-conjugated MoAbs against CD24, and PE-Cy7-conjugated MoAs against CD44 were

from Biolegend (San Diego, CA, USA).

Ethics and study design

Residual breast cancer and paired normal specimens were collected, after informed consent, from

patients undergoing surgery for breast cancer at the Azienda Ospedaliera Universitaria Federico II

(Naples, Italy). Nine patients with breast cancer were recruited. Pathological diagnosis was made

based on the histology of tumor specimens that had been examined by experienced pathologists.

Tumor histotype, size, grading and markers including ERα were determined with standard

procedures, and HER2 was determined with the Hercep Test TM (Dako, Carpintera, CA,USA). The

receptor profile of human breast tumors, namely, ER status, PgR status, and HER2 are summarized

in supplemental Table S1. The breast cancer intrinsic subtype was determined using surrogate

immunohistochemistry definitions according to Goldhirsch et al.(26).

Sample collection

Breast cancer tissue (S#) and paired normal (N#) specimens were collected using a biobanking

standard operating procedure as previously reported (27). The samples were anonymously encoded

to protect patient confidentiality and used according to protocols approved by the Azienda

Ospedaliera Universitaria Federico II Ethics Committee (Ethical Committee Approval Protocol #

107/05). The primary objective of the approved protocol was to expand human breast cancer cells to

characterize the protein expression profile of in vitro cultured cells.

Primary cultures and breast cancer stem/progenitor cells




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Within 2 hours after surgery, fragmented aliquots of fresh specimens were processed as previously

reported (28). Briefly, the samples were extensively rinsed with PBS and suspended in standard

culture media supplemented with 10% FBS. After three cycles of differential centrifugation, cells

were seeded overnight in minimal (DMEM/F12 medium (1:1) (Sigma-Aldrich), supplemented with

2 mM glutamine (Sigma-Aldrich), P+S (100 µg/ml streptomycin, 100 units/ml penicillin), 15 mM

HEPES (Sigma–Aldrich) and 5% FBS. After exposure to trypsin (0.25% in 1 mM EDTA; trypsin–

EDTA solution, Invitrogen) for 2 min at 37°C, the floating aggregates were transferred to 24 well

plates and cultured in standard medium, DMEM/F12 + 0.5% FBS, for 21-30 days at 37°C in a

humidified atmosphere of 5% CO2 air. Cells were continuously passaged with trypsin-EDTA until

only the tumor epithelial cell population remained. The epithelial origin of the cells was confirmed

by western blot with monoclonal anti-pancytokeratin antibody (Supplementary Figure S1). Multiple

vials of cells were cryopreserved. Frozen cells were thawed, allowed to adhere and harvested within

15-20 days in standard medium before sorting.

Flow cytometry and sorting

Flow cytometry experiments were performed as previously reported (24). Briefly, samples and

control cells, harvested at sub-confluence in 100-mm dishes, were dissociated by trypsin-EDTA,

counted in a hemocytometer chamber, and 2x106cells/sample were incubated for 5 min at room

temperature with 50 µL of FBS. Cells were washed twice with PBS and stained at 4°C for 20 min

with the appropriate amount of the fluorescent labeled MoAb in PBS. After staining, all samples

were washed twice with PBS, centrifuged and suspended in 0.5 ml of FACS buffer (FACS Flow

Sheat Fluid, BD Biosciences) for FACS analysis. To exclude dead cells, immediately before FACS

acquisition, cells were incubated at room temperature in the dark with a vital dye (SytoxBlue,

Invitrogen). We used a four-color flow cytometric method to measure the expression of the markers

based on a flow cytometry panel in which cells were stained with anti-CD24-Alexa Fluor 647 and

anti-CD44-PE-Cy7 MoAb, and with FITC-conjugated antibodies against CD90 (Thy1) and PE-

conjugated anti CD133 (prominin 1) (24). Fluorescence Minus One (FMO) control, was used as a




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negative control. After washing twice with PBS/0.5% bovine serum albumin (BSA), cells were

pelleted, suspended in 300 µL of PBS/0.5% BSA, and filtered through 50-micronfilters. Cell

analysis and sorting were performed with a FACS Aria flow cytometer and with the FACS Diva

software (Becton Dickinson, Franklin Lakes, NJ, USA). A total of 10,000 to 20,000 events were

recorded and analyzed in each sample run. A three gating strategy was adopted: first, to exclude

dead cells and debris, cells were gated on a two physical parameters dot plot measuring forward

scatter (FSC) versus side scatter (SSC). Then, doublets were excluded by gating cells on FSC-

Height versus FSC-Area dot plots, and, finally, SytoxBlue-negative cells were gated. The levels of

expression of surface markers were reported as percentage of positive cells in Count versus FITC-

or PE-CD histograms. For cell sorting, the cells were suspended in PBS with 2% FBS and 0.5 mM

EDTA, sequentially labeled with a cocktail of MoAb anti-CD24-AlexaFluor647and anti-CD44-PE-

Cy7, mixed with magnetic microbeads and separated using a magnet. The purity of sorted cells was

evaluated by flow cytometry. Sorted CD44+/CD24low cells were cultured with standard medium

for at least four/five passages before experiments. The steps of isolation of

CD44+/CD24low/CD90high myoepithelial precursor cells (K#) are summarized in Supplementary

Figure S2. Cultures at the fourth-fifth passage were used for the experiments.

Immunofluorescence

For immunofluorescence analysis cells were plated on glass coverslips, fixed, immunostained at 4°

overnight with rabbit polyclonal anti-Thy1 antibodies (H-110; SC-9163; Lot # B2514; Santa Cruz

Biotechnology, Inc., Santa Cruz, CA, USA) and treated for 30’ with goat anti-rabbit IgG secondary

antibody tagged with Alexa Fluor 594 (1/300; A-11012; Lot # 1420898; Life Technologies). Nuclei

were stained for 30 min at room temperature with 1:1 (v/v) Hoechst 33258 (94403, Sigma-

Aldrich)/DRAQ5TM

(ab108410; Abcam). Immunofluorescence was visualized using a Zeiss

LSM510 Meta argon/krypton laser-scanning confocal microscope.

Cumulative population doubling frequency




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Experiments were performed in triplicate in 24-well plates using 2.5 x103 cells/well. The cells,

routinely cultured in 100 mm dishes, were enzymatically detached, counted, and 2.5 x103 cells/well

were seeded with standard medium. Cells were maintained in a sterile environment, and at the times

indicated in the figures, they were trypsinized, counted in a hemocytometer chamber, and re-plated

1:2 in new wells. Cells viability was assessed by Trypan blue with paired triplicates. Lineage

continuity for Thy1 in 3D culture was assessed with immunofluorescence (representative at

Supplemental Figure S1). For 2D experiments, after trypsinization, the cells were plated over-night

in 5% FBS, allowed to adhere and then switched to 0.5% FBS. The proliferation rate of the cells

was measured by calculating the cumulative population doubling frequency (cpdf) in continuous

culture from a known number of cells using the formula Ln(No/Nn)/Ln2, where Ln is the natural

log and No and Nn are, respectively, initial and final cell numbers at each subcultivation. The sum

of the cpdf of the subcultivation periods provides the cumulative final number of total counts.

