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 HuNET are the first reported cultured pancreatic endocrine tumor cells that express and secrete VIP. In addition, they express chromogranin, synaptophysin, and synaptobrevin, all markers of a neuroendocrine phenotype. In a series of VIP tumors, Solcia and colleagues (27) reported that expression of peptide markers was not uniform. In addition to VIP, they found variable expression of chromogranin A, synaptophysin, insulin, pancreatic polypeptide, somatostatin, and other peptides. Their results suggested that VIP-secreting pancreatic tumors arise from an endocrine precursor or stem cell rather than from a strictly neuronal lineage. Additional evidence from transgenic studies suggests that the endocrine cells of the pancreas and small and large bowel may be linked through a common developmental program involving both endocrine and neuronal gene expression (28). Although VIP has been detected in mature normal islets, its expression appears restricted to neural fibers and not in endocrine cells (29). However, detection of VIP in pancreatic tumors shows a mixed pattern of expression in both neurogenic and endocrine cell types (30). Hence, there appears to be plasticity in VIP expression in gastrointestinal cells of neuronal and endocrine lineage. A similar pluripotency has been observed with cells cultured from pituitary adenomas, in which cells initially expressing a neuroendocrine phenotype gradually develop mesenchymal characteristics (31).
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.
1. Teitelman G, Alpert S, Polak JM, et al. Precursor cells of mouse endocrine pancreas coexpress insulin, glucagon and the neuronal proteins tyrosine hydroxylase and neuropeptide Y, but not pancreatic polypeptide. Development 1993; 118:1031–9.
2. Upchurch BH, Aponte GW, Leiter AB. Expression of peptide YY in all four islet cell types in the developing mouse pancreas suggests a common peptide YY-producing progenitor. Development 1994; 120:245—52.
3. Rindi G, Capella C, Solcia E. Cell biology, clinicopathological profile, and classification of gastro-enteropancreatic endocrine tumors. J Mol Med 1998; 76:413–20.
4. Gazdar AF, Chick WL, Oie HK, et al. Continuous, clonal, insulin-and somatostatin-secreting cell lines established from a transplantable rat islet cell tumor. Proc Natl Acad Sci U S A 1980; 77:3519–23.
5. Oie HK, Gazdar AF, Minna JD, et al. Clonal analysis of insulin and somatostatin secretion and L-dopa decarboxylase expression by a rat islet cell tumor. Endocrinology 1983; 112:1070–5.
6. Philippe J, Chick WL, Habener JF. Multipotential phenotypic expression of genes encoding peptide hormones in rat insulinoma cell lines. J Clin Invest 1987; 79:351–8.
7. Santerre RF, Cook RA, Crisel RM, et al. Insulin synthesis in a clonal cell line of simian virus 40-transformed hamster pancreatic beta cells. Proc Natl Acad Sci U S A 1981; 78:4339–43.
8. Efrat S, Linde S, Kofod H, et al. Beta-cell lines derived from transgenic mice expressing a hybrid insulin gene-oncogene. Proc Natl Acad Sci U S A 1988; 85:9037–41.
9. Radvanyi F, Christgau S, Baekkeskov S, et al. Pancreatic beta cells cultured from individual preneoplastic foci in a multistage tumorigenesis pathway: a potentially general technique for isolating physiologically representative cell lines. Mol Cell Biol 1993; 13:4223–32.
10. Gueli N, Toto A, Palmieri G, et al. In vitro growth of a cell line originated from a human insulinoma. J Exp Clin Cancer Res 1987; 4:281–5.
11. Cavallo MG, Dotta F, Monetini L, et al. Beta-cell markers and autoantigen expression by a human insulinoma cell line: similarities to native beta cells. J Endocrinol 1996; 150:113–20.
12. Evers BM, Ishizuka J, Townsend Jr, CM et al. The human carcinoid cell line, BON. A model system for the study of carcinoid tumors. Ann N Y Acad Sci 1994; 733:393–406.
13. Kimura N, Yamamoto H, Okamoto H, et al. Detection of multiple hormones and their mRNAs in human neuroblastoma cell line NB-1 using in situ hybridization, immunocytochemistry and radioimmunoassay. Virchows Arch 1992; 62:321–7.
14. Wollman Y, Lilling G, Goldstein MN, et al. Vasoactive intestinal peptide: a growth promoter in neuroblastoma cells. Brain Res 1993; 624:339–41.
15. Kedinger M, Duluc I, Fritsch C, et al. Intestinal epithelial-mesenchymal cell interactions. Ann N Y Acad Sci 1998; 859:1–17.
16. Muschel R, Khoury G, Reid L. Regulation of insulin mRNA abundance and adenylation: dependence on hormones and matrix substrata. Mol Cell Biol 1986; 6:337–41.
17. Wang N, Ingber D. Control of cytoskeletal mechanics by extracellular matrix, cell shape and mechanical tension. Biophys J 1994; 66:2181–9.
