Evolution of cancer stem-like cells in endocrine-resistant metastatic breast cancers is mediated by stromal microvesicles

Abstract

The hypothesis that microvesicle (MV)-mediated microRNA transfer converts non-cancer stem cells into cancer stem cells (CSCs) leading to therapy resistance remains poorly investigated. Here we provide direct evidence supporting this hypothesis, by demonstrating how MV derived from cancer associated fibroblasts (CAF) transfer miR-221 to promote hormonal therapy resistance (HTR) in models of luminal breast cancer. We determined that CAF-derived MV horizontally transferred miR221 to tumor cells and, in combination with hormone therapy activated an ER^lo/Notch^hi feed-forward loop responsible for the generation of CD133^hi CSC. Importantly, MV from patients with HTR metastatic disease expressed high levels of miR221. We further determined that the IL6-pStat3 pathway in promoted the biogenesis of onco-miR-221^hi CAF MV and established stromal CSC niches in experimental and patient-derived breast cancer models. Co-injection of patient-derived CAF from bone metastases led to de novo HTR tumors, which was reversed with IL6R blockade. Finally, we generated PDX models from patient-derived HTR bone metastases and analyzed tumor cells, stroma, and MV. Murine and human CAF were enriched in HTR tumors expressing high levels of CD133^hi cells. Depletion of murine CAF from PDX restored sensitivity to HT, with a concurrent reduction of CD133^hi CSC. Conversely, in models of CD133^neg, HT-sensitive cancer cells, both murine and human CAF promoted de novo HT resistance via the generation of CD133^hi CSC that expressed low levels of estrogen receptor alpha (ER). Overall, our results illuminate how MV-mediated horizontal transfer of genetic material from host stromal cells to cancer cells trigger the evolution of therapy-resistant metastases, with potentially broad implications for their control.

Introduction

Tumor heterogeneity and resistance to therapy may occur from MV-mediated transfer of genetic material between cells [1–3]. Thus, the characterization of this phenomenon could have important clinical ramifications most notably in the development of new therapeutically relevant compounds.

Although adjuvant hormonal-therapy (HT) improves disease free survival in luminal breast cancer patients, HT-resistant (HTR) metastatic disease commonly develops in the bones of these patients. This observation suggests that the bone microenvironment may foster estrogen receptor (ER)- independent growth of luminal breast cancer leading to HTR metastases.

The interaction of stromal cells (CAFs) with tumor cells has been shown to mediate and modulate estrogen receptor dependent (e.g. fibronectin, collagen) and independent proliferation (e.g. laminin) of luminal breast cancer cells, suggesting that stroma-tumor communication may play a pivotal role in the ER-independent self-renewal of breast cancers [4]. In the metastatic microenvironment, we hypothesize that chronic inflammation incurred by anti-estrogen therapy and the effects of disseminated tumor cells on the local microenvironment will lead to the activation of resident stromal cells or circulating mesenchymal stem cells to become CAFs. Once activated the CAFs may sustain a feed-forward circuit of self-renewal, proliferation, and differentiation of CSCs resulting in metastasis.

As tumors become more metastatic and resistant to targeted therapies, the number and types of CSCs increases, suggesting that CSCs evolve from non-CSC cells in a given tumor niche [5, 6]. The role of stroma microvesicles (MVs) in the generation of therapy-resistant cancer and the regulation of self-renewal remains poorly investigated.

Here, we investigated the hypothesis that HT and CAF-derived MVs converge to promote HT resistance and ER-independent self-renewal in luminal breast cancer. By employing patient-derived xenografts from breast cancer bone metastases and experimental models of luminal breast cancer, we uncovered a unique process of CAF-mediated resistance to hormonal therapy. Our data demonstrate the formation of therapy-resistant stromal-tumor niches via an IL6/Stat3-driven expansion of CAFs, CAF-MV mediated oncomiR 221 transfer to cancer cells leading to the expansion of Notch3^hi/ER^lo/CD133^hi CSCs. These data reinforce the concept of targeting the stromal niche to prevent both HT-resistance and metastatic progression [7–9].

Experimental procedures

Microvesicle isolation and in vivo education experiment

Plasma (10ml) was collected and processed within 4 hours from patients with metastatic disease (Table S1) and in healthy controls who were consented to an MSKCC bio specimen protocol #12-137. The plasma and conditioned media (CM) from cancer and CAF cultures was collected from 10⁷ cells grown in 5×10 cm dishes and centrifuged for 20 min at 3,000g at 4°C. The supernatant was subsequently centrifuged for 30 min at 12,500g at 4°C. The supernatant was transferred and centrifuged at 100,000g for 90 min at 4°C. The supernatant was discarded while the pellet, containing microvesicles (MVs), was resuspended in 25 ml of PBS and loaded onto a 5ml 30% sucrose cushion to deplete MVs from extracellular proteins (300 g/L sucrose, 24 g/L Tris base, pH 7.4). Samples were centrifuged at 100,000g for 90 min 4°C. 3.5ml of the cushion, containing MVs, was diluted with 10ml of PBS and centrifuged at 100,000g for 90 min at 4°C. The supernatant was discarded and the pellet resuspended in 25μl of PBS. MVs were treated with 0.1 mg/ml of DNase I solution (Epicentre) in order to eliminate contaminating DNA bound to the MVs surface or present in solution. Nanosight (Lyden laboratory, Cornell Medical Center) and electron microscopy (MSKCC EM Microscopy Core) were used to characterize the physical structures of these MVs (size and distribution). Confocal microscopy (MSKCC Microscopy Core) of cancer cells educated with pre-labeled (PKH67-Green Fluorescent Cell Linker Kit, Sigma) CAF-MVs was performed to ensure transfer and uptake. The in vivo role of CAF-MV in the promotion of hormonal therapy resistant luminal breast cancer was determined by injecting CAF-MV (Mu-CAFs, isolated from hormonal therapy resistant xenografts and cultured in vitro) and control MVs (from MCF7 cells) into the arterial circulation (retro-orbital injection, 3X10¹⁰ particles/mouse/weekly) of tumor bearing mice (MCF7 cells). Once MFP xenografts were established (after 4 months) mice were treated with HT (fulvestrant; 100μg/injection/once a week for 3 months).

Primary cultures and patient derived xenografts of endocrine resistant luminal breast cancer bone metastases

Patient derived xenografts (PDXs) were established from n=2 out of 5 hormonal therapy resistant and 1 de novo stage IV breast cancer bone metastatic tissue isolated at MSKCC (Table S2). Patients who developed metastatic breast cancer in the bone were enrolled in the study (IRB protocol #97-094). Following surgery, tissue was processed by the pathologist, 60% of the specimen was used for confirmation of diagnosis and molecular analyses (IHC, IMPACT analyses), while 40% was used for further analysis. Tissues were placed in sterile Epicult (Voden Medical), minced with sterile razor blades and incubated at 37° C for 8 to 12 h in the presence of Collagenase/Hyaluronidase enzyme mix (1,000 Units, Voden Medical). In order to grow tumor cells devoid of its cognate stroma, we performed serial centrifugations to separate epithelial cells as mammosphere cultures (MS). Secondary and tertiary MS potential (II or III-MS) was performed as follows: 7-day primary MS started to form after 4–6 days, then they were disaggregated in 1×Trypsin-EDTA (Stem Cell Technologies), washed in complete MEGM, filtered through a 40μm nylon mesh, and seeded to form second generation MS. Number of MS was assessed by counting the total number of spheres (size>100μm) from cells seeded in low attachment plates (from 100 to 1,000). To establish primary CAF cultures from patient derived tissue, MS-depleted supernatant was centrifuged at 450×g for 10min; this pellet was enriched with stromal cells was plated onto 10cm plates supplemented with DMEM 10% serum media. CAF primary cultures were expanded in vitro for n=10 passages. III-MS primary cultures were used to establish PDX: 50–100 MS (size ~100μm) were injected in the MFP of NOD/SCID mice and tumor growth was determined over a period of 5 months. At the endpoint of the experiment, xenograft tissues were collected and primary PDX cultures were established. Multiple passaged PDX were generated following repeated orthotopic injection of PDX-derived EpCAM^pos cancer cells (recognizes only human cells) in the mammary fat pad of NOD/SCID mice (from 1^st to 4^th generation). Luminal breast cancer cells expressing a vector for GFP/Luciferase were generated and used for all the in vivo experiments. Tumor growth was determined using in vivo bioluminescence technology (BLI: Xenogen, Ivis System). Luminal cancer xenografts from the co-injection of human CAFs and MCF7 cells were also generated to determine the effect of the stroma on the generation of de novo resistant endocrine tumors

