Colorectal cancer (CRC) is a leading cause of death from cancer worldwide. Thus, there is an emerging need for new experimental models that allow identification and validation of biomarkers for CRC-specific progression. In this study, we propose a repeated sphere-forming assay as a strategy to select a malignant subpopulation from a CRC line, HCT116. We validated our assay by confirming that three canonical stemness markers, Nanog, Oct4, and Lgr5, were up-regulated in the sphere state at every generation of the selection assay. The resulting line, after eight rounds of selection, exhibited an increased sphere-forming capacity in vitro and tumorgenicity in vivo. Furthermore, dipeptidase 1 (DPEP1) was identified as the major differentially expressed gene in the selected clone, and depletion of DPEP1 suppressed the elevated sphere-forming capacity in vitro and tumorgenicity in vivo. Overall, we have established an experimental strategy for the isolation of a malignant subpopulation from a CRC cell line. Results from our model also suggested that DPEP1 can serve as a promising prognostic biomarker for CRC.

Colorectal cancer (CRC) is a leading cause of cancer-associated mortality worldwide; therefore, there is an emerging need for novel experimental models that allow for the identification and validation of biomarkers for CRC-specific progression. In the present study, a repeated sphere-forming assay was used as a strategy to select a malignant subpopulation from a CRC cell line, namely HCT116. The assay was validated by confirming that canonical stemness markers were upregulated in the sphere state at every generation of the selection assay. The resulting subpopulation, after eight rounds of selection, exhibited increased sphere-forming capacity in vitro and increased tumorigenicity in vivo. Furthermore, dipeptidase 1 (DPEP1) was identified as the major differentially expressed gene in the selected clone, and its depletion suppressed the elevated sphere-forming capacity in vitro and tumorigenicity in vivo. Overall, the present study established an experimental strategy to isolate a malignant subpopulation from a CRC cell line. Additionally, results from the present model revealed that DPEP1 may serve as a promising prognostic biomarker for CRC.