Colorectal cancer (CRC) is a cold tumor with a low response rate to programmed death-ligand 1 (PD-L1) inhibitors, and the regulatory mechanism underlying its expression remains incompletely understood. Lactylation is a novel post-translational modification involved in the regulation of gene transcription and protein function. This study aims to investigate the correlation between lactylation levels and PD-L1 expression in CRC, as well as its clinicopathological significance, so as to provide a novel potential target for immunotherapy.

① The GEPIA2 database was used to analyze the expression correlation between CD274 (encoding PD-L1) and 18 glycolysis genes (including HK1, HK2, and 16 others), lactate dehydrogenase genes (LDHA, LDHB), and lactate transporter genes (SLC16A1, SLC16A4). The TCGA colon adenocarcinoma (COAD) dataset with 453 samples was obtained from the XENA website, and GSEA software was applied to analyze gene ontology_biological process (GO_BP) pathway differences between the CD274 high- and low-expression groups. The Kaplan-Meier plotter database was used to evaluate the correlation between CD274 expression and overall survival in 4 consensus molecular subtypes (CMS) of CRC. ② Human CRC cell lines HT29 and SW480 were treated with 10 mmol/L lactic acid (Lac) or 10 mmol/L sodium lactate (Nala) for 24 h, with corresponding blank control groups, or cultured in low-glucose medium (1.8 g/L glucose) or high-glucose medium (4.5 g/L glucose). The transcription and translation levels of PD-L1 were detected by RT-qPCR and Western blotting, respectively. In addition, cells were treated with gradient concentrations of sodium lactate or Oxamate (a lactate dehydrogenase inhibitor) to verify the dose-dependent regulation of lactylation on PD-L1 expression. ③ Cancer tissues and paired adjacent non-tumor mucosal tissues were collected from 35 CRC patients (aged 59±11 years, a male-to-female ratio of 2.2∶1.0) who underwent surgical treatment at Department of General Surgery of First Affiliated Hospital of Army Medical University between February 2024 and October 2025. Immunohistochemistry (IHC) was performed to detect pan-lactylation (Pan-Kla) and PD-L1 expression, expression levels were evaluated by IHC scoring, and their correlations were analyzed with clinicopathological features.

① GEPIA2 database analysis demonstrated that CD274 expression was significantly positively correlated with glycolysis and lactate dehydrogenase genes (R=0.14 to 0.47, P<0.05), including hexokinase 1 (HK1), hexokinase 3 (HK3), phosphofructokinase platelet type (PFKP), triosephosphate isomerase 1 (TPI1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), phosphoglucomutase 2 (PGM2), phosphoglucomutase 3 (PGM3), enolase 1 (ENO1), enolase 2 (ENO2), pyruvate kinase M (PKM), lactate dehydrogenase A (LDHA), and lactate dehydrogenase B (LDHB). GSEA revealed that the glycolysis pathway was significantly activated in the CD274 high-expression group, while the oxidative phosphorylation pathway showed no obvious changes. Kaplan-Meier analysis indicated that in CMS Ⅰ (microsatellite unstable) and CMS Ⅳ (mesenchymal) subtypes, the CD274 low-expression group had significantly longer overall survival, whereas no significant correlation was found in CMS Ⅱ (canonical) and CMS Ⅲ (metabolic) subtypes. ② After treatment with Lac and Nala, the mRNA and protein levels of PD-L1 were significantly increased in HT29 and SW480 cells compared with the control group. Cells cultured in low-glucose medium exhibited lower pan-lactylation and PD-L1 expression levels than those cultured in high-glucose medium. Sodium lactate upregulated PD-L1 expression in a dose-dependent manner, and Oxamate treatment reduced PD-L1 expression. ③ Pan-lactylation and PD-L1 scores were significantly higher in CRC tissues than in paired adjacent non-tumor tissues (P<0.01). A significant positive correlation was observed between them (R²=0.195 9, P=0.007 8). Clinicopathological correlation analysis showed that mucinous adenocarcinoma had significantly higher lactylation scores than non-mucinous adenocarcinoma (P=0.047).

Lactylation upregulates the transcriptional and translational levels of PD-L1 in colorectal cancer cells and is significantly positively correlated with PD-L1 expression in tumor tissues. The prognostic value of PD-L1 is CMS subtype-specific, and low PD-L1 expression indicates a better prognosis in CMS Ⅰ/Ⅳ patients.