The Prostate 34:169–174 (1998) Surgical Orthotopic Implantation Allows High Lung and Lymph Node Metastatic Expression of Human Prostate Carcinoma Cell Line PC-3 in Nude Mice Zili An,1 Xiaoen Wang,1 Jack Geller,1* A.R. Moossa,2 and Robert M. Hoffman1,2 1 AntiCancer Inc., San Diego, California Department of Surgery, University of California San Diego School of Medicine, San Diego, California 2 BACKGROUND. Prostate cancer is the second leading cause of male death in the United States. When diagnosed, nearly half the cases have metastatic lesions. An animal model of human prostate cancer demonstrating spontaneous metastasis from the orthotopic site after tumor implantation should be of great help for us to understand the disease and to formulate treatment strategy. We report here a high metastatic model of human prostate cancer PC-3. METHODS. We developed microsurgical techniques, termed surgical orthotopic implantation (SOI), to implant histologically intact tumor tissues orthotopically in immunodeficient mice. In this study intact tissue of the human prostate cancer cell line PC-3, harvested from a subcutaneous tumor in a nude mouse, was implanted to the ventral lateral lobes of the prostate gland in a series of nude mice. Mice were sacrificed when found moribund, and autopsy and histology were performed subsequently. RESULTS. A high frequency of lymph node and lung metastasis was noted upon histological examination. The extensive and widespread lung metastasis following orthotopic implantation of PC-3 is, to the best of our knowledge, the first report in the literature. CONCLUSIONS. In contrast to orthotopic injection of cell suspensions, no multiple metastatic cell selection was necessary after SOI for significant expression of the metastatic potential of PC-3. We conclude that the stromal tissue architecture maintained in the implanted tumor played a critical role in tumor growth and progression. Prostate 34:169–174, 1998. © 1998 Wiley-Liss, Inc. KEY WORDS: surgical orthotopic implantation; histologically intact tumor tissue; prostate cancer; PC-3; metastasis; nude mice INTRODUCTION Prostate cancer is the second leading cause of male death in the United States, accounting for about 40,000 deaths per year . A clinical report of over 600 patients suffering from D2 prostate cancer indicated an overall mean survival of 36 months in patients treated with combined androgen blockade . At the time of initial presentation approximately 30–50% of prostate cancer patients have evidence of metastatic disease . The mechanism controlling prostate cancer progression and effective therapeutic strategies for meta© 1998 Wiley-Liss, Inc. static prostate cancer are poorly understood. This is in part due to the lack of a suitable animal model that can mimic the clinical patterns of human prostate cancer growth and metastasis. Nonhuman mammals have very rare incidence of prostate cancer , which limit the possibilities of us- This work was performed at AntiCancer Inc. *Correspondence to: Dr. Jack Geller, AntiCancer Inc., 7917 Ostrow Street, San Diego, CA 92111. E-mail: firstname.lastname@example.org Received 18 October 1996; Accepted 10 February 1997 170 An et al. ing nonhuman mammal model to study human prostate cancer. With the first introduction of immunodeficient rodents in cancer research in the late 1960s, xenografted human cancer models are now widely used. The inability to reject many of the xenografts implanted in these animals due to defect(s) in their immune system make them the only known tool for studying in vivo human cancer growth and metastasis outside the human body . Studies in a number of laboratories have shown that orthotopic injection of human tumor cells in immunodeficient mice can produce relevant metastatic patterns in comparison to ectopic transplantation [5– 11]. Injection of tumor cells into the prostate gland of nude mice, nevertheless, requires delicate, hard-tocontrol technical conditions and spillage outside the prostate may generate rather variable results. Furthermore, some studies showed that artificial dissemination rather than spontaneous metastasis might occur following tumor cell injection, even when cells were not directly injected into the vasculature [12–14]. It has been shown that tumor cells can enter the draining lymph or blood circulation within 10 minutes to 3 hours of injection, which will eventually form distant artificial metastases [15,16]. Most importantly, a cell suspension lacks the tissue architecture which seems critical for the full expression of the spontaneous metastatic potential of the transplanted human tumors. Recently we introduced a new transplantation technique of surgical orthotopic implantation (SOI) of histologically intact tumor tissue for developing metastatic models of human cancer in immunodeficient rodents [17–21]. We previously demonstrated orthotopic growth of prostate cancer line PC-3 with subsequent lymph node metastases by using SOI technique . In the present study, a large series of animals was implanted with PC-3 by improved SOI techniques. The patterns of growth and high frequency of metastasis are reported here. The advantages of this model for the study of prostate cancer and the possible mechanisms that underlie them are also discussed. MATERIALS AND METHODS Animals Twenty male athymic nu/nu CD-1 mice (Charles River Laboratories, Wilmington, MA), 4–5 weeks old, were used in the study. They were maintained in a specific pathogen-free environment in compliance with USPHS