Hepatocellular Neoplasms Induced by Low-Number Pancreatic Islet Transplants in Autoimmune Diabetic BB/Pfd Rats

Research Article

Frank Dombrowski,1 Chantal Mathieu,2 and Matthias Evert1

1Institute of Pathology, Otto-von-Guericke-University Magdeburg, Magdeburg, Germany and 2Laboratories for Experimental Medicine and Endocrinology, Rega Institute, Catholic University Leuven, Leuven, Belgium

Abstract detailed histopathologic studies of the livers of long-term recipients It has been shown that combined high local hyperinsulinism have not yet been conducted (5–8). Thus, little is known about the and hyperglycemia after low-number islet transplantation paracrine effects of insulin on the adjacent hepatocytes, which may into the livers of streptozotocin-diabetic rats lead to the turn out to be of major clinical importance in the long term. development of hepatocellular neoplasms but a substantial On the other hand, diabetes mellitus has been identified as a risk cocarcinogenic effect of genotoxic streptozotocin could not be factor for hepatocellular carcinoma (HCC) in humans in Western ruled out completely. Thus, we herein investigated this model Europe and the United States (9–15). However, the mechanisms in BB/Pfd rats (n = 805; nine experimental groups), which by which diabetes may contribute to the formation of HCCs in develop spontaneous autoimmune diabetes similar to human humans are poorly understood. This led to a controversy about whether diabetes mellitus itself or rather associated diseases, such type 1 diabetes. After low-number islet transplantation (n = 450), the liver acini downstream of the islets show as obesity or hepatitis C, are mainly responsible for tumor insulin-induced alterations: massive glycogen and/or fat induction (11, 16, 17), and the need for experimental studies accumulation, translocation of the insulin receptor, decrease revealing the underlying mechanisms is emphasized (18). in glucose-6-phosphatase activity, increase in expression of A possible explanation for the relationship of diabetes mellitus insulin-like growth factor (IGF)-I, IGF-II/mannose-6- and liver cancer is provided by our previous studies in an animal phosphate receptor, insulin receptor substrate-1, Raf-1, and model of hormonally induced hepatocarcinogenesis in which intra- Mek-1, corresponding to clear cell preneoplastic foci of altered hepatic low number (i.e., 350-450 islets) pancreatic islet transplan- hepatocytes known from chemical hepatocarcinogenesis and tation in streptozotocin-diabetic Lewis rats seemed to be the identical to that in streptozotocin-diabetic Lewis rats. After primary trigger for carcinogenesis (19–26). Sole high number (i.e., 1,000-2,000 islets) transplantation in streptozotocin-diabetic Lewis 6 months, many altered liver acini progressed to other types h of preneoplasias often accompanied by an overexpression of rats, in which the -cells of the grafts are not maximally stimulated the glutathione-S transferase (placental form), IGF-I receptor, to secrete insulin and the resulting local hyperinsulinemia is and transforming growth factor (TGF)-A. After 12 to 15 and relatively slight, does not suffice to induce the carcinogenic process 15 to 18 months, 52% and 100% of the animals showed one (19, 21). Carcinogenesis starts with hepatocellular alterations, which, or multiple hepatocellular adenomas or hepatocellular carci- on the one hand, correspond to known insulin effects and, on nomas (HCCs), respectively. Conclusively, this study identifies the other hand, resemble the so-called clear cell focus (CCF) of combined hyperinsulinism and hyperglycemia as a carcino- preneoplastic hepatocytes, known from many other models of hepa- genic mechanism for the development of HCCs in diabetic tocarcinogenesis (27). These alterations are reflected in an increase rats. Hepatocarcinogenesis is independent from additional in glycogen and lipid storage, in an increase in cell-turnover, (i.e., genotoxic compounds (i.e., streptozotocin), but is primarily high proliferative activity and apoptotic elimination of preneoplastic triggered by increased intracellular insulin signaling via hepatocytes), as well as in characteristic alterations in the activities pathways associated with cell growth and proliferation, such of key enzymes, in particular of the carbohydrate and fatty acid metabolism (19–21, 23, 25). These include an up-regulation of as the Ras-Raf-mitogen-activated protein kinase pathway and the IGF system, and secondarily involves other growth enzymes of glycolysis (hexokinase, glyceraldehyde-3-phosphate factors, such as TGF-A. (Cancer Res 2006; 66(3): 1833-43) dehydrogenase, and pyruvate kinase), de novo lipid synthesis (fatty acid synthase), and the pentose phosphate pathway (glucose-6- phosphate dehydrogenase), whereas key enzymes of gluconeogenesis Introduction (glucose-6-phosphatase), glycogenolysis (glycogen phosphorylase), Recent clinical trials introducing new immunosuppressive regi- and adenylate cyclase activity were down-regulated (20, 23). Insulin mens and improved islet preparation techniques have shown that effects in the CCF also manifested in an overexpression of apoli- transplantation of islets of Langerhans into the liver of type 1 diabetic poprotein A-IV (25) and in an altered expression of proteins of the patients could represent an alternative to exogenous insulin treatment insulin-like growth factor (IGF) pathway in the CCF, including IGF-I and allows for the normalization of metabolic control, which cannot and its binding proteins, such as IGF binding protein (IGFBP)-1 and be achieved by administration of exogenous insulin alone (1–5). IGFBP-4 (22). Moreover, we have recently shown strongly increased Although a few cases have been published in the English literature, insulin signaling in CCF after islet transplantation, reflected in a translocation of the insulin receptor and in an overexpression of several insulin signal transduction proteins of the Ras-Raf-mitogen- Requests for reprints: Frank Dombrowski, Institut fu¨r Pathologie, Universita¨t activated protein kinase (MAPK) pathway, such as insulin receptor Magdeburg, Leipziger Strasse 44, D-39120 Magdeburg, Germany. Phone: 49-391-67- substrate-1 (IRS-1), Raf-1, and Mek-1 (24). In the beginning, the CCF 17869; Fax: 49-391-67-17952; E-mail: [email protected]. I2006 American Association for Cancer Research. were always strictly confined to the liver acini located downstream doi:10.1158/0008-5472.CAN-05-2787 of the islets but gradually expanded into the neighborhood when www.aacrjournals.org 1833 Cancer Res 2006; 66: (3). February 1, 2006

