Ori Maller

Extracellular Matrix Composition Reveals Complex and Dynamic Stromal-Epithelial Interactions in the Mammary Gland

The mammary gland is an excellent model system to study the interplay between stroma and epithelial cells because of the gland’s unique postnatal development and its distinct functional states. This review focuses on the contribution of the extracellular matrix (ECM) to stromal-epithelial interactions in the mammary gland. We describe how ECM physical properties, protein composition, and proteolytic state impact mammary gland architecture as well as provide instructive cues that influence the function of mammary epithelial cells during pubertal gland development and throughout adulthood. Further, based on recent proteomic analyses of mammary ECM, we describe known mammary ECM proteins and their potential functions, as well as describe several ECM proteins not previously recognized in this organ. ECM proteins are discussed in the context of the morphologically-distinct stromal subcompartments: the basal lamina, the intra- and interlobular stroma, and the fibrous connective tissue. Future studies aimed at in-depth qualitative and quantitative characterization of mammary ECM within these various subcompartments is required to better elucidate the function of ECM in normal as well as in pathological breast tissue.

An In-solution Ultrasonication-assisted Digestion Method for Improved Extracellular Matrix Proteome Coverage

Epithelial cell behavior is coordinated by the composition of the surrounding extracellular matrix (ECM); thus ECM protein identification is critical for understanding normal biology and disease states. Proteomic analyses of ECM proteins have been hindered by the insoluble and digestion-resistant nature of ECM. Here we explore the utility of combining rapid ultrasonication- and surfactant-assisted digestion for the detailed proteomics analysis of ECM samples. When compared with traditional overnight digestion, this optimized method dramatically improved the sequence coverage for collagen I, revealed the presence of hundreds of previously unidentified proteins in Matrigel, and identified a protein profile for ECM isolated from rat mammary glands that was substantially different from that found in Matrigel. In a three-dimensional culture assay to investigate epithelial cell-ECM interactions, mammary epithelial cells were found to undergo extensive branching morphogenesis when plated with mammary gland-derived matrix in comparison with Matrigel. Cumulatively these data highlight the tissue-specific nature of ECM composition and function and underscore the need for optimized techniques, such as those described here, for the proteomics characterization of ECM samples.

Collagen architecture in pregnancy-induced protection from breast cancer

Summary The reduction in breast cancer risk attributed to early-age pregnancy is mediated in part by changes in the mammary epithelium. Here, we address the role of the mammary stroma in this protection. Utilizing tumor cells capable of transitioning from indolent to proliferative or invasive states, we demonstrate that mammary extracellular matrix (ECM) from parous rats (parous matrix) decreases tumor growth and impedes cellular phenotypes associated with tumor cell invasion compared with that observed using nulliparous matrix. Proteomic analysis identifies an increased abundance of collagen I in parous matrix, an observation extended to breast tissue of parous women. Given the pro-tumorigenic attributes of fibrillar collagen, these results were unexpected. Second-harmonic generation imaging and atomic force microscopy revealed that the abundant collagen observed in the mammary glands of parous rats is less linearized and associated with a decrease in stromal stiffness, implicating collagen organization and stiffness in parity-induced protection. Using 3D cell culture models, we demonstrate that linearized (fibrillar) collagen I induces cellular phenotypes consistent with an invasive behavior in mammary tumor cells and alters the subcellular distribution of &bgr;1 integrin. Conversely, high-density non-fibrillar collagen I induces tumor-suppressive attributes, including increases in junctional E-cadherin in tumor cells, upregulation of genes encoding components of cell–cell junctions, and downregulation of mesenchymal-specific and metalloproteinase-encoding genes. These data show that collagen organization, rather than density alone, is a key contributor to the invasive phenotype. Furthermore, our data show that parity alters the composition and organization of mammary ECM, particularly fibrillar collagen, in a manner consistent with tumor suppression.

