Murine Model Of Prostate Cancer Research Articles

Nanotechnologies to use bisphosphonates as potent anticancer agents: the effects of zoledronic acid encapsulated into liposomes

Zoledronic acid (ZOL) is a potent amino-bisphosphonate used for the treatment of bone metastases with recently reported antitumor activity. However, the short plasma half-life and rapid accumulation in bone limits the use of ZOL as an antitumor agent in extraskeletal tissues. Therefore, we developed stealth liposomes encapsulating ZOL (LipoZOL) to increase extraskeletal drug availability. Compared to free ZOL, LipoZOL induced a stronger inhibition of growth of a range of different cancer cell lines in vitro. LipoZOL also caused significantly larger inhibition of tumor growth and increased the overall survival in murine models of human prostate cancer and multiple myeloma, in comparison with ZOL. Moreover, a strong inhibition of vasculogenetic events without evidence of necrosis in the tumor xenografts from prostate cancer was recorded after treatment with LipoZOL. We demonstrated both antitumor activity and tolerability of LipoZOL in preclinical animal models of both solid and hematopoietic malignancies, providing a rationale for early exploration of use of LipoZOL as a potential anticancer agent in cancer patients. From the Clinical EditorThe short plasma half-life and rapid accumulation in bone limits the use of zoledronic acid as an antitumor agent in extraskeletal tissues. Therefore, stealth liposomes encapsulating ZOL (LipoZOL) have been developed to increase extraskeletal drug availability.

Comparison of low-temperature-sensitive liposome encapsulated docetaxel and doxorubicin in a murine model of prostate cancer.

31 Background: Low-temperature-sensitive liposomes (LTSLs) release their encapsulated drug into targeted tissue when activated by a source of hyperthermia. The efficacy of an LTSL formulation of docetaxel (DOC) or doxorubicin (DOX) was compared against prostate cancer in a xenograft mouse model. Methods: Under an approved IACUC protocol, Luciferase transfected human prostate PC-3M-luciferase cells were inoculated (3x106 cells) subcutaneously in the right hind leg of 8 wk old female athymic nude mice. When tumors reached a volume of 200–300 mm3, mice were randomized to receive one intravenous injection of saline, Stealth liposomal DOX (5 mg/kg), LTSL DOX (5 mg/kg), or LTSL DOC (15 mg/kg), with or without hyperthermia treatment (LTSL DOX and LTSL DOC were supplied by Celsion Corp., Columbia MD). Mice undergoing hyperthermia treatment were anesthetized and stabilized in a holder that allowed for only the leg with tumor to be heated to 41–42°C that triggered LTSL drug release. Mice were monitored daily for tumor volume and body weight. Study end-points included growth of tumor to 5x the initial treatment volume or monitoring of survival for 60 days. Results: The LTSL DOC delayed tumor growth longer (> 14 days) than DOX (0 day) and LTSL DOX (1 day) with hyperthermia (P<0.05). Mice treated with LTSL DOC and hyperthermia survived longest (60 days) compared to all other mice (range 6–8 days, P<0.05). LTSL in the setting of hyperthermia demonstrated complete regression of tumor in 57% of mice. Conclusions: LTSL DOC with hyperthermia delayed tumor growth more than all other treatments. Survival studies suggest LTSL DOC is a more effective temperature sensitive delivery system against PC-3M prostate tumors. No significant financial relationships to disclose.

Use of 1,25α dihydroxyvitamin D3 as a cryosensitizing agent in a murine prostate cancer model

