Aging promotes liver and lung melanoma metastasis via tissue-infiltrating Tregs

Metastasis is the leading cause of melanoma-related deaths, yet its age-related mechanisms remain poorly understood. This gap stems partly from reliance on young mouse models (~8 weeks old, equivalent to 20 human years) in most studies. Additionally, research has largely focused on lung metastasis, even though melanoma often spreads to other sites like the liver, which is notably more resistant to immune checkpoint inhibitors (ICIs).Using syngeneic melanoma cells, we optimized intravenous (IV) injections to model lung colonization and developed a novel liver colonization model using hydrodynamic tail vein injections. Tumor cells were injected into young (8 weeks) and aged (12-16 months) male C57BL/6 mice. Spleen, lungs, and liver were harvested to assess immune infiltration of lymphocyte and myeloid populations via flow cytometry. Hematoxylin and eosin (H&E) and immunohistochemistry (IHC) staining were used to quantify metastases and examine spatial immune cell locations. Upon observation that regulatory T cells (Tregs) increased consistently in aged mice in both lung and liver metastatic sites, we treated additional aged cohorts with either anti-mouse CD25 antibody, which we show decreased Tregs while not changing CD3+, CD4+ or CD8+ T cell infiltration, or an IgG control (200 μg/mouse, intraperitoneally) to evaluate the role of Tregs and in metastasis.Aging significantly increased melanoma colonization in both the lungs and liver compared to young mice. Analysis of common changes in the immune microenvironment of both the lung and the liver revealed elevated Treg infiltration in both metastatic sites of aged mice. Notably, Tregs were not elevated in the pre-metastatic niche, suggesting an age- and tumor-specific mechanism of immune suppression. Depletion of Tregs in aged mice using anti-CD25 antibody significantly reduced lung metastases compared to IgG controls. Moreover, depletion of Tregs decreased secretion of TGF-beta and the number of myeloid cells in metastatic sites and increased the number of NK1.1 + NK cells, CD4 + and CD8 + T cells.Our findings indicate that age-associated Treg infiltration promotes metastasis to the lung and liver, suggesting that targeting Tregs could offer a promising approach to mitigate age-related metastatic progression in melanoma.