VEGFR-targeted angiogenesis imaging for therapeutic evaluation in glioblastoma

Objectives To develop a novel optical probe targeting VEGFR (vascular endothelial growth factor receptor) for imaging angiogenesis in glioblastoma, and to expand its imaging capability to evaluate anticancer therapies against the tumor.

Methods A dimeric ZD-G2 derivative of ZD6474 was synthesized and conjugated with a Cy5.5 fluorescent moiety to generate a novel optical probe targeting VEGFR. Its targeting specificity was evaluated in a human glioblastoma cell line U87-MG and a mouse xenograft model. Furthermore, to illustrate regulation of epigenetics on angiogenesis, an FDA approved drug SAHA (targeting histone deacetylases [HDACs]) was administered in the mice, followed by angiogenesis-imaging studies. Blocking, ex vivo and biodistribution studies validated the imaging efficacy of the probe.

Results Receptor binding assays indicated that ZD-G2 had a high binding affinity for VEGFR with a dissociation constant of 0.45 nM. In vitro imaging study with U87-MG cells confirmed the specificity of the probe for VEGFR. In vivo experiments using the mouse xenograft model of U87-MG showed efficient imaging capability, with specific enrichment of the probe in tumors being visible as early as 6h post-injection. The fluorescent signal peaked at 24h with a tumor-to-background ratio of 1.83±0.11 (n=3). Blocking experiments using unlabeled ZD-G2 established the binding specificity. Interestingly, prior treatment of SAHA (15mg/Kg), a HDAC pan-inhibitor, significantly reduced VEGFR-targeted imaging signals (P<0.01, n=3), suggesting VEGFR levels had been down-regulated by HDACs in the tumors. Immunohistochemistry confirmed the correlation of the levels of HDACs and VGEFR in the U87-MG tumors.

Conclusions A novel angiogenesis-imaging probe ZD-G2-Cy5.5 has been successfully developed and evaluated for its ability to visualize glioblastoma in vivo. Its potentiality of evaluating therapeutic response of anticancer treatment has been demonstrated by an HDAC-VEGFR system in vivo, which reveals an intriguing mechanism where angiogenesis may be regulated by HDACs during glioblastoma tumorigenesis.