guidelines governing the care and maintenance of experimental animals. Mice were fed with autoclaved laboratory rodent diet (Teklad LM-485, Western Research Products, Orange, CA). Surgical Orthotopic Implantation PC-3 cells were obtained initially from the American Tissue Type Culture Collection (Rockville, MD). Tumor tissue used for surgical orthotopic implantation was derived from a tumor growing subcutaneously after injection of PC-3 cells in a nude mouse. Tissue from the periphery of the tumor was harvested in log phase and necrotic tissue was carefully removed under a dissecting microscope to minimize the amount of viable tissue for implantation. The viable tissue was then cut into small cubes of 1 mm3 in standard tissue culture medium under sterile conditions. To minimize variation in subsequent tumor growth and metastasis these tumor pieces were randomly mixed and an equal amount of 5 pieces was implanted in each mouse as described below. Mice were anesthetized by isoflurane (Ohmeda Caribe Inc., Guayama, PR) and positioned supinely. An opening was made right above the pubis symphysis to expose the prostate gland. The fascia surrounding the ventral portion of the prostate was carefully isolated and the two ventral lateral lobes of the gland were separated by a small incision using a pair of fine surgical scissors. Five of the above tissue pieces were sutured into the incision using an 8-0 nylon suture. The two parts of the separated lobes were then sutured together with the tumor pieces wrapped within. The surrounding fascia was then used to wrap this portion of the gland to consolidate the incision. The abdomen was closed using a 6-0 suture. Evaluation of Tumor Growth and Metastasis Mice were euthanized if found moribund during the observation period. All mice were humanely sacrificed using CO2 inhalation three months after tumor implantation and then immersed in 10% formalin for subsequent autopsy and microscopic examination. Regional and distant lymph nodes, the lung, the liver as well as other organs suspected of metastasis were routinely embedded, sectioned, and stained with hematoxylin and eosin using standard techniques for microscopic examination. The skeletal system was carefully examined grossly under a dissecting microscope (7×) with the removal of the soft tissue for possible bone metastasis. RESULTS Orthotopic Growth of PC-3 in the Nude Mouse Prostate The well established human prostate carcinoma line PC-3 grew extensively after surgical orthotopic Metastasis of PC-3 After Surgical Orthotopic Implantation implantation (SOI) in the ventral portion of the prostate. The take rate was 95% with only 1 of 20 mice having no orthotopic growth upon autopsy. Within the 3-month period following implantation, all tumors in the prostate reached more than 2 cm in diameter and usually disfigured the shape of the lower abdomen. The survival time of the animals ranged from 9 to 12 weeks. At time of euthanization, severe signs of cachexia could be observed in all the mice. Autopsy demonstrated that the orthotopically growing tumor usually protruded into the abdominal cavity and very frequently invaded the lower abdominal wall. Distended urinary bladder and hydronephrosis due to blocked urethra were also frequently seen (Fig. 1A,B). Invasion and Metastasis The seminal vesicles, the bladder and the lower abdominal wall were often invaded by the orthotopic primary tumors (Fig. 1-A). Microscopic examination of the tissue sections demonstrated that 5 of 19 animals had lung metastases and 13 of 19 had periaortic lymph node metastases (Table I). Gross examination of the skeletal system did not demonstrate bone metastasis and subsequent histology study was not performed. Histopathology Histopathology of the primary tumor showed sheets of densely packed, anaplastic epithelial cells with abundant, foamy, eosinophilic cytoplasm. The nuclei were pleomorphic and had varying amounts of unevenly dispersed chromatin. Many abnormal mitoses could be seen. The prostate gland was almost replaced by tumor cells and very few glandular structures could be seen (Fig. 1-C). Microscopically, lymph node metastases were characterized by widespread infiltration of tumor cells in the subcapsular, the cortical and medullary area. In many of the lymph nodes analyzed, tumor cells occupied the whole node and lymphatic cells could barely be seen (Fig. 1-D). Microscopic examination of lung specimens showed that the metastases were disseminated. Small nests of tumor cells could be spotted in almost every high power field. When large tumor nests were seen, they often resided around the airway structures (Fig. 1-E). DISCUSSION Surgical orthotopic implantation of histologically intact tumor tissue into nude mice preserves the supportive stromal tissue and therefore maintains during 171 implantation, the overall tissue architecture, which refers to the three dimensional histological structure of the tumor blocks. Previous studies showed that the proliferation of tumor cells implanted in nude mice was preceded by the penetration of host stromal cells into the tumor [23,24]. Based on this important role of stroma in tumor growth, some investigators coinjected cultured fibroblasts with tumor cells and observed significant, growth-stimulating effects of the fibroblasts [25–27]. Also, many studies demonstrated enhanced growth of tumor cells following co-injection of Matrigel [28–31], an extract of basement membrane components, which primarily consists of laminin, collagen IV and