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Carcinoid syndrome, characterized by flushing, diarrhea, and valvular heart disease, can occur following carcinoid tumor metastasis to the liver, and systemic release of bioactive hormones into the systemic circulation. Treatment of this devastating disease is hampered by the lack of an in vivo model that recapitulates the clinical syndrome. Here, we have injected BON cells, a novel human carcinoid cell line established in our laboratory, into the spleens of athymic nude mice to establish liver metastases. The majority of mice injected intrasplenically with BON cells developed significant increases in plasma serotonin (5-HT) and urine 5-hydroxyindoleacetic acid (5-HIAA), and several mice exhibited mesenteric fibrosis, diarrhea, and fibrotic cardiac valvular disease reminiscent of carcinoid syndrome both by echocardiographic and histopathologic evaluation. Mice pre-treated with octreotide, a long-acting somatostatin analog, or bevacizumab, a VEGF inhibitor, developed fewer liver metastases and manifestations of carcinoid syndrome, including valvular heart disease. We have provided an important in vivo model to further delineate novel treatment modalities for carcinoid syndrome that will also be useful to elucidate the factors contributing to the sequelae of carcinoid disease (eg, mesenteric fibrosis, valvular heart disease).
Carcinoid tumors are uncommon neuroendocrine tumors. The incidence of carcinoid tumors is two cases per 100,000 patients; however, the actual incidence is thought to be higher and increasing with time (1). Carcinoid tumors demonstrate a predilection for the gastrointestinal (GI) tract, with a majority arising in the small intestine. Although generally regarded as slow-growing and indolent in nature, tumors are often multicentric and associated with an increased incidence of synchronous noncarcinoid malignancies (2, 3).
Carcinoid tumors elaborate a variety of vasoactive amines and peptides, including chromogranin A (CgA), serotonin (5-hydroxytryptamine [5-HT]), histamine, and neurotensin (NT), among others, with site of origin dictating secretory behavior (4-6). When confined to the bowel, peptides and vasoactive amines produced by the tumor are carried through the portal vein to the liver, where they are metabolized (7). However, in the presence of liver metastases or retroperitoneal disease, release of tumor products into the systemic circulation may lead to the development of the devastating sequelae known as carcinoid syndrome, characterized by flushing, diarrhea, bronchoconstriction, and valvular heart disease (7-11). Treatment has been primarily directed toward relief of the debilitating symptoms of carcinoid syndrome using the long-acting somatostatin analogue octreotide, which inhibits the release of carcinoid secretory products (12). Antiproliferative agents, such as streptozocin, 5-fluorouracil, doxorubicin, and cyclophosphamide, may be used to slow tumor growth, but their effects are limited (12, 13). The development of more effective treatment regimens for patients with carcinoid metastasis and carcinoid syndrome has been hampered by the lack of effective in vivo models which recapitulate the disease process in humans (9).
We established and characterized the BON cell line, a functioning human carcinoid cell line from a lymph node metastasis of a pancreatic carcinoid tumor (6, 14, 15). BON cells elaborate not only 5-HT, but also produce and secrete 5-hydroxytryptophan (5-HTP), NT and CgA (15-18). BON cells possess functional receptors for gastrin and somatostatin, and release biogenic amines and peptides in response to various secretagogues (18-20). When injected subcutaneously into nude mice, BON cells form xenografts which are reminiscent of the original tumor by histologic examination. We have previously demonstrated that xenograft development and growth can be inhibited by treatment with a number of agents (eg, interferon-α and octreotide) (9); however, the xenografted tumors do not metastasize, and serum levels of 5-HT and CgA are not elevated. Therefore, while subcutaneous (SQ) injection of BON tumor cells allows for assessment of tumor growth and analysis of treatment options, the SQ model does not produce the findings of carcinoid syndrome. Such a model would be beneficial not only to understand the mechanisms underlying the development of carcinoid heart disease and the systemic sequelae associated with the carcinoid syndrome, but also to aid in the development of novel agents which can effectively treat metastatic carcinoid tumors and carcinoid syndrome.
