空间多组学揭示脑转移瘤中三级淋巴结构的功能多样性

Tertiary lymphoid structures (TLS) are increasingly recognized as important drivers of anti-tumor immunity and predictors of survival and immunotherapy response in multiple non-central nervous system cancers. However, their role in brain metastases (BrM) remains poorly understood. To better advance understanding of their role in the immunosuppressive brain tumor microenvironment, we applied a multimodal approach to examine TLS prevalence, spatial organization, and clinical relevance in a retrospective cohort of 461 BrM samples from diverse primary tissues, collected between 2015 - 2022 at the University Cancer Center Frankfurt. CD20 immunohistochemistry was used to screen for lymphoid aggregates, and a subset of TLS-positive and negative tumors were subjected to comprehensive molecular and spatial profiling. For an in-depth characterization of TLS, we employed multiple spatial technologies, including 7-plex immunofluorescence imaging for spatially resolved cellular phenotyping and TLS identification, whole-transcriptome spatial profiling of regions of interest, subcellular-resolution spatial transcriptomics (468-gene panel) for whole-tissue mapping, and spatial proteomic profiling using 40 markers to assess immune and stromal interactions. TLS were detected in 50% of BrM, most frequently in lung-to-brain metastases. Notably, the majority of TLS lacked features characteristic of mature germinal centers, indicating a potentially attenuated or dysregulated immune response within the BrM microenvironment. Survival analyses revealed a favorable prognosis associated with TLS-positive tumors and an improved response to immune checkpoint inhibitors. Spatial characterization of the BrM tumor immune microenvironment revealed cellular niches that promote distinct patterns of immune cell aggregate formation and cell-cell interactions, underlying phenotypic heterogeneity within patients and across primary sites. Analysis of whole transcriptomic data further revealed remodeling of extracellular matrix and evidence of metabolic reprogramming in tumor rich areas and enrichment of processes such as antigen presentation, regulation and activation of immune response in B cell-rich niches. Integrative single-cell spatial transcriptomics and proteomics, enabled phenotyping of TLS subtypes across patients, capturing different stages of maturation and levels of organization. This spatially resolved, multimodal approach provides a comprehensive resource for understanding TLS biology in BrM and may guide microenvironment-informed strategies to improve immunotherapeutic outcomes in metastatic brain disease.