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Dissecting Tumor–Microbiome Interactions: Introducing FocuSCOPE 16S for Single Cell Insight 

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Intracellular bacteria are known to influence the development, progression, and treatment of cancer. However, studying these microbes in the tumour microenvironment has been challenging. Bulk sequencing methods fail to distinguish bacterial RNA signals at cell-type-specific level, making it difficult to identify which host cells harbour specific bacterial transcripts.

FocuSCOPE 16S is a new single cell technology designed to overcome these limitations. By capturing bacterial and host RNA from the same cell, it enables researchers to study host–microbiome interactions with greater resolution.

The Role of Intracellular Bacteria in Cancer 

Bacteria are found intracellularly across many major tumor types both in cancerous and stromal cells [1]. Studies have reported that intracellular bacteria can affect tumor growth, immune modulation, and resistance to therapy [2]. However, their presence in tumours is often at low abundance, making them difficult to detect. 

Using 16S rRNA sequencing, NGS-based bacteria detection methods have significantly improved detection sensitivity. However, current bulk sequencing methods provide only an overall bacterial composition without linking bacterial presence to specific host cells. This limits the ability to study the difference in bacteria composition in cancer, immune, or stroma tissues, the interaction between bacteria and host cells, and the mechanism of action. 

FocuSCOPE 16S: A Single Cell Approach in Intracellular Bacteria Detection

FocuSCOPE 16S allows researchers to detect bacterial ribosomal RNA (rRNA) in individual host cells while simultaneously capturing the full transcriptome from the same cells. This approach provides insights into: 

  • The microbiome profile within specific cell types. 
  • How bacterial presence and distribution correlate with tumor, immune, or stroma cell genotypes and phenotypes. 
  • Potential pathways by which bacteria populations influence tumor progression, immune response, and drug resistance. 

How It Works 

FocuSCOPE bead design: 

Figure 1. FocuSCOPE bead design. FocuSCOPE uses barcoded magnetic beads to capture RNA of interest. For FocuSCOPE 16S, each bead contains two kinds of barcode:  

  1. A poly(dT) probe that captures poly-adenylated host mRNA 
  2. Multiple  non-poly(dT) probes, designed to target bacteria rRNA binding to constant regions near hypervariable regions. 

Streamlined workflow 

Due to its unique barcode design, FocuSCOPE 16S follows the same workflow as standard single cell RNA sequencing, requiring no extra steps to capture both host mRNA and bacterial rRNA in parallel. A single experiment produces data on host gene expression and bacterial transcripts from the same cell. 

Figure 2. Overview of the FocuSCOPE 16S workflow.  

FocuSCOPE 16s Workflow Steps: 

1. Tissue or Cell Preparation

Solid tissues can be dissociated into single-cell suspensions using the sCelLiVE kit. This process can be automated using the Singleron PythoN or PythoN Junior tissue dissociator. 

2. Single-Cell Capture and Barcoding: 

Cells are loaded onto the SCOPE-chip, where barcoding beads capture host mRNA and bacterial rRNA molecules. This step can be automated using the Singleron Matrix® NEO instrument. 

3. cDNA Amplification and Library Preparation: 

The barcoded cDNA is amplified and prepared for sequencing.

4. NGS Library Construction: 

Two sequencing libraries are generated: 

  • A host transcriptomics library (for mRNA analysis). 
  • A bacterial rRNA library (for microbiome profiling). 

Potential Applications of FocuSCOPE 16s Intracellular Bacteria Detection  

Tumor Microbiome Analysis 

  • Identify bacteria present within tumor and stromal cells 
  • Determine which cell types harbor bacterial transcripts in tumor microenvironment. 
  • Compare microbial profiles between cancerous and adjacent normal tissues. 

Immune Modulation Studies 

  • Investigate how intracellular bacteria interact with immune cells. 
  • Study bacterial contributions to immune suppression   

Therapy Response Research 

  • Explore bacterial associations with tumor clones that demonstrate chemotherapy or immunotherapy resistance. 
  • Assess microbial influence on treatment outcomes in clinical samples. 

Case study 

Oral cancer is often associated with a high level of pathogenic bacteria [3]. FocuSCOPE 16S was used to analyze oral cancer tissues and demonstrated cell-type specific distribution of intracellular bacteria (Figure 3). It overcame the limitation of current analysis methods and was able to reveal the distribution of different bacteria genus in different cell type clusters (Figure 4).

Figure 3. FocuSCOPE 16S identified bacterial rRNA expression across different cell types in oral cancer tissue. Left: Cell clustering result. Right: bactera UMI count (B,D). Scale bar: Number of bacterial UMIs detected per cell. Abbreviations: ECs – Endothelial Cells; MPs – Macrophages; pDCs – plasmacytoid Dendritic Cells; SMSCs – Stromal Mesenchymal Stem Cells.

Figure 4. FocuSCOPE 16S identifies bacteria genus present in different cell type clusters.  

Conclusion

Understanding how intracellular bacteria contribute to cancer requires tools that can link microbial signals to specific host cells. FocuSCOPE 16S provides a single cell approach to studying these interactions, offering a more detailed view of the tumor microbiome.

Contact us to discuss potential applications in your research. 

References

[1] Nejman, D., et al. The human tumor microbiome is composed of tumor type–specific intracellular bacteria. Science 368, 973-980 (2020). DOI: 10.1126/science.aay9189 

[2] Chai, X., et al. Intratumor microbiome features reveal antitumor potentials of intrahepatic cholangiocarcinoma. Gut Microbes 15(1), 2156255 (2023). DOI: 10.1080/19490976.2022.2156255 
[3] Zeng, B., et al. The oral cancer microbiome contains tumor space-specific and clinicopathology-specific bacteria. Front Cell Infect Microbiol. 27(12), 942328 (2022) DOI: 10.3389/fcimb.2022.942328 

A post by Singleron team

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