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 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.

Tumor vasculature proliferates rapidly, generally lacks pericyte coverage, and is uniquely frag-ile 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 gener-ates 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 mi-croscopy 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 par-ticularly efficient and rigorous. The concept of tumor vascular disruption was proposed over 30 years ago and it remains an active area of research.

Tumor growth requires a functioning vascular network and offers an attractive therapeutic target. The invasive proliferating neovasculature of tumors lacks pericyte support and exhibits increased permeability thereby offering a unique, potentially selective target for anticancer therapy. Most vascular disrupting agents (VDAs) inhibit microtubule formation, causing rapid morphological changes in endothelia cells resulting in dramatically increased vessel permeability, cellular detachment, vessel occlusion, and vessel wall damage [doi: 10.3390/molecules26092551]. Highly vascular kidney tumors are expected to be particularly sensitive to VDAs. We are evaluating OXi8007, a water soluble indole phosphate pro-drug derivative of the first generation VDA combretastatin. The prodrug OXi8007 is stable in saline but releases active OXi8006 is the presence of alkaline phosphate. Preliminary pharmacokinetic data benchmarked using 13C-labeled OXi8006 and OXi8007 show rapid vascular clearance and conversion to the glucuronide conjugate in vivo. Doses up to 700 mg/kg were tolerated by mice. Luciferase-expressing RENCA tumor cells were implanted orthotopically in the right kidney capsule of syngeneic BALB/c mice. Bioluminescence Imaging (BLI) was applied weekly to assess tumor growth and acute response to OXi8007. Following addition of luciferin substrate subcutaneously BLI signal from the tumor reached a maximum within 10 minutes. When BLI was repeated 4 hrs after 250 or 350 mg/kg OXi8007 IP, the generated signal was <1% of baseline signal indicating acute vascular disruption. Multispectral Optoacoustic Tomography (MSOT) revealed matching acute changes in tumor vascular oxygenation and additionally showed distinct heterogeneity. An oxygen gas breathing challenge indicated lower oxygen saturation in the tumor following treatment. Histology showed massive hemorrhage following both doses. BLI signal recovered after 1 to 2 days and repeated administration of OXi8007 again caused acute vascular shutdown. These results match previous observations in breast tumor models [doi: 10.1016/j.canlet.2015.08.021] indicating opportunities for therapeutic application. Citation Format: Hashini I. Wanniarachchi, Regan Schuetze, Lorena Arango, Khagendra Hamal, Graham J. Carlson, Cyprian Pavlich, Yuling Deng, Mary L. Trawick, Kevin G. Pinney, Li Liu, Ralph P. Mason. Discerning vasculature destruction caused by novel vascular disrupting agents examined by optical and multispectral optoacoustic imaging [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6206.