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An optimization system for isolating and sequencing of single human colon cancer cells

ABSTRACT: Single cell sequencing (SCS) is a promising approach for precisely digging into the genetic heterogeneity at single cells level. Single cell whole genome/exome sequencing (scWGS/scWES) and single cell whole transcriptome sequencing (scRNA-seq) are methods of SCS and have been applied in various studies of solid tumors, e.g. liver cancer, breast cancer. However, no scRNA-seq-related studies of human colon-cancer tissue-samples have been conducted. In this study, we developed a modified and efficient colon-cancer-tissue SCSs system (named CCTSs-SCSs system) by combining four optimization technologies: tissue digestion, live and intact single-cells capture, single-cell RNA or genome amplification and sequencing, and bioinformatics analysis. This advanced system is applicable to both scRNA-seq and scWGS/scWES. Using this system, we successfully completed several single cells scRNA-seq and scWES, and found three key points of this system: an improved digestion system, FACS-based dead-cell-removal method for scRNA-seq, 20 cycles for scRNA amplification. Our CCTSs-SCSs system provides a reliable and efficient method of scRNA-seq or scWGS/scWES for human colon cancer tissue and contributes to single-cell level and precise studies for colon cancer.

ORGANISM(S): Homo sapiens

PROVIDER: GSE92432 | GEO | 2019/12/01

REPOSITORIES: GEO

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Project description:Pancreatic cancer is a complex disease with a desmoplastic stroma, extreme hypoxia, and inherent resistance to therapy. Understanding the signaling and adaptive response of such an aggressive cancer is key to making advances in therapeutic efficacy and understanding disease progression. Redox factor-1 (Ref-1), a redox signaling protein, regulates the DNA binding activity of several transcription factors, including HIF-1. The conversion of HIF-1 from an oxidized to reduced state leads to enhancement of its DNA binding. In our previously published work, knockdown of Ref-1 under normoxia resulted in altered gene expression patterns on pathways including EIF2, protein kinase A, and mTOR. In this study, single cell RNA sequencing (scRNA-seq) and proteomics were used to explore the effects of Ref-1 on metabolic pathways under hypoxia.Results: We also integrated the scRNA data analysis with the proteomic analysis and found that the differentially expressed genes and pathways identified from the scRNA-seq data are highly consistent to the significant proteins observed in the proteomics data, especially for the upregulated cell cycle and transcription pathways and downregulated metabolic, apoptosis and signaling pathways under hypoxia. Conclusion: The scRNA-seq and proteomics data consistently demonstrated down-regulated central metabolism pathways in APE1/Ref-1 knockdown vs scrambled control under both normoxia and hypoxia conditions. Experimental Methods: scRNA-seq comparing pancreatic cancer cells expressing less than 20% of the Ref-1 protein was analyzed using left truncated mixture Gaussian model. Matched samples were also collected for bulk proteomic analysis of the four conditions. scRNA-seq data was validated using proteomics and qRT-PCR. Ref-1’s role in mitochondrial function was confirmed using mitochondrial function assays and qRT-PCR. Results: We also integrated the scRNA data analysis with the proteomic analysis and found that the differentially expressed genes and pathways identified from the scRNA-seq data are highly consistent to the significant proteins observed in the proteomics data, especially for the upregulated cell cycle and transcription pathways and downregulated metabolic, apoptosis and signaling pathways under hypoxia. Conclusion: The scRNA-seq and proteomics data consistently demonstrated down-regulated central metabolism pathways in APE1/Ref-1 knockdown vs scrambled control under both normoxia and hypoxia conditions.

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An optimization system for isolating and sequencing of single human colon cancer cells

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