Single-Cell Study Uncovers Role of Kinases in Starting Colorectal Tumors

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Summary

The adenoma-carcinoma schema, a widely endorsed paradigm, delineates the ontogenesis of sporadic colorectal malignancies; however, aberrant epithelial cellular heterogeneity obfuscates the comprehensive comprehension of colorectal tissue carcinogenesis. Researchers conducted a single-cell RNA sequencing examination of 54,788 cells procured from patient-correlated specimens, characterizing cell types, transcriptional signatures, and differentially expressed genes throughout each carcinogenic phase. Precursor populations of adenoma and carcinoma were discerned and substantiated via extensive cohort biopsy analysis. Protein tyrosine kinases (PTKs) BMX and HCK emerged as putative catalysts of adenoma inception, propelling colorectal epithelial cell proliferation. Notably, within the organoid culture paradigm, BMX and HCK upregulations generated stratified, polyp-like protrusions extending into the organoid lumen, emulating pathological polyp morphologies in colorectal malignancies. This investigation enhances the understanding of colorectal epithelial evolution amid carcinogenesis at single-cell resolution, potentially informing advancements in colorectal cancer diagnostics and therapeutics.

Research Criteria

This research endeavors to enhance understanding of colorectal epithelial evolution amid carcinogenesis through single-cell resolution analysis. The investigators executed a single-cell RNA sequencing examination of 54,788 cells from patient-correlated specimens across various carcinogenic stages, characterizing cellular types, transcriptional signatures, and differentially expressed genes. Epithelial molecular signatures at normal, benign, and malignant stages were explicated, with adenoma and carcinoma precursor populations discerned, characterized, and substantiated via extensive biopsy analysis. Protein tyrosine kinases (PTKs) BMX and HCK emerged as putative adenoma initiators, promoting colorectal epithelial proliferation. This investigation augments current knowledge, potentially informing advancements in colorectal cancer diagnostics and therapeutics.

Fig.1 Experimental design. (Zheng, 2021)

Sample Type

Human blood and colon samples.

Result—Sample Collection and Quality Control

Employing a dynamic dissection approach to delineate cellular evolution and molecular signatures during the normal-adenoma-carcinoma progression, the investigators employed scRNA-seq technology to profile the transcriptome of each cellular population. Concurrently, they amassed blood, normal, peri-neoplastic, polyp, and cancer tissue samples from patients undergoing radical surgeries. Somatic short-variant analyses consistently revealed multiple mutations in patient-matched adenoma and carcinoma samples. Clonal phylogeny assessments illuminated dynamic alterations in cell populations, substantiating the evolutionary relationships. These rigorous evaluations demonstrated that biopsies from patient-correlated sequential lesions accurately recapitulated the pathological progression of colorectal tumorigenesis.

Fig.2 Single nucleotide variant and clonal evolution analyses. (Zheng, 2021)

Result—Single Cell Transcriptomic Profiling of Colorectal Normal, Adenoma, and Carcinoma Tissues

To generate a sequencing library from single-cell suspensions, researchers enzymatically digested fresh tissues using trypsin and isolated viable cells via flow cytometry, sorting propidium iodide-negative events. Utilizing the droplet-based 10x Genomics platform, they procured transcriptomes of 54,788 individual cells, visualizing cell populations with t-SNE plots based on cluster, tissue, and patient origins. Despite individual discrepancies, most cell populations were identified across all tissues and patients. Normal epithelial cells from distinct patients coalesced, exhibiting analogous expression signatures. Conversely, aberrant epithelial cells from disparate patients manifested as separate clusters, displaying pronounced heterogeneity and suggesting diversified molecular mechanisms underpinning carcinogenesis.

Fig.3 Cell type constitution of different colon tissues. (Zheng, 2021)

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Characteristics of cell populations are rarely homogeneous and synchronized. The goal of single-cell RNA sequencing is to discover transcriptome diversity in heterogeneous samples. Creative Biolabs provides complete workflows that include sample preparation, library construction, and data analysis, maximizing project flexibility, speed, and data accuracy.

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Reference

1. Zheng, X.; et al. Single-cell transcriptomic profiling unravels the adenoma-initiation role of protein tyrosine kinases during colorectal tumorigenesis. Signal Transduction and Targeted Therapy. 2022, 7(1): 60.