Abstract 4184: Change of oncological features on tumor and immune cells after anti-PD-1 antibody to pancreatic cancer orthotopic model in CD34+ HSC based humanized mice
Authors: Park Sujin , Koh Eun-young , Yoo Inha , Jeong Ji Hye , Jun Eunsung 
Published: March 2024

Abstract
(a) To investigate novel therapeutic agents in pancreatic cancer, animal models that recapitulate the features of the human immune system are necessary. We aim to analyze an orthotopic model in CD34+HSC-based humanized mice and to prove that is an indispensable model for immune-oncology therapy research.
(b) We established an orthotopic tumor model using luciferase-expressing BxPC3 cells in humanized NSG mice. After inoculation of tumor cells, anti-PD-1 Ab was administered to mice 5 times a week by I.P. injection. Tumor formation and progression were monitored using IVIS. To compare the subsets and immune-check point receptors of human immune cells in blood and tumor tissue, Flow cytometry and immunohistopathology were used. Additionally, we compared the distribution of various cells included in the tumor and the mRNA expression level of each cell through a single cell RNA sequence. We also establish primary cell lines and organoids with the humanized mouse tumor for further evaluation.
(c) After inoculation of BxPC3_Luc cell line into mice pancreas, we monitored tumor growth using a noninvasive imaging method of IVIS. When we compared the tumor growth between the anti-PD-1 treatment and control, there was no significant difference. Using FACS, we analyzed the various markers of human immune cells in mouse blood and tumor tissue. In blood, PD-1 expression of CD8+ T cells decreased, and Tim-3 expression increased in anti-PD-1 group. And, PD-1 expression of CD4+ T cells decreased in anti-PD-1 group. In tumor tissue, we also verified the decrease of PD-1 expression on tissue infiltrating CD8+ T cells and CD4+ T cells in the anti-PD-1 group. Especially in the more responsive mouse among anti-PD-1 group, activation markers in cytotoxic T cells (CD8+), helper T cells (CD4+), and regulatory T cells (CD4+FOXP3+CD25+) increased, and also, M1 macrophage (CD86+) expression was increased. Furthermore, we compared the mRNA expression levels of various cells in the tumor microenvironment according to anti-PD-1 Ab treatment through single cell RNA seq analysis. It was confirmed that the expression of genes related to proliferation and growing decreased, while the expression of genes related to apoptosis increased in cancer cells. Finally, we constructed primary cancer cells and organoids from tumor tissue and confirmed that the immune-oncological characters of these models are maintained after passaging.
(d) We successfully established a pancreatic orthotopic model in a humanized mouse and analyzed the changes in immune cells and tumor cells after anti-PD-1 treatment. In addition, by obtaining tumor tissue-based preclinical models, we laid the foundation for future immune-oncologic experiments. We hope that these attempts and results will help in the development and drug verification of new immunotherapy agents for refractory solid tumors such as pancreatic cancer.
Citation Format: Sujin Park, Eun-young Koh, Inha Yoo, Ji Hye Jeong, Eunsung Jun. Change of oncological features on tumor and immune cells after anti-PD-1 antibody to pancreatic cancer orthotopic model in CD34+ HSC based humanized mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4184.