Table 1. Experimental design, preneoplastic foci, and hepatocellular neoplasms

Inbred BB/Pfd rats (n = 805)

Spontaneous autoimmune diabetes (n = 338)

Streptozotocin administration No

Islet transplantation Low-number (n = 450) High-number (n = 1,200) No

Spontaneous reestablishment of self-tolerance Yes No Yes No Not tested

Experimental group MG (n = 148) CG 1 (n = 42) CG 2 (n = 86) CG 3 (n = 22) CG 4 (n = 40)

Blood glucose (mean F SE; mmol/L) 11.6 F 0.7 19.9 F 0.9 4.7 F 0.2 16.2 F 1.1 20.4 F 0.8

Body weight at sacrifice (mean F SE; g) 288 F 5 262 F 9 348 F 9 282 F 15 254 F 6

0-3 +++/CCF ++/CCF 0 0 0 mo 0 0 0 0 0 p.t. (15) (5) (12) (5) (2) Relative number/main type of foci 3-6 +++/CCF ++/CCF 0 0 0 mo 0 0 0 0 0 p.t. (18) (4) (8) (1) (8)

6-9 +++/CCF ++/CCF 0 0 0 mo 0 0 0 0 0 Relative no. animals bearing a p.t. (33) (13) (11) (2) (5) HCA/HCC (%) 9-12 +++/MCF ++/MCF 0 0 0 mo 14/0 0 0 0 0 p.t. (51) (11) (24) (6) (12)

12-15 +++/MCF ++/MCF +/CCF 0 0 mo 52/0 29*/0 0/0 0/0 0/0 No. investigated animals p.t. (25) (7) (22) (6) (8) (in parentheses) 15-18 +++/MCF ++/MCF +/CCF 0 0 c mo 83/50 50/0 11/0 0/0 0/0 p.t. (6) (2) (9) (2) (5)

NOTE: The number of foci was estimated semiquantitatively using three grades: +, single (sporadic) foci in single animals; ++, single number of foci in every animal; +++, numerous foci in every animal. Yellow box, significantly more tumor-bearing animals than in CG 2–5 and CG 7–8. Orange box, significantly more tumor-bearing animals than CG 5, 7, and 8 (others not tested). Abbreviations: MG, main group; CG, control group; HCA, hepatocellular adenoma; HCC, hepatocellular carcinoma; CCF; clear-cell focus; MCF, mixed cell focus, p.t., post-transplantation. *Significantly more HCA-bearing animals than in CG 2–5 and CG 7–8. All data refer to the right liver part and were tested using Fisher’s exact test and accepted if P < 0.05. cSignificantly more HCC-bearing animals than in CG 2.

undergoing neoplastic transformation (19, 21). After 15 to 22 streptozotocin to achieve diabetes. Streptozotocin is genotoxic and months, 86% and 19% of the animals developed at least one carcinogenic in rats (31, 32). Okawa and Doi (33) have reported the hepatocellular adenoma (HCA) or HCC, respectively (21). development of hepatocellular and cholangiocellular tumors in There are additional data obtained in other animal models that streptozotocin-treated Sprague-Dawley rats, although a complete showed that the phenotypes of preneoplasias can mimic responses hepatocarcinogenic potential in rats is not sufficiently proved by this to insulin action (28), and Nehrbass et al. (29, 30) have shown that study in which only 16 animals were investigated. Nevertheless, at IRS-1 overexpression is an early event in chemical hepatocarcino- least a significant cocarcinogenic contribution of streptozotocin has genesis of rats, given p.o. N-nitrosomorpholine. From these studies, to be taken into account. To finally exclude an influence of strep- it can be concluded that increased insulin action or insulinomimetic tozotocin acting as an incomplete hepatocarcinogen in this effects may constitute an interesting carcinogenic mechanism in experimental setting and for reasons of better comparability with the development of HCC in rats and possibly also in humans. the situation in human diabetes mellitus, we thus investigated in this However, a serious drawback in the interpretation of the results long-term study the local influence and the carcinogenic potential from the islet transplantation model was the administration of of insulin on the adjacent hepatocytes in a model of autoimmune

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Table 1. Experimental design, preneoplastic foci, and hepatocellular neoplasms (Cont’d)

Inbred BB/Pfd rats (n = 805)

No spontaneous diabetes (n = 467)

Streptozotocin administration No Diabetes induction by streptozotocin (65 mg/kg body weight)

Islet transplantation No Low number (n = 450) High number (n = 1,200) No

Spontaneous reestablishment of self-tolerance Tolerance never lost Tolerance never lost Tolerance never lost Tolerance never lost

Experimental group CG 5 (n = 165) CG 6 (n = 144) CG 7 (n = 118) CG 8 (n = 40)

Blood glucose (mean F SE; mmol/L) 5.0 F 0.1 10.8 F 0.6 4.2 F 0.1 17.8 F 0.6

Body weight at sacrifice (mean F SE; g) 328 F 7 296 F 6 367 F 7 289 F 8

0-3 0 +++/CCF 0 0 mo 0 0 0 0 p.t. (15) (15) (15) (0) Relative number/main type of foci 3-6 0 +++/CCF 0 0 mo 0 0 0 0 p.t. (10) (15) (15) (5)

6-9 0 +++/CCF 0 0 mo 0 0 0 0 (33) (35) (23) (7) Relative no. animals bearing a p.t. HCA/HCC (%) 9-12 0 +++/MCF 0 0 mo 0 15/0 0 0 p.t. (50) (39) (33) (12)

