MCT4-Mediated Metabolic Reprogramming in Tumor-Associated Macrophages Promotes Hepatocellular Carcinoma Progression

RMonocarboxylate transporter protein 4 (MCT4), encoded by the SLC16A3 gene, mediates lactate efflux into the extracellular microenvironment. This study aimed to characterize the MCT4 overexpression in tumor-associated macrophages (TAMs) at the single-cell level in hepatocellular carcinoma (HCC) and to investigate its role in HCC progression through metabolic reprogramming of TAMs.

In this experimental study, R software and multiple public databases, including UALCAN, TIMER2.0 and TISCH2, were used to analyze SLC16A3 expression at the single-cell level and its association with HCC prognosis. In vitro experiments were conducted and SLC16A3 knockdown was achieved using shRNAs. TAM polarization was evaluated by Western blot, quantitive real time-polymerase chain reaction (qRT-PCR) and flow cytometry. Additional bioinformatic analyses, including protein-protein interaction (PPI) network analysis, were conducted using STRING to investigate the association between SLC16A3 expression and key glycolysis genes.

Analysis of the GEO database GSE166635 demonstrated that MCT4 expression was significantly higher in macrophages than in other immune cell populations in HCC. Immunofluorescence (IF) revealed co-localization of MCT4 with the M2 TAM marker CD163. Western blot demonstrated that SLC16A3 knockdown reduced CD163 expression at protein levels to 84.1% ± 5.0% (KD-1) and 44.6% ± 8.4% (KD-3) compared to control. Flow cytometry further demonstrated that SLC16A3 knockdown increased the proportion of CD86-positive cells from approximately 13.5% (Con sh) to 43.3% (KD-1) or 43.8% (KD-3), whereas the proportion of CD163-positive cells decreased to approximately 38.2% (KD-1) or 33.4% (KD-3). Survival analysis was performed on HCC patients from TIMER2.0, including 371 HCC patients with complete survival information, further indicated that high SLC16A3 expression combined with increased M2 TAM infiltration was associated with poorer prognosis (P=0.008).

High MCT4 expression in TAMs may be associated with metabolic reprogramming toward glycolysis, and could promote M2 polarization of TAMs, thereby contributing to HCC progression and poor clinical outcomes.