In our current study, we describe a novel in vivo model of carcinoid syndrome using the BON cell line. BON cells injected into the spleens of athymic nude mice metastasize to the liver. The metastases stain positive for 5-HT, CgA, and NT. A majority of mice with BON liver metastases demonstrate cardiac valve (tricuspid and mitral) thickening and systemic sequelae of vasoactive amine production (eg, diarrhea). Furthermore, we demonstrate that the establishment of BON cell liver metastasis and subsequent tumor function is inhibited by treatment with either octreotide or the vascular endothelial growth factor (VEGF) inhibitor bevacizumab. Our findings demonstrate the successful development of a novel carcinoid syndrome model which closely recapitulates human disease. This model will be critical to better understand etiologic agents responsible for the constellation of symptoms as well as provide a preclinical model to analyze novel treatment strategies.
Rabbit monoclonal anti-NT and anti-CgA antibodies were purchased from Abcam (Cambridge, MA). Mouse monoclonal anti-serotonin antibody was purchased from Dako Corp. (Carpinteria, CA). Tissue culture media and reagents were from Mediatech (Herndon, VA). Serotonin EIA was from Biosource (Belgium). 5-hydroxyindoleacetic acide (5-HIAA) ELISA was from DRG Instruments (Germany). Octreotide was from Bedford Laboratories (Bedford, OH). Bevacizumab was from Genentech (San Francisco, CA).
BON cells are maintained in a 50:50 mixture of Dulbecco's modified Eagle's medium (DMEM) and F12K, supplemented with 5% fetal bovine serum in 5% CO2 at 37°C. To confirm liver metastases, we utilized a BON cell clone stably transfected with the plasmid pEGFP-N1 by electroporation and selected in medium containing G418 (400 μg/ml) (Cellgro); transfection was confirmed by assessment of GFP expression.
Male athymic nude mice mice (4-6 weeks; ~ 25g), were purchased from Harlan Sprague Dawley (Indianapolis, IN). Mice were housed in an American Association for Accreditation of Laboratory Animal Care-approved facility under a standard 12h light-dark cycle. They were fed standard chow (Formula Chow 5008; Purina Mills, St. Louis, MO) and tap water ad libitum and allowed to acclimate for one week. All studies were approved by the Institutional Animal Care and Use Committee of UTMB.
BON cells (passage 23) were harvested from subconfluent cultures by a 1 min treatment with 0.25% trypsin. Tumor cells were injected intrasplenically by methods previously described (21). Briefly, mice were anesthetized with isoflurane, a small left subcostal flank incision was made, and the spleen was exteriorized. Tumor cells (1 × 107 cells/200 μl) were injected into the spleen with a 27-gauge needle. The spleen was returned to the abdomen, and the wound was closed in one layer with wound clips.
(i) To first characterize the carcinoid syndrome model, we performed intrasplenic injection of BON (n = 10) or BON-GFP cells (1×107) (n = 10) into 20 athymic nude mice; intrasplenic injection of HT29 colon cancer cells into 5 mice was performed as a control. Mice injected with GFP-labeled BON cells were observed each week using the Illumatool TLS (Lightools Research, Encinitas, CA). At sacrifice, spleen, liver, and heart were harvested for analysis, and plasma was collected for 5-HT detection by EIA. (ii) To determine whether carcinoid liver metastases and the subsequent carcinoid syndrome could be attenuated, we again performed intrasplenic injections of BON cells (1×107) into 15 athymic nude mice; intrasplenic injections of HT29 colon cancer cells into 5 mice were performed as controls. Mice were then randomized into groups (n = 5) for treatment with vehicle (diH2O, 200 μl, ip, qod), octreotide (5 mg/kg, ip, qod), or bevacizumab (2.5 μg, sc, qd). Echocardiography was performed at week 12 immediately prior to sacrifice; tissues were collected for analysis as above, and plasma and urine were collected for 5-HT and 5-HIAA detection by EIA and ELISA, respectively.
