Microscope-based sorting of highly motile cancer-associated fibroblasts using a photoconvertible fluorescent protein

Abstract In the tumor microenvironment, cancer-associated fibroblasts (CAFs) are known to play key roles in promoting cancer cell invasion and cancer progression. CAFs are also highly invasive compared with normal fibroblasts in a 3-D co-culture system with lung cancer cells. Due to CAF heterogeneity, there are various differences in cell motility even within a CAF population. To classify CAFs according to their motility, we named less motile CAFs as “static CAFs” and more motile CAFs as “motile CAFs”. To separate and isolate these static CAFs and motile CAFs from a 3-D co-culture system, CAFs were transfected with a green-to-red photoconvertible fluorescent protein, Dendra2. Dendra2-transfected CAFs at specific areas (i.e., static or motile CAFs) were UV-irradiated using a confocal microscope. CAFs with red fluorescence were then isolated by flow cytometry and were subjected to RNA sequencing. In gene ontology and gene set enrichment analyses, two types of CAFs showed significant differences in global gene expression patterns. These data suggest that heterogeneous CAFs with different motility have distinct roles in the regulation of various signaling pathways in the tumor microenvironment. Citation Format: Sieun Lee, Young-Ho Ahn. Microscope-based sorting of highly motile cancer-associated fibroblasts using a photoconvertible fluorescent protein [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3861.