Low-dose TRT reshapes the microenvironment of prostate tumors to potentiate response to immunotherapy

36 Objectives: Due to a markedly immunosuppressive tumor microenvironment (TME), immunotherapy has had limited success in prostate cancer (PCa). Radiation therapy, which has shown to remodel the TME of irradiated tumors towards a more immune-susceptible phenotype even at low radiation doses of 2-3 Gy, is limited to the localized disease setting. Our goal is to demonstrate that systemically delivered targeted radionuclide therapy (TRT) using [90Y]Y-NM600 provides beneficial immunomodulatory effects that may enhance the response of PCa to immunotherapies. Methods: Male FVB mice bearing syngeneic prostate Myc-CaP tumor allografts (n=3). were administered 9.25 MBq of the positron-emitting [86Y]Y-NM600, and longitudinal static PET/CT scans were acquired at 3, 24, 48, and 72 h post-injection. A region-of-interest analysis of the PET images quantified the radiotracer uptake in Myc-CaP tumors and healthy tissues, which allowed the dosimetry estimation of [90Y]Y-NM600 TRT. In monotherapy studies, mice bearing Myc-CaP grafts (200 mm3; n=5) were administered 1.85 (low-dose) or 9.25 MBq (high-dose) [90Y]Y-NM600, delivering 4 Gy or 20 Gy to the tumor, respectively, and tumor growth and survival were monitored for 60 days. Bone marrow health was monitored periodically via complete blood count (CBC). To study the immunological effects of [90Y]Y-NM600 in the TME and lymphoid tissues (e.g., spleen and bone marrow), groups of mice (n=3) were injected with 1.85 or 9.25 MBq [90Y]Y-NM600, sacrificed at days 1, 4, 7, 14, and 21 post-injection, and Myc-CaP tumors and spleens were analyzed by flow cytometry, immunohistochemistry, and Luminex cytokine profiling. Results: PET/CT imaging revealed selective uptake and retention of [86Y]Y-NM600 in Myc-CaP tumors and gradual clearance from normal tissues. Absorbed doses per [90Y]Y-NM600 injected activity of 1.97, 1.11, and 0.53 Gy/MBq were estimated for the tumor, spleen, and bone marrow, respectively. Mice receiving a [90Y]Y-NM600 activity delivering 20 Gy, but not 4 Gy, to the tumor had significantly (p=0.004) extended survival but no durable responses. Flow cytometry analysis of splenocytes and CBC analysis showed transient lymphocyte depletion in mice injected with 9.25 MBq, with no effect in the low 1.85 MBq group. Low and high dose treatments had a similar impact on the TME showing non-specific depletion (p< 0.01) of tumor-infiltrating (CD45+) lymphocytes with a nadir at day 4 post-injection. At day 7, repopulation of the TME by activated CD8+ T-cells overexpressing the checkpoints molecules CTLA-4, PD-1, and LAG-3 began. Conversely, CD4+ T-cells remained excluded from the TME for at least 21 days after treatment. Interestingly, cytokine profiling uncovered increases in several proinflammatory cytokines and chemokines, including CXCL1, CXCL2, IL-6, RANTES, and CCL-3, that were highest in tumors receiving 4 Gy. In tumor cells, PD-L1 expression peaked (p<0.0001) on day 4, suggesting the induction of an IFN-γ related response to radiation. Conclusions: Our data indicate that [90Y]Y-NM600 immunomodulates the TME of prostate tumors by modifying TIL populations, upregulating checkpoint molecules, and promoting the release of proinflammatory cytokines. More importantly, [90Y]Y-NM600’s pro-inflammatory effects on the TME are elicited at relatively low tumor absorbed doses without incurring systemic toxicity, thus providing a strong rationale for combining low-dose TRT with immunotherapies such as checkpoint blockade.