Spatial Heterogeneity of Intratumoral Microbiota and Its Roles in Tumor-Microbiota Interactions and Therapeutic...

The intratumoral microbiota has emerged as a critical component of the tumor microenvironment (TME), with accumulating evidence indicating that its biological functions are influenced not only by microbial composition but also by their spatial organization within tumor tissues. This review summarizes the historical development and potential origins of intratumoral microbiota, and elaborates on the concept and biological significance of spatial heterogeneity. Based on recurrent spatial distribution patterns reported across different tumor types, we propose a conceptual framework comprising several putative spatial niches, including hypoxic/necrotic, immune-enriched, stromal-associated, invasive/metastatic, and intracellular niches. We further discuss the potential mechanisms contributing to the establishment and maintenance of spatial heterogeneity. The clinical significance of spatial microbial signatures is critically evaluated, alongside a comprehensive overview of spatial analytical methodologies, ranging from in situ hybridization and immunology-based approaches to emerging spatial omics and multi-omics integration strategies. Finally, we address key challenges and limitations, including contamination control, causal inference, barriers to clinical translation, and the underexplored spatial dimensions of the intratumoral mycobiome and virome. By synthesizing current knowledge and identifying critical gaps, this review aims to provide a conceptual and methodological framework for advancing spatially resolved investigations of intratumoral microbiota and facilitating their potential translational applications in precision oncology.