PLSCR3 Deficiency Triggers mtDNA‐Driven cGAS‐STING Activation to Potentiate Antitumor Immunity in Colorectal Cancer

Background Immunotherapy efficacy in colorectal cancer (CRC) is largely restricted to microsatellite instability‐high (MSI‐H) tumors, highlighting an urgent need to overcome resistance in microsatellite‐stable (MSS) CRC. Mitochondrial DNA (mtDNA) leakage activates the cGAS‐STING pathway, a potent inducer of antitumor immunity. However, endogenous regulators constraining mtDNA release in CRC, particularly within the inner mitochondrial membrane (IMM), remain poorly defined. Methods In CRC cohorts from TCGA, integrated bioinformatics analysis identified dysregulated mitochondria‐associated genes exhibiting significant differential expression and survival outcomes. Phospholipid Scramblase 3 (PLSCR3) emerged as the prime candidate. Functional validation employed siRNA knockdown and CRISPR/Cas9 knockout in human (HT29) and mouse (CT26) CRC cell lines. Mitochondrial integrity was evaluated via JC‐1 membrane potential assay, oxygen consumption rate (OCR) measurement, and cytosolic mtDNA quantification. cGAS‐STING activation was measured by 2 ′ 3 ′ ‐cGAMP ELISA, cytokine (IFN β and CXCL10) secretion, immunoblotting (p‐STING and ISGs), and RT‐qPCR. Immune cell cytotoxicity was assessed using ex vivo NK cell killing assays. In vivo antitumor immunity and response to anti‐PD‐1 therapy were evaluated in syngeneic CT26 graft models in BALB/c mice, with tumor‐infiltrating lymphocytes analyzed by flow cytometry. Results PLSCR3 was selected for further study because it fulfilled three criteria simultaneously: differential expression in CRC, significant association with survival in univariate analysis, and established mitochondrial localization. PLSCR3 deficiency disrupted mitochondrial integrity, causing membrane depolarization, impaired respiration, and significant cytosolic mtDNA accumulation. This mtDNA leakage robustly activated the cGAS‐STING pathway, evidenced by increased 2 ′ 3 ′ ‐cGAMP, IFN β /CXCL10 secretion, STING phosphorylation (Ser366), and ISG upregulation. Functionally, PLSCR3 knockdown enhanced NK cell–mediated killing of CRC cells in vitro. Critically, PLSCR3 knockout in CT26 tumors significantly potentiated the efficacy of anti‐PD‐1 therapy in vivo, overcoming inherent resistance in this MSS‐CRC model. This was associated with increased intratumoral infiltration of CD8+ and CD4+ T cells, elevated Granzyme B levels, and enhanced activation markers on CD8+ T cells within the tumor microenvironment. Conclusion Our study identifies PLSCR3 as a previously unrecognized negative regulator of mtDNA‐associated cGAS‐STING activation in CRC. By maintaining mitochondrial homeostasis and limiting mtDNA leakage, PLSCR3 constrains innate immune signaling and reduces sensitivity to anti‐PD‐1 therapy in the CT26 model. These findings establish PLSCR3 as a promising therapeutic target to enhance immunotherapy responses in CRC.