From density to spatial function: spatial multi-omics, composite biomarker design, and therapeutic engineering of tertiary lymphoid structures in solid tumors.

Background: Tertiary lymphoid structures (TLSs) have emerged as clinically relevant immune niches associated with improved survival and enhanced response to immune checkpoint inhibitors across multiple solid tumors. However, translational evaluation remains dominated by density-based scoring, which inadequately captures the functional heterogeneity of histologically similar lymphoid aggregates. This review synthesizes recent advances in spatial biology that support a shift from counting TLSs to interpreting them as spatially organized immune microenvironmental units embedded within local tumor ecosystems. Main body: Evidence from spatial transcriptomics, multiplexed imaging, single-cell profiling, computational pathology, and spatial-proteomic approaches converges on four coupled requirements for productive TLS function: stromal licensing through lymphotoxin-dependent chemokine circuits, vascular competence mediated by specialized high endothelial venules, helper T-cell programs that bridge follicular and cytotoxic immunity, and a permissive metabolic context that enables maturation. Spatial studies further show that regulatory infiltration, suppressive plasma-cell programs, and metabolic disruption can decouple structural presence from effective antitumor immunity. Across tumor types, these insights indicate that biomarker performance improves when TLS density is integrated with maturity, anatomical location, HEV coupling, immune composition, and treatment context rather than used as a standalone metric. We further discuss how spatial analyses reveal actionable pathways for inducing or reprogramming TLSs and outline a staged, module-matched composite biomarker workflow combining digital pathology, targeted multiplexed imaging, and focused molecular or metabolic augmentation. Conclusions: TLSs should be regarded as spatially organized, functionally heterogeneous immune microenvironmental units rather than biologically equivalent lymphoid aggregates. A spatial-functional framework helps explain productive, arrested, and suppressive TLS states, provides a basis for next-generation composite biomarkers, and supports module-matched therapeutic strategies tailored to the stromal, vascular, helper-cell, or metabolic bottleneck limiting TLS function in individual tumors. The immediate next step is prospective validation of a minimal TLS panel across defined treatment contexts.