Breast Cancer Stroma Research Articles

Enhancer reprogramming promotes the activation of cancer-associated fibroblasts and breast cancer metastasis.

Rationale: Cancer associated fibroblasts (CAFs) are a subpopulation of cells within the tumor microenvironment that usually promote cancer progression and metastasis. Hence it is critical to find out the driving factors and mechanisms for the development of CAFs from normal fibroblasts (NFs) in response to sustained stimulation of cancer cells. Here we perform transcriptomic and epigenomic analyses in paired NFs and CAFs associated with breast cancer metastasis to investigate the molecular mechanisms for stromal fibroblasts reprogramming. Methods: We conducted transcriptomic analyses in paired NFs and CAFs isolated from clinical specimens of breast cancer patients with metastasis. Meanwhile, genome-wide mapping of histone marks H3K4me1 and H3K27ac was also performed to characterize CAF-specific enhancer landscape. The function and mechanisms of activated JUN in stromal fibroblasts were studied using in vitro and in vivo models. Results: We have identified CAF-specific signature genes and activated enhancers, which are significantly associated with pro-metastatic programs. Among the CAF activated enhancers, FOS and JUN family of transcription factors are enriched. In line with this, we find that JUN protein is highly activated in the stroma of metastatic breast cancers. And through gain and loss-of-function studies, we demonstrate that activated JUN is necessary and sufficient to remodel enhancers and maintain the activation of CAF-specific enhancers, and thereby promotes breast cancer invasiveness in a non-cell-autonomous manner. Conclusions: Our study gets an insight into the transcriptomic features of invasive breast stroma and transcription regulatory mechanisms for stroma cell transformation, providing a proof-of-concept of stroma-targeting strategy in cancer treatment.

Complex Disease Individual Molecular Characterization Using Infinite Sparse Graphical Independent Component Analysis.

Identifying individual mechanisms involved in complex diseases, such as cancer, is essential for precision medicine. Their characterization is particularly challenging due to the unknown relationships of high-dimensional omics data and their inter-patient heterogeneity. We propose to model individual gene expression as a combination of unobserved molecular mechanisms (molecular components) that may differ between the individuals. Considering a baseline molecular profile common to all individuals, these molecular components may represent molecular pathways differing from the population background. We defined an infinite sparse graphical independent component analysis (isgICA) to identify these molecular components. This model relies on double sparseness: the source matrix sparseness defines the subset of genes involved in each molecular component, whereas the weight matrix sparseness identifies the subset of molecular components associated with each patient. As the number of molecular components is unknown but likely high, we simultaneously inferred it and the weight matrix sparseness using the beta-Bernoulli process (BBP). We simulated data from a double sparse ICA with 10/30 components with specific sparseness structures for 100/500 individuals and 500/1000/5000 genes with different noise variance levels to evaluate the reconstruction of the latent structures by our model. For all simulations, the isgICA was able to reconstruct with higher accuracy than 2 state-of-the-art methods (ica and fastICA) the number of components, the weight and source matrix sparsenesses (correlation simulated/estimated >.8). Applying our model to the expression of 1063 genes of 614 breast cancer patients, the isgICA identified 22 components. According to the source matrix, 7 of these 22 components seemed to be specifically related to 3 known molecular pathways with a prognostic effect in early breast cancer (immune system, proliferation, and stroma invasion). This proposed algorithm provides an insight into individual molecular heterogeneity to better understand complex disease mechanisms.

Adipose Stroma Accelerates the Invasion and Escape of Human Breast Cancer Cells from an Engineered Microtumor.

Approximately 20-25% of human breast tumors are found within an adipose, rather than fibrous, stroma. Adipose stroma is associated with an increased risk of lymph node metastasis, but the causal association between adipose stroma and metastatic progression in human breast cancer remains unclear. We used micropatterned type I collagen gels to engineer ~3-mm-long microscale human breast tumors within a stroma that contains adipocytes and adipose-derived stem cells (ASCs) (collectively, "adipose cells"). Invasion and escape of human breast cancer cells into an empty 120-μm-diameter lymphatic-like cavity was used to model interstitial invasion and vascular escape in the presence of adipose cell-derived factors for up to 16 days. We found that adipose cells hasten invasion and escape by 1-2 days and 2-3 days, respectively. These effects were mediated by soluble factors secreted by the adipose cells, and these factors acted directly on tumor cells. Surprisingly, tumor invasion and escape were more strongly induced by ASCs than by adipocytes. This work reveals that both adipocytes and ASCs accelerate the interstitial invasion and escape of human breast cancer cells, and sheds light on the link between adipose stroma and lymphatic metastasis in human breast cancer. The online version contains supplementary material available at 10.1007/s12195-021-00697-6.

