Amplification of interferon-γ-driven proinflammatory macrophage responses by granulocyte-macrophage colony-stimulating factor: Implications for colorectal cancer immunotherapy.

BACKGROUND

Tumor-associated macrophages in colorectal cancer predominantly exhibit an immunosuppressive M2 phenotype that contributes to tumor progression and resistance to immune checkpoint inhibition. Repolarizing M2 macrophages toward a proinflammatory M1 phenotype represents a promising immunotherapeutic strategy. While interferon-γ promotes M1 macrophage activation, the effect of combining interferon-γ with granulocyte-macrophage colony-stimulating factor remains underexplored.

METHODS

THP-1-derived M2-like macrophages were treated with interferon-γ (0-500 ng/mL) and granulocyte-macrophage colony-stimulating factor (0-1,000 ng/mL) in a dose-matrix experiment (n = 4). M1 and M2 markers and programmed cell death-ligand 1 were quantified by quantitative real-time polymerase chain reaction at 24 hours. Secreted interleukin-6, tumor necrosis factor-α, and interleukin-10 were measured by enzyme-linked immunosorbent assay. HT-29 colorectal cancer cells were treated with macrophage-conditioned media and assessed for E-cadherin, N-cadherin, vimentin, and Ki-67. Statistical analyses included t tests and analysis of variance.

RESULTS

Interferon-γ strongly induced M1 gene expression, whereas granulocyte-macrophage colony-stimulating factor alone demonstrated modest effects. Combined interferon-γ (50 ng/mL) and granulocyte-macrophage colony-stimulating factor (500 ng/mL) amplified interleukin-6, C-X-C motif chemokine ligand 10, and tumor necrosis factor-α gene expression 5.4-fold, 2.9-fold, and 3.1-fold, respectively, beyond interferon-γ alone. Two-way analysis of variance showed no significant interaction, suggesting additive rather than synergistic enhancement. Combination treatment increased the secretion of interleukin-6 and tumor necrosis factor-α and reduced interleukin-10. Conditioned media from combination-treated macrophages increased E-cadherin and reduced N-cadherin, vimentin, and Ki-67 in HT-29 cells.

CONCLUSION

Granulocyte-macrophage colony-stimulating factor augments interferon-γ-driven proinflammatory macrophage responses with protein-level changes and functional effects on colorectal cancer cells. The resulting macrophage secretome was associated with a less mesenchymal, less proliferative colorectal cancer-cell phenotype. Concurrent programmed cell death-ligand 1 upregulation provides a preclinical rationale for evaluating cytokine-mediated macrophage reprogramming in combination with programmed cell death protein 1/programmed cell death-ligand 1 checkpoint blockade in colorectal cancer.