In the new issue of Cell, Dr. Meisel’s team characterized how the orally administrated probiotic bacteria Lactobacillus reuteri (Lr) translocated from the gut to tumors in mice with melanoma.1Bender M.J. McPherson A.C. Phelps C.M. et al.Dietary tryptophan metabolite released by intratumoral Lactobacillus reuteri facilitates immune checkpoint inhibitor treatment.Cell. 2023; 186: 1846-1862.e26Abstract Full Text Full Text PDF PubMed Google Scholar Furthermore, its production of a compound called indole-3-aldehyde (I3A) derived from dietary tryptophan directly stimulates immune cells to make cancer immunotherapy more effective (Figure 1A). Here, we highlight and comment on the essential findings and provide our insights into the emerging research paradigm for future exploration of novel anti-tumor probiotics and underlying mechanisms. Microbiome translocation (MT) refers to the movement of microorganisms from one body site to another. Dr. Meisel’s team pioneered to explore the translocation mechanism of Lr, which translocated to the liver, spleen, and tumor microenvironment (TME) via vascular and lymphatic routes and modulated the TME for better anti-cancer outcomes (Figure 1A). However, the translocation mechanisms remain cryptic. (1) Lr was not identified in each host in the treatment groups, suggesting that current technologies had not sufficiently characterized its transmission, and MT might be host-dependent. (2) The exact location of Lr in the TME was still not defined. Whether the presence and abundance of Lr are controlled by the immune or stromal components in the TME should be further investigated. (3) Since Escherichia. coli (Ec) was also found in the TME, more insights could be gained if the team could indicate whether Ec also translocated to systemic tissues (liver, spleen, lymph nodes) like Lr. If Ec was present similarly in other tissues, we believed the translocation mechanisms of Lr and Ec would be comparable to a large extent, which could definitely help map out how the probiotics migrate/do not migrate to the TME. Tracking MT in hosts can be challenging due to multiple non-negligible factors. (1) Low abundance: MT events can involve small numbers of microbes and be hard to detect from tumor samples. (2) Transient nature: MT can be transient, which means that microbes are only present in the host for a short period and can contribute to (1). (3) Host specificity: MT events may be specific to certain hosts, tissues, or physiological conditions, making it difficult to generalize findings. (4) Technical limitations: traditional culture-based methods are often labor-consuming yet inadequate for detecting MT, as many invading microbes cannot be cultivated in currently available media. To overcome these challenges, researchers may use a combination of conventional and emerging techniques. (1) Isolate low-abundance probiotic bacteria using selective media (probiotic-resistant antibiotics, if any) from tumors. (2) Metagenomics allows researchers to culture-independently identify microbial functional pathways in TME associated with probiotic translocation. (3) Spatially resolved single-cell imaging techniques can characterize the spatial distribution of probiotics of interest in the TME.2Galeano Niño J.L. Wu H. LaCourse K.D. et al.Effect of the intratumoral microbiota on spatial and cellular heterogeneity in cancer.Nature. 2022; 611: 810-817Crossref PubMed Scopus (42) Google Scholar Researchers may pay more attention to the emerging spatial single-cell transcriptomic platform coupled with imaging techniques (e.g., fluorescence in situ hybridization) for spatially analyzing target probiotic bacteria, immune cells, and tumor cells within the TME. (4) A non-labeling and non-invasive single-cell sorting platform (such as a Raman-activated single-cell platform) coupled with culturomics enables isolating probiotic bacteria or its host cells and tracking the phenotypic effects on translocation events in a high-throughput manner. Collectively, a multidisciplinary approach combining the most advanced techniques is critical to accurately track and understand the MT (Figure 1B). Tryptophan is an essential amino acid that cannot be synthesized by the human body and must be obtained from diet. The human enzyme system has two main pathways of tryptophan catabolism: (1) the kynurenine (Kyn) pathway is the most critical pathway of tryptophan degradation in the body, accounting for about 95% of tryptophan degradation, and it is mainly carried out in the liver. (2) Under the action of tryptophan hydroxylase, about 5% of tryptophan is converted to 5-hydroxytryptophan, also known as serotonin. Although the proportion of the serotonin pathway is low, its metabolic intermediates are indispensable to the functions of the nervous system. Infection, changes in gut microbiota, and stress may all affect the ratio of serotonin and Kyn pathways involved in tryptophan metabolism. Therefore, the occurrence and development of some mental diseases are obviously related to immune functions, dietary and nutritional factors, and stress. In addition to the role of the human enzyme system, symbiotic microorganisms also have various tryptophan metabolic pathways. Under the action of these microorganisms, tryptophan metabolism has a third pathway: the indole/aryl hydrocarbon receptor (AhR) pathway, in which a number of gut bacteria convert tryptophan into indoles and related derivative molecules respectively.3Agus A. Planchais J. Sokol H. Gut microbiota Regulation of tryptophan metabolism in Health and disease.Cell Host Microbe. 