Analysis of the clinical significance of DCLK1+ colorectal cancer using novel monoclonal antibodies against DCLK1

Abstract

Introduction: Doublecortin-like kinase 1 (DCLK1) is considered a putative tumor stem cell (TSC) marker and a promising therapeutic target, as DCLK1⁺ progeny cells exhibit high expression in tumors. However, the biological function of DCLK1⁺ cells in tumorigenesis and tumor progression remains unclear.

Materials and methods: We generated rabbit monoclonal antibodies (mAbs) against DCLK1, DCLK1-42, and DCLK1-87 mAbs, using a novel chip-based immunospot array assay on a chip system. First, the specificity of two mAbs to DCLK1 was confirmed by Western blot, which were bound to DCLK1-long in normal colon cells and to DCLK1-short in a cancer cell line as well as colorectal cancer (CRC) cells.

Results: Precise localization analysis using immunofluorescence revealed that both mAbs had cytoplasmic signal and exhibited a high degree of overlap with microtubules. Furthermore, bacterial display technology indicated that the antigenic epitope region of DCLK1-87 mAb was consistent with that of a commercial anti-DCLK1 polyclonal antibody. In addition, DCLK1-42 mAb has the common polyclonal antibody characteristic of binding to more than one site on DCLK1. By immunohistochemistry, it was found that DCLK1-87 mAb was more specific for DCLK1⁺ cell labeling than a commercial anti-DCLK1 polyclonal antibody. DCLK1 labeled with DCLK1-87 mAb might be a potential TSC marker because the tissue expression site covers the ALDH1 area in CRC tissues. Finally, we analyzed 100 pairs of cancer tissues and matching paracancerous tissue samples from patients with CRC who received 100 months of follow-up with the DCLK1-87 mAb. The results showed that patients with high DCLK1 expression exhibited a longer survival time than that of patients with low DCLK1 expression (P=0.0029).

Discussion: Our results indicated that we successfully generated an efficient tool for the precise detection of DCLK1⁺ cells in cancer tissues. Moreover, we found that high DCLK1 expression in CRC patients appears to play a protective role against tumor progression.

Keywords: DCLK1 monoclonal antibodies; colorectal cancer; doublecortin-like kinase 1; epitope; prognosis.

Figure 1

DCLK1-42 mAb and DCLK1-87 mAb specifically bound to DCLK1. Notes: (A) DCLK1-42 mAb and DCLK1-87 mAb showed a 47 kD band for DCLK1-S in HCT116, and DCLK1-L in NCM460 was 82 kD. DCLK1 from CRC tissues was detected by DCLK1-42 and DCLK1-87 at the size of DCLK1-S. (B) Localization analysis of DCLK1-42 mAb and DCLK1-87 mAb binding to DCLK1 in HCT116 cells. Immunofluorescence staining demonstrated that DCLK1-42 mAb and DCLK1-87 mAb (green) bound to DCLK1 in the intracellular space and overlapped with microtubules (red). Negative control, no primary antibody; rabbit monoclonal negative control, an antinuclear rabbit monoclonal antibody used as a primary antibody; DCLK1-Abs, positive control, rabbit anti-DCLK1 polyclonal antibody; serum, serum from a rabbit that received a fourth immunization with DCLK1. Abbreviations: CRC, colorectal cancer; DCLK1, doublecortin-like kinase 1; DCLK1-L, DCLK1-long; DCLK1-S, DCLK1-short; DCLK1-Abs, DCLK1 antibodies; mAb, monoclonal antibody; neg. cont, negative control; polyclonal Ab, polyclonal antibody; rabbit mAb Cont, rabbit monoclonal antibody control.

Figure 2

Preliminary determination of epitopes recognized by DCLK1-42 mAb and DCLK1-87 mAb. Notes: (A) The DCLK1 cDNA was cloned into a prokaryotic expression vector with seven pieces and then transduced into bacteria. (B) The antigen epitopes of DCLK1-42 mAb and DCLK1-87 mAb were determined via flow cytometry. Serum, rabbit serum from a rabbit that received a fourth immunization with DCLK1; negative control, no primary antibody. Abbreviations: aa, amino acid; DCLK1, doublecortin-like kinase 1; mAb, monoclonal antibody.

Figure 3

Histological analysis of DCLK1⁺ cells in CRC tissues. Notes: (A) Comparison of the DCLK1-87 rabbit anti-DCLK1 mAb and the rabbit polyclonal antibody in immunohistochemistry detection. Tumor biopsies from the same patient with CRC were stained with DCLK-87 mAb or the anti-DCLK1 rabbit polyclonal antibody. The red box indicates the tumor cell area, and the black box with a solid line indicates the tissue stroma area. (B) Rabbit IgG was used as an isotype control. DCLK1-87 mAb was used as the primary antibody to detect DCLK1-expressing cells under low (4×; black box) and high (20×; right box) magnifications. A rabbit anti-ALDH1 mAb was used as the primary antibody to detect ALDH1-expressing stem cells at low (4×; black box) and high (20×; right box) magnifications. Abbreviations: Ab, antibody; CRC, colorectal cancer; DCLK1, doublecortin-like kinase 1; mAb, monoclonal antibody.

Figure 4

Overall survival and disease-free survival of CRC patients relative to low or high DCLK1 expression. Notes: (A) DCLK1 expression was detected with DCLK1-87 mAb on a tissue microarray chip containing 100 paired samples of CRC tissues. The DCLK1 expression intensity was classified into low- and high-expression groups: D0 (−) and D1 (+) indicated low DCLK1 expression; D2 (++) and D3 (+++) indicated high DCLK1 expression. (B) Kaplan–Meier overall survival curves of CRC patients in relation to the relative expression intensity of DCLK1 measured with DCLK1-87 mAb. n=100 patients. Abbreviations: Ab, antibody; CRC, colorectal cancer; DCLK1, doublecortin-like kinase 1; mAb, monoclonal antibody.