Semi-quantitative multiplex RT-PCR analysis and Real Time PCR analysis

Total RNA was isolated from sample and control cells, and from breast tumor tissues using TRIzol

Reagent (Invitrogen, Carlsbad, CA, USA) according to the producer’s instructions. Purity of RNA

was checked by measuring the absorbance ratio at 260/280 nm in a Beckman Coulter

spectrophotometer (Beckman Coulter, Fullertone, CA, USA) with appropriate purity values

between 1.8 and 2.0. RNA was stored at -80°C in aliquots of 50 ng/L. The integrity of RNA was

assessed on a standard 1% agarose/formaldehyde gel. The reverse transcription of 1.5 µg of total

RNA was performed with the Super Script III reverse transcriptase kit (Invitrogen, Carlsbad, CA,

USA) according to the manufacturer’s instructions.

Multiplex PCR was performed in 50 μL reactions using the PTC-200 Peltier Thermal Cycler (Bio-

Rad Laboratories, Hercules, CA, USA) and gene-specific sets of primers, including those for the

internal standard β-actin. Agarose gel electrophoresis and staining with 0.3 mg/ml of ethidium

bromide (Sigma, St. Louis, MO, USA) were carried out to assess template products.




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Real-time PCR amplifications were carried out on a Step One Real-Time thermocycler (Applied

Biosystems, CA, USA) using the iTaq Universal SYBR Green Supermix (Bio-Rad, CA, USA).

Experiments were performed in triplicate for each data point, and the expression of

housekeeping beta-2-microglobulin gene (B2M Forward: 5’-GCA GAA TTT GGA ATT CAT CCA

AT-3’; Reverse: 5’- CCG AGT GAA GAT CCC CTT TTT-3’) was used for normalization.

Primer sequences are listed in Supplementary Table 2 (Table S2).

Nude mice cancer xenografts

The tumorigenicity of sorted cells was assessed by injecting the harvested cells into

immunodeficient mice. Five-week-old female BALB/c athymic (null/null) mice (Charles River

Laboratories, Milan, Italy) were maintained in accordance with the institutional guidelines of the

University of Naples Animal Care Committee welfare policy (European Commission 86/609/EEC).

All the animal experiments were approved by the Ethics Committee of the University of Naples

Federico II Animal Care (ethical approval protocol # 83). Adherent harvested K197 cells were

enzymatically dissociated, counted, diluted in PBS, mixed 1:1 with 200 μl Matrigel (CBP, Bedford,

MA, USA), and injected orthotopically in the fourth mammary fat pad of triplicate mice, as

previously reported (29). We injected 1x102, 1x10

3, 1x10

4, 1x10

5, 1x10

6 of the K197 cells into the

fourth mammary fat pad of immunodeficient mice. The experiment was performed twice. Tumor

volume (cm3) was measured with calipers and calculated with the formula π/6 × largest diameter ×

(smallest diameter)2. Within 8 weeks, the tumors (size: 1-3 cm

3) were excised, digested with a

trypsin/collagenase mixture and plated for in vitro growth. The steps of isolation of

CD44+/CD24low/CD90-Thy1-low cells (Topo9) are summarized in Supplementary Figure S3. The

homogeneity of the cultures was confirmed with flow cytometry analysis of ten surface markers

(Table 1). Cultures at their fourth/fifth passage were used for the experiments.

5-Aza-2’-Deoxycytidine treatment

Thy1-positive (K197) and Thy1-low (Topo9) cells, 5x105cells/cell type, were seeded in 100mm

cell culture dishes (Corning, NY, USA) with standard medium. After an initial 24 hours of




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incubation, the cells were exposed to 5 μM 5-aza-2′-deoxycytidine (5-AZA-dC) (Sigma, St. Louis,

MO, USA) for 12, 24, 36, 48, 72 and 96 hours. The medium was renewed every 24 hrs. Control

cultures lacking 5-AZA-dC treatment were incubated in the identical culture condition. At the time

indicated, the cells were harvested for total RNA extraction.

Methylation Sensitive Amplified Polymorphism (MSAP)

Genomic DNA was extracted with phenol/chloroform technique (30). For measurement of Thy

promoter methylation status, 1µg of DNA was digested overnight at 37 C with HpaII or MspI (50

U/1µg DNA, Fermentas) restriction enzymes. DNA was recovered by phenol/chloroform extraction

and ethanol precipitation, and resuspended in DNase/RNase free water. Hpa II sensitivity was

evaluated by amplifying a 276 bp fragment containing 4 CG upstream of the Thy promoter

(position: -2328, -2297, -2259 and -2190 with respect to the first ATG). Methylation status was

assessed by analysing the efficiency of fragment amplification exposed to digestion (HpaII or MspI)

on 2% agarose gel and quantified by Real-Time PCR. Amplification was performed on a PTC-200

Peltier Thermal Cycler (Bio-Rad Laboratories, Hercules, CA, USA) through 40 PCR cycles using

the following temperature profile: 95°C for 40 sec, 61°C for 40 sec, 72°C for 1 min, and one final

elongation step at 72°C for 10 min. All reactions were preceded by a primary denaturation step at

95°C for 5 min. Real-time PCR amplifications were carried out on a Step One Real-Time

thermocycler (Applied Biosystems, CA, USA) using the iTaq Universal SYBR Green Supermix

(Bio-Rad, CA, USA). Cycling conditions were: one cycle at 95 °C for 5 min, followed by 40 cycles

of 95°C for 15 sec, 61°C for 20 sec, and 72°C for 30 sec. Experiments were performed in triplicate

for each data point. The following primers were used for Thy-1 DNA amplifications: Forward 5’-

CCAATGCGGGACCGCCTTCTCTTCC-3; Reverse 5’-GTCTTGCATGGGCGCCTGACGGCG-

3’.

Western blot

Protein preparations were obtained by lysing samples in 50 mM Tris (pH 7.5), 150 mM NaCl, 1%

Nonide tP40, 0.1% Triton, 1 mM EDTA, 10 µg/mL aprotinin, and 100 µg/mL




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phenylmethylsulfonyl-fluoride. Protein concentration was measured by the Bio-Rad protein assay

(Bio-Rad, Milan, Italy). Twenty-five-microgram aliquots were electrophoresed through from 8% to

15% SDS polyacrylamide gels. After transfer onto nitrocellulose membranes (Hybond-C pure;

Amersham Italia, Milan, Italy) the membrane was stained with Ponceau S (Sigma) to evaluate the

success of transfer, and to locate the molecular weight markers. Free protein binding sites were

blocked with non-fat dry milk and Tween-20/TBS solution. The membranes were washed, stained

with specific primary antibodies and then with secondary antisera, conjugated with horseradish

peroxidase (1:3000; Santa Cruz Biotechnology). Antibodies were: Ab anti-E Cadherin (1:1000, Cell

Signaling Technology, USA); Ab anti-Notch1 (C-20): sc-6014-R (1:200, Santa Cruz

Biotechnology, USA); Ab anti-Fibronectin (P1H11): sc-18825 (1:100, Santa Cruz Biotechnology,

USA); Ab anti-Fibronectin (EP5): sc-8422 (1:200, Santa Cruz Biotechnology, USA); Ab anti-

ERalfa (F-10):sc-8002 (1:200, Santa Cruz Biotechnology, USA). The luminescent signal was

visualized with the ECL Western blotting detection reagent kit (Amersham Italia) and quantified by

scanning with a Discover Pharmacia scanner equipped with a Sun Spark Classic Workstation.

