In Vivo Detection of Circulating Cancer-Associated Fibroblasts in Breast Tumor Mouse Xenograft: Impact of Tumor Stroma and Chemotherapy

SIMPLE SUMMARY: Cancer-associated fibroblasts (CAFs) play an important role in tumor progression. They can circulate with tumor cells to support the survival and formation of metastasis. The aim of our study was to investigate circulating CAFs and CTCs in breast cancer model to understand the effect of tumor stroma and chemotherapy on cCAFs and CTCs formation. We found that tumors with CAFs showed faster growth than tumors without CAFs, as well as more cCAFs detected. Besides, ITGA5 as a new biomarker for CAFs showed good agreement with the markers FAP and α-SMA. Furthermore, liposomal doxorubicin suppressed tumor growth, however, more cCAFs and CTCs were detected in the chemotherapy-treated mice. These findings suggest that tumor stroma benefits tumor growth and chemotherapy may increase the formation of cCAFs and CTCs, potentially promoting tumor metastases. ABSTRACT: Cancer-associated fibroblasts (CAFs) are important drivers in the tumor microenvironment and facilitate the growth and survival of tumor cells, as well as metastasis formation. They may travel together with tumor cells to support their survival and aid in the formation of a metastatic niche. In this study, we aimed to study circulating CAFs (cCAFs) and circulating tumor cells (CTCs) in a preclinical breast tumor model in mice in order to understand the effect of chemotherapy on cCAFs and CTC formation. Tumors with MDA-MB-231 human breast tumor cells with/without primary human mammary fibroblasts (representing CAFs) were coinjected in SCID mice to develop tumors. We found that the tumors with CAFs grew faster than tumors without CAFs. To study the effect of the stroma on CTCs and cCAFs, we isolated cells using microsieve filtration technology and established ITGA5 as a new cCAF biomarker, which showed good agreement with the CAF markers FAP and α-SMA. We found that ITGA5+ cCAFs shed in the blood of mice bearing stroma-rich coinjection-based tumors, while there was no difference in CTC formation. Although treatment with liposomal doxorubicin reduced tumor growth, it increased the numbers of both cCAFs and CTCs in blood. Moreover, cCAFs and CTCs were found to form clusters in the chemotherapy-treated mice. Altogether, these findings indicate that the tumor stroma supports tumor growth and the formation of cCAFs. Furthermore, chemotherapy may exacerbate the formation of cCAFs and CTCs, which may eventually support the formation of a metastasis niche in breast cancer.