Abstract Doublecortin-like kinase 1 (DCLK1) is overexpressed and marks a population of tumor-initiating cells in pancreatic ductal adenocarcinoma (PDAC). It regulates key oncogenes, pluripotency factors, angiogenic factors, and epithelial mesenchymal transition (EMT) related transcription factors. Therefore, DCLK1 may be a novel therapeutic target in PDAC. In this study we evaluated whether DCLK1-targeted monoclonal antibodies (mAbs) demonstrate therapeutic activity against pancreatic cancer. Human pancreatic cancer cells (AsPC-1) were treated with mAbs against the C-terminal domain of human DCLK1 to assess their effect on gene expression, cell proliferation, migration, invasion, and tumorigenicity. Gene expression levels were analyzed by quantitative real-time RT-PCR. The proliferative and invasive potential of cells with or without mAb treatment were compared using a MTT assay for proliferation, wound healing assay for migration, and Matrigel coated transwell assay for invasion. Tumor xenografts originated from AsPC-1 cells in nude mice were also treated with these mAbs. Here we demonstrate that treating AsPC-1 cells with mAb against DCLK1 resulted in a more than 50% decrease in DCLK1 mRNA expression. The expression levels of several key tumor suppressor microRNAs, let7a, miR144, and miR200, were upregulated in cells treated with these mAbs. The expression levels of pancreatic cancer related oncogenes KRAS, NOTCH, and REG4, angiogenic factors, VEGFR1 and VEGFR2, and EMT related genes, ZEB1, ZEB2, SNAIL, SLUG, and TWIST, were significantly downregulated (among 30-50% reduction) in the cells treated with mAb against DCLK1. Furthermore, AsPC-1 cells exhibited more than 20% decrease in migration and more than 40% decrease in invasion following anti-DCLK1 mAb treatment. Moreover, AsPC-1 xenograft tumor volumes were reduced approximately 20% following mAb treatment. These data implicate the C-terminal of DCLK1 as a potential signal transduction regulator of DCLK1 in cancer via tumor suppressor microRNA related mechanism similar to siRNA mediated knockdown of DCLK1. These results suggest that utilizing mAb targeting DCLK1 may represent a potential novel therapeutic strategy to reduce the tumor stem-like cell subpopulation in PDAC. Citation Format: Dongfeng Qu, Nathaniel Weygant, Randal May, Parthasarathy Chandrakesan, Daniel Owen, Sripathi Sureban, Courtney Houchen. DCLK1 targeted monoclonal antibodies demonstrate therapeutic potential against pancreatic ductal adenocarcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-48. doi:10.1158/1538-7445.AM2014-LB-48