Xenograft Assays and Preclinical Trials

All cancer cell lines were engineered to express a GFP positive luciferase expression vector. Prior to in vivo inoculation, cancer cells were FACS purified (for GFP) and injected bilaterally in the mammary fat pads of 5–7 weeks old non-obese diabetic/severe combined immunodeficiency mice (NOD/SCID, obtained from NCI Frederick, MD). For each in vivo experiment, cancer cells were mixed with an equal volume of Matrigel^™ (BD Biosciences) in a total volume of 50μl. Bioluminescence was used to monitor both tumour growth (weekly) and metastatic burden (at necropsy). Luminal cancer xenografts from the co-injection of human CAFs (HTR bone metastases) and MCF7 cells (10³ cells) were also generated to determine the effect of the stroma on the generation of de novo resistant endocrine tumors. Additionally, human bone marrow stromal cells HS27a, HS27shC and HS27shIL6 (100 cells/injection) were co-injected with MCF7 cells (10³ cells/injection) into the MFP. For immunostaining assays: organs were collected and fixed overnight in 4% paraformaldehyde, washed, embedded in paraffin and sectioned (Histo-Serve Core). H&E staining was performed by standard methods. For the detection of metastases at secondary sites, we performed in vivo BLI as well as immunofluorescence/immunohistochemistry staining for GFP and ER. All the surgical procedures and animal care followed the institutional guidelines and an approved protocol from our IACUC at MSKCC. For the pre-clinical studies, injectable fulvestrant (Faslodex, AstraZeneca) was given intra-muscularly in the tibialis posterior/popliteal muscles (100μg/injection/once a week) for two months. Tocilizumab (Actemra, Roche Pharmaceuticals) was diluted in PBS at a final concentration of 20 mg/ml. A dosage of 100μg/gm/mouse was administered intra-peritoneal (i.p.) every week (this is ~5-fold higher than the physiological range, patients receive 8mg/kg i.v.). Control mice received isotype control (placebo) or PBS injection.

Cell Lines and FACS

Human cancer cell lines (Namalwa -lymphoma-, Hela -cervical carcinoma-), human breast cancer cell lines (MCF7, ZR751, T47D, and BT474), human bone marrow stromal cell lines (HS5, HS27a) and human normal fibroblasts (MRC5, HMF) were purchased from the American Type Culture Collection (ATCC) and authenticated by Short Tandem Repeat (STR) DNA profiling (Genomic Core MSKCC). Murine CAFs (Mu-CAFs) were isolated from HTR xenografts and PDXs by FACS purification (GFP⁻/EpCAM⁻). Cells were mycoplasma free and maintained in MEM and RPMI (ATCC and Media Core) supplemented with 5% fetal bovine serum (Media Core), 2 mM glutamine, 100units ml⁻¹ penicillin, and 0.1mg ml⁻¹ streptomycin (Media Core). Cancer cells from xenografts were isolated from primary and metastatic tissues by enzymatic digestion (Collagenase/Hyaluronidase, Sigma-Aldrich), sorted (GFP⁺/DAPI⁻) and cultured in vitro. The following reagents: 4-hydroxytamoxifen (referred to as Tam) and fulvestrant were purchased from Sigma (Sigma-Aldrich). For FACS/Flow analyses tumors were digested in sterile Epicult media (Stem Cell Technology), minced with sterile razor blades and incubated for 3 hours in the presence of Collagenase/Hyaluronidase (1,000Units/sample). Cells were washed with sterile filtered phosphate buffer saline supplemented with 1% bovine serum albumin (PBS-BSA 1%) and filtered through a 40μM nylon mesh (BD Biosciences). For the detection of CD44 and CD133, EpCAM antigens, cells were stained in a volume of 100μl (PBS-BSA 1%) with each antibody CD44-APC (100ng/10⁶ -10⁸ cells Clone IM7, eBiosciences), CD133/1-PE (100ng/10⁶ to-10⁸ cells, clone AC133, Miltenyl Biotech) and EpCAM-FITC (250ng/10⁶ to-10⁸ cells, Clone VU-1D9, Stem Cell Technologies). Cells were labeled on ice for 30 min and analyzed (BD FACS Aria III, Flow Core). Samples were analyzed for cell population distribution and sorted for GFP/viability (GFP⁺/DAPI⁻) and CD133/CD44 expression. For flow plot analyses, samples were run using FlowJo 7.5 software (Tree Star). ShRNAs for Notch3 and IL6 were previously described [10, 11].

Microarray and microRNA analyses

Normalized gene expression values were downloaded from the GEO under accession number GSE17705 and probes were aggregated to median gene level expression. A CAF gene set from Allinen et al was used in a single sample gene set enrichment analysis (ssGSEA). ssGSEA scores were z-scored and a “CAF score” was assigned to each patient. Patients were split at the median into CAF-high and CAF-low groups. PROM1 high and PROM1 low groups were split based on PROM1 (CD133) median expression. Statistical significance for differences in PROM1 expression and ESR1 expression were assessed with a Student’s T-test. The heat map for CAF-signature gene and PROM1 was plotted in R with the heatmap.2 function. For real time PCR (qPCR), we extracted RNA using Trizol (Invitrogen). RNA concentration was determined with a NanoDrop 2000. For microarray analysis of published datasets, normalized gene expression data was downloaded from the Gene Expression Omnibus (GEO). Each gene was mean centered and scaled by standard deviation. All analyses were conducted in R. Normalized gene expression data was downloaded from the NCBI for dataset GSE69280[5]. For qPCR, 1μg of total RNA was reverse transcribed to cDNA using iScript^™ Select cDNA Synthesis Kit (Bio-Rad) following the manufacturer’s protocol. Reverse-transcription PCR (RT-PCR) analysis was performed using the following primers: ERα; Forward 5′-TGAAAGTGGGATACGAAAAGAC-3′, Reverse 5′-CAGGATCTCTAGCCAGGCACAT-3′ β2μ Forward 5′-ACCCCCACTGAAAAAGATGA-3′, Reverse 5′-ATCTTCAAACCTCCATGA-3′. DNA was isolated using phenol/chloroform technique from PDX-derived EpCAM positive/negative cells. The presence of murine and human cells was determined on 2ng of DNA by PCR for GAPDH (Murine: Forward 5′-AGCAGCCGCATCTTCTTGTGCAGTG-3′, Reverse 5′-GGCCTTGACTGTGCCGTTGAATTT-3′; Human: Forward 5′-CTCTGCTCCTCCTGTTCGAC-3′, Reverse 5′-ACGACCAAATCCGTTGACTC-3′). MiRNA expression was analyzed as described previously [12]. Briefly, miRNA were reverse-transcribed using stem-loop RT-PCR technology [13] and amplified by real-time PCR using SYBR^® Select Master Mix (Applied Biosystems) and ViiA^™ 7 Real-Time PCR System (Applied Biosystems) according to the manufacturer’s instructions. The melting curve data were collected to check PCR specificity. MiRNA expression was normalized against RNA U6 levels: (RT-miR-221) 5′-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACGAAACCC-3′; (RT-miR-222) 5′-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACACCCAGT-3′; (RT-miR-101) 5′-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACTTCAGTT-3′; (FW-miR-221) 5′-AGCTACATTGTCTGCTGGGTTTC-3′; (FW miR-222) 5′-AGCTACATCTGGCTACTGGGT-3′; (FW miR-101) 5′-GCCGCTACAGTACTGTGA-3′; (FW U6) 5′-CTTCGGCAGCACATATACT-3′; (REV U6) 5′-AAAATATGGAACGCTTCACG-3′ (REV all miRs) 5′-TGCAGGGTCCGAGGTAT-3′. All primers were purchased from Eurofins MWG Operon. MiRNA expression was analyzed as described elsewhere [12].