Tamoxifen induces pleiotrophic changes in mammary stroma resulting in extracellular matrix that suppresses transformed phenotypes

IntroductionThe functional unit of the mammary gland has been defined as the epithelial cell plus its microenvironment, a hypothesis that predicts changes in epithelial cell function will be accompanied by concurrent changes in mammary stroma. To test this hypothesis, the question was addressed of whether mammary stroma is functionally altered by the anti-oestrogen drug tamoxifen.MethodsForty female rats at 70 days of age were randomised to two groups of 20 and treated with 1.0 mg/kg tamoxifen or vehicle subcutaneously daily for 30 days, followed by a three-day wash out period. Mammary tissue was harvested and effects of tamoxifen on mammary epithelium and stroma determined.ResultsAs expected, tamoxifen suppressed mammary alveolar development and mammary epithelial cell proliferation. Primary mammary fibroblasts isolated from tamoxifen-treated rats displayed a three-fold decrease in motility and incorporated less fibronectin in their substratum in comparison to control fibroblasts; attributes indicative of fibroblast quiescence. Immunohistochemistry analysis of CD68, a macrophage lysosomal marker, demonstrated a reduction in macrophage infiltration in mammary glands of tamoxifen-treated rats. Proteomic analyses by mass spectrometry identified several extracellular matrix (ECM) proteins with expression levels with tamoxifen treatment that were validated by Western blot. Mammary tissue from tamoxifen-treated rats had decreased fibronectin and increased collagen 1 levels. Further, ECM proteolysis was reduced in tamoxifen-treated rats as detected by reductions in fibronectin, laminin 1, laminin 5 and collagen 1 cleavage fragments. Consistent with suppression in ECM proteolysis with tamoxifen treatment, matrix metalloproteinase-2 levels and activity were decreased. Biochemically extracted mammary ECM from tamoxifen-treated rats suppressed in vitro macrophage motility, which was rescued by the addition of proteolysed collagen or fibronectin. Mammary ECM from tamoxifen-treated rats also suppressed breast tumour cell motility, invasion and haptotaxis, reduced organoid size in 3-dimensional culture and blocked tumour promotion in an orthotopic xenograft model; effects which could be partially reversed by the addition of exogenous fibronectin.ConclusionsThese data support the hypothesis that mammary stroma responds to tamoxifen treatment in concert with the epithelium and remodels to a microenvironment inhibitory to tumour cell progression. Reduced fibronectin levels and reduced ECM turnover appear to be hallmarks of the quiescent mammary microenvironment. These data may provide insight into attributes of a mammary microenvironment that facilitate tumour dormancy.

Quantitative extracellular matrix proteomics to study mammary and liver tissue microenvironments

Normal epithelium exists within a dynamic extracellular matrix (ECM) that is tuned to regulate tissue specific epithelial cell function. As such, ECM contributes to tissue homeostasis, differentiation, and disease, including cancer. Though it is now recognized that the functional unit of normal and transformed epithelium is the epithelial cell and its adjacent ECM, we lack a basic understanding of tissue-specific ECM composition and abundance, as well as how physiologic changes in ECM impact cancer risk and outcomes. While traditional proteomic techniques have advanced to robustly identify ECM proteins within tissues, methods to determine absolute abundance have lagged. Here, with a focus on tissues relevant to breast cancer, we utilize mass spectrometry methods optimized for absolute quantitative ECM analysis. Employing an extensive protein extraction and digestion method, combined with stable isotope labeled Quantitative conCATamer (QconCAT) peptides that serve as internal standards for absolute quantification of protein, we quantify 98 ECM, ECM-associated, and cellular proteins in a single analytical run. In rodent models, we applied this approach to the primary site of breast cancer, the normal mammary gland, as well as a common and particularly deadly site of breast cancer metastasis, the liver. We find that mammary gland and liver have distinct ECM abundance and relative composition. Further, we show mammary gland ECM abundance and relative compositions differ across the reproductive cycle, with the most dramatic changes occurring during the pro-tumorigenic window of weaning-induced involution. Combined, this work suggests ECM candidates for investigation of breast cancer progression and metastasis, particularly in postpartum breast cancers that are characterized by high metastatic rates. Finally, we suggest that with use of absolute quantitative ECM proteomics to characterize tissues of interest, it will be possible to reconstruct more relevant in vitro models to investigate tumor-ECM dynamics at higher resolution.

