Immunohistochemical staining of the HuNET cells reveals that they have a phenotype consistent with the parent neuroendocrine tumor. As shown in Fig. 2A and C, the HuNET cells are positive for the neuroendocrine markers of synaptophysin and chromogranin A, respectively. Cytospins of HuNET cells after 3 months of culture and several passages were reacted with rabbit anti-human synaptophysin antisera or mouse anti-human chromogranin A monoclonal antibodies as described in the Methods section. HuNET cells were positive for both markers, with staining evident in every cluster of cells. Cytospins of RIN 38A cells, a rat insulinoma cell line, served as controls and were simultaneously immunostained with the HuNET cells. As shown in Fig. 2B, synaptophysin expression in the RIN 38A cells was heterogeneous, with some cells strongly positive and most cells negative. Immunostaining for chromogranin A was negative in the RIN 38A cells (Fig. 2D), which may reflect the species specificity of the monoclonal antibody used. Similar results were obtained with cytospins of HuNET cells after 18 months of culture and passaging (data not shown). In separate immunohistochemical analyses of HuNET cell cytospins, anti-rat antisera to neuroendocrine peptides were used along with a secondary Texas red–labeled antisera. As shown in Fig. 3, confocal laser microscopy demonstrated that the HuNET cells were strongly positive for synaptophysin and synaptobrevin and were weakly positive for chromogranin A and VIP. Control antisera showed detectable Texas red signal but were less intense than any of the test neuroendocrine markers (Fig. 3A). Comparison of each immunohistochemically labeled frame with each phase contrast counterpart (not shown) revealed that peptide expression is uniform among groups of HuNET cells.
The secretory functions of the HuNET cells were examined by assaying VIP and chromogranin A levels in cells and the supernatant media. In media conditioned from 24-hour exposure to the cells, approximately 5% of the total VIP and chromogranin A could be recovered in the supernatant fraction. Exposure of the HuNET cells to 1 × 10−7 mol/L phorbol 12-myristate 13-acetate (PMA) for 1 hour resulted in approximately twofold increase release of VIP and chromogranin A into the supernatant media. These results summarized in Table 2 indicate that the HuNET cells not only maintain their phenotypic expression of neuropeptides but also retain their secretory properties.
The properties of HuNET cells, namely, small size, slow growth, and matrix specificity, are suggestive that the cells are not well differentiated and may be derived from early islet cell committed progenitors or islet stem cells. It is noteworthy that embryonic islet cells grown in primary culture tend to survive longer and differentiate when culture conditions include hormonally defined medium, low serum concentration, and a biomatrix substrate (32). Although it is technically difficult to estimate the growth of stem or progenitor cell populations, most studies indicate that epithelial stem cells typically have a slow proliferation rate (33–35). It is unclear whether the HuNET cells have an intrinsically slow proliferation rate or were not presented the appropriate mix of substratum and growth factors. That the HuNET cells express several neuroendocrine markers is suggestive that they are already partially differentiated and have moved beyond the stage of a pluripotent progenitor.
The role of porous substrate in the culturing of the HuNET cells has not been fully elucidated but appears necessary for their propagation and survival. HuNET cell growth was not sustainable on impervious surfaces, such as plastic surfaces or thin biomatrix coatings on plastic. These results are consistent with those of previous reports that extracellular matrix is a key variable in survival and a major determinant of growth, fate, and expression of tissue specific genes (36,37).
Efforts to induce HuNET proliferation with growth factors were largely unsuccessful. Basic fibroblast growth factor, which has a mitogenic effect on neural progenitors, appeared to have the greatest effect, albeit modest, on the HuNET cultures. It is possible that the HuNET cells are growth factor dependent and the most appropriate combination of factors was not provided. It is likely to be complex, such as with PC12 neuroendocrine cells, where the type of growth factor receptor or duration of exposure to growth factors determine whether the cells proliferate or differentiate (38,39). The lack of response by HuNET to co-culture with 3T3 fibroblasts could reflect the requirement for developmentally stage-or tissue-specific mesenchymal cells. This raises the issue that it may be difficult to establish the appropriate microenvironment in an in vitro culture to support growth and proliferation of HuNET cells if they are comparable to islet cell progenitors. Although the mesenchymal cells appear to play an important part in determining lineage of tissues in the developing pancreas (40), what effect they have on HuNET cell growth remains to be determined.
Attempts to develop transplantable HuNET tumors in immunocompromised hosts were unsuccessful. Athymic nude mice and severe combined immunodeficient mice were inoculated with fresh tumor cells or HuNET cells by intraperitoneal or subcutaneous injections. No palpable nodules appeared in any of the mice within 6 months. Although at this time the HuNET cells have only limited utility as a culture system for neuroendocrine tumors, this experience suggests that application of methods evolving from stem cell research may provide a means of developing useful pancreatic islet cell lines.
The studies were funded by grants from NIH R29 DK49860 (L Tillotson), NIH R01-DK52851 (L Reid), Renaissance Cell Technologies (L Reid), and pilot feasibility funds and ACT Core funds from the NIH grant to the Center for Gastrointestinal and Biliary Disease Biology-CGIBD (NIH DK34987, R. Sandler, PI). Additional funding was provided by Deutsche Forschungsgeminschaft (H01288/6-1) for M. Höcker and grants from Mildred Scheel Stiftung and Verum Stiftung for B. Wiedenmann. We appreciate the helpful advice of Dr. John T. Woosley and Dr. Virginia Godfrey. Many thanks to Sheila H. Quackenbush for technical assistance with immunohistochemistry. We are greatly indebted to F. Scott Ragan for his assistance with this project.
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