Fibroblast subtypes regulate responsiveness of …...2016/10/04 · 1 Fibroblast subtypes regulate responsiveness of luminal breast cancer to estrogen. 2 Heather M. Brechbuhl 1,*

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Fibroblast subtypes regulate responsiveness of luminal breast cancer to estrogen. 1 Heather M. Brechbuhl1,*, Jessica Finlay-Schultz2, Tomomi M. Yamamoto1, Austin E. Gillen1, Diana M. 2 Cittelly2, Aik C. Tan1, Sharon Sams2, Manoj M. Pillai3, Anthony D. Elias1, William A. Robinson1, Carol A. 3 Sartorius2 and Peter Kabos1,*. 4 5 Running Title: Fibroblasts regulate estrogen response in ER+ breast cancer. 6 7 1University of Colorado Denver, Department of Medicine, Division of Medical Oncology, Aurora, Colorado 8 USA. 9 2University of Colorado Denver, Department of Pathology, Aurora, Colorado USA. 10 3Section of Hematology, Yale Cancer Center and Yale University School of Medicine, Division of 11 Hematology, New Haven, Connecticut USA. 12 * Address correspondence by email to: Peter Kabos or Heather Brechbuhl 13 University of Colorado Denver University of Colorado Denver 14 12801 E 17th Ave 12801 E 17th Ave 15 L18-8114, M.S. 8117 RC1 South 8401M, M.S. 8117 16 Aurora CO 80045 Aurora CO 80045 17 [email protected] [email protected]. 18 19 Conflict of interest: The authors have declared that no conflict of interest exists. 20 21 22 23 24

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TRANSLATIONAL RELEVANCE 25 Estrogen receptor (ER) positive breast cancer is the most common subtype. Targeting ER is an effective 26 therapy, but development of anti-endocrine resistance remains a major cause of treatment failure. Attempts 27 to uncover and therapeutically target mechanisms of anti-endocrine resistance have focused mainly on 28 tumor intrinsic traits. Here we identify two subtypes of cancer-associated fibroblasts (CAFs), based on their 29 CD146 expression. We further show that CAF subtypes differentially contribute to tumoral ER expression 30 and tamoxifen sensitivity. CD146neg CAFs enforce ER independent growth and mediate tamoxifen 31 resistance by activating receptor tyrosine kinase pathways. Furthermore, the CAF subtypes predict treatment 32 response and patient outcomes. We believe that these findings have clear clinical implications and support a 33 direct role for the tumor microenvironment in modulating response to anti-endocrine therapy. Insight into 34 CAF-tumor interactions and recognition of CAF subtypes in breast cancer could lead to further 35 improvements in personalized care. 36 37 ABSTRACT 38 Purpose: Anti-endocrine therapy remains the most effective treatment for ER+ breast cancer, but 39 development of resistance is a major clinical complication. Effective targeting of mechanisms that control 40 the loss of ER dependency in breast cancer remains elusive. We analyzed breast cancer-associated 41 fibroblasts (CAFs), the largest component of the tumor microenvironment, as a factor contributing to ER 42 expression levels and anti-endocrine resistance. 43 44 Experimental Design: Tissues from ER+ breast cancer patients were analyzed for the presence of CD146 45 positive (CD146pos) and CD146 negative (CD146neg) fibroblasts. ER dependent proliferation and tamoxifen 46 sensitivity were evaluated in ER+ tumor cells co-cultured with CD146pos or CD146neg fibroblasts. RNAseq 47 was used to develop a high confidence gene signature that predicts for disease recurrence in tamoxifen 48 treated patients with ER+ breast cancer. 49




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50 Results: We demonstrate that ER+ breast cancers contain two CAF subtypes defined by CD146 expression. 51 CD146neg CAFs suppress ER expression in ER+ breast cancer cells, decrease tumor cell sensitivity to 52 estrogen, and increase tumor cell resistance to tamoxifen therapy. Conversely, the presence of CD146pos 53 CAFs maintains ER expression in ER+ breast cancer cells and sustains estrogen-dependent proliferation and 54 sensitivity to tamoxifen. Conditioned media from CD146pos CAFs with tamoxifen-resistant breast cancer 55 cells is sufficient to restore tamoxifen sensitivity. Gene expression profiles of patient breast tumors with 56 predominantly CD146neg CAFs correlate with inferior clinical response to tamoxifen and worse patient 57 outcomes. 58 59 Conclusions: Our data suggest that CAF composition contributes to treatment response and patient 60 outcomes in ER+ breast cancer, and should be considered a target for drug development. 61




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INTRODUCTION 62 Estrogen receptor (ER) expression is the primary prognostic and predictive biomarker for patients 63

with breast cancer. Its presence defines the luminal breast cancer subtypes (A and B) and delineates 64 candidacy for anti-endocrine therapy which significantly improves survival outcomes (1, 2). Breast cancers, 65 however, commonly display high heterogeneity of ER expression, where individual cells within a tumor 66 vary in their level of ER expression. The fact that a majority of ER+ tumors contain a range of cells from 67 ER- to ER+, led to development of the Allred score for ER positivity based on overall ER presence and 68 intensity in an individual tumor (3). Clinical presentation of only one percent ER positive tumor cells 69 justifies the use of adjuvant anti-endocrine therapy (3). However, development of anti-endocrine resistance 70 remains a major clinical problem that occurs in 40 percent of patients (4). Recurrent tumors do not typically 71 demonstrate complete loss of ER expression (5), rather they show a combination of both loss of ER 72 expression and loss of ER growth dependency. 73

To date, it remains unclear, how individual tumors maintain a balance of ER positive and negative 74 cells. Intrinsic cellular factors do not fully explain the range of ER expression within a single tumor; 75 therefore, logic suggests the tumor microenvironment (TME) has a role in this phenomenon. In fact, 76 expression patterns of proteins in the stromal/fibroblast component of breast cancer, such as platelet-derived 77 growth factor receptor (PDGFRA and PDGFRB), CXCL1, CXCL14, CD10, and CD36, are prognostic of 78 patient outcomes (6-12). Furthermore, Park et. al. describes a stromal-derived profile consisting of seven 79 stromal expressed proteins that is predictive of breast cancer molecular subtypes (13). Fibroblasts represent 80 the most abundant cell type within the stroma (14) and we reasoned that in luminal breast cancer, the tumor 81 microenvironment (TME) contains functionally and phenotypically distinct fibroblast subtypes that 82 influence tumor cell ER expression and response to anti-endocrine therapy. 83

The purpose of this study was to first examine if subtypes of CAFs exist in luminal breast cancer and 84 to then determine if they have important functional roles in dictating responsiveness of breast cancer cells to 85 estrogen. Intrinsic stromal fibroblasts are known to be heterogeneous in both gene expression and function, 86