Carcinoma-associated fibroblasts derived exosomes modulate breast cancer cell stemness through exonic circHIF1A by miR-580-5p in hypoxic stress

Hypoxia is a common phenomenon in solid tumors. The roles of exosomes from hypoxic breast cancer stroma are less studied. So, the study was aimed to investigate the role of exosomes from hypoxic cancer-associated fibroblasts (CAFs) cells in breast cancer. The circRNA array analysis was performed to screen differential expressed circRNAs between hypoxic and normoxic CAFs exosomes. Candidate circHIF1A (circ_0032138) was screened out and it was confirmed that circHIF1A was up-regulated in the exosomes from hypoxic CAFs and their exosomes. Through investigating cellular functions including cell proliferation and stem cell features, it was demonstrated that hypoxic CAFs exosomes transferred circHIF1A into breast cancer cells, which played an important role in cancer stem cell properties sponging miR-580-5p by regulating CD44 expression. In a summary, circHIF1A from hypoxic CAFs exosomes played an important role in stem cell properties of breast cancer. CircHIF1A may act as a target molecule of breast cancer therapy.

Gamma-interferon inducible lysosomal thiolreductase (GILT) effect on breast tumor microenvironment through regulating CXCR6/CXCL16 axis.

e15081 Background: Gamma-interferon Inducible Lysosomal Thiolreductase (GILT) is constitutively expressed in most antigens endocytosed by antigen presenting cells (APCs), and its function is to catalyze the reduction of disulfide (S-S) bonds in protein substrates. The cytokine CXCL16 is one of the only two known plasma membrane chemokines which induces chemotaxis of activated T cells and bone marrow plasma cells in tumor microenvironment. It contains a free end folded by two sulfur bonds and therefore could also be a zymolyte of GILT. Previous studies found that specific receptor of CXCL16, CXCR6, was significantly overexpressed in breast cancer tissues and metastatic axillary lymph nodes. We suppose whether CXCR6/CXCL16 axis is regulated by GILT and affect tumor microenvironment, thereby eliciting specific anti-tumor immune responses in breast cancer (BC). Methods: GILT expression in BC was evaluated using publicly available data from The Cancer Genome Atlas (TCGA). GILT gene was analyzed in UALCAN ( http://ualcan.path.uab.edu/analysis-prot.html ) . In vitro, Immunohistochemistry (IHC) was conducted to examine the location and relation of GILT and CXCR6. Gene Set Enrichment Analysis (GSEA) was performed to mine the biological pathways involved in BC related GILT regulatory network. The expression of GILT at protein and RNA levels were detected by Western Blot and RT-PCR assay. Overexpression and knockdown of GILT in BC cell lines was carried out to further analyzed the correlation between GILT and CXCL16/CXCR6. Results: TCGA database showed that GILT expression was increased in the stroma of BC compared with normal, and was correlated to shorter BC overall survival. GSEA suggested that the expression of GILT was associated with chemotactic factors. Pearson analysis and IHC showed GILT had a strong correlation with CXCL16/CXCR6 axis in the aspect of angiogenesis and immunity. qRT-PCR and Western Blot assay also revealed that GILT had high expression in BC. Besides, patients with high expression of GILT in IHC simultaneously showed high immunoreactive to macrophage markers, which was related to neovascularization and anti-tumor immune responses. Compared with the normal breast cell line MCF-10A, GILT protein had high expression in Hs578T and low expression in MDA-MB-231 cell line. GILT was overexpressed in MDA-MB-231 and knockdown in Hs578T. Result showed that high level GILT promoted the production of CXCL16/CXCR6,while GILT siRNA knockdown inhibited the production of CXCL16/CXCR6. Conclusions: GILT could catalyze CXCL16 in BC, function as a key mechanism to affect tumor microenvironment through CXCR6/CXCL16 pathway. GILT-activated CXCL16 levels showed a strong connection with unfavorable outcomes in BC, which could be a potential biomarker of prognosis and a novel therapeutic target.