12-15 0 +++/MCF +/CCF 0 mo 0/0 54/0 0/0 0/0 No. investigated animals p.t. (32) (28) (20) (10) (in parentheses) 15-18 +/CCF +++/MCF +/CCF 0 mo 4/0 75/25 8/0 0/0 p.t. (25) (12) (12) (6)

diabetes, using autoimmune-diabetic BioBreeding (BB) rats. BB rats by a nonfasting blood glucose level higher than 22.2 mmol/L (400 mg/dL). became diabetic by an autoimmunologic disorder similar to human To avoid greater weight loss and to have normoglycemia by the time of type 1 diabetes mellitus (34), which is not completely understood but transplantation, the diabetic rats were treated with s.c. injections of insulin involves mutations of the lyp locus on chromosome 4, lymphopenia, (Insulin Protraphan HM 40, NOVO, Mainz, Germany) as described previously (21), a procedure that was immediately stopped after islet and dysregulation of inflammatory cells, including eosinophils, mast h transplantation. cells, and lymphocytes, which, in turn, lead to insulitis and -cell The spontaneous diabetic animals were finally subdivided into five destruction (35–37). The Pfd substrain of BB rats is characterized experimental groups according to whether the rats reestablished self- by the reestablishment of self-tolerance to the h-cells after previous tolerance (MG and CG 2) or not (CG 1 and CG 3) and according to the destruction of the h-cells in the pancreas (38, 39). Therefore, these modus of transplantation [low number (MG and CG 1), high-number (CG 2 animals tolerate the isologous islet grafts that were transplanted in and CG 3), or no (transplantation CG 4)], resulting in one main group (MG) the course of the experiment without the use of immunosuppression and four control groups (CG 1-4; Table 1). CG 1 and CG 3 were composed of and its possible side effects on the hepatocytes. We also included animals that never developed self-tolerance or lost it again and rejected the streptozotocin-diabetic control groups of the BB strain to compare islet grafts during the experimental course. carcinogenesis between these different types of diabetes induction Rats not having developed autoimmune diabetes were left completely untreated as normoglycemic controls (CG 5) or received a single i.v. injection and with the Lewis rat model. of streptozotocin (65 mg/kg). Two weeks later, they were again subdivided (CG 6-8) according to the respective transplantation procedure (Table 1). All animals were inspected daily. Blood glucose and body weight were Materials and Methods measured monthly and also 1 and 3 days after transplantation, as well as Animals. Eight hundred five animals were investigated in this study and immediately before and 2 days after streptozotocin treatment. Animals housed as described previously (21). Only nondiabetic littermates were showing a weight loss of more than one third of their maximal body weight paired, which also served as islet donors. Spontaneous diabetes was defined posttransplantation were given a s.c. insulin implant (Linplant, Linshin www.aacrjournals.org 1835 Cancer Res 2006; 66: (3). February 1, 2006