Cryotherapy has emerged as a primary treatment option for prostate cancer(CaP); however, incomplete ablation in the periphery of the cryogenic lesion can lead to recurrence. Accordingly, we investigated the use of a nontoxic adjunctive agent, Vitamin D3, with cryotherapy to sensitize CaP to low temperature induced, non-ice rupture related cell death. Vitamin D3 (calcitriol) has been identified as a possible adjunct in the treatment of cancer due to its anti-proliferative and anti-tumorigenic properties. This study aimed to identify the cellular responses and molecular pathways activated when vitamin D3 (calcitriol) is combined with cryotherapy in a murine prostate cancer model. Single freeze-thaw events above −15°C had little effect on cancer cell viability; however, pre-treatment with calcitriol in conjunction with cryo significantly increased cell death. The −15°C calcitriol combination increased cell death to 55% following a single freeze, compared to negligible cell loss by freezing or calcitriol alone. Repeat cryo-combination yielded90% cell death, compared to 65% in dual freeze-only cycles. Western blot analysis following calcitriol cryosensitization regimes confirmed the activation of apoptosis. Specifically, pro-apoptotic Bid and pro-caspase–3 were found to decrease at 1h following combination treatment, indicating cleavage to the active forms. A parallel in vivo study confirmed the increased cell death when combining cryotherapy with calcitriol pre-treatment. The development of an adjunctive therapy combining calcitriol and cryotherapy represents a potentially highly effective, less toxic, minimally invasive treatment option. These results suggest a role for calcitriol and cryo as a combinatorial treatment for CaP with the potential for clinical translation.

109. The use of 1,25α dihydryoxyvitamin D 3 as a cryosensitizing agent in a murine prostate cancer model

109. The use of 1,25α dihydryoxyvitamin D 3 as a cryosensitizing agent in a murine prostate cancer model

Radiation Enhances Regulatory T Cell Representation

Immunotherapy could be a useful adjunct to standard cytotoxic therapies such as radiation in patients with micrometastatic disease, although successful integration of immunotherapy into treatment protocols will require further understanding of how standard therapies affect the generation of antitumor immune responses. This study was undertaken to evaluate the impact of radiation therapy (RT) on immunosuppressive T regulatory (Treg) cells. Treg cells were identified as a CD4(+)CD25(hi)Foxp3(+) lymphocyte subset, and their fate was followed in a murine TRAMP C1 model of prostate cancer in mice with and without RT. CD4(+)CD25(hi)Foxp3(+) Treg cells increased in immune organs after local leg or whole-body radiation. A large part, but not all, of this increase after leg-only irradiation could be ascribed to radiation scatter and Treg cells being intrinsically more radiation resistant than other lymphocyte subpopulations, resulting in their selection. Their functional activity on a per-cell basis was not affected by radiation exposure. Similar findings were made with mice receiving local RT to murine prostate tumors growing in the leg. The importance of the Treg cell population in the response to RT was shown by systemic elimination of Treg cells, which greatly enhanced radiation-induced tumor regression. We conclude that Treg cells are more resistant to radiation than other lymphocytes, resulting in their preferential increase. Treg cells may form an important homeostatic mechanism for tissues injured by radiation, and in a tumor context, they may assist in immune evasion during therapy. Targeting this population may allow enhancement of radiotherapeutic benefit through immune modulation.

LL‐37 as a therapeutic target for late stage prostate cancer

The antimicrobial peptide, leucine-leucine-37 (LL-37), stimulates proliferation, angiogenesis, and cellular migration, inhibits apoptosis and is associated with inflammation. Since these functional processes are often exaggerated in cancer, the aim of the present study was to investigate the expression and role of LL-37 in prostate cancer (PCa) and establish its value as a therapeutic target. We evaluated the expression of LL-37 and the murine orthologue, cathelicidin-related antimicrobial peptide (CRAMP) in human and murine prostate tumors, respectively. Compared to normal/benign prostate tissue, both LL-37 and CRAMP were increasingly over-expressed with advancing grades of primary PCa and its metastasis in human tissues and in the transgenic adenocarcinoma mouse prostate (TRAMP) model, correspondingly. We subsequently knocked-down CRAMP in the highly tumorigenic TRAMP-C1 cell line via a RNA interference strategy to examine the importance of CRAMP on cellular proliferation, angiogenesis, invasion, apoptosis, activation of signaling pathways and tumor kinetics. Abrogation of CRAMP expression led to decreased proliferation, invasion, type IV collagenase, and the amount of phosphorylated Erk1/2 and Akt signaling in vitro. These results were paralleled in vivo. Syngenic implantation of TRAMP-C1 cells subjected to CRAMP knock-down resulted in a decreased tumor incidence and size, and the down-regulation of pro-tumorigenic mechanisms. CRAMP knock-down in a murine PCa model analogously demonstrated the tumorigenic contributions of LL-37 in PCa and its potential as a novel therapeutic target for the treatment of PCa and potentially, other cancers over-expressing the peptide.

