[CANCER RESEARCH 45,1372-1377, March 1985]

Effect of Cholecystokinin on Human Cholangiocarcinoma Xenografted into Nude Mice1

Chartes Hudd, David M. Euhus, Mane C. LaRegina, David R. Herbold, Diane C. Palmer, and Frank E. Johnson2

Departments of Surgery [C. H., D. M. E, F. E. J.], Comparative Medicine [M. C. L], and Pathology [D. R. H., D. C. P.], St. Louis University Medical Center, St. Louis, Missouri 63104

ABSTRACT manipulation, which currently has a major role in other solid tumors. Gastrointestinal polypeptide hormones regulate growth of var The polypeptide hormone CCK3 has profound metabolic ac ious normal gastrointestinal tissues as well as certain visceral tions, among which are gallbladder contraction, stimulation of cancers. Since Cholecystokinin (CCK) promotes growth of normal enzyme-rich pancreatic exocrine secretion, and growth regula biliary tract, we sought to determine whether CCK affects the tion of normal gastrointestinal tissues (17, 18). In addition to growth and metabolism of human Cholangiocarcinoma line SLU these, trophic effects on gastrointestinal cancers have been 132. Twenty-six nude mice with s.c. xenografts of this cancer reported. Townsend ef a/. (31) demonstrated that caerulein (a received either CCK octapeptide (50 ^g/kg/dose) or 0.9% NaCI CCK analogue) and secretin together stimulate growth of ham solution (saline) twice a day i.p. for 14 days. Tumor volume was ster pancreatic ductal adenocarcinoma line H2T. Lamote ef a/. calculated from Vernier caliper measurements. At sacrifice on (19) reported that acute s.c. administration of CCK increases Day 15, tumors were excised, weighed, and examined histolog- thymidine uptake by normal murine gali bladder mucosa, and ically. DNA, RNA, and protein were measured in the xenografted morphometric studies revealed gallbladder mucosal hyperplasia carcinomas. Because this Cholangiocarcinoma produces carci- after chronic infusional administration of caerulein. This trophic noembryonic antigen (CEA), we obtained serum at sacrifice for action of CCK on biliary mucosa led us to investigate whether a CEA radioimmunoassay and also tumor tissue for CEA immu- similar effect occurs in human carcinoma of biliary origin, which nolabeling with murine anti-CEA monoclonal antibody. Serum might be exploitable in the management of this tumor. CEA levels were 90% higher in the CCK-treated group. Tumor An alternative approach to improving the outcome of biliary tissue in the CCK-treated group also contained more CEA than tract cancer would be early diagnosis. This might increase the did the controls. Mean tumor volume increased significantly in likelihood of effective treatment as currently the disease is often the saline group during the 14-day treatment period, whereas far advanced when detected. We examined the effect of CCK mean tumor volume did not increase significantly in the CCK administration on an aspect of tumor metabolism which might group. Exogenous high-dose CCK thus appears to increase have clinical utility, namely, the production and release of CEA production and release of CEA from SLU-132; it also appears to into the circulation. This well-known tumor marker occurs in retard growth of this tumor line in the nude mouse. normal, preneoplastic, and neoplastic human gallbladder epithe lium (2) and is also elevated in the serum of some patients with Cholangiocarcinoma (23). INTRODUCTION

