Abstract PD9-10: Circulating CAF/cancer stem cell co-clusters bolster breast cancer metastasis
Authors: Sharma U , Miller P , Medina Saenz K , Picon-Ruiz M , Morata-Tarifa C , Spartz A , Troness B , Park DN , Seagroves TN , Slingerland JM , Lippman ME , El-Ashry D 
Published: May 2019

Background: Metastatic disease is the primary cause of breast cancer (BC) mortality. Cancer associated fibroblasts (CAFs) are the majority of stroma in BC and critical players in BC malignancy. For example, CAFs are the main source of SDF-1, a prominent chemokine in the tumor microenvironment (TME) that also imparts stem cell-like characteristics to BC cells. Metastasis occurs due to the transport of circulating tumor cells (CTC) and clusters of CTCs through the vasculature. Stem-like CTCs and clusters have a greater propensity to establish metastasis. We recently identified circulating CAFs (cCAFs) in blood from patients with BC and in spontaneous, syngeneic, and xenograft mouse models of BC. cCAFs not only circulate individually, but are also found in clusters with CTCs. In this study, we examine the role of CAFs in promoting egress of stem-like CTCs (cCSCs), determine the capacity of stem-like CTCs to cluster with CAFs, and evaluate the involvement of CTC/cCAF clustering in augmenting BC metastasis.

Methods: Our model employs NSG mice with orthotopic xenograft implantation of BC cells, primary CAF cell lines, or co-implantation of BC and CAF cell lines. We used two different BC cell lines: the non-metastatic BC cell line, MCF-7, and the highly metastatic primary BC cell line, DT28. We also employed the MMTV-PyMT spontaneous model of BC metastasis, and we used BALB/c mice injected with syngeneic 4T1 or 67nR cells to evaluate cCAFs, CTCs, and cluster egress in preclinical models. Mice were sacrificed at specific time points, and cardiac blood was collected. Blood was filtered using the faCTChecker microfluidic filtration instrument (Circulogix). Filters were stained for IF and cCAFs, CTCs, cCSCs, and clusters were enumerated. Tumors from CAF co-injected mice were evaluated for their stem cell-like phenotype and re-implanted in mice to evaluate tumorigenicity and metastasis.

Results: In spontaneous, syngeneic, and orthotopic xenograft models of BC, cCAFs, CTCs, and cCAF/CTCs co-clusters appear early in tumor development. cCAF/CTC clusters increase in correlation with tumor burden and metastasis. Co-inoculation of CAFs with BC cells resulted in a significant increase in tumor progression, metastasis, and in a substantially higher number of both individual cells and clusters in circulation. Dissociated tumor cells from CAF co-injected tumors had a higher proportion of CD44+stem cell-like cells (CSCs), enhanced ALDH-1 expression, and enhanced mammosphere formation. CD44+ CSCs, individually and in clusters, are found early on in the circulation of mice injected with dissociated tumor cells from CAF co-injected tumors. Upon re-implantation of CAF co-injected dissociated tumor cells without CAFs, dissociated tumor cells showed enhanced tumorigenicity and malignancy.

Conclusion: CAFs are highly motile and cCAFs precede CTCs into circulation and can do so independently of tumor cells. CAFs sustain egress of tumor cells by augmenting malignancy and stemness of BC cells. cCAF clusters with the highly metastatic stem cell-like subset of CTCs bolster metastatic colonization. Targeting primary CAF function and/or cCAF/cCSC co-clusters may provide novel avenues to abrogate BC metastasis.

Citation Format: Sharma U, Miller P, Medina Saenz K, Picon-Ruiz M, Morata-Tarifa C, Spartz A, Troness B, Park DN, Seagroves TN, Slingerland JM, Lippman ME, El-Ashry D. Circulating CAF/cancer stem cell co-clusters bolster breast cancer metastasis [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr PD9-10.