B7–H4 ImmunoPET Imaging Tracks Tumor-Associated Macrophage Changes in Prostate Cancer

B7-H4 is an inhibitory immune checkpoint molecule that is upregulated in various cancers and correlates with advanced tumor stages and poor clinical outcomes. This study aimed to develop an immunoPET radiotracer for noninvasive assessment of B7-H4 expression in tumors and tumor-associated macrophages (TAM) and to evaluate the radiotracer potential to monitor therapeutic responses. We generated a B7-H4-targeted immunoPET imaging tracer by radiolabeling the anti-B7-H4 monoclonal antibody (2H9) with [⁸⁹Zr], yielding [⁸⁹Zr]Zr-DFO-2H9, and assessed its biodistribution in prostate cancer xenografts to quantitatively measure B7-H4 expression in vivo. In vitro binding studies confirmed the retained immunoreactivity and specificity for B7-H4. Radiochemical purity was verified using size exclusion chromatography. In vivo evaluation of [⁸⁹Zr]Zr-DFO-2H9 was first performed in immunodeficient nude mice bearing subcutaneous DU145 human prostate tumors, with longitudinal PET imaging conducted over 7 days postinjection, followed by terminal biodistribution analysis. [⁸⁹Zr]Zr-DFO-2H9 demonstrated a good tumor-binding profile and specificity in DU145 tumor xenografts. To distinguish PET signals from tumor cells versus macrophages, immunocompetent C57BL/6 mice bearing syngeneic TRAMP-C2 prostate tumors were divided into three cohorts and treated with PBS (control), cold anti-B7-H4 mAb (for B7-H4 blockade), or clodronate liposome (for macrophage depletion). In TRAMP-C2 tumors, the PET signal was significantly reduced in both the B7-H4 blocked and macrophage-depleted group compared to controls. Immunohistochemistry revealed that B7-H4 expression differences among TRAMP-C2 treatment groups were not as clearly distinguishable as those observed in vivo via PET imaging. Multiplexed immunofluorescence staining of macrophage markers indicated that infiltrating TAMs were the major contributors to B7-H4-specific PET signals within the tumor stroma. Collectively, these results show that [⁸⁹Zr]Zr-DFO-2H9 binds B7-H4 with high affinity and specificity and reflects changes in TAM levels in vivo. The new radiotracer shows promise for detecting B7-H4 positive tumors and TAM levels, profiling the immune microenvironment, and monitoring macrophage-targeted immunotherapies.