Srivastava, Rupesh K.

Abstract Osteosarcoma (OS) is one of the top ranking and deadliest primary malignant bone tumor of youngsters and adolescents. OS has poor prognostic features due to its immunosuppressive “ cold ” tumor microenvironment, obstructing the anti-tumor effector functions of immune cells (including cytotoxic CD8⁺ T lymphocytes)․ Recent developments have focused on the role of Gut Microbiota in modulating effector immune responses in various cancers. However, the immunomodulatory role of gut microbiota and its derived gut-associated metabolites (GAMs) in OS still remains unclear. Here we report that peripheral CD8⁺ T lymphocytes in osteosarcoma patients are less frequent in circulation, hypo-producers of effector cytokines (IFN-γ and TNF-α), and have compromised metabolic fitness․ We characterized and investigated the effect of a panel of microbiota-derived GAMs on CD8⁺ T lymphocytes, confirming their immunomodulatory role with respect to CD8⁺ T lymphocytes activation, cellular metabolism and effector functions․ Indole-3-lactic acid (ILA; product of tryptophan metabolism), was observed to be the strongest immunostimulant among all the GAMs studied. ILA enhanced the anti-tumor effector functions of CD8⁺ T lymphocytes through increased glucose uptake, a higher mitochondrial bio-mass and increased production of IFN-γ and TNF-α․ Moreover, ILA-primed cytotoxic T lymphocytes exhibited considerably higher cytotoxicity and increased apoptosis of osteosarcoma cells (U2OS)․ Altogether, our data demonstrates that ILA acts as a microbial-derived immune modulator to metabolically reprogram and restore dysfunctional CD8⁺ T lymphocytes against osteosarcoma․ This study for the first time demonstrates the therapeutic potential of exploiting the nexus between “ Gut-Immune-Bone Tumor” ternary as a safe and cost-effective combinatorial immunotherapy against osteosarcoma․ Graphical Abstract Indole-3-Lactic Acid (ILA) Reinvigorates CD8 + T-Cell Immunity in Osteosarcoma Circulating CD8 + T cells from osteosarcoma patients exhibit impaired metabolic fitness, reduced effector cytokine production, and diminished tumoricidal activity. Screening of gut-associated metabolites identified the microbial tryptophan metabolite indole-3-lactic acid (ILA) as the most potent immunomodulator. ILA restores glucose uptake and mitochondrial biomass, enhances IFN-γ and TNF-α production, promotes polyfunctional CD8 + T-cell responses, and significantly improves CTL-mediated killing of osteosarcoma cells, highlighting its potential as a microbiota-derived immunometabolic therapeutic for osteosarcoma.