Rethinking the tumor microbiome: Methodological challenges and a framework for rigorous research

The intratumoral microbiome, as an essential component of the tumor microenvironment, has attracted increasing attention in recent years. The tumor microbiota displays an intricate interplay with its biological behaviors. However, emerging controversies, including the retraction of Poore et al.'s article, highlight significant challenges within this field. These issues extend beyond questions of reliability in this particular work to challenge fundamental theoretical assumptions and methodological validity across the entire field of tumor microbiome research, profoundly impacting its future direction. Here, we systematically summarize and discuss the current challenges and limitations in tumor microbiome research. We further propose a series of practical optimization strategies and solutions, from microbial quantification to functional experiments, providing a comprehensive framework covering the entire workflow of tumor microbiome analysis. With ongoing technical progress and standardized protocols, tumor microbiome research is poised to deliver novel insights into tumor biology and contribute to the development of improved therapeutic strategies.