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Supplementary Figure 1 a. Doxorubicin resistance of DOXO-R C8 cells was further validated using the cell viability assay (SRB) following 48 hrs treatment with DOXO, n=4 independent experiment, 3 replicates/experiment. **p0.6 and pval

Table S18: Two-way ANOVA

Table S1: List of regions of genes with increased CNV (>= 0.5) and gene expression (>= 0.5) in DOXO-R cells compared to parental cells Table S2: List of all fold change and pvalue of DOXO-R C1 and C8 (vs parental cells) from gene expression analysis. Table S3. GSEA using the C5 module for the 1852 genes with gene expression fold change > 1.5 (pval < 0.05) in DOXO-R C1 and C8 Table S4. GSEA using the C5 module for the 407 genes with gene expression fold change > 1.5 (pval < 0.05) and CNV change > 0.5 Table S5: Mean LOG2FC of genes commonly up-regulated in C1 and C8 Table S6: Proteins that are significantly upregulated according to the following criteria. (i) only present in one or two of the Dox resistant cell lines (**) with at least 3 and 2 unique peptides, respectively. (ii) At least 1.5-fold upregulated in both cell lines with a maximum relative standard deviation (RSD) of 31% across control replicates and 25% RSD across resistant cell line replicates. Table S7: GSEA using the C5 module for the 98 overexpressed proteins in DOXO-R vs parental cells Table S8: mRNA fold change (POST vs PRE NAC) for 9 chemoresistant TNBC patients. Table S9: GSEA using the C5 module for NEO2 up regulated genes > 2.5 FC. The complete list of genes are listed below the GSEA analysis. Table S10: GSEA using the C5 module for NEO24 up regulated genes > 2.5 FC. The complete list of genes are listed below the GSEA analysis. Table S11: GSEA using the C5 module for NEO25 up regulated genes > 2.5 FC. The complete list of genes are listed below the GSEA analysis. Table S12: GSEA using the C5 module for NEO27 up regulated genes > 2.5 FC. The complete list of genes are listed below the GSEA analysis. Table S13: GSEA using the C5 module for NEO28 up regulated genes > 2.5 FC. The complete list of genes are listed below the GSEA analysis. Table S14: GSEA using the C5 module for NEO30 up regulated genes > 2.5 FC. The complete list of genes are listed below the GSEA analysis. Table S15: GSEA using the C5 module for NEO31 up regulated genes > 2.5 FC. The complete list of genes are listed below the GSEA analysis. Table S16: GSEA using the C5 module for NEO35 up regulated genes > 2.5 FC. The complete list of genes are listed below the GSEA analysis. Table S17: GSEA using the C5 module for NEO44 up regulated genes > 2.5 FC. The complete list of genes are listed below the GSEA analysis.

The major obstacle in successfully treating triple-negative breast cancer (TNBC) is resistance to cytotoxic chemotherapy, the mainstay of treatment in this disease. Previous preclinical models of chemoresistance in TNBC have suffered from a lack of clinical relevance. Using a single high dose chemotherapy treatment, we developed a novel MDA-MB-436 cell-based model of chemoresistance characterized by a unique and complex morphologic phenotype, which consists of polyploid giant cancer cells giving rise to neuron-like mononuclear daughter cells filled with smaller but functional mitochondria and numerous lipid droplets. This resistant phenotype is associated with metabolic reprogramming with a shift to a greater dependence on fatty acids and oxidative phosphorylation. We validated both the molecular and histologic features of this model in a clinical cohort of primary chemoresistant TNBCs and identified several metabolic vulnerabilities including a dependence on PLIN4, a perilipin coating the observed lipid droplets, expressed both in the TNBC-resistant cells and clinical chemoresistant tumors treated with neoadjuvant doxorubicin-based chemotherapy. These findings thus reveal a novel mechanism of chemotherapy resistance that has therapeutic implications in the treatment of drug-resistant cancer.Implications:These findings underlie the importance of a novel morphologic–metabolic phenotype associated with chemotherapy resistance in TNBC, and bring to light novel therapeutic targets resulting from vulnerabilities in this phenotype, including the expression of PLIN4 essential for stabilizing lipid droplets in resistant cells.

Supplementary Table3 shows Top 20 regulated genes in cluster 2 (predominant liver mCAFs).

Figure S4 shows the top differentially regulated genes between mCAFs vs pCAFs

Figure S1-S8 legend

Clinical data (S1); Expression levels of SPEN and PGR as evaluated with DNA microarrays (S2); IC50 values of SPEN-overexpressing T47D clones, SPEN-silenced MCF-7 clones and their respective controls (S3); Loss of heterozysity assessment using polymorphic microsatellite repeat markers (S4); Primers sequence for the sequencing of SPEN (S5).

SPEN is mutated in T47D cells (S1); SPEN is down-regulated and mutated in breast cancer (S2); SPEN regulates cell growth and survival (S3); SPEN knockdown in triple negative breast cancer cell lines limits proliferation (S4); SPEN regulates cell death and survival (S5); SPEN regulates transcriptional activity of the Estrogen Receptor (S6); SPEN regulates response of ERα-positive breast cancer cells to tamoxifen (S7); SPEN does not affect ERα-positive breast cancer cells response to fulvestrant (S8).

Legends for Supplemental Figures S1-S8.

