Tryptophan-derived microbial metabolites activate the aryl hydrocarbon receptor in tumor-associated macrophages to suppress anti-tumor immunity

May combine two claims.

Amp treatment increases fecal microbial alpha-diversity and OTUs and reduces the relative abundance of Lactobacillus, whereas Vanc reduces overall diversity but increases Lactobacillus abundance.

“Amp treatment increased fecal microbial alpha-diversity and operational taxonomic units compared with controls (Figure S3D), while reducing the relative abundance of Lactobacillus”

• Also stated in: § Indole-producing microbiota drive suppression in the TME promoting tumor growth · plain

  “Vanc treatment, which reduced overall microbial diversity but increased the relative Lactobacillus abundance”

May combine two claims. The quote only partly states this claim.

Amp treatment, but not Vanc, reduces tumor weight and TAM AhR transcriptional activity.

“Amp treatment, but not Vanc, reduced tumor weight.”

• Also stated in: § Indole-producing microbiota drive suppression in the TME promoting tumor growth · hedged

  “the TAMs in Amp-treated mice showed increased expression of MHCII and PD-L1 (Figure 4C) and reduced expression of AhR-responsive genes (Cyp1a1, Cyp1b1, and Cyp1a2)”

May combine two claims.

AhR deletion reduces the percentage of TAMs in the CD45+ infiltrate but does not reduce their absolute numbers.

“AhR deletion reduced the percentage of TAMs in the CD45+ infiltrate but did not reduce the absolute numbers of TAMs”

May combine two claims.

Deleting AhR in tumor cells nullifies PDAC cell responses to AhR agonists in vitro but has no effect on tumor growth in vivo.

“Deletion of AhR in tumor cells by CRISPR (Figure 2E) nullified PDAC cell responses to AhR agonists in vitro (Figure 2F); however, there was no effect on tumor growth in vivo compared with mock PDAC tumors”

May combine two claims.

Combining αPD-L1 IgG and AhR inhibition extends survival 2-fold compared with control and up to 50% higher than either therapy alone.

“combining αPD-L1 IgG and AhR inhibition had the strongest effect by extending the survival 2-fold compared with the control treatment and by increasing the survival up to 50% higher than either therapy alone”

May combine two claims.

AhR deletion alters the expression of 416 genes in TAMs and decreases the immune-regulatory transcriptional signature, including reductions in Arg1 and Ido1.

“Ahr deletion altered the expression of 416 genes”

• Also stated in: § Deletion of AhR drives inflammatory polarization of TAMs and CD8+ T cell infiltration in the PDAC TME · plain

  “TAMs from Lyz2cre/+Ahrfl/fl tumors showed a decreased immune-regulatory transcriptional signature as compared with TAMs from control tumors, characterized by reductions in the expression of Arg1 and Ido1”

May combine two claims.

Removing dietary tryptophan reduces TAM AhR activity and promotes intra-tumoral accumulation of TNFα+IFNγ+CD8+ T cells.

“Removal of dietary tryptophan reduced TAM AhR activity and promoted intra-tumoral accumulation of TNFα+IFNγ+CD8+ T cells”

• Also stated in: § Trp and the indoles IAA and ILA in the diet promote immune suppression and pancreatic tumor growth · plain

  “lack of dietary Trp increased the number of TNFα+IFNγ+ and proliferating CD8+ T cells”

May combine two claims.

Mice gavaged with L. murinus have increased fecal ILA, larger tumors, decreased activated CD8+ T cells, and increased TAM Cyp1b1 and pro-tumor genes.

“mice gavaged with L. murinus had increased fecal ILA”

• Also stated in: § Indole-producing microbiota drive suppression in the TME promoting tumor growth · plain

  “Tumors in mice with L. murinus were larger than tumors in control germ-free mice”

• Also stated in: § Indole-producing microbiota drive suppression in the TME promoting tumor growth · boosted

  “TAMs sorted from 14 day tumors showed that the presence of a L. murinus microbiome increased the expression of Cyp1b1 compared with controls, demonstrating that L. murinus microbiota correlate with increased AhR activity”

May combine two claims. The quote only partly states this claim.

In Lyz2cre/+Ahrfl/fl tumors, macrophage clusters 1 and 2 show significant transcriptional changes, with enrichment of interferon response pathways.

“Macrophage clusters 1 and 2 showed a significant transcriptional change in Lyz2cre/+Ahrfl/fl tumors.”

• Also stated in: § Macrophage AhR activity shapes the transcriptional landscape in PDAC · boosted

  “iGESA analysis of macrophage cluster 2 showed an even stronger enrichment of interferon response pathways in Lyz2-cre/+Ahrfl/fl tumors, including IFNγ, IRF8, IFI16, IFNβ, and the MYD88 pathway”

May combine two claims.

