Abstract 3796: Exosomes enhance the radiation sensitivity via miR-6823-5p and modulate metastases in pancreatic cancer model
Authors: Nakaoka Ai , Nakahana Makiko , Inubushi Sachiko , Shimizu Yasuyuki , Iwashita Kazuma , Mukumoto Naritoshi , Kobayashi Kana , Ishihara Takeaki , Miyawaki Daisuke , Sasaki Ryohei 
Published: April 2023

Introduction: Pancreatic cancer (PC) is believed to be a difficult disease because of its radio-resistance and metastases. Radiotherapy reported to modify tumor microenvironment (TME) and to affect PC progression. Exosomes containing such as microRNAs (miRNAs), messenger RNAs (mRNAs), and proteins of cells, play essential roles in cell-to-cell communications. However, characteristics and mechanisms for radiation responsive exosomes remain unclear. Here, we first report mechanisms of exosomes-mediated radiation response and regulation in PC metastasis with focusing on intra/intercellular communications via miRNA.

Methods: MIAPaCa-2 cells, human pancreatic carcinoma, was used. To isolate exosomes, cell culture media was ultracentrifuged at 100,000 x g for 90 min at 4°C. Exosomes derived from non-irradiated culture media was named ‘0 Gy-Exo’ and those from irradiated with 5 Gy culture media is called ‘5 Gy-Exo’. Exosome sizes and features were evaluated by transmission electron microscopy (TEM). Intracellular reactive oxygen species (ROS) levels induced by exosomes and by X-ray irradiation were evaluated by the C-H2DCF dye staining. To detect DNA damage, cells were fixed and stained by γ-H2AX antibody. The expression of superoxide dismutase 1 (SOD1) was evaluated by using immunoblots. Total RNA was extracted from 0 Gy-Exo and 5 Gy-Exo, and then comprehensive miRNA expression analysis was performed to analyze 2,565 human miRNA sequences. The Gene Expression Omnibus database (GSE163133) and the TargetScan were used for specific miRNAs identification. Living MIAPaCa-2 cells in the presence or absence of those exosomes were injected in the spleen of Balb/c nude mice to establish liver metastatic models. Eight weeks after the injection, mice were sacrificed, and then the spleen and the liver were harvested as a whole organ. H-E staining and immunohisitochemical analyses using S100A4 and SMAD4 antibodies were performed to detect liver metastases.

Results: Isolated exosomes were shaped in the form of closed, round vesicles with a diameter of 10~100 nm. Intracellular ROS levels and the numbers of γ-H2AX foci in the cells with 5 Gy-Exo were significantly increased compared to those the control (without adding exosomes, p&lt;0.01). Intracellular SOD1 expression in the cells with the 5 Gy-Exo was decreased compared with that in the control. The miR-6823-5p was identified to have a complementary base sequence to SOD1. The average occupied proportions with MIAPaCa-2 cells in the liver was modulated according to those exosomes. The SMAD4 expression was highly observed in the liver and the spleen suggesting that the SMAD4 could be a useful marker of liver metastases.

Conclusion: Exosomes increased radiation sensitivity through increase of intracellular ROS levels via exosomal miRNAs and also could affect liver metastases in pancreatic cancer models.

Citation Format: Ai Nakaoka, Makiko Nakahana, Sachiko Inubushi, Yasuyuki Shimizu, Kazuma Iwashita, Naritoshi Mukumoto, Kana Kobayashi, Takeaki Ishihara, Daisuke Miyawaki, Ryohei Sasaki. Exosomes enhance the radiation sensitivity via miR-6823-5p and modulate metastases in pancreatic cancer model. [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 3796.