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• W1515728685 abstract "We developed a tissue slice graft (TSG) model by implanting thin, precision-cut tissue slices derived from fresh primary prostatic adenocarcinomas under the renal capsule of immunodeficient mice. This new in vivo model not only allows analysis of approximately all of the cell types present in prostate cancer within an intact tissue microenvironment, but also provides a more accurate assessment of the effects of interventions when tissues from the same specimen with similar cell composition and histology are used as control and experimental samples. The thinness of the slices ensures that sufficient samples can be obtained for large experiments as well as permits optimal exchange of nutrients, oxygen, and drugs between the grafted tissue and the host. Both benign and cancer tissues displayed characteristic histology and expression of cell-type specific markers for up to 3 months. Moreover, androgen-regulated protein expression diminished in TSGs after androgen ablation of the host and was restored after androgen repletion. Finally, many normal secretory epithelial cells and cancer cells in TSGs remained viable 2 months after androgen ablation, consistent with similar observations in postprostatectomy specimens following neoadjuvant androgen ablation. Among these were putative Nkx3.1+ stem cells. Our novel TSG model has the appropriate characteristics to serve as a useful tool to model all stages of disease, including normal tissue, premalignant lesions, well-differentiated cancer, and poorly differentiated cancer. We developed a tissue slice graft (TSG) model by implanting thin, precision-cut tissue slices derived from fresh primary prostatic adenocarcinomas under the renal capsule of immunodeficient mice. This new in vivo model not only allows analysis of approximately all of the cell types present in prostate cancer within an intact tissue microenvironment, but also provides a more accurate assessment of the effects of interventions when tissues from the same specimen with similar cell composition and histology are used as control and experimental samples. The thinness of the slices ensures that sufficient samples can be obtained for large experiments as well as permits optimal exchange of nutrients, oxygen, and drugs between the grafted tissue and the host. Both benign and cancer tissues displayed characteristic histology and expression of cell-type specific markers for up to 3 months. Moreover, androgen-regulated protein expression diminished in TSGs after androgen ablation of the host and was restored after androgen repletion. Finally, many normal secretory epithelial cells and cancer cells in TSGs remained viable 2 months after androgen ablation, consistent with similar observations in postprostatectomy specimens following neoadjuvant androgen ablation. Among these were putative Nkx3.1+ stem cells. Our novel TSG model has the appropriate characteristics to serve as a useful tool to model all stages of disease, including normal tissue, premalignant lesions, well-differentiated cancer, and poorly differentiated cancer. Over the years, significant efforts have been made to develop realistic model systems to investigate the biology of benign and malignant prostatic epithelial cells. These models are vital in prostate research and have enabled important discoveries. However, there are intrinsic limitations in these experimental models that limit their use.1Pienta KJ Abate-Shen C Agus DB Attar RM Chung LW Greenberg NM Hahn WC Isaacs JT Navone NM Peehl DM Simons JW Solit DB Soule HR VanDyke TA Weber MJ Wu L Vessella RL The current state of preclinical prostate cancer animal models.Prostate. 2008; 68: 629-639Crossref PubMed Scopus (105) Google Scholar For example, most of the immortal cell lines maintain little of the secretory differentiation that is characteristic of normal and cancerous epithelial cells of the prostate. Many do not express androgen receptor (AR) or prostate specific antigen (PSA), or express a mutated AR.2Sobel RE Sadar MD Cell lines used in prostate cancer research: a compendium of old and new lines, part 2.J Urol. 2005; 173: 360-372Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar, 3Sobel RE Sadar MD Cell lines used in prostate cancer research: a compendium of old and new lines, part 1.J Urol. 2005; 173: 342-359Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar Similarly, primary cultures derived from normal or cancer tissues typically lose functional AR and androgen-sensitive growth and gene expression.4Peehl DM Primary cell cultures as models of prostate cancer development.Endocr Relat Cancer. 