The Prostate 39:182–186 (1999) High-Malignancy Orthotopic Nude Mouse Model of Human Prostate Cancer LNCaP Xiaoen Wang, Zili An, Jack Geller, and Robert M. Hoffman* AntiCancer, Inc., San Diego, California BACKGROUND. An animal model of human prostate cancer LNCaP demonstrating high rates of spontaneous metastasis from the orthotopic site after tumor implantation would be very valuable for mechanistic and drug discovery studies. We previously developed microsurgical techniques to implant histologically intact tumor tissues orthotopically in nude mice in order to develop high metastatic mouse models of human cancer. METHODS. Intact tissue of the androgen-dependent human prostate cancer cell line, LNCaP, was implanted on the ventral lateral lobes of the prostate gland by surgical orthotopic implantation (SOI) in a series of 20 nude mice. Mice were autopsied, and histopathological examination of primary tumors and relevant organs was done to identify and quantitate micrometastasis. RESULTS. Eighteen of 20 animals transplanted with LNCaP by SOI had tumor growth. Mean primary tumor weight in the prostate was 9.24 g at time of necropsy. Sixty-one percent of the transplanted animals had lymph node metastasis. Forty-four percent had lung metastasis. Mean survival time was 72 days, indicating a high degree of malignancy of the tumor. CONCLUSIONS. The extensive and widespread lung metastasis as well as lymph node metastasis following orthotopic implantation of LNCaP in nude mice and the short survival time provide a high-malignancy nude model of the LNCaP human prostate tumor. Prostate 39:182–186, 1999. © 1999 Wiley-Liss, Inc. KEY WORDS: LNCaP; orthotopic implantation; lung metastasis; prostate cancer; lymph node metastasis; nude mouse model INTRODUCTION LNCaP is a cell line derived from a supraclavicular lymph node metastasis of human prostate carcinoma. LNCaP exhibits increased proliferation in response to androgens. When LNCaP cells were mixed with the reconstituted basement membrane Matrigel and injected subcutaneously in nude mice, tumors grew in the mice by 12 weeks without observed metastases . The tumorigenicity and incidence of metastasis of LNCaP after orthotopic (intraprostate) or subcutaneous injection in male nude mice were compared by Stephenson et al. . LNCaP cells produced tumors only when orthotopically implanted in the prostate. In a subsequent study by Pettaway et al. , 56% of mice injected orthotopically with a suspension of LNCaP cells formed prostate tumors. Twelve of 43 mice had microscopic metastases in the regional lymph nodes. In another study, orthotopic injection of LNCaP in © 1999 Wiley-Liss, Inc. nude mice resulted in local growth in 7 of 10, and lymph node metastasis in 4 of 10 nude mice . Nude and SCID mice were injected either subcutaneously or orthotopically with LNCaP cell suspensions in a comparative study by Sato et al. . LNCaP tumor incidence after subcutaneous injection was 100% in SCID mice and 80% in nude mice. No lymph node or distant metastases were observed with subcutaneous tumors. After orthotopic injection, 89% of SCID mice and 60% of athymic mice had prostate tumors. Retroperitoneal or mediastinal lymph node metastases were found in 100% of SCID mice, and microscopic pulmonary metastases were identified in 40%. Grant sponsor: U.S. National Cancer Institute; Grant number: R44 CA53963. *Correspondence to: Robert M. Hoffman, AntiCancer, Inc., 7917 Ostrow St., San Diego, CA 92111. E-mail: firstname.lastname@example.org Received 28 August 1998; Accepted 24 December 1998 Orthotopic Nude Mouse Model of LNCaP Tumors The nude mice had retroperitoneal lymph node metastases in 25% of the animals, but no other metastasis was found. 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 [6–8]. It has been shown that tumor cells can enter the draining lymph or blood circulation within 10 min–3 hr of injection, which will eventually form distant artificial metastases [9,10]. Most importantly, a cell suspension lacks the tissue architecture which seems critical for the full expression of the spontaneous metastatic potential of 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 [11–16]. We previously demonstrated orthotopic growth of prostate cancer line PC-3 with subsequent lymph node metastases by using the SOI technique . In a subsequent study, a large series of animals was implanted with PC-3 with improved SOI techniques; a high frequency of lymph nodes and lung metastases was found . In the present study, we applied SOI to LNCaP to develop a high-malignancy nude mouse model. Lymph node and lung metastases occurred frequently, with a mean survival time of 72 days for the host after transplantation. MATERIALS AND METHODS Animals Athymic male 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 (Assurance No. A38731). Mice were fed with an autoclaved laboratory rodent diet (Teklad LM-485, Western Research Products, Orange, CA). Surgical Orthotopic Implantation LNCaP tumor tissue used for surgical orthotopic implantation was derived from stock tumor growing subcutaneously in nude mice maintained in the immunodeficient-rodent facility of AntiCancer, Inc. Tissue from the periphery of the tumor was harvested in 183 log phase, and necrotic tissue was carefully removed under a dissecting microscope to maximize 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 five 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 [7,16]. Evaluation of Tumor Growth and Metastasis All dead mice were immersed in 10% formalin for subsequent autopsy and microscopic examination. Regional and distant lymph nodes, lungs, livers, and other organs where metastasis could occur were routinely embedded, sectioned, and stained with hematoxylin and eosin, using standard techniques