TIM-3 inhibition enhances breast tumor progression and metastasis: A paradoxical immune checkpoint response.

T cell immunoglobulin and mucin-domain containing-3 (TIM-3) is an emerging immune checkpoint receptor. Blocking immune checkpoint signals is a promising strategy for cancer immunotherapy. While TIM-3 blockade is currently under clinical investigation, its context-dependent role remains poorly understood. This study investigates the molecular consequences of TIM-3 inhibition using an experimental murine breast tumor model. Contrary to therapeutic expectations, administration of an anti-TIM-3 monoclonal antibody led to accelerated tumor growth and a significant increase in liver metastases. Flow cytometry revealed a paradoxical increase in tumor-infiltrating CD8(+) T cells, accompanied by a reduction in CD3(+) and Foxp3(+) T cells. Cytokine profiling showed elevated levels of IFN-γ, TNF-α, and IL-17 in the serum, with increased IL-10 and IL-1β in tumors and altered cytokine expression in the spleen. Proteomic analysis identified 1371 dysregulated proteins, and gene set enrichment analysis revealed upregulation of PI3K/Akt-mTORC signaling, which promotes CDK4-mediated proliferation and tumor stemness via B2M and CD44. Gene ontology analysis indicated suppression of autophagy and apoptosis pathways, including downregulation of the proinflammatory protein complex calprotectin (S100A8/A9). Notably, TIM-3 blockades enhanced epithelial-to-mesenchymal transition (EMT) and c-MYC signaling, potentially driven by Foxp3 downregulation. Proteomics data are available via ProteomeXchange with identifier PXD065028. This finding challenges the prevailing view that immune checkpoint blockade uniformly suppresses tumor growth. Instead, this study demonstrates that TIM-3 inhibition may paradoxically exacerbate tumor progression and metastasis.