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Animal Model |
From The Lundberg Laboratory for Cancer Research,*
the
Departments of Surgery,
Pathology,
and Radiation
Physics,
Sahlgrenska University Hospital,
University of Göteberg, Göteberg; and the Department of
Medicine,§
Malmö University Hospital,
University of Lund, Malmö, Sweden
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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 <50). Computed tomography, octreotide scintigraphy, and bone scintigraphy demonstrated metastatic spread to the liver, paraaortic and mediastinal lymph nodes, and to the skeleton. At surgery, tumor biopsies were obtained. Because the tumor burden was large, hepatic embolization therapy was considered to be a high-risk procedure so this patient was given systemic radionuclide therapy with 111In-labeled octreotide.9
Tumor Cell Culture
Tumor material from one liver metastasis was obtained at surgery. Primary cell cultures were prepared as previously described.10 In one of these primary cultures a markedly increased proliferation rate was noted after 3 months. These cells (referred to as GOT1 cells) were subsequently cultured in quantities to enable heterotransplantation to nude mice. The studies of secretory mechanisms and Ca2+ channel signaling were performed in cell cultures identically prepared from a tumor of the second generation from nude mice.
Transplantation of Tumor Cells to Nude Mice
For transplantation of tumor cells we used male BALB/cABom-nu mice, 3 to 4 weeks of age (Bomholtgaard, Ry, Denmark). The animal experiments were approved by the Ethical Committee for Animal Experiments, University of Göteborg.
Tumor Generation I
Tumor cells from cultures were harvested and grafted to 10 nude mice. Approximately 20 million tumor cells were injected subcutaneously into the back of the neck of each animal. When tumors became visible their size was estimated once a week after measurement with a caliper. Eight animals could be followed 6 to 12 months. At sacrifice, blood was collected and the tumor tissues were harvested.
Tumor Generation II to V
From the first generation of tumors new tumors were propagated by subcutaneous transplantation of two pieces of tumor tissue (1 x 1 x 2 mm each) per animal into 20 to 100 animals per generation. To date, five consecutive generations of tumors have been generated. In the fifth generation, the tumor growth rate in vivo was again estimated during a period of 3.5 months.
Cytogenetic and Fluorescence in Situ Hybridization (FISH) Analyses
Chromosome preparations were made from tumor cells obtained from a tumor of generation V. Cells were harvested after overnight exposure to colcemid followed by hypotonic treatment and fixation in methanol:acetic acid.11 Slides were subsequently G-banded and analyzed according to the guidelines of the International System for Human Cytogenetic Nomenclature.12
FISH analysis was performed on touch preparations prepared from a tumor of generation V as well as on unbanded metaphase chromosomes using whole chromosome painting probes for the human X chromosome and chromosome 1 (wcp X and 1; Vysis, Downers Grove, IL). The probes were labeled with Spectrum Green (X chromosome) and Spectrum Orange (chromosome 1) fluorophores. The conditions for hybridization and posthybridization washes were as recommended by the manufacturer. Nuclei and chromosomes were counterstained in blue with 4,6-diamino-2-phenylindole. Slides were examined in a Zeiss Axiophot epifluorescence microscope equipped with the appropriate filter sets. Fluorescence signals were digitalized, processed, and analyzed using the ProbeMaster FISH image analysis system (Perceptive Scientific International, Chester, England).
Binding and Scintigraphy of Radiolabeled Octreotide and 123I-Metaiodobenzylguanidine (MIBG)
The binding of the radiolabeled somatostatin analogue octreotide
was studied on nude mice transplanted with tumors of generation I, II,
and IV. D-Phe1-octreotide was labeled
with 111In mainly according to the manufacturer
(Mallinckrodt Medical B.V., Petten, The Netherlands). The
fraction of peptide-bound 111In exceeded 99%.
Each animal was injected intravenously with 0.25 to 4 MBq (0.1 µg) of
111In-octreotide 4 hours before killing. Tumors
and blood samples were collected and weighed, and the
111In activity was measured in a 
counter. The
activity concentration of the radionuclide was expressed as the
fraction of injected activity per unit mass of the tissue (%IA/g) and
the tumor-to-blood activity concentration ratio, T/B, was
determined.13
Before killing scintigraphy was performed
with a camera.