Human prostate cancer xenografts in lit/lit mice exhibit reduced growth and androgen‐independent progression

The growth hormone/insulin-like growth factor I (GH/IGF-I) axis has been linked to prostate cancer (PCa) risk. Although previous studies indicate that human breast cancers and a murine PCa model develop more slowly in murine hosts homozygous for a missense mutation in the GH-releasing hormone receptor (lit/lit) whose "little" dwarfed phenotype is caused by suppressed GH and IGF-I production, the role of these two hormones remains controversial. To assess how the GH/IGF-I axis influences androgen-responsive, castration-resistant (CR), and androgen-independent (AI) growth of human PCa, we compared xenograft growth of the androgen-responsive human PCa cells, LNCaP, and AI human PCa cells, PC3, in intact and castrate Nod/SCID lit/lit and lit/+ mice, and in vitro growth of these cell lines in lit/lit and lit/+ serum-containing media supplemented with GH or IGF-I. Tumor growth and PSA accumulation rates were suppressed in LNCaP tumor-bearing lit/lit mice pre- and post-castration. Growth of PC3 xenografts in lit/lit mice was also suppressed. In vitro proliferation of LNCaP and PC3 cells cultured in media containing lit/lit mouse serum was decreased as compared to growth in media containing lit/+ serum. Suppressed growth in lit/lit serum could be restored by the addition of IGF-I, and to a lesser extent, GH. Differences in growth correlated with differences in steady-state AKT and ERK1/2 activation. This study demonstrates that circulating GH and IGF-I can promote androgen-responsive growth, CR progression, and AI expansion of PTEN-deficient human PCa cell xenografts and indicates that IGF-I can promote PCa growth in a suppressed GH environment.

Constitutively active androgen receptor splice variants expressed in castration-resistant prostate cancer require full-length androgen receptor

Androgen receptor (AR) splice variants lacking the ligand binding domain (ARVs), originally isolated from prostate cancer cell lines derived from a single patient, are detected in normal and malignant human prostate tissue, with the highest levels observed in late stage, castration-resistant prostate cancer. The most studied variant (called AR-V7 or AR3) activates AR reporter genes in the absence of ligand and therefore, could play a role in castration resistance. To explore the range of potential ARVs, we screened additional human and murine prostate cancer models using conventional and next generation sequencing technologies and detected several structurally diverse AR isoforms. Some, like AR-V7/AR3, display gain of function, whereas others have dominant interfering activity. We also find that ARV expression increases acutely in response to androgen withdrawal, is suppressed by testosterone, and in some models, is coupled to full-length AR (AR-FL) mRNA production. As expected, constitutively active, ligand-independent ARVs such as AR-V7/AR3 are sufficient to confer anchorage-independent (in vitro) and castration-resistant (in vivo) growth. Surprisingly, this growth is blocked by ligand binding domain-targeted antiandrogens, such as MDV3100, or by selective siRNA silencing of AR-FL, indicating that the growth-promoting effects of ARVs are mediated through AR-FL. These data indicate that the increase in ARV expression in castrate-resistant prostate cancer is an acute response to castration rather than clonal expansion of castration or antiandrogen-resistant cells expressing gain of function ARVs, and furthermore, they provide a strategy to overcome ARV function in the clinic.

