INVADEseq to identify cell-adherent or invasive bacteria and the associated host transcriptome at single-cell-level resolution
Open accessAbstract
Single-cell RNA sequencing (scRNAseq) technologies have been beneficial in revealing and describing cellular heterogeneity within mammalian tissues, including solid tumors. However, many of these techniques apply poly(A) selection of RNA, and thus have primarily focused on determining the gene signatures of eukaryotic cellular components of the tumor microenvironment. Microbiome analysis has revealed the presence of microbial ecosystems, including bacteria and fungi, within human tumor tissues from major cancer types. Imaging data have revealed that intratumoral bacteria may be located within epithelial and immune cell types. However, as bacterial RNA typically lacks a poly(A) tail, standard scRNAseq approaches have limited ability to capture this microbial component of the tumor microenvironment. To overcome this, we describe the invasion-adhesion-directed expression sequencing (INVADEseq) approach, whereby we adapt 10x Genomics 5' scRNAseq protocol by introducing a primer that targets a conserved region of the bacterial 16S ribosomal RNA gene in addition to the standard primer for eukaryotic poly(A) RNA selection. This 'add-on' approach enables the generation of eukaryotic and bacterial DNA libraries at eukaryotic single-cell level resolution, utilizing the 10x barcode to identify single cells with intracellular bacteria. The INVADEseq method takes 30 h to complete, including tissue processing, sequencing and computational analysis. As an output, INVADEseq has shown to be a reliable tool in human cancer cell lines and patient tumor specimens by detecting the proportion of human cells that harbor bacteria and the identities of human cells and intracellular bacteria, along with identifying host transcriptional programs that are modulated on the basis of associated bacteria.
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INVADEseq targets a conserved area of bacterial 16S rRNA, enabling effective creation of cDNA libraries containing bacterial transcripts derived from human cells associated with bacteria.
“Galeano et al. introduced invasion-adhesion-directed expression sequencing (INVADEseq) targeting a conserved area of the bacterial 16 S rRNA, enabling the effective creation of cDNA libraries containing bacterial transcripts derived from human cells associated with bacteria.”
From Single-cell sequencing to multi-omics: technologies and applications (2024) · cites this paper as [159]
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Single-cell sequencing to multi-omics: technologies and applications
Wu, X., Yang, X., Dai, Y., Zhao, Z., Zhu, J., Guo, H., & Yang, R. (2024) · Biomarker Research · doi:10.1186/s40364-024-00643-4 · Cited by 195 · see citing works
“The technology is crucial to uncovering the complexity of microbiota interactions within tumor tissues [158, 159].”
Background
- Authors
- Galeano Niño, J. L., Wu, H., LaCourse, K. D., Srinivasan, H., Fitzgibbon, M., Minot, S. S., Sather, C., Johnston, C. D., & Bullman, S.
- Affiliations
- Fred Hutch Cancer Center (US)
- Corresponding author
- Johnston, Christopher D.
- Bullman, Susan
- ORCID
- Venue
- Nature Protocols
- Published
- 2023-10-03
- Volume
- 18
- Issue
- 11
- Pages
- 3355-3389
- ISSN
- 1750-2799 (ISSN-L)
- 1754-2189
- PMC
- PMC10790651
- OpenAlex
- W4387307216
- Cited by
- 36
- References
- 36
- Citations by year
- 2026: 17 · 2025: 13 · 2024: 5 · 2023: 1
- FWCI
- 0.95
- Type
- journal article
- Language
- en
- Open access
- green
- www.ncbi.nlm.nih.gov
- Licence
- Locations
- Nature Protocols (publishedVersion)
- PubMed (publishedVersion)
- PubMed Central (acceptedVersion, cc-by)
- Funders
- U.S. Department of Health & Human Services | NIH | National Cancer Institute (R00 CA229984-03)
- Cancer Research Institute (CRI4208)
- U.S. Department of Health & Human Services | National Institutes of Health (P30 CA015704)
- U.S. Department of Health & Human Services | NIH | National Institute of Dental and Craniofacial Research (R01 DE027850)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Single-cell and spatial transcriptomics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Gut microbiota and health
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Genomics and Phylogenetic Studies
- Keywords
- intratumoral bacteria, bacterial invasion, bacterial adhesion, tumor microenvironment, single-cell RNA sequencing, 16S rRNA gene, host transcriptome, host-bacteria interactions, cancer cell lines, 10x Genomics
- MeSH terms
- Animals, Bacteria, Humans, Mammals, Neoplasms, Genomics, Tumor Microenvironment, Transcriptome, Microbiota
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