Expression levels were calculated as the relative expression ratio compared to β-actin or tubulin

using Image J (ImageJ.nih.gov).

Mammosphere formation assay and growth in soft agar

Cells were dissociated and seeded, 1000 cells/well, in ultra-low attachment 24-well plates (Corning)

in DMEM/F12 plus 0.5% FCS medium, as previously reported (31). The medium was renewed

twice weekly. Mammospheres were cultured for 15 days and their diameter measured under an

Axiovert 40 C inverted microscope (Zeiss, Milan, Italy) equipped with a Canon powershot A640

camera (Zeiss). Digital images were analyzed with AxioVision software (Zeiss). For colony growth

in soft agar cells were trypsinized, counted, and 104 cells/dish were plated in 60 mm triplicate

dishes with 0.3% agar on a 0.5% agar (Type I, Sigma) underlayer DMEM/F12 containing 0.5%

FBS. Colonies, cultured for 60 days, were counted in 10 fields per dish. The fields to be counted

were ID numbered fields on a 7x7 horizontal-vertical transparency grid 60 mm in diameter. The




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same ID fields were counted for all dishes. Results are reported as mean ± SEM of three different

experiments performed in duplicate.

Cell viability assay

Cell viability experiments were performed as previously described (32). Cells were seeded at 2,500

cells/well, in 24-well plates, treated with the reported concentrations of lapatinib for 7days and

analyzed with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) assay

according to the manufacturer’s instructions (Sigma-Aldrich). The percentage of absorbance of

treated samples versus untreated is reported as a percentage of viable cells/controls. Experiments

were performed three times; values represent means±s.d. from triplicate samples for each treatment.

Kaplan–Meier curves

Kaplan–Meier curves were generated using the KM plot software and a public database of

microarray datasets (probes: 213869_x_at (Thy1-CD90);211551_at (EGFR, ERBB, ERBB1)

(http://kmplot.com/analysis) (33). Kaplan–Meier plots were generated after averaging the probes.

For the analysis, eligible patients were divided according to the median expression value, and ERα

negative/HER2-negative cases were included. P value was determined by log-rank test.

Statistical analysis

Flow cytometry, cell counting, sphere formation assay, and RT-PCR experiments were carried out

2-3 times and found to be reproducible. Human tissue samples were not pooled; each sample served

as its own control. Values are presented as mean ± s.e.m. of multiple experiments, each experiment

was performed at least in triplicate, or as mean ± s.d. of triplicates when a representative experiment

is shown. The statistical significance between two groups was determined with the Fisher exact test,

multiple group comparisons were made with ANOVA, as reported. Pearson’s correlation coefficient

was used to calculate r. GraphPad software was used for all statistical analyses.

Ethical approval



http://kmplot.com/analysis


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All procedures performed in studies involving human participants were in accordance with the

ethical standards of the institutional and/or national research committee and with the 1964 Helsinki

declaration and its later amendments or comparable ethical standards.

Availability of data and materials

The datasets used and/or analysed during the current study are available from the corresponding

author on reasonable request. The datasets generated and/or analysed during the Kaplan Meier

curves are available in the [kmplot.com] repository, [http://kmplot.com/analysis/]

Supplementary data

The Supplementary Data include supplementary tables S1 and S2, Supplementary Figures S1-S7,

Supplementary Legend to Figures S1-S7.



http://kmplot.com/analysis/


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RESULTS

CD44+/CD24

low/Thy1+ myoepithelial progenitor cells

In our previous studies we profiled heterogeneous Thy1-expressing myoepithelial phenotype in

breast carcinomas. To investigate the functional role of Thy1-expressing cells in experimental

models, we isolated breast cancer cells that displayed features of stem/progenitor cells from fresh

surgical breast tumor tissues. Nine tumor specimens (S#) were chosen from our stored breast cancer

collection of fresh frozen tissues belonging to various molecular subclasses of breast tumors: S40,

S43, S79, S88, S193 and S197 were luminal breast cancers; S66 was HER2-positive; and S77 and

S90 were triple-negative (Supplementary Table S1). Breast cancer stem cells are identified by one

or more of the following features: a CD44+/CD24

low phenotype, mammosphere formation in vitro,

and ability to form new tumors when xenografted into immunodeficient mice (34, 35). We

established nine primary cultures of breast cancer; to avoid fibroblasts, cells were continuously

passaged until only the tumor epithelial cell population remained (Supplemental Figure S1).

Immunofluorescence experiments with antibodies against Thy1/CD90 showed a mixed population

of Thy1-positive and Thy1-negative cells in the primary cultures with a percentage ranging from

27% to 58% of Thy1/CD90 stained cells versus non stained cells (Fig.1A). From each culture we

sorted the CD44+/CD24

low cells. Thy1/CD90 expression assessed by immunofluorescence on cells

harvested for 10 days after sorting, showed expression of Thy1 on 90-100% of the sorted

CD44+/CD24

low cells and on 98-100% of cells maintained in culture for three months (Fig.1A, B).

To assess the ability of the sorted cells to form spheres, we seeded 100 cells/well per each cell

culture under non-adherent conditions and found that all the nine cultures formed mammospheres in

low-attachment plates (Supplemental Figure S2). CD44/CD24 expression on cultures of cells

stabilized for 1-3 months under adherent conditions (dot plots at Supplemental Fig. S2) was

measured by calculating the mean fluorescence intensity (MFI). The analysis confirmed high

expression of CD44 molecules per cell (1153<MFI<7967) and a low signal for CD24 (1<MFI<72)




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in all the breast cancer cell cultures. The MFI for CD44/CD24 in BT474Z(16/4078), HER2-18

(123/250), MCF7 (15/79), MCF10A (914/27) and MDA-MB231 (4906/10) cell lines served as

control (Fig. 1C). To measure the doubling frequency of cells dissociated from mammospheres in

adherent (2D) and non-adherent conditions (3D), we enzymatically detached cells to obtain single-

cell suspensions, seeded in ultra-low attachment or in adherent plates and counted the cells each

month thereafter. After two months of culture, growth and propagation was arrested in breast

cancer cells in low adhesion conditions, whereas the paired adherent culture grew with a doubling

time ranging between 2 and 3 days (lowest) and 7 and 10 days (highest) across cell types. The

cumulative population doubling frequency (cpdf) at 120 days was 4.7x105 in cell cultures with a

high growth rate (K197), and 2.8x105 in cell cultures with a low growth rate (K77). The median

cpdf of the nine CD44+/CD24

low/Thy1+ cell cultures (K40, K43, K66, K77, K88, K79, K90, K193

and K197) at 120 days was 3.26 x105 for 2D cultures and 1.12x10

5 for 3D cultures (Fig. 1D). In all

cases, long-term culturing of Thy1-positive cells under non-adherent conditions delayed the

proliferation. In fact, the mean doubling time was 15-18 days during the initial two months, and was

arrested thereafter.