Mice were anesthetized with isoflurane and placed on a warming blanket in the supine position. Transthoracic echocardiographic measurements, including 2-D, M-mode, and Doppler evaluation, were obtained in mice with the use of a 15-MHz linear transducer (Acuson Sequoia Cardiac System) (22, 23).
Upon sacrifice, hearts and segments of livers and spleens were immediately placed in 10% neutral buffered formalin (NBF) for 24 h, followed by 70% EtOH for 24 h. After removal, to display cardiac valves, hearts were manually cut in a near-sagital plane at 1 mm increments (4-5 segments/heart) with emphasis on obtaining longitudinal sections of tricuspid and mitral valves.
All tissue samples were paraffin-embedded, sectioned at 5 μm and routinely deparaffinized and dehydrated. Sections of liver and spleen were stained with hematoxylin and eosin (H&E); heart sections were stained with Movat pentachrome to display collagen, elastin and glycosaminoglycans characteristic of immature connective tissue (24, 25). Immunohistochemistry (IHC) was performed on paraffin-embedded samples as previously described (21, 26) using DAKO EnVision Kit (Dako Corp., Carpinteria, CA). Sections were incubated overnight at 4°C with monoclonal antibodies diluted in 0.05M Tris-HCL + 1% BSA against F4/80 (1:100), BrdU (1:1000), PCNA (1:2000), or LC3 (1:100). After 3 washes with TBST, the sections were incubated for 30 min with secondary antibody labeled with peroxidase, then washed 3 times with TBST. Lastly, peroxidase substrate diaminobenzadine (DAB) was added for immunostaining; sections were counterstained with hematoxylin. For negative IHC controls, primary antibody was omitted from the above protocol.
Heart sections stained with Movat pentachrome were randomized, blinded to all observers, and presented to a pathologist for histologic assessment and scoring of cardiac lesions. After initial review of all specimens, a grading system for cardiac valvular lesions was devised as follows. Grade 0: normal cardiac valve; Grade 1: a single area or multiple areas of thickening involving one valve only; Grade 2: areas of thickening involving two valves; Grade 3: multiple areas of thickening involving two or more valves; Grade 4: multiple areas of thickening as in Grade 3, but with additional degenerative/secondary changes including fibrous adherence to endocardium, metaplastic change within valve structure, or endocardial/valvular thrombus formation.
EIA and ELISA were performed according to the manufacturer's instructions and as described (21). Briefly, 50 μl of supernatant was added to 50 μl of assay diluent (1:1 dilution) in a 96-well plate. After incubation, wells were washed and conjugate added. Substrate solution was then added, and the plates were incubated in the dark for 30 min. Stop solution was then added, and optical density determined at 450 nm (with a correction wavelength set at 570 nm). Experiments were performed in duplicate.
Outcomes were analyzed using Kruskal-Wallis test. Groups were assessed at the 0.05 level of significance. Multiple comparisons were conducted using Bonferroni adjustment for the number of comparisons. All statistical computations were carried out using statistical software, the SAS® system, Release 9.1 (27).
BON cells grow as tumor xenografts when injected into the flanks of athymic nude mice and demonstrate migration in vitro (28). To first determine if BON cells metastasize in vivo, single-cell suspensions of BON cells and GFP-labeled BON cells (1 × 107) were injected into the spleens of 20 nude mice, and mice were sacrificed 9 wks later. Mice injected with GFP-labeled BON cells were imaged weekly to follow tumor development (Fig. 1A). Primary tumor development was first noted by week 3, with metastatic liver lesions obvious by week 5. Following injections, 80% of the mice (n = 16/20) developed liver metastases, with all mice developing primary splenic tumors. Splenic primary tumors and metastases exhibited a similar histopathologic pattern as the patient's original tumor (9). IHC demonstrated heterogeneous, strong positive staining for CgA and 5-HT in the primary tumor and tumor metastases, consistent with previous immunocytochemical staining (9), whereas HT29 colon cancer metastases were negative for these products (Fig. 1B). Given that BON cells metastasize to the liver and produce hormones and bioactive amines characteristic of carcinoid tumors, we next determined whether these mice develop signs and symptoms which recapitulate the carcinoid syndrome in humans. Prior to sacrifice, we noted that 8 of 20 mice developed gross diarrhea (data not shown), and, at sacrifice, we noted the presence of mesenteric fibrosis in 30% (n = 3/20) of the mice by gross examination. A significant elevation of plasma 5-HT was noted in 45% (n = 9/20) of the mice (mean = 482.3 ng/ml, range = 117-841 ng/ml).
