A radiotheranostic study for strategic treatment of ovarian cancer peritoneal metastases using the all-in-one multimeric radiopeptide ⁶⁴Cu-cyclam-RAFT-c(-RGDfK-)₄

Objectives: We have successfully developed ⁶⁴Cu-cyclam-RAFT-c(-RGDfK-)₄ (⁶⁴Cu-RaftRGD) as a multimeric RGD-based radiopeptide for specifically targeting the transmembrane cell adhesion receptor, α_Vβ₃ integrin (α_Vβ₃). Ovarian cancer peritoneal metastases (OCPMs) are a pathophysiologically heterogeneous group of tumors. Despite advances in surgical cytoreduction and drug development, the 5-year survival rate for OCPM patients remains as low as <30%. The α_Vβ₃ is overexpressed on tumoral neovessels and also on ovarian cancer cells and the radioisotope ⁶⁴Cu has a suitable half-life (12.7 h) and multiple decay modes for both PET imaging and therapeutic irradiation. Hence, the present study aimed to evaluate the radiotheranostic potential of ⁶⁴Cu-RaftRGD in clinically relevant α_Vβ₃-positive OCPM small animal models.

Methods: Athymic BALB/c nude mice with intraperitoneally inoculated ovarian carcinoma IGR-OV1 and NIH:OVCAR-3 cells were used as the OCPM small animal models, and their corresponding subcutaneous xenografts were used as a reference. ⁶⁴Cu-RaftRGD was administered either intravenously or intraperitoneally to determine the optimal injection route. We performed intratumoral distribution (ITD) studies, PET/CT imaging and quantification, biodistribution assay and radiation dosimetry, and therapeutic efficacy and toxicity studies.

Results: Intraperitoneal administration (i.p.) was shown to be the efficient route for targeting ⁶⁴Cu-RaftRGD to OCPMs with excellent tumor penetration. The fluorescent surrogate Cy5.5-labeled RaftRGD and high-resolution multifluorescence imaging found that the ITD of ⁶⁴Cu-RaftRGD was colocalized with CD31-stained microvessels and spatially distinct from but complementary to that of pimonidazole-stained hypoxia. ⁶⁴Cu-RaftRGD (i.p.) PET visualized multiple OCPM deposits and ascites. The biodistribution study demonstrated an inverse correlation between the tumor size (1.2-17.2 mm) and tumor uptake levels (also absorbed doses). ⁶⁴Cu-RaftRGD at a radiotherapeutic dose (148 MBq/0.357 nmol, i.p.) showed antitumor activities by inhibiting tumor cell proliferation and inducing apoptosis detected on day 3 after therapy, and significantly prolonged the survival of mice. The toxicity evaluation of 148 MBq/0.357 nmol i.p. ⁶⁴Cu-RaftRGD in normal mice for 60 days after administration demonstrated negligible toxicity in hematology and hepatorenal functions. Conclusion: Our results demonstrate the all-in-one potential of the i.p. ⁶⁴Cu-RaftRGD as a tumor penetrating radiodrug for PET imaging-guided radiotherapy of OCPMs by targeting both tumoral neovessels and cancerous cells, with negligible toxicity. We propose that further study of intra- and intertumoral heterogeneity of the radiodrug in relation to the tumor microenvironment may provide a basis for understanding the treatment limitation and facilitate a rational combination therapy design.