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which has made it difficult to define functional subsets. CD146 (MCAM) was reported as a stromal cell 87 surface marker that defined fibroblast subtypes in the hematopoietic stem cell-niche (15). Given that all 88 tissue stromal fibroblasts are mesenchymal in origin, we speculated that breast fibroblasts also contain both 89 CD146pos and CD146neg fibroblasts, and that CD146 expression would define functional subsets of CAFs. 90 Here, we describe a hierarchical organization in tumor-associated stroma, based upon CD146 expression, 91 with implications for therapeutic sensitivity and disease progression. 92 93 MATERIALS AND METHODS 94 Cell culture. 95 The human MCF-7 (p53 wildtype, ER-positive, luminal subtype) breast cancer cell lines were cultured in 96 Modified Eagle’s Medium (MEM) supplemented with 5% fetal calf serum (FCS), nonessential amino acids, 97 L-glutamine, and HEPES buffer at 37 °C with a 5% CO2/95% atmospheric air. Human stromal cell lines 98 HS5 and HS27A and epithelial tumor cells UCD12 and T47D cells were grown in RPMI-1640 99 supplemented with 5% Fetal Calf Serum (FCS), nonessential amino acids, Penicillin (100 U/ml) and 100 streptomycin (100 mg/mL). CD146 CAF subtypes are genetically and functionally akin to HS27a and HS5 101 fibroblasts, however, unlike the HS27a and HS5 fibroblast cell lines, our CAFs have a limited number of 102 passages before they become senescent. Therefore, we used HS27a and HS5 fibroblasts in most of our 103 studies and used our primary CAFs to verify our findings in a select set of studies. Unless otherwise 104 indicated by the designation of CAF, described studies utilized HS27a and HS5 fibroblasts. MCF-7, UCD12 105 and T47D were provided by the lab of Carol Sartorius. The lab of Manoj Pillai provided HS27a and HS5 106 cell lines. All cell lines used in this manuscript were authenticated by STR profile testing in May 2016. For 107 additional methods on cell culture drug line treatments and proliferation assays please refer to the 108 supplemental methods. 109 110 Generation of Cancer Associated Fibroblasts 111




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Normal and tumor tissue samples were collected from patients at the University of Colorado Denver in 112 accordance with an IRB approved protocol. The tissue was collected into ice cold DMEM/F12 until ready 113 for processing. Finely minced tissue was placed in collagenase digestion buffer (DMEM/F12 with 10 mM 114 Hepes, 2% BSA, 5 μg/ml Insulin, 100 ng/ml hydrocortisone, 300 U/ml collagenase IV (1 mg/ml) and 100 115 U/ml hyaluronidase) overnight on a rotator at 37° C. Digestion buffer was used at a volume of 10 ml per 1 g 116 of tissue. Following digestion, any oil layer was gently aspirated off (common to normal breast tissue) and 117 the sample was filtered through a 100 μm mesh into a 50 ml conical tube and centrifuged 1000 x g, at 4° C 118 for 5 minutes to pellet the cells. The cell pellet was re-suspended in 10 ml of PBS and filtered through a 40 119 μm mesh into a new 50 ml conical tube. Differential centrifugation was used to enrich for stromal cell types. 120 A slow speed, 80 x g, 4°C for 4 minutes, was used to pellet epithelial cells. The supernatant was collected 121 for a second centrifugation step, 100 x g, 4° C for 10 minutes. The resulting pellet was enriched for stromal 122 cells and was re-suspended in DMEM/F12, 5% FBS, Insulin, NEAA, and Pen/Strep and cultured in standard 123 cell culture flasks. Non-adherent and dead cells were washed out with PBS at 4 hours, 24 hours and twice 124 weekly media changes until the cultures reached confluence in a 3 0mm dish. Confluent cell cultures were 125 immortalized with E6E7 virus as described previously (16). Following selection of transduced cells with 126 G418, limiting dilution and clonal selection was used to generate CD146pos and CD146neg fibroblast 127 subtypes from each patient sample. CD146 expression was verified by flow cytometry. For flow additional 128 methods on flow cytometry, please refer to the supplemental methods. 129 130 Animal Experiments 131 All animal experiments were conducted in an AAALAC accredited facility at the University of Colorado 132 Denver under an IACUC approved protocol. MCF-7 tumors labeled with ZS-green were established by 133 injecting 1 x10^6 cells into the mammary fat pad of NOD scid gamma (NSG) female mice. HS27a or HS5 134 cells were mixed with the tumor cells at a 1:1 ratio (N = 3-6 mice per stroma subtype). Tumors were 135 allowed to grow for at least 5 weeks prior to removal. All tumors received continuous estrogen 136




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supplementation throughout the study, as previously described (17). For the tamoxifen study, MCF-7 cells 137 mixed with HS5 or HS27a cells were randomized to either the right or left mammary fat pad of each mouse. 138 The tumors were established for 3 weeks and then the mice were randomized into groups receiving peanut 139 oil or 80 mg/kg 4-hydroxy-tamoxifen. Treatments were given 3 times per week by intraperitoneal injection 140 for 8 weeks. 141 142 Human Samples 143 Human samples were collected under an approved COMIRB protocol from a phase II clinical trial 144 performed at University of Colorado consisting of 80 patients with stage II and III newly diagnosed breast 145 cancer (both ER+ and ER-). The trial was designed to assess cellular heterogeneity in patients receiving 146 neo-adjuvant therapy. Available samples from patients with ER+ disease were used. A board certified 147 pathologist reviewed histology. The tamoxifen outcome data comes from the following GEO record number 148 GSE6532 (18-20). For additional gene expression and immunocytochemistry methods please refer to the 149 supplemental methods. 150 151 Statistical Analysis 152 Statistical analysis was completed using R-package software for the gene expression data sets and with 153 GraphPad Prism 6 analytical software (La Jolla, CA) for all other experiments. For single comparisons we 154 used unpaired two-tailed t-tests with assumptions of parametric distribution Gaussian distribution and equal 155 standard deviations. For multiple comparisons we used ordinary one-way ANOVA analysis with Tukey 156 multiple comparisons tests. Significance was set at p < 0.05. All cell culture experiments consisted of at 157 least N = 4 or more and were repeated at least once using the same ER+ breast cancer cell type, different 158 ER+ subtype. Our in vivo experiment consisted of N = 3-6 animals per stromal subtype. Outliers were 159 considered to be 2 standard deviations from the mean and data are presented as mean +/- the standard error 160 of the mean. 161




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162 RESULTS 163 CD146 expression identifies subtypes of normal and cancer associated stroma in breast tissue 164

To determine the prevalence of CD146pos/CD146neg cells in breast cancer-associated stroma, we used 165 dual immunohistochemistry (IHC) staining for CD146 and for the strongly associated stromal marker 166 vimentin. The stromal component of ER+ breast cancer-associated patient tissues contains mixed 167 populations of CD146pos and CD146neg cells (Fig. 1A). In fact, the staining revealed striking differences in 168 the intensity and frequency of CD146pos stroma between patient samples. We quantified these differences by 169 dual immunofluorescence (IF) staining for CD146 and vimentin in a cohort of 17 patient samples previously 170 scored by a board certified pathologist for ER expression. 171

In the clinical practice of pathology, cellular morphology is the standard in identifying tumor cells 172 and is the basis of pathologic diagnosis. Stromal cells are long and spindly, and bland in appearance with 173 small nuclei and fine chromatin. In contrast, breast tumor cells are easily identified as large pleomorphic 174 cells with anisonucleosis, characterized by coarse chromatin and prominent nuclei. We stained our patient 175 cohort for tumor cell marker cytokeratin 8/18 (CK8/18) and stromal marker vimentin to verify that tumor 176 cells were maintaining a distinct epithelial phenotype. All of our patient samples contained only CK8/18 177 positive, vimentin negative tumor cells and the stromal compartment was characterized by spindle shaped, 178 vimentin positive, CK8/18 negative cells (SFig. 1). Based on this combination of morphologic 179 characteristics and supporting immunohistochemical pattern, we determined that it was possible to 180 distinguish tumor cells from stromal cells with high confidence. 181

Based on our cohort size, we used Allred 6 as our cutoff point for determining if there was a 182 potential relationship between CD146 expression in the stromal component of breast cancer and the Allred 183 score for ER expression. Ten samples had Allred scores greater than 6 (high ER) and seven had scores less 184 than or equal to 6 (lower ER). We quantified our IF staining by determining the percentage of 185 vimentin+/CD146pos and vimentin+/CD146neg stroma. For this analysis, we manually excluded tumor 186




