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A Transplantable Human Carcinoid as Model for Somatostatin Receptor-Mediated and Amine Transporter-Mediated Radionuclide Uptake

2001; Elsevier BV; Volume: 158; Issue: 2 Linguagem: Inglês

10.1016/s0002-9440(10)64017-5

1525-2191

Lars Kölby, Peter Bernhardt, Håkan Ahlman, Bo Wängberg, Viktor Johanson, Annelie Wigander, Eva Forssell‐Aronsson, Sven Karlsson, Bo Åhrén, Göran Stenman, Ola Nilsson,

Ion channel regulation and function

A human midgut carcinoid tumor was successfully transplanted into nude mice and propagated for five consecutive generations (30 months) with well-preserved phenotype. Tumor cells in nude mice expressed identical neuroendocrine markers as the original tumor, including somatostatin receptors (somatostatin receptors 1 to 5) and vesicular monoamine transporters (VMAT1 and VMAT2). Because of the expression of somatostatin receptors and VMAT1 and VMAT2 the grafted tumors could be visualized scintigraphically using the somatostatin analogue 111In-octreotide and the catecholamine analogue 123I-metaiodobenzylguanidine. The biokinetics of the somatostatin analogue111In-octreotide in the tumors was studied and showed a high retention 7 days after administration. Cell cultures were re-established from transplanted tumors. Immunocytochemical and ultrastructural studies confirmed the neuroendocrine differentiation. The human origin of transplanted tumor cells was confirmed by cytogenetic and fluorescence it situ hybridization analyses. Spontaneous secretion of serotonin and its metabolite, 5-hydroxyindole acetic acid, from tumor cells was demonstrated. The tumor cells increased their [Ca2+]i in response to β-adrenoceptor stimulation (isoproterenol) and K+-depolarization. All somatostatin receptor subtypes could be demonstrated in cultured cells. This human transplantable carcinoid tumor, designated GOT1, grafted to nude mice, will give unique possibilities for studies of somatostatin receptor- and VMAT-mediated radionuclide uptake as well as for studies of secretory mechanisms. A human midgut carcinoid tumor was successfully transplanted into nude mice and propagated for five consecutive generations (30 months) with well-preserved phenotype. Tumor cells in nude mice expressed identical neuroendocrine markers as the original tumor, including somatostatin receptors (somatostatin receptors 1 to 5) and vesicular monoamine transporters (VMAT1 and VMAT2). Because of the expression of somatostatin receptors and VMAT1 and VMAT2 the grafted tumors could be visualized scintigraphically using the somatostatin analogue 111In-octreotide and the catecholamine analogue 123I-metaiodobenzylguanidine. The biokinetics of the somatostatin analogue111In-octreotide in the tumors was studied and showed a high retention 7 days after administration. Cell cultures were re-established from transplanted tumors. Immunocytochemical and ultrastructural studies confirmed the neuroendocrine differentiation. The human origin of transplanted tumor cells was confirmed by cytogenetic and fluorescence it situ hybridization analyses. Spontaneous secretion of serotonin and its metabolite, 5-hydroxyindole acetic acid, from tumor cells was demonstrated. The tumor cells increased their [Ca2+]i in response to β-adrenoceptor stimulation (isoproterenol) and K+-depolarization. All somatostatin receptor subtypes could be demonstrated in cultured cells. This human transplantable carcinoid tumor, designated GOT1, grafted to nude mice, will give unique possibilities for studies of somatostatin receptor- and VMAT-mediated radionuclide uptake as well as for studies of secretory mechanisms. Midgut carcinoid tumors are derived from the enterochromaffin cells in the small intestine. In metastatic disease these tumors can give rise to severe hormonal symptoms because of excessive production of serotonin (5-HT) and tachykinins (substance P and neurokinins). Carcinoid tumors express numerous somatostatin receptors that can be used to alleviate hormonal symptoms by treatment with the long-acting somatostatin analogue octreotide. These receptors have also been used for visualization of tumors by scintigraphy and in receptor-guided surgery and may serve as targets for radionuclide therapy.1Wängberg B Nilsson O Johanson V Kölby L Forssell-Aronsson E Andersson P Fjälling M Tisell L Ahlman H Somatostatin receptors in the diagnosis and therapy of neuroendocrine tumor.Oncologist. 