Background Treatment of solid tumors with vascular disrupting agent OXi4503 results

Background Treatment of solid tumors with vascular disrupting agent OXi4503 results in over 90% tumor destruction. the periphery with mature vessels, higher accumulation of immune cells, increased growth factor expression, minimal levels of hypoxia and increased evidence of EMT. OXi4503 treatment resulted in collapse of vessels in the tumor center; however vasculature in the periphery remained patent. Similarly, tumor apoptosis and proliferation were differentially modulated between centre and periphery after treatment. Conclusions The molecular and morphological differences between 43168-51-0 manufacture tumor periphery and center may account for the observed differential resistance to OXi4503 treatment and could provide targets for drug development to totally eliminate metastases. by serial passage in the flanks of CBA mice [15]. For passage and experimentation, subcutaneous tumors were teased, passed through a filter, treated with EDTA and washed in PBS to make a single cell suspension. Liver metastases were induced by intrasplenic injection of 5×104 tumor cells prior to splenectomy as reported previously [15]. In this model, liver metastases are fully established by 21 days following tumor induction. The tumor morphology and growth patterns in this model have Gdf5 been described previously [6,15,16]. Metastases of varying sizes are found throughout the liver. The metastasis pattern is very similar and reproducible within a group of mice. The whole liver is sliced in sections of 2 mm thickness. Cross-sections of the larger tumors are represented in more than one section. Random sections are selected to represent the entire 43168-51-0 manufacture liver and used for paraffin embedding and analysis. Each section could contain from one to several individual tumors (Additional file 1: Figure S1). Metastases seeded in close proximity often coalesce into a continuous tumor. Treatment protocol Treatment was administered sixteen days after induction of liver metastases when tumors are well established. OXi4503, kindly donated by OXiGENE (OXiGENE? Inc. South San Francisco, CA), 43168-51-0 manufacture was freshly prepared by 43168-51-0 manufacture dissolving in 0.9% sterile saline (NaCl) and protected from light. A single maximum tolerated dose of OXi4503, determined previously to be 100 mg/kg [16], was administered via intraperitoneal injection. Control groups were administered an equivalent volume of sterile saline. Tissues were collected at one hour, twenty four hours and five days following OXi4503 treatment. Definition of tumor periphery Tumor periphery in our studies consisted of the area covering the tumor-host interface and extending one hundred microns towards the tumor center. All the remaining tumor area was considered part of the tumor center. Vascular morphology Vessel morphology was examined microscopically in stained tumor sections. Immature vessels and/or vessels undergoing angiogenesis were detected by CD34 staining [17]. All CD34 positive vessels/mm2 in each tumor section were counted. Vessel stability and maturity were also assessed by pericyte coverage and angiopoetin 1(Ang1) association [18]. The presence of pericytes was visualised by SMA immunostaining and enumerated by counting of SMA positive tumor vessels in serial sections stained for SMA or CD34. Only vessels that stained for both markers were included in the enumeration. Ang1 association was determined by double immunostaining for Ang1 and CD34. Detection of tumor hypoxia Pimonidazole was used as a marker of tumor hypoxia. Pimonidazole hydrochloride was dissolved into 0.9% NaCl and administered intravenously to tumor-bearing mice in doses of 30 mg/kg. The livers were removed one hour after pimonidazole administration and fixed in 10% formalin in 0.1M phosphate buffer, pH 7.2. Hypoxic tumor regions were detected immunohistochemically as reported previously [19]. Assessment of epithelial to mesenchymal transition (EMT) The main indicators of EMT are down regulation of the cell junction protein E-cadherin, nuclear accumulation of -catenin another junctional protein, up regulation of the mesenchymal marker vimentin and up regulation of transcription inhibitors of epithelial proteins such as ZEB1.