Background <p>Prostate cancer (PCa) bone lesions are bone-forming, which contribute to an immunosuppressive bone-tumor microenvironment (bone-TME). Targeting PCa-induced bone with the radiopharmaceutical Radium-223 (Ra223) is approved for men with bone-metastatic castration-resistant PCa (bmCRPC). We studied the effect of Ra223 on spatial localization of tumor and immune cells in bmCRPC tumor biopsies to gain insights into therapeutic immunomodulation of the bone-TME.</p> Methods <p>Transiliac bone marrow biopsies were performed at baseline and end of treatment (EOT) in 24 patients with bmCRPC treated with Ra223. In three patients with paired specimens containing tumor, multiplex immunofluorescence was performed with antibodies for a tumor cell marker (CK), T-cell markers (CD3, CD4, CD8, FOXP3, and granzyme B), macrophage markers (CD68 [total macrophages] and CD206 [M2-like macrophages]).</p> Results <p>In all three patients, we observed enrichment of CD68⁺ macrophages, around tumor-induced bone at baseline. CD3 staining revealed T cell depletion where tumor cells formed large sheets but not where tumor cells were scattered. The effects of Ra223 on tumor cells and T cell subsets were heterogeneous. In one patient, tumor cells were reduced, and CD3⁺/CD8⁺ T cells were increased. In a second patient who evidenced osteolytic progression, tumor cells were increased while CD3⁺/CD8⁺ T cells were decreased. In a third patient, there was high number of tumor cells at baseline that modestly decreased and the number of CD3⁺/CD8⁺ T cells modestly increased after Ra223 treatment.</p> Conclusions <p>The changes in tumor cell numbers after Ra223 treatment seemed to be inversely correlated with changes in CD3⁺/CD8⁺ and CD4⁺/FoxP3⁺ cells, but not with other immune cells, in these 3 patients. Further characterization of immune cells in the bone-TME will be required before developing strategies to enhance immunotherapy efficacy in bmCRPC.</p> Clinical trial registration <p>This study does not report the clinical results of a clinical trial, but it does use samples from a completed clinical trial that is registered with clinicaltrials.gov (NCT02135484).</p>