Abstract 2957: Generation of a novel, mono-specific, antibody against short Isoform of DCLK1 (DCLK1-S): a novel biomarker of colon carcinogenesis in patients
Authors: Sarkar Shubhashish , Connell Malaney O. , Popov Vsevolod L. , Luthra Gurinder , Singh Pomila 
Published: July 2016

Double-Cortin Like Kinase I (DCLK1) marks colon cancer stem cells in mouse colons. Adenomas (Ads)/human colorectal cancers (hCRCs) express low to high levels of DCLK1. We recently reported that the canonical long isoform (DCLK1-L), transcribed from 5’(α)-promoter, is mainly expressed in normal human colons, while a novel short isoform (DCLK1-S), transcribed from IntronV (β)-promoter, is expressed in colonic tumors, with &gt;80% hCRCs expressing significant levels of DCLK1-S (O’Connell et al., Sci Rep, 2015). We additionally reported that CRC patients, expressing high levels of DCLK1-S, had significantly lower overall survival compared to low expressers (O’Connell et al., Sci Rep, 2015), suggesting prognostic/diagnostic value of measuring DCLK1-S levels in Ads/CRCs. Since expression of DCLK1-S by CRCs was recently discovered by us, commercial antibodies (Ab) are not available for specifically identifying DCLK1-S in Ad/Carcinoma samples of patients for diagnostic/prognostic purposes. We succeeded in generating a monospecific polyclonal Ab against the few unique amino-acid sequences of DCLK1-S. By immunohistochemistry and western blot analysis, the Ab was confirmed to be specific for DCLK1-S, with no cross-reactivity for DCLK1-L, and little or no background. The DCLK1-S-Ab did not stain normal human colonic crypts, confirming absence of DCLK1-S in normal colons. In colorectal Ads, DCLK1-S was mainly located in the cytoplasm, with &lt;10% in the nucleus, while in adenocarcinomas (AdCAs), intense staining of DCLK1-S was observed in both cytosol and nucleus, demonstrating a stage-specific increase in staining during Ad-AdCA sequence of colon carcinogenesis, with increasing localization in the nucleus. DCLK1-S-Ab and a DCLK1-L specific Ab was also used to investigate localization of the long vs short isoforms of DCLK1 in cells by immuno-electron microscopy, demonstrating important differences in their sub-cellular localization, which maybe reflective of differential biological effects. The DCLK1-S-Ab is currently being used by us to measure relative expression of DCLK1-S in Ad samples in a retrospective study, in collaboration with colleagues in the departments of pathology and biostatistics, for purposes of examining the usefulness of the Ab for prognostic purposes. Conclusion: We have generated a specific antibody against the novel short isoform of cancer stem cell marker, DCLK1, which will allow us for the first time to detect this novel cancer specific molecular biomarker in colonic tumors for prognostic purposes. Rather than lengthy and cumbersome processing, as required for most molecular markers, the Ab will allow physicians, in any setting, to make informed decisions, such as time to colonoscopy for patients, which is currently based on mostly morphological/pathological parameters, and can result in missing or over scoping patients.

Citation Format: Shubhashish Sarkar, Malaney O. Connell, Vsevolod L. Popov, Gurinder Luthra, Pomila Singh. Generation of a novel, mono-specific, antibody against short Isoform of DCLK1 (DCLK1-S): a novel biomarker of colon carcinogenesis in patients. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2957.