CD44+/CD24

low/Thy1+ cells are basal cells with intermediate epithelial-mesenchymal

phenotype

To evaluate whether Thy1 expression was stable in cells sorted for stem features, we measured

Thy1 messenger RNA (mRNA) in stabilized cultures. As shown in Fig. 2A, B, Thy1 was highly

expressed in all nine Thy1-positive cell cultures established from the CD44+/CD24

low population of

primary culture, and low in MCF7, BT474Z, HER2/18, MCF10a and MDA-MB231 cells.

Messenger RNA levels were consistent with the high expression of the protein; indeed, the

percentage of Thy1/CD90 expression, measured by immunophenotyping, ranged from 87.9% to

100% (Fig. 2C). To estimate the number of Thy1 molecules per cell, we calculated the MFI of

Thy1/CD90 expression, and found that it was low in all the cell lines BT474Z (211), HER2-18

(189), MCF7 (45), MCF10A (208) and MDA-MB231 (81) (45<MFI<211) but high in all the nine




17

cell cultures (1065<MFI<8117) (Fig. 2D). Thy1 expression per cell was significantly higher versus

the control cell lines (p=0.0078).

To investigate whether the CD44+/CD24

low/Thy1+ populations of sorted cells were

tumorigenic, we injected the K197 cells into the fourth mammary fat pad of immunodeficient mice.

As few as 1x104 CD44

+/CD24

low/Thy1+ cells generated tumors with a calculated frequency of

tumorigenic cells that resulted in 1/4326 cells. Each of the dilutions that generated tumors showed a

latency and a size when excised, at day 60, that correlated with the number of cells injected (R =

0.949, p<0.05). Indeed, the mean tumor volume of triplicates was 950+190 mm3 per 1x10

4 cells

injected, 1,500+250 mm3 per 1x10

5 cells, and 2,800+260 mm

3 per 1x10

6 cells (Fig. 3A). The

excised tumors (measuring 1-3 cm3) were minced and dissociated by enzymatic digestion, and the

cells derived were maintained in long-term culture. A signal for Thy1 mRNA was detectable in

carcinoma specimens S197 (Fig.3B, left) and absent in either normal N197 tissue (Fig.3B, middle)

and xenotransplanted specimen Topo9 (Fig.3B, right). Thy1expression levels were confirmed by

real-time PCR (Fig. 3C). We next performed flow cytometry to investigate whether the cell cultures

derived from the transplanted tumors preserved the stem cell characteristics of the implanted cells.

This analysis showed that, consistent with the implanted CD44+/CD24

low population, tumors

generated by this population recapitulated the CD44+/CD24

low profile (Fig. 3D) and the MFI (Fig.

3E). We measured the mRNA expression to compare the receptor profiles of the implanted cells

(K197 cells) with those of the cells isolated and cultured from mouse tumor tissue (Topo9 cells).

Like the parental cells, transplanted cells expressed low levels of ERα, PgR and HER2 (Fig. 3F),

which indicates subtype relationship with the implanted cells. Immunoblot for ER and HER2

(Supplemental Fig. S4) confirmed that both K197 and Topo9 cells do not express potentially

functional levels of these markers.

To further investigate the relationship between the pre-implantation population (K197) and the

population obtained from the cultivation in vitro of the CD44+/CD24low cells from implanted

tumors (Topo9), we profiled the phenotype by measuring the percentage of expressing cells and the




18

MFI of three epithelial markers (MUC1, E-caderin and EpCam), and seven stem/mesenchymal

markers (CALLA, β1integrin, α2integrin, α6integrin, β3integrin, thy1, and prominin1). As reported

in Table 1, the percentage of expressing cells and the MFI of the markers of the transplanted cells,

except Thy1, overlapped that of the parental cells. There was a strong positive correlation in

percentage terms between the transplanted and the parental cells (p<0.001), a weak correlation

between the transplanted and the MCF7 cell line, and a possible, albeit not significant correlation

between the transplanted and the MCF10A cells. MFI data were in agreement with the percentage

data, and statistical analysis confirmed the high correlation between the transplanted and the

parental cells (p<0.001). Like the parental cells, the transplanted cells preserved an α2β1 Integrin-

CD44high

/MUC1-EpCAM-CD24low

phenotype, but Thy1expression was lost (Figure 3G). To verify

this finding, we measured Thy1 mRNA expression in the cells obtained after transplantation, and

found a consistent decrease of Thy1 mRNA in the cultures derived from the transplanted tumors

versus the pre-implantation cells (Fig. 3H). qRT-PCR normalized to B2M confirmed these

differences (p<0.05) (Fig. 3I).

Xenotransplantation silences Thy1 via promoter methylation

The cellular plasticity between the epithelial and mesenchymal states is ascribed to epigenetic

changes of cancer cells that result in cellular heterogeneity (36). In metastatic breast cancer, the

Thy1 gene is silenced by methylation in those tumors that are hormone receptor-negative and basal-

like (37). To determine whether Thy1 expression is relevant in breast cancer, we interrogated

public databases. Data mining of TCGA 450K DNA methylation, at the human pan-cancer

methylation database, MethHC (https://methhc.mbc.nctu.edu.tw/ ), confirmed the enrichment of

THY1 hypermethylation in human breast carcinoma compared with normal breast tissue (p< 0.005)

(Supplemental Figure S5). To determine whether the expression of Thy1 in myoepithelial

precursors is subject to epigenetic regulation, we explored the possibility that methylation might

determine the disappearance of Thy1 when progenitors are implanted in mice. Thus, we treated the

Topo9 cells with methylation inhibitors and found that Thy1 expression was restored in a time-



https://methhc.mbc.nctu.edu.tw/


19

dependent fashion. Parental K197 cells and transplanted Topo9 cells were cultured, for various

times (Fig. 4A), with the methylation inhibitor 5-aza-20-deoxycytidine(5-AZA-dC) and subjected to

RT-PCR. Thy1 was not methylated in parental K197 cells (Fig. 4A, left blot) and highly methylated

in untreated Topo9 cells (Fig 4A, right blot, lane 0). Treatment with the demethylation agent 5-

AZA-dC time-dependently restored Thy1 gene expression in Topo9 cells. The latter effect

progressively increased from 0 to 96 hours (Fig. 4A, right blot). These data support the hypothesis

that methylation contributes to Thy1 silencing. To test this hypothesis we analyzed the Thy1

promoter using the methylation-sensitive amplified polymorphism (MSAP) technique. HpaII (CpG

methylation insensitive) digestion resulted in a 276 bp fragment in Thy1-low cells (Topo9) but not

in Thy1-positive (K197) or control cells (C) (Figure 4B). At quantitative real-time PCR analysis of

HpaII/MspI sensitivity, amplification rates (64% in Thy1-low cells vs 0.54% and 0.18% in Thy1-

positive and control cells, respectively) were significantly correlated with methylation status (Figure

4C). These results indicate that methylation occurs on the Thy1 loci in Thy1-low Topo9 cells.

