Combined treatment effects of radiation and immunotherapy: studies in an autochthonous prostate cancer model

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 colony-stimulating 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.

Figure 1. Focal radiation of autochthonous prostate tumors

A: Detection and targeting of the mouse prostate gland using the Cone-Beam Computed Tomography (CBCT) mode of the Small Animal Radiotherapy Research Platform (SARRP). Yellow rectangles represent treatment planning – a parallel-opposed, lateral beam arrangement was utilized with the central axis of each beam being localized to a point along the line that joined the pelvic bone and the posterior surface of the bladder. Yellow boxes indicate prostate situated 5.5-mm anterior to sacrum, and posterior to bladder. B, C: H2AX Immunostaining. ProHA/TRAMP mice were irradiated using the SARRP and sacrificed 30 minutes post-therapy. Untreated, age-matched non-transgenic (B10.D2) or ProHA/TRAMP served as negative controls. (B) Ventral/Dorsal prostate. (C) Bladder/Rectum.

Figure 2. Combined immunotherapy and radiation of autochthonous prostate tumors

A: Interaction between radiation and immunotherapy (T-GVAX). 12 Gy and/or immunotherapy were administered on the indicated days. Adoptively transferred, prostate-specific CD8⁺ T cells were administered on day -2, and harvested 9 days later in all groups. Prostate-specific CD8⁺ T cells were quantified from the indicated sites using flow cytometry. B, C: Optimization of combined radiation and-immunotherapy. (B) Experimental design. 12 Gy and/or immunotherapy were administered on the indicated days. Adoptively transferred, prostate-specific CD8⁺ T cells were administered on day -2, and harvested 9 days later in all groups. When co-administered on the same day, immunotherapy was given immediately following radiation. (C) Prostate-specific effector cells (lower panel), quantified using intracellular staining (ICS) for IFN-γ following ex vivo peptide stimulation (upper panel). D: Prostate infiltrating Clone 4 cells following titration of radiation dose. Radiation and immunotherapy administered on the same day. Experiments had 3–5 mice per group and were repeated twice.

Figure 3. Anti-tumor efficacy of combined immunotherapy/radiation

A: Experimental design. 12–14 week ProHA/TRAMP mice were treated with radiation, immunotherapy (T-GVAX), or combined therapy. Animals receiving vaccination were given boost vaccinations days 7 and 14 post-radiation. Animals were harvested between 22–24 weeks of age. B: Wet weight of the urogenital tract. C: Pathologic tumor score, see methods for details. D: Quantification of prostate-specific CD8⁺ T cells in the periphery. 4 weeks post-boost, HAtetramer positive CD8⁺ T cells and effector cells (CD62L^low, CD95⁺) were quantified. Experiments had 8–10 mice per group and were repeated twice.

Figure 4. Immunological mechanisms of combination radiation-immunotherapy

A: Prostate-specific CD4⁺ T cell expansion. Adoptively transferred CD4⁺ T cells from TCR transgenic donors (6.5) were harvested from indicated sites and quantified using FACS analysis. B: CD4⁺ T cell subsets. After adoptive transfer, HA-specific CD4⁺ T cells were analyzed for IFN-γ, IL-4, and IL-17 secretion by intracellular staining after ex vivo peptide stimulation. C: Regulatory T cells. Adoptively transferred prostate-specific (left panel) or endogenous (right panel) T_REG were quantified using ICS for FoxP3. D: Effector/Regulatory ratio. Ratios were calculated using absolute cell numbers of IFN-γ secreting, HA-specific T cells to FoxP3⁺ T_REG. Experiments had 5 mice per group and were repeated twice.

Figure 5. Effect of radiation and immunotherapy on metastatic disease

A: Prevention model. 20–22 week ProHA/TRAMP mice received 1×10⁶ syngeneic SWPC1 prostate tumor cells IV. Immunotherapy and radiation were administered as shown. B: Treatment model. As in panel A, with treatment initiated 3 days post tumor inoculation. Experiments had 5–8 mice per group and were repeated once.