Protein and in vitro studies

For immunoblotting assays, cells were lysed in buffer (50mmol/L Tris at pH 7.5, 150mmol/L NaCl, 5μg/mL aprotinin, pepstatin, 1% NP-40, 1mmol/L EDTA, 0.25% deoxycholate, and protease inhibitor cocktail tablet, Sigma). Proteins were separated by SDS-PAGE, transferred to PVDF membranes and blotted with specific antibodies (Table S3). For functional interference studies, anti-miR-221 and control RNA oligonucleotide were purchased from Applied Biosystems. MCF7 cells were seeded in a 6-wells plate (8×10⁵ cells/well) at 60% confluence. Following 24h, cells were transfected using Lipofectamine 2000 transfection reagent (Invitrogen) according to the manufacturer’s instructions (RNA final concentration, 200nM). After 6h of incubation at 37°C, transfection medium was replaced with 2 ml of complete medium containing 10% FCS supplemented with/without CAF-MVs. For determination of cell viability, we seeded 2,500 cells per well in 96-well plates and treated them with fulvestrant (10μM). Viable cells were determined 7–14 days after treatment using trypan blue and cell counting using bright field microscopy or DAPI staining by flow cytometry (Dako Cytomation). Crystal violet assay was performed to obtain information of relative cell density at the end point of proliferation potential experiments. IL6 ELISA assays were performed using the conditioned medium collected from five-day cultures of CAF-derived cells seeded at 200,000 cells/plate. Proliferation assay was carried out using CalceinAM technology (Invitrogen): cells were seeded in 96 wells plates treated with the pre-fluorescent compound for 20 min and fluorescence was read using a plate reader (SpectraMax plate platform). To determine the selective growth potential of cancer cells over stroma cells, we analyzed proliferation potential of luciferase positive cancer cells by in vitro BLI: cells were seeded in 96 wells plates in presence/absence of distinct CAFs/normal fibroblast (1:10 ratio of CAF’s to tumor cells) and treated with fulvestrant (10μM/weekly for 3 weeks). Conditioned media (CM) was isolated from CAFs and cancer cell lines (10⁸ cells): concentrated using Amicon Ultra-15 centrifuge tubes (Millipore) and protein levels were measured by the Lowry technique; 10μg of total extracellular protein was used for in vitro education experiments. Luciferase activity was measured weekly. Cytokine Arrays were performed on 10μg of CM-derived proteins according to manufacturer’s protocol (Antibody Array 3, RayBiotech. Inc.).

Immunostaining analysis

Serial sections of formalin-fixed paraffin-embedded samples were immunostained using monoclonal anti-CD133 diluted 1:70 (clone W6B3C1, Miltenyi Biotec, D), anti-ERα RTU (clone SP1, Ventana, USA), anti-Pankeratin RTU (clone AE1/AE3/PCK26, Ventana, USA) and polyclonal anti-Notch-3 diluted 1:400 (M-134, Santa Cruz Biotec., USA). CD133 and Notch3 immunostaining was performed as follows. Sections were de-waxed, rehydrated, and subjected to antigen retrieval treatment. Antigens were unmasked with a Tris-EDTA pH 9,0 buffer at 98°C for 20 min in a waterbath. Endogenous peroxidase activity was inhibited using a 0.5% H₂O₂ solution in methanol for 20 min at Room Temperature (RT). Sections were processed using a non-biotin amplified method (Novolink, Novocastra UK) according to the manufacturer protocols. When mouse tissue was used, a short treatment (30min at RT) with MOM blocking solution (Vector Laboratories Inc.) was conducted prior to primary antibody overnight incubation at 4°C. The reaction was visualized using the UltraView DAB Detection System. The immunological reaction was developed using a 3,3′-diaminobenzidine (DAB)/H₂O₂ Phosphate Buffer Saline pH 7.2–7.4 solution for 10 min. Sections were then washed in distilled water and counterstained in Harris Haematoxylin. Anti-ERα (ER) and Pankeratin (CK) immunostaining was performed on an automated immunostainer (Benchmark Ultra, Ventana, USA) using the UltraView DAB Detection kit according to the manufacturers protocol. Antigen retrieval was performed onboard with UltraCC1 buffer (pH 8.2–8.5) at 95°C for 52 min (ER) or 20 min (CK). Primary antibodies were incubated 28 min at 37°C (ER) or 8 min RT (CK). For CD133 and Notch3 evaluation: each section was examined at 400x. In each microscopic field, the neoplastic cells were classified according to both positive percentage and staining intensity: percentage = 0 if <1%, 1 if >1% < 25%, 2 if >25% < 50%, 3 if >50% <75%, 4 if >75%; intensity = 1 (weak), 2 (moderate), and 3 (strong). A final classification was obtained by multiplying the two mean values (percentage and intensity, IRS score). As for ER evaluation: the neoplastic population was scanned using Image Cytometry and reported as percentage of positive cells (%) (IMAGE-Pro Plus V5.0.1, Media Cybernetics Inc., USA). A detailed histologic examination of xenograft tissues was performed at the collaborating institution (Bologna, Italy). Xenograft tissue was stained with haematoxylin and eosin and examined by three independent pathologists (Claudio Ceccarelli, Donatella Santini and Massimiliano Bonafe, from the University Hospital of Bologna). For each microscope field (200X) the area occupied from cancer cells, stromal cells and necrotic components was evaluated and represented as percentage.

Characterization of CAFs

Serial sections (5 microns) of paraformaldehyde-fixed paraffin-embedded samples underwent antigen retrieval using Leica Bond ER2 Buffer (Leica Biosystems) for 20 min at 100°C before staining with 1μg/ml Desmin Rabbit polyclonal antibody (Abcam cat#ab8592) and 1μg/ml pStat3 (clone D3A7, Cell Signaling) for 1 hour using Leica Protocol F (Molecular Imaging Core facility, MSKCC). Quantification of Desmin/pStat3 staining was performed using ImageJ/FIJI (NIH). At least 19 fields at 400X were randomly selected and evaluated. The results were expressed as percentage of immunostained cells/over total area of tissue. To discriminate between cancer and stromal cells fortified H&E staining was also performed (HistoServ Inc, Germantown, MD). A color deconvolution algorithm was then used, with RGB vectors for the stromal component and counterstain/background stain created from ROIs drawn from example images (Molecular Imaging Core facility, MSKCC). Appropriate thresholds were then set for each cell type of interest and area measurements were taken for all images. To rule out possible non-CAFs/non-cancer cells component, specific staining for CAFs (desmin -murine CAFs) and cancer cells (human pankeratin) was also performed in serial section slides. Stroma-tumor niches were evaluated as area of tissue slide with the co-presence of pankeratin positive cells and stroma cells.

ALDEFLUOR assay

Aldefluor analysis was performed using the ALDEFLUOR Kit (Stem Cell Technologies) according to the manufacturer’s protocol. Cancer cells from PDX primary cultures were washed with 5ml 10% PBS supplemented with Accumax (Innovative Cell Technologies) and, single cell suspensions were first stained with anti-CD133-PE conjugated antibody for 20 min, washed twice with PBS-BSA (5%) and then incubated with Aldefluor reagent.

Conditioned Media preparation and phenotypic assays

Conditioned media (CM) was isolated from CAFs and cancer cell lines (10⁸ cells): concentrated using Amicon Ultra-15 centrifuge tubes (Millipore) and protein levels were measured by the Lowry technique; 10μg of total extracellular protein was loaded for zymographic/protein (MMP-2, MMP-9) and in vitro studies (invasion capacity). Cell growth of co-cultured cancer cells with CAFs was determined with and without anti- Jagged1/Notch3 blocking antibody (AF1277, R&D Systems 500ng/every 72h). Briefly luciferase positive breast cancer cells (MCF7) grown with CAFs (1:50) were seeded in 96 well plate and treated with fulvestrant (10μM/weekly) in the presence of mouse anti-Jagged1 blocking antibody. BLI signals was measured every 48h and growth curves were generated accordingly.

Luciferase assays

Cells were plated in 6-well plates at a density of 2×10⁵ cells per well. Cells were transfected with 0.3μg of promoter luciferase (CD133) [14] and the activated form of Notch3 (pNICD3 2μg) [10]. To normalize transfection efficiency, cells were also co-transfected with 0.1μg of the pRL-CMV (Renilla luciferase, Promega). Forty-eight hours after transfection, luciferase activity was measured using the Dual-Luciferase Assay kit (Promega). Three independent experiments were performed, and the calculated means and standard deviations are presented.