To determine whether mice with BON cell liver metastases develop cardiac valvular abnormalities as a result of tumor development, mice were imaged with transthoracic echocardiography as described in Methods. Limitations of these studies included an inability to clearly image the tricuspid and pulmonic valves; additionally, control athymic nude mice were noted to have slight enlargement of the right ventricular diastolic volume relative to control Swiss-Webster or C57BL6 mice of similar age, leading to an inability to utilize right ventricular size as a potential marker of tricuspid or pulmonic valve pathology. Despite these limitations, 6 of 15 mice with BON cell liver metastases clearly showed valvulopathy or functional cardiac impairment when compared with control mice or mice injected with HT29 colon cancer cells. Figure 2A demonstrates a normal Doppler, M-mode, and 2-D ECHO study of a mouse injected with HT29 cancer cells; right and left ventricular sizes, appearance of mitral and aortic valves, and laminar flow through the mitral and aortic valves are grossly normal. In contrast, 6 mice with BON cell liver metastases developed valvular or ventricular abnormalities which were visualized by ECHO, including irregular thickening of the mitral valve leaflets (Fig. 2B), significant right and left ventricular dilatation with irregularity of mitral valve leaflets associated with arrhythmia (Fig. 2C), as well as left ventricular hypertrophy, mitral valve thickening, and aortic regurgitation (Fig. 2D). (Video of echocardiographic studies is available in supplemental data).
In initial screening studies of mice injected with BON cells, histopathologic lesions of cardiac valves were noted in 20% (n = 4/20) of mice; these lesions affected the tricuspid and/or mitral valve as noted by H&E and Movat pentachrome stains (pulmonic and aortic valve lesions were not observed). Subsequent double-blinded studies in which the heart was multiply sectioned to maximize visualization of valves and endocardial structures, as described in Methods, showed that 100% of the mice (n = 5/5) with BON cell metastases treated with vehicle exhibited valvular lesions of two or more cardiac valves (grade 2 - 4). In contrast to normal mitral and pulmonic valves (Fig. 3A, right and left panels, respectively), histopathologic lesions consisted of marked thickening of valve leaflets by spindly fibroblast-like cells and extensive deposition of blue-green mucopolysaccharides noted by Movat Pentachrome stain (Fig. 3B, right and left panels). Although the cardiac valves were sampled equally in multiple sections, lesions were noted most frequently in the tricuspid (91% of hearts with lesions) and mitral (90%) valves; in comparison, aortic and pulmonic valves showed a lower incidence of lesion development (55% and 33%, respectively). In more advanced lesions (Grade 2-3), thickened areas were large, covered extensive areas of the valve leaflets and involved multiple valves. In addition, advanced lesions (Grade 4) showed attachment of valve leaflets to the underlying endocardium (Fig. 3B, left panel). In areas of endocardial, valvular, and subendocardial fibrosis, rounded cells within lacunae were seen, indicative of cartilaginous metaplasia (Fig. 3C). Additionally, thrombus formation on the valvular surface was occasionally noted in advanced lesions (Fig. 3D). It was subsequently noted that all mice exhibiting advanced valvular histopathologic lesions (grades 3-4) had significant valvular and/or ventricular wall motion abnormalities upon ECHO evaluation, providing a strong correlation between advanced histopathologic grade and valvular heart disease.
