The role of different microbiota

The role of different microbiota in metastatic brain tumors Golnaz Morad, Ashish Damania, Matthew C. Wong, Sarah B. Johnson, Pranoti Sahasrabhojane, Nadim J. Ajami, Sherise D. Ferguson, Jennifer A. Wargo The University of Texas MD Anderson Cancer Center, Houston, TX

METHODS

ABSTRACT

RESULTS

Figure 4. Antibiotic treatment was associated with alterations in the peripheral immune profile in non-tumor bearing mice. G ut microbiome depletion increased the circulating Treg population (A) and the CTLA4+ CD4 and CD8 populations (B).

Background: Metastatic brain tumors are associated with significant morbidity and mortality. The current limited understanding of the mechanisms underlying brain metastasis has hindered the development of efficient diagnostics and therapeutics for this disease. The microbiota has emerged as a novel hallmark of cancer, with a prominent role in tumorigenesis, tumor immunity, and response to treatment. However, the role of different microbial communities in tumor metastasis, and in particular brain metastasis, is poorly understood. We hypothesize that distinct microbial communities can alter the immune microenvironment in the brain and affect brain metastasis development. Methods: To evaluate the role of different microbial communities in brain metastasis, matched stool, saliva, tumor, and plasma samples were collected prospectively from patients with metastatic brain tumors who underwent surgical tumor resection at the University of Texas MD Anderson Cancer Center. Stool and saliva samples were collected using the OMNIgene microbiome collection and stabilization kits (DNAgenotek, Kit # OM-200 and OM-505, respectively). Tumor samples were flash frozen in sterile conditions. Stool and saliva samples from 30 patients and tumor and plasma from 15 patients were sequenced via metagenomic shotgun and 16S rRNA sequencing, respectively. Microbiome profiling was conducted through established computational pipelines reported previously by our group. To further explore the mechanistic role of the gut microbiota in brain metastasis, we depleted gut microbiota in conventionally raised mice using a broad- spectrum non-absorbable antibiotic regimen. Melanoma tumor cells were subsequently injected intracranially to evaluate the effect of gut microbiota depletion and associated immune changes on tumor growth. Tumor growth was measured through in vivo bioluminescent imaging and histology. Peripheral and tumor immune profiling was conducted through flow cytometry and immunohistochemistry. Results: In all types of microbiota evaluated in this study, distinct signatures were identified to be associated with brain metastasis compared to other types of brain tumors. Interestingly, we demonstrated an overlap between the tumor microbiome and the oral microbiome but not with the gut microbiome. These findings suggest a direct contribution of the oral microbiome and the potential indirect contribution of the gut microbiome to the development of brain metastasis. Our mechanistic studies on the role of gut microbiota in brain metastasis demonstrated that depletion of the gut microbiota in mice decreased tumor growth in the brain. Evaluation of the peripheral and tumor immune profiles suggested the underlying mechanisms to involve alterations in the circulating cytokine profiles and an increase in anti-tumor T cell activity. Conclusion: Our clinical studies suggest the association of distinct microbial communities with brain metastasis. Our pre-clinical findings demonstrate that the absence of gut microbiota can modulate the regulation of T cell activity to induce an anti-tumor response in the brain. Further studies, currently in progress, will determine the individual and collective role of different microbial communities in the development of brain metastasis.

P= 0.0024

Figure 1. Schematics demonstrating the experimental design of (A) clinical and (B) animal studies.

P= 0.02

Antibiotic Control

P=0.045

Antibiotic Control

P=0.0002

CD4 CTLA4+ CD8 CTLA4+ 0

Treg

2000000

P= 0.0298

1500000

1000000

500000

Lactobacillus Anaerostipes Actinomyces Clostridium Ruminococcaceae_unclassified Lachnoclostridium Candidatus_Stoquefichus Lactobacillus Roseburia

Antibiotic

Antibiotic Control

Control

Figure 5. Depletion of the gut microbiome was associated with a decrease on melanoma tumor growth in the brain. Tumor size at the site of injection was evaluated by histology (A) and quantified using ImageJ (B). Peripheral immune profiling demonstrate a decrease in Tregs (C) and the CTLA4+ CD4 cells (D). Antibiotic Control