The dietary isothiocyanate sulforaphane modulates gene expression and alternative gene splicing in a PTEN null preclinical murine model of prostate cancer

BackgroundDietary or therapeutic interventions to counteract the loss of PTEN expression could contribute to the prevention of prostate carcinogenesis or reduce the rate of cancer progression. In this study, we investigate the interaction between sulforaphane, a dietary isothiocyanate derived from broccoli, PTEN expression and gene expression in pre malignant prostate tissue.ResultsWe initially describe heterogeneity in expression of PTEN in non-malignant prostate tissue of men deemed to be at risk of prostate cancer. We subsequently use the mouse prostate-specific PTEN deletion model, to show that sulforaphane suppresses transcriptional changes induced by PTEN deletion and induces additional changes in gene expression associated with cell cycle arrest and apoptosis in PTEN null tissue, but has no effect on transcription in wild type tissue. Comparative analyses of changes in gene expression in mouse and human prostate tissue indicate that similar changes can be induced in humans with a broccoli-rich diet. Global analyses of exon expression demonstrated that sulforaphane interacts with PTEN deletion to modulate alternative gene splicing, illustrated through a more detailed analysis of DMBT1 splicing.ConclusionTo our knowledge, this is the first report of how diet may perturb changes in transcription induced by PTEN deletion, and the effects of diet on global patterns of alternative gene splicing. The study exemplifies the complex interaction between diet, genotype and gene expression, and the multiple modes of action of small bioactive dietary components.

Role of Vitamin D 3 as a Sensitizer to Cryoablation in a Murine Prostate Cancer Model: Preliminary In Vivo Study

Calcitriol has been reported to have antitumor efficacy in several cancers. In this study, we hypothesized that calcitriol may potentially function as a cryosensitizer that can enhance cryoablation, and we investigated several molecular marker changes in a murine model of prostate cancer. Murine prostate tumors (RM-9) were grown in male C57BL/6J mice subcutaneously with neoadjuvant intratumoral injection of calcitriol followed by cryoablation. The microenvironmental changes after cryoablation alone and in combination with calcitriol were analyzed in a comparative fashion using immunohistochemistry and Western blot analyses. Both cryoablation and the combination group could suppress tumor growth after treatment compared with the control. At final pathologic assessment, a larger necrotic area was seen in the combination group (P = .026). Although microvessel density (CD31) and the area of hypoxia (pimonidazole) was not different between the control and combination groups, cell proliferation (Ki-67) significantly decreased in the combination treatment (P = .035). In Western blot analyses, several markers for apoptosis were expressed significantly higher with the combination treatment. The synergistic effect of calcitriol with cryoablation was demonstrated because of enhanced antitumor efficacy by increasing necrosis and apoptosis and reduced cell proliferation. This study suggests that calcitriol is a potentially applicable reagent as a freeze sensitizer to cryoablation.

Defective NKT Cell Activation by CD1d+ TRAMP Prostate Tumor Cells Is Corrected by Interleukin-12 with alpha-Galactosylceramide

Numerical and functional defects of invariant natural killer T cells (iNKT) have been documented in human and mouse cancers, resulting in a defect in IFN production in several malignancies. iNKT cells recognize glycolipids presented on CD1d molecules by dendritic and related cells, leading to their activation and thereby regulating immune reactions. Activated iNKT cells cytokine secretion and cytotoxicity can inhibit existing and spontaneous tumor growth, progression, and metastasis. We have identified functional iNKT cell defects in the murine TRAMP prostate cancer model. We found that iNKT cells show the ability to migrate into TRAMP prostate tumors. This infiltration was mediated through CCL2: CCR5 chemokine: receptor interaction. Prostate tumor cells expressing CD1d partially activated iNKT cells, as appreciated by up-regulation of CD25, PD-1 and the IL-12R. However, despite inducing up-regulation of these activation markers and, hence, delivering positive signals, prostate tumor cells inhibited the IL-12-induced STAT4 phosphorylation in a cell-cell contact dependent but CD1d-independent manner. Consequently, tumor cells did not induce secretion of IFNγ by iNKT cells. Blocking the inhibitory Ly49 receptor on iNKT cells in the presence of α-GalCer restored their IFNγ production in vivo and in vitro. However, Ly49 blockade alone was not sufficient. Importantly, this defect could be also be reversed into vigorous secretion of IFNγ by the addition of both IL-12 and the exogenous CD1d ligand alpha-galactosylceramide, but not by IL-12 alone, both in vivo and in vitro. These data underscore the potential to optimize iNKT-based therapeutic approaches.