The PI3-kinase/AKT/mTOR pathway plays a central role in cancer signaling. While p110 alpha is the catalytic alpha-subunit of PI3-kinase and a major drug target, PTEN is the main negative regulator of the PI3-kinase/AKT/mTOR pathway. PTEN is often down-regulated in cancer, and there are conflicting data on PTEN's role as breast cancer biomarker. PTEN and p110 alpha protein expression in tumors is commonly analyzed by immunohistochemistry, which suffers from poor multiplexing capacity, poor standardization, and antibody crossreactivity, and which provides only semi-quantitative data. Here, we present an automated, and standardized immuno-matrix-assisted laser desorption/ionization mass spectrometry (iMALDI) assay that allows precise and multiplexed quantitation of PTEN and p110 alpha concentrations, without the limitations of immunohistochemistry. Our iMALDI assay only requires a low-cost benchtop MALDI-TOF mass spectrometer, which simplifies clinical translation. We validated our assay's precision and accuracy, with simultaneous enrichment of both target proteins not significantly affecting the precision and accuracy of the quantitation when compared to the PTEN- and p110 alpha-singleplex iMALDI assays (

Trastuzumab is integral to HER2+ cancer treatment, but its therapeutic index is narrowed by the development of resistance. Phosphorylation of the translation initiation factor eIF2α (eIF2α-P) is the nodal point of the integrated stress response, which promotes survival or death in a context-dependent manner. Here, we show an anti-tumor function of the protein kinase PKR and its substrate eIF2α in a mouse HER2+ breast cancer model. The anti-tumor function depends on the transcription factor ATF4, which upregulates the CDK inhibitor P21CIP1 and activates JNK1/2. The PKR/eIF2α-P arm is induced by Trastuzumab in sensitive but not resistant HER2+ breast tumors. Also, eIF2α-P stimulation by the phosphatase inhibitor SAL003 substantially increases Trastuzumab potency in resistant HER2+ breast and gastric tumors. Increased eIF2α-P prognosticates a better response of HER2+ metastatic breast cancer patients to Trastuzumab therapy. Hence, the PKR/eIF2α-P arm antagonizes HER2 tumorigenesis whereas its pharmacological stimulation improves the efficacy of Trastuzumab therapy., The HER2 monoclonal antibody, Trastuzumab, is the current standard treatment for HER2+ cancers but resistance to therapy occurs. Here, the authors show that activation of the PKR/eIF2α-P pathway exhibits anti-tumor effects in HER2+ cancer and is required for the response to Trastuzumab.

The tumor suppressor PTEN is the main negative regulator of PI3K/AKT/mTOR signaling and is commonly found downregulated in breast cancer (BC). Conflicting data from conventional immunoassays such as immunohistochemistry (IHC) has sparked controversy about PTEN's role as a prognostic and predictive biomarker in BC, which can be largely attributed to the lack of specificity, sensitivity, and interlaboratory standardization. Here, we present a fully standardized, highly sensitive, robust microflow immuno-MRM (iMRM) assay that enables precise quantitation of PTEN concentrations in cells and fresh frozen (FF) and formalin-fixed paraffin-embedded (FFPE) tissues, down to 0.1 fmol/10 μg of extracted protein, with high interday and intraday precision (CV 6.3%). PTEN protein levels in BC PDX samples that were determined by iMRM correlate well with semiquantitative IHC and WB data. iMRM, however, allowed the precise quantitation of PTEN-even in samples that were deemed to be PTEN negative by IHC or western blot (WB)-while requiring substantially less tumor tissue than WB. This is particularly relevant because the extent of PTEN downregulation in tumors has been shown to correlate with severity. Our standardized and robust workflow includes an 11 min microflow LC-MRM analysis on a triple-quadrupole MS and thus provides a much needed tool for the study of PTEN as a potential biomarker for BC.

Background: Her2+ breast cancer accounts for 15% of breast cancer cases and is primarily treated with a combination of Her2-targeted agents and chemotherapy. While use of targeted agents has substantially improved survival of these patients, clinical drug resistance is prevalent. DNA repair defects are a feature of cancer that has been exploited by targeted agents such as inhibitors of poly-ADP ribose polymerase (PARP). PARP inhibitors have been shown to be effective in patients with germline mutations in other DNA repair factors such as BRCA1/2. There is limited data for the use of PARP inhibitors in HER2+ breast cancers, and in tumors without BRCA1/2 mutations. Using a collection of HER2+ and HER2-low expressing breast cancer patient-derived xenografts (PDXs) that have differential responses to the Her2-targeted agents Trastuzumab, Pertuzumab, and T-DM1, we tested the combination of these agents with Talazoparib (Talzenna™), a clinically approved Parp inhibitor. Methods: Orthotopically engrafted PDX mouse models resistant to either Trastuzumab or T-DM1 were randomized into treatment arms including Trastuzumab or T-DM1 in combination with Talazoparib, and the single agents alone. Mice are monitored for body weight and change in tumour volume every 2-3 days, then sacrificed at endpoint for survival analysis. We have also generated PDX-derived cells (PDCs) from one model with which to validate in vivo results and perform mechanistic studies. All models are being characterized for copy number changes, somatic mutations, and protein expression by immunohistochemistry. Results: The combination of Talazoparib and either Trastuzumab or T-DM1 was effective in 3 PDX models, including a Trastuzumab-resistant HER2+ PDX, a T-DM1-resistant HER2+ PDX and a T-DM1-resistant HER2-low expressing PDX. The combinations resulted in significant delays in tumour growth (p Conclusion: Our results suggest that combining HER-targeted agents with Talazoparib may benefit patients with advanced HER2-therapy resistant HER2+ and low HER2+ breast cancers. Exploratory biomarker analysis and mechanistic studies using PDCs are currently underway in our laboratory. Citation Format: Kathryn Bozek, Marguerite Buchanan, Cathy Lan, Josiane Lafleur, Cedric Darini, Urszula Krzemien, Adriana Aguilar-Mahecha, Mark Basik. The combination of Talazoparib with anti-her2 drugs shows efficacy in drug resistant Her2+ and low Her2 PDX models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1794.

