Autochthonous Prostate Cancer Model Research Articles

Therapeutic Radiation in Genetically Engineered Mouse Models of Cancer

Some drugs that target tumor metabolism are minimally effective in vitro but limit proliferation in vivo. Our goal was to develop a preclinical platform enabling us to test drugs that target cancer cell metabolism in combination with radiation therapy in vivo. Here we report the initial results of preclinical studies in autochthonous mouse models of prostate and colorectal cancer using radiation as monotherapy.A dose escalation study was performed in C57BL/6 wild-type mice using 1-5 fraction regimens with a BED of 90, 130, 180, and 250 (a/b = 3). Flank xenograft models of prostate cancer (22Rv1 and PC3 cells) were treated with 24 Gy in 4 fractions or sham irradiated (n = 10 animal per group, 40 animals total). Pb-Cre;Ptenfl/fl;p53fl/fl mice (autochthonous prostate cancer model) age 5 months were randomly assigned to two dose regimens (37.5 Gy in 5 fractions or 45 Gy in 5 fractions) or sham irradiation (n = 11-14 per group). Survival was estimated by Kaplan-Meier Analysis. Colorectal tumors were induced in male and female Villin-CreER;Apcfl/fl mice by intramucosal tamoxifen injection. Mice were assigned 2:1 to 37.5 Gy or sham irradiation (n = 10-15 per group). Treatment effect was measured as percent survival 6 months after tumor induction. In all cases pathologic response rate was determined using serial histologic sections and proliferation by Ki-67 immunohistochemistry.For a 2 cm field directed at the low abdomen and pelvis of mice, the maximum tolerated dose of radiation was 45 Gy delivered in 5 fractions. The intestinal tract was identified as the dose limiting organ for abdominopelvic radiation. In irradiated flank xenografts, growth delay was observed for all tumors compared with 100% progression in unirradiated controls. Pathologic complete response was observed in 1 of 10 (10%) 22Rv1 xenografts and 4 of 10 (40%) PC3 xenografts. In the autochthonous prostate cancer model, median survival was significantly improved in the 45 Gy (236 days, P < 0.0001) and 37.5 Gy (227 days, P = 0.0007) arms relative to control (206 days). Irradiated tumors were also smaller relative to control (mean difference -2.637 grams [95% CI -4.617 to -0.6563, P = 0.0153); however, all mice eventually died of local tumor progression. In the autochthonous colorectal cancer model, survival at 6 months was significantly improved in the radiation group relative to control (100% vs 40%, P = 0.0012). In both prostate and colorectal cancer autochthonous models the proliferation index at time of death (prostate) or 6 months after tumor induction (colorectal) was similar in irradiated and control cohorts.Hypofractionated radiation therapy can be safely administered to the abdominopelvic region in mice and improves survival in autochthonous mouse models of prostate and colorectal cancer. Radiation can induce pathologic complete responses in flank xenografts, but not autochthonous tumors, suggesting the presence of radioresistant persister cells in these models.

Abstract 4890: Preclinical analysis of combination therapy using radiation and cellular immunotherapy

Abstract Purpose: To optimize the combination of ionizing radiation and cellular immunotherapy using a preclinical autochthonous model of prostate cancer. Methods and Materials: Transgenic mice expressing a model antigen under a prostate-specific promoter were treated using a platform that integrates cone-beam CT imaging with 3-dimensional conformal therapy. Using this technology we investigated the immunologic and therapeutic effects of combining ionizing radiation with granulocyte/macrophage colonystimulating factor-secreting cellular immunotherapy for prostate cancer in mice bearing autochthonous prostate tumors. Results: The combination of ionizing radiation and immunotherapy resulted in a significant decrease in pathologic tumor grade and gross tumor bulk that was not evident with either single-modality therapy. Furthermore, combinatorial therapy resulted in improved overall survival in a preventive metastasis model and in the setting of established micrometastases. Mechanistically, combined therapy resulted in an increase of the ratio of effector-to regulatory T cells for both CD4 and CD8 tumor-infiltrating lymphocytes. Conclusions: Our preclinical model establishes a potential role for the use of combined radiation-immunotherapy in locally advanced prostate cancer, which warrants further exploration in a clinical setting. Citation Format: Satoshi Wada. Preclinical analysis of combination therapy using radiation and cellular immunotherapy. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4890.

