Innovative technologies and future perspectives in tumor microbiomics.

Microorganisms within the tumor microenvironment represent an important but still underexplored dimension of cancer biology, influencing genomic stability, immune responses, metabolism, and therapeutic efficacy. Although recent findings underscore the significance of the tumor microbiome, distinguishing causal "drivers" from non-functional "passengers" remains a major challenge, particularly in the context of extremely low microbial biomass and high contamination risk. Advances in next-generation sequencing, single-cell genomics, and spatial transcriptomics are now transforming the field, enabling profiling that extends beyond descriptive catalogs toward functional and spatial resolution. Here, we introduce a unified, contamination-aware analytical framework tailored to the unique constraints of tumor microbiome studies, integrating standardized sampling, library construction, host-depletion strategies, and multi-layer computational analysis. We also evaluate emerging technologies that couple microbial identity with host-cell states at single-cell and spatial resolution, offering new opportunities to functionally map tumor-associated microorganisms. Furthermore, we propose that the integration of orthogonal validation strategies-combining imaging, spatial transcriptomics, in situ microbial profiling, and sequencing-is poised to define the next stage of mechanistic tumor microbiome research. Together, these perspectives outline key methodological inflection points and future directions toward a mechanistic understanding of tumor-microbe interactions and their therapeutic potential.