Carcinoma-associated fibroblasts (CAFs) can remodel the extracellular matrix to promote cancer cell invasion, but the paracrine signaling between CAFs and cancer cells that regulates tumor cell migration remains to be identified. To determine how the interaction between CAFs and cancer cells modulates the invasiveness of cancer cells, we developed a 3-dimensional co-culture model composed of breast cancer (BC) MDA-MB-231 cell spheroids embedded in a collagen gel with and without CAFs. We found that the crosstalk between CAFs and cancer cells promotes invasion by stimulating the scattering of MDA-MB-231 cells, which was dependent on RhoA/ROCK/phospho MLC signaling in cancer cells but independent of RhoA in CAFs. The activation of RhoA/ROCK in cancer cells activates MLC and increases migration, while the genetic-down-regulation of RhoA and pharmacological inhibition of ROCK reduced cell scattering and invasion. Two distinct mechanisms induced the activation of the RhoA/ROCK pathway in MDA-MB-231 cells, the secretion of IGF-1 by CAFs and the upregulation of PAI-1 in cancer cells. In an orthotopic model of BC, IGF-1R inhibition decreased the incidence of lung metastasis, while Y27632-inhibition of ROCK enhanced the lung metastasis burden, which was associated with an increased recruitment of CAFs and expression of PAI-1. Thus the crosstalk between CAFs and BC cells increases the secretion of IGF-1 in CAFs and PAI-1 activity in cancer cells. Both IGF1 and PAI-1 activate RhoA/ROCK signaling in cancer cells, which increases cell scattering and invasion.

Purpose: The microenvironment of metastatic breast cancer is incompletely characterized, despite prior evidence that it plays a key role in the biology of metastasis. A major component of the tumor stroma is the carcinoma-associated fibroblast (CAF), which has been shown to communicate with other stromal and cancer cells to create a protumorigenic milieu. Our study was designed to characterize human CAFs from different metastatic sites. Experimental Design: We collected eight carcinoma-associated fibroblasts (mCAFs) from different metastatic sites and compared them with CAFs from primary tumors (pCAFs) and with normal breast fibroblasts (NFs). Molecular profiles and effects on breast cancer cell growth, on response to doxorubicin and on T-cell proliferation were compared. Results: We observed marked differences in mCAFs compared with pCAFs and NFs with respect to in vitro proliferation and effects on breast cancer cell migration, spheroid growth, invasion, response to doxorubicin, and in vivo tumor growth. We found marked transcriptomic differences between mCAFs and pCAFs, including increased expression of IFN-related genes and IGF2 in the former. Cluster analysis revealed two groups of mCAFs, with the liver mCAFs clustering together, with increased PDGFA expression. Treatment with an antibody against insulin-like growth factors (BI836845) inhibited growth of mixed mCAF-tumor cell xenografts in vivo. Also, mCAFs had a suppressive effect on T-cell proliferation. Conclusions: This is the first comparative analysis of a set of CAFs from metastatic sites in breast cancer. It revealed a marked protumorigenic effect in these mCAFs, which occurs in part through increased expression of IGF2.

The major obstacle in successfully treating triple-negative breast cancer (TNBC) is resistance to cytotoxic chemotherapy, the mainstay of treatment in this disease. Previous preclinical models of chemoresistance in TNBC have suffered from a lack of clinical relevance. Using a single high dose chemotherapy treatment, we developed a novel MDA-MB-436 cell-based model of chemoresistance characterized by a unique and complex morphologic phenotype, which consists of polyploid giant cancer cells giving rise to neuron-like mononuclear daughter cells filled with smaller but functional mitochondria and numerous lipid droplets. This resistant phenotype is associated with metabolic reprogramming with a shift to a greater dependence on fatty acids and oxidative phosphorylation. We validated both the molecular and histologic features of this model in a clinical cohort of primary chemoresistant TNBCs and identified several metabolic vulnerabilities including a dependence on PLIN4, a perilipin coating the observed lipid droplets, expressed both in the TNBC-resistant cells and clinical chemoresistant tumors treated with neoadjuvant doxorubicin-based chemotherapy. These findings thus reveal a novel mechanism of chemotherapy resistance that has therapeutic implications in the treatment of drug-resistant cancer. Implications: These findings underlie the importance of a novel morphologic–metabolic phenotype associated with chemotherapy resistance in TNBC, and bring to light novel therapeutic targets resulting from vulnerabilities in this phenotype, including the expression of PLIN4 essential for stabilizing lipid droplets in resistant cells.