Thy1-low cells activate the Notch1-EGFR program

We examined Thy1-low cells for cellular functions (EMT-marker expression, growth in 3D) that

are critical when cells are allowed to adhere to a substrate (38, 39). The protein expression profile

of the EMT markers CK18, CK19, CK5, vimentin, αSMA and fibronectin (Fig. 5A) showed that

Thy1-low cells acquired a partial epithelial phenotype. In fact, these cells expressed CK18, CK19,

and vimentin, reduced fibronectin but they did not express CK5 or αSMA.

To shed light on the growth features of Thy1-low cells and to evaluate whether the cells

exerted sphere-forming activity, we seeded the Topo9 cells under adherent and non-adherent

conditions. In 2D cultures, Thy1-low cells were smaller than Thy1-positive cells and had and an

epithelial-like morphology (Supplementary Figure S3), the median cpdf was 6.3x105 at 120 days,

that was double that of Thy1-positive cells (reported in Fig. 1D). The 3D-harvested Thy1-low cells

in low-adhesion formed aggregates in suspension instead of mammospheres (Fig. 5B). To




20

understand whether the loss of Thy1 affects growth on substrate, we performed soft agar

experiments. After seeding in semisolid medium for 3 weeks, Thy1-low Topo9 cell frequency was

four-fold higher than Thy1-positive K197 cell (Fig. 5C), which confirms the greater propensity of

Thy1-low cells to proliferate on adhesion to a semisolid substrate. These observations prompted us

to investigate on pathways involved in EMT.

To evaluate whether Thy1-silencing in the grafted cells signals a transition phenotype, we

searched for signaling pathways through which cells interact during development and tissue

homeostasis. Molecular analysis had demonstrated that both parental K197, Thy1-positive, and

transplanted Topo9, Thy1-low cells are triple-negative (Fig. 3 F). Triple-negative breast cancers

generally express EGFR (40), which cross talks with the Notch pathway in this setting (41). We

measured the levels of EGFR and Notch mRNA in Thy1-positive and Thy1-low cells (Fig. 5D). At

densitometry, EGFR mRNA was three times higher in Topo9 cells than in parental K197cells (grey-

scale: 0.62 vs 1.93, respectively). Moreover, Topo9 cells expressed Notch1 mRNA, whereas K197

parental cells did not (Fig. 5D). Immunoblots for EGFR and Notch1 (NICD) confirmed that Topo9

cells express potentially functional levels of these protein (Fig. 5E). Further mRNA and protein

analysis of Topo9 cells rescued after 48 hours with 5-AZA-dC showed a concomitant reduction of

EGFR levels together with Thy1 induction (Fig. 5F-G).

Having identified activation of signaling pathways susceptible to targeting, we investigated the

ability of Thy1-low cells to respond to tyrosine kinase inhibition. In gastric cancers, Thy1 is a

cancer stem cell marker and trastuzumab (humanized anti-HER2 antibody) treatment of high

tumorigenic gastric primary tumor models reduces the Thy1 population in the tumor mass thereby

suppressing tumor growth when combined with chemotherapy (42). As HER2 has no ligand,

antibodies against this receptor inhibit its activation by preventing heterodimerization (43) with

other members of the HER2 family (44). Heterodimerization results in intrinsic kinase activation.

We treated K197 and Topo9 cells, and BT474, MCF7 and MDA-MB231 cells with increasing

concentrations (0.7-2-5-10 µg/ml) of trastuzumab and measured cell viability with the MTT assay




21

4, 5 and 7 days later. Trastuzumab did not alter the viability of either the K197 or Topo9 cells (% of

inhibition = 0). Because Thy1-low Topo9 cells express the EGFR (HER1), we tested the effect of

lapatinib, a reversible tyrosine kinase inhibitor that binds intracellularly and inhibits both the EGFR

and HER2 activity. We found that lapatinib, in a concentration-dependent manner, significantly

reduced the viability of Thy1-low cells (Fig. 5H). Indeed, at concentrations of 0.1 µM and 1 µM,

lapatinib reduced cell viability by 75% and 85%, respectively (P = 0.0011). These results suggest

that Thy-low cell proliferation is sustained by tyrosine kinase activation.

To determine whether Thy1 overepression might have clinical relevance in conjunction with

EGFR and Notch1 expression status we analyzed our series of breast carcinoma and performed real

time PCR experiments on the carcinoma tissue samples paired with those used to establish primary

cultures of Thy1-positive cells. At qRT-PCR we examined six tissue specimens and found that

Thy1 was highly expressed in breast carcinoma tissues (range 0.82-1.9), while transcript levels for

EGFR and Notch1 were very low (range 0.003-0.1) (Figure 5I).

We interrogated the Pancancer Analysis of Whole Genomes (PCAWG)

(https://www.ebi.ac.uk/gxa/) for the TCGA RNA-sequencing data, to compare combined expression

of Thy1, EGFR and Notch1 in breast adenocarcinoma, normal-adjacent to breast adenocarcinoma,

invasive lobular carcinoma and normal breast. Thy1 was more highly expressed in invasive lobular

(24FPKM) and invasive adeno carcinoma (18FKM) than in tissue adjacent to breast carcinoma

(11FPKM) or normal breast (7FPKM). Interestingly, the levels of EGFR and Notch1 appear to be

directly related each other, but inversely related to Thy1. Indeed, EGFR in normal tissues (13

FPKM) was more than 3 fold higher than in breast tumors (3/4 FPKM), as well Notch1 had higher

levels (11 FPKM) in normal than in tumors (6/6 FPKM) (Figure 6A).

Furthermore, to situate our model within the context of a broad spectrum of “in vitro” cell

lines, we interrogated Oncomine (https://www.oncomine.org) for Neve collection of cultured cells

(Supplemental Figure S6). Also in this case, the results support the notion of an inverse



https://www.ebi.ac.uk/gxa/

https://www.oncomine.org/




22

relationship between Thy1 and EGFR; as for Topo9 cells, the pattern with low THY1 and high

EGFR is largely represented in most of the cell lines broadly used to model breast cancer.

We then performed a Kaplan-Meier analysis to measure relapse-free survival in subjects

categorized ER- and HER2-negative based on high/low Thy1 and EGFR expression. At the time of

analysis, the median follow-up was 200 months (range, 0.1–200 months). In this setting, high Thy1

expression (Thy1-high vs Thy1-low P = 0.062) (Fig. 6B) as well as low EGFR expression (EGFR-

high vs EGFR-low P = 0.061) (Fig. 6C) identifies those patients with a worst relapse-free survival.