2005; 12: 19-47Crossref PubMed Scopus (135) Google Scholar Their limited life span also makes it difficult to generate sufficient cells for long-term studies.4Peehl DM Primary cell cultures as models of prostate cancer development.Endocr Relat Cancer. 2005; 12: 19-47Crossref PubMed Scopus (135) Google Scholar Moreover, the interactions between the epithelial, stromal, and vascular compartments of the prostate, which have been demonstrated to be essential for normal prostate development and function as well as in the development and progression of prostate cancer (PCa), are also missing in these in vitro model systems.1Pienta KJ Abate-Shen C Agus DB Attar RM Chung LW Greenberg NM Hahn WC Isaacs JT Navone NM Peehl DM Simons JW Solit DB Soule HR VanDyke TA Weber MJ Wu L Vessella RL The current state of preclinical prostate cancer animal models.Prostate. 2008; 68: 629-639Crossref PubMed Scopus (105) Google Scholar Commonly used in vivo models of human PCa such as xenografts generated from cell lines suffer similar drawbacks of lack of expression of wild-type AR and/or the complex biochemical and physical interactions between the various cellular, tissue, and hormonal compartments that characterize human PCa. Animal models of PCa such as transgenic and knock-out mouse models maintain an intact prostate architecture; however, these systems do not model the progression of human PCa because the single molecule transgenic and knock-out mice either rarely develop a pathology beyond hyperplasia or prostatic intraepithelial neoplasia or rapidly progress to poorly differentiated cancer.5Lamb DJ Zhang L Challenges in prostate cancer research: animal models for nutritional studies of chemoprevention and disease progression.J Nutr. 2005; 135: 3009S-3015SPubMed Google Scholar In addition, differences in the anatomy and physiology between the rodent and human prostate also make it difficult to generalize conclusions obtained by using these models.6Shappell SB Thomas GV Roberts RL Herbert R Ittmann MM Rubin MA Humphrey PA Sundberg JP Rozengurt N Barrios R Ward JM Cardiff RD Prostate pathology of genetically engineered mice: definitions and classification. The consensus report from the Bar Harbor meeting of the Mouse Models of Human Cancer Consortium Prostate Pathology Committee.Cancer Res. 2004; 64: 2270-2305Crossref PubMed Scopus (484) Google Scholar Xenografts derived from direct implantation of small pieces of tumors freshly taken from patients into mice, so-called “tumorgrafts,” are thought to be the most realistic experimental models of human cancer because they recapitulate the parent tumors microscopically as well as molecularly.7Garber K From human to mouse and back: “tumorgraft” models surge in popularity.J Natl Cancer Inst. 2009; 101: 6-8Crossref PubMed Scopus (115) Google Scholar In several malignancies, tumorgrafts have gained popularity recently because they are very predictive of drug response.8Garber K Personal mouse colonies give hope for pancreatic cancer patients.J Natl Cancer Inst. 2007; 99: 105-107Crossref PubMed Scopus (21) Google Scholar The ability to generate prostate tumorgrafts has been demonstrated by several groups.9Presnell SC Werdin ES Maygarden S Mohler JL Smith GJ Establishment of short-term primary human prostate xenografts for the study of prostate biology and cancer.Am J Pathol. 2001; 159: 855-860Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar, 10Wang Y Revelo MP Sudilovsky D Cao M Chen WG Goetz L Xue H Sadar M Shappell SB Cunha GR Hayward SW Development and characterization of efficient xenograft models for benign and malignant human prostate tissue.Prostate. 