Tumor vasculature proliferates rapidly, generally lacks pericyte coverage, and is uniquely fragile making it an attractive therapeutic target. A subset of small-molecule tubulin binding agents cause disaggregation of the endothelial cytoskeleton leading to enhanced vascular permeability generating increased interstitial pressure. The resulting vascular collapse and ischemia cause downstream hypoxia, ultimately leading to cell death and necrosis. Thus, local damage generates massive amplification and tumor destruction. The tumor vasculature is readily accessed and potentially a common target irrespective of disease site in the body. Development of a therapeutic approach and particularly next generation agents benefits from effective non-invasive assays. Imaging technologies offer varying degrees of sophistication and ease of implementation. This review considers technological strengths and weaknesses with examples from our own laboratory. Methods reveal vascular extent and patency, as well as insights into tissue viability, proliferation and necrosis. Spatiotemporal resolution ranges from cellular microscopy to single slice tomography and full three-dimensional views of whole tumors and measurements can be sufficiently rapid to reveal acute changes or long-term outcomes. Since imaging is non-invasive, each tumor may serve as its own control making investigations particularly efficient and rigorous. The concept of tumor vascular disruption was proposed over 30 years ago and it remains an active area of research.

The selective disruption of tumor-associated vasculature represents an attractive therapeutic approach. We have undertaken the first in vivo evaluation of KGP265, a water-soluble prodrug of a benzosuberene-based tubulin-binding agent, and found promising vascular-disrupting activity in three distinct tumor types. Dose escalation in orthotopic MDA-MB-231-luc breast tumor xenografts in mice indicated that higher doses produced more effective vascular shutdown, as revealed by dynamic bioluminescence imaging (BLI). In syngeneic orthotopic 4T1-luc breast and RENCA-luc kidney tumors, dynamic BLI and oxygen enhanced multispectral optoacoustic tomography (OE-MSOT) were used to compare vascular shutdown following the administration of KGP265 (7.5 mg/kg). The BLI signal and vascular oxygenation response (ΔsO2) to a gas breathing challenge were both significantly reduced within 2 h, indicating vascular disruption, which continued over 24 h. A correlative histology confirmed increased necrosis and hemorrhage. Twice-weekly doses of KGP265 caused significant growth delay in both MDA-MB-231 and 4T1 breast tumors, with no obvious systemic toxicity. A combination with carboplatin produced significantly greater tumor growth delay than carboplatin alone, though significant carboplatin-associated toxicity was observed (whole-body weight loss). KGP265 was found to be effective at low concentrations, generating long-term vascular shutdown and tumor growth delay, thus providing strong rationale for further development, particularly in combination therapies.

The vascular disrupting activity of a promising tubulin-binding agent (OXi6196) was demonstrated in mice in MDA-MB-231 human breast tumor xenografts growing orthotopically in mammary fat pad and syngeneic RENCA kidney tumors growing orthotopically in the kidney. To enhance water solubility, OXi6196, was derivatized as its corresponding phosphate prodrug salt OXi6197, facilitating effective delivery. OXi6197 is stable in water, but rapidly releases OXi6196 in the presence of alkaline phosphatase. At low nanomolar concentrations OXi6196 caused G2/M cell cycle arrest and apoptosis in MDA-MB-231 breast cancer cells and monolayers of rapidly growing HUVECs underwent concentration-dependent changes in their morphology. Loss of the microtubule structure and increased bundling of filamentous actin into stress fibers followed by cell collapse, rounding and blebbing was observed. OXi6196 (100 nM) disrupted capillary-like endothelial networks pre-established with HUVECs on Matrigel®. When prodrug OXi6197 was administered to mice bearing orthotopic MDA-MB-231-luc tumors, dynamic bioluminescence imaging (BLI) revealed dose-dependent vascular shutdown with >80% signal loss within 2 h at doses ≥30 mg/kg and >90% shutdown after 6 h for doses ≥35 mg/kg, which remained depressed by at least 70% after 24 h. Twice weekly treatment with prodrug OXi6197 (20 mg/kg) caused a significant tumor growth delay, but no overall survival benefit. Similar efficacy was observed for the first time in orthotopic RENCA-luc tumors, which showed massive hemorrhage and necrosis after 24 h. Twice weekly dosing with prodrug OXi6197 (35 mg/kg) caused tumor growth delay in most orthotopic RENCA tumors. Immunohistochemistry revealed extensive necrosis, though with surviving peripheral tissues. These results demonstrate effective vascular disruption at doses comparable to the most effective vascular-disrupting agents (VDAs) suggesting opportunities for further development.

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