Canada, Scarborough, ON, Canada) to prevent death by diabetes. Animal ase placental form (dilution 1:100, no pretreatment; Biogenex, San Ramon, treatment was in line with the guidelines of the Society for Laboratory CA), anti-TGF-a (final concentration 10 Ag/mL; Oncogen Sciences, Cam- Animal Service and the strict German Animal Protection Law. bridge, MA), as well as anti-insulin, antiglucagon, and antisomatostatin Transplantation procedure. Animals were anesthetized (100 mg/kg (dilution 1:200; all from DAKO, Hamburg, Germany). The anti-BrdUrd ketamin and 4 mg/kg xylazin) and islets of Langerhans were isolated from antibody (dilution 1:100; DAKO) was monoclonal. Negative controls without nondiabetic littermates and transplanted into the liver via the portal vein as usage of the primary antibody were done in each run. Further details of the described in detail previously (19). During infusion, the branch supplying staining procedures, including antigen retrieval methods, secondary anti- the left part of the liver (i.e., left lobe and left part of the middle lobe) was bodies, blocking of endogenous peroxidase, counterstaining, and mounting clamped, thus making sure that the transplants were embolized only into of the tissue sections, were described previously (21, 22, 24). the right part of the livers (i.e., right lobe, right part of the middle lobe, caudate lobe, and anterior and posterior papillary processus) and the left part served as an intraindividual control. Ischemia for the left part lasted for Results f1 minute. Rats of the MG, CG 1, and CG 6 received a low number (n =450) Blood glucose and body weight. Spontaneous diabetes of islet grafts; CG 2, CG 3, and CG 7 animals received a high number manifested in f10% of male and in 2% of female 3- to 6-month- (n = 1,200) of islet grafts; and CG 4, CG 5, and CG 8 were not transplanted. old rats. All animals of CG 4 (and also animals of CG 1 and CG 3) V Animal sacrifices and 5-bromo-2 -deoxyuridine application. Animals developed hyperglycemia (27-33 mmol/L), which was so severe that were killed because of severe complications or were matched in time groups insulin depots had to be implanted to prevent death. Thus, blood (Table 1). Rats were anesthetized, the aorta was cannulated, the inferior caval vein was cut, and the vessels were then rinsed for 2 minutes with glucose of CG 4 was lowered to a mean of 20.4 mmol/L but was Ringer’s solution, mixed with 0.5% procain and 4% dextran, followed by still higher than in untreated streptozotocin diabetic rats (CG 8: perfusion fixation using a cocktail of aqua dest containing 4% dextran, 17.8 mmol/L). Animals after low-number transplantation persisted 0.5% glutaraldehyde, and 3% paraformaldehyde. All animals received a in a mild diabetic state as was intended (MG, mean blood glucose single dose of BrdUrd i.p. (50 mg/kg) 1 hour before sacrifice as described 11.6 mmol/L). This was the result of previous autoimmunologically previously (21). induced self-destruction of the h-cells in the pancreas and the Tissue sampling and processing. After fixation, the livers were removed, subsequently transplanted and, owing to the reestablished self- f cut into slices ( 0.5 mm), and examined with a stereomicroscope. All tolerance now tolerated, low number of intrahepatic islet grafts. macroscopic liver lesions and at least 10 additional slices, as well as specimens However, 22% of these animals failed to reestablish long-lasting self- from the heart, lung, kidneys, adrenal glands, small intestine and colon, tolerance and resumed h-cell destruction during the experimental pancreas, spleen, thyroid gland, muscle, and pituitary gland were embedded in paraffin. Serial sections (2-3 Am) of the liver specimens were stained with H&E course, this time in the intrahepatic islet grafts, as revealed by and with the periodic acid-Schiff (PAS) reaction. Additional sections were postmortem liver examination. The mean blood glucose level in made for immunohistochemistry. The other organs were stained with H&E. these animals was 19.9 mmol/L. They had to be excluded from the The morphologic classification of lesions was done as described MG and formed CG 1. As expected, high-number transplantation previously (21). Briefly, preneoplastic foci composed exclusively of glycogen- initially established normoglycemia in all animals but only 80% and fat-storing cells were classified as CCF; a glycogen-depleted, exclusively stayed normoglycemic owing to the reestablishment of self-tole- basophilic cell population as basophilic cell foci; and those with both cell rance in these animals (CG 2, mean blood glucose: 4.7 mmol/L). types as mixed cell foci. HCA extended beyond the original liver acini, were Twenty percent resumed h-cell destruction in the islet grafts, sharply limited, and compressed the surrounding liver parenchyma. Tumors proved by postmortem examination, and became diabetic again being larger than 5 mm in diameter, exhibiting trabeculae thicker than three 1 to 6 months posttransplantation. They had to be excluded from cell layers in at least two separate areas, and showing a higher number of mitotic figures, vascular invasion, or metastases were classified as HCC. CG 2 and formed CG 3 (mean blood glucose: 16.2 mmol/L). Insulinomas were diagnosed if they fulfilled the following criteria: On average, the blood glucose level in the streptozotocin-treated intrahepatic islet graft larger than 2 mm, immunohistochemical positivity animals was 7% to 13% lower when compared with the corres- for insulin, mitotic activity of insulin-positive cells, and severe hypoglycemia ponding autoimmune diabetic groups (Table 1) and they did not [blood glucose lower than 1.11 mmol/L (<20 mg/dL)]. need insulin implants. They also showed mild hyperglycemia after For electron microscopy, appropriate tissue specimens of 2 2 mm size low-number transplantation (CG 6, mean blood glucose: 10.8 were postfixed in OsO4 and embedded in Epon. Semithin sections were mmol/L) and normoglycemia after high-number transplantation stained according to Richardson et al. (40); ultrathin sections were stained (CG 7, mean blood glucose: 4.2 mmol/L). Completely untreated rats with uranyl acetate and lead citrate. The examination was done using a without spontaneous diabetes stayed normoglycemic throughout Phillips (Einthoven, the Netherlands) CM10 electron microscope. the entire experiment (CG 5, mean blood glucose: 5.0 mmol/L). For enzyme histochemistry, the middle lobes of selected livers were removed Single animals of the MG and CG 6 reached normoglycemia after before fixation, cut into slices, and immediately frozen in 80jCcoldiso- pentane. Cryostat sections of 6 Am thickness were cut, stained according to several months owing to hyperplasia of the islet grafts. The three Benner et al. (41) for glucose-6-phospatase activity, and semiquantitavely animals that developed graft insulinomas (see below) were hypo- evaluated by comparison with the adjacent unaltered liver parenchyma. Addi- glycemic; the lowest blood glucose measured was 0.4 mmol/L. tional sections were also stained with the PAS reaction and fat stain (Sudan red). Body weight was inversely correlated with blood glucose; that is, Immunohistochemistry. Paraffin sections were incubated with the it was highest in normoglycemic animals of the CG 2, CG 5, and CG following primary polyclonal rabbit antibodies: anti-insulin receptor (A1314, 8, and lowest in the completely untreated diabetic animals of CG 4 0.5 mg/mL, kindly provided by Dr. J.W. Unger, Department of Anatomy, (Table 1). University of Munich, Munich, Germany), anti-IRS-1 (dilution: 1:50; Upstate Intercurrent diseases and causes of spontaneous deaths. Biotechnology, Inc., New York, NY), anti-Raf-1 (dilution 1:50; Santa Cruz Some rats were affected by other severe diseases that were not Biotechnology, Heidelberg, Germany), anti-Mek-1 (dilution 1:100; Santa Cruz located in the liver and had no effect on hepatocarcinogenesis. Biotechnology), anti-IGF-I (dilution 1:250; DSL, Webster, TX), anti-IGF-I receptor (Santa Cruz Biotechnology), anti-IGF-II/mannose-6-phosphate- When there were no complications of the diabetes, they were receptor [purified from rat liver (42) and kindly provided by Dr. J.G. Scharf, equally distributed between the experimental groups. They Division of Gastroenterology and Endocrinology, Department of Medicine, included malignant lymphomas, pituitary gland adenomas, and, University of Go¨ttingen, Go¨ttingen, Germany], anti-gluthathione S-transfer- most frequently, a transmural eosinophilic inflammation of the

