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Uncovering the Role of Cancer Associated Fibroblasts in Facilitating Breast Cancer Metastasis

Periodic Reporting for period 3 - MetCAF (Uncovering the Role of Cancer Associated Fibroblasts in Facilitating Breast Cancer Metastasis)

Reporting period: 2018-07-01 to 2019-12-31

Mortality from breast cancer is almost exclusively a result of tumor metastasis.
Since advanced metastatic cancers are usually fatal, understanding the biology of tumor metastasis is the most significant challenge in cancer research today.
It has become clear that the microenvironment of tumors is crucial in supporting tumor growth. Nevertheless, the role of the metastatic microenvironment in facilitating metastatic colonization is largely unknown.
We recently uncovered a novel role for Cancer-Associated Fibroblasts (CAFs) in mediating tumor-promoting inflammation. However, the role of CAFs in the formation of a permissive metastatic niche that enables the growth of disseminated tumor cells is unresolved.
In this project, we are systematically investigating, for the first time, the molecular changes in CAFs that facilitate metastases formation, which may lead to the discovery of novel targets for cancer therapeutics.
To achieve this goal, we integrate tumor biology methodologies, including novel mouse models of spontaneous lung metastasis of breast cancer imitating the clinical setting, with multi-transgenic reporter mice that enable origin tracing and unbiased analysis of fibroblast sub-populations.
By performing a comprehensive molecular and functional characterization of fibroblast co-evolution during metastases formation and analysis of breast cancer patients, we uncovered the dynamic changes in CAFs at the metastatic niche, identified the origin of metastatic CAFs, and elucidated some of the central signaling pathways that govern their functional role in breast cancer progression and metastasis.

Molecular understanding of the early stages of tumor metastasis is an essential prerequisite for the discovery of novel therapeutic targets. Achievement of the proposed goals will shed light on a central conundrum in cancer biology and open new horizons for the development of novel therapeutics that will transform cancer into a chronic, yet manageable disease.

The overall objectives of this study are:
Aim 1: Characterize the dynamic transcriptional changes in lung fibroblasts during breast cancer metastasis. Utilizing our novel mouse models of spontaneous lung metastasis combined with transcriptome analysis and functional assays, we analyzed the dynamic co-evolution of lung fibroblasts during pulmonary metastases.
Aim 2: Define the origin of CAFs at the lung metastatic niche and elucidate their role in mediating inflammation. By combining adoptive BM transplantations and multi-transgenic reporter mice we defined the origin of CAFs at the metastatic niche, and will follow up by dissecting their distinct functional contributions to lung metastasis.
Aim 3: Elucidate the role of CAF-mediated fibrosis in fostering lung metastasis. We analyzed the link between enhanced collagen deposition and pulmonary metastasis, and identified tumor-derived factors that induce pro-fibrogenic activation of lung fibroblasts.
The main focus of this project is uncovering the role of cancer-associated fibroblasts in facilitating breast cancer metastasis, with emphasis on the early changes that precede clinically manifested metastatic relapse. In this reporting period, we have made substantial progress in all the specific aims of the project as detailed below:
Aim 1: Characterize the dynamic transcriptional changes in lung fibroblasts during breast cancer metastasis.
1. Utilizing our novel mouse models of spontaneous lung metastasis we performed RNA-Seq transcriptome analysis of fibroblasts isolated from normal lungs, lungs with micrometastases and lungs with macrometastases. We performed in-depth bioinformatics analysis of the transcriptome data obtained from the three populations and found that macrometastases-associated fibroblasts are significantly distinct from normal lung fibroblasts in their gene expression. Moreover, we identified several functional tasks that are upregulated in metastases-associated fibroblasts based on their transcriptional upregulation. Specifically, the main pathways identified are related to inflammation, immune response, and extracellular matrix remodelling. These results imply that fibroblasts undergo a process of co-evolution along the development of lung metastases. We are currently validating the results, and performing functional assays with selected candidates. A manuscript describing the RNA-Seq results and analyses is in preparation for submission.


3. We found that CAFs in mouse and in human breast carcinomas upregulated the NLRP3 inflammasome pathway, associated with recognition of tissue damage. We demonstrated that fibroblasts recognize damage-associated molecular patterns (DAMPs), resulting in activation of pro-inflammatory signaling and secretion of IL-1β. CAF-derived inflammasome signaling facilitated mammary carcinogenesis and lung metastasis, which was attenuated when CAF-derived NLRP3 or IL-1β were ablated. Functionally, CAF-derived inflammasome promoted tumor progression and metastasis by modulating the tumor microenvironment toward an immune suppressive milieu and by upregulating the expression of adhesion molecules on endothelial cells. Thus, our findings suggest that the physiological damage response of fibroblasts is ‘hijacked’ in breast cancer to promote tumor growth and metastasis, and reveal a novel functional role for CAFs that links sensing of tissue damage with with tumor-promoting inflammation.

A manuscript describing these results is currently under revision (“Activation of the NLRP3 inflammasome in cancer-associated fibroblasts links tissue damage with tumor-promoting inflammation in breast cancer progression and metastasis”
Ershaid et al. 2018.