The treatment of breast cancer has benefitted tremendously from the generation of estrogen receptor-α (ERα)–targeted therapies, but disease relapse continues to pose a challenge due to intrinsic or acquired drug resistance. In an effort to delineate potential predictive biomarkers of therapy responsiveness, multiple groups have identified several uncharacterized cofactors and interacting partners of ERα, including Split Ends (SPEN), a transcriptional corepressor. Here, we demonstrate a role for SPEN in ERα-expressing breast cancers. SPEN nonsense mutations were detectable in the ERα-expressing breast cancer cell line T47D and corresponded to undetectable protein levels. Further analysis of 101 primary breast tumors revealed that 23% displayed loss of heterozygosity at the SPEN locus and that 3% to 4% harbored somatically acquired mutations. A combination of in vitro and in vivo functional assays with microarray-based pathway analyses showed that SPEN functions as a tumor suppressor to regulate cell proliferation, tumor growth, and survival. We also found that SPEN binds ERα in a ligand-independent manner and negatively regulates the transcription of ERα targets. Moreover, we demonstrate that SPEN overexpression sensitizes hormone receptor–positive breast cancer cells to the apoptotic effects of tamoxifen, but has no effect on responsiveness to fulvestrant. Consistent with these findings, two independent datasets revealed that high SPEN protein and RNA expression in ERα-positive breast tumors predicted favorable outcome in patients treated with tamoxifen alone. Together, our data suggest that SPEN is a novel tumor-suppressor gene that may be clinically useful as a predictive biomarker of tamoxifen response in ERα-positive breast cancers. Cancer Res; 75(20); 4351–63. ©2015 AACR.

Great advances in analytical technology coupled with accelerated new drug development and growing understanding of biological challenges, such as tumor heterogeneity, have required a change in the focus for biobanking. Most current banks contain samples of primary tumors, but linking molecular signatures to therapeutic questions requires serial biopsies in the setting of metastatic disease, next-generation of biobanking. Furthermore, an integration of multidimensional analysis of various molecular components, that is, RNA, DNA, methylome, microRNAome and post-translational modifications of the proteome, is necessary for a comprehensive view of a tumor's biology. While data using such biopsies are now regularly presented, the preanalytical variables in tissue procurement and processing in multicenter studies are seldom detailed and therefore are difficult to duplicate or standardize across sites and across studies. In the context of a biopsy-driven clinical trial, we generated a detailed protocol that includes morphological evaluation and isolation of high-quality nucleic acids from small needle core biopsies obtained from liver metastases. The protocol supports stable shipping of samples to a central laboratory, where biopsies are subsequently embedded in support media. Designated pathologists must evaluate all biopsies for tumor content and macrodissection can be performed if necessary to meet our criteria of >60% neoplastic cells and

SummaryStroma in the tumor microenvironment plays a critical role in cancer progression, but how it promotes metastasis is poorly understood. Exosomes are small vesicles secreted by many cell types and enable a potent mode of intercellular communication. Here, we report that fibroblast-secreted exosomes promote breast cancer cell (BCC) protrusive activity and motility via Wnt-planar cell polarity (PCP) signaling. We show that exosome-stimulated BCC protrusions display mutually exclusive localization of the core PCP complexes, Fzd-Dvl and Vangl-Pk. In orthotopic mouse models of breast cancer, coinjection of BCCs with fibroblasts dramatically enhances metastasis that is dependent on PCP signaling in BCCs and the exosome component, Cd81 in fibroblasts. Moreover, we demonstrate that trafficking in BCCs promotes tethering of autocrine Wnt11 to fibroblast-derived exosomes. This work reveals an intercellular communication pathway whereby fibroblast exosomes mobilize autocrine Wnt-PCP signaling to drive BCC invasive behavior.

CXCR4 is a chemokine receptor implicated in the homing of cancer cells to target metastatic organs, which overexpress its ligand, stromal cell-derived factor (SDF)-1. To determine the efficacy of targeting CXCR4 on primary tumor growth and metastasis, we used a peptide inhibitor of CXCR4, CTCE-9908, that was administered in a clinically relevant approach using a transgenic breast cancer mouse model. We first performed a dosing experiment of CTCE-9908 in the PyMT mouse model, testing 25, 50 and 100 mg/kg versus the scrambled peptide in groups of 8-16 mice. We then combined CTCE-9908 with docetaxel or DC101 (an anti-VEGFR2 monoclonal antibody). We found that increasing doses of CTCE-9908 alone slowed the rate of tumor growth, with a 45% inhibition of primary tumor growth at 3.5 weeks of treatment with 50 mg/kg of CTCE-9908 (p = 0.005). Expression levels of VEGF were also found to be reduced by 42% with CTCE-9908 (p = 0.01). In combination with docetaxel, CTCE-9908 administration decreased tumor volume by 38% (p = 0.02), an effect that was greater than that observed with docetaxel alone. In combination with DC101, CTCE-9908 also demonstrated an enhanced effect compared to DC101 alone, with a 37% decrease in primary tumor volume (p = 0.01) and a 75% reduction in distant metastasis (p = 0.009). In combination with docetaxel or an anti-angiogenic agent, the anti-tumor and anti-metastatic effects of CTCE-9908 were markedly enhanced, suggesting potentially new effective combinatorial therapeutic strategies in the treatment of breast cancer, which include targeting the SDF-1/CXCR4 ligand/receptor pair.

