Cytoplasmic Expression of DCLK1-S, a Novel DCLK1 Isoform, Is Associated with Tumor Aggressiveness and Worse Disease-Speci�c Survival in Colorectal Cancer

Background: Oncogenic role of doublecortin-like kinase 1 (DCLK1) as a putative cancer stem cell (CSC) marker has been clari�ed in colorectal cancer (CRC). Isoform-speci�c function of DCLK1 has highlighted key role of the DCLK1-S (short isoform) in tumor maintenance, progression, and invasion. Considering the lack of commercial anti-DCLK1-S antibody suitable for immunohistochemical (IHC) application, this study was conducted to produce and validate an anti-DCLK1-S polyclonal antibody in order to speci�cally evaluate expression pattern and clinical signi�cance of short isoform of DCLK1 in colorectal cancer tissues. Methods: Rabbit immunization was performed against a synthetic peptide corresponding to the published six speci�c amino acid sequences of DCLK1-S, and production of antibody was evaluated by enzyme-linked immunosorbent assay (ELISA). After IHC assessment of the puri�ed anti-DCLK1-S polyclonal antibody, it was used to undertake a de�nitive study for determining prognostic signi�cance of DCLK1-S expression in a well-de ﬁ ned tissue microarray (TMA) series including 348 CRC and 51 adjacent normal tissues with a follow-up period of 108 months. Results: Positive immunoreactivity of DCLK1-S was found in 84.2% of CRC samples. Cytoplasmic expression was the main localization of DCLK1-S compared to nuclear and membranous area of tumor cells. Expression of DCLK1-S in CRC samples was signi�cantly higher compared to adjacent normal samples (P <0.001). A positive signi�cant association was found between high cytoplasmic expression of DCLK1-S and advanced tumor, nodes, and metastases (TNM) stage (P<0.001) as well as the increased tumor differentiation (P= 0.02). Moreover, the patients with CRC whose tumors showed higher cytoplasmic expression of DCLK1-S had worse disease-speci�c survival (DSS) (log-rank test, P = 0.03) and 5-year DSS rate (P= 0.01). Additionally, the improved prognostic value was seen in the patients with CRC with high DCLK1-S expression versus moderate expression (HR: 2.70, 95% CI: 0.98-7.38; p =0.04) by multivariate analysis. Conclusions: Our �ndings strongly supported that DCLK1-S isoform may play a crucial role in invasion, tumor aggressive behavior, and worsened DSS of the patients with CRC. Importantly, high cytoplasmic expression of DCLK1-S compared to moderate expression could be considered as an independent prognostic factor in�uencing DSS. Taken together, DCLK1-S can be a candidate as a promising prognostic and targeted-therapeutic indicator for effective treatment of CRC. Production of by enzyme-linked immunosorbent assay (ELISA) Anti-DCLK1-S antibodies puri�ed by the peptide coupled to Sepharose-4B. Purity and reactivity of the puri�ed antibodies were assessed by sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and ELISA techniques, respectively. Immunoreactivity pattern of the generated antibody was compared with available commercial anti-DCLK1 antibody; ab31704 (which is commonly used to detect long and short DCLK1 isoforms; Abcam, UK) in CRC tissues by IHC method.