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Prostate cancer has been modeled in various species, including dogs, mice, and rats. A common method involves the use of transplanted prostate cancer cell lines. The Lobund-Wistar (LW) rat model of prostate cancer allows for the use of models involving either autochthonous prostate cancer or transplantation of a cell line (PAIII), originally derived from a LW rat. The origin of autochthonous prostate tumors in the LW rat is unknown, though suspected to originate from the seminal vesicles. Likewise, the prostatic origin of the PAIII cell line is also uncertain. To determine if the PAIII cell line is derived from the prostate, subcutaneous PAIII tumors from LW rats underwent immunohistochemical labeling for prostate specific antigen (PSA), a prostate-specific serine protease. All 5 PAIII tumors demonstrated labeling for PSA, supporting a prostatic origin for the PAIII cell line.
Le cancer de la prostate a été modélisé chez différentes espèces, incluant les chiens, les souris et les rats. Une méthode usuelle implique l’utilisation de la transplantation de lignées cellulaires du cancer de la prostate. Le modèle du cancer de la prostate du rat Lobund-Wistar (LW) permet l’utilisation de modèles impliquant soit un cancer de la prostate autochtone ou la transplantation d’une lignée cellulaire (PAIII), dérivée à l’origine d’un rat LW. L’origine des tumeurs de la prostate autochtones chez le rat LW est inconnue, mais il est suspecté provenir des vésicules séminales. L’origine prostatique de la lignée cellulaire PAIII est également incertaine. Afin de déterminer si la lignée cellulaire PAIII est dérivée de la prostate, des tumeurs sous-cutanées de PAIII chez des rats LW ont été soumis à un marquage immuno-histochimique pour l’antigène spécifique de la prostate (PSA), une sérine protéase spécifique à la prostate. Les 5 tumeurs ont montré un marquage pour PSA, confirmant l’origine prostatique de la lignée cellulaire PAIII.
(Traduit par Docteur Serge Messier)
Cancer of the prostate gland is the most commonly diagnosed cancer in men and the second most common cancer resulting in death of men, on an age-adjusted basis. In the United States, over 220 000 new cases and 30 000 deaths are recorded annually. In an effort to better understand the pathogenesis of the disease and to develop therapeutic approaches, animal models have proved invaluable. Relatively few species spontaneously develop prostate cancer, with dogs and some strains of rats being exceptions (1,2). Furthermore, several transgenic and knockout mouse models, such as the TRAMP and the Nkx3.1 strains, have been created to manifest various aspects of the disease (3). In addition, xenografts of human prostate cancer cell lines into immunodeficient rodents have also commonly served to model the disease (4).
As with any animal model, those for prostate cancer should optimally demonstrate characteristics similar to those seen in the human disease. In this regard, there are no perfect animal models of prostate cancer; all have fallen short in one way or another. Prostate cancer in the Lobund-Wistar (LW) rat closely follows many aspects of that of humans. Features, such as progression through androgen dependence to androgen independence; metastasis to distant sites (lungs); heritability; and development of the disease in an immuno-competent host, all distinguish the LW rat as an excellent model of prostate cancer (5). Methods have been developed that allow either development of authochthonous prostate tumors or production of subcutaneous tumors using the PAIII prostate cancer cell line in the LW rat. Use of this cell line has allowed for testing of a number of compounds against cancer (6,7).
The PAIII cell line was derived from an autochthonous prostate tumor in a LW rat (8). When transplanted subcutaneously into LW rats, large adenocarcinomas develop within 40 d. The PAIII tumors are hormone-independent and metastasize to the lungs via the lymphatic system.
While autocthonous prostate tumors in the LW rat typically involve the dorsal prostate, tumors are also often found in the seminal vesicles, leading to speculation that the tumor arises not from the prostate but from the seminal vesicles (9), although another study indicated that early neoplastic lesions were confined to the prostate and not the seminal vesicles (10). One line of evidence that discounts the possibility of seminal vesicle origin is the expression of prostate specific antigen (PSA) in autocthonous prostate tumors in LW rats (11). Since PSA is predominantly expressed by prostate epithelium (12), detection of PSA expression by LW tumors involving the prostate and seminal vesicle complex suggests a prostatic origin. In contrast, no studies have similarly established the PAIII cell line as having originated in the prostate gland. The study described here was undertaken to determine if subcutaneous PAIII prostate tumors express detectable PSA, which would support a prostatic origin for this cell line.
Five male LW rats, 3 to 4 mo of age were obtained from the LW rat-breeding colony at the University of Notre Dame. The animals were documented to be serologically free of common rodent pathogens and were maintained in an individually ventilated caging system with hardwood bedding. Free access was provided to drinking water and feed (2016 Rodent Chow; Harlan Teklad, Madison, Wisconsin, USA). Rats were euthanized by carbon dioxide inhalation followed by sectioning of the diaphragm. All animal studies were approved by the University of Notre Dame Institutional Animal Care and Use Committee and were conducted in a facility accredited by the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC), International.
The PAIII cell line was maintained as passaged subcutaneous tumors in LW rats. To acquire tumors for the study, a passaged tumor was harvested immediately following euthanasia of a LW rat. The tumor was then finely minced in 5-mL of modified Eagle’s media (MEM). The tumor tissue suspension was then injected (0.3 mL) subcutaneously into the rear flank of 5 rats. Typical for this model, palpable tumors were present in the rats within 10 d and were allowed to grow for a total of 28 d prior to euthanasia and of the tumors harvest. When harvested, tumor tissue was fixed in 10% neutral buffered formalin for 24 h, and then changed to 70% ethanol. Fixed tissue was then embedded in paraffin prior to sectioning.
