Isolation, Maintenance, and Characterization of Human Pancreatic Islet Tumor Cells Expressing Vasoactive Intestinal Peptide
Tillotson, Loyal G.*; Lodestro, Cynthia†; Höcker, Michael¶; Wiedenmann, Bertram¶; Newcomer, Christian E.§; Reid, Lola M.†‡
*Division of Digestive Diseases and Nutrition, Department of Medicine, †Department of Cell and Molecular Physiology, ‡Program in Molecular Biology and Biotechnology and §Division of Laboratory Animal Medicine, University of North Carolina School of Medicine, Chapel Hill, North Carolina, U.S.A.; ¶Medizinische Klinik mit Schwerpunkt Hepatologie und Gastroenterologie, Universitätsklinikum Charité, Campus Virchow-Klinikum, Humboldt Universität Berlin, Germany
Manuscript received October 21, 1999;
revised manuscript accepted April 10, 2000.
Address correspondence and reprint requests to Dr. L. G. Tillotson, Division of Digestive Diseases and Nutrition, CB#7038, University of North Carolina, Chapel Hill, NC 27599-7038, U.S.A. E-mail: email@example.com
Tissue from a vasoactive intestinal peptide (VIP)-secreting human tumor has been used to establish and characterize human neuroendocrine primary cell cultures from which permanent, clone-derived cell lines have been established. Viable cells were obtained by enzymatic and mechanical dissociation of freshly resected pancreatic islet tumor and hepatic metastatic tumor tissues. Aliquots of tumor cells were established ex vivo under culture conditions including porous substrata coated with type IV collagen and laminin and a low serum, hormonally defined culture medium. The small (<10 μm) rounded, grape-like cells had a very slow growth rate of doubling times estimated at several weeks or more. After several passages, morphologically uniform cells were derived that strongly expressed neuroendocrine markers of synaptophysin and synaptobrevin. Although chromogranin A and VIP had somewhat weaker expression, both demonstrated phorbol ester-stimulated secretion. The morphologic and secretory properties were maintained by the cells for nearly 2 years in culture. The establishment of this novel VIP-secreting human neuroendocrine cell line (HuNET) makes available a culture model with which to study a transformed version of this pancreatic islet cell type and offers approaches by which to establish islet tumor cell lines.
During fetal development, progenitor cells of pancreatic islets rapidly proliferate and express several peptide hormones including gastrin, secretin, and insulin. At birth, the islets organize, cease proliferation, and undergo terminal differentiation. The mature islets express insulin (β cells), somatostatin (δ cells), glucagon (α cells), and pancreatic polypeptide (PP cells), but no gastrin or secretin (1,2). Like the embryonic islet progenitor cells, pancreatic islet tumors proliferate, invade, and express peptide hormones such as insulin, gastrin, or vasoactive intestinal peptide (VIP). Although the symptoms associated with islet tumors vary depending on which peptides are expressed, the tumors themselves typically are well differentiated and indolent. The overall incidence of islet tumors is approximately 1/106, with VIP-secreting tumors even more uncommon at only 1/107 tumors (3). The rarity of incidence and the typically small size of islet tumors have greatly limited research. Despite numerous attempts to culture islet tumor cell lines from various species, relatively few cell lines have been established. A number of rat insulinoma (RIN) cell lines have been isolated from radiation-induced tumors (4). The RIN cells characteristically have pleotropic peptide expression that can be unstable after numerous passages (5,6). Viral transformation has been another approach taken to develop cultured islet cell lines. Simian virus 40 (SV40) transformation of hamster islet cells (7) or targeted expression of SV40 large T antigen in transgenic mice (8) have yielded hamster insulinoma tumor (HIT) and β tumor cells (βTC) cell lines, respectively. The latter transgenic approach has been extended to isolating immortal cell lines from hyperplastic islet foci, which better approximate the physiological behavior of murine β cells (9). Only two human islet-like cell lines, designated CM and BON, have been developed and characterized to date. CM cells were derived from ascites cells from a patient with an insulinoma (10). These cells do secrete small amounts of insulin and possess some antigenic markers for human β cells (11). The BON cell line, originally cloned from a lymph node with metastatic carcinoid tumor, has a neuroendocrine phenotype and expresses serotonin and chromogranin A (12). However, neither cell line expresses VIP, gastrin, glucagon, somatostatin, or pancreatic polypeptide. In contrast, greater success has been achieved in developing human neuroblastoma cell lines that express and secrete VIP (13). Furthermore, VIP appears to have growth stimulatory properties for these neuronal cell lines (14).
Efforts to culture progenitor cells from hepatic and intestinal tissues indicate that in many instances, proliferation of these progenitor cells depends on certain growth factors and substratum components that can be distinct from the conditions for their mature counterparts (15,16). However, once cells begin to differentiate, they lose their proliferative capacity. This decline in cell proliferation poses a challenge when attempting to develop differentiated islet tumor cell lines. Hence, the relative lack of success in culturing islet tumor cells may result from the inherent difficulty in growing well-differentiated cells, as well as a failure to supply the specific mesenchymal and extracellular matrix factors for islet cell growth.
