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

Periodic Reporting for period 4 - MetCAF (Uncovering the Role of Cancer Associated Fibroblasts in Facilitating Breast Cancer Metastasis)

Reporting period: 2020-01-01 to 2021-06-30

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. In this project, we systematically investigated, 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.

The overall objectives of this study are:
Aim 1: Characterize the dynamic transcriptional changes in lung fibroblasts during breast cancer metastasis.
Aim 2: Define the origin of CAFs at the lung metastatic niche and elucidate their role in mediating inflammation.
Aim 3: Elucidate the role of CAF-mediated fibrosis in fostering lung metastasis.

The overall conclusions of this study are:
We elucidated the heterogeneity and functional plasticity of CAFs in breast cancer progression and metastasis and demonstrated their functional role in mediating inflammation, tissue-damage response and fibrosis that facilitate breast cancer metastasis.
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. 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 transcriptome analysis of fibroblasts isolated from lungs at distinct metastatic stages. Employing multiple knowledge-based platforms of data analysis we demonstrated that fibroblasts in lung metastases are transcriptionally dynamic and plastic, and revealed stage-specific gene signatures that imply functional tasks. Furthermore, we identified Myc as a central regulator of fibroblast rewiring and found that stromal upregulation of Myc transcriptional networks is associated with worse survival in human breast cancer (Shani et al. eLife 2021).
2. We found that IL-33 is upregulated in metastases-associated fibroblasts in mouse models of spontaneous breast cancer metastasis and in patients with breast cancer with lung metastasis. Upregulation of IL-33 instigated type 2 inflammation in the metastatic microenvironment and mediated recruitment of eosinophils, neutrophils, and inflammatory monocytes to lung metastases. Targeting of IL-33 in vivo resulted in inhibition of lung metastasis and significant attenuation of immune cell recruitment and type 2 immunity (Shani et al Cancer Res. 2020).
3. We demonstrated that CAFs in mouse and in human breast carcinomas upregulated the NLRP3 inflammasome pathway, associated with recognition of tissue damage. We showed 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. 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 (Ershaid et al. Nature Comm. 2019).
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, and communicating with macrophages in the tumor microenvironment (Cohen et al. Oncogene 2017).

Aim 2: Define the origin of CAFs at the lung metastatic niche and elucidate their role in mediating inflammation.
We discovered a subpopulation of bone marrow (BM)-derived CAFs that are recruited to mammary tumors and lung metastases, and characterized their functional role. 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. 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 (Raz et al. J Exp Med 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 macrometastases from breast cancer. Proteomic analysis of serum from normal and tumor-bearing mice revealed that Activin A is significantly upregulated in sera from tumor-bearing mice.
We showed that ActA levels are gradually upregulated in mouse serum in correlation with tumor progression. Genetic targeting of Activin A in breast cancer cells resulted in attenuated metastasis. (Cohen et al. 2021, in preparation).
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.


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.

4. We showed for the first time that fibroblasts can sense tissue damage in the tumor microenvironment, leading to activation of pro-inflammatory signaling.

5. We demonstrated the role of CAF-derived IL-33 in the formation of a hospitable lung metastatic niche.

In summary, our project provided comprehensive characterization of the plasticity and functional heterogeneity cancer-associated fibroblasts in breast cancer progression and metastasis.
A subpopulation of Col1α+ CAFs in mammary tumors and lung metastases are BM-derived