The binding and scintigraphy of MIBG (10 to 12 MBq) was studied in eight nude mice transplanted with tumors of generation IV at 4 hours (n = 2), 24 hours (n = 3), and 48 hours (n = 3) after injection.
Biokinetics of Radiolabeled Octreotide
Each animal was injected into a tail vein with 2 MBq (0.1 µg) of
111In-octreotide. The 111In
activity in the syringe was measured before and after injection of the
radiopharmaceutical in a well-type ionization chamber to obtain the
injected activity for each animal. These animals were killed 0.5 hours
(n = 5), 24 hours (n =
5), 3 days (n = 5), and 7 days
(n = 5) after injection. The
111In activity in tumor, blood, and whole body
was measured using a Wallace 1480 counter (WIZARD 3"; Wallace,
Oy, Finland). The 111In-activity concentration in
the tissue was expressed as the fraction of the injected activity per
unit mass of the tissue (%IA/g).
Assays of 5-HTP, 5-HT, and 5-HIAA
The 5-HT precursor 5-hydroxytryptophan (5-HTP), serotonin (5-HT), and the main metabolite 5-hydroxyindole acetic acid (5-HIAA) in conditioned culture media were determined by reverse-phase HPLC with electrochemical detection.6 For measurement of 5-HTP, 5-HT, and 5-HIAA in nude mice, whole blood was lysated in sterile water. Proteins were denatured with ZnSO4 and NaOH. The blood samples were thereafter centrifuged at 3,500 x g for 30 minutes and aliquots (20 µl) of the supernatant were injected onto the column.
Immunocytochemistry
Tumor tissues (primary tumor and tumors from nude mice) were fixed
in buffered formalin for 4 to 24 hours and embedded in paraffin wax.
Sections were incubated with primary antibodies overnight (Table 1)
. Bound antibodies were visualized by
an indirect immunoperoxidase technique. Cultures were fixed in 4%
paraformaldehyde in phosphate-buffered saline at pH 7.4 for 4 hours and
incubated for indirect immunofluorescence using primary antibodies
listed in Table 1
. Control cultures were incubated identically, except
for the primary antibodies, which were replaced by normal rabbit
serum/mouse IgG. Cultures were examined with confocal laser scanning
microscopy using a Zeiss Laser Scanning Microscope (LSM
410).5
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Tumor tissue was fixed in 2.5% glutaraldehyde in 0.1 mol/L sodium cacodylate buffer (pH 7.2) for 24 to 48 hours and postfixed for 1 hour in 1% OsO4 in the same buffer. After dehydration the specimens were embedded in epoxy resin (TAAB 812). Ultrathin sections were contrasted with uranyl acetate and lead citrate and examined in a Philips CM 12 electron microscope.
Northern Analysis
Cells from primary cultures and tumor tissues from nude mice were harvested and total RNA was prepared by acid guanidinum thiocyanate-phenol-chloroform extraction.14 Electrophoresis and hybridization with 32P-labeled antisense RNA probes for the five somatostatin receptor subtypes was performed as previously described.15 As performed this analysis gave quantitative information of the expression of somatostatin receptor subtypes.
Intracellular Calcium Determinations
Cells attached to coverslips were washed and loaded for 30 minutes with FURA-2Am (1 µmol/L; Sigma) in the presence of 0.01% pluronic F-127 (Sigma) in a modified HEPES medium supplemented with 11.1 mmol/L glucose as previously described.16 After loading, the cells were washed and the coverslips mounted in a temperature-controlled (37°C) chamber (volume, 110 µl) placed over a 100 X Fluor objective (Nikon, Tokyo, Japan) on the stage of an inverted Nikon microscope (DIAPHOT-TMD); 75 W xenon lamp. The cells were superinfused (flow rate of 1 ml/min) in the HEPES medium supplemented with 11.1 mol/L glucose and 0.5% bovine serum albumin to which was added isoproterenol, carbachol, or KCl (all from Sigma) according to the experimental protocol. The fluorescence of FURA-2 was recorded with dual wavelength excitation spectrophotofluorometry (emission wavelength, 510 nm; excitation wavelengths, 350 and 380 nm) and the [Ca2+]i was calculated according to Grynkiewicz and colleagues.17
Statistical Analysis
The tumor size doubling time, 5-HT concentration in peripheral blood of nude mice, 111In-octreotide and 123I-MIBG concentration in tumor tissues, and [Ca2+]i are given as mean ± SEM. For statistical analysis the Students t-test was used.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The primary tumor was a single lesion in the distal ileum
measuring 2.5 cm in diameter. Light microscopic appearance was that of
a typical ileal carcinoid with predominantly insular growth pattern.