1997; 2: 50-58PubMed Google Scholar Studies of receptor mechanisms and secretory processes in these tumors have been hampered by the lack of suitable experimental models. Heterografting of human carcinoid tumors into privileged sites, eg, the anterior eye-chamber of immunosuppressed rats, has been tried but is restricted to the study of very small tissue pieces.2Nilsson O Wängberg B Kölby L Dahlström A Ahlman H Intraocular transplantation and primary cell cultures as experimental models for the study of human carcinoid disease.Ann NY Acad Sci. 1994; 733: 380-392Crossref PubMed Scopus (2) Google Scholar Midgut carcinoids can also be studied in primary cell cultures, but tumor cells proliferate slowly and survive only for limited periods of time.3Ahlman H Wigander A Mölne J Nilsson O Karlsson JE Theodorsson E Dahlström A Presence of nerve growth factor-like immunoreactivity in carcinoid tumour cells and induction of a neuronal phenotype in long-term culture.Int J Cancer. 1989; 43: 949-955Crossref PubMed Scopus (35) Google Scholar, 4Debons-Guillemin MC Launay JM Roseto A Peries J Serotonin and histamine production by human carcinoid cells in culture.Cancer Res. 1982; 42: 1513-1516PubMed Google Scholar, 5Nilsson O Wängberg B Theodorsson E Skottner A Ahlman H Presence of IGF-I in human midgut carcinoid tumours—an autocrine regulator of carcinoid tumour growth?.Int J Cancer. 1992; 51: 195-203Crossref PubMed Scopus (67) Google Scholar, 6Westberg G Ahlman H Nilsson O Illerskog A Wängberg B Secretory patterns of tryptophan metabolites in midgut carcinoid tumor cells.Neurochem Res. 1997; 22: 977-983Crossref PubMed Scopus (15) Google Scholar Cell lines are ideal for studies of receptor mechanisms and secretory mechanisms, however, few carcinoid tumor cell lines are available today (ie, KRJ-I and BON).7Pfragner R Wirnsberger G Niederle B Behmel A Rinner I Mandl A Wawrina F Luo JS Adamiker D Hoeger H Ingolic E Schauenstein K Establishment of a continuous cell line from a human carcinoid of the small intestine (KRJ-I): characterization and effects of 5-azacytidine on proliferation.Int J Oncol. 1996; 8: 513-520PubMed Google Scholar, 8Evers BM Townsend Jr, CM Upp JR Allen E Hurlbut SC Kim SW Rajaraman S Singh P Reubi JC Thompson JC Establishment and characterization of a human carcinoid in nude mice and effect of various agents on tumor growth.Gastroenterology. 1991; 101: 303-311Abstract PubMed Scopus (0) Google Scholar The most widely used cell line, BON, was derived from a lymph node metastasis of a highly malignant pancreatic carcinoid. BON cells have been used to investigate responses to secretagogues and cytotoxic agents and express both epithelial markers (cytokeratin) and multiple amines and peptides [5-HT, chromogranin A (CgA), neurotensin, and pancreastatin]. In this report we present a transplantable human midgut carcinoid tumor that can be propagated in nude mice for extended periods of time with well-preserved neuroendocrine phenotype, including expression of somatostatin receptors and VMAT1 and VMAT2. A 55-year old woman presented with a severe midgut carcinoid syndrome associated with elevated urinary levels of 5-HIAA (630 μmol/24 hours; reference 95% of the cells analyzed were of human origin. Hybridization with human painting probes showed that the majority of nuclei contained two X chromosome-specific and two chromosome 1-specific signals (Figure 3). The few cells that were negative for the human wcp probes had generally larger nuclei than did those that were positive for the probes. Cytogenetic analysis of a 17-hour culture confirmed that >95% of the metaphases had a human chromosome complement. The chromosome counts of all