Multivariate analysis of ESR1 and HER2 expression (stages I and II vs stage III; hazard ratio,

2.815; 95% confidence interval, 1.022–7.751; P=0.045) excluded interaction of Thy1 and EGFR

with ER and HER2, respectively. The analysis suggests that combined detection of Thy1 and/or

EGFR expression might help to better identify subclasses of patients with breast cancer of basal

origin.

DISCUSSION

Tumor cell heterogeneity, induced by a combination of genetic and epigenetic events that lead to

cancer cell plasticity, is one of the cancer features responsible for drug resistance and treatment

failure (45). Gene expression profiling has revealed inter-tumor heterogeneity and identified five

intrinsic breast cancer subtypes: luminal A and B (ERα- and/or PgR-positive and HER2-negative),

HER2-enriched (HER2-positive), basal-like (which includes triple negative) and normal-like, that

differ in biologic, prognostic and predictive features (2, 3, 46). In routine clinical practice,

immunohistochemistry is used to identify the molecular subclasses (26). Within the subclasses,

triple negative (ER- and PgR- and HER2-negative), which accounts for 15%-20% of all invasive

breast cancers, is the most heterogeneous (47), has a higher risk of relapse and responds poorly to

targeted therapy (48). Eighty percent (80%) of triple negative breast cancers harbor a signature that

coincides with a high proportion of cells with the basal/myoepithelial phenotype (4). The




23

“phenotypic” plasticity of cancer cells favors intra-tumor heterogeneity that, through the EMT,

promotes the invasion and dissemination of cells, while the MET counterpart confers fitness

advantage which enables cells to return to a highly proliferative state and mediates tumor relapse at

metastatic sites (45). In tumor xenografts, the expansion of subclones with fitness advantages is

ascribed to co-segregating genomic factors, such as methylation, that, as determinants of fitness,

lead to reproducible clonal dynamics (49). DNA methylation is a dynamic process that contributes

to tumor heterogeneity (50). Because DNA methylation is often altered in early cancer

development, candidate methylation markers may serve as prognostic or predictive factors (37).

In this context, we investigated the relevance of Thy1 as a biomarker of myoepithelial progenitor to

gain insight into the role of basal myoepithelial cells in breast cancer heterogeneity.

Here we isolated and harvested, from human breast cancer tissues, Thy1-expressing cells with

phenotypic and functional stem cell characteristics. When we transplanted the (α2β1integrin-

CD44)high

(MUC1-EpCAM-CD24)low

Thy1-positive myoepithelial progenitors in nude mice, Thy1

expression was lost. Recent studies on invasive breast cancer report that Thy1 is highly methylated

in the group of HRnegative-Basal-like-p53mutant (37). To evaluate whether epigenetic changes

modified Thy1 expression, as occurs in patients with metastatic basal-like tumors, we treated the

cells with methylation inhibitors and found that they time-dependently restored Thy1 mRNA

expression. This result suggested the emergence of epigenetic-induced transiting phenotypes. In

agreement with data obtained with different approaches (7, 34, 51), in our model of human

myoepithelial progenitors, Thy1-silenced cells displayed the alternative MET phenotype, which

resulted in a better propensity to proliferate and differentiate. This behavior, together with the

finding that Thy1-low cells activated the Notch1-EGFR program may explain the rarity of tumors

displaying myoepithelial features notwithstanding the ubiquity of myoepithelial cells in breast

tissue. The Notch signaling network is an evolutionarily conserved intercellular signaling pathway

that regulates interactions between physically adjacent cells. In mouse mammary cells, Notch

activation increases the proliferation potential of both bipotent and myoepithelial progenitor cells




24

(52, 53). In normal breast, Notch1 mRNA is expressed in luminal cells and its effect on lineage

commitment is irreversible (52). In breast cancer, Notch signaling pathways crosstalk with EGFR.

Forced overexpression of Notch1 by transfection increases EGFR expression (53), although

inhibition of EGFR or Notch signaling alone is not sufficient to suppress human breast cancer cell

survival and proliferation (54). Our data suggest that Thy1 methylation signals the acquisition of a

cycling epithelial phenotype (CK18-CK19-positive/CK5-αSMA-negative) that activates the

Notch1-EGFR pathway. Thy1 suppression, through methylation, at metastatic sites might be

required for implantation and growth of clones with fitness advantages. Intriguingly, examination

on disease progression, of the Sorlie Breast 2 dataset at Oncomine (https://www.oncomine.org )

comparing expression status of 107 breast carcinomas at primary site vs 5 metastasis showed a

decreased Thy1 expression at metastatic site (Supplemental Figure S7). This observation might

have clinical implications because high Thy1 expression might identify, within the heterogeneous

basal-like (and TNBC) subclass of breast cancer, a subset of primary tumors originating from

precursors/bipotent myoepithelial cells that have worst prognosis. If Thy1 is methylated and the loss

of Thy1 coincides with the expression of EGFR, patients might have a better relapse free survival.

The tumors that we obtained after xenotransplantation have low levels of Thy1and high levels of

EGFR and resemble those EGFR overexpressing tumors with better prognosis. Finally, since

lapatinib inhibits the viability of Thy1-low cells, but not that of Thy1-positive cells, the Thy1-

methylated/EGFR-expressing (Thy-/EGFR+) phenotype might define a subtype, within the basal-

like, that may benefit from tyrosine kinase inhibition. Further studies are needed to determine the

relative impact of these processes during cancer evolution.

CONCLUSIONS

In our in vitro/in vivo model of stable basal breast cancer progenitor cells in culture, Thy1

expression tracks the myoepithelial lineage. Quiescent myoepithelial progenitor cells are





25

components of luminal and basal subtypes of breast cancer tissues, and can be identified based on

their expression of Thy1. Thy1-expressing myoepithelial progenitors are quiescent in 3D culture

and display a stable phenotypic profile of (α2β1integrin-CD44)high

(MUC1-EpCAM-CD24)low

that,

in vivo, might be responsible for attachment of cells to the extracellular matrix. Upon

transplantation, Thy1 expression is silenced by methylation parallel to activation of Notch/EGFR

signaling. This process marks the emergence of differentiated clones, namely,

CK18/CK19/vimentin-positive/ CK5/αSMA-negative clones. The latter proliferate extensively, and,

notably, might be arrested by tyrosine kinase inhibition. Collectively, our results suggest that Thy1

methylation may track the shift of Thy1-positive bipotent progenitors into Thy1-low differentiated

cells. This behavior may explain the rarity of tumors with myoepithelial features, and considered to

be of "basal" origin, such as some metaplastic carcinomas, notwithstanding the ubiquity of

myoepithelial cells in breast tissue. An understanding of the dynamics of cellular states in breast

cancer evolution can lead to a more accurate definition of the subtypes of breast cancer, and opens

the way to new therapeutic strategies.

List of Abbreviations

EGFR, epidermal growth factor receptor; HER2, epidermal growth factor receptor 2;CK,

cytokeratin; ERα, Estrogen Receptor alpha; PgR, progesterone receptor; TNBC, triple negative

breast cancer; EMT, epithelial-mesenchymal transition; MET, mesenchymal-epithelial transition.

