Repetitive fasting-refeeding enhances metformin-induced CXCR6+ CD8+ T cell tumor infiltration via VCAM-1 upregulation on normalized vasculature during refeeding.

Fasting is known to alter the circulation dynamics of immune cells, including T cells, by shifting them from peripheral tissues to the bone marrow (BM), where they enter a quiescent state to avoid starvation stress and acquire apoptosis resistance through upregulation of BCL2. Upon refeeding, these T cells exit the BM and return to circulation. In solid tumors, fasting-refeeding not only affects the trafficking of CD8+ T cells between tumors and their draining lymph nodes (dLNs); but also modulates the antitumor immune response. In this study, we investigated how metformin's antitumor responses are affected by repeated fasting-refeeding cycles. Metformin administration combined with weekly 48-hour fasting showed a synergistic antitumor effect, which was abolished by in vivo depletion of CD8+ T cells. Immunohistofluorescence staining showed that fasting reduced CD8+ T cells in tumors and dLNs while increasing their presence in the BM; refeeding reversed this distribution. Refeeding also increased the expression of Ifng, Gzmb, Tnf, and Tbx21 in tumors. Likewise, Cxcr6, Cxcl16, and Vcam1 expression levels were elevated only upon refeeding. Notably, CXCR6 was exclusively expressed on CD62L- effector memory T cells (TEM). The antitumor effect induced by the combinational therapy was abolished by administration of an anti-VCAM-1 neutralizing antibody. Our findings demonstrate that combining metformin with fasting exerts a synergistic antitumor effect by recruiting CD8+ T cells-relocated to the BM during fasting-back to the tumor during refeeding, facilitated by enhanced VCAM-1 expression on normalized tumor vasculature.