INFLUENCE OF THE PERITUMORAL MATRIX ON COLON CANCER

Abstract INTRODUCTION Several publications have highlighted the importance of the peritumoral matrix in various epithelial tumors. Our team has pointed out the value of ColXIα1 in the stroma of breast, colon and pancreatic cancer. The possible prognostic factor of the stroma in colon cancer is debated. Our aim is to analyze the prognostic role of the stroma and ColXIα1 in colon adenocarcinoma. MATERIAL AND METHODS Samples of 189 patients with colon cancer were studied by image analysis. The matrix was stained with Gomori green trichrome and the CAFs with the mouse monoclonal anti-proColXIα1 antibody. The samples were evaluated with an optical microscope, obtaining data from the area stained with proColXIα1, the area stained with Gomori and the number of pro ColXIα1 positive cells. 35 Gb of images were analyzed with Image J-FIJI, which measures the number of cells and the total area of marked regions, and also quantifies the number and area of unlabeled cells. Statistical analysis by MedCalc v1o and SpSS v15. RESULTS Colon cancer stages (%): I, 13; II, 40; III, 29; IV, 18. Recurrences (%), 32; by stages: I, 0; II, 21; III, 44; IV, 86. Global survival: median, 28 months; S5, 33%. Total stromal cells, number of CAFs and its area: higher in early stages. Gomori area, smaller in early stages and in cases without recurrences. CONCLUSIONS The number of CAFs and the extension of the stromal area could have influence in the development of the cancer colon, and may be related to the prognosis of disease.

Abstract PO065: Single cell transcriptomics of triple negative breast cancer allografts following chemotherapy treatment reveals increased T cell abundance in doxorubicin-sensitive tumors

Abstract The contribution of stromal cells on drug response in primary tumors remains unclear. To determine how individual cells within the stroma of triple negative breast cancer (TNBC) allografts respond to chemotherapy, we used single cell sequencing to profile cells present in murine tumors with or without exposure to doxorubicin. Doxorubicin is a chemotherapeutic agent commonly used to treat TNBC by inhibiting cancer cell proliferation through intercalation of DNA and preventing topoisomerase II activity. In this study, murine TNBC 4T1 cell line was utilized to generate allograft tumors in immunocompetent BALB/c mice. Syngeneic 4T1 tumors exhibit a range of responsiveness to doxorubicin treatment. Tumor growth rates were monitored throughout the chemotherapeutic regiment then stratified into sensitive or resistant response. Cellular composition and behavior were then analyzed via single cell RNA sequencing (scRNA-seq) and flow cytometry 8 days post-doxorubicin chemotherapeutic administration mimicking clinical treatment. ScRNA-seq revealed decreases in tumor infiltrating lymphocytes after doxorubicin exposure. Furthermore, an increase in T cell abundance was discovered in tumors sensitive to doxorubicin treatment. This finding was further supported by flow cytometric analysis showing a tumor specific increase in all, CD4+, and CD8+ T cell populations relative to resistant tumors. Additionally, T-cell differentiation, exhaustion, and activation states were further examined from scRNA-seq data providing insights into functional properties of the tumor residing T cell populations undergoing chemotherapeutic treatment. Future work will focus on analyzing prognostic value of specific T-cell populations in disease regression following doxorubicin treatment. This study received funding from LLNL LDRD grant 19-SI-003. This work was conducted under the auspices of the USDOE by LLNL (DE-AC52-07NA27344). Citation Format: Nicholas R. Hum, Aimy Sebastian, Sean F. Gilmore, David M. Gravano, Naiomy D. Rios-Arce, Kelly A. Martin, Elizabeth K. Wheeler, Matthew A. Coleman, Gabriela G. Loots. Single cell transcriptomics of triple negative breast cancer allografts following chemotherapy treatment reveals increased T cell abundance in doxorubicin-sensitive tumors [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2020 Oct 19-20. Philadelphia (PA): AACR; Cancer Immunol Res 2021;9(2 Suppl):Abstract nr PO065.

Novel quantitative signature of tumor stromal architecture: polarized light imaging differentiates between myxoid and sclerotic human breast cancer stroma.