The use of patient derived models for pre-clinical drug testing provides a unique opportunity for testing non-conventional therapies and novel drug combinations to overcome drug resistance in cancer. We have established 22 models from breast cancer, 20 PDXs were engrafted in house and two were generated from live tissue obtained from PDXs established in a partner lab. The overall engraftment rate in house was of 24%. Engraftment varied according to BC subtype: 38% (3/8) for ER-HER2+ and 33% (17/51) for TNBC. No PDXs could be generated from ER+PR+Her2- tumors (0/13) or ER+HER2+ breast cancers (0/11). Post-chemotherapy drug resistant tumors engrafted with better success (18/55) compared to non-treated tumors (2/28). In 3 patients, we established PDX models with tissues collected at different anatomical sites, enabling the study of tumor heterogeneity. For one patient, 2 PDX models were established from serial samples after different lines of chemotherapy. Molecular profiling at the copy number and mutation level has confirmed the molecular stability of our models over engraftment passages. Drug studies were performed to validate clinical drug response and models were also treated with unconventional drugs and drug combinations to overcome drug resistance. Drugs that resulted in significant tumor regression in drug resistant TNBC models included irinotecan as well as olaparib in combination with an angiogenesis inhibitor. We successfully established one PDX model from a recurrent Her2+ gastric tumor resistant to trastuzumab. In this model, treatment with T-DM1 alone as well as with pertuzumab/trastuzumab resulted in complete tumor regression, while everolimus significantly inhibited tumor growth. This PDX study provided an alternative clinical management for the patient who subsequently received T-DM1 and showed clinical response, validating the results obtained in the PDX model. To develop a more agile model, we also established conditionally reprogrammed cell lines directly from patient tumors or from PDX models. Drug resistant conditionally reprogrammed cells from Her2+ breast cancers harboring a FGFR1 amplification were found to be sensitive to FGFR inhibitors, chloroquine, and a CDK4/6 inhibitor. Correlation of drug responses between conditionally reprogrammed cells and matched PDX models will be presented. These live tumor models enable timely and clinically pertinent drug testing, which has the potential to expand the known therapeutic arsenal as well as provide truly personalized therapy to cancer patients. Citation Format: Marguerite Buchanan, Catherine Chabot, Josiane Lafleur, Urszula Krzemien, Cathy Lan, Olga Savichtcheva, Jean-François Boileau, Cristiano Ferrario, Paul Savage, Morag Park, Gerald Batist, Adriana Aguilar-Mahecha, Mark Basik. Use of PDXs and patient-derived cell lines to uncover unconventional drug therapies and combinations for the treatment of drug-resistant cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2149.

Carcinoma-associated fibroblasts (CAFs) can remodel the extracellular matrix to promote cancer cell invasion, but the paracrine signaling between CAFs and cancer cells that regulates tumor cell migration remains to be identified. To determine how the interaction between CAFs and cancer cells modulates the invasiveness of cancer cells, we developed a 3-dimensional co-culture model composed of breast cancer (BC) MDA-MB-231 cell spheroids embedded in a collagen gel with and without CAFs. We found that the crosstalk between CAFs and cancer cells promotes invasion by stimulating the scattering of MDA-MB-231 cells, which was dependent on RhoA/ROCK/phospho MLC signaling in cancer cells but independent of RhoA in CAFs. The activation of RhoA/ROCK in cancer cells activates MLC and increases migration, while the genetic-down-regulation of RhoA and pharmacological inhibition of ROCK reduced cell scattering and invasion. Two distinct mechanisms induced the activation of the RhoA/ROCK pathway in MDA-MB-231 cells, the secretion of IGF-1 by CAFs and the upregulation of PAI-1 in cancer cells. In an orthotopic model of BC, IGF-1R inhibition decreased the incidence of lung metastasis, while Y27632-inhibition of ROCK enhanced the lung metastasis burden, which was associated with an increased recruitment of CAFs and expression of PAI-1. Thus the crosstalk between CAFs and BC cells increases the secretion of IGF-1 in CAFs and PAI-1 activity in cancer cells. Both IGF1 and PAI-1 activate RhoA/ROCK signaling in cancer cells, which increases cell scattering and invasion.

Breast cancer is one of the most common causes of cancer-related death in women in the world. Although most research is focused on the tumor cells, it is important to elucidate the molecular nature of the tumor-stromal relationship to truly understand the biology of breast tumor growth. Cancer-associated fibroblasts (CAFs) are the major cellular constituents in the tumor microenvironment. CAFs are in direct contact with the adjacent cancer cells and crosstalk with them through soluble cytokines, growth factors and exosomes during carcinogenesis in human breast cancer. To determine for the first time the function of CAFs in metastatic sites of breast cancer, we collected CAFs from human patients of metastatic sites (m-CAFs) and compared them with CAFs from primary tumor (p-CAFs) and normal fibroblasts (NFs). We found that m-CAFs expressed higher levels of α-smooth muscle actin (SMA) compared with p-CAFs and NFs. The proliferation of MDA-MB-436 breast cancer cells was significantly increased by treatment with conditioned medium (CM) from m-CAFs compared with p-CAFs in vitro. Also, CM by m-CAFs induced paclitaxel resistance in MDA-MB-436 cells, suggesting that soluble factors produced by m-CAFs promote chemo-resistance, to a greater extent than CM from p-CAFs. The migration and invasion ability of MDA-MB-436 cells co-cultured with m-CAFs CM was significantly greater than that of the p-CAFs CM. m-CAFs outperformed the p-CAFs and NFs in terms of their ability to promote sphere-forming activity in a 3D in vitro culture model. Furthermore, m-CAFs protected MDA-MB-436 cells from the cytotoxic effects of doxorubicin more than p-CAFs in this spheroid 3-D culture. When MDA-MB-436 cells were sorted from these spheroids and grew in the respective CMs, they showed higher expression of the stemness markers of CD44+CD24- compared with cells obtained from p-CAFs/MDA-MB-436 spheroids. Also these cells from the co-culture with m-CAFs experienced with the cadherin switch. Nanog has been demonstrated to promote chemoresistant and epithelial-mesenchymal transition (EMT). The results displayed that MDA-MB-436 cells incubated with the CM from the m-CAFs had a higher expression of Nanog compared with that of p-CAFs. RNA-seq results showed that IGF2 is one of the most different genes between m-CAFs and p-CAFs, its expression was more significant in m-CAFs co-cultured with MDA-MB-436 cells than p-CAFs. The signaling pathway in MDA-MB-436 cells were comparable between co-cultured with m-CAFs CM and the IGF2 stimulation at 2 hours. All these data illustrates that fibroblasts isolated from metastatic site differed in terms of their ability to promote breast cancer cells progression compared to p-CAFs. Our data suggests that m-CAFs are more potent than p-CAFs in inducing the proliferation, migration, drug resistance and cancer stemness of breast cancer cells in an in vitro model. Citation Format: Yirui Gui, Adriana Aguilar-Mahecha, Marguerite Buchanan, Mark Basik. Stromal fibroblasts from metastatic breast cancer promote proliferation and migration of breast cancer cell and regulate its stemness [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5894. doi:10.1158/1538-7445.AM2017-5894