Acknowledgements

Breast cancer tissue specimens were obtained from the Breast Cancer Tissue Bank developed under

the auspices of the BIONCAM (Biobanca Oncologica Campania) Project and maintained by the

CRPO (Centro Regionale Prevenzione Oncologica), University of Naples “Federico II”. We thank

Jean Ann Gilder (Scientific Communication srl) for editing the text.




26

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Table 1. Surface marker percentage expression (%) and mean fluorescence intensity (MFI) in

myoepithelial progenitors before (K197) and after transplantation (Topo9) and in cell lines.

K197§ Topo9§ MCF7* MCF10A§

% MFI % MFI % MFI % MFI

CD 227-

MUC1

0.0 49 0.6 41 90.2 605 42.6 227

CD 324-

Ecadherin

0.5 45 0.8 52 6.0 90 3.3 148

CD 326-

EpCAM

2.0 32 1.9 51 100.0 1377 72.1 425

CD 10-

CALLA

8.4 65 15.0 20 18.7 8 82.4 388

CD 29-

β1Integrin

100.0 1474 100.0 1373 100.0 1300 100.0 3526

CD 49b-

α2Integrin

100.0 597 100.0 960 99.9 560 99.9 1996

CD 49f-

α6Integrin

100.0 519 100.0 554 23.8 66 100.0 6044

CD 61-

β3Integrin

60.0 11 33.0 18 0.4 6 25.2 121

CD 90-Thy1 100.0 1065 11.0 76 0.2 45 58.6 208

CD 133-

Prominin1

16.0 35 18.0 25 68.0 29 28.0 70

Numbers of positive cells are mean percentage of average of triplicates of three§ or

two* experiment; SD, not reported, was < 10%.

Percentage (%) Pearson

R score

P-value Significance

at p<0.001

Correlation

K197 vs Topo9

0.9818 1.5E-05 yes Strong

positive

K197 vs MCF7

0.3412 0.3688 no Weak

K197 vs MCF10A

0.7603 0.0174 no Positive

MFI Pearson

R score

P-value Significance

at p<0.001

Correlation

K197 vs Topo9

0.9659 9.7E-05 yes Strong

positive

K197 vs MCF7

0.3263 0.3914 no Weak

K197 vs MCF10A

0.5006 0.1698 no Moderate

Positive




31

FIGURE LEGENDS

Figure 1. Breast tissue specimens contain Thy1 expressing cells.

(A) Percentage of Thy1/CD90-positive cells as assessed by immunofluorescence in primary

cultures, in CD44+/CD24

low cells cultured for 10 days after sorting and in long-term (3 months)

cultures. Numbers of positive cells were counted from 7-10 representative fields of three slides per

cell culture (n=9 cultures). (B) Immunofluorescence of Thy1/CD90 on two representative cell

cultures (K43 and K197) at three months after sorting; scale bar 50 µm. (C) Mean fluorescence

intensity of CD24 (light grey) and CD44 (dark grey) markers expressed on the cell surface of breast

cancer stem/progenitor cells (K40, K43, K66, K77, K88, K79, K90, K193, K197) and cell lines

(BT474Z, HER2-18, MCF7, MCF10A and MDA-MB231) (log scale range: 1-10,000); the standard

error, not reported on graph, was < 10%. For the analysis, cells were cultured for 15-20 days in

standard medium, then detached, counted, and 1-3x106 cells were used. For each tube, 20,000

events were recorded and analyzed. (D) Cumulative population doubling frequency (cpdf) of Thy1-

positive cells. The median cpdf of the nine CD44+/CD24

low/Thy1+ cell cultures (K40, K43, K66,

K77, K88, K79, K90, K193 and K197 is plotted. Triplicate dishes of each cell culture, plated at 2.5

x103 cells/well, were cultured on adherent dishes (light grey) and as floating mammospheres on

non-adherent wells (dark grey) and counted in a hemocytometer chamber for the time indicated.

After counting, the cells, maintained in a sterile environment, were re-plated 1:2 in new wells. The

cpdf was calculated with the formula Ln(No/Nn)/Ln2. Statistical analysis was done by Chi-squares

(p=0.069). Error bars indicate the SEM of triplicates.

Figure 2. Thy1 expression in CD44+/CD24

low breast cancer cells.

(A) RT-PCR of Thy1 mRNA (235 bp) expression of the K40, K43, K66, K77, K88, K79, K90,

K193, and K197 myoepithelial progenitor cells. Cell lines MCF7, MCF10a, BT474, HER2/18 and

MDA-MB231 served as references. Standardization was based on β-actin cDNA levels. (B)




32

Densitometry RT-PCR for Thy1 in MCF7, MCF10a, BT474, HER2/18, and MDA-MB231, and in

the K40, K43, K66, K77, K88, K79, K90, K193, and K197 cells. (C) Percentage of surface protein

expression of Thy1-CD90 in MCF7, MCF10a, BT474, HER2/18, and MDA-MB231, and in the

myoepithelial progenitor cells K40, K43, K66, K77, K88, K79, K90, K193, and K197. The levels

of expression of surface markers were reported as percentage of positive cells in Count versus

FITC- or PE-CD histograms. (D) Protein expression of Thy1-CD90 calculated as mean

fluorescence intensity (MFI) on breast cancer stem/progenitor cells (K40, K43, K66, K77, K88,

K79, K90, K193, and K197) and cell lines (MCF7, BT474Z, HER2-18, MCF10A and MDA-

MB231); (log scale range: 1-10000) is plotted. The SEM of triplicates, not reported on the graph,

was < 10%. Statistical analysis of differences of breast cancer stem/progenitor cells vs control cells

was done by one-way ANOVA (p=0.0078).

Figure 3. Thy1 expression in xenotransplanted cells.

(A) CD44+/CD24

low Thy1-positive K197 cells were assayed for the ability to form tumors after

injection into mice. Five-week-old female BALB/c athymic (null/null) mice were injected

orthotopically in the fourth mammary fat pad, with K197 cells resuspended in Matrigel/PBS. Tumor

volume (mm3) was measured by calculating π/6 × largest diameter × (smallest diameter)

2, and

correlated with the number of cells injected (R = 0.949, p<0.05). Within 60 days, tumors were

excised (250 mm3), digested with trypsin/collagenase mixture and mouse-derived tumor cells were

harvested for in vitro growth (Topo9 cells). (B) RT-PCR of Thy1 mRNA (235 bp) expression in

carcinoma specimens S197 (left), paired normal N197 (middle) and xenotransplanted specimen

Topo9 (right) tissue. Standardization with β-actin cDNA levels. (C) qRT-PCR of Thy1 expression

in carcinoma specimens S197, paired normal N197 and xenotransplanted specimen Topo9 tissue (S-

Topo9). Experiments were performed in triplicate for each data point, and the expression of

housekeeping beta-2-microglobulin gene (B2M) was used for normalization. (D) Cells derived from

xenotransplanted tumors recapitulate the CD44+/CD24

low profile of the grafted stem/progenitor




33

cells. Dot plots of flow cytometry analysis of parental K197 and Topo9 cells. Cells were cultured

for 15days in standard medium, and 1x106 cells were stained for the flow cytometric analysis of

CD44PE-Cy7A and CD24-AlexaFluor647. The expression of each antigen is represented on a

frequency distribution histogram (count vs FITC or PE signal). The expression of the two markers

is presented on a biparametric dot plot CD44-PE-Cy7 vs CD24-AlexaFluor647 for each cell type.