As a leading cause of death in women, breast cancer is a global health concern for which personalized therapy remains largely unrealized, resulting in over- or under-treatment. Recently, tumor stroma has been shown to carry important prognostic information, both in its relative abundance and morphology, but its current assessment methods are few and suboptimal. Herein, we present a novel stromal architecture signature (SAS) methodology based on polarized light imaging that quantifies patterns of tumor connective tissue. We demonstrate its ability to differentiate between myxoid and sclerotic stroma, two pathology-derived categories associated with significantly different patient outcomes. The results demonstrate a 97% sensitivity and 88% specificity for myxoid stroma identification in a pilot study of 102 regions of interest from human invasive ductal carcinoma breast cancer surgical specimens (20 patients). Additionally, the SAS numerical score is indicative of the wide range of stromal characteristics within these binary classes and highlights ambiguous mixed-morphology regions prone to misclassification. The enabling polarized light microscopy technique is inexpensive, fast, fully automatable, applicable to fresh or embedded tissue without the need for staining and thus potentially translatable into research and/or clinical settings. The SAS metric yields quantifiable and objective stromal characterization with promise for prognosis in many types of cancers beyond breast carcinoma, enabling researchers and clinicians to further investigate the emerging and important role of stromal architectural patterns in solid tumors.

Single-Cell Transcriptomic Analysis of Tumor-Derived Fibroblasts and Normal Tissue-Resident Fibroblasts Reveals Fibroblast Heterogeneity in Breast Cancer.

Cancer-associated fibroblasts (CAFs) are a prominent stromal cell type in solid tumors and molecules secreted by CAFs play an important role in tumor progression and metastasis. CAFs coexist as heterogeneous populations with potentially different biological functions. Although CAFs are a major component of the breast cancer stroma, molecular and phenotypic heterogeneity of CAFs in breast cancer is poorly understood. In this study, we investigated CAF heterogeneity in triple-negative breast cancer (TNBC) using a syngeneic mouse model, BALB/c-derived 4T1 mammary tumors. Using single-cell RNA sequencing (scRNA-seq), we identified six CAF subpopulations in 4T1 tumors including: 1) myofibroblastic CAFs, enriched for α-smooth muscle actin and several other contractile proteins; 2) ‘inflammatory’ CAFs with elevated expression of inflammatory cytokines; and 3) a CAF subpopulation expressing major histocompatibility complex (MHC) class II proteins that are generally expressed in antigen-presenting cells. Comparison of 4T1-derived CAFs to CAFs from pancreatic cancer revealed that these three CAF subpopulations exist in both tumor types. Interestingly, cells with inflammatory and MHC class II-expressing CAF profiles were also detected in normal breast/pancreas tissue, suggesting that these phenotypes are not tumor microenvironment-induced. This work enhances our understanding of CAF heterogeneity, and specifically targeting these CAF subpopulations could be an effective therapeutic approach for treating highly aggressive TNBCs.

MiR-18a and miR-18b are expressed in the stroma of oestrogen receptor alpha negative breast cancers

BackgroundPreviously, we have shown that miR-18a and miR-18b gene expression strongly correlates with high proliferation, oestrogen receptor -negativity (ER−), cytokeratin 5/6 positivity and basal-like features of breast cancer.MethodsWe investigated the expression and localization of miR-18a and -18b in formalin fixed paraffin embedded (FFPE) tissue from lymph node negative breast cancers (n = 40), by chromogenic in situ hybridization (CISH). The expression level and in situ localization of miR-18a and -18b was assessed with respect to the presence of tumour infiltrating lymphocytes (TILs) and immunohistochemical markers for ER, CD4, CD8, CD20, CD68, CD138, PAX5 and actin. Furthermore, in two independent breast cancer cohorts (94 and 377 patients) the correlation between miR-18a and -18b expression and the relative quantification of 22 immune cell types obtained from the CIBERSORT tool was assessed.ResultsCISH demonstrated distinct and specific cytoplasmic staining for both miR-18a and miR-18b, particularly in the intratumoural stroma and the stroma surrounding the tumour margin. Staining by immunohistochemistry revealed some degree of overlap of miR-18a and -18b with CD68 (monocytes/macrophages), CD138 (plasma cells) and the presence of high percentages of TILs. CIBERSORT analysis showed a strong correlation between M1-macrophages and CD4+ memory activated T-cells with mir-18a and -18b.ConclusionsOur study demonstrates that miR-18a and miR-18b expression is associated with ER- breast tumours that display a high degree of inflammation. This expression is potentially associated specifically with macrophages. These results suggest that miR-18a and miR-18b may play a role in the systemic immunological response in ER− tumours.

