Original articles

Improving exploration of biological sample pretreatment in single-cell transcriptome sequencing of gastrointestinal tumors

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  • Department of General Surgery, Shanghai Institute of Digestive Surgery, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China

Received date: 2022-09-13

  Online published: 2023-01-29

Abstract

Objective: To explore the method of increasing the proportion of epithelial cells in the single-cell suspension of tissue in single-cell transcriptome sequencing in gastrointestinal epithelial tumors for improving the quality of test samples. Methods: The proportion of epithelial cell in the single-cell transcriptome sequencing data set of common gastrointestinal epithelial tumors (gastric cancer, colorectal cancer and pancreatic cancer) in GEO public database was analyzed. The difference in proportion and activity of epithelial cells in single-cell suspension between traditional enzyme digestion method and mechanical physical blowing method (improved method). Results: The data analysis in public database showed that the proportion of epithelial cells in 4 samples of normal gastric epithelium or gastric cancer was 17.05%, 6.11%, 8.93% and 14.66% respectively. The proportion of epithelial cells in 2 samples of colorectal cancer was 3.86% and 16.60%. The proportion of epithelial cells in 2 samples of pancreatic cancer was 3.22% and 21.37%. For two freshly collected gastric cancer samples, the cell agglomeration rate of single-cell suspension prepared by traditional enzyme digestion method was 56.26%±1.98% and 38.34%±1.26%, and the percentage of living cells in cell activity test was 98.43%±0.56% and 97.24%±0.48%, respectively. However, in the single-cell suspension treated by traditional enzyme digestion and mechanically blown with 0.33 mm insulin syringe for 30 seconds before being put on the machine, the epithelial cell agglomeration rates in 2 test samples were reduced to 22.78%±1.38% and 14.46±0.92%(P<0.000 1), and the proportion of living cells was 95.16%±0.42% and 93.52%±0.82% respectively(P<0.05). Conclusions: The pretreatment of tissue samples with traditional enzyme digestion and mechanical physical blowing could reduce epithelial cell agglomeration and increase the proportion of epithelial cells in single-cell suspension. The proportion of living cells decreased slightly, while it could meet the requirements of subsequent sequencing.

Cite this article

YANG Ruixin, DU Yutong, YAN Ranlin, ZHU Zhenggang, LI Chen, YU Yingyan . Improving exploration of biological sample pretreatment in single-cell transcriptome sequencing of gastrointestinal tumors[J]. Journal of Diagnostics Concepts & Practice, 2022 , 21(05) : 567 -574 . DOI: 10.16150/j.1671-2870.2022.05.004

References

[1] Cancer Genome Atlas Research Network. Comprehensive molecular cha-racterization of gastric adenocarcinoma[J]. Nature, 2014, 513(7517):202-209.
[2] Tang F, Barbacioru C, Wang Y, et al. mRNA-Seq whole-transcriptome analysis of a single cell[J]. Nat Methods, 2009, 6(5):377-382.
[3] Fu K, Hui B, Wang Q, et al. Single-cell RNA sequencing of immune cells in gastric cancer patients[J]. Aging (Albany NY), 2020, 12(3):2747-2763.
[4] Zhang Y, Song J, Zhao Z, et al. Single-cell transcriptome analysis reveals tumor immune microenvironment heterogenicity and granulocytes enrichment in colorectal cancer liver metastases[J]. Cancer Lett, 2020, 470:84-94.
[5] Elyada E, Bolisetty M, Laise P, et al. Cross-species single-cell analysis of pancreatic ductal adenocarcinoma reveals antigen-presenting cancer-associated fibroblasts[J]. Cancer Discov, 2019, 9(8):1102-1123.
[6] Zhang M, Hu S, Min M, et al. Dissecting transcriptional heterogeneity in primary gastric adenocarcinoma by single cell RNA sequencing[J]. Gut, 2021, 70(3):464-475.
[7] Zhang P, Yang M, Zhang Y, et al. Dissecting the single-cell transcriptome network underlying gastric premalignant lesions and early gastric cancer[J]. Cell Rep, 2019, 27(6):1934-1947.
[8] Park J, Shrestha R, Qiu C, et al. Single-cell transcriptomics of the mouse kidney reveals potential cellular targets of kidney disease[J]. Science, 2018, 360(6390):758-763.
[9] Kim J, Park C, Kim KH, et al. Single-cell analysis of gastric pre-cancerous and cancer lesions reveals cell lineage diversity and intratumoral heterogeneity[J]. NPJ Precis Oncol, 2022, 6(1):9.
[10] Guo W, Zhang C, Wang X, et al. Resolving the difference between left-sided and right-sided colorectal cancer by single-cell sequencing[J]. JCI Insight, 2022, 7(1):e152616.
[11] Lee JJ, Bernard V, Semaan A, et al. Elucidation of tumor-stromal heterogeneity and the ligand-receptor inte-ractome by single-cell transcriptomics in real-world pancreatic cancer biopsies[J]. Clin Cancer Res, 2021, 27(21):5912-5921.
[12] Mei Y, Xiao W, Hu H, et al. Single-cell analyses reveal suppressive tumor microenvironment of human colorectal cancer[J]. Clin Transl Med, 2021, 11(6):e422.
[13] Zheng C, Zheng L, Yoo JK, et al. Landscape of infiltra-ting T cells in liver cancer revealed by single-cell sequencing[J]. Cell, 2017, 169(7):1342-1356,e16.
[14] Sathe A, Grimes SM, Lau BT, et al. Single-cell genomic characterization reveals the cellular reprogramming of the gastric tumor microenvironment[J]. Clin Cancer Res, 2020, 26(11):2640-2653.
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