The frequency and number of CD8+ T cells increase within the tumor microenvironment of Lyz2cre/+Ahrfl/fl mice, and these cells shift from a naive to an effector/memory phenotype.

“the frequency and number of CD8+ T cells were increased within the TME of Lyz2cre/+Ahrfl/fl mice”

• Also stated in: § Deletion of AhR drives inflammatory polarization of TAMs and CD8+ T cell infiltration in the PDAC TME · plain

  “intra-tumoral CD8+ T cells from Lyz2cre/+Ahrfl/fl mice exhibited a change from a naive (CD62L+CD44lo) to an effector/memory (CD62LnegCD44hi) phenotype”

May combine two claims.

Depletion of CD8+ T cells abrogates the effect of macrophage Ahr deletion on tumor size, and IFNγ blockade rescues tumor growth to control levels.

“Depletion of CD8+ T cells abrogated the effect of macrophage Ahr deletion on tumor size, showing that CD8+ T cells are needed for control of tumor growth”

• Also stated in: § Macrophage AhR activity shapes the transcriptional landscape in PDAC · plain

  “IFNγ blockade in Lyz2cre/+Ahrfl/fl tumor-bearing mice rescued tumor growth to control levels”

May combine two claims.

In patients with pancreatic ductal adenocarcinoma, high AHR expression associates with rapid disease progression, mortality, and an immune-suppressive TAM phenotype.

“In patients with PDAC, high AHR expression associated with rapid disease progression and mortality, as well as with an immune-suppressive TAM phenotype”

May combine two claims. The quote only partly states this claim.

L. reuteri and L. murinus cultures produce ILA and IAld, while L. johnsonii and L. intestinalis do not.

“neither L. johnsonni nor L. intestinalis produced IAld or another AhR ligand indole lactic acid (ILA)”

• Also stated in: § Indole-producing microbiota drive suppression in the TME promoting tumor growth · plain

  “ILA and IAld were produced by both L. reuteri and L. murinus cultures, with L. murinus showing higher relative production of both the metabolites compared with L. reuteri”

May combine two claims.

Amp treatment increases infiltration of activated CD8+ T cells and intra-tumoral IFNγ+TNFα+CD8+ T cells 5-fold compared with Vanc.

“Amp treatment increased infiltration of activated CD8+ T cells”

• Also stated in: § Indole-producing microbiota drive suppression in the TME promoting tumor growth · plain

  “intra-tumoral IFNγ+TNFα+CD8+ T cells increased 5-fold in Amp-treated mice compared with Vanc-treated groups”

May combine two claims.

Bifidobacterium species were not a significant part of the microbiome in the authors' mice, but the Bifidobacterium genus was enriched in short-term survivor human pancreatic ductal adenocarcinoma microbiome samples, though less than other taxa.

“Bifidobacterium species were not a significant constituent of the microbiome in our mice; however, the Bifidobacterium genus was enriched in the microbiome of the STS in our human PDAC microbiome analysis, albeit to a much lesser extent when compared with other taxa.”

May combine two claims.

Loss of the gut microbiome or removal of dietary tryptophan phenocopied macrophage Ahr deletion, and providing indoles rescued the effect.

“In this report, we demonstrated that loss of the gut microbiome, or removal of dietary Trp phenocopied macrophage Ahr deletion; importantly, the effect could be rescued by provision of indoles.”

May combine two claims.

IAA and ILA supplementation abrogate the effect of a Trpneg diet, resulting in tumor weight comparable to Trp+ controls.

“both IAA and ILA supplementation abrogated the effect of a Trpneg diet, resulting in tumor weight that was comparable with that of the dietary Trp+ controls”

May combine two claims. The quote only partly states this claim.

In the dietary Trpneg group, TAMs decrease as a percentage of the immune infiltrate and show decreased MHCII expression, which is rescued by IAA or ILA supplementation.

“In the dietary Trpneg group, TAMs decreased as a percentage of the immune infiltrate and showed decreased expression of MHCII compared with controls”

• Also stated in: § Trp and the indoles IAA and ILA in the diet promote immune suppression and pancreatic tumor growth · plain

  “Supplementation of the diet with IAA or ILA rescued this effect”

May combine two claims.

iGSEA showed that indole-treated macrophage cultures increased enrichment of pathways linked to pancreatic ductal adenocarcinoma growth and metastasis and reduced enrichment of genes linked to negative regulation of pancreatic ductal adenocarcinoma proliferative potential.