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Figure 1. Stereomicroscopic aspects of unstained liver slices. A to H, stepwise development from altered liver acini downstream of the transplanted islet (arrows)in(A) to HCC (H) in main group livers. Arrowheads, terminal hepatic venules. In (A and B), the borders of the liver acini are respected, but in (C and D) the altered tissue grows beyond. The yellow-white color is the result of excessive glycogen and fat storage in the altered hepatocytes. Mixed cell foci (E) developing later are irregular in color and shape as a result of admixed glycogen-rich and glycogen-poor hepatocytes. HCAs (F and G) show expansive growth. The time course is as follows: A, 3 weeks; B, 3 months; C and D, 7 months; E, 9 months; F, 14 months; G, 15 months; H, 16 months. The increasing size of the lesions is reflected by the increasing length of the lower edge of the panel: 1.6 mm in (A); 1.9 mm in (B); 2.7 mm in (C); 3 mm in (D); 2.3 mm in (E); 4.6 mm in (F); 4.6 mm in (G); and 10.2 mm in (H). I, a liver slice of CG1 at 14 months after islet transplantation. All islets were rejected in this animal; nevertheless, some foci of altered hepatocytes persisted (small white spots) and one HCA has developed. The lower edge represents 7.2 mm.

colon and sometimes also the small intestine, leading to fibrosis, were clamped during infusion of the islets. Focal white lesions stenosis, and consecutive megacolon and massive obstipation as were observed on the liver surface and on the cut surface of reported by Meehan et al. (43). These animals were usually killed in liver slices, identified under a stereomicroscope as yellow-white time to prevent further suffering and spontaneous deaths. liver acini draining the blood from the transplanted islet grafts Unfortunately, owing to this high morbidity, only a small number (Fig. 1A). In the first 3 months, they were always confined to the of animals stayed alive for 15 to 18 months after transplantation anatomic borders of the respective liver acinus and limited by and prolongation of the experiment exceeding 18 months was not the draining hepatic venules at the border to the adjacent acini possible. As reported in previous studies (35, 44), some animals also (Fig. 1B). With increasing time, they expanded around the showed signs of an autoimmune enteropathy (i.e., decreased villous hepatic venules (Fig. 1C), extended into the neighboring liver and enlarged crypt length) as well as an increase in the number of parenchyma (Fig. 1D), and became inhomogenous in color and intraepithelial lymphocytes; however, this was not the focus of our irregular in shape (Fig. 1E). Tumors of several millimeters in size study. We also observed several renal cell carcinomas showing a began to develop after 9 months (Fig. 1F-I). strong predilection for the diabetic groups, indicating diabetes Light and electron microscopy of the liver parenchyma. mellitus to be a risk factor also for renal cell carcinomas. The Light microscopy confirmed that in the first 3 months, the relationship between diabetes mellitus and renal carcinogenesis hepatocellular alterations were strictly limited to the liver acini, will be investigated in detail in a future study. draining the blood from the transplanted islets. The hepatocytes Macroscopy and stereomicroscopy of the livers. With the at this stage were of uniformly clear cell morphology in the H&E exception of very few sporadic CCF in the control livers of late- stain and showed strong purple staining in the PAS reaction, stage animals, all focal liver alterations were observed in the resulting from a high amount of glycogen (Fig. 2A). Electron right part of the livers of the MG, CG 6, and, to a lesser extent, microscopy revealed that glycogen was deposited in a-particles in of CG 1. This was the result of the transplantation procedure, as the cytoplasm (Fig. 2E). In addition, these hepatocytes displayed the branches of the portal vein that supply the left liver part an increase in fat storage in the form of single or multiple lipid www.aacrjournals.org 1837 Cancer Res 2006; 66: (3). February 1, 2006

Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 2006 American Association for Cancer Research. Cancer Research vacuoles within the cytoplasm (Fig. 2B and E). These CCF were as strong as in CCF (Table 2). It is noteworthy that part of the also characterized by an increase in mitotic activity and in preneoplastic foci in the MG and CG 6 persisted, although the apoptotic elimination. In addition, they showed a mean 39-fold respective animals became normoglycemic and the insulin increase in BrdUrd-positive nuclei when compared with the unal- secretion of the respective islet graft probably strongly decreased. tered liver parenchyma (Fig. 2C; Table 2). Approximately 6 months Moreover, we also observed persistence of some preneoplasias in posttransplantation, a subpopulation of altered hepatocytes lost CG 1, although the islet grafts were rejected, became fibrotic, and their glycogen and fat, now displaying a basophilic cytoplasm the h-cells completely vanished (Fig. 1I,3G, and 4I). (Fig. 3C). In addition to the reduced glycogen content, these cells The first hepatocellular neoplasms developed after 9 months. were ultrastructurally characterized by abundant ribosomes and Most HCA and HCC in this study were composed of a mixed rough endoplasmic reticulum. In the beginning, these basophilic population of clear and basophilic cells possibly originating from cells were intermingled with hepatocytes that retained the clear cell mixed cell foci. Some small HCA were purely clear cell. Pure morphology and such lesions were classified as mixed cell foci. basophilic or clear cell carcinomas were rare (Fig. 3D-F). They were However, some of the lesions gradually progressed to pure found to contain remnants of islet grafts in most cases. HCAs were basophilic foci. Mixed cell foci and basophilic cell foci showed an characterized by expansive growth into the neighborhood without increase in mitotic activity and BrdUrd-labeled hepatocyte nuclei cytologic atypia. HCCs were highly differentiated, showed a when compared with the adjacent unaltered liver tissue, albeit not trabecular growth pattern with trabecules thicker than three cell

