Background: Radium-223 is a targeted alpha-therapy that improves survival in men with mCRPC. The biologic basis for radium-223 efficacy is not completely understood. We hypothesized that PC osteomimicry, a form of epithelial plasticity leading to an osteoblastic phenotype, may contribute to the intralesional deposition of radium-223 and subsequent irradiation of the tumor microenvironment. Methods: We conducted a pharmacodynamic study (NCT02204943) of radium-223 in men with bone metastatic CRPC to investigate genomic and phenotypic alterations in circulating tumor cells (CTCs), ctDNA, and metastases. Prior to and 3 & 6 months after radium, we collected liquid and metastatic biopsies including CTCs for phenotypic characterization and CTC/ctDNA genomic analysis. The primary objective was to describe the prevalence of CTC bone alkaline phosphatase (BAP) over time. We measured radium-223 decay products in tumor and surrounding normal bone during treatment. Results: We enrolled 20 men with heavily pre-treated symptomatic bone predominant mCRPC and treated with radium-223 over a median of 6 doses; 55% had elevated serum BAP. PFS was 5.5 mo; OS was 13.3 mo; 13% had unfavorable (≥5) to favorable ( < 5) Cellsearch CTC conversion; 5% had ≥30% PSA decline. We found evidence of persistent BAP+CTCs in the majority of men over time during radium-223 therapy despite serum BAP normalization in 53% of men. We identified genomic gain of key osteomimicry regions in CTC DNA, including gains of BAP, osteopontin, and OB-cadherin. We observed greater gamma emission from radium-223 from tumor biopsies than adjacent normal bone. CTC DNA and matched ctDNA studies suggested persistence of aggressive genomic alterations such as AR, FOXA1, and MYC/N-MYC gain and PTEN, GRHL2, FGFR2, and BRCA1 loss. We established several prostate CTC cultures exhibiting evidence of epithelial plasticity and BAP expression. Conclusions: Osteomimicry may contribute to the uptake of Radium-223 within bone metastases and may thereby enhance the therapeutic benefit of radium-223. We found genomic and phenotypic evidence of osteomimicry in CTCs from men with mCRPC. Clinical trial information: NCT02204943