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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

Data S2

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

High-grade serous ovarian cancers (HGSOC) have been subdivided into molecular subtypes. The mesenchymal HGSOC subgroup, defined by stromal-related gene signatures, is invariably associated with poor patient survival. We demonstrate that stroma exerts a key function in mesenchymal HGSOC. We highlight stromal heterogeneity in HGSOC by identifying four subsets of carcinoma-associated fibroblasts (CAF-S1-4). Mesenchymal HGSOC show high content in CAF-S1 fibroblasts, which exhibit immunosuppressive functions by increasing attraction, survival, and differentiation of CD25+FOXP3+ T lymphocytes. The beta isoform of the CXCL12 chemokine (CXCL12β) specifically accumulates in the immunosuppressive CAF-S1 subset through a miR-141/200a dependent-mechanism. Moreover, CXCL12β expression in CAF-S1 cells plays a crucial role in CAF-S1 immunosuppressive activity and is a reliable prognosis factor in HGSOC, in contrast to CXCL12α. Thus, our data highlight the differential regulation of the CXCL12α and CXCL12β isoforms in HGSOC, and reveal a CXCL12β-associated stromal heterogeneity and immunosuppressive environment in mesenchymal HGSOC., Cancer-associated fibroblasts (CAFs) are an important part of the tumor microenvironment. Here the authors characterize four subsets of CAFs across human samples of ovarian cancer subtypes and show in the mesenchymal subtype a specific CAF-S1 population that attracts immunosuppressive Tregs via CXCL12β.

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.

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.