Cancer-associated fibroblasts (CAFs) are a key component of the tumour microenvironment with diverse functions, including matrix deposition and remodelling, extensive reciprocal signalling interactions with cancer cells and crosstalk with infiltrating leukocytes. As such, they are a potential target for optimizing therapeutic strategies against cancer. However, many challenges are present in ongoing attempts to modulate CAFs for therapeutic benefit. These include limitations in our understanding of the origin of CAFs and heterogeneity in CAF function, with it being desirable to retain some antitumorigenic functions. On the basis of a meeting of experts in the field of CAF biology, we summarize in this Consensus Statement our current knowledge and present a framework for advancing our understanding of this critical cell type within the tumour microenvironment.

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A framework for advancing our understanding of cancer-associated fibroblasts

The extracellular matrix is a fundamental, core component of all tissues and organs, and is essential for the existence of multicellular organisms. From the earliest stages of organism development until death, it regulates and fine-tunes every cellular process in the body. In cancer, the extracellular matrix is altered at the biochemical, biomechanical, architectural and topographical levels, and recent years have seen an exponential increase in the study and recognition of the importance of the matrix in solid tumours. Coupled with the advancement of new technologies to study various elements of the matrix and cell-matrix interactions, we are also beginning to see the deployment of matrix-centric, stromal targeting cancer therapies. This Review touches on many of the facets of matrix biology in solid cancers, including breast, pancreatic and lung cancer, with the aim of highlighting some of the emerging interactions of the matrix and influences that the matrix has on tumour onset, progression and metastatic dissemination, before summarizing the ongoing work in the field aimed at developing therapies to co-target the matrix in cancer and cancer metastasis.

The matrix in cancer.

The epithelial-to-mesenchymal transition (EMT) is a cell biological program that confers mesenchymal traits on carcinoma cells and drives their metastatic dissemination. It is unclear, however, whether the activation of EMT in carcinoma cells can change their susceptibility to immune attack. We demonstrate here that mammary tumor cells arising from more epithelial carcinoma cell lines expressed high levels of MHC-I, low levels of PD-L1, and contained within their stroma CD8+ T cells and M1 (antitumor) macrophages. In contrast, tumors arising from more mesenchymal carcinoma cell lines exhibiting EMT markers expressed low levels of MHC-I, high levels of PD-L1, and contained within their stroma regulatory T cells, M2 (protumor) macrophages, and exhausted CD8+ T cells. Moreover, the more mesenchymal carcinoma cells within a tumor retained the ability to protect their more epithelial counterparts from immune attack. Finally, epithelial tumors were more susceptible to elimination by immunotherapy than corresponding mesenchymal tumors. Our results identify immune cells and immunomodulatory markers that can be potentially targeted to enhance the susceptibility of immunosuppressive tumors to various therapeutic regimens. Cancer Res; 77(15); 3982-9. ©2017 AACR.

Epithelial-to-Mesenchymal Transition Contributes to Immunosuppression in Breast Carcinomas

The Application of Ferroptosis in Diseases

Natural products targeting the PI3K-Akt-mTOR signaling pathway in cancer: A novel therapeutic strategy.

The ability of breast cancer cells to interconvert between epithelial and mesenchymal states contributes to their metastatic potential. As opposed to cell autonomous effects, the impact of epithelial–mesenchymal plasticity (EMP) on primary and metastatic tumor microenvironments remains poorly characterized. Herein we utilize global gene expression analyses to characterize a metastatic model of EMP as compared to their non-metastatic counterparts. Using this approach, we demonstrate that upregulation of the extracellular matrix crosslinking enzyme tissue transglutaminase-2 (TG2) is part of a novel gene signature that only emerges in metastatic cells that have undergone induction and reversion of epithelial–mesenchymal transition (EMT). Consistent with our model system, patient survival is diminished when primary tumors demonstrate enhanced levels of TG2 in conjunction with its substrate, fibronectin. Targeted depletion of TG2 inhibits metastasis, while overexpression of TG2 is sufficient to enhance this process. In addition to being present within cells, we demonstrate a robust increase in the amount of TG2 and crosslinked fibronectin present within extracellular vesicle (EV) fractions derived from metastatic breast cancer cells. Confocal microscopy of these EVs suggests that FN undergoes fibrillogenesis on their surface via a TG2 and Tensin1-dependent process. Upon in vivo administration, the ability of tumor-derived EVs to induce metastatic niche formation and enhance subsequent pulmonary tumor growth requires the presence and activity of TG2. Finally, we develop a novel 3D model of the metastatic niche to demonstrate that conditioning of pulmonary fibroblasts via pretreatment with tumor-derived EVs promotes subsequent growth of breast cancer cells in a TG2-dependent fashion. Overall, our studies illustrate a novel mechanism through which EMP contributes to metastatic niche development and distant metastasis via tumor-derived EVs containing aberrant levels of TG2 and fibrillar FN.

