Influence of AMIGO2 on CAFs/MD231 Co-clusters and Extravasation of Cancer Cells
Breast cancer (BC) is the most commonly diagnosed cancer and the primary cause of cancer mortality in women because of the tumor’s metastatic features. Cancer-associated fibroblasts (CAFs) promote the BC metastasis phenotypes and accommodate the tumor microenvironment (TME) by secreting cytokines and chemokines. These growth factors help CAF/BC cells to form co-clusters and bind to endothelial cells for extravasation. Our lab recently discovered through Single Cell RNA sequencing that CAFs induce the expression of Amphoterin-induced gene and open reading frame 2 (AMIGO2) in triple-negative breast cancer (TNBC) cell lines that may aid in extravasation of BC/CAFs co-clusters versus mono-clusters. AMIGO2 is an adhesion transmembrane protein that has been studied to promote the attachment of liver cancer cells to endothelial cells. This study aimed to observe AMIGO2 gene expression in BC cells as they bind to endothelial cells during metastasis. Through immunofluorescence staining and RT-PCR on MDA-MB-231 TNBC cells treated with AMIGO2 siRNA led to the discovery that AMIGO1 protein compensates when AMIGO2 expression decreases. Analysis of RT-PCR of MDA-MB-231 cells treated individually with AMIGO2 siRNA, AMIGO1 siRNA, and a combination of both confirmed that this compensatory effect was statistically significant (p<0.01). The percent of MDA-MB-231 cells treated with siRNA that were bound to endothelial cells during endothelial binding assay was significantly lower (p<0.01) compared to the samples not treated with AMIGO siRNA. Future directions will be to determine if the extravasation of CAF/MDA-MB-231 co-clusters and BC models is dependent on AMIGO proteins.