Abstract P4-10-16: Reactive stroma and trastuzumab resistance in HER2-positive early breast cancer

Abstract Purpose We investigated the value of reactive stroma as a predictor for trastuzumab resistance in patients with early HER2-positive breast cancer receiving adjuvant therapy. Experimental Design The pathological reactive stroma and the mRNA gene signatures that reflect reactive stroma in 209 HER2-positive breast cancer samples from the FinHer adjuvant trial were evaluated. Levels of stromal gene signatures were determined as a continuous parameter, and pathological reactive stromal findings were defined as stromal predominant breast cancer (SPBC; ≥50% stromal) and correlated with distant disease-free survival (DDFS). Results Gene signatures associated with reactive stroma in HER2-positive early breast cancer (N=209), were significantly associated with trastuzumab resistance in estrogen receptor (ER)-negative tumors (HR=1.27 p-interaction=0.014 [DCN], HR=1.58, p-interaction=0.027 [PLAU], HR=1.71, p-interaction=0.019 [HER2STROMA, novel HER2 stromal signature]), but not in ER-positive tumors (HR=0.73 p-interaction=0.47 [DCN], HR=0.71, p-interaction=0.73 [PLAU], HR=0.84; p-interaction=0.36 [HER2STROMA]). Pathological evaluation of HER2-positive/ER-negative tumors suggested an association between SPBC and trastuzumab resistance. Reactive stroma did not correlate with tumor-infiltrating lymphocytes (TILs), and the expected benefit from trastuzumab in patients with high levels of TILs was pronounced only in tumors with low stromal reactivity (SPBC <50%). Conclusions Reactive stroma in HER2-positive/ER-negative early breast cancer tumors may predict resistance to adjuvant trastuzumab therapy. Citation Format: Amir Sonnenblick, Mali Salmon-Divon, Roberto Salgado, Noam Pondé, Sibylle Loibl, Carsten Denkert, Heikki Joensuu, Pirkko-Liisa Kellokumpu-Lehtinen, Amos Azaria, Sherene Loi, Stefan Michiels, Christos Sotiriou. Reactive stroma and trastuzumab resistance in HER2-positive early breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P4-10-16.

Reactive stroma and trastuzumab resistance in HER2-positive early breast cancer.

We investigated the value of reactive stroma as a predictor for trastuzumab resistance in patients with early HER2-positive breast cancer receiving adjuvant therapy. The pathological reactive stroma and the mRNA gene signatures that reflect reactive stroma in 209 HER2-positive breast cancer samples from the FinHer adjuvant trial were evaluated. Levels of stromal gene signatures were determined as a continuous parameter, and pathological reactive stromal findings were defined as stromal predominant breast cancer (SPBC; ≥50% stromal) and correlated with distant disease-free survival. Gene signatures associated with reactive stroma in HER2-positive early breast cancer (N = 209) were significantly associated with trastuzumab resistance in estrogen receptor (ER)-negative tumors (hazard ratio [HR] = 1.27 p interaction = 0.014 [DCN], HR = 1.58, p interaction = 0.027 [PLAU], HR = 1.71, p interaction = 0.019 [HER2STROMA, novel HER2 stromal signature]), but not in ER-positive tumors (HR = 0.73 p interaction = 0.47 [DCN], HR = 0.71, p interaction = 0.73 [PLAU], HR = 0.84; p interaction = 0.36 [HER2STROMA]). Pathological evaluation of HER2-positive/ER-negative tumors suggested an association between SPBC and trastuzumab resistance. Reactive stroma did not correlate with tumor-infiltrating lymphocytes (TILs), and the expected benefit from trastuzumab in patients with high levels of TILs was pronounced only in tumors with low stromal reactivity (SPBC <50%). In conclusion, reactive stroma in HER2-positive/ER-negative early breast cancer tumors may predict resistance to adjuvant trastuzumab therapy.

Quantitative mast cell analysis and hormone receptor study (ER, PR and HER2/neu) in invasive carcinoma of breast.