4. We characterized the role of CAF-derived Chitinase 3-like 1 (Chi3L1) and found that fibroblasts drive an immunosuppressive and growth-promoting microenvironment in breast cancer via secretion of Chi3L1. we characterized a novel signaling axis between fibroblasts, cancer cells and immune cells in breast tumors that drives an immunosuppressive microenvironment, mediated by CAF-derived Chi3L1. We demonstrated that Chi3L1 is highly upregulated in CAFs isolated from mammary tumors and pulmonary metastases of transgenic mice, and in the stroma of human breast carcinomas. Genetic ablation of Chi3L1 in fibroblasts in vivo attenuated tumor growth, macrophage recruitment and reprogramming to an M2-like phenotype, enhanced tumor infiltration by CD8+ and CD4+ T cells, and promoted a Th1 phenotype. These results indicate that CAF-derived Chi3L1 promotes tumor growth and shifts the balance of the immune milieu towards type 2 immunity. Taken together, our findings implicated fibroblast-derived Chi3L1 as a novel key player in the complex reciprocal interactions of stromal cells that facilitate tumor progression and metastasis, and suggest that targeting Chi3L1 may be clinically beneficial in breast cancer.

These findings were published in Oncogene: Fibroblasts drive an immunosuppressive and growth-promoting microenvironment in breast cancer via secretion of Chitinase 3-like 1. Cohen N, Shani O, Raz Y, Sharon Y, Hoffman D, Abramovitz L and Erez N. Oncogene. 2017 Aug;36(31):4457-4468. doi: 10.1038/onc.2017.65. Epub 2017 Apr 3.

Aim 2: Define the origin of CAFs at the lung metastatic niche and elucidate their role in mediating inflammation. We have combined adoptive BM transplantations and multi-transgenic reporter mice and discovered a subpopulation of BM derived CAFs that are recruited to mammary tumors and lung metastases, and characterized their functional role. We demonstrated that bone marrow (BM)-derived mesenchymal stromal cells (MSCs) are specifically recruited to primary breast tumors and to spontaneous lung metastases, and differentiate in the tumor microenvironment to a phenotypically and functionally distinct subpopulation of CAFs. We showed that BM-derived CAFs are functionally important for tumor growth and are more potent in inducing angiogenesis than resident CAFs, via upregulation of Clusterin. Moreover, we demonstrated that BM-derived fibroblasts are transcriptionally reprogrammed in a tissue-specific manner. Using newly generated transgenic mice and adoptive BM transplantations that enable unbiased tracking and characterization of specific CAF populations, we showed that BM-derived fibroblasts are a substantial source of CAFs in the tumor microenvironment. Unlike resident CAFs, BM-derived CAFs do not express PDGFR, a known fibroblasts marker. As a result of BM recruitment, the percentage of PDGFR expressing cells gradually decreased during tumor progression. Strikingly, this decrease was evident in breast cancer patients and associated with worse prognosis, suggesting that BM-derived CAFs may have deleterious effects on survival. Therefore, PDGFR expression distinguishes two functionally unique CAF populations in mammary tumors and lung metastases and may have important implications for patient stratification and precision therapeutics in breast cancer.

A manuscript describing these results is currently under revision (“Bone marrow-derived fibroblasts are a functionally distinct stromal cell population in breast cancer and lung metastases”. Raz et al. 2018.

Aim 3: Elucidate the role of CAF-mediated fibrosis in fostering lung metastasis. We found that collagen deposition is an early event in the formation of the pre-metastatic niche in lungs, that precedes the formation of mactometastses from breast cancer. To identify tumor-derived factors that may induce fibrosis by systemic signaling we performed proteomic analysis of serum from normal and tumor-bearing mice revealed that multiple factors are significantly upregulated in sera from tumor-bearing mice. We focused on a candidate factor and demonstrated that its expression of is upregulated in human breast cancer and in multiple other human cancers. Moreover, we showed that its levels are gradually upregulated in mouse serum in correlation with tumor progression, in two different mouse model of breast cancer. In vitro, this candidate factor activated fibroblasts and induced the expression of a pro-fibrogenic gene signature. We are currently performing functional in vivo studies to characterize its role in inducing a fibrotic microenvironment in the lung metastatic niche.
It is increasingly appreciated that the metastatic microenvironment is crucial in supporting metastases formation.
Nevertheless, while years of research have revealed the well-documented role of the microenvironment in supporting tumor growth at the primary tumor site, the role of the metastatic microenvironment and the molecular crosstalk between disseminated cancer cells and stromal cells at the metastatic site that enable metastatic colonization are poorly characterized.

In this project, we were able to go beyond the state of the art and demonstrate for the first time the following:

1. We performed transcriptome profiling of lung fibroblasts at distinct stages of breast cancer metastases, analyzed the dynamic changes and characterized the main pathways that were upregulated in CAFs during their co-evolution. We identified novel CAF-derived factors that may be functionally important for the metastases-promoting activity of CAFs.

By the end of the project, we expected to complete detailed functional characterization of selected candidates, and validate their importance in human samples.

2. We defined bone marrow (BM) mesenchymal stromal cells as an important source of CAFs in both the primary tumors and the metastatic site, and characterized them functionally. We found that PDGFRa is a marker of resident fibroblasts, and is not expressed on BM-derived CAFs. Therefore, we showed for the first time that PDGFRa expression distinguishes two functionally unique CAF populations in mammary tumors and lung metastases and may have important implications for patient stratification and precision therapeutics in breast cancer.

3. We found that metastases-promoting fibrosis in lungs is instigated by systemic signaling from the primary tumor, and identified Activin A as a candidate tumor-derived factor.

By the end of the project, we expect to characterize in vivo the importance of tumor-derived Activin A in instigating pro-fibrogenic activation of lung fibroblasts, and to validate the importance of Activin A as a predictive marker for lung metastasis in breast cancer patients.