Combined Treatment Effects of Radiation and Immunotherapy: Studies in an Autochthonous Prostate Cancer Model

To optimize the combination of ionizing radiation and cellular immunotherapy using a preclinical autochthonous model of prostate cancer. Transgenic mice expressing a model antigen under a prostate-specific promoter were treated using a platform that integrates cone-beam CT imaging with 3-dimensional conformal therapy. Using this technology we investigated the immunologic and therapeutic effects of combining ionizing radiation with granulocyte/macrophage colony-stimulating factor-secreting cellular immunotherapy for prostate cancer in mice bearing autochthonous prostate tumors. The combination of ionizing radiation and immunotherapy resulted in a significant decrease in pathologic tumor grade and gross tumor bulk that was not evident with either single-modality therapy. Furthermore, combinatorial therapy resulted in improved overall survival in a preventive metastasis model and in the setting of established micrometastases. Mechanistically, combined therapy resulted in an increase of the ratio of effector-to-regulatory T cells for both CD4 and CD8 tumor-infiltrating lymphocytes. Our preclinical model establishes a potential role for the use of combined radiation-immunotherapy in locally advanced prostate cancer, which warrants further exploration in a clinical setting.

Prostate carcinoma in transgenic Lewis rats - a tumor model for evaluation of immunological treatments.

Transgenic rodent models of prostate cancer have served as valuable preclinical models to evaluate novel treatments and understand malignant disease progression. In particular, a transgenic rat autochthonous model of prostate cancer using the SV40 large T antigen expressed under a prostate-specific probasin promoter was previously developed as a model of androgen-dependent prostate cancer (TRAP). In the current report, we backcrossed this strain to the Lewis strain, an inbred rat strain better characterized for immunological analyses. We demonstrate that Lewis transgenic rats (Lew-TRAP) developed prostate adenocarcinomas with 100% penetrance by 25 weeks of age. Tumors were predominantly androgen-dependent, as castration prevented tumor growth in the majority of animals. Finally, we demonstrate that Lew-TRAP rats could be immunized with a DNA vaccine encoding a human prostate tumor antigen (prostatic acid phosphatase) with the development of Lewis strain-specific T-cell responses. We propose that this Lew-TRAP strain, and prostate tumor cell lines derived from this strain, can be used as a future prostate cancer immunotherapy model.

The androgen receptor: a biologically relevant vaccine target for the treatment of prostate cancer

The androgen receptor (AR) plays an essential role in the development and progression of prostate cancer. However, while it has long been the primary molecular target of metastatic prostate cancer therapies, it has not been explored as an immunotherapeutic target. In particular, the AR ligand-binding domain (LBD) is a potentially attractive target, as it has an identical sequence among humans as well as among multiple species, providing a logical candidate for preclinical evaluation. In this report, we evaluated the immune and anti-tumor efficacy of a DNA vaccine targeting the AR LBD (pTVG-AR) in relevant rodent preclinical models. We found immunization of HHDII-DR1 mice, which express human HLA-A2 and HLA-DR1, with pTVG-AR augmented AR LBD HLA-A2-restricted peptide-specific, cytotoxic immune responses in vivo that could lyse human prostate cancer cells. Using an HLA-A2-expressing autochthonous model of prostate cancer, immunization with pTVG-AR augmented HLA-A2-restricted immune responses that could lyse syngeneic prostate tumor cells and led to a decrease in tumor burden and an increase in overall survival of tumor-bearing animals. Finally, immunization decreased prostate tumor growth in Copenhagen rats that was associated with a Th1-type immune response. These data show that the AR is as a prostate cancer immunological target antigen and that a DNA vaccine targeting the AR LBD is an attractive candidate for clinical evaluation.