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epithelial cells (vimentin negative, CK8/18 positive) and obvious vessel structures (vimentin positive). 187 Patients who had an Allred score greater than 6 had a mean of 68% CD146pos stroma, whereas Allred scores 188 equal to or less than 6 had a mean of 18% CD146pos stroma (Fig. 1B). The range of vimentin+/CD146pos 189 stroma cells in our 17 samples was 2-87% (Fig. 1C-D). These data imply that threshold expression for 190 breast tumor ER is correlated to the CD146pos fibroblast subtype. 191

We next tested normal breast tissue and cancer-associated breast tissue for the presence of CD146pos 192 and CD146neg stromal subtypes. Primary human cancer associated and normal breast stroma cells were 193 isolated from 11 ER+ breast cancer patient samples, enriched for fibroblasts and established as primary cell 194 lines using immortalization and clonal expansion. We determined stromal cell enrichment by flow analysis 195 and found that our stromal isolation method resulted in a highly enriched fibroblast fraction (95% VIMpos+, 196 92% FSP1+), and was depleted of epithelial (3.9% CD136pos) and endothelial (CD31pos) cells, with minimal 197 contribution of possible pericytes (0% CD31pos/NG2pos, 4.4% FSP1neg/NG2pos) (SFig. 2A-B, SFig. 3A). We 198 further validated that our cells expressed vimentin by gene expression and immunofluorescence staining 199 (SFig. 3B-D). Our method utilized several wash steps to deplete the sample of poorly adherent 200 hematopoietic cells. Finally, we flow sorted our clonal cell lines for expression of CD146, and we identified 201 two subtypes of normal fibroblasts (NBFs) and cancer associated fibroblasts (CAFs) in both normal breast 202 and breast cancer associated patient tissues (Fig. 1E). Taken together, these data demonstrate that the breast 203 TME is composed of at least two CAF subtypes, which can be identified according to CD146 expression 204 and are common to both normal breast stroma and breast cancer-associated stroma. Patients with lower ER 205 expression based on Allred scores (≤ 6) have significantly decreased expression of CD146pos CAF compared 206 to patients with high ER (Allred scores > 6). 207

208 CD146pos CAFs are functionally and phenotypically akin to CD146pos fibroblasts found in normal bone 209 marrow 210




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We next used the cobble stone area assay (CSA) to determine if the CAF subtypes we derived from 211 human ER+ breast cancer tissue had similar functionality to the CD146pos and CD146neg bone marrow 212 derived HS27a and HS5 fibroblasts. A description of this assay can be found in the supplemental methods. 213 MNC/HS27a co-cultures had 5.8-fold more CSA than MNC/HS5 co-cultures (SFig. 4A-B). Similar to the 214 results with normal fibroblasts, MNCs co-cultured with CD146pos compared to CD146neg CAF cells had 215 significantly more CSA (3-fold greater) (SFig. 4A-B). These results show that our CAFs promote equivalent 216 MNC behavior as normal bone marrow-derived fibroblasts when stratified by CD146 expression. 217

To determine if CAF cell isolates were phenotypically akin to HS27a and HS5 normal fibroblasts we 218 used gene expression signatures and hierarchical clustering analysis. The gene expression signatures of 219 CD146neg CAFs demonstrated significant similarity by clustering in the same family with normal HS5 220 fibroblasts (SFig. 4C). Likewise, CD146pos CAFs clustered with HS27 fibroblasts (SFig. 4C). Our CAF cell 221 lines have similar expression levels for genes associated with activated fibroblasts, including pro-collagen 222 type 1 alpha, smooth muscle actin and fibroblast activation protein alpha (STable 1). These data support the 223 assertion that our human cancer derived stromal subtypes have a fibroblast gene signature that is similar to 224 the human bone marrow-derived HS27a and HS5 normal fibroblasts. 225 226 CAF subtypes differentially influence ER expression in breast cancer cells 227

To pursue a functional role for CAFs in distinguishing tumor characteristics, we compared the 228 phenotype and growth of ER+ breast cancer cells (BCCs) grown in conjunction with the two fibroblast 229 subtypes. ER+ MCF-7 BCCs were co-cultured with CD146pos and CD146neg fibroblasts in estrogen depleted 230 media for 5 days and stained for ER using immunofluorescence (Fig. 2A). Cytokeratin 18 (CK18) was used 231 to positively identify tumor cells. ER expression was significantly higher in MCF-7 cells when they were 232 co-cultured with CD146pos fibroblasts (74% vs. 37% ER+ cells, Fig. 2B). Similar results were observed 233 when we co-cultured BCCs with our primary CAF subtypes (SFig. 5A). 234




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To assess if CAF subtypes would similarly affect ER in tumors, we grew ER+ MCF-7 cells co-235 implanted with the fibroblast subtypes as xenografts. A 1:1 mixture of MCF-7 cells was injected with 236 CD146pos or CD146neg fibroblasts. Tumors were established and allowed to grow with estrogen 237 supplementation for 4 weeks prior to collection. Tumor sizes at 4 weeks were not significantly different 238 between fibroblast subtypes. We co-stained the tumors with ER plus CK8/18 to identify tumor cells. MCF-7 239 xenograft tumors mixed with CD146pos fibroblasts expressed higher levels of ER (Fig. 2C) compared to 240 MCF-7/CD146neg mixed tumors (38% vs. 24%, Fig. 2D). These data show that CD146neg fibroblasts drive 241 decreased ER expression in BCCs and suggest one possible mechanism for stroma-induced development of 242 anti-endocrine resistance. 243 244 ER+ breast cancer cells use ER growth dependent pathways when stimulated by CD146pos fibroblasts. 245

To determine if CD146 positive or negative fibroblasts influence estrogen dependent proliferation in 246 ER+ breast cancer cells, we used co-culture and conditioned media experiments. We analyzed proliferation 247 rates using a total protein assay or live cell imaging using Incucyte Zoom. MCF-7 cell cultures were grown 248 in estrogen-starved conditions for 72 hours prior to treatment with conditioned media (CM). RT-PCR 249 analysis verified ER expression, in absence of estrogen, was decreased simply by treating MCF-7 cells with 250 conditioned media from CD146neg fibroblasts (Fig. 3A). In vehicle treated samples after 72 hours, 251 conditioned media from both CD146pos and CD146neg fibroblasts increased MCF-7 BCC proliferation 5-fold 252 (Fig 3B). Treatment with estrogen (17β-estradiol, E2) significantly increased proliferation of BCCs grown 253 in unconditioned or CD146pos CM (2.9-fold and 1.3-fold respectively) (Fig. 3B). Estrogen did not alter 254 proliferation of BCCs treated with CD146neg CM (Fig. 3B). Tamoxifen significantly inhibited estrogen-255 induced proliferation of BCCs in unconditioned or CD146pos fibroblasts CM by over 7-fold (Fig. 3B). 256 However, proliferation of BCCs with CD146neg fibroblast CM was not significantly changed with tamoxifen 257 (Fig. 3B). Similar results were obtained using co-cultures of MCF-7 with fibroblasts instead of CM, and 258 with two other ER+ BCCs, UCD12 (SFig 5B) and T47D (SFig 5C-D). These data demonstrate that ER+ 259




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BCCs influenced by CD146pos fibroblasts remain estrogen responsive and anti-estrogen sensitive; however, 260 influence from CD146neg fibroblasts renders ER+ BCCs estrogen unresponsive and tamoxifen insensitive. 261