heparan sulfate proteoglycan . Current concepts suggest that Matrigel serves as a supportive matrix for the tumor cells. It collects tumor cells, allows paracrine factors to take effect, activates the release of angiogenic factors and stimulates the production of proteases and motility of tumor cells, thus facilitating growth and progression [33,34]. Our studies with human bladder and stomach cancer, on the other hand, demonstrated that orthotopic implantation of histologically intact tumor tissue by the techniques of SOI, rather than cell suspensions, resulted in much greater metastatic expression of the implanted tumor in a head-to-head comparison [35– 37]. The histologically intact tumor tissue used in SOI possesses a large amount of supportive stromal cells, which are essential for maintaining the threedimensional tumor architecture. The tissue architecture is believed to contribute to the difference in metastatic expression resulting from SOI and orthotopic injection of tumor cell suspensions, which have no three-dimensional architecture. Stephenson et al.  reported lymph node metastasis but no lung metastasis of PC-3 after orthotopic injection of cell suspension in nude mice. Since as proven by other previous studies that the injected tumor cells may enter the lymphatic stream very shortly after the injection [12–16], there even exists a question of whether the lymph node metastasis is an artificial dissemination. Shevrin et al.  demonstrated lung metastasis of PC-3 cells, but this was achieved by injecting tumor cells directly into the tail vein of the nude mice, in which several steps of the metastatic process that naturally occur in patients were bypassed [40,41,42]. More recently, Waters et al.  reported rather high lung metastasis of PC-3 cells after urinary bladder wall injection of cell suspensions. However, they concluded, on the other hand, that lung metastasis of PC-3 from orthotopic tumor cell injection was infrequent (1 of 10 mice). In this study, we report, for the first time, the high 172 An et al. Fig. 1. A: Gross picture of primary tumor of PC-3 growing in the prostate gland of a nude mouse after orthotopic implantation (SOI) (solid arrow). The hollow arrow indicates the distended bladder due to urethra obstruction from tumor growth. Note that the bladder wall was invaded by the primary tumor. B: Solid arrow shows the hydronephrosis from urinary tract obstruction. Hollow arrows indicate enlarged periaortic lymph nodes due to metastasis (The primary tumor was removed at autopsy). C: Histopathology of primary tumor of PC-3 after SOI. a: Tumor cells; b: the capsule of the prostate gland. Arrows indicate the compressed glandular acini of the prostate gland. D: Lymph node metastasis of PC-3 after SOI. a: Residual cluster of lymphocytes; b: tumor cells. Arrow indicates the capsule of the lymph node. E: Lung metastasis of PC-3 after SOI. a: Metastatic tumor nests; b: airway structure. rate of spontaneous lung and lymph node metastasis of human prostate cancer line PC-3 from the orthotopic site. Although it is possible that genetic drift and differences in individual laboratory technique may influence the comparative result between cell suspensions and intact-tissue transplantation, such a high incidence of spontaneous metastasis from the orthotopic site after SOI observed in this study still brings this model of human prostate cancer closer to the clinical profile. The model is thus a unique and valuable tool for the study of and for development of new thera- peutics for metastatic, hormone-independent prostate cancer. Future development will include a bonemetastasis model. In a very recent report, Pettaway et al.  demonstrated that cells derived from the metastatic lesions of the orthotopically injected parental PC-3, as well as the androgen-dependent cell line LNCaP, became increasingly metastatic after multiple selection cycles of orthotopic injection, isolation of metastatic cells and orthotopic reinjection. Even so, after a number of cycles of selection PC-3 still did not metastasize to the Metastasis of PC-3 After Surgical Orthotopic Implantation TABLE I. Metastatic Sites of Human Prostate Cancer Cell Line PC-3 After Surgical Orthotopic Implantation* No. of mice autopsied 19 Incidence and sites of metastasis Lymph node† Lung† 13/19 5/19 *PC-3 tumor tissue fragments derived from a subcutaneous tumor in nude mice, which grew after cell suspension injection were implanted in the ventral lateral lobes of the prostate of nude mice by surgical orthotopic implantation (SOI) (see text for details). Lymph nodes and the lungs, collected during autopsy, were routinely fixed, embedded, sectioned, and stained by using standard procedures of H&E staining for microscopic examination. † Number of positive mice/number of mice explored. lung from the orthotopic site. Lung metastasis was eventually achieved by iv injection of the selected metastatic cells. Our techniques of SOI should eliminate such a need to select highly metastatic cells through complex procedures from a parental tumor for the purpose of establishing metastatic animal models of human cancer. Moreover, the SOI metastatic model more closely mimics the clinical pattern, thus providing a better tool to study the biology of cancer metastasis and for evaluating novel cancer therapeutics. CONCLUSIONS To our knowledge, this is the first report demonstrating high lung metastasis of PC-3 after orthotopic implantation. 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