Carcinoma of the biliary tract remains a challenge to the MATERIALS AND METHODS clinician. Diagnosis is usually late, and survival statistics are Animals. Thirty 6 to 8-week-old male nude mice (athymic nu/nu; unfavorable regardless of treatment (7). Surgical excision is often Harlan-Sprague-Dawley, Indianapolis, IN) were used. Animals were indicated and offers the best chance of tumor eradication. Failing housed in shared cages and kept in a laminar flow cabinet (Laboratory this, relief of jaundice by palliative surgical bypass or prosthesis Products, Madison, Wl). Husbandry was according to the National Re insertion is possible. However, since these patients are fre search Council's Guide for the Care and Use of the Nude (Thymus quently elderly, sick, and ill-prepared for surgery, operative mor Deficient) Mouse in BiomédicalResearch (14), and research procedures tality and complications are common. This has led to a search were according to current guidelines set by the NIH. Free access was for alternative modes of therapy. Although conventional radiation allowed to sterilized diet (Autoclavable Rodent Chow 5010; Ralston therapy has only a modest role in palliation of bile duct cancer, Purina, St. Louis, MO) and sterile water. Mice were identified by ear improved delivery techniques (particularly brachytherapy) are clipping. promising and may have curative potential (12). Chemotherapy Tumor. A specimen of tumor tissue was obtained from a liver metas tasis in a 65-year-old Caucasian male with Cholangiocarcinoma and is often used, but there is no firmly established standard regimen successfully xenografted into nude mice. After the tumor line had under (22). Palliative percutaneous biliary drainage provides relief of gone 3 passages in the nude mouse with stable doubling times and jaundice but is associated with appreciable morbidity and mor retention of its original histological appearances, it was designated SLU- tality (4). New approaches are clearly required if treatment results 132. The line was passaged 7 additional times prior to this study and are going to improve. One such approach might be hormonal continues to exhibit the same features. Since serum levels of CEA were 1This work was supported by NIH Grant CA 33389 from the National Cancer not measured in the patient, we performed CEA immunofluorescence studies on slides from the original specimen and demonstrated consid Institute. 2To whom requests for reprints should be addressed, at Department of Surgery, erable tumor tissue content of this substance. In a preliminary study, we St. Louis University Medical Center, 1325 South Grand Boulevard, St. Louis, MO 63104. 'The abbreviations used are: CCK, Cholecystokinin; CEA, carcinoembryonic Received May 10,1984; accepted December 4,1984. antigen; PBS, phosphate-buffered saline.

