Abstract
SummaryBackgroundTumor heterogeneity of human colorectal cancer (CRC)-initiating cells (CRCICs) in cancer tissues often represents aggressive features of cancer progression. For high-resolution examination of CRCICs, we performed single-cell whole-exome sequencing (scWES) and bulk cell targeted exome sequencing (TES) of CRCICs to investigate stemness-specific somatic alterations or clonal evolution.MethodsSingle cells of three subpopulations of CRCICs (CD133+CD44+, CD133−CD44+, and CD133+CD44− cells), CRC cells (CRCCs), and control cells from one CRC tissue were sorted for scWES. Then, we set up a mutation panel from scWES data and TES was used to validate mutation distribution and clonal evolution in additional 96 samples (20 patients) those were also sorted into the same three groups of CRCICs and CRCCs. The knock-down experiments were used to analyze stemness-related mutant genes. Neoantigens of these mutant genes and their MHC binding affinity were also analyzed.FindingsClonal evolution analysis of scWES and TES showed that the CD133+CD44− CRCICs were the likely origin of CRC before evolving into other groups of CRCICs/CRCCs. We revealed that AHNAK2, PLIN4, HLA-B, ALK, CCDC92 and ALMS1 genes were specifically mutated in CRCICs followed by the validation of their functions. Furthermore, four predicted neoantigens of AHNAK2 were identified and validated, which might have applications in immunotherapy for CRC patients.InterpretationAll the integrative analyses above revealed clonal evolution of CRC and new markers for CRCICs and demonstrate the important roles of CRCICs in tumorigenesis and progression of CRCs.FundingA full list of funding bodies that contributed to this study can be found in the Acknowledgements section.
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