Abstract P2-21-12: Targeting ADAM8 dependent maintenance of the myCAF phenotype in the breast cancer tumor microenvironment

Abstract

Abstract Background: Cancer associated fibroblasts (CAFs) regulate breast cancer growth, differentiation and metastasis. Targeting the signaling pathways between myofibroblast CAFs (myCAF) and cancer cells required for myCAF maintenance provides a mechanism to reverse engineer the tumor microenvironment to inhibit cancer growth and metastasis. Methods and Results: We co-cultured human triple negative breast cancer (MDA-MB-231) with human mesenchymal stem cells (MSC). After 72h, MSC mRNA expression of the myCAF markers, alpha-smooth muscle actin (α-SMA)/vimentin (Vim)/tenascin C (Ten-C) are all significantly increased. In parallel, MB-231 mRNA expression of stemness markers, SRY-Box Transcription factor 2 (Sox2)/Octamer-binding transcription factor 4 (Oct4)/Nanog, are also significantly increased. Migration assay and morphology imaging confirm the induction of the myCAF phenotype to promote MB-231 cancer stemness. shRNA lentivirus was used to knock down Sox2 gene expression in MB-231. We performed RNA-seq in MB-231 first comparing MB-231 vs. MB-231+MSC and then MB-231(-/-sox2)+MSC vs. MB-231+MSC. There are 104 overlapping genes in MB-231+MSC from the two analyses indicating upregulation with myCAF differentiation and cancer stemness. Using the Human Cancer Secretome Database, 9 genes are either membrane or secreted proteins. Using antibody blockade in co-culture, antibody depleted co-culture medium, and adding activated proteins to culture, A Disintegrin And Metalloprotease 8 (Adam8) gene was found to be required for myCAF maintenance. Using SELEX, we isolated 5 RNA aptamers targeting the human Adam8 soluble MP domain. Using in vitro quantification of Adam8 MP activity with human Adam8 fluorogenic peptide substrate and recombinant human Adam8 protein, Adam8 Apt-1 with IC50=33nM and Kd=29nM was selected. APt-1 does not inhibit human Adam 10 or 17 MP activity and remains extracellular. The core inhibitory domain of Apt-1 aptamer contains 23nt. Following induction of the myCAF phenotype and cancer stemness in MB-231+MSC coculture, addition of Apt-1 significantly decreased myCAF and cancer stemness markers. In murine NOD/scid xenograft model, 10^6 luciferase-MB-231 and 10^6 MSC were implanted in the R3 position at week 0. Apt1 was administered (10mg/kg via tail vein every 2 days) from week 3 to week 6. MB-231 volume (bioluminescence) was significantly decreased following Apt1. (Table) Conclusion: Adam8 is expressed in MB-231 breast cancer cells in the context of MSC/myCAF coculture and MB231 stemness and is required for MB-231 cancer growth. Aptamer ablation of Adam8 extracellular activity reverses MB-231 growth. MB-231 and MSC Murine Xenograft Model In murine NOD/scid xenograft model, 10^6 luciferase-MB-231 and 10^6 MSC were implanted in the R3 position at week 0. Apt1 was administered (10mg/kg via tail vein every 2 days) from week 3 to week 6. Citation Format: Zhiyong Mi, Paul C. Kuo. Targeting ADAM8 dependent maintenance of the myCAF phenotype in the breast cancer tumor microenvironment [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-21-12.