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Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1985 American Association for Cancer Research. CCK AND CHOLANGIOCARCINOMA measured CEA and a-fetoprotein levels in serum from mice bearing SLU- next incubated with each tissue section for 30 min. After PBS washes, 132 and found appreciable amounts of CEA but no a-fetoprotein. CEA the sections were reacted for 30 min with fluorescein isothiocyanate- values in non-tumor-bearing mice (0.65 ng/ml) were similar to those labeled F(ab')2 rabbit anti-mouse IgG (Cappel, West Chester, PA), cov- obtained in similar work by Schmitz ef al. (26). Preliminary ¡mmunofluo- erslipped in glycerohPBS, and examined with a Leitz Dialux 20 fluores rescence labeling of xenografted nodules of SLU-132 taken from nude cence microscope. The intensity of CEA immunolabeling was assessed mice also showed appreciable CEA but no a-fetoprotein. This led us to for each tumor and graded from 0 to 4+ by one of us (D. C. P.) who had also study CEA in greater detail. For the purposes of this study, the no knowledge of treatment group. tumor line was expanded by taking nodules of SLU-132 from 10 tumor- Serum CEA. Serum (200 to 400 pi) was obtained from each animal. bearing mice and finely mincing these together in a sterile Retri dish. CEA determinations were performed in the St. Louis University Hospitals Histology was performed on samples from the 10 nodules and confirmed Radioimmunoassay Laboratory by a research biochemist without knowl that all were identical to the original tumor. Thorough mixing was carried edge of treatment group. Serum (150 *tl) was diluted with 300 ¿ilofzero out using a vortex mixer (S8223; American Scientific Products, McGaw standard solution prior to the addition of 0.9 ml of buffer as approved by Park, IL), and 0.15 ml of tumor tissue was injected s.c. into the shoulder the manufacturer. This proved to be a reliable method of dealing with region of each mouse with a 15-gauge 1.25-cm trocar. small volumes of blood. Serum CEA determinations were performed CCK. Synthetic sulfated C-terminal octapeptide (SQ19,844, a gift from using the Abbott CEA radioimmunoassay kit (Abbott, North Chicago, IL). Squibb) was used. In order to ensure sterility, this was filtered through This methodology uses a solid-phase radioimmunoassay based on the Acrodisc micropore filters (0.22-fim pore size; Gelman Sciences, Ann "sandwich" principle. CEA present in serum specimens is bound to solid Arbor, Ml) into 10-ml aliquots which were stored at -70°. An equal beads coated with guinea pig anti-CEA. After appropriate washings, the number of CCK and 0.9% NaCI solution (saline) vials was prepared. beads are incubated with goat anti-CEA labeled with 125I. Unbound These were coded by a person not involved in the experimental manip radiolabeled antibody is removed, and the bound radioactivity is deter ulations; this blinding prevented the observer from identifying treatments mined by counting in a gamma counter. Bound radioactivity is pro when measuring tumors. Prior to each day's injections, fresh sterile portional to the concentration of the CEA in the specimen. Five standards aliquots were thawed at 25°.Animals were given injections i.p. of CCK are used in this analysis: 0; 1.5; 3; 10; and 20 ng/ml. Four different (50 Mg/kg/dose) twice daily or, in the control group, an equal volume of positive CEA controls were analyzed with each test run (one from Abbott; saline. 3 from Hyland Diagnostics, Malvem, PA). Treatment Groups and Tumor Measurement. When most tumor RNA, DNA, and Protein Analysis. Tissue was thawed and homoge nodules measured 0.5 cm along the longest axis, the mice were divided nized (Polytron; Brinkmann Instruments, Westbury, NY). RNA, DNA, and into 2 groups and paired according to tumor size and body weight. Mice protein were precipitated by adding 0.4 N perchloric acid to the homog whose tumors had failed to grow were excluded at this time, leaving a enized tissue. The supernatant was discarded, and the precipitate was total of 26 mice for study. This day was designated Day 0. On Days 0, solubilized by the addition of 0.3 N potassium hydroxide. Samples were 2, 5, 8, 11, and 14, animals were weighed to ±0.1 g on a triple-beam heated at 37°for 90 min, and DNA and protein were again precipitated balance (Dial O Gram; Ohaus Scale Corp., Florham Park, NJ), and tumor by the addition of 10% perchloric acid. After cooling for 45 min at 4°, size was measured in 2 linear dimensions using sliding-jaw Vernier samples were centrifugea at 4000 rpm for 15 min. The RNA in the calipers (PN1350-05; Ohaus Scale Corp.) accurate to 0.1 mm. The supernatant was determined using the orcinol reaction (8). The precipi maximum longitudinal and transverse dimensions were measured. The tate was solubilized by the addition of 10% perchloric acid and heated formula in a water bath at 70°for 20 min. This was then cooled to 4°for 45 min and centrifugea for 15 min at 4000 rpm. DNA in the supernatant was (length) (width)2 assayed using the diphenylamine reaction (6, 13). The protein in the Volume 2 pellets was solubilized in N sodium hydroxide and measured using the biuret reaction (27). was used. This correlates well with volume determined by water displace Statistics. Results are all expressed as mean ± S.E. Significant differences between the 2 groups were evaluated using Student's i test ment (11) and has been shown to be reliable when compared to other formulae (5). Since we have shown that interobserver error is significant for paired data and, in the case of the tumor tissue CEA, the 2-sample when measuring small tumors in the nude mouse (11), one observer binomial test (28). underwent a period of training and performed ail measurements. Pathology. On Day 15, animals were killed in a CO2 chamber, weighed RESULTS on an electronic balance (1500D; Ohaus Scale Corp.), and exsanguinated by cardiac puncture. Blood was allowed to clot for 30 min and centri- Twenty-six animals were included in and completed the study. fuged. Separated serum was stored at -70° prior to estimation of CEA. As a result of pairing, there were no significant differences Tumors were excised, trimmed, and weighed; samples were taken for between groups in initial tumor volume or body weight. Body histology (hematoxylin:eosin), tissue CEA immunofluorescence staining, and RNA, DNA, and protein estimations. In order to ensure that the CCK weight did not change significantly during the course of the used was biologically active, we utilized pancreas growth as a bioassay. experiment (Table 1). Mean pancreas weights at necropsy (ex The pancreas was excised, carefully trimmed of fat with the aid of pressed as a percentage of total body weight) were 1.00 ±0.03 magnifying glasses, and weighed on an electronic balance (AC100; in the saline group and 1.21 ±0.02 in the CCK group, which Mettler Instrument Co., Hightstown, NJ) sensitive to 0.0001 g. Micro represents a 21% increase in the CCK group (p < 0.001) and is scopic examination of tumor nodules, noting grade, presence of necrosis, consistent with trophic effects of CCK on this organ reported and number of mitoses per high-power field, was performed by one of previously. us (D. R. H.) who had no knowledge of treatment group. Tumors grew in both groups as discrete, multilobulated For CEA immunofluorescence staining, tumor tissue sections cut at 5 /i m were heated for 1 hr at 60°, deparaffinized in xylene, and then masses which were encapsulated and showed no gross invasion of surrounding tissues. No métastaseswere seen in any animal. hydrated through decreasing concentrations of ethanol to distilled water. Treatment in 0.10% trypsin (Sigma Chemical Co., St. Louis, MO) for 20 The morphological features of the tumors were consistent in all min at 37°was then followed by washes in PBS (0.13 M NaCI:0.005 M animals. The tumors generally had well-differentiated glandular Na2HPO4:0.0015 M KhfcPO«,pH 7.2). Monoclonal murine anti-human elements with variable amounts of mucin secretion and a fibre- CEA (Boehringer-Manheim, Indianapolis, IN) at 1:5 dilution in PBS was vascular stroma with reactive sclerosis. In foci within the tumor