The administration of ex vivo culture-expanded mesenchymal stromal cells (MSCs) has been shown to reverse symptomatic neuroinflammation observed in experimental autoimmune encephalomyelitis (EAE). The mechanism by which this therapeutic effect occurs remains unknown. In an effort to decipher MSC mode of action, we found that MSC conditioned medium inhibits EAE-derived CD4 T cell activation by suppressing STAT3 phosphorylation via MSC-derived CCL2. Further analysis demonstrates that the effect is dependent on MSC-driven matrix metalloproteinase proteolytic processing of CCL2 to an antagonistic derivative. We also show that antagonistic CCL2 suppresses phosphorylation of AKT and leads to a reciprocal increased phosphorylation of ERK associated with an up-regulation of B7.H1 in CD4 T cells derived from EAE mice. CD4 T cell infiltration of the spinal cord of MSC-treated group was robustly decreased along with reduced plasma levels of IL-17 and TNF-α levels and in vitro from restimulated splenocytes. The key role of MSC-derived CCL2 was confirmed by the observed loss of function of CCL2−/− MSCs in EAE mice. In summary, this is the first report of MSCs modulating EAE biology via the paracrine conversion of CCL2 from agonist to antagonist of CD4 Th17 cell function.

The clinical use of trastuzumab (Herceptin™), a humanized antibody against the HER2 growth factor receptor, has improved survival in patients with breast tumors with ERBB2 amplification and/or over-expression. However, most patients with advanced ERBB2 amplified breast cancers whose tumors initially respond to trastuzumab develop resistance to the drug, leading to tumor progression. To identify factors responsible for acquired resistance to trastuzumab, gene expression profiling was performed on subclones of an ERBB2 amplified breast cancer cell line, BT474, which had acquired resistance to trastuzumab. The most overexpressed gene in these subclones was PPP1R1B, encoding the DARPP-32 phosphatase inhibitor. Western analysis revealed that only the truncated isoform of the DARPP-32 protein, t-Darpp, was overexpressed in the trastuzumab resistant cells. Using gene silencing experiments, we confirmed that t-Darpp over-expression was required for trastuzumab resistance in these cells. Furthermore, transfecting t-Darpp in parental BT-474 cells conferred resistance to trastuzumab, suggesting that t-Darpp expression was sufficient for trastuzumab resistance. We also found that t-Darpp over-expression was associated with Akt activation and that the T75 residue in t-Darpp was required for both Akt activation and trastuzumab resistance. Finally, we found that full-length DARPP-32 and t-Darpp are expressed in a majority of primary breast tumors. Over-expression of full-length DARPP-32 can also confer resistance to trastuzumab and, moreover, is associated with a poor prognostic value in breast cancers. Thus, t-Darpp and DARPP-32 expression are novel prognostic and predictive biomarkers in breast cancer.

Detecting proteins that are present at lower levels in human plasma, for the identification of potential disease biomarkers, is complicated by a few highly abundant proteins. One promising strategy is the removal of these abundant proteins interfering with the analysis of plasma content by proteomic techniques. This study compared three affinity-based methods to remove the most abundant proteins in human plasma. Two of them, based on antibodies, which depletes the six or the 12 most abundant proteins, demonstrated the highest efficiency in enriching less abundant plasma proteins. Comparison of two anticoagulant treatments for plasma preparation, EDTA and CTAD, showed that this treatment influenced the patterns of lower-abundance proteins visible on 2-dimensional (2-D) gels. Several staining procedures including two fluorescent dyes, Sypro Ruby and Deep Purple, were also compared with a very sensitive silver staining method for the visualization of lower-abundance proteins on 2-D gels. Furthermore, treatments of lower-abundance plasma proteins with hydroxyethyl disulfide enhanced protein sharpness and resolution. The purpose of all these systematic comparisons was to select the most reliable methods in different steps of plasma preparation and handling as well as in analysis of proteins by 2-D gels to obtain highly reproducible patterns of lower-abundance plasma proteins. Importantly, the lower-abundance plasma proteins enriched by these optimized conditions were further analyzed by antibody microarrays allowing the identification of 61 proteins using 350 antibodies directed against signalling proteins suggesting that this proteomic strategy is a valuable approach for detecting potential plasma biomarkers.

