Abstract 2044: CD44 facilitates metastasis by promoting co-clustering of breast cancer cells and cancer associated fibroblasts
Authors: Troness Benjamin , Spartz Angela , Sharma Utsav , Miller Philip , Saenz Kelsie Medina , Lippman Marc , McCarthy James , El-Ashry Dorraya 
Published: August 2019

Abstract
Background: Transport of tumor cells via the circulation (CTCs) is a key step in metastasis. CTC clusters and in particular, cancer stem cell-like cell (cCSC) clusters exhibit enhanced metastatic capacity over individual CTCs or cCSCs. Cancer associated fibroblasts (CAFs) are critical players in the breast tumor microenvironment (TME) and are also found in metastases. In clinical and pre-clinical studies, we have identified circulating CAFs (cCAFs) and cCAF/CTC co-clusters as potential biomarkers for breast cancer progression and metastasis. Our data suggest that the ability of breast cancer cells (BCCs) to cluster with CAFs depends on their intrinsic metastatic capacity, and specifically, their CSC-like phenotype, characterized by expression of the CD44 cell surface marker: both in vivo and in in vitro cluster assays, only metastatic BCCs could form co-clusters while non-metastatic BCCs could not. Further, both in vivo and in vitro, co-clustering potential was marked by a stem cell-like phenotype, including increased CD44 expression. We hypothesize that cCAF/cCSC co-clusters are the functional units of metastasis. The goal of this current study is to identify mechanisms that influence co-clustering between CTCs and cCAFs and specifically to investigate the role of CD44 in co-clustering.
Methods: Co- and mono-clusters of CAFs and BCCs were established in vitro by plating equal numbers of CAFs/BCCs into ultra-low attachment plates. After 24 hrs, clusters were collected and injected into female NSG mice via tail vein. Metastases was monitored by IVIS and confirmed by IHC. Mice were sacrificed at specific time points and blood collected by cardiac puncture. In vitro-generated clusters and blood were filtered using the faCTCheker microfluidic filtration instrument (Circulogix) to capture circulating cells with an 8-micron pore filter. Filters were stained by immunofluorescence for tumor cells, CAFs, and CD44. Hyaluronin (HA) was visualized with biotinylated HA binding protein. anti-CD44 siRNA (Dharmacon smart pool) was used to knockdown CD44 in BCCs or in CAFs
Results: Injection of CAF/BCC co-clusters resulted in accelerated metastases compared to mono-cluster injection. In both co-clusters captured from mouse blood and established in vitro, HA, a CD44 ligand secreted by CAFs as well as produced by cancer cells, was observed. Elimination of HA in CAFs and BCCs reduced co-cluster formation. Knockdown of CD44 expression in BCCs via siRNA abrogated BCC co-clustering ability.
Conclusion: In vitro and in vivo models of induced and conditional knockout of CD44 are defining the role of cancer cell-autonomous or CAF-autonomous CD44 in cCAF/CTC co-clustering and BC metastasis. These studies implicate CD44 as a driver of co-clustering between BCCs and CAFs. Inactivation of CD44 may abrogate co-clustering as well as impair the CSC phenotype, thus impacting critical pathways in BCC metastasis.
Citation Format: Benjamin Troness, Angela Spartz, Utsav Sharma, Philip Miller, Kelsie Medina Saenz, Marc Lippman, James McCarthy, Dorraya El-Ashry. CD44 facilitates metastasis by promoting co-clustering of breast cancer cells and cancer associated fibroblasts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2044.