Cancer-associated fibroblasts (CAFs) reside within the tumor microenvironment, facilitating cancer progression and metastasis via direct and indirect interactions with cancer cells and other stromal cell types. CAFs are composed of heterogeneous subpopulations of activated fibroblasts, including myofibroblastic, inflammatory, and immunosuppressive CAFs. In this study, we sought to identify subpopulations of CAFs isolated from human lung adenocarcinomas and describe their transcriptomic and functional characteristics through single-cell RNA sequencing (scRNA-seq) and subsequent bioinformatics analyses. Cell trajectory analysis of combined total and THY1 + CAFs revealed two branching points with five distinct branches. Based on Gene Ontology analysis, we denoted Branch 1 as “immunosuppressive”, Branch 2 as “neoantigen presenting”, Branch 4 as “myofibroblastic”, and Branch 5 as “proliferative” CAFs. We selected representative branch-specific markers and measured their expression levels in total and THY1 + CAFs. We also investigated the effects of these markers on CAF activity under coculture with lung cancer cells. This study describes novel subpopulations of CAFs in lung adenocarcinoma, highlighting their potential value as therapeutic targets.

Cancer-associated fibroblasts (CAFs) promote EMT (epithelial-mesenchymal transition), invasion, and metastasis of cancer cells in the tumor microenvironment. To identify microRNAs (miRNAs) associated with the reprogramming of normal lung fibroblasts (LFs) into CAFs, global miRNA expression was profiled in LFs and CAFs through Nanostring nCounter. miR-224 was one of the increased miRNAs in CAFs, and its expression was higher in tumors than in normal tissues in dbDEMC and TCGA_lung adenocarcinoma data. miR-224 levels were then checked by qRT-PCR in paraffine-embedded tumor samples from patients with lung adenocarcinoma. Kaplan-Meier survival analysis showed that high miR-224 expression was related to poor survival. These data show that lung cancer progression is promoted by miR-224. To discover the roles of increased miR-224 in CAFs, miR-224 was overexpressed by lentiviral infection in LFs. LF_miR-224 overexpression enhanced the expression of CAF activation markers including Fap, S100a4, and Vim. In spheroid invasion assay using co-culture of LFs and cancer cells, as an experimental model of the tumor microenvironment, LF_miR-224 promoted invasion of lung cancer cells compared with control LFs. In transwell migration assay, LF_miR-224 stimulated migration of lung cancer cells more than control LFs. Collectively, these results indicate that CAFs reprogrammed by miR-224 promote the migration and invasion of lung cancer cells. Citation Format: Seonyeong Oh, Inyoung Cheon, Young-Ho Ahn. miR-224 activates cancer-associated fibroblasts to promote cancer cell invasion in lung tumor microenvironment. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5843.

Background: Cancer-associated fibroblasts (CAFs) reside within the tumor microenvironment, facilitating cancer progression and metastasis via direct and indirect interactions with cancer cells as well as other stromal cell types. CAFs are comprised of heterogeneous subpopulations of activated fibroblasts, including myofibroblastic, inflammatory, or immunosuppressive CAFs. In this study, we sought to identify subpopulations of CAFs isolated from human lung adenocarcinomas and describe their transcriptomic as well as functional characteristics through single-cell RNA sequencing (scRNA-seq) and subsequent bioinformatics analyses. Methods: We isolated and cultured two types of CAFs. Total CAFs were isolated after negative selection using cell-type-specific antibodies against lymphocytes (CD45), macrophages (CD68), endothelial cells (CD31), and epithelial cells (Ep-CAM). THY1+ CAFs were isolated after additional selection based on CAF-specific marker THY1. Total CAFs (n=5,139 cells) and THY1+ CAFs (n=4,038 cells) were subjected to 10X Genomics scRNA-seq. scRNA-seq data were then integrated and analyzed using the Seurat method. Results: Cell trajectory analysis of combined total and THY1+ CAFs revealed two branching points with five distinct branches. Based on Gene Ontology analysis, we denoted Branch 1 as “immunosuppressive”, Branch 2 as “neoantigen-presenting”, Branch 4 as “myofibroblastic”, and Branch 5 as “proliferative” CAFs. We selected representative branch-specific markers and measured their expression levels in total and THY1+ CAFs. We also investigated the effects of these markers on CAF activity under co-culture with lung cancer cells. Conclusions: This study describes novel subpopulations of CAFs in lung adenocarcinoma, highlighting their potential value as therapeutic targets. Citation Format: Daeseung Kim, Jeong Seon Kim, Seoree Kim, Sang Hoon Chun, Jae Jun Kim, Inyoung Cheon, Sieun Lee, Jung Sook Yoon, Soon Auck Hong, Hye Sung Won, Keunsoo Kang, Young-Ho Ahn, Yoon Ho Ko. Identification and characterization of cancer-associated fibroblast subpopulations in lung adenocarcinoma [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 LB043.

Cancer-associated fibroblasts (CAFs) promote cancer progression and metastasis in the tumor microenvironment. CAFs are regulated by diverse processes including regulation by microRNAs (miRNAs). To profile miRNAs involved in the regulation of CAF activation, we performed the NanoString nCounter® assay in mouse CAFs and normal lung fibroblasts (LFs). Among the miRNAs differentially expressed between CAFs and LFs, miR-200 family members were significantly downregulated in CAFs. Overexpression of miR-200 in CAFs suppressed the migration and invasion of lung cancer cells in co-culture systems (transwell migration and spheroid invasion assays). Next, we tried to identify novel miR-200 target genes based on RNA sequencing data obtained from control and miR-200-overexpressed CAFs, lung adenocarcinoma TCGA data, and TargetScan. We are now studying on these candidates to see whether they are involved in the interaction between CAFs and cancer cells.On the basis of these findings, we conclude that miR-200 suppresses the interaction between CAFs and lung cancer cells in the tumor microenvironment. Keywords: cancer-associated fibroblasts, miR-200, lung cancer, tumor microenvironment, invasion Citation Format: Inyoung Cheon, Sieun Lee, Young-Ho Ahn. Overexpression of miR-200 in cancer-associated fibroblasts suppresses the migration and invasion of cancer cells in co-culture systems [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 3872.

No abstract