Most women with estrogen receptor expressing breast cancers receiving anti-estrogens such as tamoxifen may not need or benefit from them. Besides the estrogen receptor, there are no predictive biomarkers to help select breast cancer patients for tamoxifen treatment. CCND1 (cyclin D1) gene amplification is a putative candidate tamoxifen predictive biomarker. The RSF1 (remodeling and spacing factor 1) gene is frequently co-amplified with CCND1 on chromosome 11q. We validated the predictive value of these biomarkers in the MA.12 randomized study of adjuvant tamoxifen vs. placebo in high-risk premenopausal early breast cancer. Premenopausal women with node-positive/high-risk node-negative early breast cancer received standard adjuvant chemotherapy and then were randomized to tamoxifen (20 mg/day) or placebo for 5 yrs. Overall survival (OS) and relapse-free survival (RFS) were evaluated. Fluorescent in-situ hybridization was performed on a tissue microarray of 495 breast tumors (74% of patients) to measure CCND1 and RSF1 copy number. A multivariate Cox model to obtain hazard ratios (HR) adjusting for clinico-pathologic factors was used to assess the effect of these biomarkers on Os and RFS. 672 women were followed for a median of 8.4 years. We were able to measure the DNA copy number of CCND1 in 442 patients and RSF1 in 413 patients. CCND1 gene amplification was observed in 8.7% and RSF1 in 6.8% of these patients, preferentially in estrogen receptor-positive breast cancers. No statistically significant interaction with treatment was observed for either CCND1 or RSF1 amplification, although patients with high RSF1 copy number did not show benefit from adjuvant tamoxifen (HR = 1.11, interaction p = 0.09). Unlike CCND1 amplification, RSF1 amplification may predict for outcome in high-risk premenopausal breast cancer patients treated with adjuvant tamoxifen.

Introduction: Personalized medicine (PM) is a concept that has raised high expectations amongst scientists, clinicians, and patients. An emerging approach is to examine tumor biopsy material for genomic changes that are known targets of currently available therapeutic agents, with the assumption that a clinical benefit will be observed if the target is inhibited. While striking anecdotal reports are predictable from this approach, the clinical impact of these agents is limited by the inevitable development of therapeutic resistance. Our focus is on the design of parallel research programs using both in vitro and in vivo strategies, in an effort to delay or inhibit resistance. We present here preliminary data for a signature of resistance to standard first-line treatment - fluorouracil, folinic acid, oxaliplatin and bevacizumab (FOLFOX/B) using cell line models of resistance to this regimen. In parallel, we are conducting a prospective study to identify biomarkers of clinical resistance to first-line therapy in patients with metastatic colorectal cancer (CRC) (NCT00984048). Methods: Ten established CRC cell lines were treated with FOLFOX/B and categorized as resistant or sensitive based on IC50 values. In parallel, patients who consented to an initial biopsy and one at disease progression following an initial response were identified as intrinsically resistant or as having acquired resistance during treatment. CRC cell lines that were initially sensitive were rendered resistant to mimic the acquired resistance in patients, by serial passages with gradual increases in concentration of the combination regimen. We compared microarray data from three sensitive and three resistant cell lines. Results: We found a different expression pattern from microarray data comparing sensitive and resistant cell lines, thereby indicating a potential signature of resistance to FOLFOX/B. Interestingly, we found that the Src family kinase Lyn was overexpressed in resistant cells lines. Treating cells with non-cytotoxic concentration of dasatinib, a dual Src family kinase and Abl inhibitor, sensitized both the parental sensitive cells and the cells with acquired resistance to FOLFOX/B, thereby suggesting that combination treatment with dasatinib may be effective in delaying or inhibiting resistance. We have thus far collected needle core biopsies from liver metastases from forty patients who agreed to partake in this multi-center trial. Eligible patients have confirmed metastatic CRC, measurable disease, and consent to three needle-core biopsies (NCBs) of a non-resectable liver metastasis before treatment and at resistance, as well as serial blood collection throughout the study. Using standard operating procedures developed for this trial, we were able to both preserve morphology and obtain high-quality genomic material from biopsy tissue. We will determine if the resistance signature and overexpression of Lyn observed in the resistant CRC cell lines are similarly demonstrated in patients that were intrinsically resistant to FOLFOX/B. Conclusions: We have designed parallel in vitro and in vivo experiments to study resistance to standard first-line treatment for mCRC. These studies provide insight on metastatic signatures of resistance and suggest combination therapies to delay or inhibit therapeutic resistance in patients.

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Introduction: The biomarker discovery process requires patient tissue samples from which histology is verified and high-quality genomic material is isolated. Several methods have been developed to either preserve tissue morphology or extract sufficient quality and quantity of RNA and DNA for downstream discovery efforts. As clinical trials incorporate patient biopsies for biomarker discovery or validation, it is becoming increasingly important to ensure quality material and identify methods that allow for preservation of morphology and stabilization of molecular content concurrently. We assessed, in liver needle-core biopsies, different sampling, fixation, and genomic isolation methods to maintain morphology and obtain high-quality genomic material for a multi-center prospective study to identify biomarkers of clinical resistance to first-line therapy in metastatic colorectal cancer. Four sampling methods (snap freezing, RNAlater, frozen RNAlater, formalin), two different fixation protocols for histological studies (10% formalin, RNAlater followed by OCT embedding and freezing) and two RNA isolation procedures (Triazol and AllprepDNA/RNA isolation) were evaluated. Results: Keeping in mind site feasibility, we report that the ideal condition to both preserve morphology and obtain high-quality genomic material of patient liver biopsy samples is to collect biopsies in RNAlater for shipping to a Central Pathology core, followed by washing with cold PBS (on dry ice) to permit proper RNA preservation during OCT embedding and cryostat sectioning for histological verification. Simultaneous extraction of DNA and RNA from the same biopsy core yields nucleic acids of optimal concentration and quality for downstream genomic applications. Conclusion: The collection of biospecimens using pre-determined protocol-specific standard operating procedures (SOPs) is essential to control for pre-analytical variability inherent to multicenter trials. Furthermore, histological control of percent tumor cells in each biopsy is absolutely necessary to ensure optimal representation of tumor (>70%) in the specimen. The above conditions were used in the multicenter Q-CROC-01 study ([NCT00984048][1]), where three needle core biopsies are collected from liver metastases of patients with colorectal cancer. One biopsy is collected in formalin and is set aside for downstream immunohistochemistry experiments. Two biopsies are collected in RNAlater and verified for histology. If they pass quality control, both DNA and RNA are isolated concurrently and sent to discovery platforms (DNA: array comparative genomic hybridization (aCGH), methylation profiles, RNA: gene expression profiles, RT-PCR, microRNA profiles, alternative splicing profiles). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3389. doi:1538-7445.AM2012-3389 [1]: /lookup/external-ref?link_type=CLINTRIALGOV&access_num=NCT00984048&atom=%2Fcanres%2F72%2F8_Supplement%2F3389.atom

