Single-cell profiling methods compared for gut biopsy analysis

Research on gastrointestinal diseases, especially cancer, has mainly focused on epithelial cells, which line the surfaces of organs, are important for various functions, and are believed to be the cells that go awry to cause these diseases. However, studying these cells using single-cell RNA sequencing (scRNA) methods is tricky because the cells are delicate and can be damaged when taken out of their natural environment in the mucosal lining of the gut.

Droplet-based scRNA (D-scRNA) methods have provided many insights into these cell environments, but this approach can stress the epithelial cells and requires a lot of resources. Picowell-based (P-scRNA) platforms have emerged as a potentially gentler and more resource-efficient alternative to D-scRNA methods. However, no direct comparison between D-scRNA and P-scRNA methods for studying human mucosal biopsies, which are crucial for gastrointestinal research, has been done yet.

In our study published in Cellular and Molecular Gastroenterology and Hepatology, we directly compared these two approaches.

Our results highlight that there are unique advantages and disadvantages to each of the platforms. These variations pose significant challenges in maintaining rigor and reproducibility across studies that use different techniques. We anticipate that this study will help researchers more efficiently approach projects employing scRNA on human biopsy samples.

Human colon biopsies were collected and prepared using identical methods to allow for the analysis of the same set of cells with both D-scRNA and P-scRNA to directly compare the results. Factors such as the number of cells, gene coverage and mitochondrial DNA contamination were included in our analysis to better understand the strengths and limitations of each method.

We found clear differences between the D-scRNA and P-scRNA platforms and that each method has significant differences in the types of cells captured and the genes measured within them.

D-scRNA appears to provide more detailed insights into the environment of the mucosal lining and within individual cells, by capturing more cells and providing higher gene coverage. However, it requires more resources (i.e. time, money), adds stress to the cells and there was a higher rate of contamination from mitochondrial DNA, both of which may influence results if not carefully accounted for.

P-scRNA is gentler on cells, more resource efficient and better at preserving cell quality in terms of mitochondrial contamination. However, it isn’t as effective at capturing large quantities of cells or the full, wide array of cell types present in the mucosal lining, especially in rare cell-types, which may have clear importance for the disease a researcher is studying.

Our results can help scientists better understand the context for interpreting their scRNA findings when using either D-scRNA or P-scRNA methods. Additionally, by sharing these strengths and weaknesses, we believe we can help researchers determine the appropriate platform to use for their own studies without them requiring additional testing on their own.

We seek to apply this knowledge to optimize our approach for analyzing biopsies collected from patients enrolled in our precision prevention clinical trials. These clinical trials aim to better understand the effects of potential anti-cancer and protective agents, including aspirin, omega-3-fatty acids, coffee and incretin mimetics (GLP1-receptor agonists), on the colon tissue microenvironment and prevent the occurrence of early-onset colorectal cancer and gastrointestinal cancer.