Cuproptosis in microsatellite stable colon cancer cells affects the cytotoxicity of CD8+T through the WNT signaling pathway

Abstract

The microsatellite stable (MSS) colon cancer (CC) has long been considered resistant to immunotherapy. Cuproptosis, as a novel form of cell death, may interact with tumor immunity. This project focused on the impact of cuproptosis on the cytotoxicity of CD8⁺T in MSS CC, aiming to provide effective clues for improving the treatment strategy of MSS CC. The study developed an MSS CC cuproptosis model using 50 nM elesclomol and 1 μM CuCl₂. Cuproptotic SW480 cells were directly co-cultured with CD8⁺ T cells. Cuproptosis levels were assessed via intracellular copper ion detection, Western blot, and confocal laser scanning microscopy. CCK-8, Hochest/PI staining, CFSE cell proliferation assay, LDH cytotoxicity detection, and ELISA were used to evaluate CD8⁺ T cell immune activity and cytotoxicity. Transcriptome sequencing and bioinformatics analysis identified regulated signals in cuproptotic SW480 cells. A rescue experiment utilized a WNT pathway activator (BML-284). PD-L1 expression in cells/membranes was analyzed using qRT-PCR, Western blot, and flow cytometry. NSG mice were immunoreconstituted, and the effects of cuproptosis on immune infiltration and cancer progression in MSS CC mice were assessed using ELISA and immunohistochemistry (IHC). Treatment with 50 nM elesclomol and 1 μM CuCl₂ significantly increased cuproptosis in SW480 cells. Co-culture with CD8⁺ T cells enhanced their cytotoxicity. Sequencing revealed cuproptosis-mediated modulation of immune and inflammatory pathways, including WNT signaling. Rescue experiments showed downregulation of WNT signaling in cuproptotic SW480 cells. Indirectly, CD8⁺ T cell immune function was enhanced by reducing PD-L1 expression. In mice, cuproptosis resulted in increased infiltration of CD8⁺ T cells in tumor tissue, leading to delayed cancer progression compared to the control group. Cuproptosis in MSS CC cells enhances the cytotoxicity of CD8⁺ T cells, which may be achieved through downregulation of the WNT signaling pathway and decreased expression of PD-L1. In the future, drugs that can induce cuproptosis may be a promising approach to improve MSS CC immunotherapy.