**7. Cancer cell surface expression of eAGR2 and tumor localization**

To demonstrate cancer cell surface expression, we used flow cytometry with our obtained monoclonal antibodies. The mouse Agr2+ pancreatic cell line DT6606 [44], derived from an engineered C57BL/6 mouse strain to develop pancreatic cancer [45], was incubated with P3A5 followed by dye-conjugated anti-mIgG2a. Antibody binding to the cell surface was indicated by fluorescence shift (*vs*. isotypematched control) [22]. Most available anti-human AGR2 antibodies, like P3A5, recognize both human AGR2 and murine Agr2 as the two proteins share a high degree of sequence homology. Human pancreatic cells were previously shown to have cell surface expression of eAGR2 using a different antibody [37].

To demonstrate tumor localization, 111In-radiolabeled P3A5 was injected into mice carrying DT6606 tumors. At 48 h post-injection, the implanted tumors showed intense labeling (**Figure 6**). There was minimal labeling of the iAgr2<sup>+</sup> bladder or lung, or elsewhere [22]. The imaging confirmed cancer cell surface localization as well as cancer cell specificity of eAgr2. The cross-reactivity between human AGR2 and mouse Agr2 allowed one to speculate that a similar result would be obtained in human patients, i.e., specific localization to eAGR2<sup>+</sup> pancreatic (or other solid) tumors and not to iAGR2<sup>+</sup> organs. With cancer-specific localization, there is a strong likelihood that ant-AGR2 would be highly effective against cancer with minimal reactivity towards non-cancer tissue.