Taqman gene expression profile and RT PCR

qPCR was performed on 100ng of cDNA using TaqMan pre-custom probes (Applied Biosystems, ERα Hs00174860 62pb, GATA3 Hs00231122 80bp, FOXA1 Hs0418755 59bp, GREB1 Hs00536409 67bp, EGR3 Hs00231780 91bp, CCL5 Hs00174575 63bp, PGR Hs01556702 77bp) and SYBR Green technique (α-sma forward 5′-CAGGGCTGTTTTCCCATCCAT-3′, reverse 5′-GCCATGTTCTATCGGGTACTTC-3′; SDF-1α forward 5′-CCATGAACGCCAAGGTCGTG-3′, reverse 5′-CCAGGTACTCCTGAATCCAC-3′; Vimentin forward 5′-TGGCACGTCTTGACCTTGAAA-3′, reverse 5′-GGTCATCGTGATGCTGAGAA -3′; Slug 5′-AGATGCATATTC GGACCCACA-3′, reverse 5′-CCTCATGTTTGTGCAGGAGA-3′; CD44 forward 5′-CAGCAACCCTACTGATGATGACG-3′, reverse 5′-GCCAAGAGGGATGCCAAGATGA -3′). ViiATM 7 Real-Time PCR System was used (Applied Biosystems) in accordance with the manufacturer’s instructions. For analysis, ΔCt method was applied and fold change was calculated (2−ΔΔCt). All values were normalized to GAPDH expression (TaqMan, Hs02758991). RT-PCR for Notch3 (forward 5′-TCAGGCTCTCACCCTTGG-3′,reverse 5′-AGTCACTGGCACGGTTGTAG-3′), Jagged1 (forward 5′-TCGCTGTATCTGTCCACCTG-3′, reverse 5′-AGTCACTGGCACGGTTGTAG-3′) and β2μ as internal control 5′-ACCCCCACTGAAAAAGATGA-3′, reverse 5′-ATCTTCAAACCTCCATGA-3′ was performed in MCF7 cells control and shNotch3 and mCAFs/fibroblasts cell lines.

Statistical analysis

Statistical analysis was performed by SPSS (SPSS Incorporation). Continuous variables were analyzed by unequal variance t-test, paired t-test (samples, n=2), general linear model (GLM) Anova or GLM for repeated measures (samples, n>2). Mann-Whitney and Wilcoxon tests were used to analyze ordinal variables. P values were adjusted for multiple comparisons according to Bonferroni correction. Association among quantitative variables was quantified by Pearson correlation coefficient. Categorical variables were analyzed by Monte Carlo χ2 test. All the tests were two-sided. P<0.05 was considered significant. Elda software was used to measure the statistics of limiting dilution experiments (bioinf.wehi.edu.au/<http://bioinf.wehi.edu.au/>software/elda/).

Results

MVs from CAFs mediated hormonal therapy resistance

The presence of CAFs have been assessed as prognosticators in breast cancer and an “active stromal signature” in normal fibroblasts exhibits a tumor promoting phenotype [15]. Many stromal secreted factors including IL6, SFD-1α, and HFG participate in the communication between CAFs and tumor cells within the tumor microenvironment.

Stromal MVs have also been implicated in tumor progression in glioblastomas and ovarian cancers [16, 17]. However, the molecular and pathological relevance of CAF-derived MVs in luminal breast cancer remains unclear.

In order to study tumor progression in luminal breast cancer, we established long-term xenografts of highly tumorigenic MCF7 and ZR751 cells [5]. Following tumor establishment (1cm), all mice received HT (fulvestrant a selective estrogen receptor degrader commonly given to patients with ER+ metastatic disease, 10μM) for 3 months. Although the majority of xenografts displayed sensitivity to HT (HTS, stable disease or remission), ~10% of the xenografts (data not shown) grew in the presence of therapy (Fig. 1A, HTR -resistance to HT-). Interestingly, the histological analysis of these tissues revealed the enrichment of CAFs in the HTR xenografts (Fig. 1B and Fig. S1A). Furthermore, we could isolate and in vitro passage CAFs from HTR-derived xenograft tissues. These CAFs cell lines displayed the up-regulation of CAF markers by western blot analysis and the capability of growth for multi passages (more than 20 passages) (Fig. 1B and data not shown). Although we were able to isolate CAFs from HTS lesions in a small fraction of xenografts (5%, n=3 out of 60), we could not propagate them in culture for more than 2 passages (2 weeks). Therefore no CAF-cell lines (0%) were established from HTS xenografts.

Figure 1. MVs from CAFs mediated hormonal therapy resistance.

(A), Generation of hormonal therapy resistant (HTR, purple) and sensitive (HTS, green) xenografts in luminal breast cancer: highly tumorigenic luminal breast cancer cells (MCF7 and ZR751 Luciferase positive) were injected in the mammary fat pad, xenograft bearing mice were treated with fulvestrant starting at 4 months for 3 months (HT, fulvestrant intra-muscular injection 100μg/mouse/weekly). Some tumors grew in presence of HT (HTR), while the majority of them were sensitive to HT (HTS). Data are reported as error bars mean bioluminescence (BLI value ± s.e.m.) at the end point of the experiment (7 months, n=10 mice/group);

(B), HTR cultures were enriched with murine CAFs (Mu-CAFs,red):representative fortified H&E staining of HTR xenografts (from panel A, MCF7, scale bar 50μm, yellow denotes stroma desmoplastic reaction), bright field images of primary cultures from HTS and HTR xenografts and western blot analysis of FACS purified cancer cells (Hu-HTR) and murine cells (Mu-CAFs);

(C), Schematic of the experiment: HT naïve cancer cells (GFP⁺/Luciferase⁺) were co-cultured with FACS purified Mu-CAFs (purple), human BMSC/CAF HS27a cells (red) or in the absence of stromal cells (green). Cells were treated with or without HT (fulvestrant, 10μM/weekly).

(D), Proliferation potential (after 2 weeks, BLI) was determined ±HT (fulvestrant, 10μM). Data are reported as error bars, mean±s.d. of n=3 independent experiments. *P<0.05 (Student’s t-test);

(E), Electron microscopy images and quantification (number by nanosight) of CAF-derived MVs (murine and human, HS27a). Scale bar 200nm; Data are reported as error bars, mean±s.d. of number of particles/ml for 10⁶ cells. *P<0.05 (Student’s t-test) of n=3 independent experiments; (F), Schematic of the experiment: Mice were injected in the MFP with HTS cells (MCF7) and subsequently injected weekly with either Mu-CAFs MVs or control MVs (MCF7 derived) (see experimental procedures). After 4 months, HT was administered for 3 months (fulvestrant intra-muscular injection 100μg/mouse/weekly); (G), Dot plot of tumor growth of HTS cells (luciferase/p/s) grown in the MFP of mice educated with CAF EVs or CTRL EVs (MCF7) in the presence/absence of HT (fulvestrant see panel F; MVs, retro-orbital injection, 3X10¹⁰ particles/mouse/weekly): error bars, mean ± s.e.m of the last point of the growth curve before (4 months) and after HT (7 months). *P<0.05 values refer 2-way Anova (G).

To further characterize these tumor-associated stromal cells, we cultured HTR tumors and isolated stromal cells by FACS (negative selection with EpCAM, which recognizes epithelial cells) and determined that EpCAM^neg cells were morphologically spindle shaped, were murine in origin (expressed murine genomic DNA, data not shown) and expressed markers of activated cancer-associated fibroblasts (CAFs) including Fap, vimentin, fibronectin and activated Stat3 (Fig. 1B, phospho tyrosine 705 Stat3 -pStat3-).

Next, we asked whether these CAFs could promote de novo HTR disease. We co-cultured murine CAFs and human-HS27a “CAF” like cells (bone marrow derived immortalized mesenchymal cells) with HT (Luciferase^pos) naive cancer cells in the presence/absence of HT (fulvestrant, 10μM/weekly) and cancer cell growth was analysed by in vitro bioluminescence after 2 weeks (Fig. 1C). We found that CAFs promoted tumor cell growth following HT, whereas no difference was found in the absence of therapy (Fig. 1D and Fig. S1B). In contrast to HS27a cells and murine CAFs, normal fibroblasts (mammary and lung) did not confer resistance to HT in co-cultures (Fig. S1C).