Figure 2. Histochemical and immunohistochemical alterations of liver acini downstream of transplanted islets and HCCs in the main group. A, strongly positive PAS reaction in the liver acini downstream of the transplanted islet (arrow) owing to increased glycogen storage. Arrowheads, hepatic venules at the border to the neighboring unaltered liver acini. Other altered liver acini show predominant storage of fat droplets (B). Glycogen particles and fat droplets are also visible using electron microscopy (E). Glycogenosis in altered liver acini is due to a strong decrease in the activity of glucose-6 phosphatase (D, islet in the center). Increase in proliferative activity of the altered hepatocytes (and islet cells) is shown by BrdUrd immunostaining in (C): left, islet; middle, altered acinus; right third of the panel, unaltered acinus. The insulin receptor (IR) is translocated from the cell membrane into the cytoplasm of the altered glycogen-storing hepatocytes (hyperinsulinemic acinus in the left part of F) and is increased in HCCs (I). CCF are defined by their clear appearance in H&E stain (G). As a consequence of insulin action, IGF-I is up-regulated (H). Signal transduction proteins IRS-1, Raf-1, and Mek-1 are up-regulated in the altered acini (J, PAS; K, M, and N) and are increased in glycogen-storing HCCs (L). Whereas all these phenomena are present a few days after transplantation, TGF-a is detectable not earlier than 3 months after transplantation, beginning in acinar zone 3, and is stronger in mixed cell foci (O). In addition to TGF-a, the placental form of glutathione S-transferase (GST-P)is also strongly increased in glycogen-poor neoplasms, such as this HCA (P and Q). Time after transplantation and length of the lower edge of the panel: A, 3 weeks, 2.2 mm; B, 3 weeks, 360 Am; C, 3 months, 720 Am; D, 2 months, 2.2 mm; E, 1 month, 10.6 Am; F, 1 month, 140 Am; G and H, 3 months, 3.6 mm; I, 18 months, 560 Am; J and K, 4 months, 2.9 mm; L (same tumor as Fig. 1H), 16 months, 1.1 mm; M and N, 1 month, 1.5 mm; O, 3 months, 900 Am; P and Q, 14 months, 1.5 mm. A, B, and D, frozen sections; E, electron micrograph of an ultrathin section; all others are paraffin sections.

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Table 2. Expression of insulin-related proteins and BrdUrd index in preneoplasias and hepatocellular tumors

CCF MCF BCF cHCA bHCA cHCC bHCC

Insulin receptor Membranous #(=) = (#)= = = = = Intracytoplasmic ""(=) = (#) "" =(#) "" =(#) IRS-1 "" " (=) = (#) "" =(#) "" =(#) Raf-1 "" " (=) = (#) "" =(#) "" =(#) Mek-1 "" " (=) = (#) "" =(#) "" =(#) IGF-I "" " = " = "#(=) IGF-I receptor = = = " (=) " (=) " (=) " (=) IGF-II/M6P receptor "" " " "" " "" " TGF-a = " "" " "" " "" GST-P = " (=) ""(=) "" " "" Glucose-6-phosphatase ## # N.D. N.D. N.D. N.D. N.D. Fatty acid synthase* "" N.D. N.D. N.D. N.D. N.D. N.D. Increase of BrdUrd 39 F 415F 625F 336F 468F 565F 480F 6 c labeling index

NOTE: The intensity of the immunohistochemical variables in focal lesions was estimated semiquantitatively compared with unaltered liver tissue of the same sections using the following grades: ##, strong decrease; #, decrease; = equal; ", increase; "", strong increase. If staining was not uniform in one lesion, the secondary pattern is given in parentheses. Abbreviations: BCF, basophilic cell focus; cHCA/cHCC, clear cell HCA/clear cell HCC; bHCA/bHCC, basophilic HCA/basophilic HCC; GST-P, glutathione S-transferase (placental form); N.D., not determined. *Data obtained from a previous study (see ref. 23). cX-fold increase when compared with the adjacent unaltered liver tissue.

layers in at least two separate areas, and showed mild to moderate insulin effects (i.e., overexpression of the fatty acid synthase and cytologic atypia. down-regulation of the glucose-6-phophatase; Fig. 2D). The In addition to the MG and CG 6, a significant increase in HCAs insulin receptor was translocated from the membrane into the was noted in the low-number-transplantation CG 1 (Table 1). One cytoplasm (Fig. 2F), triggering increased intracellular signaling via single sporadic HCA occurring after 15 to 18 months also devel- the Ras-Raf-MAPK pathway as reflected in the overexpression of oped in CG 2, CG 5, and CG 7. HCCs emerged only in the right part IRS-1, Raf-1, and Mek-1 (Fig. 2J, K, M, and N). Altered expressions of the livers of the MG and CG 6 and did not develop before 15 were also found for proteins of the IGF axis (Fig. 2G and H). months after transplantation. BrdUrd labeling was strongly Clear-cell HCAs and HCCs retained many of these alterations increased when compared with the unaltered parenchyma, ranging but additionally showed a moderate expression of the IGF-I between a 36-fold increase in clear cell HCAs and an 80-fold receptor and TGF-a. Corresponding to the proportion of baso- increase in basophilic HCCs (Table 2). Vascular invasion or philic cells, mixed cell foci, basophilic cell foci, and basophilic metastases were not observed. Between 15 and 18 months, 100% tumors displayed not only a gradual normalization of the of the MG animals (6 of 6) and 83% of CG 6 animals (10 of 12) increased insulin receptor signaling but also a strong overexpres- developed at least one hepatocellular neoplasm. Unfortunately, the sion of TGF-a (Fig. 2O and P). diabetic BB/Pfd rats did not survive longer than 18 months Islet graft morphology. Islet grafts can already be identified posttransplantation as most HCCs had developed between 18 to 24 using a stereomicroscope (Fig. 1A and B). Histologically, they were months in the Lewis rats in previous studies. Some proliferative found within terminal portal venules (Fig. 2A and 3A-D) and cholangiocellular lesions and cholangiomas, but no cholangiocel- consisted of different types of endocrine cells, which were immu- lullar carcinomas, also developed in the draining area of the islets in nohistochemically discriminated using antibodies directed against the MG and were more pronounced in the CG 6. insulin, glucagon, and somatostatin (Fig. 4E and F). They were In summary, the carcinogenic process was restricted to the right richly vascularized and sometimes showed small ductular pro- liver part containing the islet grafts in animal groups that were liferations at the border to the neighboring hepatocytes (Fig. 4A). diabetic and that received a low number of islets (i.e., MG, CG 1, Some islets displayed mild lymphocytic infiltration (Fig. 3A). and CG 6). Tumorigenesis was only weak in animals of CG 1, which Distinct differences were found regarding the ultrastructure of rejected most of their islet grafts (Fig. 4G-I) but was equally strong a- and h-cells in the hyperglycemic animals but not in the and showed no differences in the modus of progression in the MG normoglycemic rats. The stimulated h-cells were enlarged, showed and CG 6. massive hyperplasia of the rough endoplasmic reticulum and the Enzyme histochemistry and immunohistochemistry. Enzyme Golgi complex, and were nearly completely degranulated (Fig. 4C). and immunohistochemical results are given in detail in Table 2. By contrast, the a-cells (and also the y-cells) were atrophic, The results were identical in autoimmune and streptozotocin were reduced in number, and stored many electron-dense secretion diabetic BB/Pfd rats and were in line with previous results in granules (Fig. 4C). At later stages, islets were often observed in the streptozotocin diabetic Lewis rats (20–22, 24). Briefly, CCF were center of small hepatocellular tumors that had originated from a biochemically characterized by alterations reflecting typical downstream preneoplastic focus. In larger HCAs and in HCCs, the www.aacrjournals.org 1839 Cancer Res 2006; 66: (3). February 1, 2006

Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 2006 American Association for Cancer Research. Cancer Research integrity of the islets became disrupted and small clusters or single independent from the previously important hyperglycemia. We islet cells were scattered within the tumor. did not observe a topographic relation between insulinomas and In the few animals of the MG, CG 1, and CG 6, which became hepatocellular tumors. normoglycemic after z10 months posttransplantation, the overall number of islets was reduced but the remaining islets were considerably enlarged and consisted predominantly of large h- Discussion cells (Fig. 4J). In addition, we observed two and one insulinomas In this experiment, we confirmed previous findings made in that had originated 15 to 18 months after transplantation from streptozotocin diabetic Lewis rats (19–25), showing that low- grafts in the MG and CG 6, respectively (Fig. 4K and L). No number transplantation of pancreatic islets into the liver exerts a insulinomas in the pancreas have emerged. The liver tissue strong carcinogenic influence on the downstream hepatocytes in surrounding the insulinomas showed small areas of glycogen streptozotocin diabetic BB/Pfd rats (CG 6-8). This study is the first storage; however, owing to the systemic hypoglycemia in these to show that the same carcinogenic process can be induced in animals, this was not as pronounced and is much more variable spontaneously autoimmune diabetic BB/Pfd rats (MG and CG1), than in the CCF of hyperglycemic animals. Nevertheless, these providing conclusive evidence that hepatocarcinogenesis in this animals had classic preneoplastic foci in other parts of the liver that model is a result of islet-derived factors and is independent from were obviously progressed to a state in which they became streptozotocin.

Figure 3. Histomorphology of altered liver acini, hepatocellular neoplasms, and control livers. A, in CG 2, animals stayed normoglycemic owing to the high number of transplanted islets; thus, no hepatocellular alterations emerged downstream of the islets. At the transplantation site, a few lymphocytes were often visible (top right part of the islet in A), even when the transplants were not rejected. B to F, stepwise development to a HCC in the MG, beginning with a slightly progressed CCF (B), early mixed cell focus with areas of glycogen depletion (C), early clear cell HCA (D), large clear cell HCA (E), and basophilic (glycogen-poor) HCC (F). Arrows (B-D), islet grafts belonging to the respective liver lesion. In CG 1 (G and H), the transplanted islets were rejected after low-number transplantation, but the respective hepatocellular lesions persisted; arrows, mononuclear cell infiltrate in the area of the rejected islet. On serial sections of these animals, no vital islet epithelial cells were present. G, mixed cell focus; H, a very early HCA with nodular expansive growth and mitotic figures (arrowheads). A to H, PAS reaction. Time after transplantation and length of the lower edge of the panel: A, 12 months, 1.1 mm; B, 7 months, 1.8 mm; C, 7 months, 1.5 mm; D, 14 months, 2.2 mm; E, 14 months, 720 Am; F, 18 months, 900 Am; G, 8 months 720 Am; H, 13 months, 720 Am.

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Figure 4. Morphology of transplanted islets (A-F), islet grafts during rejection (G-I), and development of insulinomas (J-L). After high-number islet transplantation and resulting normogylcemia, the transplants remained more compact (CG2 in A; CG7 not shown), whereas after low-number transplantation (MG in B-F) the islet cells intermingled with the liver parenchyma (B). Activated h-cells showed degranulation and hyperplasia of smooth endoplasmic reticulum and Golgi fields in electron microscopy (C). Note the atrophy of the a-cell, packed with electron-dense secret granules (top right part of C). D to F, the same lesion showing an islet graft surrounded by a CCF of altered hepatocytes (E and F, insulin immunostain). In CG 1 (G and I) and CG 3 (H), mononuclear inflammatory infiltrates and rejection of the islets were visible. H, an insulin immunostain showing a few h-cells during rejection. I, a fibrotic scar marks the former place of a rejected islet in the center of a glycogenotic CCF (note strong PAS reactivity in the surrounding hepatocytes); thus, this CCF persists without depending on continuing insulin action. Insulinomas occurred only in MG and CG 6. The h-cells of the transplanted islets in these groups proliferated until the animal became normoglycemic at 10 months or later. Then, single large islets were visible (J). In single animals, these large islets continued to proliferate and secrete insulin, resulting in the formation of insulinomas and severe hypoglycemia (K and L show the same insulinoma). A, B, and G, semithin sections stained according to Richardson; C, electron micrograph of an ultrathin section; D and K, unstained liver slices; E, F, H, and L, insulin immunostain; I and J, PAS stain. Time after transplantation and length of the lower edge of the panel: A, 13 months, 360 Am; B, 9 months, 360 Am; C, 5 months, 9.6 Am; D-F (same lesion), 11 months, D, 3.6 mm, E, 2.9 mm, F, 570 Am; G, 8 months, 360 Am; H, 1 month, 280 Am; I, 13 months, 360 Am; J, 13 months, 1.1 mm; K and L (same lesion), 14 months, K, 14.5 mm, L, 1.1 mm.

