Abstract 3763: Identification of gpA33 as a marker expressed on colon cancer stem cell lines.

Introduction: Recent studies have implied that solid tumors including colon cancer, can arise from tissue-specific stem cells. Defined serum-free culture conditions, employed to select for fetal colon epithelial progenitor cells, were utilized to isolate and expand a sub-population of epithelial cells from human tumors resected from a repertoire of colon cancer patients (Roberts 2011, AACR #5211). These cancer stem-like cell (CSLC) lines are stable homogenous populations that, when cultured under conditions that promote colon crypt differentiation, differentiate into organoids containing the 3 principal cell types seen in the colon and in differentiated colorectal tumors. In vivo they form tumors that fully recapitulate the morphologic and phenotypic characteristics of the patients’ original tumors. While any consistent definition of cancer stem cell properties remains elusive, these CSLC providing unique tools to interrogate the biology of CSC or tumor initiating cells, and identify CSC-expressed surface antigens. Methods: Cell immunizations were performed with colon CSLC. mAbs were analyzed by IHC to determine their reactivity toward normal and cancer tissues as well as a panel of cancer lines including colon CSLC. Antitumor bioactivity screens were followed by antigen identification of promising candidates. Affymetrix U133 micro-array analysis was performed to profile gene expression and Taqman analyses performed to identify differentially expressed genes compared to non-CSLC control populations. Results: mAbs from CSLC immunizations included a subset that displayed uniform binding across the complete panel of CSLC lines while displaying high penetrance of expression on both primary and metastatic colon cancer tissues. Expression cloning identified the antigen as glycoprotein A33 (gpA33), which was confirmed by IP and SPR analyses. Transcriptional profiling confirmed elevated gpA33 expression across the panel of colon CSLC in addition to revealing enhanced LGR5, ASCL2 and SOX9 expression, consistent with the CSLC having a colon stem cell origin. The uniform expression of gpA33 on putative cancer stem cell populations is distinguished from other cell surface molecules generally regarded as “canonical” CSC markers, including CD133 and CD44, which are variably expressed. Conclusion: gpA33 is an antigen with excellent expression penetrance in colon cancer and colon cancer-derived CSLC. As shown here, immunizations with CSLC lines can yield antibodies specific for colon cancer antigens that may also represent CSC expressed markers. This provides an example how new therapeutic antibody candidates can be generated to recognize and target both differentiated cancer cells as well as their associated CSC population. Citation Format: Jonathan Li, Claudia B. Fieger, Penny Roberts, Doug Smith, Monica Licea, Jeff Hooley, Francine Chen, Kathy King, Jennie Mather, Ezio Bonvini, Deryk Loo, Paul A. Moore. Identification of gpA33 as a marker expressed on colon cancer stem cell lines. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3763. doi:10.1158/1538-7445.AM2013-3763