Advances in cell biology and extracellular matrix chemistry have led to improvements in culture methods for epithelial cells (17,18). With the aim to establish islet cell lines using innovative techniques, this project was initiated when a surgical specimen from a pancreatic islet VIP-secreting tumor became available. Culture conditions specifically designed for epithelial cell progenitors were employed (19,20). Expectations for this project were that the development of transplantable human islet tumors in mice, as well as well-characterized human islet cell lines would provide an important resource for research on islet tumorigenesis and treatment.
MATERIALS AND METHODS
Hormonally defined medium
The HDM is comprised of Ham's F12 base medium (GIBCO, Grand Island, NY, U.S.A.) supplemented with the following: penicillin and streptomycin (10,000 U/mL), nicotinamide (10 mmol/L), bovine serum albumin (1%), insulin (5 μg/mL), iron-saturated transferrin (10 μg/mL), epidermal growth factor (50 ng/mL), IGF II (10 ng/mL), basic fibroblast growth factor (10 ng/mL), high-density lipoprotein (5 μg/mL), triiodothyronine (1 nmol/L), a mixture of free fatty acids bound to human serum albumin (7.6 μEq/L), CuSO4 (10−7 mol/L), H2SeO3 (3 × 10−10 mol/L), and ZnS04 (1 × 10−8 mol/L). All additives were obtained from Sigma (St. Louis, MO, U.S.A.) with the exception of growth factors supplied by Collaborative Biomedical Products (Becton Dickinson Labs, Bedford, MA, U.S.A.). When serum supplementation was used at all, it was not more than 2% fetal bovine serum ([FBS] certified, GIBCO).
Initial processing of the tumor
Samples from the primary of a pancreatic islet tumor were minced immediately, whereas those from the hepatic metastatic lesion were sterilized by a 15-second immersion in 95% ethanol, minced on ice, and passed through a Bellco Cellector. The extruded cells were rinsed and suspended in a hormonally defined medium (HDM).
Ex vivo expansion of human islet cells
Those cells placed into culture were seeded approximately 500,000 cells/35-mm well onto Nytex membranes (nylon with pore size of 0.45 mm) coated with type IV collagen and laminin (both from Collaborative Biomedical Products). Later experiments employed six-well Transwell plates (Falcon, Becton Dickinson) also coated with type IV collagen and laminin. The cultures were submerged in HDM. Half of the cultures were serum free and half received further supplementation with 1% FBS. The medium was changed approximately every 3 days. All other growth factors tested were obtained from Collaborative Biomedical Products and added to HDM at a final concentration of 25 ng/mL. Relative cell counts were determined every 2 weeks from the same location on each well with a microscopic grid.
The cells were suspended in HDM + 2% FBS + 10% dimethylsulfoxide (DMS0), slowly frozen over several hours, and stored in liquid nitrogen vapor phase. Cell recovery from frozen stocks averaged 50% of total cells.
Cytospins (800 g for 10 minutes) of the cells were fixed on glass slides by methanol for 5 minutes and acetone for 10 seconds at −20°C. Immunostaining for paraffin-embedded tumor (Fig. 1) and cytospins of cells (Fig. 2) were accomplished by established methods (21). The primary antisera included a monoclonal antibody to human chromogranin A at 5 μg/mL (Boehringer Mannheim, Indianapolis, IN, U.S.A.) and polyclonal rabbit anti-human synaptophysin affinity-isolated antisera at 1:100 dilution (DAKO, Hamburg, Germany). After incubation and washing of the primary antisera, binding was detected with a biotinylated secondary antibody, streptavidin peroxidase system according to the method of Hsu et al. (22). Positive (neuronal cells in human colon sections) and negative (pancreatic exocrine tumor cell line, BxPC3 cells) controls were run with each immunostaining preparation.
Immunofluorescence microscopy for Fig. 3 was performed as previously described (23,24). In brief, slides with cytospins were rinsed with 37°C phosphate-buffered saline (PBS) and (pH 7.4), fixed in methanol (at −20°C), and air dried after acetone fixation for 10 seconds at −20°C. After rehydration in PBS, slides were incubated with primary antibodies for 30 minutes in a wet chamber at the following concentrations: polyclonal anti-human synaptophysin antisera at 1:200 dilution (DAKO), monoclonal anti-human chromogranin A antisera at 1:400 dilution (Camon, Wiesbaden, Germany), polyclonal anti-human synaptobrevin antisera at 1:200-300 dilution (T. Rapoport, Boston, MA, U.S.A.), and polyclonal anti-human VIP antisera at 1:200 dilution (DAKO). After incubation with the primary antisera, slides were washed with PBS and incubated with Texas red secondary antibodies (Dianova, Hamburg, Germany). Secondary antibodies were pretreated with 5% low-fat milk powder/PBS solution (vol/vol) for 5–10 minutes. After three washes with PBS, slides were dehydrated in ethanol, dried, and covered with polyvinyl alcohol. Fluorescence was detected using a Zeiss LSM 310 laser scan microscope (Carl Zeiss, Oberkochen, Germany), operating with a helium-neon laser at 543 nm and an argon laser at 488 nm.