Vertical and horizontal markers delineate the quadrants used to identify the CD44/CD24 subsets

and were set with the appropriate FMO control. (E) The histogram reports the mean fluorescence

intensity (MFI) of CD24 (light grey) and CD44 (dark grey) expressed on the cell surface of the

indicated cells (log scale range: 1-10,000); standard error, not reported on the graph, was < 10%.

(F) Topo9 cells, cultured from xenotransplanted tumors, are triple-negative, as the grafted K197

Thy1-positive cells. RT-PCR for mRNA of ERα (441 bp), PgR (121 bp) and HER2 (420 bp) of

K197 and Topo9 cells. Standardization was based on β-actin cDNA levels. Protein expression

determined with Western blot at Supplemental Fig. S4. (G) Percentage of Thy1/CD90 surface

protein as determined by flow cytometry with CD90-FITC. (H) RT-PCR of Thy1 mRNA (235 bp)

expression in K197 (left), and xenotransplanted Topo9 (right) cells. Standardization with β-actin

cDNA levels. (I) qRT-PCR of Thy1 expression in MDA-MB231 cell line, in K197 and

xenotransplanted Topo9 cells. Experiments were performed in triplicate for each data point, and the

expression of housekeeping beta-2-microglobulin gene (B2M) was used for normalization.

Figure 4. Methylation on the Thy1 loci in transplanted cells.

(A) Thy1-positive (K197) and Thy1-low (Topo9) cells were cultured, for the time indicated, with

the methylation inhibitor 5-aza-20-deoxycytidine (5-AZA-dC) and qRT-PCR for Thy1 (235 bp)

was performed. Treatment with the demethylation agent 5-AZA-dC did not affect the methylation

status of Thy1 in parental K197 Thy1-positive cells (left blot) whereas it restored the expression of

Thy1 mRNA in Topo9 Thy1-low cells ( right blot). This effect progressively increased from 0 to 96

hours. Standardization with β-actin. (B) Methylation analysis of the THY1 promoter in K197 and




34

Topo9 cells and control cells. After HpaII/Msp I digestion (blocked/unblocked by CpG methylation,

respectively), only methylated (undigested) genomic DNA produced a fragment. PCR-based assay

of HpaII/MspI sensitivity showed a 276 bp fragment only in low expressing/ hypermethylated

samples (Thy1-low Topo9 cells) and not in high expressing / hypomethylated controls (Thy-

positive K197 cells and control cells); no product is seen in MspI digestions. (C) Real-time analysis

of HpaII/MspI sensitivity. The differences in amplification rates (64% in Thy-cells versus 0.54%

and 0.18% in Thy1-positive cells and control cells, respectively) relates to the methylation (mC)

status. * indicates the difference between Thy-low cells vs Thy1-positive cells and control cells

with P < 0.05; ** indicates the difference between control cells vs Thy1-positive cells with P <

0.01.

Figure 5. Thy1 methylated cells are Notch1-EGFR expressing cells.

(A) Western blot for EMT markers CK18, CK19, CK5, vimentin and αSMA; Thy1-low cells

(Topo9) acquired CK18, CK19 and vimentin, and lost CK5 and αSMA. Standardization with

tubulin. (B) Representative phase-contrast microphotographs of cultures in 3D of mammospheres of

Thy1-positive (K197, left) and aggregates of Thy1-low (Topo9, right) cells. Scale bar 100µm. (C)

Colony growth in soft agar of Thy1-positive (K197) and Thy1-low (Topo9) cells. Colonies were

counted in ten fields per dish on a horizontal/vertical grid. The mean results of two experiments of

triplicates dishes are plotted. (D) RT-PCR for EGFR (348 bp) and Notch1 (520 bp) in K197 and

Topo9 cells. Standardization with β-actin. (E) Western blot for EGFR and cleaved Notch1 (NICD).

Standardization with tubulin. (F) RT-PCR for Thy1 mRNA (235 bp) and EGFR (348 bp) and (G)

immunoblots for Thy1 and EGFR protein expression in K197 and Topo9 cells treated, 24 hours

after seeding, without (--) and with (+) 5 μM 5-AZA-dC for 48 hours. Standardization with β-actin

and tubulin, respectively. (H) Lapatinib inhibited the proliferation of EGFR-expressing Thy1-low

(Topo9) cells. The effect of treatment on the survival of control cells (BT474, MCF7 and MDA-

MB231), parental Thy1-positive (K197) and xenotransplanted Thy1-low (Topo9) cells is expressed




35

as percentage of viable cells over control. All cells were seeded at 2500 cells/well, treated with

lapatinib 0.1 µM (grey) and 1 µM (black) for seven days and analyzed by the MTT assay. The

percentage of absorbance of treated samples versus untreated samples is reported as a percentage of

viable cells/controls. Values represent means±s.d. from triplicate samples for each treatment. Error

bars indicate s.d. values. Asterisks indicate statistical significance, as determined by two tailed

Fisher’s exact test (**, two-sided P=0.0011). (I) real-time RT-PCR quantification of Thy1, Notch1

and EGFR expression in six carcinoma tissues paired with tissue used to obtain primary cultures of

Thy1-positive cells; All samples were run in triplicate and normalized to B2M housekeeping.

Figure 6. Low EGFR-high THY1 expression in human breast correlates with poor prognosis

(A) Distribution of various phenotypes with different ratio of Notch1-EGFR/ Thy1 expression in a

large collection of tumors derived from cancer databases. TCGA RNA-sequencing data were

extracted from the Pancancer Analysis of Whole Genomes (PCAWG) website

(https://www.ebi.ac.uk/gxa/) to compare combined expression of Thy1, EGFR and Notch1 in

breast adenocarcinoma (IDC), normal-adjacent to breast adenocarcinoma, invasive lobular

carcinoma (ILC) and normal breast. (B, C) Kaplan-Meier analysis of relapse-free survival on

subjects with ERα-negative HER2-negative breast cancer on the basis of Thy1 (B) and EGFR (C).

P value was determined by log-rank test. The group with the lowest Thy1expression were more

likely to have a relapse-free survival. The group with highest EGFR expression were more likely to

have a relapse-free survival. Multivariate analysis with ER (ESR1) and HER2 (ERBB2) was

performed. The plots were generated using http://kmplot.com (probes: 213869_x_at (Thy1-

CD90);211551_at (EGFR, ERBB, ERBB1)(33).



https://www.ebi.ac.uk/gxa/


Published OnlineFirst September 21, 2018.Mol Cancer Res Micaela Montanari, Maria R Carbone, Luigi Coppola, et al. CD44+/CD24low/CD90+ myoepithelial cellsNotch1-EGFR signaling in a xenograft model of Epigenetic silencing of THY1 tracks the acquisition of the

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