SLC16A3 in lung adenocarcinoma regulates glycolysis and lactate release to facilitate M2 polarization of tumor-associated macrophages : Short Title: SLC16A3 reinforces macrophage M2 polarization through glycolysis.

BACKGROUND: SLC16A3 is considered to affect the malignant progression of lung adenocarcinoma (LUAD), but its mechanism remains elusive. Lactate secretion can facilitate the M2 polarization of macrophages, which are essential components of the tumor immune microenvironment (TIME). METHODS: Based on the Cancer Genome Atlas (TCGA) database, differential expression analysis of SLC16A3 in LUAD was undertaken and the Pearson correlation analysis was on SLC16A3 and targets of M2 macrophages. Pathway enrichment analysis on SLC16A3 was achieved by utilizing the gene set enrichment analysis (GSEA). The expression of SLC16A3 in cells was examined by qPCR and Western blot (WB). The levels of glycolysis marker proteins in cells were tested by WB. The Glucose test kit, lactate test kit, Seahorse energy metabolism analyzer, and pHrodo™ Green AM intracellular indicator reagent kit were applied in assessing cellular glycolysis levels. CCK-8, scratch assay, Transwell assay, and flow cytometry were conducted to evaluate the malignant phenotype and apoptosis level of cancer cells. Flow cytometry and Enzyme-linked immunosorbent assay (ELISA) were utilized to assess the polarization of macrophages. Finally, a mouse model of allograft tumors was created, and the effects of SLC16A3 on glycolysis and M2 polarization of macrophages in vivo were evaluated by tracking tumor growth and detecting related protein distribution through Immunohistochemistry. RESULTS: SLC16A3 was greatly upregulated in LUAD. Knocking down SLC16A3 remarkably repressed the malignant phenotype of LUAD cells and reinforced apoptosis. The results derived from GSEA manifested that SLC16A3 had a higher enrichment in the glycolysis pathway. SLC16A3 positively modulated the extracellular and intracellular levels of lactate and glycolysis. Pearson correlation analysis uncovered a positive linkage between SLC16A3 and M2 macrophage markers. According to the rescue experiment, glycolysis inhibitors were observed to greatly reduce the enhancement in M2 polarization of macrophages caused by overexpression of SLC16A3. The final mouse experiment demonstrated that SLC16A3 boosted tumor growth in vivo and enhanced tumor glycolysis level and M2 macrophage infiltration in the TIME. CONCLUSION: SLC16A3 in LUAD modulates the glycolysis pathway to facilitate M2 polarization of macrophages.