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Table 1 Nude mice body weights and tumor volumeat beginning (Day 0) and end (Day 14) of experiment There were no significant differences in body weights (p > 0.30). Significant increasein tumor volume occurred in the saline group (p < 0.025) but not in the CCK group (p > 0.30). Body wt (g) Tumor volume (cu cm) GroupCCKSalinep024.4 1424.9 valueNS" 140.25 valueNS ±0.5a ±0.4 + 0.02 ±0.02 24.8 +0.4NSDay 25.4 ±0.4NSp NSDayO0.21 0.19±0.02NSDay0.27 ±0.03NSp <0.025C

valueDay a Mean ±S.E. 6 NS, not significant. c Significant increasein tumor volume occurring in the saline group.

Table2 Tumorconcentration and total tumor content of DNA,RNA,and protein No significant difference exists between groups for any measurement(p > 0.05). Concentration (mg/g tumor) Total tumor content (mg/tumor) DNA RNA Protein DNA RNA Protein 4 + 0.6" CCKSaline11. ±1.4 ±4.2 + 0.26 ±0.44 ±1.6 13.1 ±1.111.7 11.8 + 1.154.3 65.8 ±4.02.17 2.23 ±0.272.40 2.11 ±0.2910.1 11.2±1.6 ' Mean + S.E. masses, the tumor cells had a more aggressive growth pattern mones are also seen on certain cancers. Addition of pentagastrin and invaded the sclerotic stroma as small nests of cells and as to culture medium leads to increased uptake of tritiated thymidine single tumor cells. Vascular invasion was not seen nor was in human gastric carcinoma cell lines (3). Administration of gastrin infiltration noted beyond the sclerotic margins of the tumor mass. s.c. to nude mice, each bearing one of several gastric cancer Mean tumor volume increased from 0.19 to 0.27 cu cm in the lines, enhances growth in certain lines while retarding it in others saline group, representing a 42% increase over 14 days (p < (24). 0.025). In the CCK group, mean tumor volume changed from Townsend ef al. (31) demonstrated that caerulein, a homo 0.21 to 0.25 cu cm, representing a 19% increase over 14 days logue of CCK, when given with secretin, stimulates the growth (not significant; p > 0.30) (Table 1). There were no significant of pancreatic ductal adenocarcinoma line H2T. Given alone, differences between groups in the DNA, RNA, and protein con caerulein or secretin has no effect, suggesting synergism be tent of the tumors (Table 2). tween these hormones. We know of no studies examining the Serum CEA levels were 90% higher in the CCK group (1.9 ± effect of CCK on biliary tract cancer in animals or humans, an 0.3 versus 1.0 ±0.1 ng/ml; p < 0.02). Tissue immunolabeling obvious area of study, as growth of normal biliary tract is for immunoreactive CEA was present in all tumors, both along modulated by CCK (19). Our experiments were designed to the luminal surface of the neoplastic glandular formations and study the effect of CCK on carcinoma of the biliary tract. Since lying freely within glandular spaces. Occasionally, weak cyto- there is no suitable animal model of spontaneous or induced plasmic labeling was also noted, and background fluorescence cholangiocarcinoma, we chose to use a human tumor trans was always faint. Two different patterns of tumor CEA expres planted into the nude mouse. This animal is widely used for the sion emerged from this study which varied significantly (p < 0.04) study of various exogenous agents on tumor xenografts. Since between the CCK-treated and control groups (Chart 1). Tumors this model permits experimentation on human cancers (25), the from the CCK-treated group were generally characterized by practical and ethical dilemmas of work in humans are avoided. large distended glandular spaces filled with intensely staining Clear isolation of variables is possible while retaining the advan (3+) CEA-positive material (Fig. 1, A and B). In contrast, many tages of an in vivo model. Tumor measurement s.c. is easy and, of the tumors from the control group had smaller, less distended though animals are small, blood sampling for tumor marker glandular spaces (Fig. 1C) and showed less intense (1 to 2+) substances can be performed. SLU-132, the human cholangio CEA immunofluorescence (Fig. 1D). carcinoma line which we used in this study, has exhibited stable doubling times during repeated passages in the mouse and has DISCUSSION retained its original histológica! appearances, makes appreciable amounts of CEA, and thus is ideal for our purposes. The CCK Some trophic effects of gastrointestinal hormones on normal we used is commercially available, stable, and easy to administer. digestive tract epithelia are well known. In the Zollinger-Ellison This synthetic material is not contaminated with other gastroin syndrome, hypergastrinemia is associated with hyperplasia of testinal hormones which confounded some prior studies. Since acid-secreting mucosa. Conversely, antrectomy, with its attend its effect on pancreas size is obvious, this makes for a reliable ant fall in gastrin, leads to mucosal atrophy of the remaining index of hormone activity. We have demonstrated a maximum stomach (20). On the other hand, massive small bowel resection trophic effect of CCK on pancreas of the non-tumor-bearing is associated with mucosal hypertrophy and hyperplasia (33). nude mouse at a dosage of 50 ^g/kg/dose twice a day (16). As This is currently believed to be hormonally mediated. Growth- the serum half-life of exogenous CCK is only a few min, we modulating effects of several gastrointestinal polypeptide hor chose this high dose to minimize the chances of overlooking an