Differential Requirement for CD70 and CD80/CD86 in Dendritic Cell-Mediated Activation of Tumor-Tolerized CD8 T Cells

A major obstacle to efficacious T cell-based cancer immunotherapy is the tolerizing-tumor microenvironment that rapidly inactivates tumor-infiltrating lymphocytes. In an autochthonous model of prostate cancer, we have previously shown that intratumoral injection of Ag-loaded dendritic cells (DCs) delays T cell tolerance induction as well as refunctionalizes already tolerized T cells in the tumor tissue. In this study, we have defined molecular interactions that mediate the effects of DCs. We show that pretreating Ag-loaded DCs with anti-CD70 Ab abolishes the ability of DCs to delay tumor-mediated T cell tolerance induction, whereas interfering with 4-1BBL, CD80, CD86, or both CD80 and CD86 had no significant effect. In contrast, CD80(-/-) or CD80(-/-)CD86(-/-) DCs failed to reactivate already tolerized T cells in the tumor tissue, whereas interfering with CD70 and 4-1BBL had no effect. Furthermore, despite a high level of programmed death 1 expression by tumor-infiltrating T cells and programmed death ligand 1 expression in the prostate, disrupting programmed death 1/programmed death ligand 1 interaction did not enhance T cell function in this model. These findings reveal dynamic requirements for costimulatory signals to overcome tumor-induced tolerance and have significant implications for developing more effective cancer immunotherapies.

Adaptive immune response initiated by high mobility group box 1 promotes tumor progression (48.36)

Abstract Cancers are often observed to be associated with infiltrating lymphocytes. However, the role of lymphocytes in regulating pro-tumor versus anti-tumor immune responses remains controversial. We report that lymphocytes are essential for tumor progression in an autochthonous prostate cancer model. Activated T lymphocytes gradually accumulate inside the tumor during the process of de novo spontaneous tumor development driven by the tissue-specific expression of an oncogene. T cell infiltration is essential for the increased CD11b+Gr-1-F4/80+ macrophages inside the tumor. Intriguingly, blockade of high mobility group box 1 (HMGB1) drastically inhibits tumor-antigen-specific T cell activation, infiltration and tumor progression. Our study has demonstrated that “endogenous danger signals” like HMGB1 produced by a growing tumor are essential to bridge innate and adaptive immune responses, which could further promote tumor progression over time. Our finding may have important implications for future cancer prevention and therapy.

Cutting Edge: Delay and Reversal of T Cell Tolerance by Intratumoral Injection of Antigen-Loaded Dendritic Cells in an Autochthonous Tumor Model

The tumor environment exerts a powerful suppressive influence on infiltrating tumor-reactive T cells. It induces tolerance of adoptively transferred effector T cells as they enter tumors and maintains the tolerance of persisting tumor-infiltrating T cells. In an autochthonous prostate cancer model, in which tumor-reactive CD8 T cells are trackable, we demonstrate that both depletion of endogenous dendritic cells (DCs) and intratumoral injection of Ag-loaded mature DCs delayed the tolerization of tumor-infiltrating effector CD8 T cells. Intratumoral injection of Ag-loaded DCs also reactivated tolerized CD8 T cells in the tumor tissue. The observed effects lasted as long as the injected DCs persisted. These findings are consistent with a critical role of DCs in modulating T cell reactivity in the tumor environment. They also suggest new potential strategies to extend the functionality of transferred effector T cells and to restore function to tolerized tumor-infiltrating T cells for cancer immunotherapy.

Cyclophosphamide Augments Antitumor Immunity: Studies in an Autochthonous Prostate Cancer Model

To study the immune response to prostate cancer, we developed an autochthonous animal model based on the transgenic adenocarcinoma of the mouse prostate (TRAMP) mouse in which spontaneously developing tumors express influenza hemagglutinin as a unique, tumor-associated antigen. Our prior studies in these animals showed immunologic tolerance to hemagglutinin, mirroring the clinical situation in patients with cancer who are generally nonresponsive to their disease. We used this physiologically relevant animal model to assess the immunomodulatory effects of cyclophosphamide when administered in combination with an allogeneic, cell-based granulocyte-macrophage colony-stimulating factor-secreting cancer immunotherapy. Through adoptive transfer of prostate/prostate cancer-specific CD8 T cells as well as through studies of the endogenous T-cell repertoire, we found that cyclophosphamide induced a marked augmentation of the antitumor immune response. This effect was strongly dependent on both the dose and the timing of cyclophosphamide administration. Mechanistic studies showed that immune augmentation by cyclophosphamide was associated with a transient depletion of regulatory T cells in the tumor draining lymph nodes but not in the peripheral circulation. Interestingly, we also noted effects on dendritic cell phenotype; low-dose cyclophosphamide was associated with increased expression of dendritic cell maturation markers. Taken together, these data clarify the dose, timing, and mechanism of action by which immunomodulatory cyclophosphamide can be translated to a clinical setting in a combinatorial cancer treatment strategy.