YAP forces fibroblasts to feel the tension.

Cancer-associated fibroblasts (CAFs) may contribute to tissue tension and cancer progression by increasing extracellular matrix (ECM) deposition and remodelling. However, how CAFs become activated and their roles in tumour mechanics have remained unclear. YAP is now identified as a tension-stimulated CAF activator that promotes malignancy through a mechanically reinforced feed-forward loop.

The Rodent Liver Undergoes Weaning-Induced Involution and Supports Breast Cancer Metastasis

Patients with postpartum breast cancer are at increased risk for metastasis compared with age-matched nulliparous or pregnant patients. Here, we address whether circulating tumor cells have a metastatic advantage in the postpartum host and find the postlactation rodent liver preferentially supports metastasis. Upon weaning, we observed liver weight loss, hepatocyte apoptosis, extracellular matrix remodeling including deposition of collagen and tenascin-C, and myeloid cell influx, data consistent with weaning-induced liver involution and establishment of a prometastatic microenvironment. Using intracardiac and intraportal metastasis models, we observed increased liver metastasis in post-weaning BALB/c mice compared with nulliparous controls. Human relevance is suggested by a ∼3-fold increase in liver metastasis in patients with postpartum breast cancer (n = 564) and by liver-specific tropism (n = 117). In sum, our data reveal a previously unknown biology of the rodent liver, weaning-induced liver involution, which may provide insight into the increased liver metastasis and poor prognosis of women diagnosed with postpartum breast cancer. SIGNIFICANCE We find that patients with postpartum breast cancer are at elevated risk for liver metastasis. We identify a previously unrecognized biology, namely weaning-induced liver involution, that establishes a prometastatic microenvironment, and which may account in part for the poor prognosis of patients with postpartum breast cancer. Cancer Discov; 7(2); 177-87. ©2016 AACR.This article is highlighted in the In This Issue feature, p. 115.

A tension-mediated glycocalyx–integrin feedback loop promotes mesenchymal-like glioblastoma

Glioblastoma multiforme (GBMs) are recurrent lethal brain tumours. Recurrent GBMs often exhibit mesenchymal, stem-like phenotypes that could explain their resistance to therapy. Analyses revealed that recurrent GBMs have increased tension and express high levels of glycoproteins that increase the bulkiness of the glycocalyx. Studies showed that a bulky glycocalyx potentiates integrin mechanosignalling and tissue tension and promotes a mesenchymal, stem-like phenotype in GBMs. Gain- and loss-of-function studies implicated integrin mechanosignalling as an inducer of GBM growth, survival, invasion and treatment resistance, and a mesenchymal, stem-like phenotype. Mesenchymal-like GBMs were highly contractile and expressed elevated levels of glycoproteins that expanded their glycocalyx, and they were surrounded by a stiff extracellular matrix that potentiated integrin mechanosignalling. Our findings suggest that there is a dynamic and reciprocal link between integrin mechanosignalling and a bulky glycocalyx, implying a causal link towards a mesenchymal, stem-like phenotype in GBMs. Strategies to ameliorate GBM tissue tension offer a therapeutic approach to reduce mortality due to GBM.Barnes et al. report a dynamic and reciprocal crosstalk between tissue tension and glycocalyx bulkiness that promotes a mesenchymal, stem-like phenotype in GBM.