Since our data indicated that CD146 fibroblast subtypes influence BCC response to tamoxifen, we 262 next tested the response of tamoxifen resistant MCF-7/TAMR-1 (TAMR-1) cells to tamoxifen when grown 263 under the influence of CD146pos or CD146neg fibroblasts. TAMR-1 cells are a tamoxifen resistant derivative 264 of MCF-7 BCCs (21). We cultured TAMR-1 cells in unconditioned or conditioned media from CD146pos or 265 CD146neg fibroblasts and treated with 10 nM E2 alone or with 100 nM 4-hydroxy-tamoxifen (Tamoxifen). 266 Tamoxifen treatment had no effect on proliferation of BCCs in unconditioned or CD146neg fibroblasts CM 267 (Fig 3C). However, TAMR-1 cells cultured in CD146pos fibroblast CM had significantly decreased 268 proliferation with tamoxifen (29% compared to control) (Fig 3C), suggesting TAMR-1 cells gained 269 sensitivity to tamoxifen. 270

To substantiate our in vitro results we established MCF7 tumors mixed with CD146 positive or 271 negative fibroblasts in the mammary fat pad of NSG mice and then treated the mice with tamoxifen. 272 Animals were given a 1 mg subcutaneous estrogen pellet that remained in place throughout the experiment. 273 Tumors were established for 3 weeks and then randomized into groups receiving TAM or vehicle (average 274 tumor size = 54 mm3) (Fig. 4A). Two weeks post tamoxifen treatment, MCF-7/CD146neg tumors were 275 significantly larger than MCF-7/CD146pos fibroblasts (p < 0.001; 326 ± 67 mm3 versus 73 ± 39 mm3 276 respectively). Vehicle treated MCF-7/CD146neg fibroblasts were significantly larger than MCF-7/CD146pos 277 tumors by 7 weeks (p < 0.001; 151 ± 84 mm3 versus 151 ± 84 mm3 respectively). MCF-7/CD146neg mixed 278 tumors did not respond to tamoxifen treatment (Fig. 4A-B). However, tamoxifen treated MCF-7/CD146pos 279 tumors were significantly smaller than vehicle treated MCF-7/CD146pos tumors (p < 0.03; 0.4 mm3 versus 280 0.17 mm3) (Fig. 4A-B). Although MCF-7/CD146pos tumors were significantly smaller than MCF-281 7/CD146neg tumors, all of the tumors contained dense regions of CK8/18 positive tumors with vimentin 282 positive stroma present throughout (SFig. 6A). These data support the hypothesis that fibroblast subtypes 283 can influence tamoxifen sensitivity of ER+ BCCs. 284




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285 ER+ breast cancer cells activate receptor tyrosine kinase pathways when stimulated by CD146neg 286 fibroblasts. 287

Evidence suggests that early development of endocrine resistance involves cross-talk between ER, 288 EGFR/HER2 and IGF1R pathways (22). Therefore, we next determined if growth of ER+ BCCs influenced 289 by CD146neg fibroblasts were sensitive to EGFR inhibition. We co-cultured GFP labeled MCF-7 BCCs with 290 CD146pos or CD146neg fibroblasts in serum reduced media (2.5% FBS) for 48 hours and then treated the 291 cells with the EGFR specific inhibitor gefitinib (10 μM). Proliferation was unchanged for MCF-7 cells co-292 cultured with CD146pos fibroblasts and treated with gefitinib, whereas it was reduced by 60% in gefitinib 293 treated BCCs co-cultured with CD146neg fibroblasts (Fig 5A). 294

To further examine this effect, we performed a dose response experiment using CM from MCF-7 295 cells or CM from fibroblasts and treatment with 0-25 μM gefitinib (EGFR inhibitor) or 0-50 μg/mL 296 trastuzumab (HER2 specific inhibitor). Proliferation was measured with a total protein assay. At the 297 maximum concentration of gefitinib (2.5 μM), MCF-7 cells had a growth reduction of 17%, 25% and 46% 298 when grown in CM from MCF-7 cells, CD146pos fibroblasts or CD146neg fibroblasts respectively (Fig. 5B). 299 Trastuzumab treatment caused MCF-7 growth reduction between 3-12% in MCF-7 CM, 15% in CD146pos 300 CM and 24-31% in CD146neg CM (Fig. 5C). Western blot analysis of MCF7 cells treated with CM from 301 MCF-7, CD146pos or CD146neg fibroblasts demonstrates that MCF7 cells grown in all conditions express a 302 small amount of EGFR (SFig. 6B) and that only CM from the fibroblasts result in detectable phosphorylated 303 EGFR protein (Fig. 5D). These data suggest that CD146pos fibroblasts stimulate EGFR and CD146neg 304 fibroblasts stimulate both EGFR and HER2 in ER+ BCCs. 305

To further examine which tyrosine kinase pathways might be active in CM cultures, we used a 306 human phospho-RTK array panel. As previously demonstrated by western blot, MCF-7 cells plus CD146pos 307 or CD146neg CM were positive for phospho-EGFR, but not phospho-ERBB2/HER2 (Fig. 5D). Interestingly, 308 the cultures grown with CD146neg fibroblasts were also positive for phosphorylated IGF1R, which has been 309




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implicated in promoting tamoxifen resistance (23-25). Our data suggest that BCCs influenced by CD146neg 310 fibroblasts escape estrogen dependent proliferation and exhibit tamoxifen resistance through activation of 311 EGFR, HER2 and IGF1R. 312 313 CD146 expression in fibroblasts correlates with patient outcomes 314

Previous analysis by Finak et al (13) demonstrated that the gene signature from the total stromal 315 component of breast cancer patients accurately identifies normal versus cancer tissue and predicts patient 316 outcomes (13). We compared the gene signatures of HS27a, HS5, CD146pos CAFs and CD146neg CAFs to 317 the published gene expression data of normal and breast cancer-associated stroma. We verified considerable 318 overlap of expressed annotated genes in all four cell types (HS27a, HS5, CD146pos and CD146neg) with the 319 gene set used to identify breast stromal origin and to generate predictions of breast cancer patient outcomes 320 (128 of the 163 stromal genes identified by Finak et. al. (13). We then used the 128 identified stromal genes 321 in our data set to determine if CD146pos or CD146neg fibroblasts clustered with normal breast stroma or 322 breast cancer-associated stroma in the published data set. 323 Because HS5 cells and our CD146neg CAFs cluster in a single family, and HS27a cells cluster with our 324 CD146pos CAFs, we pooled the genes from each subtype together for our comparison. The gene expression 325 profile from HS5 (CD146neg) fibroblasts and our breast cancer patient-derived CD146neg CAFs aligned with 326 the Finak et. al. gene profile pattern for breast cancer associated stroma, whereas a CD146pos gene profile 327 from HS27a or our CD146pos CAFs aligned with normal breast associated stroma (Fig. 6A). Furthermore, 328 CD146neg CAFs predicted poor/mixed clinical outcomes for patients with ER+ breast cancer compared to 329 CD146pos CAFs, which were aligned with better clinical outcomes (Fig. 6B). These data demonstrate that 330 CD146 expression is a distinguishing characteristic of stromal fibroblasts in the normal and diseased breast 331 that mimics the fibroblast hierarchy present in the hematopoietic system, demarcates normal vs. tumor 332 associated stroma, and is predictive of disease outcomes. 333 334




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CAF induced gene expression signature predicts breast cancer recurrence in patients treated with 335 tamoxifen. 336