Transglutaminase-2 facilitates extracellular vesicle-mediated establishment of the metastatic niche.

Both epithelial-mesenchymal transition (EMT) and mesenchymal-epithelial transition (MET) are linked to metastasis via their ability to increase invasiveness and enhance tumor-initiating capacity. Growth factors, cytokines, and chemotherapies present in the tumor microenvironment (TME) are capable of inducing EMT, but the role of the extracellular matrix (ECM) in this process remains poorly understood. Here, a novel tessellated three-dimensional (3D) polymer scaffolding is used to produce a fibrillar fibronectin matrix that induces an EMT-like event that includes phosphorylation of STAT3 and requires expression of β1 integrin. Consistent with these findings, analysis of the METABRIC dataset strongly links high-level fibronectin (FN) expression to decreased patient survival. In contrast, in vitro analysis of the MCF-10A progression series indicated that intracellular FN expression was associated with nonmetastatic cells. Therefore, differential bioluminescent imaging was used to track the metastasis of isogenic epithelial and mesenchymal cells within heterogeneous tumors. Interestingly, mesenchymal tumor cells do not produce a FN matrix and cannot complete the metastatic process, even when grown within a tumor containing epithelial cells. However, mesenchymal tumor cells form FN-containing cellular fibrils capable of supporting the growth and migration of metastatic-competent tumor cells. Importantly, depletion of FN allows mesenchymal tumor cells to regain epithelial characteristics and initiate in vivo tumor growth within a metastatic microenvironment.Implications: In contrast to the tumor-promoting functions of fibronectin within the ECM, these data suggest that autocrine fibronectin production inhibits the metastatic potential of mesenchymal tumor cells. Mol Cancer Res; 16(10); 1579-89. ©2018 AACR.

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Autocrine Fibronectin Inhibits Breast Cancer Metastasis.

In breast cancer (BC), tissue stiffening via fibronectin (FN) and collagen accumulation is associated with advanced disease progression at both the primary tumor and metastatic sites. Here, we evaluate FN production in 15 BC cell lines, representing a variety of subtypes, phenotypes, metastatic potentials, and chemotherapeutic sensitivities. We demonstrate that intracellular and soluble FN is initially lost during tumorigenic transformation but is rescued in all lines with epithelial-mesenchymal plasticity (EMP). Importantly, we establish that no BC cell line was able to independently organize a robust FN matrix. Non-transformed mammary epithelial cells were also unable to deposit FN matrices unless transglutaminase 2, a FN crosslinking enzyme, was overexpressed. Instead, BC cells manipulated the FN matrix production of fibroblasts in a phenotypic-dependent manner. In addition, varied accumulation levels were seen depending if the fibroblasts were conditioned to model paracrine signaling or endocrine signaling of the metastatic niche. In the former, fibroblasts conditioned by BC cultures with high EMP resulted in the largest FN matrix accumulation. In contrast, mesenchymal BC cells produced extracellular vesicles (EV) that resulted in the highest levels of matrix formation by conditioned fibroblasts. Overall, we demonstrate a dynamic relationship between tumor and stromal cells within the tumor microenvironment, in which the levels and fibrillarization of FN in the extracellular matrix are modulated during the particular stages of disease progression.

The Dynamic Relationship of Breast Cancer Cells and Fibroblasts in Fibronectin Accumulation at Primary and Metastatic Tumor Sites.

Metastatic breast cancer (MBC) is an extremely recalcitrant disease capable of bypassing current targeted therapies via engagement of several growth promoting pathways. SH2 containing protein tyrosine phosphatase-2 (SHP2) is an oncogenic phosphatase known to facilitate growth and survival signaling downstream of numerous receptor inputs. Herein, we used inducible genetic depletion and two distinct pharmacological inhibitors to investigate the therapeutic potential of targeting SHP2 in MBC. Cells that acquired resistance to the ErbB kinase inhibitor, neratinib, displayed increased phosphorylation of SHP2 at the Y542 activation site. In addition, higher levels of SHP2 phosphorylation, but not expression, were associated with decreased survival of breast cancer patients. Pharmacological inhibition of SHP2 activity blocked ERK1/2 and AKT signaling generated from exogenous stimulation with FGF2, PDGF, and hGF and readily prevented MBC cell growth induced by these factors. SHP2 was also phosphorylated upon engagement of the extracellular matrix (ECM) via focal adhesion kinase. Consistent with the potential of SHP2-targeted compounds as therapeutic agents, the growth inhibitory property of SHP2 blockade was enhanced in ECM-rich 3D culture environments. In vivo blockade of SHP2 in the adjuvant setting decreased pulmonary metastasis and extended the survival of systemic tumor-bearing mice. Finally, inhibition of SHP2 in combination with FGFR-targeted kinase inhibitors synergistically blocked the growth of MBC cells. Overall, our findings support the conclusion that SHP2 constitutes a shared signaling node allowing MBC cells to simultaneously engage a diversity of growth and survival pathways, including those derived from the ECM.