The sequence of hepatocellular alterations in the BB/Pfd rats, exception, are typical insulin effects, and we showed increased beginning with the early formation of CCF even a few days after islet intracellular insulin signaling in these cells via IRS-1 and the Ras- transplantation and progressing partly via mixed cell foci and Raf-MAPK pathway. Thus, CCF must be interpreted as adaptive basophilic cell foci to HCAs and HCCs, was qualitatively and alterations resulting from increased insulin action, although minor quantitatively not different from our previous results (19–25). In the additional effects of other islet hormones cannot be ruled out. This beginning, CCF were always confined to the anatomic borders of the is also corroborated by the fact that these alterations in preneo- liver acini that drain the hyperinsulinemic blood from the islet plasias and the subsequent development into hepatocellular tumors grafts. Moreover, all metabolic and morphologic alterations, without only took place in the right part of the liver in which the islet www.aacrjournals.org 1841 Cancer Res 2006; 66: (3). February 1, 2006

Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 2006 American Association for Cancer Research. Cancer Research grafts were transplanted. Similar to streptozotocin diabetic Lewis series of cases that describe focal, mostly steatotic, or glyco- rats, preneoplastic CCF virtually did not develop in animals after genotic alterations in the livers of islet transplant recipients high-number islet transplantation, which fully compensates the that are strikingly similar to our observations (5–7, 49). The diabetic state and establishes normoglycemia, illustrating that macroscopic descriptions and the histopathologic depictions of hyperglycemia is also of relevance for tumor development. However, these alterations, as well as the clinical data (recurrence of hyper- even high-number islet transplantation in conjunction with glycemia, high fasting glucose levels) in these patients, are virtually normoglycemia has at least cocarcinogenic potential, as it strongly identical to the alterations seen in our rats, illustrating obvious promotes hepatocarcinogenesis in Lewis rats, initiated by admin- similarities in the metabolic situation and its influence on the istration of the hepatocarcinogen N-nitrosomorpholine (26), hepatocytes in our model and in a group of clinically transplanted indicating a dose-dependent effect of insulin and glucose levels. patients. Preneoplastic foci did not regress in late-stage animals of the MG or The increased incidence of HCC in human diabetic patients CG 6 that became normoglycemic or even hypoglycemic owing to reported in epidemiologic and case-control studies is not well excessive insulin production by hyperplastic transplants or graft understood. We suggest, although not always being clearly stated, insulinomas, which corroborates similar observations formerly that these patients are suffering from type 2 diabetes mellitus, made in Lewis rats (45). However, a new and interesting finding was which is usually characterized by hyperglycemia and hyper- that CCF also did not regress in animals of CG 1, which showed a insulinemia. On the one hand, the metabolic situation in these rejection of islet grafts after several months of tolerance and local patients is similar to that in the altered liver acini of our model hyperinsulinism (Fig. 1I and 4I). These observations clearly indicate and some researchers have indeed proposed that insulin and that at this time point, the primary adaptive nature of these CCF has glucose may directly be involved in the carcinogenic process in already changed and that neoplastic transformation no longer humans (11, 14). On the other hand, the occurrence of depends on insulin action. In this context, the overexpression of hepatocellular CCF in human livers that resemble preneoplastic other tumorigenic growth factors or their receptors, such as TGF-a, CCF known from a variety of animal models has been shown (50), which has been shown to promote hepatocarcinogenesis in and even indications for their involvement in human hepatocarci- transgenic mice (46, 47), in the late stage lesions and neoplasms nogenesis have been found (51). Therefore, our results may help to of the present model is interesting. The point of transformation of understand how the combination of insulin action with the the purely adaptive alterations of the liver acini into genetically or, diabetic state can alter the expression of growth factors and their probably initially more likely, epigenetically fixated preneoplasias receptors, intracellular signaling, enzyme activities, morphology, that did no longer spontaneously regress must lie between 3 and 12 and proliferative activity of hepatocytes, thus inducing and/or months after transplantation. The clarification of this important promoting hepatocarcinogenesis. In addition, they may also help biological alteration and the underlying mechanisms is one of the to explain the increase in HCC incidence in human type 2 diabetic most interesting aims for future studies. patients and warrant a careful observation of liver alterations in To the best of our knowledge, no detailed histopathologic patients having undergone clinical islet transplantation. studies of human recipient livers in clinical islet transplantation have been done nor has the occurrence of hepatocellular neo- Acknowledgments plasms been reported. However, Hirshberg et al. (48) conducted Received 8/5/2005; revised 10/14/2005; accepted 11/16/2005. a histopathologic study of livers in a nonhuman primate model Grant support: Deutsche Forschungsgemeinschaft (German Research Foundation) and showed glycogenotic CCF, which only developed in one grant Do622/1-5. The costs of publication of this article were defrayed in part by the payment of page single animal that was insufficiently treated by a too low num- charges. This article must therefore be hereby marked advertisement in accordance ber of functioning islet grafts and that stayed hyperglycemic. with 18 U.S.C. Section 1734 solely to indicate this fact. Histologic reports in humans generally deal with the islet graft We thank Gabriele Becker, Jo¨rg Bedorf, Danuta Chrobok, Mariana Dombrowski, Mathilde Hau-Liersch, Regine Landeck, and Claudia Miethke for technical assistance; morphology and do not describe the liver morphology in detail Yvonne Fischer and Kurt Ru¨del for animal care; and Bernd Wu¨sthoff for editing the (8). However, there are a few recent single case reports or small manuscript.

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Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 2006 American Association for Cancer Research. Hepatocellular Neoplasms Induced by Low-Number Pancreatic Islet Transplants in Autoimmune Diabetic BB/Pfd Rats

Frank Dombrowski, Chantal Mathieu and Matthias Evert

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