681 REDUCED ANDROGEN DEPENDENT AND INDEPENDENT GROWTH OF HUMAN PROSTATE CANCER XENOGRAFTS IN GROWTH HORMONE RELEASING HORMONE RECEPTOR MUTANT HOSTS

You have accessJournal of UrologyProstate Cancer: Basic Research V1 Apr 2010681 REDUCED ANDROGEN DEPENDENT AND INDEPENDENT GROWTH OF HUMAN PROSTATE CANCER XENOGRAFTS IN GROWTH HORMONE RELEASING HORMONE RECEPTOR MUTANT HOSTS Kiyoshi Takahara, Howard Tearle, Tabitha Tombe, Martin E. Gleave, and Michael E. Cox Kiyoshi TakaharaKiyoshi Takahara More articles by this author , Howard TearleHoward Tearle More articles by this author , Tabitha TombeTabitha Tombe More articles by this author , Martin E. GleaveMartin E. Gleave More articles by this author , and Michael E. CoxMichael E. Cox More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2010.02.1080AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Signaling through the growth hormone/insulin-like growth factor (GH/IGF) axis has been linked to prostate cancer (PCa) risk. Previous studies have indicated that human breast cancer xenografts and murine PCa models develop more slowly in little murine hosts. The little (lit/lit) phenotype is due to a D60G missense mutation in the GH-releasing hormone receptor (GHRHR) resulting in loss of pituitary GHRHR function and suppression of GH and IGF-1 expression. We hypothesized that the GH/IGF axis influences PCa growth and castration-resistant (CR) progression of human PCa xenografts. METHODS To determine whether PCa growth in vivo models is influenced by the host GH/IGF axis, we performed in vivo growth studies using the androgen-responsive human PCa cell line, LNCaP, and CR human PCa cell line, PC3, in Nod/SCID lit/lit mice and compared them to growth and CR progression in mice heterozygous for the lit allele (lit/+). To assess whether suppressed GH/IGF levels affected androgen-responsive growth of PCa xenografts, we monitored tumor size and serum PSA levels before and after castration of LNCaP tumor-bearing lit/lit and lit/+ mice. We also monitored growth rate of PC3 xenografts in intact mice to assess the impact of GH/IGF axis deficiencies on CR PCa. Growth of LNCaP and PC3 cells in vitro using serum from lit/lit mice or lit/+ mice serum was examined to complement the in vivo experiments. RESULTS In intact mice, androgen-responsive growth rate of LNCaP xenografts was significantly reduced in lit/lit mice as compared to lit/+ mice [mm3/day (mean±SD) 56.0±8.5 vs 93.2±16.3, p < 0.05]. CR progression of LNCaP xenografts was also significantly delayed in lit/lit mice as compared to lit/+ mice [mm3/day (mean±SD) 65.2±12.8 vs 147.2±47.3, p < 0.1]. After castration, serum PSA levels in lit/lit mice was significantly lower than in lit/+ mice [ng/ml/day (mean±SD) 18.0±6.5 vs 60.0±33.0, p < 0.1]. Growth of CR PC3 xenografts was significantly delayed in lit/lit mice (n=19) as compared to lit/+ mice (n=17) [mm3/day (mean±SD) 234.5±24.1 vs 441.6±42.4, p < 0.001]. LNCaP and PC3 cells grown in vitro with serum from lit/lit mice showed decreased proliferation as compared with serum from lit/+ mice. CONCLUSIONS The GH/IGF axis appears to be an important stimulator of both androgen-responsive growth and CR progression of these PCa xenograft models. The results motivate clinical trials of novel hormonal treatment strategies that target the GH/IGF axis for PCa patients. Vancouver, Canada© 2010 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 183Issue 4SApril 2010Page: e266 Advertisement Copyright & Permissions© 2010 by American Urological Association Education and Research, Inc.MetricsAuthor Information Kiyoshi Takahara More articles by this author Howard Tearle More articles by this author Tabitha Tombe More articles by this author Martin E. Gleave More articles by this author Michael E. Cox More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

