Abstract P2-01-10: Circulating CAF/CTC complexes and breast cancer metastasis
Authors: Miller P , Sharma U , Medina-Saenz K , Yeasky T , Picon-Ruiz M , Morata-Tarifa C , Seagroves T , Slingerland J , Lippman M , El-Ashry D 
Published: February 2018

Abstract
Background: Metastatic disease in breast cancer (BC) is the leading cause of cancer-related mortality among women worldwide. Synergy between cancer cells and non-cancer cells of the tumor microenvironment (TME) are vital for disease progression. Cancer associated fibroblasts (CAFs) are the major cell type in the stroma of BC and are critical mediators of tumor progression and metastasis. Transport of circulating tumor cells (CTCs) and CTC clusters through the vasculature seeds metastasis and clinical and preclinical studies demonstrate that CTC clusters have a higher metastatic potential than individual CTCs. More recently, circulating cancer stem cells (cCSCs) have been implicated as more metastatic than non-CSC CTCs. In our lab, we have demonstrated that CAFs also circulate (cCAFs). We have observed cCAFs in peripheral blood from breast cancer patients and in murine models of breast cancer. Furthermore, we have observed that cCAFs are present in circulation as both individual cells and as well as in complexes with CTCs. Given the integral role of CAFs in BC metastasis, we hypothesize that cCAFs complex with CTCs/cCSCs to bolster BC metastasis.
Methods: cCAF/CTC clusters were identified and enumerated from peripheral blood of patients with BC, and associations with clinical features and disease outcomes were evaluated. Blood was collected by cardiac puncture from PyMT mice from 4 weeks through to the presence of metastases (10 weeks) and cCAF/CTC clusters enumerated. We co-injected CAFs with MCF-7 cellsl into NSG mice, blood collected by cardiac puncture, and cCAF/CTC clusters were enumerated. At time of final sacrifice, tumors were removed and assessed for presence of CSCs. Using our established model of cCAF/CTC clustering in vitro we interrogated cCAF/CTC complexing with both metastatic and poorly metastatic BC cells.
Results: Circulating cCAFs/CTCs clusters are significantly increased in the blood of patients with advanced stage BC and associate not only with severity of disease but also with poorer clinical outcomes. In the spontaneous PyMT mouse model, the appearance of circulating cCAF/CTC clusters increased significantly as tumors grew but prior to metastasis. We demonstrate that metastatic BC cells form clusters with CAFs in vitro while non-metastatic BC cells do not form complexes with CAFs in vitro. Enriching for stem cells from MCF7 mammospheres, resulted in CAF/CSC clusters in vitro. In mice that were co-injected with non-metastatic MCF7 cells and CAFs from a TNBC/Basal-like BC (CAF23) we observed disease metastasis, an enrichment for cancer stem cell (CSC)-like CTCs, and the presence of circulating cCAF/MCF7-CSC clusters.
Conclusions: Circulating clusters of CTCs and cCAFs are characteristic, and potentially causative, of BC metastasis. Observations of cCAF/CTC clusters from preclinical and clinical samples are corroborated by our determination that the ability of BC cells to form complexes with CAFs in vitro is related to the intrinsic metastatic ability of the breast cancer cells. Both in vitro and in circulation, the BC cells in cCAF/cBC clusters are CSCs, so cCAF/cCSC clusters. Disrupting the formation of cCAF/CTC complexes may be a potential strategy to reduce treat or prevent breast cancer metastasis.
Citation Format: Miller P, Sharma U, Medina-Saenz K, Yeasky T, Picon-Ruiz M, Morata-Tarifa C, Seagroves T, Slingerland J, Lippman M, El-Ashry D. Circulating CAF/CTC complexes and breast cancer metastasis [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P2-01-10.