“iGSEA enrichment analysis of the differentially expressed genes in the PDO co-cultured with control macrophages versus IAA-treated macrophages showed that indole-treated macrophage cultures increased enrichment of pathways associated with PDAC growth and metastasis and reduced enrichment of genes associated with negative regulation of PDAC proliferative potential”

• cites Lourenço and Coffer, 2017Not checked yet

  Not checked in the free run.

May combine two claims.

When AHR expression was divided into quartiles, the lowest quartile had the best overall survival, while the other quartiles did not differ in overall survival.

“we observed the best OS in the lowest AHR quartile (Q1), whereas the other expression quartiles did not show differential OS”

May combine two claims.

In the Riquelme intra-tumoral microbiome dataset, L. reuteri prevalence was higher in short-term survivors than long-term survivors, with 5 of 21 short-term survivors showing relative abundance above 2% and no long-term survivors showing detectable L. reuteri after filtering.

“We found an increased prevalence of L. reuteri in STS over LTS, with 5/21 STS patients displaying a relative abundance of L. reuteri above 2%, whereas none of the LTS patients displayed any detectable relative abundance of L. reuteri upon filtering.”

May combine two claims.

Lack of dietary Trp increases the number of TNFα+IFNγ+ and proliferating CD8+ T cells, an effect reversed by dietary IAA or ILA.

“lack of dietary Trp increased the number of TNFα+IFNγ+ and proliferating CD8+ T cells, an effect that was reversed by dietary IAA or ILA”

May combine two claims.

Indole-producing Lactobacilli greatly increased tumor size, increased MDSC numbers, and reduced activated CD8+ T cells and TNFα production.

“The fact that indole-producing Lactobacilli drastically increase tumor size, with an increase in MDSC numbers and a reduction in activated CD8+ T cells and TNFα production”

May combine two claims.

Macrophage AhR function increased Arg1 and Il10 expression in tumor-associated macrophages and reduced IFNγ expression in CD8+ T cells.

“We found that macrophage AhR function promotes the expression of Arg1 and Il10 in TAMs and inhibits IFNγ expression in CD8+ T cells.”

May combine two claims.

AhR is a central driver of TAM function and suppression of inflammatory T cell maturation, and its inhibition improves responses to immune checkpoint blockade.

“Hence, the data show that AhR is a central driver of TAM function, and suppression of inflammatory T cell maturation, and its inhibition improves responses to immune checkpoint blockade.”

May combine two claims.

AhR is a key driver of the TAM pro-tumor phenotype, and its abrogation shifts TAM polarization to a proinflammatory state.

“Thus, the data suggest that AhR is a key driver of the TAM pro-tumor phenotype, and when AhR function is abrogated, TAM phenotypic polarization is shifted to a proinflammatory state.”

May combine two claims.

The microbiota can promote or inhibit tumor progression in pancreatic ductal adenocarcinoma and affect the cellular composition of the tumor microenvironment.

“Thus, the microbiota can promote or inhibit tumor progression in PDAC, impacting the TME cellular composition.”

• cites Riquelme et al., 2019 sourceNot checked yet

  Not checked in the free run.

May combine two claims.

Indole-producing bacteria increase AhR transcriptional responses and promote an immunosuppressive tumor microenvironment in pancreatic ductal adenocarcinoma.

“the data show that indole-producing bacteria increase AhR transcriptional responses and promote an immunosuppressive TME in PDAC”

May combine two claims.

Amp-sensitive microbes may promote tumor growth and immune suppression in pancreatic ductal adenocarcinoma.

“These data suggest that Amp-sensitive microbes may promote tumor growth and immune suppression in PDAC.”

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

Indoles are essential for mucosal barrier integrity and suppression of inflammation through activation of AhR.

“Indoles are essential for mucosal barrier integrity and suppression of inflammation by activation of the transcriptional regulator AhR”

• cites Monteleone et al., 2011 sourceNot checked yet

  Not checked in the free run.

May combine two claims.

The presence of indole-producing bacteria in the gastrointestinal tract contributes to tumor growth, and increased tumor weight in L. m/r mice is due to TAM AhR activity.

“suggesting that the presence of indole-producing bacteria in the GIT contributes to tumor growth”

• Also stated in: § Indole-producing microbiota drive suppression in the TME promoting tumor growth · hedged

  “suggesting that the increased tumor weight in L. m/r mice is due to TAM AhR activity”

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

Metabolites with peak heights below 5000 or retention-time variability above 30% CV were excluded from further metabolomic analysis.

“Metabolites with low peak heights (<5000) and peaks with variable retention times (>30% CV) were excluded from further analysis.”