Breast cancer constitutes nearly one third of cancers among women. Immune responses caused by neoplastic cells lead to the accumulation of inflammatory cells like mast cells (MCs), macrophages, lymphocytes, and plasma cells around the tumor tissue forming the tumor microenvironment. The study aims at quantifying the role of MCs in different grades of invasive carcinoma of breast with respect to estrogen receptor (ER), progesterone receptor (PR), and Human Epidermal growth factor Receptor 2 (HER2/neu). This study included 60 cases of invasive carcinoma of breast. Toluidine blue staining was used for quantitative MC analysis and correlated with immunohistochemistry analysis for hormonal markers' positivity-ER, PR and HER2/neu. The mean age was 52 years (range: 25-75 years). The average number of MCs in Grade I, II, and III were 24.05, 18.4, and 7.9, respectively, with a significant P value. ER, PR, and HER2/neu positivity was found in 60%, 55%, and 32% of the cases, respectively. ER positivity with mean MC count of 23.55 was found in 36 cases, and 33 cases were positive for PR with a mean MC count of 24.18 and a significant P value. HER2 positive cases were 28 with a mean MC count of 20.82. The presence of MCs in breast cancer is inversely proportional to the grade of tumor, i.e., a maximum number of MCs were seen in low grade tumors. In addition, there is a positive correlation between ER and PR receptor positivity with the presence of MCs in the stroma of breast cancer.

Integrin α11β1 is expressed in breast cancer stroma and associates with aggressive tumor phenotypes

Cancer‐associated fibroblasts are essential modifiers of the tumor microenvironment. The collagen‐binding integrin α11β1 has been proposed to be upregulated in a pro‐tumorigenic subtype of cancer‐associated fibroblasts. Here, we analyzed the expression and clinical relevance of integrin α11β1 in a large breast cancer series using a novel antibody against the human integrin α11 chain. Several novel monoclonal antibodies against the integrin α11 subunit were tested for use on formalin‐fixed paraffin‐embedded tissues, and Ab 210F4B6A4 was eventually selected to investigate the immunohistochemical expression in 392 breast cancers using whole sections. mRNA data from METABRIC and co‐expression patterns of integrin α11 in relation to αSMA and cytokeratin‐14 were also investigated. Integrin α11 was expressed to varying degrees in spindle‐shaped cells in the stroma of 99% of invasive breast carcinomas. Integrin α11 co‐localized with αSMA in stromal cells, and with αSMA and cytokeratin‐14 in breast myoepithelium. High stromal integrin α11 expression (66% of cases) was associated with aggressive breast cancer features such as high histologic grade, increased tumor cell proliferation, ER negativity, HER2 positivity, and triple‐negative phenotype, but was not associated with breast cancer specific survival at protein or mRNA levels. In conclusion, high stromal integrin α11 expression was associated with aggressive breast cancer phenotypes.

Phosphodiesterase 5 (PDE5) Is Highly Expressed in Cancer-Associated Fibroblasts and Enhances Breast Tumor Progression.

The overexpression of phosphodiesterase (PDE) 5 is frequently found in various human cancers, such as those of the breast. However, PDE5’s role in the tumor microenvironment is still unknown. As PDE5 represents a high-value therapeutic target, we investigated whether the expression and function of PDE5 in breast cancer-associated fibroblasts (CAFs) may be clinically relevant to malignant progression. PDE5 expression was increased in human breast cancer stroma compared with normal stroma and was correlated to a shorter overall survival. Treatment of CAFs, isolated from breast tumor biopsies, with selective PDE5 inhibitors inhibited their proliferation, motility, and invasiveness, and negatively controlled tumor–stroma interactions in both ‘in vitro’ and ‘in vivo’ models. PDE5 stable overexpression transformed immortalized mouse embryonic fibroblasts (MEFs) towards an activated fibroblast phenotype, impacting their intrinsic characteristics and paracrine effects on breast cancer cell growth and migration through an enhanced production of the C-X-C motif chemokine 16 (CXCL16). On the other hand, CAF exposure to PDE5 inhibitors was associated with reduced CXCL16 expression and secretion. Importantly, CXCL16 levels in breast cancer stroma showed a strong correlation with PDE5 levels and poor patient outcomes. In conclusion, PDE5 is overexpressed in breast cancer stroma, enhances the tumor-stimulatory activities of fibroblasts, and impacts clinical outcomes; thus, we propose this enzyme as an attractive candidate for prognosis and a potential target for treatments in breast cancer patients.