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SHP2 is a multifunctional therapeutic target in drug resistant metastatic breast cancer

The ability of breast cancer cells to interconvert between epithelial and mesenchymal states contributes to their metastatic potential. As opposed to cell autonomous effects, the impact of epithelial-mesenchymal plasticity (EMP) on primary and metastatic tumor microenvironments remains poorly characterized. Herein we utilize global gene expression analyses to characterize a metastatic model of EMP as compared to their non-metastatic counterparts. Using this approach, we demonstrate that upregulation of the extracellular matrix crosslinking enzyme tissue transglutaminase-2 (TG2) is part of a novel gene signature that only emerges in metastatic cells that have undergone induction and reversion of epithelial-mesenchymal transition (EMT). Consistent with our model system, patient survival is diminished when primary tumors demonstrate enhanced levels of TG2 in conjunction with its substrate, fibronectin. Targeted depletion of TG2 inhibits metastasis, while overexpression of TG2 is sufficient to enhance this process. In addition to being present within cells, we demonstrate a robust increase in the amount of TG2 and crosslinked fibronectin present within extracellular vesicle (EV) fractions derived from metastatic breast cancer cells. Confocal microscopy of these EVs suggests that FN undergoes fibrillogenesis on their surface via a TG2 and Tensin1-dependent process. Upon in vivo administration, the ability of tumor-derived EVs to induce metastatic niche formation and enhance subsequent pulmonary tumor growth requires the presence and activity of TG2. Finally, we develop a novel 3D model of the metastatic niche to demonstrate that conditioning of pulmonary fibroblasts via pretreatment with tumor-derived EVs promotes subsequent growth of breast cancer cells in a TG2-dependent fashion. Overall, our studies illustrate a novel mechanism through which EMP contributes to metastatic niche development and distant metastasis via tumor-derived EVs containing aberrant levels of TG2 and fibrillar FN. HighlightsO_LICells that undergo EMT and MET return to a phenotype that is unique from the original state C_LIO_LIExpression of transglutaminase 2 (TG2) is part of this secondary epithelial signature C_LIO_LITG2 is necessary for fibronectin crosslinking on extracellular vesicles (EVs) C_LIO_LITG2 and fibrillar fibronectin enhance EV-mediated metastatic niche formation C_LI SummaryExtracellular vesicle (EV) production and content change as epithelial tumor cells undergo epithelial-mesenchymal transition (EMT) and mesenchymal-epithelial transition (MET). Prior to EMT, epithelial tumor cells produce EVs that lack the presence of fibronectin (FN), transglutaminase 2 (TG2), tensin1 (TNS1), and particular integrins. Upon induction of EMT upregulation of integrins and FN allows tumor cells to produce EVs containing high levels of globular fibronectin (G-FN). Upon completion of MET, expression of TG2 allows tumor cells to produce EVs that contain crosslinked FN leading to the formation of fibrillar FN (F-FN) via tensin1 (TNS1)-mediated integrin clustering. Equipped with these molecular components tumor cell-derived EVs have an increased capacity to modulate the metastatic niche to support secondary tumor formation. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=98 SRC="FIGDIR/small/875948v1_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@8731bdorg.highwire.dtl.DTLVardef@1d2719dorg.highwire.dtl.DTLVardef@1211f41org.highwire.dtl.DTLVardef@18b09e9_HPS_FORMAT_FIGEXP M_FIG C_FIG

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Sarah Libring

Papers

Transglutaminase-2 facilitates extracellular vesicle-mediated establishment of the metastatic niche.

Autocrine Fibronectin Inhibits Breast Cancer Metastasis.

The Dynamic Relationship of Breast Cancer Cells and Fibroblasts in Fibronectin Accumulation at Primary and Metastatic Tumor Sites.

SHP2 is a multifunctional therapeutic target in drug resistant metastatic breast cancer

Transglutaminase-2 facilitates extracellular vesicle-mediated establishment of the metastatic niche