Advances in Spatial Heterogeneity and Cellular Localization Specificity of Intratumoral Bacteria

Bacteria in tumors are increasingly recognized to be a part of the tumor microenvironment and their biological function depends on the location and relative abundance of a given microorganism or microbial community. Genetic sequencing data directly from tissue samples to detection of active metabolic signals from microorganisms, cell-microbiome interactions, ultrastructural localization of microorganisms; these are all different kinds of inferences about the functional role of the microbiome, and the evidence for "microbial presence and function" grows in a stepwise fashion. Furthermore, most current microbial detection methods do not, alone, predict the viability, biological activity, or functional properties of microorganisms. In this review, we compare different existing microbial detection techniques and distinguish three spatial paradigms: organized communities in tissue, intracellular bacteria, and extracellular bacteria. We summarize how each paradigm influences signal transduction, metabolism, metastatic spread, treatment resistance, antitumor immunity, and immunotherapy responses. We focus on low-biomass contamination, batch effects, negative controls, absolute quantification, and orthogonal validation. Finally, we classify results according to localization, biological role, clinical evidence, application, and applications, while distinguishing observations of human patients from mechanistic and therapeutic preclinical studies. Spatial microbiology provides useful information about tumor-microbial interactions, but clinically actionable conclusions require standard analysis, reproducible localization, viability or activity, and functional disruption experiments to demonstrate microbial effects and independent clinical validation.