HuNET cells received a change of HDM (1% FBS) 48 hours before the secretion study. For phorbol 12-myristate 13-acetate (PMA) treatment cells were washed twice with Krebs-Ringer buffer (pH 7.4), followed by incubation for 1 hour in buffer with 1 × 10−7 mol/L final PMA. The supernatant medium was aspirated from the cultured HuNET and snap frozen with freshly prepared phenylmethylsulfonyl fluoride (PMSF, 100 ng/mL). Cells were then suspended into lysis buffer Krebs-Ringer (pH 7.4 containing 0.2% Triton X-100 detergent and 100 ng/mL PMSF). The cell lysis suspension was snap frozen. Conditioned medium was obtained at 24 hours from untreated cells (basal) and the cell lysates prepared as noted above.
VIP and chromogranin A peptide determinations
Chromogranin A determinations were performed using a commercially available enzyme-linked immunosorbent assay kit (K 0025, DAKO, Glostrup, Denmark) according to the manufacturer's recommendations. In this assay, a 23-kDa C-terminal fragment of the human chromogranin A peptide served as a control. Measurement of VIP concentrations were assessed by radioimmunoassays according to a previously described protocol (25). In this assay, synthetic human VIP (Peninsula Laboratories, Merseyside, U.K.) was used as internal standard. Both peptide assays were conducted on duplicate samples measured in triplicate (intra-assay standard deviation noted in Table 2). Total peptide amounts were calculated from the sum of the supernatant medium and lysate.
Fresh tissue samples were obtained from surgical resection of a VIP-secreting pancreatic endocrine tumor in a 32-year-old man. Before surgery, he experienced chronic diarrhea and had an elevated spot serum VIP level (365 pg/mL; normal, <70 pg/mL). Sections from a 2-cm pancreatic primary tumor and a 6-cm hepatic metastasis were processed as described in the Methods section. As seen in the hematoxylin and eosin–stained section from a hepatic metastatic lesion shown in Fig. 1A, sheets of small, uniform neoplastic cells replaced the hepatic parenchyma. The cells had a course granular, aminophilic cytoplasm with a high nuclear:cytoplasmic ratio. The cells strongly stained for neuroendocrine markers of synaptophysin (Fig. 1B) and chromogranin A (not shown). These markers are nearly universally positive in neuroendocrine tumors and rarely in nonendocrine neoplasms of the gastrointestinal tract (21,26). The metastatic tumor was highly vascular as evidence of endothelia and red blood cells was prominent in both photomicrographs. Tissue from the pancreatic primary tumor was densely fibrotic and upon extraction yielded <10% of the comparable amount of cells extracted from the hepatic lesions.
Ex vivo maintenance of the islet cells
Attempts to develop tumor explants in immunocompromised mice from injected tumor cell suspensions were unsuccessful. Cultures proved a more successful approach for establishing the islet tumor cells ex vivo. Those plated on tissue culture plastic or on matrix-coated tissue culture dishes did not survive. Survival occurred only for cells plated on the Nytex filters coated with type IV collagen and laminin mimicking the requirement for porous substrata and matrix components seen for other progenitor cell populations (19,20). Initially, two cell types were observed: a bipolar/multipolar cell that was typically squamous in appearance and a rounded, grape-like cell that was positioned on top of the bipolar cell. After several weeks, the larger cells disappeared leaving a homogeneous population of rounded cells, which we have designated HuNET, of approximately 5–10 μm in diameter shown in Fig. 4A. The HuNET cells tended to grow in grape-like clusters, but occasionally some tubular clusters were observed. Aggregates of these small cells were loosely adherent to the collagen coated Nytex membranes (Fig. 4B) and were eventually passaged to type I or IV collagen gels (Fig. 4A). HuNET cells were tolerant of freezing in DMSO-containing HDM and could be recovered from frozen stocks. Their growth rate was exceedingly slow, with an estimated doubling time of several weeks. The slow division rate for the cells inspired a series of studies with the intent of making the cells proliferate faster. Attempts with co-cultures in transwell plates with 3T3 fibroblasts, HepG2 hepatocytes or AR42J pancreatic exocrine cell lines, and primary liver cells proved futile and had no apparent effect on the cells. However, media supplementation with growth factors had a slight positive effect on the proliferation of HuNET cells. As shown in Table 1, basic fibroblast growth factor and hepatocyte growth factor/scatter factor stimulated proliferation, whereas most other factors had no effect. Leukemia inhibitory factor appeared to accelerate cell death and had a negative effect. In addition, several factors were used in combinations but had no appreciable supplementary effects.
Immunohistochemical studies of the cells
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.
Secretatory activity 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.
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