Microniches of the intratumoral microbiota influence spatial and cellular heterogeneity in cancer

Microniches of the intratumoral microbiota influence spatial and cellular heterogeneity in cancer

In a recently published article in Nature The researchers mapped spatial, cellular and molecular interactions of host and tumor-associated bacteria within the tumor microenvironment (TME). They used in situ spatial profiling technologies and single-cell ribonucleic acid sequencing (scRNA-seq), focusing on gastrointestinal cancers, particularly oral squamous cell carcinoma (OSCC) and colorectal cancer (CRC).

Lernen: Wirkung der intratumoralen Mikrobiota auf die räumliche und zelluläre Heterogenität bei Krebs. Bildquelle: Kristalllicht/Shutterstock

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In vitro and preclinical studies using animal models have provided molecular evidence for the role of tumor-associated bacteria in at least 33 major cancers and metastases. Imaging data have shown co-localization of bacterial markers with epithelial and immune cell targets, suggesting that the intratumoral microbiota may be intracellular.

Furthermore, intratumoral host microbiota play a role in immune surveillance and chemoresistance. However, studies have not revealed the identity of these tumor cell-associated organisms within the TME and the host cell types with which they interact in patient tumors. Furthermore, whether their spatial distribution and interactions with the host influence different functional capabilities within the TME.

About studying

In the present study, researchers first assessed the composition of the intratumoral microbiota at the phylum and genus levels. Next, they visually confirmed the heterogeneous spatial distribution of the identified bacterial communities, including Fusobacterium nucleatum. To do this, they performed 16S ribosomal RNA gene sequencing on 44 pieces of tissue from the tumors of 11 patients with CRC. In addition, they targeted RNAscope fluorescence in situ hybridization (RNAscope-FISH) imaging of densely populated compartments with bacterial cell biomass and bacteria-negative areas within the same tumor sample. In addition, they quantified the transcriptional load of the tissues of specific organisms using the Unique Molecular Identifiers (UMI) metric.

In addition, researchers quantified the expression profile of 77 proteins associated with anti-tumor immunity and cancer progression using a digital spatial profiling (DSP) platform. Next, they developed the INVADEseq method to study the interaction between bacteria and host cells within the TME and the effects on host cell transcriptomics. This method enabled the generation of complementary DNA (cDNA) libraries containing bacterial transcripts from the bacteria-associated human cells. In other words, it helped researchers map bacterial reads to individual human cells.

A reductionist in vitro coculture approach allowed researchers to assess the direct interactions of a dominant member of the intratumoral microbiota with immune or epithelial cancer cells. They cocultured CRC epithelial spheroids with an F. nucleatum CRC isolate, followed by embedding in collagen matrices containing neutrophils evenly distributed throughout the gel. Next, they used live-cell confocal microscopy to visualize, track, and compare the embedded neutrophils in F. nucleatum-infected spheroids with uninfected spheroids.

Study results

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RNAscope-FISH identified bacterial transcripts in 46% and 28% of capture spots in CRC and OSCC tumors, respectively. Bacterial genera identified per capture site ranged from one to 42 and one to 31, with a median of eight and two for the OSCC and CRC tumors, respectively. In OSCC tumor, the UMI metric identified Parvimonas, Peptoniphilus and Fusobacterium as the most dominant bacterial genera. Conversely, the dominant genera in the CRC sample were Fusobacterium and Bacteroides. Interestingly, the latter had an order of magnitude more reads and UMIs compared to the OSCC specimen. The colocalization of communities of isolated genera and multiple different genera within trapping sites highlighted the complexity of intratumoral microbiota interactions between these two cancer types.

Bacteria-positive tissues in tumors showed a significant increase in CD11b+ and CD66b+ myeloid cells, but a lower density of CD4+ and CD8+ T cells. The case with neighboring bacteria-negative regions was different; this suggested that the tumor-associated microbiota had a highly localized effect. Possibly, invasive bacteria recruited myeloid cells to induce an inflammatory response through Janus kinase (JAK) signal transducer and activator of transcription (STAT) signaling. It promoted T cell exclusion and tumor growth by secreting specific interleukins and chemokines into the environment. Remarkably, intracellular bacteria generated gene signatures consistent with cancer-induced cell invasion, metastasis, DNA damage repair, and cell quiescence by activating transcription factors from the Jun and Fos families.

Mapping bacterial metrics from INVADEseq analysis to annotated single cells showed that Fusobacterium and Treponema in these patient tumors were predominantly associated with the epithelial and monocyte-derived macrophages (cell clusters), with an overall bacterial infection rate of 25% and 52%, respectively.

Gene set enrichment analysis (GSEA) confirmed that the cells within the bacteria-dominated “epithelial cell cluster 3” were cancer cells, with upregulation of signaling pathways involved in cancer progression (e.g. epithelial-mesenchymal transition or EMT pathway). The recruitment and retention of neutrophils in the F. nucleatum-infected cancer cell spheroids suggested that the intratumoral microbiota played an active role in the enrichment of neutrophils in bacterial-colonized microniches of patient tumors.

The F. nucleatum-infected CRC epithelial cells detached from the spheroid mass and migrated as individual epithelial cells into the surrounding collagen gel. In contrast, uninfected cancer epithelial cells spread through the gel as a spheroid mass with an average expansion rate of 1.34 × 105 µm3 h−1. Invasive bacteria promoted cancer cell invasion but also altered the movement patterns of infected cancer cells, thereby promoting cell heterogeneity at the functional level.

Conclusions

The study found that the distribution of intratumoral microbiota was heterogeneous in human tumors. Despite their heterogeneity, the distribution of microbiota within a tumor was not random but highly organized, and microniches with immune and epithelial cell functions stimulated cancer actively. Overall, it altered the biology of specific cellular compartments and influenced anti-tumor immunity and cancer epithelial cell migration. According to the authors, the intratumoral microbiota in 33 major cancers discovered to date could be analyzed using the same tools and technologies.

Reference:

• Galeano Niño, JL, Wu, H., LaCourse, KD et al. (2022). Wirkung der intratumoralen Mikrobiota auf die räumliche und zelluläre Heterogenität bei Krebs. Natur. doi: https://doi.org/10.1038/s41586-022-05435-0 https://www.nature.com/articles/s41586-022-05435-0

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