Probably not a substantial claim: may not pass S3 (load-bearing).

Other researchers have reported that pancreatic ductal adenocarcinoma tumor cells can produce L-Kyn through an IDO- or TDO-dependent mechanism that drives AhR activity.

“others have reported PDAC tumor cells can produce L-Kyn via an IDO- or TDO-dependent mechanism driving AhR activity (Wang et al., 2020)”

• cites Wang et al., 2020 sourceNot checked yet

  Not checked in the free run.

May combine two claims.

IAA activates AhR and promotes an immunosuppressive tumor-associated macrophage phenotype.

“suggesting that IAA activates AhR and drives an immunosuppressive TAM phenotype (Figure 5F)”

Classified as probably not a substantial claim.

Cell types were assigned to clusters using known surface markers uniquely expressed in the cluster and significant at FDR < 0.05.

“Different cell types were assigned to clusters based on known surface markers uniquely expressed in the particular cluster and found as a significant marker (FDR < 0.05).”

Probably not a substantial claim: may not pass S3 (load-bearing).

For flow cytometric analysis, at least 105 events were collected per sample.

“For flow cytometric analysis at least 105 events were collected on the LSR Fortessa flow cytometer (BD Biosciences).”

Probably not a substantial claim: may not pass S3 (load-bearing).

The discrepancy between the authors' findings and reports of IDO/TDO-driven AhR activity likely reflects variable contributions of the microbiome and IDO to the tumor microenvironment in heterogeneous pancreatic ductal adenocarcinoma.

“This dichotomy with our current study is likely reflective of the diverse and variable role of the microbiome versus IDO in shaping the TME in a heterogeneous disease, such as PDAC.”

May combine two claims.

AHR expression correlates with poor outcomes and reduced immune signatures in human pancreatic ductal adenocarcinoma, and indole-exposed human macrophages support pancreatic ductal adenocarcinoma organoid growth in an AhR-dependent way, suggesting analogous function in human disease.

“The observation that AHR expression correlates with poor outcomes and reduced immune signatures in human PDAC and indole-exposed human Mφ support PDAC organoid growth in an AhR-dependent mechanism suggests analogous function in human disease.”

May combine two claims.

Macrophage AhR is a central driver of tumor-associated macrophage function, responding to multiple inputs to produce an immune-suppressive phenotype in the tumor microenvironment.

“This study, along with the data from our paper, strongly supports the prediction that macrophage AhR is a central driver of TAM function responding to multiple inputs to drive an immune-suppressive phenotype in the TME.”

Probably not a substantial claim: may not pass S3 (load-bearing).

A single fecal microbiota transplant from melanoma patients responsive to αPD-1 therapy into αPD-1-refractory recipients promoted CD8+ T cell accumulation and acquired responsiveness to αPD1 therapy.

“a recent report has shown that a single FMT from melanoma patients responsive to αPD-1 therapy to αPD-1 refractive recipients promoted the accumulation of CD8+ T cells and acquired responsiveness to αPD1 therapy (Davar et al., 2021)”

• cites Davar et al., 2021 sourceNot checked yet

  Not checked in the free run.

Probably not a substantial claim: may not pass S3 (load-bearing).

Microbiome data were filtered to remove OTUs with low counts or variance using default parameters.

“Data were filtered to remove OTUs with low counts or variance using default parameters.”

Probably not a substantial claim: may not pass S3 (load-bearing).

Metabolite peak heights were normalized using the mean of the nearest quality control samples.

“Metabolite peak heights were normalized using the mean of nearest QC samples.”

Probably not a substantial claim: may not pass S3 (load-bearing).

Bacterial species were identified by 16S sequence alignment against NCBI with at least 99.5% identity.

“Bacterial species were identified by alignment of the 16S sequence using BLASTn against the NCBI database with an alignment of at least 99.5%.”

May combine two claims.

Low AHR expression may be beneficial for survival, while even moderate AHR expression may worsen outcomes.

“the relationship of AHR to OS suggests that low AHR expression may be beneficial for survival, but even moderate expression may negatively impact outcomes”

May combine two claims.

Targeting immune responses to the microbiome could meaningfully affect therapy, either by augmenting microbiome manipulation or by inhibiting responses to microbial metabolites.

“Our results suggest that targeting immunologic responses to the microbiome could have meaningful impact on therapy, augmenting microbiome manipulation approaches or potentially bypassing the need to manipulate the microbiome by precise inhibition of the response to microbial metabolites.”

May combine two claims.

AHR is highly expressed in subsets of alternatively activated tumor-associated macrophages in pancreatic ductal adenocarcinoma and is linked to myeloid gene set enrichment across tissues.

