Spatial-Resolved Transcriptomics Reveals Immune Landscape Variations in Primary Central Nervous System Lymphoma (PCNSL) and Diffuse Large B-Cell Lymphoma (DLBCL)

Primary central nervous system lymphoma (PCNSL) is a rare and aggressive non-Hodgkin lymphoma with a high risk of recurrence, posing significant clinical challenges due to its morbidity. While PD1-based immunotherapy has shown promise in some PCNSL cases, it has been less effective in diffuse large B-cell lymphoma (DLBCL). Understanding the tumor microenvironment factors that distinguish PCNSL from DLBCL and those mediating PCNSL relapse after standard chemotherapy is crucial for integrating immunotherapy into treatment. In this study, we employed digital spatial profiling (DSP), an advanced technique for spatially resolved transcriptomics, to capture the whole transcriptome atlas (WTA) with over 18,000 RNA targets across specific cell types. Using DSP, we profiled the WTA of macrophages, T cells, and B cells in tumor tissue samples from patients with DLBCL (n = 64) and PCNSL (n = 16), encompassing 409 areas of interest (AOIs). Selective collection of UV-cleavable probes from distinct masks, generated by immunofluorescent staining of the morphology markers CD68, CD3, and CD20, accurately captured the WTA of respective cell types in their native tissue environment. We identified many differentially expressed genes (DEGs) significantly upregulated in DLBCL or PCNSL (adjusted P value < 0.05 and |log2FC| > 0.58). In PCNSL tumor cells, immune checkpoints such as LAG-3 and chemokines (CXCL13, CCL3, CCL5) were actively upregulated. Pathway enrichment analysis revealed that inflammatory response, complement, and IL-2/STAT5 signaling were enriched in PCNSL tumor cells, indicating a more pronounced immune response within the tumor microenvironment, potentially contributing to the preferential sensitivity to PD1 therapy. Significant differences were also noted in the macrophage compartment between DLBCL and PCNSL. DEGs such as SPP1 and CD163 were highly expressed in PCNSL macrophages. Metabolic pathways such as hypoxia and glycolysis were enriched in PCNSL macrophages, suggesting that metabolic reprogramming may create a pro-tumorigenic microenvironment that supports tumor progression. Utilizing MoMac-VERSE, the largest single-cell transcriptomic meta-analysis of human monocytes and macrophages, we projected the top DEGs of the CD68 mask and found that PCNSL macrophage DEGs overlapped with the TREM2 macrophage cluster. This cluster has been shown to be associated with a poor prognosis and attenuate responses to PD-1 antibodies in various solid cancers, indicating that this specific macrophage subpopulation in PCNSL may have unique functions in shaping the tumor microenvironment and affecting the response to immune checkpoint inhibitors. In summary, leveraging DSP, we present a comprehensive understanding of the distinct immune landscapes in DLBCL and PCNSL. Our findings highlight the importance of spatial transcriptomics in elucidating the unique characteristics of these lymphoma subtypes and may have implications for novel immunotherapeutic treatment strategies.