Untargeted metabolomics in microbiome-immune crosstalk in cancer: challenges and recommendations for functional interpretation

The gut microbiome plays a pivotal role in modulating responses to immune checkpoint blockade (ICB) through its influence on host immunity. While several studies have identified individual bacterial species associated with ICB efficacy, findings remain inconsistent, and the underlying mechanisms are poorly defined. As microbial metabolic capacity is better conserved across individuals than taxonomic composition and is associated with ICB response, there is growing interest in investigating the role of gut-derived metabolites in response to ICB. Untargeted metabolomics provides an opportunity to study this in an unbiased manner but poses important technical and interpretive challenges. Using untargeted fecal metabolomics from patients with mismatch repair-deficiency/microsatellite instable-high (MMR-d/MSI-H) metastatic cancers receiving ICB, we examine challenges in metabolite annotation and identification, defining reliable response associations, and validating their potential immunomodulatory effects. Based on these data, we outline key lessons and recommendations that may inform future studies linking metabolomic profiling to microbiome-immune dynamics.