2018; 23: 716-724Abstract Full Text Full Text PDF PubMed Scopus (1024) Google Scholar The authors demonstrated a critical microbial-host crosstalk between probiotic-released AhR agonist I3A and CD8+ T cells within the TME that facilitates immune checkpoint inhibitor (ICI) efficacy in preclinical melanoma. Lr-secreted I3A was both necessary and sufficient to drive anti-tumor immunity, and loss of AhR signaling within CD8+ T cells abrogated Lr’s anti-tumor effects. Further, a tryptophan-enriched diet potentiated both Lr- and ICI-induced anti-tumor immunity, dependent on CD8+ T cell AhR signaling. The authors further evaluated the impact of systemic I3A levels on progression-free survival (PFS) and overall survival. They found that patients with high systemic I3A at baseline exhibited significantly prolonged PFS and overall survival compared with patients with low I3A levels (Figure 1A). AhR can be activated by environmental xenobiotic toxic chemicals, for instance, 2,3,7,8-tetrachlorodibenzo-p-dioxin (dioxin), and endogenous ligands, such as Kyn.4Opitz C.A. Litzenburger U.M. Sahm F. et al.An endogenous tumour-promoting ligand of the human aryl hydrocarbon receptor.Nature. 2011; 478: 197-203Crossref PubMed Scopus (1323) Google Scholar Bender et al. (2023)1Bender M.J. McPherson A.C. Phelps C.M. et al.Dietary tryptophan metabolite released by intratumoral Lactobacillus reuteri facilitates immune checkpoint inhibitor treatment.Cell. 2023; 186: 1846-1862.e26Abstract Full Text Full Text PDF PubMed Google Scholar showed that I3A binding to AhR was necessary and sufficient to drive anti-tumor immunity. Another recent study reported that the endogenous AhR ligand Kyn and the microbial AhR ligands indole-3-acetic acid (3-IAA) displayed pro-cancerogenic properties in pancreatic ductal adenocarcinoma (PDAC), while I3A failed to affect tumor growth in this model. The mechanism is unclear, but the data suggest that AhR ligands act on myeloid cells.5Hezaveh K. Shinde R.S. Klötgen A. et al.Tryptophan-derived microbial metabolites activate the aryl hydrocarbon receptor in tumor-associated macrophages to suppress anti-tumor immunity.Immunity. 2022; 55: 324-340.e8Abstract Full Text Full Text PDF PubMed Google Scholar Overall, these findings reveal that microbial AhR agonists impact cancer development in a ligand-dependent, cell type-, and cancer type-specific manner and underscore the urgent need to better understand the role of microbiota-produced AhR ligands in the context of cancer. The seemingly paradoxical role of AhR—oncogene or tumor suppressor—remains unresolved. Therefore, we suggest systemically screening and determining the biological functions and mechanisms of AhR endogenous and exogenous ligands in tumorigenesis and/or tumor therapy. More importantly, it is essential to elucidate the structure of the AhR ligand-binding domain through crystallography and/or artificial intelligence. Additionally, we should be cautious about the functions of different AhR ligands. Especially, people are suggested to prevent the production (endogenous) or intake (exogenous) of oncogenic AhR ligands. Contradictory effects caused by the same exogenous or endogenous substances via AhR have been reported. For example, I3A may induce different effects via AhR under different conditions. Firstly, such contradictory findings may be due to the interactions between AhR agonists and antagonists, such as synergic or antagonistic effects. Therefore, the systematic and comprehensive evaluation of such interplays is highly recommended to get a full picture of the combined effects of exogenous or endogenous substances via AhR. Furthermore, AhR-targeted therapeutics should be developed based on host individual variations and gene-environment interactions, the essence of precision medicine. Other than Lr, many other probiotic bacteria, including Lactobacillus acidophilus, Lactobacillus murinus, and Bifidobacterium Breve, can also produce I3A using tryptophan. Whether these microbes harboring I3A production ability can enhance ICI treatment in clinical tumor warrants further investigations. Besides, further insights into the TME and tumor microbiota would help identify other microbes or molecules promoting anti-tumor immunity. In the future, the precise integration of specific probiotics and diet would provide outstanding anti-tumor effects in vivo and supplement/contribute to the current anti-tumor treatment strategy, which might prolong cancer patient survival. Moreover, metabolically engineered wild-type probiotics that produce anti-tumor molecules would be promising for personalized treatment for cancer patients. In the original paper, the authors did not clarify how they first realized and pinpointed the I3A pathway as the primary supporting mechanism for the Lr’s anti-tumor function and performed the downstream functional validation with genetic manipulation. Given that more probiotic bacteria would have fit into this research paradigm, how to identify such pathway(s) is worth careful thinking. We suggest researchers conduct comprehensive multi-omics studies on the interested probiotic bacteria using the original strain, tumor-derived strain(s), and stool-derived strain(s) to characterize probiotic genetic evolution during cross-body-site transmission. The integrated analysis of genomic, transcriptomic, metabolomic, and even methylation of isolated probiotics will certainly facilitate researchers in rationally devising strategies for genetic manipulation for validating critical pathways involved in the functional effects of probiotics. Notably, testing if such (epi-)genetic changes of translocated probiotic bacteria are host dependent would be fascinating. Finally, a randomized controlled trial should be further designed to establish causality between the probiotic and host phenotypes (Figure 1B). The authors acknowledge the financial support for this research received from the Health and Medical Research Fund of Hong Kong (10212276), National Natural Science Foundation of China (Nos. 32071301, 32111530179, 32270886, and 32070827), National Key R&D Program of China (2022YFA1106400 and 2020YFA0803201), Key laboratory tasks (LG202103-01-07), Youth Innovation Promotion Association of CAS (2021264), Shanghai Natural Science Foundation (22ZR1469800), Outstanding Youth Reserve Talent Project (IDF201370/074), and Scientific Research Foundation for the introduction of talent (JIF201035Y). The authors declare no competing interests.