18. Reid L. Stem cell/lineage biology and lineage-dependent extracellular matrix chemistry: keys to tissue engineering of quiescent tissues such as liver. In: Lanza R, Langer R, Chick W, eds. Principles of tissue engineering. New York: Landes Press, 1996:481–514.
19. Brill S, Zvibel I, Reid L. Maturation-dependent changes in the regulation of liver-specific gene expression in embryonal versus adult primary liver cultures. Differentiation 1995; 59:95–102.
20. Reid L. Stem cell-fed lineages and gradients of signals: relevance to epithelial differentiation. Mol Biol Rep 1996; 23:21–33.
21. Wiedenmann B, Franke WW, Kuhn C, et al. Synaptophysin: a marker protein for neuroendocrine cells and neoplasms. Proc Natl Acad Sci U S A 1986; 83:3500–4.
22. Hsu SM, Raine L, Fanger H. A comparative study of the peroxidase-antiperoxidase method and an avidin-biotin complex method for studying polypeptide hormones with radioimmunoassay antibodies. Am J Clin Pathol 1981; 75:734–8.
23. Leube RE, Leimer U, Grund C, et al. Sorting of synaptophysin into special vesicles in nonneuroendocrine epithelial cells. J Cell Biol 1994; 127:1589–601.
24. Hoecker M, John M, Anagnostopoloulos I, et al. Molecular dissection of regulated secretory pathways in human gastric enterochromaffin-like cells: an immunohistochemical analysis. Histochem Cell Biol 1999; 112:205–14.
25. Schmidt W, Creutzfeldt W, Hoecker M, et al. Cholecystokinin receptor antagonist loxiglumide modulates plasma levels of gastro-entero-pancreatic hormones in man. Eur J Clin Invest 1991; 21:501–11.
26. Nobels FR, Kwekkeboom DJ, Bouillon R, et al. Chromogranin A: its clinical value as marker of neuroendocrine tumours. Eur J Clin Invest 1998; 28:431–40.
27. Solcia E, Capella C, Riva C, et al. The morphology and neuroendocrine profile of pancreatic epithelial VIPomas and extrapancreatic, VIP-producing, neurogenic tumors. Ann N Y Acad Sci 1988; 527:508–17.
28. Lopez MJ, Upchurch BH, Rindi G, et al. Studies in transgenic mice reveal potential relationships between secretin-producing cells and other endocrine cell types. J Biol Chem 1995; 270:885–91.
29. De Giorgio R, Sternini C, Anderson K, et al. Tissue distribution and innervation pattern of peptide immunoreactivities in the rat pancreas. Peptides 1992; 13:91–8.
30. Dawiskiba S, Pour PM, Stenram U, et al. Immunohistochemical characterization of endocrine cells in experimental exocrine pancreatic cancer in the Syrian golden hamster. Int J Pancreatol 1992; 11:87–96.
31. Weil RJ, Huang S, Pack S, et al. Pluripotent tumor cells in benign pituitary adenomas associated with multiple endocrine neoplasia type 1. Cancer Res 1998; 58:4715–20.
32. Rawdon BB, Andrew A. Development of embryonic chick insulin cells in culture: beneficial effects of serum-free medium, raised nutrients, and biomatrix. In Vitro Cell Dev Biol Anim 1997; 33:774–82.
33. Potten CS. Stem cells in gastrointestinal epithelium: numbers, characteristics and death. Philos Trans R Soc Lond B Biol Sci 1998; 353:821–30.
34. Cheshier SH, Morrison SJ, Liao X, et al. In vivo proliferation and cell cycle kinetics of long-term self-renewing hematopoietic stem cells. Proc Natl Acad Sci U S A 1999; 96:3120–5.
35. Morrison SJ, Shah NM, Anderson DJ. Regulatory mechanism in stem cell biology. Cell 1997; 88:287–298.
36. Cybulsky AV, McTavish AJ, Papillon J, et al. Role of extracellular matrix and Ras
in regulation of glomerular epithelial cell proliferation. Am J Pathol 1999; 154:899–908.
37. Zvibel I, Brill S, Halpern Z, et al. Hepatocyte extracellular matrix modulates expression of growth factors and growth factor receptors in human colon cancer cell. Exp Cell Res 1998; 245:123–31.
38. Scharfmann R, Tazi A, Polak M, et al. Expression of functional nerve growth factor receptors in pancreatic beta-cell lines and fetal rat islets in primary culture. Diabetes 1993; 42:1829–36.
39. Goi T, Rusanescu G, Urano T, et al. Ral-specific guanine nucleotide exchange factor activity opposes other Ras
effectors in PC12 cells by inhibiting neurite outgrowth. Mol Cell Biol 1999; 19:1731–41.
40. Gittes GK, Galante PE, Hanahan D, et al. Lineage-specific morphogenesis in the developing pancreas: role of mesenchymal factors. Development 1996; 122:439–47.