Abstract 2157: Generation of Rab27ako and PD-L1ko tumor cells to evaluate potential suppression of exosomal PD-L1 on host immune components involved in cancer immunotherapy
Authors: Qin You , Lyu Kexing , Yu Sifei , Lin Ming , Wicha Max , Chang Alfred E. , Li Qiao 
Published: September 2020

Abstract
Exosome is a kind of tiny membrane vesicles containing small molecules such as mRNA, miRNA, and proteins. It was showed that exosomes participate in cancer progression and metastasis by transferring bioactive molecules between cancer and various cells in the tumor microenvironment. We isolated exosomes from murine melanoma D5 tumor cell culture media by differential ultracentrifuge, and verified the isolated exosomes by transmission electron microscopy and nanoparticle tracking analysis as well as by exosome markers, e.g. CD63, HRS, ALIX, and TSG101. Using these isolated exosomes, we tested their ability to promote tumor growth. C57BL/6J mice were inoculated with D5 cells on day 0, and were then divided into two groups: treated (i.v.) with PBS vs. PBS containing isolated exosomes every 3 days starting from day 7 for 2 weeks. We found that D5-derived exosomes considerably promoted the tumor growth and notably reduced animal survival. It was reported recently that exosomes could function as immunosuppressive factors in immune responses. To test this in our animal models, we generated Rab27ako D5 and F10 cells to delete exosomes by CRISPR/Cas9 gene editing system. Rab27a knockout significantly decreased the exosome production from D5 and F10 tumor cells as demonstrated by nanoparticle tracking analysis. In addition, it was also reported that exosomes mediate immune suppression via its expression of PD-L1. To address this issue in our animal models, we also generated PD-L1ko D5 and F10 cells. While PD-L1 was expressed in both wild type tumor cells and tumor-derived exosomes, it was absent in the PD-L1ko D5 and F10 cells and the exosomes isolated from the PD-L1ko cells as evident by western blot and flow cytometry. Furthermore, IFNγ failed to augment the PD-L1 expression in D5 and F10 cells treated with PD-L1-trageted CRISPR/Cas9, confirming the knockout of PD-L1 gene in these tumor cells. Deletion of Rab27a or PD-L1 did not affect the proliferation of the tumor cells. Knockout of PD-L1 did not influence exosome secretion, nor did the knockout of Rab27a influence cell-surface PD-L1 expression. Although the immune suppression of exosome on T cells is well-known, such suppression of exosome on other immune cells is largely unknown. Based on our initial experimental results as described above, utilizing the Rab27ako and PD-L1ko D5 and F10 cells generated in our lab will allow us to evaluate the potential suppression of exosomal PD-L1 on host T cells as well as other immune components involved in cancer immunotherapy.
Citation Format: You Qin, Kexing Lyu, Sifei Yu, Ming Lin, Max Wicha, Alfred E. Chang, Qiao Li. Generation of Rab27ako and PD-L1ko tumor cells to evaluate potential suppression of exosomal PD-L1 on host immune components involved in cancer immunotherapy [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 2157.