Doublecortin-like kinase 1 exhibits cancer stem cell-like characteristics in a human colon cancer cell line

Abstract

Objective: Colon cancer stem cells (CSCs) are implicated in colorectal cancer carcinogenesis, metastasis, and therapeutic resistance. The identification of these cells could help to develop novel therapeutic strategies. Doublecortin-like kinase 1 (DCLK1) has been viewed as a marker for gastrointestinal stem cells that fuel the self-renewal process, however others view them as a marker of Tuft cells or as an enteroendocrine subtype. The purpose of this study was to use a colon cancer cell line to identify and characterize the stem-like characteristics of the DCLK1+ cell population.

Methods: To enrich stem-like cells, HCT116 cells (derived from colon adenocarcinomas) were cultured using serum-free media to form spheres under both normal oxygen and hypoxia condition. DCLK1 transcript expression in the adherent parental cells and spheroids was quantified using quantitative real time reverse transcription- polymerase chain reaction [(q)RT-PCR]. DCLK1 protein expression was determined using flow cytometry. Self-renewal capability from adherent parental cells and spheroids was determined using extreme limiting dilution analysis (ELDA).

Results: Under both normal oxygen and hypoxia condition, the adherent parental cells were composed of cells that express low levels of DCLK1. However, spheroids exhibited an increased frequency of cells expressing DCLK1 on both mRNA and protein levels. Cells derived from spheroids also possess stronger self-renewal capability.

Conclusions: The higher fraction of DCLK1+ cells exhibited by spheroids and hypoxia reflects the stem-like characteristics of these cells. DCLK1 may represent an ideal marker to study and develop effective strategies to overcome chemo-resistance and relapse of colon cancer.

Keywords: Doublecortin-like kinase 1 (DCLK1); cancer stem cells; colorectal cancer; spheroids; stem cell marker.

Figure 1

DCLK1 is up-regulated in the HCT116 spheroids under normal oxygen condition determined by (q)RT-PCR and FACS. A. Melt curve for DCLK1 examined by the designed primers. A pair of primers which can recognize DCLK1 isoform_1, 2, and 3 were designed. Genomic DNA from HCT116 cells (the only line with a smaller peak and shifted temperature, indicated by *) and cDNA from spheroids (lines with a similar sharp peak, indicated by #) were used as template in the (q)RT-PCR to test the specificity of the primers; B. DCLK1 expression in cells derived from spheroid formed under normal oxygen conditions was determined by (q)RT-PCR (Column labeled as mRNA) and FACS (Column labeled as Protein). For the (q)RT-PCR, DCLK1 expression was normalized to beta-actin before fold change calculation. DCLK1 fold change was calculated by comparing DCLK1 expression in the spheroids to the parental adhesive cells under the same culture condition. Data was expressed as mean±SEM from 5 [(q)RT-PCR]and 2 (FACS) independent experiments. For the statistical analysis, DCLK1 fold change in the spheroids was compared to adhesive parental cells, which was given as 1. *P<0.01 vs. adhesive parental cells; C. FACS data for determination of DCLK1 and CD44 expression in the adherent parental cells and spheroids under normal oxygen condition

Figure 2

DCLK1 is up-regulated in the HCT116 spheroids under hypoxia condition determined by (q)RT-PCR and FACS. A. Fold change of DCLK1 expression in spheroids compared to parental adhesive cells cultured under hypoxia condition. Results from (q)RT-PCR was labeled as Hypoxia mRNA (5% O₂). Results from FACS were labeled as Hypoxia protein (1% O₂). For the (q)RT-PCR, DCLK1 expression was normalized to beta-actin before fold change calculation. DCLK1 fold change was calculated by comparing DCLK1 expression in the spheroids to the parental adhesive cells under the same culture condition. Data was expressed as mean±SEM from 2 independent experiments for both (q)RT-PCR and FACS . For the statistical analysis, DCLK1 fold change in the spheroids was compared to adhesive parental cells, which was given as 1. *P<0.05 vs. the adherent parental cells, **P<0.01 vs. the adherent parental cells; B. FACS data for determination of DCLK1 and CD44 expression in the adherent parental cells and spheroids under hypoxia condition

Figure 3

Cells derived from spheroids possess stronger self-renewal capability. Extreme limiting dilution analysis was used to measure self-renewal capability of cells derived from parental adhesive cells and spheroids under normal oxygen condition. Each cell concentration has 24 replicates in 96-well plate. Progeny spheroid number formed from spheroids was compared to that from parental adhesive cells. *P<0.001 vs. cells derived from the adherent parental