We used RNA-seq to determine gene expression changes that occur in BCCs with fibroblast 337 subtypes we co-cultured MCF-7 cells with CD146pos and CD146neg fibroblasts. Analysis revealed that MCF-338 7 cells co-cultured with CD146neg fibroblasts had increased expression of transcripts from 21 genes 339 identified in the literature as up regulated in tamoxifen resistance (26-32) (STable 2). Conversely, MCF-7 340 cells co-cultured with CD146pos fibroblasts had increased expression of 15 genes identified in the literature 341 as down-regulated in tamoxifen resistance (STable 3). From these lists we identified a set of 9 genes directly 342 influenced by fibroblast co-cultures (5 up-regulated genes in MCF-7 cells co-cultured with CD146neg 343 fibroblasts and 4 genes up-regulated in MCF-7 cells co-cultured with CD146pos fibroblasts) that produced a 344 high confidence gene signature that reliably predicts recurrence-free survival in patients treated with 345 tamoxifen (training set 181 patients p = 3x10-5 and validation set 87 patients p = 0.00147) (Fig. 6C). 346 Importantly, the 9-gene set was not predictive of recurrence-free survival in a set of 125 patients that were 347 not treated with tamoxifen, suggesting it is linked to tamoxifen resistance and not a mere association with 348 poor patient prognosis (SFig. 6C). Furthermore, a signature composed of all 36 genes is not predictive of 349 recurrence free survival in the tamoxifen treated groups (SFig. 6D). Because CD146 correlates with Allred 350 score, we asked if our gene set was predictive of recurrence-free survival simply due to the fact that ER 351 expression correlates with better patient outcomes in patients treated with tamoxifen. Consistent with this 352 idea, we split the same data cohorts, along the median, into high and low ER expression and assessed 353 recurrence-free survival. If our CD146 based gene signature, and, by extension, the stromal composition, 354 were only a surrogate for ER expression, then a significant survival advantage would be expected in patients 355 with higher ER expression. However, high ER gene expression was not predictive of recurrence-free 356 survival in tamoxifen treated or untreated patients (SFig. 6E-G), and we conclude that the stromal 357 composition significantly influences tamoxifen response in a novel way that does not resolve simply on ER 358 expression. These data show that influence from CD146pos fibroblasts is predictive of improved recurrence 359




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free survival after tamoxifen treatment, whereas influence from CD146neg fibroblasts produces a tumor gene 360 signature predictive of poorer prognosis in tamoxifen treated breast cancer patients. 361 362 DISCUSSION 363

This study demonstrates how functionally distinct subtypes of cancer associated fibroblasts can 364 directly affect ER expression and growth dependency in luminal breast cancers. We identified two subtypes 365 of CAFs that contribute to tumor ER heterogeneity influence tumor response to anti-endocrine therapy. 366 Development of tamoxifen resistant cell lines, such as TAMR-1 cells, requires escalating doses and long-367 term agent exposure (up to one year) (33, 34); strikingly, in this study we show that a similar phenotype can 368 be achieved by a short co-culture (five days) with CD146neg CAFs. Equally important, we can reverse the 369 tamoxifen resistant phenotype by a short co-culture with CD146pos CAFs. These data strongly support the 370 need to consider CAFs in order to further elucidate mechanisms of anti-endocrine resistance. As proof of 371 principle, we were able to identify an epithelial gene signature enforced by CAF subtypes that accurately 372 predicted recurrence-free survival in tamoxifen treated patients. 373

Development of anti-endocrine resistance has been linked to activation of signaling pathways that 374 converge on PI3K/AKT/mTOR signaling, including EGFR, HER2 and IGF1R(24, 35, 36). Although there is 375 reasonable controversy regarding the role of IGF1R signaling in conditions of sustained anti-endocrine 376 resistance (37), several studies demonstrate IGF1R as a modifying factor (23-25, 35). Our data focuses on 377 short-term cultures and demonstrates phosphorylation of EGFR and IGF1R as well as decreased 378 proliferation in response to the HER2 inhibitor trastuzumab in ER+ BCCs influenced by CD146neg 379 fibroblasts. Discoveries of RTK activation in tamoxifen resistance prompted several clinical trials utilizing 380 single receptor tyrosine kinase inhibitors (RTKIs) in combination with anti-endocrine therapy as a way to 381 delay endocrine resistance (38-42). Unfortunately, the variable outcome in many of these trials led to 382 conclusions that targeting RTKs was largely ineffective for ER+ breast cancer (42, 43). 383




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Our data demonstrates that fibroblast subtypes, present in the TME, can dictate which RTKs are 384 active in ER+ BCCs. For example, both CD146pos and CD146neg fibroblasts promote EGFR phosphorylation 385 in ER+ BCCs, but CD146neg fibroblasts also promote IGF1R phosphorylation and sensitize ER+ BCCs to 386 the HER2 inhibitor trastuzumab. These data would suggest that a single EGFR RTKI combined with anti-387 endocrine therapy might work for tumors with a high percentage of CD146pos fibroblasts in the TME, but a 388 use of a broad RTKI that can target EGFR, HER2 and/or IGF1R simultaneously may be a better choice for 389 tumors expressing mostly CD146neg fibroblasts in the TME. In fact, recent efforts suggest that combining 390 multiple RTKIs, or using a broad spectrum RTK inhibition approach with AKT/mTOR inhibitors in 391 combination with anti-endocrine therapy is a more effective approach (44-50). Our data suggest that the 392 examination of TME and fibroblast subtypes, may lead to improvements in personalization of care. 393 Specifically in luminal breast cancers, ER serves as both a prognostic and predictive marker in 394 patients and forms the basis of clinical decision-making (1, 2). However, efficacy of treatment is limited by 395 development of anti-endocrine resistance that leads to treatment failure and disease recurrence and 396 progression. Although ER expression is correlated with better patient outcomes, it does not predict for 397 which patients will have recurrent disease after tamoxifen treatment. Here, we have identified an epithelial 398 gene signature based on stromal influence that is predictive of recurrent disease after tamoxifen treatment. 399 This signature could be used to guide aggressive treatment from the time of diagnosis in patients with the 400 CD146neg signature. Minimally, our data presents a new paradigm for considering CAFs as a heterogeneous 401 population that has significant impact on endocrine response. However, whether the ratio of 402 CD146pos/CD146neg cells is host dependent remains to be answered. Prior studies have shown recruitment of 403 stromal components to tumors and it is therefore conceivable that some tumors are more apt at recruiting 404 distant stroma into the microenvironment than others (51-53). Furthermore, it is unclear if recruited CAFs 405 have a given CD146 phenotype/signature prior to arriving or if this is a programmable state. 406

In summary, we have shown that CAFs do not represent a homogeneous cell population, but contain 407 at least two distinct cellular subtypes that differentially influence breast cancer cells with respect to their 408




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molecular characteristics, phenotypic behavior in disease progression, and markers of therapeutic response. 409 Our data support the hypothesis that tumors hijack normal stromal components of the tissue 410 microenvironment and use it to their advantage. The generation of CAF cell lines and their study with a 411 broader range of metastatic transplant models will also provide a model system to functionally define the 412 breast cancer microenvironment. We believe that studies of CAF biology and improved targeting of their 413 interactions with tumor cells will enhance our ability to deliver personalized therapy. 414 415 GRANT SUPPORT 416 This work was supported in part by National Institutes of Health (NCI CA164048, CA205044 to P. Kabos 417 and HL104070 to M. Pillai and CA140985 to C. Sartorius), Grohne Stem Cell Fund (to P. Kabos) and the 418 Cancer League of Colorado (to P. Kabos). Additional support in part by the University of Colorado Cancer 419 Center’s Flow Cytometry Shared Resource funded by NCI grant P30CA046934. 420 421 ACKNOWLEDGEMENTS 422 We would like to acknowledge Veronica Wessells for assistance with tissue processing, the animal core 423 facility, and the Biochemistry and Molecular Genetics high throughput-sequencing core (School of 424 Medicine) at the University of Colorado, Denver. 425