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12- cross the blood-brain barrier or that the mouse is relatively 11- resistant to this effect of CCK. Final body weights did not differ between groups, thus suggesting the effects on the tumor were 10- not related to dietary differences. Many provocative tests are used in clinical practice, ranging 9- from exercise electrocardiogram to the glucose tolerance test. 8- Examples of valuable provocative tests in cancer diagnosis would be the calcium and secretin infusion tests for gastrinoma. 7 Provocation of CEA release by secretin and cholecystokinin has been suggested in the diagnosis of pancreatic disease (21), but 6- results were disappointing with pancreatic carcinoma. To our 5- knowledge, this has not been attempted previously in biliary tract cancer. In this study, CCK increased serum CEA levels by 90%. 4- Tumor tissue immunolabeling showed that more CEA was con centrated along the luminal surface and within the spaces of the 3- neoplastic glands in the CCK-treated group. It could be argued 2- that the increase in serum CEA was due to its production by normal murine tissue rather than tumor, but since immunofluo- •* rescence studies revealed more tumor tissue CEA in the CCK group than in the control group, we believe that CCK may have 0 1-2+ 3+ 1-2+ 3+ affected the biology of SLU-132. Naturally, this requires confir mation by in vitro studies in order to exclude the presence of an Salín« CCK intermediary messenger. Perhaps, CCK challenge will be useful Chart 1. Tumor CEA immunolabeling. More intense CEA expression (3+) was as a diagnostic test for high-risk patients, either in seeking early seen in the CCK-treated group (*, p < 0.04). detection or in following for residual disease. We are currently pursuing these possibilities in tissue culture experiments and effect of this agent on the tumor. also in human patients with cholangiocarcinoma. As mentioned previously, some studies have shown that hor We note the apparent paradox of growth retardation concom monal treatment stimulates growth of some cancers while inhib itant with increased CEA levels in tumor and blood. It is interest iting others. A familiar example is breast cancer, where estrogen ing to speculate that treatment with CCK has led to a uniquely therapy can cause either flare or regression. This study suggests altered metabolism in favor of exportable protein (CEA) at the a retardation in growth of SLIM 32 by CCK. Delivery of CCK expense of structural protein (tumor size). The intracellular octapeptide by a different route, schedule, duration, dose, or events which accompany CCK binding are incompletely under vehicle (e.g., slow release) might affect tumor growth kinetics stood in normal tissues and virtually unknown in transformed differently, as might using another molecular species of CCK. It cells. Our preliminary study clearly awaits independent confir may be that CCK affects the tumor by modulating the release of mation. However, if this is a real effect, further studies are other endogenous substances, but the current work obviously indicated to examine why this paradox occurs. cannot answer this question. The best way to determine that a hormone acts directly on its target cell is the in vitro demonstra tion of its effects, although this does not rule out involvement of REFERENCES intracellular second messengers. Clearly, further and independ 1. Adrian, T. E., Pasquali, C., Péseosla,F.. Bacarese-Hamilton, A. J., and Bloom, ent studies along these lines are called for. It may then be S. R. Soya-induced pancreatic hypertrophy and rise of circulating cholecysto possible to determine whether response is uniform among can kinin (abstract). Gut, 23: A889.1982. 2. Albores-Saavedra, J., Nadji, M., Morales, A. R., and Henson, D. E. 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CCK AND CHOLANGIOCARCINOMA

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Charles Hudd, David M. Euhus, Marie C. LaRegina, et al.

Cancer Res 1985;45:1372-1377.

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