Donor-cell HKDC1 contributes to glycolysis-associated macrophage-like responses induced by HCC-derived ectosome-enriched fractions

Macrophages are abundant in the tumor microenvironment and contribute to cancer progression through cytokine secretion and intercellular communication. This study combined paired transcriptomic and metabolomic screening with functional analyses to investigate the relationship between hexokinase domain-containing 1 (HKDC1) and macrophage-related responses in hepatocellular carcinoma (HCC). HKDC1 was preferentially detected in an HCC-cell-derived ectosome-enriched fraction under the present isolation conditions. Exposure of THP-1 cells to fractions derived from control HCC cells increased glucose uptake, lactate release, macrophage-associated markers, and an M2-associated gene profile, whereas these effects were attenuated when HKDC1 was silenced in the donor HCC cells. The inhibitory effects of 2-deoxy-d-glucose supported the involvement of recipient-cell glycolytic activity in the observed phenotype. GATA1 contributed to the transcriptional regulation of HKDC1 in HCC cell lines. In addition, HCC-derived extracellular fractions increased CCL1 secretion from THP-1 cells, and CCL1 treatment increased extracellular HKDC1 abundance without a corresponding increase in cellular HKDC1, suggesting a putative feed-forward relationship. HKDC1 was also detected in plasma extracellular-particle-enriched preparations from an HCC mouse model and patients with HCC, and tumor HKDC1 expression was associated with macrophage-related markers and poorer survival. Collectively, these findings support an association between donor-cell HKDC1 and the macrophage-modulating activity of HCC-derived ectosome-enriched fractions. Direct HKDC1 transfer, HKDC1-specific enzymatic activity in recipient cells, and the diagnostic or treatment-predictive utility of circulating HKDC1 remain to be established.