Abstract 5827: Hydroxychloroquine increases the tumor killing efficiency via elevating the membrane MHC-I protein levels of tumor cells
Authors: Man Zhang, Chen Huang, Xingming Ma, Wenjia Zhuang, Huajun Jin
Journal: Cancer Research (2025)

Abstract Background: Hydroxychloroquine (HCQ) was firstly developed as an antimalarial drug, while recently it has been used to treat various types of cancer due to its molecular mechanism in inhibiting autophagy process, induction of apoptosis, and the inhibition of CXCL12/CXCR4 pathway in cancer cells. Previous studies found that autophagy promotes immune evasion of pancreatic cancer by degrading major histocompatibility complex class I (MHC-I), so we supposed HCQ treatment might restore or even elevate the MHC-I levels of tumor cells through inhibiting autophagy and then benefit tumor infiltrating lymphocytes (TILs) immunotherapy. Methods: Malignant melanoma cell line A-375, non-small cell lung cancer (NSCLC) cell line NCI-H2122, ovarian cancer OVCAR-8 and glioblastoma U-87 MG were subjected to HCQ treatment for 48 hours and the protein levels of membrane MHC-I and programmed cell-death ligand 1 (PD-L1) were measured by flow cytometry. IFN-γ was set as a positive control for its well-known function in elevating MHC-I levels. Western blots were carried out to verify the autophagy inhibition. Real-time cell analysis (RTCA) was used to evaluate the in vitro cytotoxicity for transgenic hmTCRT cells specifically recognizing NY-ESO-1 epitope and targeting HCQ treated or non-treated NY-ESO-1 positive A-375 cells. Using the same concentrations as treating tumor cells, 15µM, 20µM HCQ treatment was carried out on TILs in rapid expansion phase to determine whether their proliferation would be affected. Results: HCQ treatment can significantly elevate the protein levels of membrane MHC-I for tumor cell lines A-375, NCI-H2122, OVCAR-8 and U-87 MG (P value of t-test &amp;lt;0.01), and the elevation can sustain for 72 hours after removing HCQ from the culture system. After HCQ treatment, the surface PD-L1 levels of aforementioned four cell lines didn’t change significantly compared to DPBS negative control group, contrarily PD-L1 levels of IFN-γ treatment groups increased significantly. Western blot results indicated that levels of LC3II and p62 increased significantly after HCQ treatment, suggesting that the autophagy process was inhibited. HCQ pre-treatment can significantly elevate the killing efficiency of hmTCRT cells targeting NY-ESO-1 positive A-375 cells. 15µM, 20µM HCQ treatment didn’t affect TILs’ proliferation, which were the same concentrations as treating tumor cells. Conclusions: Our studies indicated that HCQ can significantly increase the tumor killing efficiency via elevating the membrane MHC-I protein levels of tumor cells while it doesn't affect TILs’ propagation. In addition, its effect on surface PD-L1 is not significant, which is more advantageous than IFN-γ. These results can provide preclinical evidences for HCQ pretreatment before the adoptive cell therapy of TILs in our clinical trials (NCT06375187). Citation Format: Man Zhang, Chen Huang, Xingming Ma, Wenjia Zhuang, Huajun Jin. Hydroxychloroquine increases the tumor killing efficiency via elevating the membrane MHC-I protein levels of tumor cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 5827.