Tumor Desmoplastic Stroma - Its Utility as A Prognostic Marker for Breast Carcinoma

Background Breast cancer is the second most common malignancy in women, affecting more than 1 million worldwide annually and is the most frequent cause of cancer related deaths in women. Dynamic interactions between neoplastic epithelial cells and the surrounding stroma can select stromal cells that modulate tumor behavior. The interplay between a tumor and its environment is exemplified by the morphological changes observed in the stroma of human breast cancer. Hence in this study we tried to evaluate a grading system for tumor desmoplasia and its role as a prognostic marker for breast cancer. Methods This is a retrospective study including 102 primary invasive breast malignancy diagnosed at a tertiary care hospital between January 2015 to September 2017. The cases with prior chemotherapy and radiotherapy were excluded. In all the cases hematoxylin & eosin stained slides were retrieved and assessed for desmoplastic stromal categorization, tumor grade and axillary lymph nodal involvement. The status of immunohistochemistry for ER, PR &, Her2 neu were tabulated. The association of desmoplastic stroma was evaluated with other known prognostic factors. p value calculated, p < 0.05 considered significant. Results: There was statistically significant association of the type of desmoplastic stroma with the tumor grade, tumor size and metastatic axillary lymphnode. Conclusion: This emphasizes the role of stromal desmoplasia in tumor progression and prognosis. Hence we conclude that a special mention to be made on the type of desmoplasia in the histopathology report of all breast cancer.

Extra domain A-containing fibronectin expression in Spin90-deficient fibroblasts mediates cancer-stroma interaction and promotes breast cancer progression.

Cancer-associated fibroblasts (CAFs) in the tumor microenvironment play major roles in supporting cancer progression. A previous report showed that SPIN90 downregulation is correlated with CAF activation and that SPIN90-deficient CAFs promote breast cancer progression. However, the mechanisms that mediate cancer-stroma interaction and how such interactions regulate cancer progression are not well understood. Here, we show that extra domain A (EDA)-containing fibronectin (FN), FN(+)EDA, produced by mouse embryonic fibroblasts (MEFs) derived from Spin90-knockout (KO) mice increases their own myofibroblast differentiation, which facilitates breast cancer progression. Increased FN(+)EDA in Spin90-KO MEFs promoted fibril formation in the extracellular matrix (ECM) and specifically interacted with integrin α4β1 as the mediating receptor. Moreover, FN(+)EDA expression by Spin90-KO MEFs increased proliferation, migration, and invasion of breast cancer cells. Irigenin, a specific inhibitor of the interaction between integrin α4β1 and FN(+)EDA, significantly blocked the effects of FN(+)EDA, such as fibril formation by Spin90-KO MEFs and proliferation, migration, and invasion of breast cancer cells. In orthotopic breast cancer mouse models, irigenin injection remarkably reduced tumor growth and lung metastases. It was supported by that FN(+)EDA in assembled fibrils was accumulated in cancer stroma of human breast cancer patients in which SPIN90 expression was downregulated. Our data suggest that SPIN90 downregulation increases FN(+)EDA and promotes ECM stiffening in breast cancer stroma through an assembly of long FN(+)EDA-rich fibrils; moreover, engagement of the Integrin α4β1 receptor facilitates breast cancer progression. Inhibitory effects of irigenin on tumor growth and metastasis suggest the potential of this agent as an anticancer therapeutic.

Cancer-associated fibroblast-derived Gremlin 1 promotes breast cancer progression

BackgroundBone morphogenetic proteins (BMPs) have been reported to maintain epithelial integrity and to antagonize the transforming growth factor β (TGFβ)-induced epithelial to mesenchymal transition. The expression of soluble BMP antagonists is dysregulated in cancers and interrupts proper BMP signaling in breast cancer.MethodsIn this study, we mined the prognostic role of BMP antagonists GREMLIN 1 (GREM1) in primary breast cancer tissues using in-house and publicly available datasets. We determined which cells express GREM1 RNA using in situ hybridization (ISH) on a breast cancer tissue microarray. The effects of Grem1 on the properties of breast cancer cells were assessed by measuring the mesenchymal/stem cell marker expression and functional cell-based assays for stemness and invasion. The role of Grem1 in breast cancer-associated fibroblast (CAF) activation was measured by analyzing the expression of fibroblast markers, phalloidin staining, and collagen contraction assays. The role of Grem1 in CAF-induced breast cancer cell intravasation and extravasation was studied by utilizing xenograft zebrafish breast cancer (co-) injection models.ResultsExpression analysis of clinical breast cancer datasets revealed that high expression of GREM1 in breast cancer stroma is correlated with a poor prognosis regardless of the molecular subtype. The large majority of human breast cancer cell lines did not express GREM1 in vitro, but breast CAFs did express GREM1 both in vitro and in vivo. Transforming growth factor β (TGFβ) secreted by breast cancer cells, and also inflammatory cytokines, stimulated GREM1 expression in CAFs. Grem1 abrogated bone morphogenetic protein (BMP)/SMAD signaling in breast cancer cells and promoted their mesenchymal phenotype, stemness, and invasion. Moreover, Grem1 production by CAFs strongly promoted the fibrogenic activation of CAFs and promoted breast cancer cell intravasation and extravasation in co-injection xenograft zebrafish models.ConclusionsOur results demonstrated that Grem1 is a pivotal factor in the reciprocal interplay between breast cancer cells and CAFs, which promotes cancer cell invasion. Targeting Grem1 could be beneficial in the treatment of breast cancer patients with high Grem1 expression.