“Thus, AHR is highly expressed in subsets of alternatively activated TAMs in the PDAC TME and is associated with gene set enrichment in myeloid cells across tissue types.”

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

The 16S rRNA gene sequencing run included a single-species control, a mock community, and a template-free negative control.

“A single-species (Pseudomonas aeruginosa DNA), a mock community (Zymo Microbial Standard: https://www.zymoresearch.de/zymobiomics-community-standard) and a template-free negative control were included in the sequencing run.”

Probably not a substantial claim: may not pass S3 (load-bearing).

The local tumor microbiota in pancreatic ductal adenocarcinoma influences survival independently of immune therapy.

“The local tumor microbiota in PDAC influences survival independent of immune therapy (Riquelme et al., 2019).”

• cites Riquelme et al., 2019 sourceNot checked yet

  Not checked in the free run.

Probably not a substantial claim: may not pass S3 (load-bearing).

Quality control samples for metabolomics were prepared by pooling 10 μl from each sample.

“Quality control (QC) samples were prepared by pooling 10 μl of each sample.”

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

All 16S rRNA gene amplification reactions were performed in triplicate, pooled, and verified on a 1% agarose TBE gel.

“All amplification reactions were done in triplicate, pooled together, and checked on a 1% agarose TBE gel.”

Probably not a substantial claim: may not pass S2 (checkable), S3 (load-bearing).

The link between AhR, immune function, indoles, and microbiome constituents may provide useful prognostic information for predicting patient outcomes.

“The link we identified between AhR, immune function, indoles, and microbiome constituents may provide useful prognostics for predicting patient outcomes.”

Probably not a substantial claim: may not pass S3 (load-bearing).

All RNA samples used for sequencing had RIN values above 8.

“All samples had RIN above 8.”

Probably not a substantial claim: may not pass S3 (load-bearing).

The use of defined, cultured mouse feces-isolated bacteria may affect dissemination from the gut to the tumor.

“our use of defined, cultured mouse feces-isolated bacteria may impact dissemination from the gut to the tumor”

May combine two claims.

AhR gene signatures correlate with fewer T cell transcriptional signatures and a cold tumor, whereas reduced AhR signatures predict more cytolytic CD8+ T cells and inflammation.

“Thus, the data show that AhR gene signatures correlate with a paucity of T cells transcriptional signatures in the TME and a cold tumor overall, whereas reduced AhTSs predicts increased cytolytic CD8+ T cell presence and inflammation.”

Probably not a substantial claim: may not pass S3 (load-bearing).

Cells with fewer than 200 detected genes or more than 5% mitochondrial transcripts were excluded from single-cell analysis.

“We excluded cells with less than 200 detected genes or > 5% mitochondrial transcripts.”

Probably not a substantial claim: may not pass S3 (load-bearing).

A prior report suggested that the microbiome can promote tumor growth through TLR-dependent local innate inflammation with Bifidobacterium enrichment.

“A recent report suggested that the microbiome can promote tumor growth by TLR-dependent stimulation of innate inflammation locally with enrichment of Bifidobacterium (Pushalkar et al., 2018).”

• cites Pushalkar et al., 2018 sourceNot checked yet

  Not checked in the free run.

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

In glioblastoma multiforme, tumor-associated macrophage AhR function drives CD39 expression and impairs T cell responses.

“Recently, a role for TAM AhR function was also identified in glioblastoma multiforme (GBM), driving CD39 expression and impairing T cell responses (Takenaka et al., 2019).”

• cites Takenaka et al., 2019 sourceNot checked yet

  Not checked in the free run.

Probably not a substantial claim: may not pass S3 (load-bearing).

Microbiome data were scaled using total sum scaling to remove variability between samples based on sequencing depth.

“Data were scaled using Total sum scaling (TSS) to remove variability between samples based on sequencing depth.”

May combine two claims.

Fecal microbiota transplantation from long-term survivor pancreatic ductal adenocarcinoma patients reduced tumor burden in mice, whereas transplantation from short-term survivors had a detrimental effect on tumor burden.

“FMT experiments suggested that, although microbiota from LTS patients reduced tumor PDAC burden in mice, FMTs from patients who survived less than 5 years post-surgery (matched STS) had a detrimental effect on tumor burden (Riquelme et al., 2019).”

• cites Riquelme et al., 2019 sourceNot checked yet

  Not checked in the free run.

Probably not a substantial claim: may not pass S3 (load-bearing).

The animal protocols were approved by the Princess Margaret Cancer Centre Animal Care Committee.

“Protocols were approved by the Princess Margaret Cancer Centre Animal Care Committee.”