In addition to growth factors, stromal cells have been shown to secrete microvesicles (MVs) which can horizontally transfer numerous pro-survival factors and confer resistance to radiation therapy [18]. Here we determined that the number of MVs produced by murine CAFs and HS27a cells was much greater than MCF7 tumor cells (Fig. 1E). To determine whether these MVs could confer a pro-tumorigenic advantage, we set up an in vivo model (Fig. 1F). MFP xenografts from HT naïve cells (MCF7) were established; CAF-MVs (3X10¹⁰) and control MVs (tumor-derived MVs, MCF7) were injected retro-orbitally weekly for 7 months. Once tumors were established (after 4 months) all mice received HT (fulvestrant weekly). Although there was no difference in tumor growth before HT, those animals treated with CAF-MVs had tumors resistant to HT (HTR) while MCF7-MVs provided no benefit as tumors regressed with HT (Fig. 1G). Overall, these data demonstrated that circulating stromal MVs can induce resistance to HT in vivo.

CAF-derived microvesicle transfer of oncomiR 221/222 to cancer cells promotes de-novo hormonal therapy resistance

Distinct genes and pathways have been associated with resistance to HT including: the activation of mutations in the ESR1 gene [19], increased Her2 expression [20], decreased ER levels and ER transcriptional signatures [21], [22], increased expression of oncomiRs including the ER-repressor mir221/222 [23] and, more recently, increased Notch signaling in CSCs [5, 24].

Since a reduction in ER expression is associated with resistance to HT [21], and CD133^hi CSCs have lower ER levels (mRNA and protein) as compared to CD133^lo/CD44^lo cells, we reasoned that the suppression of ER signaling could be a mechanism of stroma-mediated expansion of therapy resistant cancer stem cells (CD133^hi/Notch3^hi).

To test our hypothesis we first demonstrated that the conditioned media (CM) from CAFs (murine and human), but not normal fibroblasts, led to a decrease in ER protein expression and ER-dependent transcripts (e.g. GATA3, FOXA1, GREB1, EGR3, CCL5, PGR) in MCF7 cells (Fig. 2A and Fig. S2A). Since the suppression of ER protein occurred with CM from both murine and human CAFs, we hypothesized that rather than soluble factors (which can be typically species-specific) [25], CAF-derived microRNAs might be mediating the down-regulation of ER expression. MVs have been suggested to be mediators of nucleic acid transfer including microRNAs [26]. Amongst different microRNAs, the forced over-expression of oncomiR 221/222 in luminal breast cancer cells has been found to reduce ER expression and promote HT resistance [23]. Additionally increased plasma levels of mir221 were found in ER negative breast cancer patients[27]. The administration of 10⁸ MVs from mCAFs to ER+ cancer cells (MCF7) reduced ER levels after 48 hours (Fig. 2B). We showed that oncomiR 221/222 sequences are conserved between human and mouse species, suggesting possible functional cross-species interactions (Fig. 2C). Importantly, miR221 expression was found in circulating MVs from patients with HTR metastatic disease (independent of tumor burden) as compared to healthy controls (Fig. 2D, n=11; Table S1).

Figure 2. Microvesicle-mediated OncomiR 221 transfer from CAFs to cancer cells promotes an ER^lo phenotype leading to HTR resistance.

(A), Western blot of ERα protein in MCF7 cells following treatment with the conditioned media (CM) of human normal mammary fibroblasts (HMF), mCAFs (red font) and HS27a cells (blue font, 48h);

(B), Western blot of ERα protein in MCF7 cells treated with and without mCAF derived MVs (10⁸ particles, 48h); (C), Image showing the OncomiR 221 sequence conservation in Mus Musculus and Homo Sapiens; (D), MV-miR221 expression as determined by qPCR as fold increase from patients with HTR disease and healthy controls (reference MCF7-MVs was used and normalized to total RNA expression, n=3 replicates; Table S1). Patients with high volume disease (>10% of organ involvement) denoted in red and low volume disease (<1% of organ involvement) denoted in blue; (E), Bar graphs representing oncomiR 221 expression (qPCR) in MVs from indicated sources (10⁸ cells). Data are reported as fold increase (221/U6 expression) ± s.d of n=3 replicates (MV-MCF7 is used as reference); (F), RT-PCR analysis of miR 221 and 101 in MCF7, mCAFs and mCAF derived MVs (RNA was isolated from 10¹⁵ particles) and bar graph showing oncomiR 221 expression (qPCR, Fold, normalized to U6 values) in mCAFs and HS27a cells and their respective MVs. The expression of oncomiR 221 in MCF7 was used as a reference control; (G), Bar graph of oncomiR 221 (qPCR, Fold) in MCF7 cells following chronic mCAF-MV education (one month, 10⁸ particles weekly). MVs were isolated from MRC5 cells (normal lung fibroblasts) and used as control MVs (CT-MVs); (H), Western blot of ERα protein in MCF7 cells treated with mCAF-MVs (10⁸ particles, 48h) previously transfected (24h before MV education) with the antimiR 221 or control (CT) (see experimental procedures); (I), Bar graph representing Cell Death (by trypan blue, %) of MCF7 cells transfected with antimiR 221 and controls, treated with fulvestrant (10μM, 7days) in the presence/absence of mCAF and MRC5 MVs (10⁸ particles every 48h). miR221 negative MVs were used as control MVs and were obtained from normal lung fibroblasts (MRC5). Representative images of Crystal violet staining of MCF7 cells at the endpoint of the experiment descried in panel H. Data are reported as mean ± s.d. of three independent experiments (n=3). NS, stands for not significant. P values refer to Student t-test (D–G, I).

We determined that CAF-derived MVs were enriched for miR221 compared to normal fibroblasts and distinct cancer cells lines (breast, cervical and lymphoma), suggesting that stroma derived MV could account for the increased level of mir221 expression in MVs (Fig. 2E). Accordingly, miR 221—but not a control miRNA (miR101)—was highly expressed (100–200 fold enrichment) in MVs from CAFs as compared to MVs from normal fibroblasts, CAFs and cancer derived cell lines (Fig. 2F and Fig. S2B). Administration of murine CAF-MVs compared to normal fibroblast-MVs (MRC5) to MCF7 cells (oncomiR 221 negative) led to the transfer of oncomiR 221 (Fig. 2G, 20-fold increase in expression in educated cancer cells).

In order to address the consequences of MV-derived oncomiR 221 transfer in mediating HT resistance, antimiR 221 was transfected in cancer cells before MVs administration. We demonstrated that antimiR 221 transfection abrogated mCAF-MVs dependent downregulation of ER protein (Fig. 2H) and HT resistance following chronic mCAF-MV education (Fig. 2I). Restored sensitivity to HT in antimiR 221 transfected cells was associated with an increase in ER expression/activity (Fig. S2C, D). These data suggest that CAF-MV-mediated HT resistance occurs via the transfer of oncomiR 221 promoting an ER^lo phenotype.

CAF-mediated expansion of CD133^hi CSCs via an OncomiR 221^hi/ER^lo/Notch3^hiloop

Increased expression of Notch and downstream signalling events as well as a higher number of cancer stem cells (CSCs) are found in hormonal therapy resistant breast cancer [28, 29]. Amongst Notch proteins, Notch3 and Notch4 are crucial mediators of resistance to hormonal therapy in distinct models of luminal breast cancers [5, 24, 30, 31].

Given the pivotal role of CAF-derived MV in promoting the switch from sensitive to resistant disease (HTS to HTR), we asked whether these stromal MVs could also modulate Notch3 expression. We cultured MCF7 cells with fulvestrant (10μM/weekly) for two weeks (see schematic Fig. 1C) with MVs (10⁸ particles/weekly) from either MRC5 or mCAFs. Although no effect was observed in the absence of HT (no treatment), mCAF-MVs restored Notch3 expression and activity (Hes1, Hey 1 mRNAs) following fulvestrant which associated with increased growth (Fig. 3A and Supplementary Fig. S3A, HTR cells see Fig. 2I). Conversely, MRC5-MVs administered cells did not overcome ER-dependent downregulation of Notch3 expression and activity (Hes1, Hey 1 mRNAs) as well as suppression of growth (Fig. 3A, HTS cells see Fig. 2I). The transfection of antimiR 221 led to decreased Notch3 protein levels and restored sensitivity to HT (fulvestrant) of CAF-MV treated cancer cells (Fig. 3B and see Fig. 2I). These data demonstrate that miR221 in CAF-MVs can block HT-mediated down-regulation of Notch3 expression.

