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BackgroundEarly luminal breast cancer (BC) represents 70% of newly diagnosed BC cases. Among them, small (under 2 cm) BC without lymph node metastasis (classified as T1N0) have been rarely studied, as their prognosis is generally favorable. Nevertheless, up to 5% of luminal T1N0 BC patients relapse with distant metastases that ultimately prove fatal. The aim of our work was to identify the mechanisms involved in metastatic recurrence in these patients.MethodsOur study addresses the role that autonomous and non-autonomous tumor cell features play with regard to distant recurrence in early luminal BC patients. We created a cohort of T1N0 luminal BC patients (tumors between 0.5–2 cm without lymph node metastasis) with metastatic recurrence (“cases”) and corresponding “controls” (without relapse) matched 1:1 on main prognostic factors: age, grade, and proliferation. We deciphered different characteristics of cancer cells and their tumor micro-environment (TME) by deep analyses using immunohistochemistry. We performed in vitro functional assays and highlighted a new mechanism of cooperation between cancer cells and one particular subset of cancer-associated fibroblasts (CAF).ResultsWe found that specific TME features are indicative of relapse in early luminal BC. Indeed, quantitative histological analyses reveal that “cases” are characterized by significant accumulation of a particular CAF subset (CAF-S1) and decrease in CD4+T lymphocytes, without any other association with immune cells. In multivariate analysis, TME features, in particular CAF-S1 enrichment, remain significantly associated with recurrence, thereby demonstrating their clinical relevance. Finally, by performing functional analyses, we demonstrated that CAF-S1 pro-metastatic activity is mediated by the CDH11/osteoblast cadherin, consistent with bones being a major site of metastases in luminal BC patients.ConclusionsThis study shows that distant recurrence in T1N0 BC is strongly associated with the presence of CAF-S1 fibroblasts. Moreover, we identify CDH11 as a key player in CAF-S1-mediated pro-metastatic activity. This is independent of tumor cells and represents a new prognostic factor. These results could assist clinicians in identifying luminal BC patients with high risk of relapse. Targeted therapies against CAF-S1 using anti-FAP antibody or CDH11-targeting compounds might help in preventing relapse for such patients with activated stroma.

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 TGFβ 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., Cancer associated fibroblasts are known to promote the progression of cancer. Here, the authors show that two particular subsets of cancer associated fibroblasts induce metastasis but work via distinct mechanisms including, chemokine signalling and Notch signalling.

A subset of cancer-associated fibroblasts (FAP+/CAF-S1) mediates immunosuppression in breast cancers, but its heterogeneity and its impact on immunotherapy response remain unknown. Here, we identify 8 CAF-S1 clusters by analyzing more than 19,000 single CAF-S1 fibroblasts from breast cancer. We validate the five most abundant clusters by flow cytometry and in silico analyses in other cancer types, highlighting their relevance. Myofibroblasts from clusters 0 and 3, characterized by extracellular matrix proteins and TGFβ signaling, respectively, are indicative of primary resistance to immunotherapies. Cluster 0/ecm-myCAF upregulates PD-1 and CTLA4 protein levels in regulatory T lymphocytes (Tregs), which, in turn, increases CAF-S1 cluster 3/TGFβ-myCAF cellular content. Thus, our study highlights a positive feedback loop between specific CAF-S1 clusters and Tregs and uncovers their role in immunotherapy resistance. Significance: Our work provides a significant advance in characterizing and understanding FAP+ CAF in cancer. We reached a high resolution at single-cell level, which enabled us to identify specific clusters associated with immunosuppression and immunotherapy resistance. Identification of cluster-specific signatures paves the way for therapeutic options in combination with immunotherapies. This article is highlighted in the In This Issue feature, p. 1241

A major challenge in cancer research is the complexity of the tumor microenvironment and the necessity to take into account the host immunological setting. An innovative way to address these problems is to exploit the emerging technology of organ-on-chip to achieve co-cultures in microfluidic devises that recapitulate ex vivo the tumor ecosystem. We generated tumors-on-chip integrating four cell populations (cancer, immune, endothelial cells and fibroblasts) in a 3D collagen matrix, reconstituting HER2 breast cancer ecosystem. By time-lapse microscopy, differential live staining and a novel automated analysis method, we visualized, and quantified the complex dynamics of this ecosystem, in absence or in presence of the drug trastuzumab (Herceptin), a targeted antibody therapy directed against the HER2 receptor. We uncovered the capacity of the drug trastuzumab to specifically promote long cancer-immune interactions (≳ 60 min), recapitulating an anti-tumoral ADCC (antibody-dependent cell-mediated cytotoxicity) immune response. Cancer-associated fibroblasts (CAFs) on the contrary inhibited cancer-immune interactions, antagonizing the action of the trastuzumab. These observations constitute a proof-of-concept that tumors-on-chip are novel powerful platforms that can be exploited to study ex vivo immunocompetent tumor microenvironments, to characterize ecosystem-level responses to anti-cancer drugs, and to dissect the roles of the various cellular components of the stroma.