Stromal Markers of Breast Cancer Progression: A Review of Recent Findings

Breast cancer research has long focused on breast epithelium in cancer development. This traditional view is being challenged by more recent work which suggests that stromal components such as fibroblasts, immune cells, endothelial cells, and others may play a central role in cancer development. The purpose of this review is to investigate the elements of the breast stroma and their interaction with the epithelium in creating the necessary pro-tumorigenic environment. Several studies have revealed that breast cancer stroma differs significantly from normal breast stroma, with altered cellular and molecular factors that may serve as potential therapeutic targets in breast cancer treatment. Studies of the complex interactions within the cancer ecosystem have revealed tumor-associated stromal changes that promote cell growth and invasiveness. They also reveal specific targets for novel strategies to inhibit inflammation, immunosuppression, angiogenesis, and distant invasion.

Abstract 130: Characterization of the tumor microenvironment using single cell transcriptomics of triple negative breast cancer allografts treated with doxorubicin

Abstract It is currently unclear how stromal components affect drug response and the emergence of drug resistance, in primary tumors. To determine how individual cells within the stroma of triple negative breast cancer (TNBC) allografts respond to chemotherapy, we used single cell sequencing to profile individual cells present in murine tumors with or without exposure to doxorubicin (Dox). Dox is a common chemotherapeutic agent used to treat breast cancer which inhibits breast cancer proliferation by intercalating into DNA and preventing topoisomerase II activity. Several autonomous processes have been implicated in the development of chemoresistance yet the impact of stromal and immune cells on tumor progression is still poorly understood. In this study, TNBC 4T1 cell line were utilized to generate murine allograft tumors in immunocompetent BALB/c mice. Tumor composition was analyzed via single cell RNA sequencing after 3 and 7 days of doxorubicin chemotherapeutic regiment mimicking clinical treatment. Using Cell Ranger single cell software suite and Seurat R toolkit, single cell transcriptomic analysis identified the cellular composition of tumors through expression of cell-type specific genes. Stromal cell types such as endothelial, fibroblast and epithelial cells were assessed and quantified in the tumor microenvironment. Immune cell types including neutrophils, monocytes, macrophages, T-cells and B-cells were also identified in the stroma and the responses to doxorubicin treatment was determined based on the gene expression changes. In this study, cancer-associated fibroblasts and non-canonical tumor associated macrophage subpopulations are of particular interest. As expected, we found both qualitative and quantitative changes in specific subpopulations of stromal cells in response to Dox exposure. Identification of stromal and immune cell sub-types could also lead to improved diagnostic capabilities and tumor susceptibilities. Future studies modulating non-cancerous cells in the tumor microenvironment may increase efficacy of chemotherapeutics. Further elucidating the specific cellular subpopulations within the tumor microenvironment that shift in response to drug exposure may provide new therapeutic avenues. Understanding changes in cell populations within the drug exposed tumor microenvironment can aid in future drug development to specifically target cells least sensitive to chemotherapy exposure. This study received funding from LLNL LDRD grant 19-SI-003. This work was conducted under the auspices of the USDOE by LLNL (DE-AC52-07NA27344). Citation Format: Nicholas Hum, Aimy Sebastian, Sean Gilmore, Elizabeth K. Wheeler, Matthew A. Coleman, Gabriela G. Loots. Characterization of the tumor microenvironment using single cell transcriptomics of triple negative breast cancer allografts treated with doxorubicin [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 130.