Mice injected intrasplenically with HT29 colon cancer cells demonstrated liver metastases but did not exhibit valvulopathy, mesenteric fibrosis, diarrhea, or elevation of plasma 5-HT, demonstrating that the metabolic sequelae and valvular abnormalities were specific for BON metastases and not a nonspecific consequence of liver metastases.
Next, we determined whether BON carcinoid liver metastases and subsequent carcinoid syndrome could be attenuated by administration of octreotide, commonly used in the symptomatic management of carcinoid syndrome, or bevacizumab, which is currently being evaluated in clinical trials as adjuvant treatment for carcinoid tumors (29), but has nonetheless demonstrated effectiveness in limiting the growth of other types of tumors, including cancers of the colon, thyroid, lung, and brain (30-33). Following intrasplenic injection of BON cells, mice were randomized to groups of 5 to receive treatment with vehicle (diH2O, 200 μl, ip, once every other day), octreotide (5 mg/kg, ip, once every other day), or bevacizumab (2.5 μg, sc, every day) within 2 h of splenic injection. Following BON or HT29 cell injections, 100% of mice (20/20) developed liver metastases. Mice treated with either octreotide or bevacizumab (n = 5/group) demonstrated significantly less liver metastasis and tumor burden compared with mice treated with vehicle (Figs. 4A, B).
Similarly, whereas all vehicle-treated mice developed significant histopathologic lesions of the heart valves (as described above), octreotide- or bevacizumab-treated mice developed significantly fewer lesions (Fig. 5A). This data correlated with ECHO findings; mice with advanced valvular histopathologic score (3-4) displayed significant cardiac valvular and ventricular wall motion abnormalities as in the previous study. Mesenteric fibrosis and diarrhea were noted in 20% of mice (3/15) (data not shown). Elevation of plasma 5-HT was noted in 100% of vehicle-treated, 80% of octreotide-treated, and 20% of bevacizumab-treated mice (mean = 486.6 ng/ml, range = 7.3 - 1497 ng/ml) (Fig. 5B); elevation of urinary 5-HIAA was noted in 60% of vehicle-treated and none of the octreotide- or bevacizumab-treated mice (mean = 9.2 ng/ml, range = 3.6 - 19.2 ng/ml) (Fig. 5B). Although there was a trend suggesting increased 5-HT and 5-HIAA levels in vehicle-treated mice relative to octreotide- or bevacizumab-treated mice, the range of values was high and sample size was small; a larger sample size is therefore needed to confirm these findings. Taken together, our results demonstrate that vehicle-treated mice developed more liver metastases, elevated plasma 5-HT levels, and had a higher incidence of carcinoid-related sequelae, such as diarrhea, mesenteric fibrosis, and valvulopathy; treatment with either octreotide or bevacizumab significantly inhibited BON liver metastasis and manifestations of the carcinoid syndrome.
Our understanding of carcinoid tumors and the clinical manifestation of the carcinoid syndrome as well as the development of better treatment options has been hampered by the absence of an appropriate in vivo model. Other investigators, including those in our laboratory who have used subcutaneous xenograft placement, have described in vivo models of carcinoid tumor development, but have failed to demonstrate in vivo manifestations of carcinoid syndrome (9, 34-36). In our current study, we describe for the first time a novel in vivo model of carcinoid syndrome which recapitulates many of the clinical sequelae noted in humans. When dispersed BON cells are injected into the spleen of athymic nude mice, the majority of the mice develop multiple multilobar liver metastases which express CgA, 5-HT, and NT, as confirmed by immunohistochemical staining. Additionally, several mice develop sequelae which mimic human carcinoid syndrome, such as flushing, diarrhea, mesenteric fibrosis, and cardiac valvulopathy; these findings occur in mice with significant tumor burden and elevated plasma 5-HT.
