Cancer-associated fibroblast heterogeneity in axillary lymph nodes drives metastases in breast cancer through complementary mechanisms.

Although fibroblast heterogeneity is recognized in primary tumors, both its characterization in and its impact on metastases remain unknown. Here, combining flow cytometry, immunohistochemistry and RNA-sequencing on breast cancer samples, we identify four Cancer-Associated Fibroblast (CAF) subpopulations in metastatic lymph nodes (LN). Two myofibroblastic subsets, CAF-S1 and CAF-S4, accumulate in LN and correlate with cancer cell invasion. By developing functional assays on primary cultures, we demonstrate that these subsets promote metastasis through distinct functions. While CAF-S1 stimulate cancer cell migration and initiate an epithelial-to-mesenchymal transition through CXCL12 and TGFß pathways, highly contractile CAF-S4 induce cancer cell invasion in 3-dimensions via NOTCH signaling. Patients with high levels of CAFs, particularly CAF-S4, in LN at diagnosis are prone to develop late distant metastases. Our findings suggest that CAF subset accumulation in LN is a prognostic marker, suggesting that CAF subsets could be examined in axillary LN at diagnosis.

A new biomimetic assay reveals the temporal role of matrix stiffening in cancer cell invasion.

Tumor initiation and growth is associated with significant changes in the surrounding tissue. During carcinoma progression, a global stiffening of the extracellular matrix is observed and is interpreted as a signature of aggressive invasive tumors. However, it is still unknown whether this increase in matrix rigidity promotes invasion and whether this effect is constant along the course of invasion. Here we have developed a biomimetic in vitro assay that enabled us to address the question of the importance of tissue rigidity in the chronology of tumor invasion. Using low concentrations of the sugar threose, we can effectively stiffen reconstituted collagen I matrices and control the stiffening in time with no direct effect on residing cells. Our findings demonstrate that, depending on the timing of its stiffening, the extracellular matrix could either inhibit or promote cancer cell invasion and subsequent metastasis: while matrix stiffening after the onset of invasion promotes cancer cell migration and tumor spreading, stiff matrices encapsulate the tumor at an early stage and prevent cancer cell invasion. Our study suggests that adding a temporal dimension in in vitro models to analyze biological processes in four dimensions is necessary to fully capture their complexity.

Author Correction: Cancer-associated fibroblasts induce metalloprotease-independent cancer cell invasion of the basement membrane.

In the original version of this Article, financial support and contributions in manuscript preparation were not fully acknowledged. The PDF and HTML versions of the Article have now been corrected to include the following:'M.P. and P.O. would like to thank Prof. Roderick Y.H. Lim for advice during manuscript preparation and for providing the laboratory and microscopy infrastructure.… [We also thank] the NanoteraProject, awarded to the PATLiSciII Consortium (M.P and P.O)…'.

Cancer-associated fibroblasts induce metalloprotease-independent cancer cell invasion of the basement membrane.

At the stage of carcinoma in situ, the basement membrane (BM) segregates tumor cells from the stroma. This barrier must be breached to allow dissemination of the tumor cells to adjacent tissues. Cancer cells can perforate the BM using proteolysis; however, whether stromal cells play a role in this process remains unknown. Here we show that an abundant stromal cell population, cancer-associated fibroblasts (CAFs), promote cancer cell invasion through the BM. CAFs facilitate the breaching of the BM in a matrix metalloproteinase-independent manner. Instead, CAFs pull, stretch, and soften the BM leading to the formation of gaps through which cancer cells can migrate. By exerting contractile forces, CAFs alter the organization and the physical properties of the BM, making it permissive for cancer cell invasion. Blocking the ability of stromal cells to exert mechanical forces on the BM could therefore represent a new therapeutic strategy against aggressive tumors.Stromal cells play various roles in tumor establishment and metastasis. Here the authors, using an ex-vivo model, show that cancer-associated fibroblasts facilitate colon cancer cells invasion in a matrix metalloproteinase-independent manner, likely by pulling and stretching the basement membrane to form gaps.