2005; 64: 149-159Crossref PubMed Scopus (147) Google Scholar, 11Gray DR Huss WJ Yau JM Durham LE Werdin ES Funkhouser Jr, WK Smith GJ Short-term human prostate primary xenografts: an in vivo model of human prostate cancer vasculature and angiogenesis.Cancer Res. 2004; 64: 1712-1721Crossref PubMed Scopus (45) Google Scholar, 12Huss WJ Gray DR Werdin ES Funkhouser Jr, WK Smith GJ Evidence of pluripotent human prostate stem cells in a human prostate primary xenograft model.Prostate. 2004; 60: 77-90Crossref PubMed Scopus (41) Google Scholar, 13Staack A Kassis AP Olshen A Wang Y Wu D Carroll PR Grossfeld GD Cunha GR Hayward SW Quantitation of apoptotic activity following castration in human prostatic tissue in vivo.Prostate. 2003; 54: 212-219Crossref PubMed Scopus (44) Google Scholar The take rate of prostate tumorgrafts under the renal capsule of immunodeficient mice is >90% using a recently optimized protocol.10Wang Y Revelo MP Sudilovsky D Cao M Chen WG Goetz L Xue H Sadar M Shappell SB Cunha GR Hayward SW Development and characterization of efficient xenograft models for benign and malignant human prostate tissue.Prostate. 2005; 64: 149-159Crossref PubMed Scopus (147) Google Scholar This model has been used to compare angiogenesis in PCa versus benign prostate and to quantify apoptotic activity after castration in human prostate tissue.11Gray DR Huss WJ Yau JM Durham LE Werdin ES Funkhouser Jr, WK Smith GJ Short-term human prostate primary xenografts: an in vivo model of human prostate cancer vasculature and angiogenesis.Cancer Res. 2004; 64: 1712-1721Crossref PubMed Scopus (45) Google Scholar, 13Staack A Kassis AP Olshen A Wang Y Wu D Carroll PR Grossfeld GD Cunha GR Hayward SW Quantitation of apoptotic activity following castration in human prostatic tissue in vivo.Prostate. 2003; 54: 212-219Crossref PubMed Scopus (44) Google Scholar Androgen signaling plays a key role not only in the growth and function of normal prostate, but also in the development and progression of PCa.14Richter E Srivastava S Dobi A Androgen receptor and prostate cancer.Prostate Cancer Prostatic Dis. 2007; 10: 114-118Crossref PubMed Scopus (58) Google Scholar Androgen deprivation therapy is a common treatment for men with advanced PCa.15Sharifi N Gulley JL Dahut WL Androgen deprivation therapy for prostate cancer.JAMA. 2005; 294: 238-244Crossref PubMed Scopus (794) Google Scholar Prostate tissue grafts have been used to determine the response of benign and cancerous tissues to androgen deprivation.12Huss WJ Gray DR Werdin ES Funkhouser Jr, WK Smith GJ Evidence of pluripotent human prostate stem cells in a human prostate primary xenograft model.Prostate. 2004; 60: 77-90Crossref PubMed Scopus (41) Google Scholar, 13Staack A Kassis AP Olshen A Wang Y Wu D Carroll PR Grossfeld GD Cunha GR Hayward SW Quantitation of apoptotic activity following castration in human prostatic tissue in vivo.Prostate. 2003; 54: 212-219Crossref PubMed Scopus (44) Google Scholar It was reported that benign glandular structures in postcastration grafts were populated by basal, secretory, and squamous cells, whereas cancer glands in the grafts resembled the original cancer tissue.12Huss WJ Gray DR Werdin ES Funkhouser Jr, WK Smith GJ Evidence of pluripotent human prostate stem cells in a human prostate primary xenograft model.Prostate. 2004; 60: 77-90Crossref PubMed Scopus (41) Google Scholar However, it is not clear whether androgen signaling is decreased in castrated grafts and whether the pathway activity can resume after androgen restoration. We have modified the “tumorgraft” model by the use of thin, precision-cut tissue slices. This protocol extends previous applications of such tissue slices in vitro.16Kiviharju-af Hallstrom TM Jaamaa S Monkkonen M Peltonen K Andersson LC Medema RH Peehl DM Laiho M Human prostate epithelium lacks Wee1A-mediated DNA damage-induced checkpoint enforcement.Proc Natl Acad Sci USA. 2007; 104: 7211-7216Crossref PubMed Scopus (60) Google Scholar, 17Blauer M Tammela TL Ylikomi T A novel tissue-slice culture model for non-malignant human prostate.Cell Tissue Res. 2008; 332: 489-498Crossref PubMed Scopus (16) Google Scholar, 18Parrish AR Gandolfi AJ Brendel K Precision-cut tissue slices: applications in pharmacology and toxicology.Life Sci. 1995; 57: 1887-1901Crossref PubMed Scopus (221) Google Scholar This new in vivo model not only allows analysis of approximately all of the cell types present in PCa within an intact tissue