RESULTS Oral microbiome

P= 0.019

P= 0.02

Gut microbiome

Tumor microbiome

Antibiotic Control

P= 0.02 P=0.045

Antibiotic Control

GBM BM:Tumor samples

Anaerostipes Actinomyces Clostridium Ruminococcaceae_unclassified Lachnoclostridium Candidatus_Stoquefichus Lactobacillus Roseburia

P=0.0002

Corynebacterium

Streptococcus

Streptobacillus

Citrobacter Turicimonas Weissella Catenibacterium

BM GBM Brain metastasis Glioma

Campylobacter

Anaerococcus

Actinomyces

Streptococcus

− 0.4 0.0 0.4 tumor_category_LFC

Porphyromonas

Aggregatibacter

Citrobacter Turicimonas Weissella Catenibacterium

BM GBM

Mogibacterium

BM GBM

0 CD4 CTLA4+ CD8 CTLA4+ 0

Gemella

CD4 CTLA4+ CD8 CTLA4+ 0

Granulicatella

Monoglobus Eubacterium

Treg

Aggregatibacter

Phyllobacterium

Monoglobus Eubacterium

Peptostreptococcus

Cloacibacterium

0 Slackia Bacteroides Bifidobacterium

Slackia Bacteroides Bifidobacterium

Capnocytophaga

Aquabacterium

Prevotella

GBM

− 2

− 1 2 Log fold change by cancer type in Stool samples Ancombc coefficient 0 1

− 1 2 Log fold change by cancer type in Saliva samples Ancombc coefficient 1

Ancombc Coefficient Ancombc coefficient

CONCLUSIONS • We identified distinct gut, oral, and tumor bacterial signatures that were enriched in brain metastasis patients compared to primary tumors. • Our findings suggest a direct contribution of the oral microbiome and the potential indirect contribution of the gut microbiome to the development of brain metastasis. • Our pre-clinical findings demonstrate that the absence of gut microbiota can regulate T cell activity to induce an anti-tumor response in the brain.

− 1 2 Log fold change by cancer type in Stool samples 0 1

Figure 2. Distinct microbial signatures in the gut, oral, and tumor microbiome are enriched in brain metastasis compared to primary brain tumors. Analysis of Compositions of Microbiomes with Bias Correction (ANCOM-BC) demonstrating enrichment of taxa in samples from brain metastasis patients (blue) and primary brain tumors (orange).

BACKGROUND

Figure 3. Tumor microbiome overlaps with oral microbiome, suggesting the possibility of direct contribution. A lluvial plots demonstrating the overlap between bacterial taxa in the tumor microbiome and gut (left) or oral (right) microbiome.

100

CBOP ouar i rt bype arhni cyi bt i r ecao or cmi cut eocmruni sua ms

Acinetobacter Haemophilus Anaerococcus

REFERENCES

Alishewanella Acinetobacter Anaerococcus Alloprevotella

• Gut and oral microbiome contributes to different aspects of tumor progression 1,2 ; however, their role in primary and metastatic brain tumors remains unknown. • The gut-brain axis is the bidirectional communication between the gut microbiota and the brain and contributes to a variety of neurological disorders 3 .

Actinomyces Staphylococcus Lautropia Capnocytophaga Haemophilus Megasphaera

Streptococcus

Staphylococcus

1. Gopalakrishnan et al., Science 2018 2. Helmink et al., Nature Medicine 2019 3. Morais et al., Nature Reviews microbiology 2021

Lachnospiraceae_unclassified

Eubacterium Clostridium Faecalibacterium Akkermansia

Neisseria Rothia Alishewanella Streptococcus

Neisseria

Streptococcus

Lactobacillus

Streptococcus

Staphylococcus

Ruminococcus

Prevotella

Alistipes Bifidobacterium Blautia

Rothia

Neisseria

ACKNOWLEDGEMENTS

Lachnoclostridium

Parabacteroides

Pseudomonas

Prevotella

National Institute of Health F32CA260769

Bacteroides

Veillonella

We hypothesize that distinct gut and oral microbial signatures contribute to the development of metastatic brain tumors.

Pseudomonas Stool stool

saliva Saliva

tumor Tumor

site

Glioblastoma Moon Shot ® Program

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