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References 426 427 1. Lozowski MS, Mishriki Y, Chao S, Grimson R, Pai P, Harris MA, et al. Estrogen receptor 428 determination in fine needle aspirates of the breast. Correlation with histologic grade and comparison 429 with biochemical analysis. Acta cytologica. 1987;31:557-62. 430 2. Puhalla S, Bhattacharya S, Davidson NE. Hormonal therapy in breast cancer: a model disease 431 for the personalization of cancer care. Molecular oncology. 2012;6:222-36. 432 3. Harvey JM, Clark GM, Osborne CK, Allred DC. Estrogen receptor status by 433 immunohistochemistry is superior to the ligand-binding assay for predicting response to adjuvant 434 endocrine therapy in breast cancer. Journal of clinical oncology : official journal of the American 435 Society of Clinical Oncology. 1999;17:1474-81. 436 4. Ring A, Dowsett M. Mechanisms of tamoxifen resistance. Endocr Relat Cancer. 2004;11:643-58. 437 5. Kuukasjarvi T, Kononen J, Helin H, Holli K, Isola J. Loss of estrogen receptor in recurrent breast 438 cancer is associated with poor response to endocrine therapy. Journal of clinical oncology : official 439 journal of the American Society of Clinical Oncology. 1996;14:2584-9. 440 6. DeFilippis RA, Chang H, Dumont N, Rabban JT, Chen YY, Fontenay GV, et al. CD36 repression 441 activates a multicellular stromal program shared by high mammographic density and tumor tissues. 442 Cancer Discov. 2012;2:826-39. 443 7. Frings O, Augsten M, Tobin NP, Carlson J, Paulsson J, Pena C, et al. Prognostic significance in 444 breast cancer of a gene signature capturing stromal PDGF signaling. The American journal of 445 pathology. 2013;182:2037-47. 446 8. Makretsov NA, Hayes M, Carter BA, Dabiri S, Gilks CB, Huntsman DG. Stromal CD10 expression 447 in invasive breast carcinoma correlates with poor prognosis, estrogen receptor negativity, and high 448 grade. Mod Pathol. 2007;20:84-9. 449 9. Paulsson J, Sjoblom T, Micke P, Ponten F, Landberg G, Heldin CH, et al. Prognostic significance 450 of stromal platelet-derived growth factor beta-receptor expression in human breast cancer. The 451 American journal of pathology. 2009;175:334-41. 452 10. Sjoberg E, Augsten M, Bergh J, Jirstrom K, Ostman A. Expression of the chemokine CXCL14 in 453 the tumour stroma is an independent marker of survival in breast cancer. Br J Cancer. 454 2016;114:1117-24. 455 11. Vo TN, Mekata E, Umeda T, Abe H, Kawai Y, Mori T, et al. Prognostic impact of CD10 expression 456 in clinical outcome of invasive breast carcinoma. Breast Cancer. 2015;22:117-28. 457 12. Zou A, Lambert D, Yeh H, Yasukawa K, Behbod F, Fan F, et al. Elevated CXCL1 expression in 458 breast cancer stroma predicts poor prognosis and is inversely associated with expression of TGF-beta 459 signaling proteins. BMC Cancer. 2014;14:781. 460 13. Finak G, Bertos N, Pepin F, Sadekova S, Souleimanova M, Zhao H, et al. Stromal gene expression 461 predicts clinical outcome in breast cancer. Nature medicine. 2008;14:518-27. 462 14. Pietras K, Ostman A. Hallmarks of cancer: interactions with the tumor stroma. Experimental 463 cell research. 2010;316:1324-31. 464 15. Iwata M, Sandstrom RS, Delrow JJ, Stamatoyannopoulos JA, Torok-Storb B. Functionally and 465 phenotypically distinct subpopulations of marrow stromal cells are fibroblast in origin and induce 466 different fates in peripheral blood monocytes. Stem cells and development. 2014;23:729-40. 467 16. Roecklein BA, Torok-Storb B. Functionally distinct human marrow stromal cell lines 468 immortalized by transduction with the human papilloma virus E6/E7 genes. Blood. 1995;85:997-469 1005. 470 17. Kabos P, Finlay-Schultz J, Li C, Kline E, Finlayson C, Wisell J, et al. Patient-derived luminal 471 breast cancer xenografts retain hormone receptor heterogeneity and help define unique estrogen-472 dependent gene signatures. Breast cancer research and treatment. 2012;135:415-32. 473 Research.

on December 21, 2020. © 2016 American Association for Cancerclincancerres.aacrjournals.org Downloaded from



20

18. Loi S, Haibe-Kains B, Desmedt C, Wirapati P, Lallemand F, Tutt AM, et al. Predicting prognosis 474 using molecular profiling in estrogen receptor-positive breast cancer treated with tamoxifen. BMC 475 Genomics. 2008;9:239. 476 19. Loi S, Haibe-Kains B, Majjaj S, Lallemand F, Durbecq V, Larsimont D, et al. PIK3CA mutations 477 associated with gene signature of low mTORC1 signaling and better outcomes in estrogen receptor-478 positive breast cancer. Proceedings of the National Academy of Sciences of the United States of 479 America. 2010;107:10208-13. 480 20. Mishiro S, Hoshi Y, Takeda K, Yoshikawa A, Gotanda T, Takahashi K, et al. Non-A, non-B 481 hepatitis specific antibodies directed at host-derived epitope: implication for an autoimmune process. 482 Lancet. 1990;336:1400-3. 483 21. Cittelly DM, Das PM, Spoelstra NS, Edgerton SM, Richer JK, Thor AD, et al. Downregulation of 484 miR-342 is associated with tamoxifen resistant breast tumors. Mol Cancer. 2010;9:317. 485 22. Nicholson RI, Hutcheson IR, Harper ME, Knowlden JM, Barrow D, McClelland RA, et al. 486 Modulation of epidermal growth factor receptor in endocrine-resistant, oestrogen receptor-positive 487 breast cancer. Endocr Relat Cancer. 2001;8:175-82. 488 23. Fan P, Agboke FA, Cunliffe HE, Ramos P, Jordan VC. A molecular model for the mechanism of 489 acquired tamoxifen resistance in breast cancer. Eur J Cancer. 2014;50:2866-76. 490 24. Fox EM, Kuba MG, Miller TW, Davies BR, Arteaga CL. Autocrine IGF-I/insulin receptor axis 491 compensates for inhibition of AKT in ER-positive breast cancer cells with resistance to estrogen 492 deprivation. Breast cancer research : BCR. 2013;15:R55. 493 25. Fox EM, Miller TW, Balko JM, Kuba MG, Sanchez V, Smith RA, et al. A kinome-wide screen 494 identifies the insulin/IGF-I receptor pathway as a mechanism of escape from hormone dependence in 495 breast cancer. Cancer research. 2011;71:6773-84. 496 26. Huber-Keener KJ, Liu X, Wang Z, Wang Y, Freeman W, Wu S, et al. Differential gene expression 497 in tamoxifen-resistant breast cancer cells revealed by a new analytical model of RNA-Seq data. PloS 498 one. 2012;7:e41333. 499 27. De Placido S, De Laurentiis M, Carlomagno C, Gallo C, Perrone F, Pepe S, et al. Twenty-year 500 results of the Naples GUN randomized trial: predictive factors of adjuvant tamoxifen efficacy in early 501 breast cancer. Clinical cancer research : an official journal of the American Association for Cancer 502 Research. 2003;9:1039-46. 503 28. Jansen MP, Foekens JA, van Staveren IL, Dirkzwager-Kiel MM, Ritstier K, Look MP, et al. 504 Molecular classification of tamoxifen-resistant breast carcinomas by gene expression profiling. 505 Journal of clinical oncology : official journal of the American Society of Clinical Oncology. 506 2005;23:732-40. 507 29. Kalyuga M, Gallego-Ortega D, Lee HJ, Roden DL, Cowley MJ, Caldon CE, et al. ELF5 suppresses 508 estrogen sensitivity and underpins the acquisition of antiestrogen resistance in luminal breast cancer. 509 PLoS Biol. 2012;10:e1001461. 510 30. Mohseni M, Cidado J, Croessmann S, Cravero K, Cimino-Mathews A, Wong HY, et al. MACROD2 511 overexpression mediates estrogen independent growth and tamoxifen resistance in breast cancers. 512 Proceedings of the National Academy of Sciences of the United States of America. 2014;111:17606-11. 513 31. Oosterkamp HM, Hijmans EM, Brummelkamp TR, Canisius S, Wessels LF, Zwart W, et al. USP9X 514 downregulation renders breast cancer cells resistant to tamoxifen. Cancer research. 2014;74:3810-20. 515 32. Elias D, Vever H, Laenkholm AV, Gjerstorff MF, Yde CW, Lykkesfeldt AE, et al. Gene expression 516 profiling identifies FYN as an important molecule in tamoxifen resistance and a predictor of early 517 recurrence in patients treated with endocrine therapy. Oncogene. 2015;34:1919-27. 518 33. Kilker RL, Hartl MW, Rutherford TM, Planas-Silva MD. Cyclin D1 expression is dependent on 519 estrogen receptor function in tamoxifen-resistant breast cancer cells. J Steroid Biochem Mol Biol. 520 2004;92:63-71. 521 Research.