microenvironment, but also provides a more accurate assessment of the effects of interventions when tissues from the same specimen with similar cell composition and histology are used as control and experimental samples. The thinness of the slices ensures that sufficient samples can be obtained for large experiments as well as permits optimal exchange of nutrients, oxygen, and drugs between the grafted tissue and the host. Using this model, we investigated the responses of prostate tissue to androgen ablation and restoration. Two histologically confirmed cancer tissues from radical prostatectomy specimens were used to generate tissue slice grafts (TSGs). Both tissues contained cancer of Gleason grade 4. Neither patient had prior chemical, hormonal, or radiation therapy. Putative cancers were grossly identified in two radical prostatectomy specimens obtained immediately after surgery under an Institutional Review Board-approved protocol. With an automated coring device (Alabama Research and Development, Mundford, AL) under aseptic conditions, 5-mm diameter cores were bored from the putative cancers and submerged in ice-cold HEPES-buffered saline. A Krumdieck tissue slicer (Alabama Research and Development) was used to prepare precision-cut tissue slices according to the manufacturer’s instructions. Specifically, each tissue core was encased in 3% sterile agarose (EMD Chemicals Inc., Hawthorne, NY) inside a mold-plunger assembly specifically devised for tissue embedding before slicing. The embedded tissues were then transferred into the slicer filled with precooled HEPES-buffered saline, and 300-μm slices were cut one at a time. Each slice was collected and submerged in ice-cold HEPES-buffered saline in serially numbered tubes. All animal studies were done in compliance with the regulations for animal studies at Stanford University. Each male recombination activating gene-2 (RAG2)−/−γC−/− mouse between 6 and 8 weeks of age was anesthetized and maintained under a heating lamp during the procedure. Sterile practices were followed throughout the surgical procedure. The skin at the incision site was shaved and sterilized with three scrubs of povidone iodine. A short incision was made with a scalpel immediately over the kidney. The kidney was gently popped through the incision and kept moist with sterile physiological saline. A small hole in the renal capsule was made with forceps and raised to create a small pocket between the capsule and the underlying kidney tissue. Into this pocket, a tissue slice (5 mm × 0.3 mm) was inserted with forceps. The renal capsule was released and allowed to cover the inserted tissue slice. The kidney was gently popped back through the incision, and the body wall was sutured. The skin incision was sealed with wound clips. A 25-mg testosterone pellet was inserted into a small incision made under the skin between the shoulder blades. The release rate of such pellets is approximately 0.2 mg/day.19Kochakian C The rate of absorption and effects of testosterone propionate pellets on mice.Endocrinology. 1941; 28: 478-484Crossref Scopus (5) Google Scholar, 20Ibrahim L Wright EA Effect of castration and testosterone propionate on mouse vibrissae.Br J Dermatol. 1983; 108: 321-326Crossref PubMed Scopus (9) Google Scholar The skin of the scrotal sacs was shaved and sterilized with three scrubs of povidone iodine. Both testes were pushed down into the scrotal sacs by gently applying pressure to the abdomen, and an incision (∼1 cm) was made through the skin along the midline of the scrotal sac. The midline wall between the testes sacs under the covering membranes was then located and a 5-mm incision in the membrane on the left side of the midline was made. The testis was carefully pushed out. A 4–0 Vicryl absorbable suture was used to ligate the vessels going to and from the testis. The fat pad that adheres to the testis was grasped with blunt forceps, and the testis was dissected away and removed from the fat pad. After pushing back the fat pad into the scrotal sac, the incision was closed with 4–0 Vicryl absorbable suture. The remnant of the testosterone pellet was then located and removed from the mouse. Mice were sacrificed and host kidneys carrying tissue slice grafts were fixed