15 Defining Systemic and Tumor-Intrinsic Biomarkers of Immune Suppression in Renal Cell Carcinoma

Vollständiger Abstract

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Abstract Background Renal cell carcinoma (RCC) is characterized by a highly immunosuppressive tumor microenvironment enriched in tumor-associated macrophages (TAMs) and regulatory T cells (Tregs), both of which correlate with poor clinical outcomes. TAMs in RCC commonly exhibit an immunosuppressive phenotype, marked by production of TGFb, IL-10 and IL-6, which suppress cytotoxic T cell responses and promote tumor progression. Despite advances in immunotherapy, metastatic RCC remains highly lethal, underscoring the need for improved biomarkers and therapeutic strategies that overcome immune suppression. Methods To define systemic and tumor-intrinsic immune alterations in RCC, we prospectively collected 26 fresh surgical RCC tumors with associated normal tissue and peripheral blood, along with peripheral blood from 20 healthy donors. Peripheral blood mononuclear cells (PBMCs) were analyzed by 25-color flow cytometry and single-cell RNA sequencing, while serum samples were profiled using high-throughput proteomics platforms (Olink and Illumina), enabling quantification of over 5,000–9,000 circulating proteins. Tumor specimens were analyzed by single-cell RNA sequencing and immunohistochemistry (IHC) to characterize immune composition and cellular interactions within the TME. Results Our analyses reveal systemic and intratumoral features consistent with coordinated immunosuppressive programming. RCC patient PBMCs exhibited a significant expansion of Tregs alongside a reduction in circulating CD14+HLA-DR+ monocytes, suggesting depletion of activated, antigen-presenting myeloid populations. Serum proteomics identified increases in proteins associated with macrophage activation and immune regulation, including increased levels of IL-6, as well as ADGRG1 and HAVCR1. Gene set enrichment analysis (GSEA) of serum proteomic data revealed significant upregulation of metabolic and transport-related pathways, including fatty acid binding and lipid transmembrane transport, alongside enrichment of L-amino acid metabolic processes, mitochondrial membrane organization, extracellular vesicles and exosomes, and regulation of lymphocyte-mediated immunity. Conversely, pathways related to cell adhesion, cytoskeletal organization, and tissue repair processes were downregulated. Together, these findings suggest systemic alterations in myeloid and T cell populations that reinforce immunosuppressive programs within RCC tumors, while also reflecting pathways associated with kidney injury and tumor stemness. To functionally interrogate therapeutic strategies targeting myeloid-driven immune suppression, we established ex vivo tumor slice cultures from primary RCC specimens. These cultures preserve tumor architecture and immune contexture, enabling direct assessment of therapeutic responses. We evaluated the neutralization of IL-34, a tumor-derived cytokine associated with macrophage proliferation and poor survival in ccRCC, and of phosphoinositide 3-kinase gamma (PI3Kγ), a key regulator of immunosuppressive myeloid cell polarization and trafficking. Pharmacologic PI3Kγ inhibition (IPI-549) reprograms macrophages toward a pro-inflammatory phenotype and enhances anti-tumor immunity. We hypothesize that these interventions will restore anti-tumor immune responses and kill and or reduce tumor burden. To test this, naïve and treated tumor slices were analyzed by immunohistochemistry (CD3, CD68, PanCK, cleaved caspase-3) and single-cell sequencing. These approaches are enabling us to define whether macrophage-targeted therapies can reshape myeloid and T cell states, tumor cell survival, and immune composition within intact ccRCC tumors. Conclusions Together, this integrated approach combining systemic immune profiling, serum proteomics, tumor single-cell analysis, and functional ex vivo modeling identifies candidate biomarkers of immune suppression and therapeutic response in RCC. These findings support the development of biomarker-guided strategies targeting macrophage-driven immune dysfunction and may inform combination therapies to improve clinical outcomes in RCC patients.