but one of the 46 metaphases analyzed were in the hypodiploid region with a modal number of 44 chromosomes (range, 38 to 45 chromosomes). All metaphases had clonal structural rearrangements including several marker chromosomes. No cell with a normal human karyotype was observed. Only a small percentage (<5%) of cells had mouse karyotype. Microscopic examination of tumors demonstrated a characteristic pattern of insular growth with tumor cells positive for CgA, 5-HT, VMAT1, and VMAT2 (Figure 4). One tumor from each of the first four generations was examined. The neuroendocrine differentiation was well maintained over the four generations (Table 2). Electron microscopy confirmed presence of numerous electron-dense granules in the cytoplasm of the tumor cells. The average 5-HT concentration in peripheral blood of nude mice with generation II tumors was 5,103 ± 795 μmol/L (n = 7) versus 1,784 ± 225 μmol/L (n = 8) in animals without tumor (P < 0.001). Northern analysis revealed expression of all five somatostatin receptor subtypes in tumor cells from the primary cell culture. The somatostatin receptor expression was also investigated in tumors from nude mice and all five somatostatin receptor subtypes were expressed in all tumor generations investigated (generations I, II, and III). The only exception was somatostatin receptor 2, which was not detectable in generation III. The transcript sizes were as expected (Figure 5). The concentration of 111In-octreotide in tumor at 4 hours after injection was very high for tumor generation I with 13% IA/g (n = 1), and lower values for generation II with 0.87 ± 0.32% IA/g (n = 3) and generation IV with 0.40 ± 0.08% IA/g (n = 10). The corresponding T/B values were 250, 63 ± 39, and 20 ± 4. Scintigraphy of the nude mice with tumor generation I showed high uptake in the tumor (Figure 6), but also uptake/distribution of the radionuclide in the kidneys and urinary bladder. The concentration of 123I-MIBG in tumor tissue of generation IV was 0.93 ± 0.06% IA/g (n = 2) at 4 hours, 1.1 ± 0.1% IA/g (n = 3) at 24 hours, and 0.54 ± 0.08% IA/g (n = 3) at 48 hours after injection. The corresponding T/B values were 12 ± 2, 28 ± 5, and 83 ± 13. Scintigraphy at 24 hours showed high uptake in the tumor, but also some uptake/distribution of the radionuclide in the salivary glands and urinary bladder (Figure 6). The tumors accumulated 111In after intravenous injection of 111In-octreotide. After a rapid release during the first 24 hours the decline in111In-activity concentration was very slow for the tumor whereas it was considerably faster for blood and whole body. Seven days after the injection 17% of the initial111In-activity concentration still remained in the tumors versus 0.05% and 3.3% in blood and whole body, respectively (Figure 7). The concentration of 111In-activity in tumor tissue was 1.25 ± 0.14% IA/g (n = 5) at 0.5 hours, 0.33 ± 0.09% IA/g (n = 5) at 24 hours, 0.29 ± 0.02% IA/g (n = 5) at 3 days, and 0.22 ± 0.06 (n = 5) at 7 days after injection. The spontaneous secretion of 5-HTP, 5HT, and 5-HIAA into culture medium was followed up to 72 hours after change of culture medium. 5-HT levels started to rise 4 hours after the change of medium and was elevated eightfold at 72 hours. The medium levels of 5-HTP and 5-HIAA started to rise at 24 hours after change of medium with very high levels of 5-HIAA (Figure 8). The baseline [Ca2+]iwas 37 ± 4 μmol/L (n = 4). Isoproterenol (10 μmol/L) slightly increased [Ca2+]i, whereas carbachol (100 μmol/L) had no additional effect. Depolarization by KCl (20 mmol/L) rapidly increased [Ca2+]i in a reversible manner (Figure 9). Increased understanding of the biology of carcinoid tumor cells and development of new approaches for diagnostic and therapeutic attempts require reliable human cell lines. In this study we present a human midgut carcinoid tumor that was transplanted to nude mice and successfully propagated for five consecutive generations with well-preserved neuroendocrine differentiation. The human origin of the cells was confirmed by