in 10% buffered formalin overnight and embedded in paraffin. Five-micron sections were cut from the blocks. Tissue sections were deparaffinized with xylene and rehydrated with ethanol. Antigen retrieval was performed for 20 minutes by heating in a citrate buffer (pH 6.0), followed by a 20-minute cool-down. Endogenous peroxidase activity was blocked by incubation in methanol containing 0.3% hydrogen peroxide. After pre-incubation with 10% horse serum for 20 minutes at room temperature to block nonspecific binding of antibodies, the tissues were incubated overnight at 4°C with primary antibodies against cytokeratin 18 (K18), AR, high molecular weight cytokeratins (HMWK), PSA, p63, Ki-67, cleaved caspase 3 (Biocare Medical, Concord, CA), Ku70 (Abcam, Cambridge, MA), human specific-CD31 (Dako Corp., Carpinteria, CA), mouse specific-CD68 (Abcam), and mouse specific-IgE receptor (eBioscience, San Diego, CA). Nkx3.1 antibody was a gift from Dr. Edward P. Gelmann at Columbia University, New York. The slides were then washed and incubated in a biotinylated secondary antibody at room temperature for 30 minutes, washed, and incubated again at room temperature for another 30 minutes in peroxidase-conjugated streptavidin. Color was developed with 3,3-diaminobenzidine (DakoCytomation California, Inc., Carpinteria, CA). Counter staining was performed with hematoxylin. For double immunofluorescence staining, Alexa 488 goat anti-mouse (Invitrogen, Carlsbad, CA) and Alexa 555 goat anti-rabbit (Invitrogen) were used as secondary antibodies. Proliferation or apoptotic index, defined as percentages of proliferating or apoptotic cells, was established by counting the number of Ki-67- or cleaved caspase 3-positive cells as well as the total number of epithelial cells in ten 40× microscopic fields for each tissue. Mouse serum testosterone concentrations were measured by using the Testosterone EIA kit (Cayman Chemical Company, Ann Arbor, MI). The assay was performed according to the manufacturer’s instructions. Briefly, 50 μl of each mouse serum sample in duplicate were incubated at room temperature with 50 μl of Testosterone Antiserum and 50 μl of Testosterone AChE Tracer on an orbital shaker for 2 hours. After washing, 200 μl of Ellman’s Reagent were added to each well for color development. The plate was read at a wavelength of 405 nm. Testosterone concentration, in picogram per milliliter, was calculated and determined by using the formula provided by the manufacturer. Statistical significance was determined by using Student’s t-test. TSGs were established from human prostate tissues obtained from two radical prostatectomy specimens by precision-cutting and subrenal implantation. We used a new severe combined immunodeficiency mouse model developed by crossing mice lacking the common cytokine receptor γ chain with mice lacking the RAG2, which has proven experimentally useful for supporting in vivo growth of cancer cells traditionally difficult to establish as xenografts because these mice lack not only T and B cells but also natural killer cells.21van Rijn RS Simonetti ER Hagenbeek A Hogenes MC de Weger RA Canninga-van Dijk MR Weijer K Spits H Storm G van Bloois L Rijkers G Martens AC Ebeling SB A new xenograft model for graft-versus-host disease by intravenous transfer of human peripheral blood mononuclear cells in RAG2−/− gammac−/− double-mutant mice.Blood. 2003; 102: 2522-2531Crossref PubMed Scopus (146) Google Scholar, 22Prince ME Sivanandan R Kaczorowski A Wolf GT Kaplan MJ Dalerba P Weissman IL Clarke MF Ailles LE Identification of a subpopulation of cells with cancer stem cell properties in head and neck squamous cell carcinoma.Proc Natl Acad Sci USA. 2007; 104: 973-978Crossref PubMed Scopus (1784) Google Scholar Fifteen tissue slices (300-μm thick and 5-mm in diameter) were obtained from each specimen. As outlined in Figure 1A, in each experiment, 15 tissue slices were implanted into the left kidneys of (RAG2)−/−γC−/− mice (1 slice/mouse) supplemented with testosterone at the time of implantation. TSGs were allowed to establish for a month, at which time three mice were sacrificed as controls to examine the viability, histology, and protein expression of TSGs. Nine of the remaining 12 mice were castrated, and