Spatial multi-omics reveal distinct immunosuppressive lipid-laden macrophages in primary CNS lymphoma compared to systemic DLBCL

Abstract Introduction Primary central nervous system lymphoma (PCNSL) is an aggressive B-cell lymphoma exhibiting unique central nervous system (CNS) tropism, and high recurrence rates despite sharing morphological and molecular features with systemic Diffuse Large B-Cell Lymphoma (DLBCL). Given the unique immune landscape of the CNS and the critical role of macrophages in neural tissue, we hypothesized that PCNSL may be sustained by a CNS-specific macrophage program distinct from DLBCL, representing a targetable mechanism underlying immune evasion, CNS confinement, and treatment resistance. While tumor-associated macrophages (TAMs), particularly CD163+ M2-like macrophages, are known to be enriched in PCNSL tumor microenvironment (TME), prior spatial studies have focused predominantly on tumor-intrinsic features and T-cell dysfunction, leaving macrophage organization and functional programming poorly characterized. Methods We employed cutting-edge spatial multi-omics using four complementary platforms to comprehensively profile macrophage heterogeneity. Formalin-fixed paraffin-embedded (FFPE) samples in TMA format (26 PCNSL, 89 DLBCL) were analyzed using a Xenium 380-gene immuno-oncology panel. GeoMx digital spatial profiling whole transcriptome analysis (DSP-WTA) was performed in 82 cases (17 PCNSL, 65 DLBCL) using CD3, CD20, and CD68 cell masks. Macrophage signatures identified from DSP were validated across independent single-cell RNA sequencing (scRNA-seq) datasets (PCNSL N=28, DLBCL N=17, reactive lymph nodes N=2), distinguishing microglia from monocyte-derived macrophages. CellScape high-plex imaging was used to confirm phenotypes at the protein level and assess spatial proximity and interactions in 24 PCNSL and 5 tonsil samples using 40 architecture, immune and macrophage markers. Results Xenium spatial profiling revealed significantly higher macrophage abundance in PCNSL versus DLBCL, confirmed by DSP-WTA (p=0.0002). DSP further demonstrated that PCNSL TAMs upregulate immunosuppressive genes (CRYAB, SPP1) while downregulating T-cell recruitment genes (CCL19, IGSF6), with enrichment of glycolysis, cholesterol homeostasis, and peroxisome pathways. The CXCL9:SPP1 expression ratio, a validated macrophage polarity biomarker of prognosis in cancer, was significantly reduced in PCNSL macrophages across both DSP and scRNA-seq datasets (p=0.027). Of specific interest, differentially expressed gene (DEG) projection and BayesPrism deconvolution of CD68+ regions of interest (ROIs) revealed enrichment of TREM2+ macrophages (p=0.00066) and elevated GPNMB+ lipid-laden macrophage (LLM) signatures (p=0.004) in PCNSL. scRNA-seq confirmed higher LLM signatures in PCNSL versus DLBCL (p=0.0052) and lymph nodes (p=1.2e-06), identifying a monocyte-derived subset enriched in cholesterol metabolism and high-density lipoprotein (HDL) binding pathways. Cell-cell communication analysis revealed enhanced interactions between LLMs and regulatory T-cells via secreted phosphoprotein 1 (SPP1), apolipoprotein E (APOE), and intercellular adhesion molecule 1 (ICAM1) signaling axes. CellScape imaging confirmed the presence of GPNMB+CD163+CD274+ macrophages in PCNSL, with spatial analysis showing that LLM-T cell proximity correlated with treatment response. Conclusion We define a CNS-specific macrophage program characterized by TREM2+ GPNMB+ lipid-laden macrophages that may foster CNS tropism in PNCSL, along with immune evasion through metabolic reprogramming and regulatory T-cell activation. This first comprehensive spatial multi-omics characterization of macrophage heterogeneity distinguishing PCNSL from DLBCL identifies TREM2, SPP1 and lipid metabolism as potential therapeutic targets for macrophage-directed immunotherapy in this disease of unmet clinical need.