Comment inBreast cancer: Fibroblast subtypes alter the microenvironment. [Nat Rev Clin Oncol. 2018]; International audience; Carcinoma-associated fibroblasts (CAF) are key players in the tumor microenvironment. Here, we characterize four CAF subsets in breast cancer with distinct properties and levels of activation. Two myofibroblastic subsets (CAF-S1, CAF-S4) accumulate differentially in triple-negative breast cancers (TNBC). CAF-S1 fibroblasts promote an immunosuppressive environment through a multi-step mechanism. By secreting CXCL12, CAF-S1 attracts CD4+CD25+ T lymphocytes and retains them by OX40L, PD-L2, and JAM2. Moreover, CAF-S1 increases T lymphocyte survival and promotes their differentiation into CD25HighFOXP3High, through B7H3, CD73, and DPP4. Finally, in contrast to CAF-S4, CAF-S1 enhances the regulatory T cell capacity to inhibit T effector proliferation. These data are consistent with FOXP3+ T lymphocyte accumulation in CAF-S1-enriched TNBC and show how a CAF subset contributes to immunosuppression.

Summary: A major challenge in cancer research is the complexity of the tumor microenvironment, which includes the host immunological setting. Inspired by the emerging technology of organ-on-chip, we achieved 3D co-cultures in microfluidic devices (integrating four cell populations: cancer, immune, endothelial, and fibroblasts) to reconstitute ex vivo a human tumor ecosystem (HER2+ breast cancer). We visualized and quantified the complex dynamics of this tumor-on-chip, in the absence or in the presence of the drug trastuzumab (Herceptin), a targeted antibody therapy directed against the HER2 receptor. We uncovered the capacity of the drug trastuzumab to specifically promote long cancer-immune interactions (>50 min), recapitulating an anti-tumoral ADCC (antibody-dependent cell-mediated cytotoxicity) immune response. Cancer-associated fibroblasts (CAFs) antagonized the effects of trastuzumab. These observations constitute a proof of concept that tumors-on-chip are powerful platforms to study ex vivo immunocompetent tumor microenvironments, to characterize ecosystem-level drug responses, and to dissect the roles of stromal components. : Inspired by the emerging technology of tumor-on-chip, Nguyen et al. reconstituted ex vivo a human tumor microenvironment (HER2+ breast cancer), characterized the ecosystem-level responses to the drug trastuzumab (Herceptin), and dissected the roles of stromal components (immune cells and fibroblasts), demonstrating the power of immunocompetent tumors-on-chip for preclinical drug studies. Keywords: tumor microenvironment, organ-on-chip, tumor-on-chip, trastuzumab, HER2+ breast cancer, cancer-associated fibroblasts, live cell imaging, microfluidics, pre-clinical models, immunotherapy

Stress has an emerging role in cancer and targeting stress-related β-adrenergic receptors (AR) has been proposed as a potential therapeutic approach in melanoma. Here we report that β3-AR expression correlates with melanoma aggressiveness. In addition, we highlight that β3-AR expression is not only restricted to cancer cells, but it is also expressed in vivo in stromal, inflammatory and vascular cells of the melanoma microenvironment. Particularly, we demonstrated that β3-AR can (i) instruct melanoma cells to respond to environmental stimuli, (ii) enhance melanoma cells response to stromal fibroblasts and macrophages, (iii) increase melanoma cell motility and (iv) induce stem-like traits. Noteworthy, β3-AR activation in melanoma accessory cells drives stromal reactivity by inducing pro-inflammatory cytokines secretion and de novo angiogenesis, sustaining tumor growth and melanoma aggressiveness. β3-ARs also play a mandatory role in the recruitment to tumor sites of circulating stromal cells precursors, in the differentiation of these cells towards different lineages, further favoring tumor inflammation, angiogenesis and ultimately melanoma malignancy. Our findings validate selective β3-AR antagonists as potential promising anti-metastatic agents. These could be used to complement current therapeutic approaches for melanoma patients (e.g. propranolol) by targeting non-neoplastic stromal cells, hence reducing therapy resistance of melanoma.