the other three mice were left intact as controls. One month after castration, three castrated mice were sacrificed and TSGs were harvested. Meanwhile, testosterone pellets were implanted under the skin of another three castrated mice to restore androgen. At the end of the experiment (2 months after castration), the remaining mice were sacrificed including castrated mice with or without androgen restoration and mice that were intact as controls. At 1 month after implantation, TSGs were approximately the same size as before implantation (Figure 1B). Vascularization of the TSGs was apparent (Figure 1C). Immunohistochemistry using human-specific CD31 antibody demonstrated that a considerable amount of the vasculature present in TSGs after 1 month of implantation was lined by endothelial cells of human origin. This observation is consistent with a previous report that the majority of the vessels in primary xenografts of benign and malignant prostate tissue were lined with human endothelial cells throughout a 30-day study period.11Gray DR Huss WJ Yau JM Durham LE Werdin ES Funkhouser Jr, WK Smith GJ Short-term human prostate primary xenografts: an in vivo model of human prostate cancer vasculature and angiogenesis.Cancer Res. 2004; 64: 1712-1721Crossref PubMed Scopus (45) Google Scholar Interestingly, similar results were observed in TSGs even after 3 months of implantation, demonstrating the persistence of human endothelial cells in prostate TSGs. The level of testosterone in mice 1 month after castration decreased 96% compared with that in intact mice, from a mean of 1522 pg/ml in intact animals to 52 pg/ml in castrated mice (Figure 2). One month after restoration of androgen pellets, testosterone levels in castrated mice became similar to those in intact mice, whereas in mice without androgen restoration, testosterone levels remained low (Figure 2). No significant difference in testosterone levels was observed between intact mice and mice castrated and subsequently implanted with androgen pellets. The testosterone level in castrated mice was ∼10-fold lower than the most commonly used cut-off point of testosterone values to define castration in humans (500 pg/ml).23Pathak AS Pacificar JS Shapiro CE Williams SG Determining dosing intervals for luteinizing hormone releasing hormone agonists based on serum testosterone levels: a prospective study.J Urol. 2007; 177 (discussion 2135): 2132-2135Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar, 24de Jong IJ Eaton A Bladou F LHRH agonists in prostate cancer: frequency of treatment, serum testosterone measurement and castrate level; consensus opinion from a roundtable discussion.Curr Med Res Opin. 2007; 23: 1077-1080Crossref PubMed Scopus (18) Google Scholar In addition, androgen restoration increased the level of testosterone to 1292 pg/ml, comparable with that in intact mice with androgen supplementation. We examined the histology and protein expression of cell type-specific markers in TSGs harvested 1 month after implantation. As shown in Figure 3, the implanted tissue slices contained both normal and cancerous components. The normal glands showed two layers of epithelial cells, the inner secretory and the outer basal cells. The inner layer of cells expressed classical secretory cell markers including K18, AR, and PSA. Specifically, strong K18 staining was observed in the cytoplasm (Figure 3, A and B). These cells also showed nuclear staining of AR (Figure 3, D and E) and cytoplasmic PSA staining (Figure 3, G and H). In contrast, the outer layer of cells was positive for typical basal cell markers including p63 and HMWK recognized by antibody 903, which is widely used to differentiate benign glands from malignant glands in prostate needle biopsies based on the fact that cancerous glands lose basal cells. The intense signal of HMWK was seen in the cytoplasm of the outer cell layer (Figure 3, M and N), whereas p63 staining in these cells was nuclear (Figure 3, P and Q). In addition, antibody against Ku70 was used to distinguish human versus mouse cells. Both the inner and outer epithelial cell layers displayed strong nuclear staining of Ku70, whereas mouse cells showed background level staining in the cytoplasm (Figure 3, J and K). It was noted