Tumor Recognition and Self-Recognition Induce Distinct Transcriptional Profiles in Antigen-Specific CD4 T Cells

Tumors express a wide variety of both mutated and nonmutated Ags. Whether these tumor Ags are broadly recognized as self or foreign by the immune system is currently unclear. Using an autochthonous prostate cancer model in which hemagglutinin (HA) is specifically expressed in the tumor (ProHA x TRAMP mice), as well as an analogous model wherein HA is expressed in normal tissues as a model self-Ag (C3HA(high)), we examined the transcriptional profile of CD4 T cells undergoing Ag-specific division. Consistent with our previous data, transfer of Ag-specific CD4 T cells into C3HA(high) resulted in a functionally inactivated CD4 T cell profile. Conversely, adoptive transfer of an identical CD4 T cell population into ProHA x TRAMP mice resulted in the induction of a regulatory phenotype of the T cell (Treg) both at the transcriptional and functional level. Interestingly, this Treg skewing was a property of even early-stage tumors, suggesting Treg induction as an important tolerance mechanism during tumor development.

Blocking the regulatory T cell molecule LAG-3 augments in vivo anti-tumor immunity in an autochthonous model of prostate cancer

2573 Background: LAG-3 is a CD4 homolog expressed on activated T cells, NK cells, tumor infiltrating lymphocytes (TIL), and plasmacytoid dendritic cells. Recently, we showed that LAG-3 was relatively overexpressed in specific T cells rendered unresponsive in vivo by the presence of cognate self-antigen. These anergic T cells display regulatory function both in vitro and in vivo, and blockade of LAG-3 with a non-depleting monoclonal antibody significantly mitigates their regulatory T cell activity. Methods: Using a novel model of prostate cancer in which a tumor-specific antigen is expressed in autochthonous tumors, we tested whether treatment with a non-depleting anti-LAG-3 antibody affected trafficking and function of tumor-specific T cells. Results: LAG-3 blockade significantly augments specific CD8 T cell trafficking to antigen-expressing tumors, but not to normal tissue. Most significantly, LAG-3 blockade functionally reversed CD8 T cell tolerance as assayed by an in vivo cytotoxic T lymphocyte (CTL) assay. Combining LAG-3 blockade with specific anti-tumor vaccination results in a dramatic increase in activated CD8 T cells in the tumor parenchyma. Conclusions: Taken together, these data support the concept that treatment with a LAG-3 blocking antibody may significantly delay disease progression in patients with cancer. We have recently generated a panel of monoclonal antibodies directed against human LAG-3; several of these antibodies significantly augment human T cell responses in vitro. No significant financial relationships to disclose.

LONGITUDINAL COHORT ANALYSIS OF LETHAL PROSTATE CANCER PROGRESSION IN TRANSGENIC MICE

Purpose Human prostate cancer is variably lethal, shows heterogeneous progression, and exhibits a spectrum of histopathology. Traditional rodent models of prostate cancer lack these characteristics. An alternative, autochthonous model of prostate cancer consists of transgenic mice which develop prostate cancer due to prostatic expression of SV40 T antigen. Lethal progression of such cancers in individual mice has not been previously characterized. Studies were undertaken to characterize the longitudinal progression of prostate cancers in these transgenic mice. Methods A prospective longitudinal cohort study was undertaken to characterize prostate cancer volume, progression, lethality, and histological heterogeneity in a transgenic mouse model of prostatic adenocarcinoma. Fifty-one transgenic mice were followed prospectively to determine the age at onset of palpable tumor and age at cancer-related death. Tumor volume was followed longitudinally by magnetic resonance imaging (MRI) in a subset of these mice and lethal cancers were evaluated by histopathology. Results Primary tumors became palpable at 10-38 weeks of age. Palpable tumors always preceded lethal progression. Cancer death followed 2-9 weeks later, and age at cancer death varied from 24 to 39 weeks of age. The histopathological changes were heterogeneous. Primary tumors were detectable by MRI before they became detectable by palpation. MRI showed that, analogous to human prostate cancers, volume of early stage primary tumors did not necessarily predict age at cancer death. Conclusion Prostate cancer in transgenic mice mimics heterogeneic tumor progression in human prostate cancer, providing a uniquely relevant pre-clinical model. Tumor detection by MRI and palpation are valid surrogate measures of tumor progression in this model.