on December 21, 2020. © 2016 American Association for Cancerclincancerres.aacrjournals.org Downloaded from



21

34. Madsen MW, Reiter BE, Lykkesfeldt AE. Differential expression of estrogen receptor mRNA 522 splice variants in the tamoxifen resistant human breast cancer cell line, MCF-7/TAMR-1 compared to 523 the parental MCF-7 cell line. Mol Cell Endocrinol. 1995;109:197-207. 524 35. Ojo D, Wei F, Liu Y, Wang E, Zhang H, Lin X, et al. Factors Promoting Tamoxifen Resistance in 525 Breast Cancer via Stimulating Breast Cancer Stem Cell Expansion. Curr Med Chem. 2015;22:2360-74. 526 36. Morrison G, Fu X, Shea M, Nanda S, Giuliano M, Wang T, et al. Therapeutic potential of the dual 527 EGFR/HER2 inhibitor AZD8931 in circumventing endocrine resistance. Breast cancer research and 528 treatment. 2014;144:263-72. 529 37. Fagan DH, Uselman RR, Sachdev D, Yee D. Acquired resistance to tamoxifen is associated with 530 loss of the type I insulin-like growth factor receptor: implications for breast cancer treatment. Cancer 531 research. 2012;72:3372-80. 532 38. Gee JM, Harper ME, Hutcheson IR, Madden TA, Barrow D, Knowlden JM, et al. The 533 antiepidermal growth factor receptor agent gefitinib (ZD1839/Iressa) improves antihormone 534 response and prevents development of resistance in breast cancer in vitro. Endocrinology. 535 2003;144:5105-17. 536 39. Burstein HJ, Cirrincione CT, Barry WT, Chew HK, Tolaney SM, Lake DE, et al. Endocrine therapy 537 with or without inhibition of epidermal growth factor receptor and human epidermal growth factor 538 receptor 2: a randomized, double-blind, placebo-controlled phase III trial of fulvestrant with or 539 without lapatinib for postmenopausal women with hormone receptor-positive advanced breast 540 cancer-CALGB 40302 (Alliance). Journal of clinical oncology : official journal of the American Society 541 of Clinical Oncology. 2014;32:3959-66. 542 40. Kaufman B, Mackey JR, Clemens MR, Bapsy PP, Vaid A, Wardley A, et al. Trastuzumab plus 543 anastrozole versus anastrozole alone for the treatment of postmenopausal women with human 544 epidermal growth factor receptor 2-positive, hormone receptor-positive metastatic breast cancer: 545 results from the randomized phase III TAnDEM study. Journal of clinical oncology : official journal of 546 the American Society of Clinical Oncology. 2009;27:5529-37. 547 41. Johnston S, Pippen J, Jr., Pivot X, Lichinitser M, Sadeghi S, Dieras V, et al. Lapatinib combined 548 with letrozole versus letrozole and placebo as first-line therapy for postmenopausal hormone 549 receptor-positive metastatic breast cancer. Journal of clinical oncology : official journal of the 550 American Society of Clinical Oncology. 2009;27:5538-46. 551 42. Chen HX, Sharon E. IGF-1R as an anti-cancer target--trials and tribulations. Chin J Cancer. 552 2013;32:242-52. 553 43. Johnston SR. New strategies in estrogen receptor-positive breast cancer. Clinical cancer 554 research : an official journal of the American Association for Cancer Research. 2010;16:1979-87. 555 44. Bachelot T, Bourgier C, Cropet C, Ray-Coquard I, Ferrero JM, Freyer G, et al. Randomized phase 556 II trial of everolimus in combination with tamoxifen in patients with hormone receptor-positive, 557 human epidermal growth factor receptor 2-negative metastatic breast cancer with prior exposure to 558 aromatase inhibitors: a GINECO study. Journal of clinical oncology : official journal of the American 559 Society of Clinical Oncology. 2012;30:2718-24. 560 45. Baselga J, Campone M, Piccart M, Burris HA, 3rd, Rugo HS, Sahmoud T, et al. Everolimus in 561 postmenopausal hormone-receptor-positive advanced breast cancer. N Engl J Med. 2012;366:520-9. 562 46. Johnston SR. Enhancing Endocrine Therapy for Hormone Receptor-Positive Advanced Breast 563 Cancer: Cotargeting Signaling Pathways. J Natl Cancer Inst. 2015;107. 564 47. Finn RS, Crown JP, Lang I, Boer K, Bondarenko IM, Kulyk SO, et al. The cyclin-dependent kinase 565 4/6 inhibitor palbociclib in combination with letrozole versus letrozole alone as first-line treatment of 566 oestrogen receptor-positive, HER2-negative, advanced breast cancer (PALOMA-1/TRIO-18): a 567 randomised phase 2 study. Lancet Oncol. 2015;16:25-35. 568




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48. Jerusalem G, Bachelot T, Barrios C, Neven P, Di Leo A, Janni W, et al. A new era of improving 569 progression-free survival with dual blockade in postmenopausal HR(+), HER2(-) advanced breast 570 cancer. Cancer Treat Rev. 2015;41:94-104. 571 49. Bachelot T, McCool R, Duffy S, Glanville J, Varley D, Fleetwood K, et al. Comparative efficacy of 572 everolimus plus exemestane versus fulvestrant for hormone-receptor-positive advanced breast 573 cancer following progression/recurrence after endocrine therapy: a network meta-analysis. Breast 574 cancer research and treatment. 2014;143:125-33. 575 50. Yardley DA, Noguchi S, Pritchard KI, Burris HA, 3rd, Baselga J, Gnant M, et al. Everolimus plus 576 exemestane in postmenopausal patients with HR(+) breast cancer: BOLERO-2 final progression-free 577 survival analysis. Adv Ther. 2013;30:870-84. 578 51. Jung Y, Kim JK, Shiozawa Y, Wang J, Mishra A, Joseph J, et al. Recruitment of mesenchymal stem 579 cells into prostate tumours promotes metastasis. Nat Commun. 2013;4:1795. 580 52. Ishii G, Sangai T, Ito T, Hasebe T, Endoh Y, Sasaki H, et al. In vivo and in vitro characterization 581 of human fibroblasts recruited selectively into human cancer stroma. Int J Cancer. 2005;117:212-20. 582 53. Dong J, Grunstein J, Tejada M, Peale F, Frantz G, Liang WC, et al. VEGF-null cells require PDGFR 583 alpha signaling-mediated stromal fibroblast recruitment for tumorigenesis. EMBO J. 2004;23:2800-10. 584 585 586