POD-04.02: Non-Myeloablative Allogeneic Bone Marrow Transplantation Inhibited Tumor Growth and Prolonged Recipients' Survival Time in Murine Prostate Cancer Model

POD-04.02: Non-Myeloablative Allogeneic Bone Marrow Transplantation Inhibited Tumor Growth and Prolonged Recipients' Survival Time in Murine Prostate Cancer Model

Inactivation of LGI1 expression accompanies early stage hyperplasia of prostate epithelium in the TRAMP murine model of prostate cancer

The LGI1 gene has been implicated in tumor cell invasion through regulation of the ERK pathway. To determine whether human prostate cancer cells (PC3, 22RV, Du145) are similarly affected by exposure to LGI1, we conducted scratch wound assays and demonstrated that the secreted LGI1 protein can reduce cell motility, an essential component of invasion and metastasis. These studies have now been extended to an in vivo mouse model of prostate cancer. Using a BAC transgenic mouse expressing a GFP reporter gene under the control of cis regulatory elements, we demonstrated that LGI1 is highly expressed in the normal prostate epithelium. To determine whether loss of LGI1 expression is associated with development and progression of murine prostate cancer, we bred the GFP reporter BAC transgenic mice with TRAMP mice which undergo early hyperplasia and progressive stages of prostate cancer. In the F1 animals, although the surrounding normal prostate epithelium expressed high levels of LGI1 in the double transgenic mice, the LGI1 gene had been inactivated even at the earliest stages of hyperplasia. This observation supports the suggestion that inactivation of LGI1 in certain cell types is related to tumor progression. Taken together these results suggest that LGI1 may be an important molecule for the arrest of prostate cancer cell invasion and possibly as a biomarker for early detection of prostate hyperplasia.

The role of murine models of prostate cancer in drug target discovery and validation

Background: Mice provide us with an excellent preclinical model of human prostate cancer. They have expanded our understanding of the molecular pathways involved in prostate carcinogenesis as well as allowing us to explore both novel and traditional treatment regimes based on the molecular profile of these lesions. Continuing refinement of the transgenic prostate models has proven challenging since no one model seems to represent the entire continuum of the disease, thus currently limiting its applicability to the human condition. This platform may potentially have major impact in validation of drug targeting specific biological process of prostate carcinogenesis, supplementing (or even replacing) many of the current in vitro and in vivo assays with the in vivo environment that transgenic prostate models provide. Objective/method: This review focuses primarily on the current state of murine model systems as a preclinical therapeutic platform for the treatment of prostate cancer, as well as hope for the future of the field. Conclusion: Much of the work in the drug discovery field has been done with the PTEN-/- and TRAMP models of prostate cancer. Despite their limitations they have contributed much to our understanding of the pathophysiology of the disease. There is, however, a need for transgenic models that better reflect the stepwise progression found in the human condition. We feel that they will prove to be invaluable as a preclinical platform regarding efficacy and tolerability of various anticancer agents, which ultimately allows us to translate these findings to the clinical setting to prognosticate and ultimately render cancer patients disease-free.

γ‐Tocopherol‐enriched mixed tocopherol diet inhibits prostate carcinogenesis in TRAMP mice

