Single-Cell Sequencing Reveals CD133+CD44 - – Originating Evolution and Stemness-Variants in AHNAK2, PLIN4, HLA-B, ALK, CCDC92 and ALMS1 of Human Colorectal Cancer Stem Cells

Tumor heterogeneity and internal clonal evolution of human colorectal cancer stem cells (CRCSCs) within cancerous tissues often represents an aggressive feature of cancer progression. For a high-resolution examination of CRCSCs, we performed single cell whole-exome sequencing (scWES) of sorted cells, including three groups of CRCSCs: CD133+CD44+, CD133-CD44+, or CD133+CD44- cells, colorectal cancer cells (CRCCs) lacking these stem-cell markers (EpCAM+), plus one group of normal control cells. We examined and validated the scWES data for stem-specifically somatic alterations, clonal evolution, etc. Clonal evolution analysis showed that all CRCSCs were located at the main clone, and that the CD133+CD44- CRCSCs, which harbored the lowest number of mutations, were the likely origin of colorectal cancer (CRC) before evolving into other groups of CRCSCs/CRCCs. These results were validated by cell-lines culture experiments. Functional analyses validated that stem cell-specific mutated genes, such as GOLGA8J, ARHGAP39, LURAP1L, CDH1, were positively associated with stemness and knocking down each of them could increase the proportion of CD133+ cells. To validating mutations, especially stem cell-related genes, we performed targeted exome sequencing using a panel of all mutated genes to analyze the distribution of them in 96 samples (20 patients) those were also sorted into the same three groups of CRCSCs and CRCCs. We found two mutational sites of AHNAK2 gene (Chr14_105410531 and Chr14_105410533) were stem-cell specific in both scWES data and target sequencing data. PLIN4, HLA-B, ALK, CCDC92 and ALMS1 genes were specifically mutated in stem cells in both data. Another 33 genes tended to mutate in stem cells from all data. In all stem cell-related genes, AHNAK2, PTPRF and NIPPA5 which subcellularly located at plasma membrane had totally eleven predicted neoantigens. These neoantigens might be useful for some CRC patients in immunotherapy. All integrative analyses above suggested new markers for CRCSCs and showed the important roles of CRCSCs in tumorigenesis and progression of CRCs.