Tissue blocks were cut into 3-μm sections and mounted on coated slides. To stop endogenous peroxidase activity, deparaffinized and rehydrated sections were submersed in 0.3% hydrogen peroxide for 20 min at room temperature. Antigen retrieval was performed by autoclaving the slides in a pressure cooker (21°C for 20 min) in Target Retrieval Solution (Dakocytomation, Carpinteria, California, USA). Slides were rinsed 3 times with Tris-buffer and were then loaded into an autostrainer (Artisan Autostainer; Dakocytomation). The autostainer applied pre-diluted primary PSA antibody (rabbit polyclonal antibody, 1 μg/mL; Dakocytomation) to the slides for 20 min at 35°C followed by 4 Tris-buffer rinses. A secondary pre-diluted biotinylated immunoglobulin (Ig) link [consisting of 0.05% horse anti-mouse IgG, 0.05% goat anti-mouse IgM, 0.017% goat anti-rabbit IgG in sodium phosphate buffer (PBS) containing 0.3% carrier protein; Dakocytomation] was applied to the slides for 20 min at 35°C and was followed by 4 Tris-buffer rinses. The slides were then incubated with streptavidin-horseradish peroxidase (Sigma Chemical Company, St. Louis, Missouri, USA) for 20 min at 35°C and followed by 4 Tris-buffer rinses. Diaminobenzidine tetrachloride (DAB) chromogen (0.2% DAB in PBS; Dakocytomation) and DAB substrate (0.02% hydrogen peroxide in PBS; Dakocytomation) were then applied for 20 min at 35°C, followed by 4 wash solution rinses. Afterward, copper enhancer for DAB (5% cupric sulfate in deionized water) was applied for 20 min at room temperature followed by four wash solution rinses. For counterstaining, Mayer’s hematoxylin was applied for 15 min at room temperature, followed by 4 Tris-buffer rinses and 2 wash solution rinses. Normal LW rat prostate and seminal vesicle tissue samples and an appropriate positive control using normal human prostate gland were included.
Microscopically, tumors were poorly differentiated adenocarcinomas with abundant fibrous stroma. In some areas of tumors, there was necrosis and infiltration with polymorphonuclear leukocytes. Labeling for PSA was focally present both in adenocarcinoma epithelial cells and spindle sarcomatous stromal cells in all PAIII tumor samples (Figure 1). Approximately 5% of the viable tumor cells were labeled for PSA in both membrane and cytoplasm. Necrotic tumor debris in the center of the tumor was strongly labeled in a diffuse pattern; however, this likely represented non-specific precipitation of chromogen, which is commonly seen in necrotic debris. Normal LW rat prostate and seminal vesicle tissues did not demonstrate any labeling for PSA (Figure 2).
Prostate specific antigen, a 33-kDa serine protease belonging to the kallikrein family of proteases, has been reported to be generated from epithelial cells lining the ducts of the prostate (13). Because secreted levels of PSA have been reported to correlate directly with prostate size, PSA levels have been used as a biomarker for prostate cancer.
The kallikreins are encoded by a multigene family, which has been extensively documented in both rats (14), and humans (15). In the rat, prostate kallikrein expression is androgen-dependent (16). Interestingly, conserved androgen-responsive elements directing prostate transcription of kallikrein genes have been demonstrated only in humans and dogs, among non-rodent mammals (17), and in the rat (18), all of which develop prostate cancer with higher frequency compared with other species.
There is a great need for animal models that replicate aspects of prostate cancer in man. Among relevant aspects is the elaboration of a biomarker, such as PSA. In dogs with prostate adenocarcinoma, only 2 out of 31 cases were found to demonstrate labeling for PSA (19). Further, xenotransplants of some human prostate cancer cell lines, such as LNCaP, express PSA (20). With respect to PSA, the LW rat is the only immunocompetent small animal model of prostate cancer in which PSA expression has been demonstrated. An approximately 5-fold increase in serum PSA and 15-fold increase in prostate tissue PSA were demonstrated in LW rats with autochthonous prostate cancer compared to controls and these increases were moderated by γ-linoleic acid, a compound shown to suppress the growth of tumor cells (11). The anti-PSA antibody used in the present study is a specific reagent that reacts with normal prostate and primary and metastatic human prostate tumor tissue. Normal and hyperplastic prostate tissues and well-differentiated prostatic carcinomas have shown a high incidence of PSA-immunoreactive cells, while poorly differentiated prostate adenocarcinoma tissues demonstrated much greater variability, with a lower incidence of immunoreactive cells within and between tumors (21). Similarly, the poorly differentiated prostate PAIII adenocarcinomas examined from LW rats in this study demonstrated a relatively low percentage of PSA-immunoreactive cells. It is important to note that normal prostate and seminal vesicle tissues from LW rats were not immunoreactive for PSA. Taken together, these data suggest that PSA-labeling occurs in a small percentage of the poorly differentiated PAIII tumor cells, but not in normal LW rat prostate or seminal vesicle tissues.
In summary, focal immunohistochemical PSA labeling of the PAIII tumors examined in this study supports the idea that the PAIII cell line has a prostatic origin.
The in vivo work was conducted at the University of Notre Dame and the PSA analysis was conducted at the Goshen Center for Cancer Care.