Figure 3. CAF-mediated expansion of CD133^hi CSCs via an OncomiR 221^hi/ER^lo/Notch3^hiloop.

(A), Western blot of Notch3 protein from MCF7 cells in the presence/absence of mCAF and MRC5 MVs (10⁸ particles every 48h) ± Fulv (10μM for seven days). Tubulin is a loading control; (B), Representative western blot of Notch3 protein from MCF7 cells transfected with the antimiR 221/CT (1μg/well) and treated with fulvestrant and mCAF-MVs (48h). Tubulin is a loading control; (C), Schematic of co-culture experiments: control (shCT) and Notch3 knocked-down MCF7 GFP/Luciferase+ cells (shN3) were cultured with PDX-derived mCAFs (1:10; mCAFs:cancer cells) in the presence/absence of fulvestrant (Fulv 10μM, weekly administration for a month); growth of MCF7 shCT/shN3 cells in co-cultures were then determined. Data are reported as mean (luciferase, p/s -photons/seconds-) ± s.e.m. of the last time point of the growth curve or 28 days (3 biological replicates with 3 technical replicates each); (D), Fold Increase in CD133^hi CSCs (Flow Analysis) from MCF7 shCT/shN3 cell co-cultures treated with fulvestrant (1 month, endpoint panel C). Data are reported as total number of CD133 positive cells (mean ± s.d. of fold change) of n=3 different specimens (as a reference total numbers of CD133^hi cells from 10⁹ naïve MCF7 cells was used). NS, stands for not significant. P values refer to Student t-test (C, D).

We recently described the enrichment of CD133^hi/ER^lo/Notch3^hi cancer stem cells (CSCs) in hormonal therapy resistant tumors which also expressed high levels of Notch regulated genes such as Hey1 and Hes1 (GSE69280[5]).

Given the role of CAF or stromal MVs in promoting a mir221^hi/ER^lo/Notch3^hi phenotype, we tested the hypothesis that CAFs could promote the in vivo expansion of CD133^hi cells via Notch3 up-regulation. The selective reduction of Notch3 expression in cancer cells (shNotch3) and activity (using an anti-Jagged1 blocking antibody) abrogated CAF-mediated HT-resistance and the expansion of CD133^hi cancer cells (Fig. 3C, D and Fig. S3B). In agreement with the knock down experiment, over-expression of the activated form of Notch3 (pNICD3) in MCF7 cells led to an increase in CD133 promoter luciferase activity (pCD133) with HT (fulvestrant) in association with a reduction in ER protein levels. These data suggest that higher Notch3 activation could promote a feed-forward ER^lo/CD133^hi loop necessary for the generation of CD133^hi CSCs (Fig. S3C, D). Overall our data describe a novel mechanism of hormonal therapy resistance: CAF-MV-mediated transfer of the oncomiR 221 leading to reduced ER expression and Notch3 up-regulation.

IL6/Stat3 activity is required for CAF-CSC niche formation

Since the biogenesis of oncomiR 221/222^hi MVs occurs preferentially in CAFs (not normal fibroblasts), we hypothesized that the abrogation of a CAF phenotype would interfere with the generation of HT resistant cancer stem cells. To investigate this hypothesis, we examined possible candidates responsible for CAF growth. Compared to breast cancer cells, the conditioned media (CM) of CAFs (HS27a cells) expressed higher levels of chemokines (e.g. IL8, MIP-1δ, CCL5) and cytokines, including IL6 an activator of Stat3 (Fig. 4A). These findings were further supported by evidence of high pStat3 levels in murine CAFs from HTR derived xenografts (Fig. 4B). Differently from other signaling pathways (HER, PI3K, ER), pStat3 activity was required for CAFs proliferation as well as the generation of oncomiR 221^hi MVs (Fig. 4C and Fig. S4A). In concordance with these data, reduced IL6 expression in HS27a cells (using an IL6-shRNA) lowered secreted IL8/IL6R/CCL5 levels as well as the expression of CAF markers including pStat3, vimentin and CD44 (Fig. 4D, E). Additionally, compared to HS27shCT cells, HS27shIL6 cells had reduced proliferative and invasive potential (Fig. S4B, C) as well as lowered MMP2/9 expression and activity, indicating a loss of characteristic CAF features (Fig. S4D). Moreover, MVs from HS27shIL6 cells had essentially no expression of oncomiR 221 compared to HS27shCT cells with no change in MV production (Fig. 4F, protein content as a surrogate marker of MV yield). These data suggest that IL6/pStat3 signaling is crucial for the proliferation of CAFs and the production of oncomiR 221+ MVs.

Figure 4. IL6/Stat3 signaling from CAFs promotes the expansion of CD133^hi CSCs.

(A), Cytokine array expression of the CM from MCF7 and HS27a cells (2μg total protein). Highlighted are over-expressed cytokines and chemokines (IL6, IL6sR, IL8, MIP-1δ and CCL5); (B), Dot plot showing phospho-tyrosine 705 Stat3 (pStat3) IHC quantification in HTR derived primary tumor tissues (see Fig. 1B) in both the stroma and tumor compartments. Quantification of pStat3 was performed using ImageJ/FIJI (NIH) of n=19 fields from n=5 different tumors. The results were expressed as ratio of pStat3 IHC value/total tissue area. A representative IHC image is shown. Scale bar 25μm; (C), Bar graph of the proliferation capacity (CalceinAM, fluorescence) of xenograft-derived mCAFs isolated from HTR xenografts (Fig. 1) and cultured in the presence of vehicle (Placebo) or signaling pathways inhibitors including ER (fulvestrant, 10μM) or PI3K (BYL, 100nM), HER (lapatinib, 100nM) or JAK/pStat3 (AZD1480, 500nM). Data are reported as mean (fluorescence) ± s.e.m. of the last time point of the growth curve (14 days. 3 biological replicates with 3 technical replicates each);

(D), Cytokine array expression from the CM (2μg) of HS27shIL6 versus HS27a cells. Highlighted are over-expressed cytokines and chemokines (IL6, IL6sR, IL8, MIP-1δ and CCL5);

(E), Western blot of pStat3, Stat3, CD44, Vimentin, Caveolin1 and Tubulin protein levels in HS27a cells CT and shIL6;

(F), Bar graph of OncomiR 221 expression in MVs derived from 10⁸ HS27a and HS27shIL6 cells (qPCR, Fold as reference MCF7 MVs was used, normalized to U6 expression). Bar graph of protein levels (μg) in 10⁸ MVs isolated from HS27a and HS27shIL6 cells is also shown;

(G, H), Tumor growth and metastatic burden (luciferase, BLI) in MCF7/HS27a and MCF7/HS27shIL6 xenografts: 10³ cancer cells were inoculated in both 4^th inguinal MFP with 10² HS27a cells (CT or shIL6). Tumor growth was examined over 20 weeks. Data are reported as mean BLI value ± s.e.m. (log scale) for each time point (n=4/5 mice/group). Metastatic burden is mean BLI value ± s.e.m. of signal from metastatic tissues including lymph-nodes, lungs and bones. A representative image of primary tumors from MCF7/HS27a versus MCF7/HS27shIL6 is shown;

(I), Bar graph representing immunohistochemical quantification (IRS score) of ERα, CD133 and Notch3 in tumor derived tissues from MCF7/HS27a or MCF7/HS27shIL6 xenografts (panel G). Data are reported as mean ± s.d. of n=10 tissue sections for each group;

(J), Bar graph showing percentage (H&E, %) of stroma-tumor niches versus tumor compartment in primary tumor tissues derived from MCF7/HS27a and MCF7/HS27shIL6 xenografts (panel G);

(H), Representative Pankeratin staining of tissues slides from panel G depicting stroma-tumor niches (cancer cells surrounded by stromal cells). Data are reported as mean ± s.d. of n=10 tissue slides for each group. P values refer to t-test (B, F, H, I), Wilcoxon two sample test (J), multiple comparisons corrected post-hoc t-test after GLM Anova (C), repeated measures GLM Anova (G).

To address the phenotypic consequences of decreasing IL6 signaling in CAFs, we co-injected MCF7 cells with HS27shCT and HS27shIL6 CAFs into the MFP of mice. Compared to controls (MCF7/HS27), the co-injection of MCF7/HS27shIL6 cells resulted in impaired tumor growth, lower metastatic burden and decreased expression of CD133^hi/Notch3^hi/ERα^lo CSCs (Fig. 4G–I and Fig. S4E). In agreement with the loss of CD133^hi/Notch3^hi CSCs, MCF7/HS27shIL6 derived tumors had fewer stroma-tumor niches (Fig. 4J and K) and decreased pStat3 expression (Fig. S4F). Overall, our data suggests that IL6 mediated generation of stromal niches is required for the expansion of CD133^hi CSCs.

