In recent years, several studies have shown the importance of both the gut and the tumor microbiome in cancer progression and immune checkpoint inhibitors (ICI) response. Thus, microbiomes appear as a new source of therapeutic strategies to expand the efficacy and adherence to ICI treatments. In parallel, probiotic self-administration is increasing in cancer patients starting immunotherapy treatments. In their study, Bender and colleagues explored the mechanism through which the probiotic Lactobacillus reuteri increases ICI efficacy in a melanoma preclinical model and provided evidence that the same type of mechanisms may occur in advanced melanoma patients.
Lactobacillus reuteri induces antitumor immunity and increases ICI efficacy
Four frequently used probiotic bacteria Bifidobacterium longum, Lactobacillus reuteri, Lactobacillus johnsonii, and Escherichia coli were administered daily to a preclinical melanoma model. Administration, one day post tumor cell engraftment, of B. longum, L. reuteri, and E. coli restrained tumor growth and increased survival. The administration of L. johnsonii failed to show similar effects, which implies species-specific abilities in reducing tumor growth. Out of the 4 probiotic strains, L. reuteri showed the most potent tumor-suppressive ability. The daily oral administration of L. reuteri led to a significant increase in its relative abundance in the small intestine but neither in the caecum nor the colon, and did not substantially affect the overall composition of the gut microbiota.
The treatment with L. reuteri skewed the tumor microenvironment (TME) toward an antitumorigenic, immunostimulatory environment, characterized by an expansion of interferon-γ (IFNγ)-producing CD4 Th1 and CD8 Tc1 cells that actively proliferate. Combinatorial treatment with L. reuteri and αPD-L1 showed significantly improved tumor size reduction and showed the most pronounced TME Tc1 response. Interestingly, combinatorial treatment with L. reuteri and α-CTLA-4 also resulted in an additive effect on tumor reduction, suggesting that L. reuteri may improve responses to various ICI therapies.
Lactobacillus reuteri translocates to and colonizes the tumor, where it mediates antitumoral effects
The presence of a tumor microbiome was shown in several intestinal and non-intestinal cancers, and live bacteria have been cultivated from breast and pancreatic human tumors. However, whether intratumoral bacteria are active participants that impact tumor development remains elusive. Using a broad-spectrum culturomics approach, a diverse array of viable bacteria was detected within tumors of control mice. Tumors of L. reuteri-treated mice had reduced bacterial diversity driven by an enrichment of L. reuteri. The tumor-cultivated colonies isolated from L. reuteri-treated mice displayed high similarity to the orally administered L. reuteri strain, unlike the isolates from the control mice, which likely represent endogenous strains. Surprisingly, neither tumor formation nor L. reuteri administration was found to increase intestinal permeability during early (day 9), intermediate (day 11), and late (day 14) stages of tumor formation. L. reuteri was also shown to persist within the TME after the intratumoral injection of a single dose. This confirmed that following oral administration, L. reuteri translocates to, colonizes, and dominates the TME.
Lactobacillus reuteri catabolizes dietary tryptophane into I3A, which activates the AhR within CD8 T cells
Lactobacillus reuteri can release several immunomodulatory metabolites, including indole-3-aldehyde. To determine, whether L. reuteri effect on tumor growth and survival is linked to I3A production, experiments with a mutant strain of L. reuteri were performed. The mutant strain, L. reuteri DArAT, lacks the ability to catabolize dietary tryptophan into I3A. The oral administration of L. reuteri DArAT failed to suppress tumor growth, increase survival, or trigger potent antitumor Tc1 immunity. Both L. reuteri strains WT and DArAT were found present and viable at similar levels in tumor isolates, thus the failure of the mutant strain to induce tumor suppression was not due to an inability to translocate to the tumor. Furthermore, L. johnsonii, which cannot naturally produce I3A, failed to suppress tumor growth. I3A alone, through oral administration or intratumoral injection, was also able to suppress tumor growth and increase survival in a dose-dependent manner, thus confirming I3A critical role.
I3A has been shown to activate the aryl hydrocarbon receptor (AhR), a ubiquitously expressed transcription factor, which plays a pivotal role in skewing CD4 T cell differentiation toward Th17 or Treg cell lineage in a ligand-dependent fashion. However, the impact of microbial AhR ligands on Tc1 function remains undefined. Cultures of splenic naive CD8 T cells with supernatant derived from L. reuteri WT or DArAT showed that only the supernatant of the WT strain induces the production of IFNγ. Thus, L. reuteri-released I3A acts directly on CD8 T cells to promote Tc1 function.
To investigate further, mice were placed either on a tryptophan-enriched (Trp-high, 1.19%) or tryptophan-depleted (Trp-low, 0.19%) diet for 4 weeks before tumor cell implantation and stayed on their respective diet for the entirety of the experiment. Although the Trp-low diet did not fully abolish the tumor-suppressing properties of L. reuteri, mice on the Trp-high diet showed significant tumor suppression and increased survival. Interestingly, the Trp-high diet alone was sufficient to slow the tumor growth and prolong survival, as well as to potentiate αPD-L1 efficacy. Trp-enriched diet being sufficient to reduce tumor growth indicates that other Trp-catabolizing members of the microbiota can contribute to antitumor immunity.
Evidence for the role of I3A in promoting ICI responses and survival in advanced melanoma patients
The potential influence of I3A on ICI efficacy was investigated in advanced, stage-IV melanoma patients that either responded (n=19) or not (n=23) to combinatorial IFNα and αPD1 immunotherapy. I3A serum levels were significantly higher in ICI responders than in non-responders. Furthermore, patients with high systemic I3A levels at baseline exhibited significantly prolonged progression-free survival and overall survival in contrast to patients with low I3A levels.
In their study, Bender and colleagues showed the role of L. reuteri in tumor regression and ICI response. They demonstrated that L. reuteri translocates to, colonizes, and persists within tumors where it locally promotes antitumor Tc1 immunity via the release of I3A, an AhR agonist acting on CD8 T cells. They also provided early evidence for the I3A role in promoting ICI response in melanoma patients. This study reinforces the importance of investigating microbiomes in cancer research by providing a rational mechanistic basis to design novel dietary and probiotic combinatorial therapeutic strategies.
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Dietary tryptophan metabolite released by intratumoral Lactobacillus reuteri facilitates immune checkpoint inhibitor treatment
Bender MJ, McPherson AC, Phelps CM, Pandey SP, Laughlin CR, Shapira JH, Medina Sanchez L, Rana M, Richie TG, Mims TS, Gocher-Demske AM, Cervantes-Barragan L, Mullett SJ, Gelhaus SL, Bruno TC, Cannon N, McCulloch JA, Vignali DAA, Hinterleitner R, Joglekar AV, Pierre JF, Lee STM, Davar D, Zarour HM, Meisel M. Cell. 2023 Apr 27;186(9):1846-1862.e26. doi: 10.1016/j.cell.2023.03.011. Epub 2023 Apr 6. PMID: 37028428; PMCID: PMC10148916.
Keywords :melanoma, immune checkpoint inhibitor, aryl hydrocarbon receptor, Lactobacillus reuteri, indole-3-aldehyde, tryptophan, microbial AhR ligands, tumor microbiome, microbial-host crosstalk.
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