Gamma-tocopherol (gamma-T) alone or in combination with alpha-tocopherol has been shown to suppress biomarkers of oxidative stress in asthamatics and human subjects with metabolic syndrome. Oxidative stress has been implicated as a key event in prostate carcinogenesis. Hence, the purpose of this study was to examine the effects of gamma-tocopherol-enriched mixed tocopherol diet on prostate carcinogenesis in a murine prostate cancer model (TRAMP). 8 week old TRAMP males were fed 0.1% gamma-T-enriched mixed tocopherol diet that contained 20-fold higher levels of gamma-tocopherol, and roughly 3-fold higher levels of alpha-tocopherol. The effect of such diet on tumor and PIN development was observed. The expression of phase II detoxifying, antioxidant enzymes and Nrf2 mRNA and protein were determined by RT-PCR, immunohistochemistry and western blotting techniques. Treatment with gamma-T-enriched mixed tocopherols significantly suppressed the incidence of palpable tumor and Prostate Intraepithelial Neoplasia (PIN) development without affecting the expression of the transgene (SV-40). Tumor progression occurred with a significant suppression of antioxidant enzymes such as catalase, superoxide dismutase, glutathione peroxidase, heme-oxygenase-1 and phase II detoxifying enzymes. Treatment with gamma-T-enriched mixed tocopherol diet upregulated the expression of most detoxifying and antioxidant enzymes. Nrf2-a redox sensitive transcription factor known to mediate the expression of phase II detoxifying enzymes, was also significantly upregulated following treatment with gamma-T-enriched mixed tocopherol diet. Gamma-T-enriched mixed tocopherols significantly up-regulated the expression of Nrf2 and its related detoxifying and antioxidant enzymes thereby suppressing PIN and tumor development.

PTEN Knockout Prostate Cancer as a Model for Experimental Immunotherapy

Testing immunotherapeutic strategies for prostate cancer has been impeded by the lack of relevant tumor models in immunocompetent animals. This opportunity is now provided by the recent development of prostate specific PTEN knockout mice, which show spontaneous development of true adenocarcinoma arising from prostate epithelium and more faithfully recapitulate the human disease than any previous model. We investigated the feasibility of using tumor cells derived from this model to test tumor vaccination and adoptive immunotherapeutic strategies for prostate cancer. PTEN-CaP8 adenocarcinoma cells derived from the biallelic PTEN knockout prostate cancer model were used to vaccinate nontumor bearing litter mates. Tumor specific effector cells were generated from splenocytes of vaccinated mice by mixed lymphocyte-tumor reactions, and antiproliferative effects and cytokine generation were examined in vitro. The effect of vaccination or adoptive immunotherapy on luciferase marked PTEN-CaP8 subcutaneous tumors was monitored by tumor volumetric measurements and noninvasive bioluminescence imaging. Vaccination of litter mate mice with irradiated PTEN-CaP8 cells showed a significant prophylactic effect against the subsequent tumor challenge. Effector cells harvested from vaccinated litter mates showed significant interferon-gamma secretion upon co-incubation with PTEN-CaP8 target cells and they were capable of efficient target cell growth inhibition in vitro. Intratumor adoptive transfer of effector cells resulted in significant growth inhibition of preestablished prostate tumors in vivo. The PTEN knockout model serves as a highly useful model in which to investigate tumor cell vaccination and adoptive immunotherapeutic strategies in the context of true adenocarcinoma of the prostate. This model should accelerate efforts to develop effective immunotherapies for human prostate cancer.

Radiotherapy augments the immune response to prostate cancer in a time‐dependent manner

Cancer immunotherapy refers to an array of strategies intended to treat progressive tumors by augmenting a patient's anti-tumor immune response. As immunotherapy is eventually incorporated into oncology treatment paradigms, it is important to understand how these therapies interact with established cancer treatments such as chemotherapy or Radiotherapy (RT). To address this, we utilized a well-established, autochthonous murine model of prostate cancer to test whether RT could augment (or diminish) the CD4 T cell response to a tumor vaccine. Transgenic mice that develop spontaneous prostate cancer (TRAMP) which also express a unique tumor associated antigen (Influenza hemagglutinin) under the control of a prostate-specific promoter were given local RT in combination with immunotherapy. The immunological outcome of this combinatorial strategy was assayed by monitoring the effector response of adoptively transferred, prostate-specific CD4 T cells. Neither RT nor immunotherapy alone was capable of priming an anti-tumor immune response in animals with evolving tumors. The combination of immunotherapy with RT resulted in anti-tumor T cell activation--this effect was profoundly dependent on the relative timing of RT and immunotherapy. Anti-tumor immune responses occurred when immunotherapy was administered 3-5 weeks post-RT, but such responses were undetectable when immunotherapy was administered either earlier (peri-radiotherapy) or later. The therapeutic temporal window of immunotherapy post-RT suggests that highly aggressive, immuno-suppressive tumors might be most sensitive to immunotherapy in a fairly narrow time window; these results should help to guide future development of clinical combinatorial strategies.