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FIGURE LEGENDS 587 588 Figure 1. Two subtypes of fibroblasts are present in normal and breast cancer associated stroma based 589 on CD146 expression and increased ratios of CD146pos fibroblasts correlates with high ER expression. (A) 590 Immunohistochemistry staining of patient tissue demonstrating the presence of both CD146pos (A, vector red 591 stain, blue arrowhead) and CD146neg (A, DAB stain, yellow arrowhead) stroma. (B) Quantification 592 demonstrating that patients with Allred > 6 have significantly increased CD146pos stroma compared to Allred ≤ 593 6. (C-D) Immunofluorescence staining of tissue from ER+ breast cancer patients demonstrates a decreased ratio 594 of CD146pos (C-D, red stain) to vimentin (C-D, green stain) expressing stroma in patients with low Allred (C) 595 compared to high Allred (D) scores. (E) Representative histograms demonstrating the presence of both 596 CD146pos and CD146neg stroma in patient derived normal and breast cancer associated tissue. N = 14 patient 597 tumor stroma samples and N = 11 matched normal breast tissue samples. Scale bars: 100 μm. T, tumor; V, 598 endothelial vessels. 599 600 Figure 2. CD146pos CAFs sustain ER expression in ER+ breast cancer cells, whereas CD146neg CAFs 601 promote decreased ER expression. (A) Representative immunofluorescence staining for ER (red) and the 602 tumor cell maker CK18 (green) in co-cultures of MCF-7 cells with HS27a (CD146pos) or HS5 (CD146neg) 603 fibroblasts showing decreased ER expression in CD146neg co-cultures compared to CD146pos co-cultures. (B) 604 Analysis of 4 replicates imaged in 3 positions and quantified for the percent of ER positive tumor cells 605 demonstrates a significant reduction in ER expression in co-cultures with CD146neg fibroblasts. (C) Mixed 606 MCF-7/fibroblast (HS27a or HS5) tumors were established in NSG mice and harvested for 607 immunofluorescence staining for ER (red) and CK18 (green) showing decreased ER expression in tumors 608 mixed with CD146neg fibroblasts. (D) Analysis of 5 animals per group, imaged in 3 position and quantified for 609 the percent of ER positive tumor cells demonstrates a significant reduction in ER expression in tumors mixed 610 with CD146neg fibroblasts. Percent ER+ cells = (Triple positive ER+/CK18+/DAPI+)/(All 611 CK18+/DAPI+))Outliers were considered to be 2 standard deviations from the mean and excluded. Scale bars: 612 20 μm. 613 614 Figure 3. Influence of CD146neg CAFs programs ER+ breast cancer cells to bypass estrogen dependent 615 proliferation and decrease tamoxifen sensitivity. (A) MCF-7 cells cultured for 5 days in conditioned media 616 from CD146pos (HS27a) fibroblasts have significantly more ER gene expression than MCF-7 cells grown in 617 conditioned media from CD146neg (HS5) fibroblasts. (B) SRB total protein analysis of MCF-7 cells cultured in 618 conditioned media from CD146pos (HS27a) fibroblasts have significantly increased proliferation in response to 619 estrogen treatment and significantly decreased proliferation in response to treatment with 4-OH-tamoxifen. In 620 contrast, conditioned media from CD146neg fibroblasts renders MCF-7 cells unresponsive to estrogen and 4-621 OH-tamoxifen treatment. Data are normalized against an estrogen withdrawn (EWD) untreated control 622 collected at the time of treatment. (C) SRB total protein analysis of tamoxifen resistant TAMR-1 cells cultured 623 in conditioned media from CD146pos (HS27a) fibroblasts have significantly decreased proliferation when 624 treated with 4-OH-tamoxifen. Data are normalized against an estrogen withdrawn (EWD) untreated control 625 collected at the time of treatment. (***p < 0.001; ****p < 0.0001.) 626 627 Figure 4. CD146neg CAFs promote tamoxifen resistance in ER+ breast cancer tumors. Mixed MCF-628 7/fibroblast (HS27a or HS5) tumors were established in NSG mice for 3 weeks and then treated with 4-629 hydroxy-tamoxifen (4-OH-TAM) or peanut oil (vehicle) for 8 weeks. (A) Tumors established with CD146pos 630 (HS27a) fibroblasts had significantly smaller tumors by 6 weeks after treatment with tamoxifen. In comparison, 631 tumors established with CD146neg (HS5) fibroblasts did not respond to tamoxifen treatment. (B) Tumors 632 established with CD146pos (HS27a) fibroblasts and treated with tamoxifen weighed significantly less at excision 633 compared to vehicle. In comparison, tumor established with CD156neg (HS5) fibroblasts did not respond to 4-634 OH-TAM. N = 6 mice per group. 635 636




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Figure 5. Influence of CD146neg CAFs program ER+ breast cancer to express tyrosine kinase receptor 637 pathways shown to promote tamoxifen resistance. (A) Live cell imaging and quantification of MCF-7 638 proliferation in response to the specific EGFR inhibitor gefitinib demonstrates that CD146neg fibroblasts (HS5) 639 cause significantly decreased proliferation in MCF-7 cells, whereas proliferation of MCF-7 cells co-cultured 640 with CD146pos (HS27a) fibroblasts is not significantly changed. (B) SRB total protein analysis of MCF-7 cells 641 cultured in conditioned media with 2.5% reduced charcoal stripped serum from MCF-7 cells or either fibroblast 642 subtype (HS27a and HS5) have significantly decreased proliferation in response to gefitinib treatment. (C) SRB 643 total protein analysis of MCF-7 cells cultured in conditioned media with 2.5% reduced charcoal stripped serum 644 from CD146neg (HS5) fibroblasts have significantly decreased proliferation in response to trastuzumab 645 treatment. (D) Western blot analysis of MCF-7 cells demonstrates that only MCF-7 cells grown in CM from 646 CD146pos (HS27) or CD146neg (HS5) fibroblasts express pEGFR protein. Receptor tyrosine kinase arrays 647 demonstrate phosphorylated EGFR protein (pEGFR) in MCF-7 cells influenced by both fibroblast subtypes 648 (HS27a and HS5). Phospho-IGF1R is present only in MCF-7 cells influenced by CD146neg

fibroblasts (HS5). 649 (** p < 0.01 ***p < 0.001; ****p < 0.0001.) 650 651 Figure 6. CD146neg CAF gene signature predicts poorer clinical outcomes and produces an epithelial 652 signature that is predictive of decreased recurrence-free survival in tamoxifen treated patients. (A) 653 Hierarchical clustering of Affymetrix gene expression data for normal fibroblasts (HS27a and HS5) and our 654 primary CAFs compared to Finak et. al. data demonstrates that CD146pos fibroblasts cluster with normal breast 655 stroma, whereas CD146neg fibroblasts cluster with tumor associated stroma. (B) Hierarchical clustering of 656 Affymetrix gene expression data for normal fibroblasts (HS27a and HS5) and our primary CAFs compared to 657 Finak et. al. data demonstrates that CD146pos fibroblasts predict for better patient outcomes and CD146neg 658 fibroblasts for poorer patient outcomes. Triplicate samples were used for gene expression array analysis. (C) 659 Patient training and validation sets for a gene signature consisting of 5 genes (PRKCA, MACROD2, 660 SMARCA4, BNIP3, MYO1B) predicted to be up and 4 genes (RPLP1, CDC42EP4, MAP2K4 and SIAH2) 661 predicted to be down in the epithelial component of ER+ breast cancer was generated from RNAseq data in 662 MCF-7/CAF co-cultures. The gene signature demonstrates significant predictive power of increased recurrence-663 free survival in patients with the CD146pos signature. *** p < 0.001; **** p < 0.0001. 664 665






















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Fibroblast subtypes regulate responsiveness of …...2016/10/04 · 1 Fibroblast subtypes regulate responsiveness of luminal breast cancer to estrogen. 2 Heather M. Brechbuhl 1,*