Anti-galectin-9 therapy synergizes with EGFR inhibition to reprogram the tumor microenvironment and overcome immune...

Despite the remarkable clinical outcomes of epidermal growth factor receptor (EGFR)-targeted therapies in patients with lung cancer, therapeutic resistance eventually develops. This study elucidates the role of galectin-9 (Gal-9), a TIM-3 immune checkpoint ligand, in facilitating tumor immune escape during EGFR tyrosine kinase inhibitor (TKI) therapy, and evaluates the therapeutic potential of combined EGFR-TKI and Gal-9 blockade in preclinical models.

EGFR-TKI-mediated Gal-9 regulation was systematically investigated through multianalysis including RNA-seq transcriptomics, quantitative reverse transcription-PCR, immunoblotting, ELISA, flow cytometry, and immunohistochemical validation across human and murine lung/colorectal cancer cell lines, murine tumor tissues, and paired patient tumor tissues/serum samples. Therapeutic efficacy was evaluated in two syngeneic murine models, with comprehensive immune monitoring of tumor microenvironment (TME), tumor-draining lymph nodes (tdLNs), and splenic compartments. Mechanistic investigations employed CD8⁺ T-cell/macrophage depletion strategies (anti-CD8α monoclonal antibodies (mAbs)/PLX-3397), type I interferon (IFN-I) pathway inhibition (anti-IFNAR1 mAbs), and lymph node retention approaches (FTY720 administration).

EGFR-TKI treatment significantly induced Gal-9 expression in both tumor cells and host immune cells, particularly myeloid cells. Clinical validation revealed elevated Gal-9 levels in EGFR-TKI-treated patient with lung cancer tumor tissues and serums, correlating with reduced progression-free survival. Mechanistically, EGFR-TKIs triggered DNA damage-potentiated cytosolic double-stranded DNA accumulation and activated tumor-intrinsic STING-IFN-I innate immune pathway that transcriptionally regulated Gal-9 expression. Notably, Gal-9-neutralizing antibodies synergized with EGFR-TKI to markedly inhibit tumor growth in two syngeneic mouse models, including the poorly immunogenic LLC lung tumor model unresponsive to programmed cell death protein-1/programmed death-ligand 1 blockade. The combination therapy remodeled myeloid landscapes toward antigen-presenting phenotypes, promoted dendritic cell accumulation in the tdLN and enhanced CD8⁺ T response in the TME. Depleting CD8⁺ T cells or macrophages/monocytes abrogated the therapeutic benefits. Blocking the IFN-I pathway attenuated Gal-9 expression and enhanced the antitumor immunity of afatinib in the LLC tumor model.

These findings identify Gal-9 upregulation as a key mechanism mediating immune evasion and limiting EGFR-TKI efficacy, providing a promising combinational therapeutic strategy of EGFR-TKI and Gal-9 blockade for the treatment of EGFR-driven cancers.