cytogenetic and FISH analyses. Touch preparations from generation V showed that ≥95% of the cells were of human origin using painting probes for the human X chromosome and chromosome 1. Cytogenetic analysis revealed that the vast majority of metaphases had a human chromosome complement. The tumor had a hypodiploid stemline with several clonal structural and numerical abnormalities. The neuroendocrine features of the transplanted tumors were those of a well-differentiated endocrine tumor, ie, strongly immunoreactive for general endocrine markers such as CgA and synaptophysin. Furthermore, the tumor cells expressed markers characteristic for midgut carcinoids, eg, 5-HT. Comparison of the immunohistochemical profiles and ultrastructural features confirmed a high degree of similarity between the original primary carcinoid and the transplanted tumors of all generations.111In-octreotide showed high uptake of the radionuclide and high T/B values in the tumors and Northern blot analysis showed expression of all five somatostatin receptor subtypes in the tumors. These tumors can therefore be used for diagnostic visualization by octreotide scintigraphy and also as a model for studies of somatostatin receptor-mediated radiation therapy. Quantitative analysis of the somatostatin receptor expression levels needs to be performed to give further information about the mechanism for 111In-octreotide uptake. To date biokinetic studies of radiolabeled somatostatin analogues in animal models have been restricted to somatostatin receptor-expressing exocrine pancreatic tumors, ie, CA20948 and AR4-2J.18Breeman WA Hofland LJ Bakker WH van der Pluijm M van Koetsveld PM de Jong M Setyono-Han B Kwekkeboom DJ Visser TJ Lamberts SW Krenning EP Radioiodinated somatostatin analogue RC-160: preparation, biological activity, in vivo application in rats and comparison with [123I-Tyr3]octreotide.Eur J Nucl Med. 1993; 20: 1089-1094Crossref PubMed Scopus (43) Google Scholar, 19Mäcke HR Behe M Froidevaux S Heppeler A Jermann E DOTA-D-Phe(1)-Tyr(3)-octreotide (DOTATOC). A unique somatostatin receptor ligand for labeling with a variety of metallic radionuclides.J Nucl Med. 1997; 38: 18PGoogle Scholar The strong accumulation and slow release of 111In in the transplanted tumors of our model closely resembles the biokinetics of111In of the original midgut carcinoid tumor, demonstrating the relevance of this new model.20Andersson P Forssell-Aronsson E Grétarsdóttir J Johanson V Wängberg B Nilsson OMF Ahlman H Biokinetics and dosimetry after repeated injections of 111In-DTPA-D-Phe1-octreotide.Sixth International Radiopharmaceutical Dosimetry Symposium. Tennessee, Gatlinburg1996Google Scholar For therapeutic purposes it may be of interest to modulate somatostatin receptor expression, eg, selective up-regulation of somatostatin receptor 2 for optimal binding to octreotide, which today is the analogue most widely used for targeted radionuclide therapy. Such receptor up-regulation can probably be induced by steroid hormones.21Visser-Wisselaar HA Hofland LJ van Uffelen CJ van Koetsveld PM Lamberts SW Somatostatin receptor manipulation.Digestion. 1996; 57: 7-10Crossref PubMed Scopus (21) Google Scholar The uptake of radiolabeled MIBG has been shown to be high in pheochromocytomas, expressing both VMAT1 and VMAT2, but reduced to one third in midgut carcinoids, which often lack expression of VMAT2.22Kimmig BN Radiotherapy for gastroenteropancreatic neuroendocrine tumors.Ann NY Acad Sci. 1994; 733: 488-495Crossref PubMed Scopus (17) Google Scholar In vitro experiments have shown that MIBG may act as a substrate for chromaffin granules.23Henry JP Gasnier B Desnos C Scherman D Krejci E Massoulie J The catecholamine transporter of adrenal medulla chromaffin granules.Ann NY Acad Sci. 1994; 733: 185-192Crossref PubMed Scopus (7) Google Scholar VMAT2 may thus be important for MIBG transport and tumor visualization. In this model we found that the transplanted tumors, cultured tumor cells and the original primary ileal carcinoid expressed both VMAT1 and VMAT2. 