that many Ku70-positive cells were present in the stroma of TSGs maintained in the host for 1 month (Figure 3, K and L) as well as for 3 months (Figure 4, B and C), suggesting that human prostatic stromal cells survived implantation similar to the epithelial cells. Overall, these results indicated that normal prostate glands maintained appropriate histomorphology, cell composition, and protein expression in TSGs at 1 month postimplantation.Figure 4Expression of cell-type specific markers in TSGs harvested at three months after implantation by immunohistochemistry. A, D, G, J, M, and P were low magnification (×4) images of the same area of the TSG in serial sections stained with different antibodies. The junction of TSG versus mouse kidney was clearly identifiable. Magnification for the rest of the images was ×40. Cells of human origin were distinguished from host cells by strong nuclear presence of Ku70 in cancer cells (B) and normal glands (C). Cytoplasmic staining of K18 and PSA was observed in cancer cells (E and K, respectively) and secretory cells of the normal glands (F and L, respectively). Nuclear staining of AR was detected in secretory cells of cancer cells (H) and the normal glands (I). Basal epithelial cell markers, HMWK and p63, were expressed in the basal epithelial cells of normal glands (O and R, respectively), but not in cancer cells (N and Q, respectively).View Large Image Figure ViewerDownload Hi-res image Download (PPT) High grade cancer (Gleason grade 4) was also identifiable in this TSG (Figure 3), appearing as an irregular mass of neoplastic cells with little or no gland formation. These cancer cells were strongly positive for K18 and Ku70 (Figure 3, C and L). In addition, these cells showed nuclear staining of AR and cytoplasmic PSA (Figure 3, F and I). Moreover, these cells were negative for the basal epithelial cell markers HMWK and p63 (Figure 3, O and R). These results demonstrated that grade 4 PCa maintained its histopathological and molecular characteristics in our TSG model. We further examined TSGs maintained for 3 months under the renal capsule of (RAG2)−/−γC−/− mice. Cancer (Gleason grade 4) surrounded by normal glands was present in the graft shown in Figure 4. Cells within this cancer were stained intensely with antibodies against Ku70 (Figure 4, A and B), K18 (Figure 4, D and E), and AR (Figure 4, G and H). These cells also showed high expression of PSA (Figure 4, J and K), but no staining for p63 (Figure 4, M and N) or HMWK (Figure 4, P and Q). The lack of staining for HMWK and p63 was not due to technical failure because the normal glands adjacent to the cancer were strongly positive for p63 (Figure 4N) and HMWK (Figure 4Q) in the basal epithelial cells. As expected, the normal glands in this TSG consisted of secretory epithelial cells expressing K18 (Figure 4F), AR (Figure 4I), and PSA (Figure 4L), and basal epithelial cells expressing p63 (Figure 4O) and HMWK (Figure 4R). These results demonstrated that our TSG model preserved the histological and molecular characteristics of normal prostate epithelial cells and high grade PCa for up to 3 months in intact mice. To evaluate inflammatory infiltrates in TSGs, we performed immunocytochemistry to identify macrophages using a monoclonal antibody against mouse CD68 and mast cells using anti-IgE receptor. Neither macrophages nor mast cells were detected in TSGs at any time points (data not shown). In addition, no neutrophils were present in TSGs as determined by Giemsa staining (data not shown). Our results indicated little if any inflammatory infiltrates in this TSG model. We performed immunohistochemistry by using antibodies against Ki-67 and cleaved caspase-3 to evaluate proliferation and apoptosis, respectively, at the different points of the experiments. As shown in Table 1, benign epithelial cells showed a proliferation index (percentage of Ki-67-positive cells) of 1.28 and 0.55 at 1 and 3 months after implantation, respectively. Consistent with previous findings,25Tamboli P Amin MB Schultz DS Linden MD Kubus J Comparative analysis of the nuclear proliferative index (Ki-67) in benign prostate, prostatic intraepithelial neoplasia, and prostatic" @default.
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