Sapra, Leena

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.

Cellular senescence is a heterogeneous phenotype characterized primarily in mesenchymal cells, but the extent to which immune cells differ in their senescence phenotype, or “senotype,” is unclear. Here, we applied single-cell approaches alongside both global and cell-specific genetic senolytic mouse models to evaluate the senotype of immune cells in the bone marrow of aging mice. We found that myeloid-lineage cells exhibited the highest expression of p16 and senescence-associated secretory phenotype markers among all immune cell types. In contrast with clearance of p16 + senescent mesenchymal cells, targeted clearance of p16 + myeloid cells in aged mice had only minor effects on age-related bone loss in male mice, with no effects in females. In more detailed analyses, p16 + myeloid cells were only acutely cleared, being repopulated back to basal levels within a short time. This led to a lack of long-lasting reduction in senescent cell burden, unlike when targeting bone mesenchymal cells. In vitro, myeloid-lineage cells differed markedly from mesenchymal cells in the development of a senescent phenotype. Collectively, our findings indicate that aged bone marrow myeloid cells do not achieve the fully developed senescent phenotype originally described in mesenchymal cells, justifying further characterization of senotypes of immune cells across tissues.