Immunocytochemical analysis demonstrated 5-HT- and substance
P-production by tumor cells. The majority of tumor cells were positive
for VMAT1 and VMAT2. General markers for neuroendocrine differentiation
were also strongly positive (CgA, SV2, and NCAM) (Figure 1
and Table 2
).
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The epithelial phenotype was confirmed by positive
immunocytochemical reactions for cytokeratin and E-cadherin, and
negative reactions for vimentin and neurofilament. Immunocytochemical
analysis demonstrated a strong expression of several neuroendocrine
markers. A strong positive reaction was obtained for 5-HT, substance P,
and for VMAT1 and VMAT2. In the cytoplasm of tumor cells SV2
immunoreactivity was abundant, whereas NCAM was located on the plasma
membrane (Table 2)
. Electron microscopy showed numerous secretory
granules in the cytoplasm of tumor cells. These granules had the
appearance typical for midgut carcinoids, ie, pleiomorphic, dense-core
granules, measuring 100 to 400 nm in diameter.
Growth of Tumor Cells in Nude Mice
Tumor Generation I
Out of the 10 injected animals, eight were followed for 6 to 12
months after grafting of tumor cells. One animal was lost at
transplantation and one was sacrificed before development of tumors,
because of infectious complications. Gross tumors developed in 6 of 8
animals (take rate = 75%). The first tumors were observed 6
months after tumor cell injection. The doubling time for these tumors
was estimated to 17.5 ± 2.2 days (n = 6;
range, 8.9 to 22.7 days) (Figure 2)
.
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In these generations the first tumors were observed already after
4 to 6 weeks. The take rates were 67% in generation II, 89% in
generation III, 98% in generation IV, and 100% in generation V. The
estimated doubling time in generation V was 15.6 ± 0.6 days
(n = 31; range, 5.4 to 21.5) (Figure 2)
. The
tumors have now been continuously propagated for 30 months in nude
mice.
The Transplanted GOT1 Tumor Cells Are of Human Origin
FISH analysis of the touch preparation revealed that
>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.
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Immunocytochemical Properties and Amine Production of Transplanted Tumors
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.
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Somatostatin Receptors in Transplanted Tumors
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)
.
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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.
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. Biokinetics of Radiolabeled Octreotide
The tumors accumulated 111In after
intravenous injection of 111In-octreotide. After
a rapid release during the first 24 hours the decline in
111In-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 initial
111In-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.
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Spontaneous Secretion of 5-HTP, 5HT, and 5-HIAA by Tumor Cells
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)
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The baseline [Ca2+]i
was 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)
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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.18,19 The strong accumulation and slow release of 111In in the transplanted tumors of our model closely resembles the biokinetics of 111In of the original midgut carcinoid tumor, demonstrating the relevance of this new model.20 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.21
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 VMAT2.22 In vitro experiments have shown that MIBG may act as a substrate for chromaffin granules.23 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.6
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 islets24 and chromaffin PC12 cells25 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 A25,26 and K+ depolarization. In contrast, the muscarinic agonist carbachol had no effect on [Ca2+]i, which is different from other cell systems27 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.28 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.
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Acknowledgements |
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Footnotes |
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Supported by The Swedish MRC (5220 and 6834), The Swedish Cancer Society (2690, 3427 and 3911), I.B. and A. Lundberg Research Foundation, Assar Gabrielsson Foundation, The Swedish Society of Medicine, The Swedish Society for Medical Research, The Göteborg Medical Society, The King Gustav V Jubilee Clinic Cancer Fund, Göteborg, Sahlgrenska University Hospital Research Funds, Gunvor and Josef Anérs Stiftelse, Axel Linders Stiftelse, Gunvor, Arvid and Elisabet Nilssons Stiftelse, Serena Ehrenströms donationsfond, Wilhelm och Martina Lundgrens Vetenskapsfond, and Malmö University Hospital Foundation for the Prevention and Treatment of Cancer.
Accepted for publication October 24, 2000.
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