Fig. 3. Lactate treatment increases infiltrating CD8⁺ T cells in MC38 tumors.

a Experimental design of single cell transcriptomic analysis of anti-PD-1 with or without lactate. C57BL/6 mice were inoculated with 1 × 10⁶ MC38 tumor cells and treated with anti-PD-1 (10 mg/kg, day 14 and 17) and lactate (1.68 g/kg, s.c. daily from day 15 to 19). Tumor and tumor draining lymph nodes were harvested on day 20 and analyzed by single cell RNA sequencing using the 10x platform. b tSNE plot of T cell clusters with location and cell type information analyzed with Seurat v3.0.1. DLN: tumor draining lymph nodes. c Distribution of T cells from different treatments and expression of marker genes. d Significantly upregulated pathways in tumor infiltrating CD8⁺ T cells after lactate treatment by unbiased gene set enrichment analysis (gene set database: c2.cp.kegg.v7.2.symbols). e Validation of increased tumor infiltrating CD8⁺ T cells by flow cytometry. C57BL/6 mice (n = 5) were inoculated with 1 × 10⁶ MC38 tumor cells and treated with anti-PD-1 (10 mg/kg, day 14 and 17) and lactate (1.68 g/kg, s.c. daily from day 15 to 19). MC38 tumors were harvested on day 20 and analyzed by flow cytometry. f Analysis of apoptosis markers of CD8⁺ T cells in tumor microenvironment by flow cytometry. Data are shown as means ± SEM. P value was determined by two-tail unpaired t-test (e, f). Source data are provided in Source Data file.