The clinical effect of octreotide, and other long-acting somatostatin analogues, in patients with carcinoid syndrome remains controversial. Classically used to inhibit the release and action of multiple hormones and attenuate exocrine secretion, its effects on tumor proliferation remain unclear (7, 37). We have shown that octreotide treatment inhibits the growth of subcutaneous BON carcinoid xenografts placed in the flanks of athymic nude mice (9). Consistent with these findings, our present study demonstrates that the administration of octreotide leads to significantly less metastatic tumor burden within the liver, indicating an antiproliferative effect of octreotide. While carcinoid tumors exhibit differential somatostatin receptor expression based upon site of origin, often leading to a discrepancy in tumoral response to octreotide therapy (7, 11), BON cells are known to highly express the somatostatin receptor, likely contributing to the significant decrease in tumor progression with octreotide treatment.
VEGF is a potent endothelial cell-specific mitogen that promotes endothelial cell growth from pre-existing vasculature (40). While there is clear evidence that treatment with bevacizumab attenuates the development of colon, thyroid, lung, and brain cancer in vivo by inhibiting tumoral blood vessel development (30-33), its use has only recently been suggested in the treatment of carcinoid tumors (29). Elevated expression of VEGF appears to correlate with decreased progression-free survival among patients with neuroendocrine tumors (41). In our current study, we found that treatment with bevacizumab significantly inhibited tumor growth, and tumors which did develop were far smaller in size, consistent with findings described by Zhang et al (41).
Because survival of patients with carcinoid tumors has improved with the availability of supportive medications such as octreotide, the clinical manifestations of fibrosis are now the leading causes of morbidity and mortality associated with this disease (7, 11). Carcinoid-related mesenteric fibrosis may lead to intestinal obstruction, vascular occlusion and intestinal ischemia, or ureteral obstruction with subsequent hydronephrosis and renal failure (42-44). Carcinoid heart disease, characterized by fibrous, plaque-like thickening of the endocardium of the tricuspid and pulmonic valves most commonly, is a serious complication occurring in approximately two thirds of patients with carcinoid syndrome, leading to death in as many as one third of the cases (7, 45). The cardiac manifestations of carcinoid syndrome in humans were essentially reproduced in our model, with extensive fibrosis and thickening involving all cardiac valves, with greatest frequency noted in the tricuspid and mitral valves. Also observed were endocardial degenerative changes of cartilaginous metaplasia, an alteration that is normally noted in aging rats near or in the aortic valve ring but is also described in other forms of endocardial fibrosis (46).
Although the association of carcinoid disease with fibrosis has been well documented, the mechanism is poorly understood. Although classically attributed to the systemic effects of elevated 5-HT, 5-HT does not promote fibroblast secretion or proliferation in vitro, nor do anti-serotonergic agents prevent the development of fibrosis in patients (7, 47, 48). Therefore, focus has recently shifted to growth factors such as transforming growth factor β, connective tissue growth factor, and platelet derived growth factor as potential etiologic agents. The establishment of an in vivo model of carcinoid syndrome is central to the elucidation of factors contributing to this disease process. In the future, we plan to develop knock-out or knock-in BON cell lines to better delineate the precise factors which promote carcinoid-associated fibrosis.
In conclusion, we describe a unique in vivo model of carcinoid syndrome which results in liver metastases, systemic sequelae of increased 5-HT and valvular heart disease. Our findings suggest that treatment with either octreotide or bevacizumab significantly inhibits carcinoid tumor metastasis with bevacizumab the most effective. This model provides an important in vivo model to further delineate novel treatment modalities for carcinoid syndrome and will also be useful to elucidate the factors contributing to the sequelae of carcinoid disease, such as mesenteric fibrosis and valvular heart disease.
The authors would like to thank Karen Martin for manuscript preparation, Tatsuo Uchida for statistical analysis, and Dr. Blaise Carabello for thoughtful suggestions and recommendations.
This work was supported by National Institutes of Health grants R37 AG10885, R01 DK48489, T32DK07639, R01 CA104748 and P01 DK35608
*This paper was presented, in part, at the annual meeting of the American Gastroenterological Association's Digestive Disease Week (May 19-23, 2007, Washington D.C.).