Phenotype-Specific CpG Island Methylation Events in a Murine Model of Prostate Cancer

Aberrant DNA methylation plays a significant role in nearly all human cancers and may contribute to disease progression to advanced phenotypes. Study of advanced prostate cancer phenotypes in the human disease is hampered by limited availability of tissues. We therefore took advantage of the Transgenic Adenocarcinoma of Mouse Prostate (TRAMP) model to study whether three different phenotypes of TRAMP tumors (PRIM, late-stage primary tumors; AIP, androgen-independent primary tumors; and MET, metastases) displayed specific patterns of CpG island hypermethylation using Restriction Landmark Genomic Scanning. Each tumor phenotype displayed numerous hypermethylation events, with the most homogeneous methylation pattern in AIP and the most heterogeneous pattern in MET. Several loci displayed a phenotype-specific methylation pattern; the most striking pattern being loci methylated at high frequency in PRIM and AIP but rarely in MET. Examination of the mRNA expression of three genes, BC058385, Goosecoid, and Neurexin 2, which exhibited nonpromoter methylation, revealed increased expression associated with downstream methylation. Only methylated samples showed mRNA expression, in which tumor phenotype was a key factor determining the level of expression. The CpG island in the human orthologue of BC058385 was methylated in human AIP but not in primary androgen-stimulated prostate cancer or benign prostate. The clinical data show a proof-of-principle that the TRAMP model can be used to identify targets of aberrant CpG island methylation relevant to human disease. In conclusion, phenotype-specific hypermethylation events were associated with the overexpression of different genes and may provide new markers of prostate tumorigenesis.

Provision of tumor‐specific CD4+ T cells to enhance tumor immunity in a murine model of prostate cancer

T cell tolerance to tumor antigens is a considerable challenge to cancer immunotherapy. Our laboratory studies T cell tolerance to tumor antigens using the TRansgenic Adenocarcinoma of the Mouse Prostate (TRAMP) model in combination with adoptive transfer of TcR transgenic T cells with specificity for a TRAMP tumor antigen. We previously reported that adoptive transfer of CD8+ (TcR‐I) cells into TRAMP mice resulted in rapid tolerization of the cells. The objective of the current study was to test the ability CD4+ TcR‐II cells to prevent or reverse TcR‐I cell tolerance. Naïve TcR‐II cells transferred into TRAMP mice proliferate in the lymph nodes, are transiently activated, and subsequently traffic to the prostate where they become tolerant by 10 days after transfer. We next tested whether the transient activation of TcR‐II cells was sufficient to prevent TcR‐I cell tolerization. Co‐transfer of naïve TcR‐II cells with TcR‐I cells initially enhanced the frequency, activation, survival and function of TcR‐I cells but this effect was not durable, as TcR‐I cells were eventually tolerized by 3 weeks after co‐transfer with TcR‐II cells. Furthermore, our preliminary data suggest that TcR‐II cells transferred 10 days after TcR‐I cells were unable to rescue TcR‐I cells from tolerization. Taken together, these data demonstrate that the powerful immunosuppressive environment of the developing TRAMP tumor can exert its effect on T cells, irrespective of provision of T cell help. On‐going studies will test whether conditioning TcR‐II cells affects their ability to prevent or reverse TcR‐I tolerance. By studying the ability of CD4+ T cells to enhance anti‐tumor CTL activity, we hope to elucidate novel approaches to stimulate a more potent anti‐tumor immune response.Supported in part by NCI IRP and DOD PCRP.