Temporal dynamics of mesenchymal stem cell administration influence immune modulation in a 4T1 breast cancer model

Mesenchymal stem cells (MSCs) are recognized for their capacity to modulate immune responses, including those directed against tumors. In this study, we investigated the temporal effects of MSCs administration on anti-tumor immunity in a murine 4T1 breast cancer model. BALB/c mice were intraperitoneally injected with MSCs either 24 h (MSC1d) or 14 days (MSC14d) after orthotopic implantation of 4T1 mammary carcinoma cells. Early MSC administration (MSC1d) exhibited changes in immune cell phenotypes consistent with enhanced antitumor potential, including increased activity of natural killer (NK) cells, dendritic cells (DCs), macrophages, and T lymphocytes. These immunological changes correlated with reduced tumor growth and prolonged survival. Mice in the MSC1d group exhibited elevated serum levels of pro-inflammatory and anti-tumor cytokines (TNF-α, IFN-γ, IL-6, and IL-17), alongside decreased concentrations of immunosuppressive cytokines (TGF-β and IL-10). Tumor tissue analysis revealed increased infiltration of NK cells expressing markers associated with antitumor activity (IFN-γ-producing CD178⁺), CD80⁺/CD86⁺/I-A⁺ TNF-α-producing DCs, Th1-type CD4⁺ T cells, and Granzyme B-expressing CD8⁺ cytotoxic T lymphocytes (CTLs). Additionally, spleens of MSC1d-treated mice displayed significantly elevated populations of CD11c⁺ DCs, TNF-α/IFN-γ-secreting NK cells, CD4⁺ Th1 and Th17 cells, and CD8⁺ CTLs expressing markers associated with cytotoxic function (TNF-α, IFN-γ, and IL-17). Conversely, late MSCs administration (MSC14d) was associated with immunosuppression. Tumors from MSC14d-treated mice showed a decreased presence of IFN-γ⁺ and IL-17⁺ NK1.1⁺ cells, F4/80⁺ macrophages, IL-12⁺ DCs, and cytotoxic T cells. Spleens from these mice revealed a significant expansion of regulatory T cell (Treg)-like populations, including CD25⁻, FoxP3⁻, CD25⁺FoxP3⁻ cells, and TGF-β/IL-10-producing CD3⁺ and CD4⁺ T cells. Furthermore, serum levels of immunosuppressive mediators TGF-β and vascular endothelial growth factor (VEGF) were significantly elevated in the MSC14d group. Collectively, these findings demonstrate that the immunomodulatory effects of MSCs on breast cancer are highly dependent on the timing of their administration. Mesenchymal stem cells delivered during early tumor development enhance phenotypes consistent with antitumor potential and suppress tumor progression, whereas administration during later stages promotes immune evasion and tumor growth.