Approximately 15-20% of all invasive breast carcinomas overexpress the HER-2 receptor, a membrane surface-bound receptor tyrosine kinase upstream of critical proliferation and cell survival pathways. Trastuzumab, a humanized monoclonal antibody to the extracellular domain of HER-2, has proven to be a beneficial treatment for patients diagnosed as HER-2+, but does have its limitations. A majority of advanced HER-2 positive breast cancer patients will develop resistance to the therapy within the first year while many others do not respond to the drug when used alone. The purpose of this study was to determine whether AZD8931, a reversible pan-ERBB inhibitor that equipotently inhibits the tyrosine kinase-activity of EGFR, HER-2, and HER-3, could reverse this resistance in established trastuzumab-resistant BT474 and SKBR3 cell lines. To account for tumor heterogeneity in HER-2+ tumors, response to AZD8931 was examined in both ER-positive (BT474) and ER-negative (SKBR3) cell lines. Proliferation assays were used to assess the efficacy of AZD8931 alone and in combination with trastuzumab. Results showed significantly diminished cell growth in all tested cell lines, including those resistant to trastuzumab, at clinically relevant concentrations of AZD8931 ( Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 770. doi:1538-7445.AM2012-770

EphrinB transmembrane ligands and their cognate EphB receptors critically contribute to vascular development through cell-to-cell signaling by the EphB receptor (forward signaling) and by the EphrinB ligand (reverse signaling). Previous studies demonstrated that mutant mice lacking ephrinB2 or EphB4 and a proportion of mice lacking EphB2 and EphB3 display severe defects in the embryonic vascular system and die at the embryo stage likely due to these vascular defects. Previously, we showed that EphB2, EphB4 and EphrinB2 are required for mature endothelial cell assembly into capillary structures. In the currently studies, we observed that silencing EphrinB2 expression in different types of primary human endothelial cells causes significant and time-dependent apoptotic cell death, as measured by cleavage of Poly(ADP-ribose) Polymerase (PARP), caspase-3 and caspase-8. VEGF, bFGF and SDF-1 variously modulated apoptosis induced by loss of EphrinB2 expression in endothelial cells. Gene expression profiling revealed that EphrinB2 silencing is associated with reduced expression of genes linked to regulation of the cell cycle progression and cytoskeleton organization, and with increased expression of pro-apoptotic genes. Breast cancer tissue from a patient treated with bevacizumab, a humanized monoclonal antibody that neutralizes VEGF, displayed evidence of endothelial cell apoptosis, which was associated with selectively reduced EphrinB2 expression, suggesting a link between VEGF signaling, EphrinB2 expression and cell death. These studies show that EphrinB2 expression is critical to endothelial cell survival in vitro and suggest that EphrinB2 contributes to blood vessel integrity in vivo. Thus, in addition to its well-characterized function as a regulator of cell-to-cell adhesion and repulsion, EphrinB2 is an endothelial pro-survival molecule, which may serve as a novel target for anti-angiogenic therapies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3469. doi:10.1158/1538-7445.AM2011-3469

Background Cancer-associated fibroblasts (CAFs) play an important role in breast cancer pathogenesis by paracrine regulation of breast cancer cell biology. Several in vitro and mouse models have characterized the role of cell contact and cytokine molecules mediating this relationship, although few reports have used human CAFs from breast tumors. Methods Primary breast CAF cultures were established and gene expression profiles analysed in order to guide subsequent co-culture models. We used a combination of colorimetric proliferation assays and gene expression profiling to determine the effect of CAFs on the MCF-7 breast cancer cell in an indirect co-culture system. Results Using gene expression profiling, we found that a subgroup of breast CAFs are positive for a type one interferon response, confirming previous reports of an activated type one interferon response in whole tumor datasets. Interferon positive breast cancer patients show a poor prognostic outcome in an independent microarray dataset. In addition, CAFs positive for the type one interferon response promoted the growth of the MCF-7 breast cancer cell line in an indirect co-culture model. The addition of a neutralizing antibody against the ligand mediating the type one response in fibroblasts, interferon-β, reverted this co-culture phenotype. CAFs not expressing the interferon response genes also promoted the growth of the MCF-7 breast cancer cell line but this phenotype was independent of the type one fibroblast interferon ligand. Conclusions Primary breast CAFs show inter-patient molecular heterogeneity as evidenced by interferon response gene elements activated in a subgroup of CAFs, which result in paracrine pro-proliferative effects in a breast cancer cell line co-culture model. Electronic supplementary material The online version of this article (doi:10.1186/s12885-015-1117-0) contains supplementary material, which is available to authorized users.