Anti-IL6 therapy abrogates CAF-mediated de novo resistance to HT

To extend our results to clinical specimens, we established primary cultures of stromal cells from patient-derived bone metastases (Table S2, BM-CAFs). IL6 is a well-known pleiotropic cytokine, secreted at high levels from the bone marrow microenvironment and CAFs [32, 33]. We isolated and cultured CAFs from bone metastases (Fig. S5A and data not shown) and detected high levels of IL6 protein from the conditioned media of these primary cultures (Fig. 5A). These levels are similar to those found from the conditioned media of HS27a cells. Consistent with the HS27 model, the abrogation of IL6 signaling, using the anti-IL6R-IL6 antibody (tocilizumab), abrogated the growth in 70% of CAF primary cultures (Fig. 5B), reduced IL6 secretion and the expression of CAF markers (Fig. 5C and Fig. S5B), this finding suggests that autocrine IL6 maintains the CAF phenotype of these cells.

Figure 5. Autocrine CAF-derived IL6 triggers endocrine resistant disease.

(A), Bar graph showing IL6 levels measured by ELISA from the conditioned media (2μg total protein) of primary CAF cultures (BM-CAFs) isolated from patient bone derived breast cancer metastases (Table S2). IL6 levels from MCF7 and HS27a are also shown;

(B), Growth (cell number) of BM-CAFs (specimen 5, Table S2) treated with an anti IL6-IL6R antibody tocilizumab (500ng/ml every 48h) versus IgG control;

(C), Bar graph of IL6 protein (ELISA) from the conditioned media (2μg total protein) of BM-CAFs treated with tocilizumab or IgG for a week (500ng/ml every 48h);

(D), Bar graph of oncomiR 221 expression by qPCR (Fold, normalized on U6 level) in MVs isolated from BM-CAF cultures. MVs isolated from MCF7 cells were used as a reference control;

(E), Tumor burden (luciferase) of MCF7 xenografts alone (black) and co-injected with BM-CAFs (specimen 6, Table S2) treated with fulvestrant and Tocilizumab. Briefly MCF7 xenografts were established in the MFP of NOD/SCID mice alone (10³ cells) or in combination with CAFs (10² cells): when tumors reached ~1 cm (3 months), mice were randomized (n=4/group) to receive either fulvestrant (1mg/weekly) or fulvestrant and tocilizumab (100μg/gm/mouse) for 4 months. Data are reported as mean BLI value ± s.e.m. at the endpoint of the experiment (7 months). Data are reported as mean ± s.d. of three independent experiments (n=3). P values refer to Student T-test (C, D, E), multiple comparisons corrected post-hoc t-test after repeated measures GLM Anova (B).

Although miR221/222 expression is very low in luminal breast cancer tissues and cells (Fig. 2), high levels of miR 221 was found in circulating MVs from HTR patients (Fig. 2D). Next, we reasoned that -in agreement with other investigators- CAFs would be the major source of miR221 [34]. We then isolated MVs from CAF primary cultures derived from bone metastases and found increased levels of oncomiR 221 as compared with tumor MVs (Fig. 5D); these results were similar to those found in HS27a cells and mCAFs (Fig. 4). We subsequently demonstrated that the co-injection of BM-CAFs with MCF7 cells promoted HTR tumor growth (treated with fulvestrant) in an IL6-dependent manner, as combination fulvestrant/tocilizumab abrogated tumor growth (Fig. 5E).

CAF-mediated expansion of CD133^hi cancer stem cells

We and others have recently demonstrated the enrichment of CSCs expressing CD133 or ALDH^hi activity in experimental luminal breast cancers following HT [5, 24]. Additionally, expression of Prominin1 (CD133) was identified in tumors from patients who progressed on adjuvant HT [22]. In agreement with other investigators, CD133^hi and CD44^hi cells are functionally cancer stem cells as they were capable of engrafting with low cell numbers (<1000, Fig. S6A). We previously demonstrated that differently from CD44^hi cells, CD133^hi/CD44^lo cells expressed an embryonic stem cell signatures and increased expression of ABCG2, a cancer stem cell gene associated with therapy-resistance [5, 35] (GSE69280). Here we determined that CD133^hi/CD44^lo cells expressed normal stem cell signatures by gene set enrichment analysis GSEA [5](Fig. S6B, GSE69280). Additionally, compared to CD133^lo/CD44^lo and CD44^hi/CD133^lo cells, the injection of CD133^hi/CD44^lo cells gave rise to slow-growing tumors (Fig. S6C) with an increased capacity to disseminate to the bone marrow (Fig. S6D). These data suggest that the CD133^hi phenotype is a distinct CSC population [36].

Next, we investigated the hypothesis that extrinsic (stromal heterogeneity) factors could regulate the evolution of therapy resistant niches leading to metastatic progression [37, 38]. We demonstrated high PROM1 (encoding CD133) expression by microarray (GSE17705) was associated with increased levels of a mammary CAF gene signature in the setting of human HT-resistant primary tumors (Fig. 6A, p<1.82×10⁻⁴ and Fig. S7A).

Figure 6. CAF-mediated expansion of CD133^hi cancer stem cells in patient derived xenografts from hormonal therapy resistant breast cancer bone metastases.

(A), Box plot of PROM1 expression (log₂) separated into “low” (black) and “high” (red) groups by the median CAF score as described in the experimental procedure section using the GSE17705 data set[22]. Box plots are drawn such that horizontal lines indicate the sample median, the box spanning the interquartile distance (IQD), whiskers extending to 1.5xIQD, and remaining outlying points shown as open circles;

(B), Tumor take percentage (%) of PDX’s following three sequential passages in vivo. Briefly, cancer cells were isolated from bone metastases (Table S2, sample shown B-Met3), grown in culture dishes for 2 weeks and then injected into the MFP of NOD/SCID mice (1^st n=1/5; 2^nd n=3/10; 3^rd n=40/40) treated with HT (fulvestrant,, 1mg/weekly). Following 5 months tumor tissues were digested and cancer cells were cultured in vitro and FACS-sorted for EpCAM positivity (to eliminate murine cells) before re-inoculating (10⁵ EpCAM^pos cells) these into new cohorts of mice;

(C), Number of CD133^hi/CD44^lo cancer cells (EpCAM^pos) and stroma cells (EpCAM^neg) derived from 1^st and 3^rd generation PDXs (Flow Analysis, Fold);

(D), Schematic of the in vitro PDX culture established in presence/absence of CAFs (CAF^pos or CAF^neg): unsorted (cancer cells and CAFs) or previously FACS purified EpCAM^pos cancer cells from PDX primary cultures were grown for 6 months (6 mo), then EpCAM^pos cancer cells were FACS-purified from EpCAM^neg cells and treated with fulvestrant (Fulv, 10μM 14 days). Scale bar 10μm;

(E), Bar graphs of cell growth (CalceinAM, left panel): data are reported as mean (fluorescence) ± s.e.m. after 14 days (3 biological replicates with 3 technical replicates each). The number of CD133^hi cells (Flow Analysis -%-, right panel) is also shown after 14 days of culturing;

(F), Images and quantification of secondary MS growth of FACS purified CD133^hi and CD133^lo cancer cells from experiment panel E. Scale bar 100μm;

(G), Schematic and growth of MCF7 (GFP+/Luciferase+) cells, using BLI -luciferase- grown with and without mCAFs (PDX derived). A 1:10 mCAF to MCF7 cell ratio in the presence/absence of fulvestrant (Fulv, 10μM, added weekly for 30 days);

(H), Representative CD133/CD44 expression by flow analysis of fulvestrant resistant MCF7 cancer cells (FulvR) in the presence/absence of mCAFs at the endpoint of the experiment described in panel G;

(I), RT-PCR analysis of ERα in FACS isolated CD133^hi and CD133^lo cancer cells from experiment panel G and H. β2μ as a loading control is shown;

(J), Box plot depicting ESR1 expression (log₂) separated into “low” (white) and “high” (purple) groups by the median PROM1 expression using the GSE17705 data set [22]. Box plots are drawn with a horizontal line indicating the sample median, box spanning IQD, whiskers extending to 1.5x IQD, and outlying points shown as open circles. Data are reported as mean ± s.d. of three independent experiments (n=3). P values refer to Monte Carlo χ2 test (A), Student T-test (B, C, E, J), t test after GLM Anova (G), GLM Anova for repeated measures (E, left panel).

