Spatial proteomics of T-cell infiltrates in melanoma reveals differential regulation of TOR signaling

ABSTRACT: The tumor microenvironment determines the clinical response of patients to therapeutic immune checkpoint inhibition (ICI), but a comprehensive understanding of the underlying immune-regulatory proteome is still lacking. To better understand and assess targetable promising biological processes that specifically determine the tumor T cell infiltrate (TiLs), we mapped the spatial distribution of proteins in TiL-enriched vs. low compartments in melanoma by combining microscopy, MALDI-MSI-based imaging and liquid chromatography-mass spectrometry (LC-MS/MS), and computational data mining. Using a spatial proteome algorithm and gene ontology-based enrichment analysis, we identified >145 proteins that were differentially expressed in CD8high tumor compartments including negative regulators of the mTOR signaling such as sirtuin 1 (SIRT1). Multiplexed immunohistochemistry confirmed that SIRT1 protein was higher expressed in CD8high compared to CD8low compartments and present in both CD8+ TiLs and CD8- cell types. Complementary analysis of bulk and single cell RNAseq data suggested expression of SIRT1 by different lymphocyte subpopulations (CD8+ T, CD4+ T and B cells) in close proximity to melanoma cells. We demonstrated in a syngeneic ICI-sensitive mouse melanoma model that the SIRT1 inhibitor EX-527 reduces the anti-tumor effect of α-PD1 ICI accompanied by decreased CD4+ and CD8+ TiLs in the tumor margin. In silico analysis of large transcriptional data cohorts showed that SIRT1 is positively associated with the proinflammatory T-cell chemokines CXCL-9, -10, and IFN-γ, and prolonged overall survival of melanoma patients. Our study concludes that spatial proteomics by MALDI-MSI is a promising technology to characterize the functional tumor microenvironment providing an important mechanistic layer of biologic information for the identification of protein candidates with important prognostic and therapeutic implications.

INSTRUMENT(S): Orbitrap Fusion

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Skin

SUBMITTER: Hannah Voß  

LAB HEAD: Prof. Hartmut Schlüter

PROVIDER: PXD035502 | Pride | 2025-03-04

REPOSITORIES: Pride