for microscopic examination. RESULTS AND DISCUSSION Orthotopic Growth of LNCaP in the Nude Mouse Prostate LNCaP grew extensively after surgical orthotopic implantation (SOI) in the ventral portion of the prostate. Eighteen of 20 implanted mice had orthotopic growth. Tumors in the prostate had a mean weight of 9.2 g (Table I), which disfigured the shape of the lower abdomen (Fig. 1A). Autopsy demonstrated that the orthotopically-growing tumors usually protruded into the abdominal cavity and very frequently invaded the lower abdominal wall. Distended urinary bladders and hydronephrosis due to blocked urethras were also frequently seen. Invasion and Metastasis The seminal vesicles, the bladder, and the lower abdominal wall were often invaded by orthotopic pri- 184 Wang et al. TABLE I. Tumor Growth, Metastases, and Survival of SOI Model of LNCaP Mouse number Survival time (days) Tumor size (mm) Tumor weight (g) Lymph node metastasis Lung metastasis 67 56 44 68 63 66 71 78 120 88 71 137 119 46 48 46 50 54 28.6 × 31.4 24.1 × 32 14.1 × 22.4 25 × 29.9 23.8 × 32.3 29.8 × 31.3 28.7 × 31.3 25 × 29.4 26.3 × 32.3 24.6 × 29.8 21.1 × 31.8 31 × 38.4 27.4 × 31.1 23.9 × 35.7 25.1 × 36.9 25.3 × 31.4 25.6 × 27.1 20 × 23.2 11.72 9.08 2.02 9.68 10.48 9.04 9.57 11.85 7.74 9.35 6.3 14.29 8.89 10.7 11.05 9.47 9.4 5.65 + − − − + − + + + − + − − + + + + + + − − − + − − − − − + − + − + + + + 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 TABLE II. Comparison of Orthotopic Injection of Cell Suspensions and Surgical Orthotopic Implantation of Tumor Fragments of LNCaP in Nude Mice Reference Stephenson et al. a Pettaway et al. b Sato et al.  Rembrink et al.  Present study Orthotopic implantation method Mean size of primary tumor Lymph node metastasis (number of positive animals/total) Lung metastasis (percent of positive animals) Average survival time Cell suspension Cell suspension Cell suspension Cell suspension Fragment 0.23 g 1.2 g ND 0.71 gc 9.24 g 0 12/43 3/12 5/10 11/18 0 0 0 0 44 ND 100 days ND ND 72 days a Study with orchiectomized animals not included. Study with selected metastatic variants not included. c Includes prostate and seminal vesicles + bladder. Normal average weight was 0.35–0.45 g. b mary tumors. Microscopic examination of the tissue sections demonstrated that 8 of 18 animals had lung metastases, and 11 of 18 had periaortic lymph node metastases (Fig. 1, Table I). Microscopic examination of lung specimens showed that the metastases were disseminated in small nests of tumor cells (Fig. 1D). Survival Histopathology Histopathology of the primary tumor showed densely packed, poorly differentiated cells. The prostate gland was almost replaced by tumor cells, and very few glandular structures could be seen (Fig. 1B). Microscopically, lymph node metastases involved almost the whole node (Fig. 1C). The mean survival time of the animals was 72 days (Fig. 2). Our studies with human bladder and stomach cancer demonstrated that orthotopic implantation of histologically intact tumor tissue by the techniques of SOI, when directly compared to cell suspensions, resulted in much greater metastatic expression of the implanted tumor after SOI [18–20]. Orthotopic Nude Mouse Model of LNCaP Tumors 185 Fig. 1. Gross and histologic appearance of LNCaP after orthotopic implantation in the ventral prostate lobes of nude mice as intact tissue fragments. A: Gross photograph of primary tumor of LNCaP 60 days after implantation (arrow). The tumor has protruded into the abdominal cavity. B: Histologic appearance of primary tumor showed a group of anaplastic epithelial cells with pleomorphic nuclei and prominent nucleoli. C: Microscopic appearance of regional lymph node metastases. The metastatic tumor cells (indicated by solid arrow) replaced most of the normal lymph node structure (indicated by open arrow). D: Microscopic appearance of lung metastases. Arrow indicates the metastatic tumor, which is near a vascular structure. 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 compared to orthotopic injection of tumor cell suspensions, which have no three-dimensional architecture. In this study, we report on the high rate of spontaneous lung and lymph node metastasis and short survival time of nude mice after SOI of LNCaP in nude mice. The high incidence of spontaneous metastasis and malignancy of LNCaP after SOI observed in this study demonstrates a high-malignancy model of androgen-dependent human prostate cancer. The model is thus a unique and valuable tool for the study of and for development of new therapeutics for metastatic, hormone-dependent prostate cancer. Pettaway et al.  demonstrated that cells derived from the metastatic lesions of the orthotopically injected parental line of LNCaP became increasingly metastatic after multiple selection cycles of orthotopic injection, isolation of metastatic cells, and orthotopic reinjection in nude mice. Even so, after a number of cycles of selection, LNCaP still did not metastasize to the lung from the orthotopic site. Lung metastasis was 186 Wang et al. 4. 5. 6. 7. Fig. 2. Survival curve of LNCaP model after orthotopic implantation in the ventral prostate lobes of nude mice as intact tissue fragments. The average survival time for this group was 72 days. only achieved by iv injection of the selected metastatic cells. Our techniques of SOI eliminate the 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. 8. 9. 10. 11. 12. 13. CONCLUSIONS This report demonstrates a high malignancy and short survival model of LNCaP after orthotopic implantation in nude mice. The technique of SOI of histologically intact tumor tissue preserves the stromal tissue and overall tumor architecture during the implantation. 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