Abstract 5117: CD44 and its ligand, hyaluronic acid, mediate co-clustering between breast cancer cells and cancer associated fibroblasts
Authors: Troness Benjamin Charles , Spartz Angela , Sharma Utsav , McCarthy James , El-Ashry Dorraya 
Published: September 2020

Abstract
Background: Cancer associated fibroblasts (CAFs) help form the tumor microenvironment and we have demonstrated that circulating CAFs (cCAFs) from primary tumors are found in breast cancer patients and mouse models. cCAFs form heterotypic co-clusters with circulating cancer cells (CTCs) that exhibit enhanced metastatic seeding potential. CTC/cCAF co-clusters appear in the early stages of disease before the appearance of overt metastases. We have shown that the ability of CTCs and CAFs to form co-clusters is linked to the CTCs' intrinsic metastatic capacity, particularly the cancer stem cell (CSC)-like phenotype. Metastatic breast cancer cells, such as MDA-MB-231s and DT28s, readily form co-clusters with CAFs in vivo and in vitro while non-metastatic breast cancer cells, such as MCF7s, do not. CD44 is a cell surface adhesion receptor commonly associated with the CSC phenotype. CD44 is abundantly expressed by CAFs and binds a number of ligands with established roles in cancer, including hyaluronic acid (HA) and osteopontin. Here, we demonstrate that CD44 and its primary ligand, HA, mediate the co-clustering of CTCs and CAFs.
Methods: MDA-MB-231 and DT28 (Primary TNBC) cells were treated with CD44 siRNA to knock down CD44 expression. Control and siRNA-treated MDA-MB-231 or DT28 cells were seeded in ultra-low attachment plates with CAF23s (Primary CAFs from TNBC). After 24 hours, cells were captured on microfilters using a faCTChecker microfluidic filtration instrument (Circulogix). Cancer cells and CAFs were stained by immunofluorescence, co-clusters were counted and their size was measured. To test the role of HA in co-clustering, MDA-MB-231, DT28, and CAF23 cells were treated for 24hrs with 1mM 4-methylumbelliferone (4-MU), then with 2000 U/mL bovine HAase for 1hr. Clustering assays between treated and untreated CTCs and cCAFs were performed as described above with the addition of 1mM 4-MU to the culture.
Results: Knock down of CD44 in both MDA-MB-231 and DT28 cells significantly reduced the number and size of co-clusters that were formed with CAF23 cells. In the co-clusters that were formed by CD44-kd 231s and DT28s, CD44 expression returned in these cells while un-clustered 231s and DT28s remained CD44-negative. The removal of HA also significantly reduced the number and size of co-clusters that formed. The reduction in co-clustering caused by HA removal was less than that caused by CD44-kd, suggesting that other ligands or the activity of CD44 is important in co-clustering.
Conclusions: These results suggest that co-clustering between CTCs and cCAFs is mediated through CD44 and its ligand, HA. The role of other ligands, such as osteopontin, is currently being tested. The targeting of CD44 and its ligands in breast cancer patients may disrupt co-cluster formation and communication between CTCs and CAFs, reduce the number of circulating CSCs, and prevent the development of metastases.
Citation Format: Benjamin Charles Troness, Angela Spartz, Utsav Sharma, James McCarthy, Dorraya El-Ashry. CD44 and its ligand, hyaluronic acid, mediate co-clustering between breast cancer cells and cancer associated fibroblasts [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5117.