Cancer-associated fibroblasts lead tumor invasion through integrin-ß3-dependent fibronectin assembly.

Cancer-associated fibroblasts (CAFs) are the most abundant cells of the tumor stroma. Their capacity to contract the matrix and induce invasion of cancer cells has been well documented. However, it is not clear whether CAFs remodel the matrix by other means, such as degradation, matrix deposition, or stiffening. We now show that CAFs assemble fibronectin (FN) and trigger invasion mainly via integrin-αvß3. In the absence of FN, contractility of the matrix by CAFs is preserved, but their ability to induce invasion is abrogated. When degradation is impaired, CAFs retain the capacity to induce invasion in an FN-dependent manner. The level of expression of integrins αv and ß3 and the amount of assembled FN are directly proportional to the invasion induced by fibroblast populations. Our results highlight FN assembly and integrin-αvß3 expression as new hallmarks of CAFs that promote tumor invasion.

Microfluidic-Based Generation of 3D Collagen Spheres to Investigate Multicellular Spheroid Invasion.

During tumor progression, cancer cells acquire the ability to escape the primary tumor and invade adjacent tissues. They migrate through the stroma to reach blood or lymphatics vessels that will allow them to disseminate throughout the body and form metastasis at distant organs. To assay invasion capacity of cells in vitro, multicellular spheroids of cancer cells, mimicking primary tumor, are commonly embedded in collagen I extracellular matrix, which mimics the stroma. However, due to their higher density, spheroids tend to sink at the bottom of the collagen droplets, resulting in the spreading of the cells on two dimensions. We developed an innovative method based on droplet microfluidics to embed and control the position of multicellular spheroids inside spherical droplets of collagen. In this method cancer cells are exposed to a uniform three-dimensional (3D) collagen environment resulting in 3D cell invasion.

SHARPIN regulates collagen architecture and ductal outgrowth in the developing mouse mammary gland.

SHARPIN is a widely expressed multifunctional protein implicated in cancer, inflammation, linear ubiquitination and integrin activity inhibition; however, its contribution to epithelial homeostasis remains poorly understood. Here, we examined the role of SHARPIN in mammary gland development, a process strongly regulated by epithelial-stromal interactions. Mice lacking SHARPIN expression in all cells (Sharpincpdm), and mice with a stromal (S100a4-Cre) deletion of Sharpin, have reduced mammary ductal outgrowth during puberty. In contrast, Sharpincpdm mammary epithelial cells transplanted in vivo into wild-type stroma, fully repopulate the mammary gland fat pad, undergo unperturbed ductal outgrowth and terminal differentiation. Thus, SHARPIN is required in mammary gland stroma during development. Accordingly, stroma adjacent to invading mammary ducts of Sharpincpdm mice displayed reduced collagen arrangement and extracellular matrix (ECM) stiffness. Moreover, Sharpincpdm mammary gland stromal fibroblasts demonstrated defects in collagen fibre assembly, collagen contraction and degradation in vitro Together, these data imply that SHARPIN regulates the normal invasive mammary gland branching morphogenesis in an epithelial cell extrinsic manner by controlling the organisation of the stromal ECM.

The hallmarks of CAFs in cancer invasion.

The ability of cancer cells to move out of the primary tumor and disseminate through the circulation to form metastases is one of the main contributors to poor patient outcome. The tumor microenvironment provides a niche that supports cancer cell invasion and proliferation. Carcinoma-associated fibroblasts (CAFs) are a highly enriched cell population in the tumor microenvironment that plays an important role in cancer invasion. However, it remains unclear whether CAFs directly stimulate cancer cell invasion or they remodel the extracellular matrix to make it more permissive for invasion. Here we discuss paracrine communication between cancer cells and CAFs that promotes tumor invasion but also stimulates CAFs to remodel the matrix increasing cancer dissemination.