Clinical significance of “S” isoform of DCLK1 in different gastric cancer subtypes using newly produced monoclonal antibody

BackgroundDoublecortin-like kinase 1 (DCLK1) isoforms play distinct roles in the progression of gastrointestinal cancers. For the first time ever, the current study aimed to generate DCLK1-S-specific monoclonal antibodies (mAbs) to evaluate the clinical value of DCLK1-S (short isoform) in gastric cancer (GC).Materials and methodsMice were immunized with a unique 7-mer synthetic peptide of DCLK1-S conjugated with keyhole limpet hemocyanin (KLH). Immunoreactivity of hybridomas and mAbs was determined by ELISA assays and immunohistochemistry (IHC). DCLK1-S expression in two GC cell lines was assessed by flow cytometry. After characterization, the expression pattern of DCLK1-S was investigated in different histological subtypes of GC (n=217) and adjacent normal tissues (n=28) using IHC on tissue microarrays. The association of clinical prognostic values with DCLK1-S expression was also investigated.ResultsELISA findings demonstrated that the generated monoclonal antibody (mAb) exhibited strong immunoreactivity towards the immunizing peptide. Positive control tissues, including GC and colorectal cancer, showed strong positive immunoreactivity with anti-DCLK1-S mAb whereas negative reagent control sections represented no staining, demonstrating the specificity of produced mAb. Flow cytometry analysis confirmed that the newly developed mAbs effectively recognized DCLK1-S on the cell surface. A mixture pattern of membranous, cytoplasmic, and nuclear DCLK1-S expression in the GC cells was observed. A significant and inverse association was identified between the expression DCLK1-S in the cell membrane and cytoplasm and PT stage, muscolarispropia, subserosa, and perineural invasion in intestinal subtype, respectively. In signet ring cell type, however, nuclear DCLK1-S expression was adversely associated with tumor size and PT stage. Furthermore, patients with low DCLK1-S expression had a shorter survival than patients with high expression, however, without a statistically significant association.ConclusionAn efficient and precise tool for detecting DCLK1-S in cancer tissues has been developed. Moreover, DCLK1-S overexpression might be considered a favorable clinical factor in GC patients.