NLRP3 inflammasome in fibroblasts links tissue damage with inflammation in breast cancer progression and metastasis.

Cancer-Associated Fibroblasts (CAFs) were shown to orchestrate tumour-promoting inflammation in multiple malignancies, including breast cancer. However, the molecular pathways that govern the inflammatory role of CAFs are poorly characterised. In this study we found that fibroblasts sense damage-associated molecular patterns (DAMPs), and in response activate the NLRP3 inflammasome pathway, resulting in instigation of pro-inflammatory signalling and secretion of IL-1ß. This upregulation was evident in CAFs in mouse and in human breast carcinomas. Moreover, CAF-derived inflammasome signalling facilitated tumour growth and metastasis, which was attenuated when NLRP3 or IL-1ß were specifically ablated. Functionally, CAF-derived inflammasome promoted tumour progression and metastasis by modulating the tumour microenvironment towards an immune suppressive milieu and by upregulating the expression of adhesion molecules on endothelial cells. Our findings elucidate a mechanism by which CAFs promote breast cancer progression and metastasis, by linking the physiological tissue damage response of fibroblasts with tumour-promoting inflammation.

Tumor-derived osteopontin reprograms normal mammary fibroblasts to promote inflammation and tumor growth in breast cancer.

Breast tumors are characterized by an extensive desmoplastic stroma, abundantly populated by fibroblasts. Cancer-associated fibroblasts (CAF) support tumorigenesis by stimulating angiogenesis, cancer cell proliferation, and invasion. CAF also orchestrate tumor-promoting inflammation in multiple tumor types, including breast cancer. However, the mechanisms through which normal tissue fibroblasts are reprogrammed to tumor-promoting CAFs are mainly obscure. Here, we show that mammary fibroblasts can be educated by breast cancer cells to become activated to a proinflammatory state that supports malignant progression. Proteomic analysis of breast cancer cell-secreted factors identified the secreted proinflammatory mediator osteopontin, which has been implicated in inflammation, tumor progression, and metastasis. Osteopontin was highly secreted by mouse and human breast cancer cells, and tumor cell-secreted osteopontin activated a CAF phenotypes in normal mammary fibroblasts in vitro and in vivo. Osteopontin was sufficient to induce fibroblast reprogramming and neutralizing antibodies against osteopontin-blocked fibroblast activation induced by tumor cells. The ability of secreted osteopontin to activate mammary fibroblasts relied upon its known receptors CD44 and αVß3 integrin. Strikingly, osteopontin silencing in tumor cells in vivo attenuated stromal activation and inhibited tumor growth. Our findings establish a critical functional role for paracrine signaling by tumor-derived osteopontin in reprograming normal fibroblasts into tumor-promoting CAFs.

Isolation of normal and cancer-associated fibroblasts from fresh tissues by Fluorescence Activated Cell Sorting (FACS).

Cancer-associated fibroblasts (CAFs) are the most prominent cell type within the tumor stroma of many cancers, in particular breast carcinoma, and their prominent presence is often associated with poor prognosis. CAFs are an activated subpopulation of stromal fibroblasts, many of which express the myofibroblast marker α-SMA. CAFs originate from local tissue fibroblasts as well as from bone marrow-derived cells recruited into the developing tumor and adopt a CAF phenotype under the influence of the tumor microenvironment. CAFs were shown to facilitate tumor initiation, growth and progression through signaling that promotes tumor cell proliferation, angiogenesis, and invasion. We demonstrated that CAFs enhance tumor growth by mediating tumor-promoting inflammation, starting at the earliest pre-neoplastic stages. Despite increasing evidence of the key role CAFs play in facilitating tumor growth, studying CAFs has been an on-going challenge due to the lack of CAF-specific markers and the vast heterogeneity of these cells, with many subtypes co-existing in the tumor microenvironment. Moreover, studying fibroblasts in vitro is hindered by the fact that their gene expression profile is often altered in tissue culture. To address this problem and to allow unbiased gene expression profiling of fibroblasts from fresh mouse and human tissues, we developed a method based on previous protocols for Fluorescence-Activated Cell Sorting (FACS). Our approach relies on utilizing PDGFRα as a surface marker to isolate fibroblasts from fresh mouse and human tissue. PDGFRα is abundantly expressed by both normal fibroblasts and CAFs. This method allows isolation of pure populations of normal fibroblasts and CAFs, including, but not restricted to α-SMA+ activated myofibroblasts. Isolated fibroblasts can then be used for characterization and comparison of the evolution of gene expression that occurs in CAFs during tumorigenesis. Indeed, we and others reported expression profiling of fibroblasts isolated by cell sorting. This protocol was successfully performed to isolate and profile highly enriched populations of fibroblasts from skin, mammary, pancreas and lung tissues. Moreover, our method also allows culturing of sorted cells, in order to perform functional experiments and to avoid contamination by tumor cells, which is often a big obstacle when trying to culture CAFs.