A transplantable human carcinoid as model for somatostatin receptor-mediated and amine transporter-mediated radionuclide uptake

Abstract

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 analogue 111In-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 beta-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.

Figure 1.

Morphology of the primary ileal carcinoid. Tumor cells grow in an insular pattern with abundant eosinophilic cytoplasm. A positive immunocytochemical reaction for CgA, serotonin (5-HT), and VMAT1 is observed in a majority of tumor cells. Scale bars, 30 μm.

Figure 2.

Diagrams showing the increase in tumor size throughout time in tumor generation I (top, n = 6) and tumor generation V (bottom, n = 27). The doubling times were 17.5 ± 2.2 days (range, 8.9 to 22.7 days) in tumor generation I and 15.6 ± 0.6 days (range, 5.4 to 21.5 days) in tumor generation V.

Figure 3.

FISH analysis of a touch preparation made from a transplanted tumor of generation V. Hybridization with wcp probes specific for the X chromosome (green signal) and chromosome 1 (red signal) reveals human chromosome-specific signals in the vast majority of cell nuclei (blue staining). Scale bar, 30 μm.

Figure 4.

Immunocytochemical characterization of carcinoid tumors grown in nude mice. Tumor cells are positive for 5-HT, CgA, and VMAT1 and VMAT2. The immunohistochemical profile in grafted tumor cells was the same as in the primary midgut carcinoid tumor. Scale bars, 30 μm.

Figure 5.

Quantitative analysis of somatostatin receptor expression. Northern analysis was performed on carcinoid tumor cells in primary culture before transplantation (A), tumor cells in cell culture derived from a transplanted tumor from nude mice (B) and tumors from generation I to III (I, II, and III). All five somatostatin receptor subtypes were expressed by the tumor cells in culture as well as tumors in nude mice. The expression of somatostatin receptors in the three generations was identical except for somatostatin receptor 2 in generation III, which was not detectable. G3PDH was used as housekeeping gene to confirm the integrity of the blotted mRNA.

Figure 6.

Octreotide scintigraphy (left) of a nude mouse with a carcinoid tumor located in the back of the neck (anteroposterior view). The mouse was examined in a γ camera 10 minutes after injection of ¹¹¹In-octreotide. Uptake in the tumor (T) is indicated as well as uptake in the kidneys (K) and the urinary bladder (B). MIBG scintigraphy (right) of a nude mouse with a carcinoid tumor growing in the back of the neck (lateral view). The mouse was examined in a γ camera for 30 minutes 24 hours after the injection of ¹²³I-MIBG. Uptake in the tumor (T) is indicated as well as uptake in the kidneys (K) and the urinary bladder (B).

Figure 7.

¹¹¹In-activity concentration (%IA/g) in tumors transplanted to nude mice, blood, and whole body. After a rapid decline during the first 24 hours the decline in ¹¹¹In-activity concentration was very slow for the tumor. Still 7 days after the injection 17% of the ¹¹¹In-activity concentration at day 0 remained in the tumors whereas only 0.05% and 3.3% remained in the blood and whole body, respectively.

Figure 8.

Spontaneous secretion of 5-HTP, 5HT, and 5-HIAA into medium of cell cultures established from a transplanted tumor of generation II. The medium levels of 5-HTP and 5-HIAA started to rise 24 hours after change of medium with very high levels of 5-HIAA indicating a rapid turnover of 5-HT by the tumor cells.

Figure 9.

Effect of isoproterenol (10 μmol/L), carbachol (100 μmol/L), and depolarization by KCl (20 mmol/L), respectively, on [Ca²⁺]_i in single superinfused tumor cells prepared from heterografted tumors of generation II. The trace is representative of three different experiments.