In order to examine the importance of CD133^hi cells in clinically relevant models of metastatic breast cancer, we generated PDXs from hormonal therapy resistant (HTR) luminal breast cancer bone metastases (Fig. 6B, Table S2). Cultured tumor cells were serially transplanted into the mammary fat pad (MFP) of immunocompromised mice in the presence of HT (fulvestrant: a selective estrogen receptor degrader). After 3 sequential passages, the enhanced tumorigenic capacity (% tumor take) of PDXs was associated with increased Notch3 expression (Fig. S7B), enrichment of CD133^hi (~40-fold) cancer cells and murine stromal cells (~30 fold) which had infiltrated the tumor (Fig. 6B, C and Fig. S7C, D). We further determined that the PDX-derived CD133^hi cells had low ALDH activity suggesting different and unique stem cell populations arising through a stroma-mediated metastatic transition within ER+ breast cancer (Fig. S7E).

Given the co-enrichment of CD133^hi cells with CAFs, we hypothesized that CAFs could directly promote the expansion of HT-resistant CD133^hi cells. To address this hypothesis, we depleted HT-resistant PDX primary cultures from CAFs (by FACS for EpCAM^pos) and maintained these cancer cells in culture for several months. When long-term depleted for murine CAFs, EpCAM^pos cells from PDX primary cultures were growth inhibited by HT as compared to tumor cells freshly isolated from co-cultures (sorting for EpCAM^pos, Fig. 6D, E). Moreover, acquired sensitivity to HT resulted in decreased numbers of self-renewing CD133^hi cells (Fig. 6E, F).

To determine whether CAFs could confer de-novo HT resistance and promote the biogenesis of CD133^hi cells, we cultured HT-sensitive MCF7 cells with murine CAFs, isolated from xenografts, with/without HT (fulvestrant) for 4 weeks (Fig. 6G). When cultured with CAFs, MCF7 cancer cells became resistant to HT (fulvestrant), as compared to MCF7 cells alone (Fig. 6G). Concomitant with HTR cancer cell growth, by FACS we observed a marked (30-fold) increase in CD133^hi CSCs as compared to MCF7 cells alone (Fig. 6H). Further characterization of the CD133^hi cells revealed that ERα mRNA levels were lower in these cells than in CD133^lo/CD44^lo cells (Fig. 6I). In agreement with these data, higher CD133/PROM1 expression was associated with decreased ESR1 mRNA expression and activity in HT-resistant primary tumors (Fig. 6J, p<0.0001). Overall, these findings demonstrate that CAFs, in the presence of HT, can promote the de-novo formation of CD133^hi CSCs and HTR disease.

In summary, our data led us to propose the following model: IL6 up-regulation drives a CAF phenotype leading to the generation of stroma-tumor niches in vivo. The presence of active CAFs promotes a skewing of the cancer cell population towards a CD133^hi/Notch3^hi/ER^lo CSC phenotype. This occurs via the production/action of CAF-derived MV which reduces ER activity leading to HT resistant disease (Fig. 7)[39].

Figure 7. Stroma microvesicle mediated cancer stem cell evolution in endocrine resistant metastatic breast cancer.

Schematic of the proposed model of CAF-mediated endocrine resistant disease in luminal breast cancer. Autocrine IL6/Stat3 signaling drives the proliferation of CAFs and the biogenesis of oncomiR221/222^hi MVs; these MVs are taken up by ER^pos breast cancer cells and in conjunction with HT leads to the potent suppression of ER signaling promoting Notch3 up-regulation which in turn sustains the self-renewal of CD133^hi CSCs in an ER-independent manner.

Discussion

Significant progress has been made in identifying tumor cell-specific factors (e.g. tumor secretome and gene signatures) that promote cancer cell survival and proliferation in the bone microenvironment [26, 40]. Additionally, cross-talk between bone marrow derived myeloid cells, osteoclasts and osteoblasts with tumor cells has identified many growth factors, chemokines and miRNAs as critical regulators of bone metastasis [41]. A recent manuscript by Luo et. al. has also demonstrated that increased osteoblast-derived IL6 promotes tumor cell seeding and bone metastases in a model of breast cancer cells injected in the arterial circulation [42]. IL6 is a pleiotropic cytokine, which is secreted from several cell types, including CAFs and plays a crucial role in the expansion of cancer stem cells [43, 44] as well as in the proliferation potential of CAFs [45, 46].

The presence of CAFs have been assessed as a poor prognostic feature in breast cancer [47] and an “active stromal signature” in normal fibroblasts exhibits a tumor promoting phenotype [15]. Moreover, PDX tumor tissue was shown to be enriched with host CAFs [48]; but the molecular and pathological relevance of any given CAF^hi phenotype in PDXs remains unclear.

The majority of breast cancers (~70%) are of the estrogen receptor (ER) positive or luminal subtype. Although, the suppression of ER activity with hormonal therapies (HT) has led to improved survival, when these cancers recur they preferentially metastasize to the bone, and eventually acquire resistance to HT and are thus incurable [49, 50].

The up-regulation of distinct pathways, including Her2, PI3K and/or overactive estrogen signaling, are found in metastatic cancer cells escaping tumor dormancy from adjuvant HT. Although targeting these pathways in the metastatic setting leads to clinical responses [51], resistance to anti-Her2/estrogens/PI3K regimens invariably occurs [26, 52]. We and others have reported the clinical relevance of high numbers of CD133^hi cells in “therapy” resistant cancers including lung cancer and hormonal therapy resistant luminal breast cancer metastases [5, 53].

Recently a crucial role for stromal MV in tumor progression has been suggested [17, 18]. However, whether CAF-MVs could promote therapy resistant breast cancer and if these MVs could recapitulate the phenotypic role of CAFs in the tumor microenvironment remain under debate.

Although HT itself suppresses ER signaling, HT alone cannot be a unique trigger of metastatic disease in luminal breast cancer. We propose that communication between CAFs and tumor cells promotes an ER-dependent to an ER-independent switch in metastatic disease. This can occurs via the genetic transfer of microRNAs (221/222), leading to the post-transcriptional downregulation of ER and the expansion of HT resistant tumors. Recently, the over-expression of miR221/222 was demonstrated to promote mammosphere generation in T47D cells. Additionally, a manuscript from Shah et al has proved the presence of miR221 high MVs from CAFs [34]. However, the phenotypic relevance of these CAF-MVs in the context of hormonal therapy resistance disease was not investigated.

In this manuscript we have demonstrated that the expansion of CD133^hi CSCs are functionally associated with the expansion of CAFs in experimental and patient derived HTR disease. We developed the hypothesis that CAF-derived MVs could generate de-novo HTR disease via a miR221-mediated conversion of non-CSCs (ER^hi) into therapy resistant CSCs (ER^lo). We generated PDX models of luminal breast cancer and isolated CAFs from HTR bone metastases (Table S2) and through their analysis uncovered a step-wise process of CAF-MV-mediated HT resistance: the 1) IL6-pStat3 dependent activation of CAFs, 2) the biogenesis of oncomiR 221/222 high MVs, 3) the transfer of these oncomiRs from CAF-MVs to ER^hi cancer cells, 4) the suppression of ER signaling, Notch3 activation and the generation of CD133^hi/ER^lo/Notch^hi CSCs.

Given the pivotal role of IL6 in CAF cell growth and the generation of stromal-tumor niches, we identified combination IL6R-IL6 blockade and HT as a therapeutic intervention to abrogate the establishment of stromal-tumor niches and endocrine resistance in metastatic luminal breast cancer (Fig. 7). Taken together our data suggests a novel pathological role of stromal IL6 in luminal breast cancer: the secretion of oncomiR 221/222 high MVs leading to the evolution of therapy resistant stromal-tumor niches. We characterized the cellular components of this stroma-tumor niche: “CD133^hi CSCs and CAFs” and determined the molecular machinery responsible for the niche generation: autocrine IL6 in CAFs and CAF-derived MV-dependent downregulation of ER in cancer cells.