123I-MIBG scintigraphy visualized the tumors in nude mice and determination of the radionuclide concentrations in tumor tissue and blood showed very high T/B, which clearly indicates that radiolabeled MIBG may be used therapeutically in GOT1 tumors. In cell cultures very high levels of the main 5-HT metabolite, 5-HIAA, were found. Because midgut carcinoid tumor cells contain both the deaminating enzymes monoamine oxidase A and B, the high 5-HIAA/5-HT ratio indicates a high turnover of 5-HT and complete intracellular metabolism with rapid release of the metabolite into the culture medium.6Westberg G Ahlman H Nilsson O Illerskog A Wängberg B Secretory patterns of tryptophan metabolites in midgut carcinoid tumor cells.Neurochem Res. 1997; 22: 977-983Crossref PubMed Scopus (15) Google Scholar This study further shows that the tumor cells have mechanisms for homeostatic regulation of [Ca2+]i, which are similar to other neuroendocrine cells, eg, endocrine cells of the normal pancreatic islets24Hellman B Gylfe E Bergsten P Grapengiesser E Lund PE Berts A Dryselius S Tengholm A Liu YJ Eberhardson M Chow RH The role of Ca2+ in the release of pancreatic islet hormones.Diabete Metab. 1994; 20: 123-131PubMed Google Scholar and chromaffin PC12 cells25Ämmälä C Ashcroft FM Rorsman P Calcium-independent potentiation of insulin release by cyclic AMP in single β-cells.Nature. 1993; 363: 356-358Crossref PubMed Scopus (350) Google Scholar in regard to baseline [Ca2+]i and the [Ca2+]i responses to isoproterenol, which is explained by opening of plasma membrane Ca2+ channels through activation of cyclic AMP and protein kinase A25Ämmälä C Ashcroft FM Rorsman P Calcium-independent potentiation of insulin release by cyclic AMP in single β-cells.Nature. 1993; 363: 356-358Crossref PubMed Scopus (350) Google Scholar, 26Kaspar SP Pelzer DJ Modulation by stimulation rate of basal and cAMP-elevated Ca2+ channel current in guinea pig ventricular cardiomyocytes.J Gen Physiol. 1995; 106: 175-201Crossref PubMed Scopus (8) Google Scholar and K+depolarization. In contrast, the muscarinic agonist carbachol had no effect on [Ca2+]i, which is different from other cell systems27Gilon P Henquin JC Influence of membrane potential changes on cytoplasmic Ca2+ concentration in an electrically excitable cell, the insulin-secreting pancreatic B-cell.J Biol Chem. 1992; 267: 20713-20720Abstract Full Text PDF PubMed Google Scholar and suggests lack of functional muscarinic receptors in these cells or lack of a significant phospholipase C-dependent formation of inositol 1,4,5-trisphosphate. A previous study on human midgut carcinoid cells in a primary cell culture revealed the existence of voltage-gated Ca2+ currents through L-type Ca2+ channels studied by a patch-clamp technique.28Glassmeier G Strubing C Riecken EO Buhr H Neuhaus P Ahnert-Hilger G Wiedenmann B Scherubl H Electrophysiological properties of human carcinoid cells of the gut.Gastroenterology. 1997; 113: 90-100Abstract Full Text PDF PubMed Scopus (29) Google Scholar Therefore, the GOT1 cells respond with changes in [Ca2+]i in response to formation of cyclic AMP and depolarization as other neuroendocrine cells. This study presents evidence that this transplantable human GOT1 carcinoid expresses all recognized somatostatin receptors, the amine transporters VMAT1 and VMAT2 both in vitro and in vivo, bind and internalize radiolabeled somatostatin analogue and MIBG and exhibits neuroendocrine characteristics of handling [Ca2+]i. This cell line is therefore suited to understand biological aspects of human midgut carcinoids and to evaluate new modalities for therapy. We thank Graeme I. Bell (University of Chicago, Chicago, IL), Friedrich Raulf (Preclinical Research, Novartis, Basle, Switzerland), and Susumo Seino (Chiba University School of Medicine, Japan) for generously supplying somatostatin receptor probes; and Siv Tuneberg, Pernilla Andersson, Annki Illerskog, Lena William-Olsson, Ellinor Andersson, Malin Bengtsson, Gülay Altiparmak, Kerstin Knutsson, and Ragnar Alm for expert technical assistance.