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Synergistic Repolarization of M2 to M1 Macrophages by Cytokine Combination Therapy: Implications for Colorectal Cancer Immunotherapy
Shalmali Dharmadhikari
*1, Robert Beal
2, Brayden Martinez
2, Cara Gable
2, Susan Galandiuk
11Hiram C. Polk Jr. MD Department of Surgery, University of Louisville, Louisville, KY; 2Price Institute of Surgical Research, University of Louisville, Louisville, KY
Objective:
A major challenge in colorectal cancer (CRC) treatment is the fact that only 15% of patients respond to immunotherapy. Tumor-associated macrophages(TAMs) in CRC predominantly exhibit an M2(immunosuppressive) phenotype, contributing to immune evasion and tumor progression. Repolarizing M2 macrophages toward an M1(pro-inflammatory) phenotype which is susceptible to immunotherapy, represents a promising immunotherapeutic strategy. IFN-γ is known to promote M1 polarization; however, the effect of combining IFN-γ with GM-CSF (granulocyte-macrophage colony-stimulating factor) remains unexplored in the context of M2 repolarization.
Methods:
M2 macrophages were generated from THP-1 cells using IL-4/IL-13 and treated with varying doses of IFN-γ(0-500 ng) and GM-CSF(0-1000 ng) in a dose-matrix experiment (N=4 replicates). Gene expression of M1 markers (IL-6, CXCL10, TNF-α, HLA-DR, IL-1β), M2 markers (IL-10, TGF-β, SPP1[Secreted phosphoprotein-1]), and immune checkpoint molecules (PD-L1) were quantified by qRT-PCR at 24 hours. Statistical significance was assessed using t-tests.
Results:
IFN-γ alone(100ng) induced robust M1 marker expression (IL-6: 63-fold, CXCL10: 17,656-fold) and PD-L1 upregulation (14-fold), while GM-CSF alone(200ng) demonstrated modest M2 repolarization capacity (maximum 15-fold for CXCL10)
The combination of 50ng IFN-γ with 500ng GM-CSF produced synergistic enhancement of M1 markers compared to 50ng IFN-γ alone with very high CXCL10 induction (25,901-fold, p=0.03), IL-6 upregulation (207-fold, p=0.001), and robust TNF-α expression (15-fold, p=0.02). This combination also achieved M2 marker suppression (IL-10: 4.6-fold decrease, p=0.32; SPP1: 2.5-fold decrease, p=0.10) and PD-L1 upregulation (30-fold increase, p=0.19). The synergistic pattern was dose-dependent,with maximal effects observed at the 1:10
IFN-γ: GM-CSF ratio.
Conclusions:
The robust CXCL10 induction indicates strong STAT1-IRF pathway activation, a key mechanism driving inflammatory macrophage polarization. These findings suggest that combination IFN-γ/GM-CSF therapy at specific dose ratios may effectively enhance pro-inflammatory signaling in the tumor microenvironment. The concurrent PD-L1 upregulation suggests rational combination with anti-PD-1/PD-L1 checkpoint inhibitors, potentially converting immunologically "cold" tumors into checkpoint blockade-responsive microenvironments. Further studies including functional validation, mechanistic pathway confirmation, and in vivo models are warranted to assess the therapeutic potential of this combination strategy for reprogramming the immunosuppressive environment in CRC.
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