Abstract A098: A role for fibrosis in promoting pro-tumor immune response in breast cancer

We established a positive correlation between a fibrotic phenotype in human breast tumors — especially the Her2 and basal-like breast cancer subtypes — and CD45 and CD68 positive immune cell infiltration. We were interested in elucidating how this fibrotic phenotype may influence the immune response. To address this question, we examined if matrix stiffness alters the function of STAT3, a central regulator of tumor inflammation. We hypothesize that tissue fibrosis promotes STAT3 signaling in mammary tumor cells and alter the cytokine milieu to induce a pro-tumor immune response. We found that ECM stiffness directly enhanced STAT3 phosphorylation in tumor cells both in vitro and in vivo. Our data suggest the fibrotic phenotype promotes STAT3 activity, enhancement of which may drive a pro-tumor immune response. Indeed, we observed several alterations in cytokines and immune cell populations upon STAT3 ablation consistent with anti-tumor immune response. Interestingly, our data also suggest STAT3 knockout in tumor cells doesn9t necessary influence immune cell infiltration, but rather their differentiation in mammary tumors. Finally, we investigated if matrix stiffness has potentiated macrophage differentiation when cultured with specific immunosuppressive cytokines. Overall, our work reveals a novel mechanistic insight into how a pro-tumor immune response stems from the interplay between fibrosis and STAT3 signaling in tumor cells. As such, our findings may stimulate an interest in exploring combinational treatment options with anti-fibrotic agents and immunotherapy. Citation Format: Ori Maller, Luke Cassereau, Allison Drain, Brian Ruffell, Irene Acerbi, Miranda Broz, Jennifer Munson, Melody Swartz, Matthew Krummel, Lisa Coussens, Valerie Weaver. A role for fibrosis in promoting pro-tumor immune response in breast cancer [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A098.

Abstract B090: Collagen organization implicated in tumor dormancy

It is known that tumor cells can reside in a dormant state for decades. Data from numerous studies strongly suggest that extracellular matrix (ECM) proteins can impart a dormant phenotype, however little is known regarding how ECM regulates tumor cell dormancy. Given the technical difficulties in studying tumor cell dormancy in the context of secondary lesions (i.e., the searching for a needle in a haystack problem), here we use tumor suppression at the primary site as a model to investigate the role of ECM in breast cancer dormancy. Specifically, we hypothesize that insight into tumor dormancy can be obtained by studying the response of the mammary ECM to tamoxifen treatment and parity, two conditions that protect the mammary epithelium from tumorigenesis. Utilizing breast cancer cell lines capable of transitioning from indolent to proliferative/invasive states, we demonstrate that ECM isolated from tamoxifen treated or parous rat mammary glands decreases tumor growth and impedes invasive phenotypes compared to nulliparous mammary matrix, using both in vitro and in vivo models. Collagen stained tissues and proteomic analysis identified increased abundance of collagen I in tamoxifen treated and parous mammary stroma, an observation extended to breast tissue of parous women. These results are unexpected given the known pro-tumorigenic attributes of fibrillar collagen. Second harmonic generation imaging and atomic force microscopy reveals that the abundant collagen observed in the mammary glands of parous rats is less linearized and has decreased stromal stiffness compared to nulliparous mammary glands. These data implicate collagen organization rather than density in tumor suppression. Using 3D cell culture models, we demonstrate that linearized fibrillar collagen I induces an invasive phenotype in mammary tumor cells through a mechanism dependent on β1 integrin endocytic recycling. Conversely, high density, non-fibrillar collagen I decreases β1 endocytic integrin recycling, increases junctional E-cadherin staining, upregulates adherens and tight junction genes, and downregulates EMT transcription factors and metalloproteinase genes, all attributes consistent with tumor suppression. These data show that collagen organization, rather than collagen density, is a key contributor to the tumor cell invasive phenotype. In conclusion, we demonstrate that collagen | organization is dominant over density in driving tumor cell invasion, an observation that may have implications in understanding how mammographic breast density determines breast cancer risk, as well as how collagen organization contributes to tumor cell dormancy at distant sites. Citation Format: Ori Maller, Kirk C. Hansen, Valerie Weaver, Rytis Prekeris, Pepper Schedin. Collagen organization implicated in tumor dormancy. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr B090.