fosbretabulin and Fibrosarcoma

Tumour vasculature is notoriously sinusoidal and leaky, and is hence susceptible to vascular disruption. Microtubule destabilising drugs such as the combretastatins form the largest group of tumour vascular disrupting agents and cause selective shutdown of tumour blood flow within minutes to hours, leading to secondary tumour cell death. Targeting the tumour vasculature is a proven anticancer strategy but early treatment response biomarkers are required for personalising treatment planning. Protein induction following treatment with combretastatin A4-phosphate was examined in a mouse fibrosarcoma model (fs188), where tumour cells express only the matrix-bound isoform of vascular endothelial growth factor A (VEGF188). These tumours are relatively resistant to vascular disruption by combretastatin A4-phosphate and hence a study of protein induction following treatment could yield insights into resistance mechanisms. The distribution of a number of proteins induced following treatment were visualised by MALDI-mass spectrometry imaging. Responses identified were validated by LC-ESI-MS/MS and immunohistochemical staining. Significant changes in proteins connected with necrosis, cell structure, cell survival and stress-induced molecular chaperones were identified. Protein-protein interactions were identified using STRING 9.0 proteomic network software. These relationship pathways provided an insight into the activity of the active tumour milieu and a means of linking the identified proteins to their functional partners.

Topics: Animals; Fibrosarcoma; Gene Expression Regulation, Neoplastic; Humans; Mice; Neovascularization, Pathologic; Protein Interaction Maps; Proteomics; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Stilbenes; Vascular Endothelial Growth Factor A

Based on the soil-to-seeds principle, we explored the small-molecular sequential dual-targeting theranostic strategy (SMSDTTS) for prolonged survival and imaging detectability in a xenograft tumor model.. Thirty severe combined immunodeficiency (SCID) mice bearing bilateral radiation-induced fibrosarcoma-1 (RIF-1) subcutaneously were divided into group A of SMSDTTS with sequential intravenous injections of combretastatin A4 phosphate (CA4P) and (131)I-iodohypericin ((131)I-Hyp) at a 24 h interval; group B of single targeting control with CA4P and vehicle of (131)I-Hyp; and group C of vehicle control (10 mice per group). Tumoricidal events were monitored by in vivo magnetic resonance imaging (MRI) and planar gamma scintiscan, and validated by ex vivo autoradiography and histopathology. Besides, 9 mice received sequential intravenous injections of CA4P and (131)I-Hyp were subjected to biodistribution analysis at 24, 72 and 120 h.. Gamma counting revealed fast clearance of (131)I-Hyp from normal organs but intense accumulation in necrotic tumor over 120 h. After only one treatment, significantly prolonged survival (p<0.001) was found in group A compared to group B and C with median survival of 33, 22, and 21 days respectively. Tumor volume on day 15 was 2.0 ± 0.89, 5.66 ± 1.66, and 5.02 ± 1.0 cm(3) with tumor doubling time 7.8 ± 2.8, 4.4 ± 0.67, and 4.5 ± 0.5 days respectively. SMSDTTS treated tumors were visualized as hot spots on gamma scintiscans, and necrosis over tumor ratio remained consistently high on MRI, autoradiography and histology.. The synergistic antitumor effects, multifocal targetability, simultaneous theranostic property, and good tolerance of the SMSDTTS were evident in this experiment, which warrants further development for preclinical and clinical applications.

Topics: Administration, Intravenous; Animals; Anthracenes; Antineoplastic Agents; Disease Models, Animal; Fibrosarcoma; Histocytochemistry; Humans; Iodine Radioisotopes; Magnetic Resonance Imaging; Male; Mice; Mice, SCID; Perylene; Radiography; Radionuclide Imaging; Stilbenes; Survival Analysis; Transplantation, Heterologous; Treatment Outcome

Characterising the protein signatures in tumours following vascular-targeted therapy will help determine both treatment response and resistance mechanisms. Here, mass spectrometry imaging and MS/MS with and without ion mobility separation have been used for this purpose in a mouse fibrosarcoma model following treatment with the tubulin-binding tumour vascular disrupting agent, combretastatin A-4-phosphate (CA-4-P). Characterisation of peptides after in situ tissue tryptic digestion was carried out using Matrix-Assisted Laser Desorption/Ionisation-Mass Spectrometry (MALDI-MS) and Matrix-Assisted Laser Desorption/Ionisation-Ion Mobility Separation-Mass Spectrometry Imaging (MALDI IMS-MSI) to observe the spatial distribution of peptides. Matrix-Assisted Laser Desorption/Ionisation-Ion Mobility Separation-Tandem Mass Spectrometry (MALDI-IMS-MS/MS) of peaks was performed to elucidate any pharmacological responses and potential biomarkers. By taking tumour samples at a number of time points after treatment gross changes in the tissue were indicated by changes in the signal levels of certain peptides. These were identified as arising from haemoglobin and indicated the disruption of the tumour vasculature. It was hoped that the use of PCA-DA would reveal more subtle changes taking place in the tumour samples however these are masked by the dominance of the changes in the haemoglobin signals.

Topics: Animals; Antineoplastic Agents, Phytogenic; Biomarkers, Tumor; Fibrosarcoma; Mice; Peptide Mapping; Proteins; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Stilbenes

Tubulin-binding vascular-disrupting agents (VDA) are currently in clinical trials for cancer therapy but the factors that influence tumor susceptibility to these agents are poorly understood. We evaluated the consequences of modifying tumor vascular morphology and function on vascular and therapeutic response to combretastatin-A4 3-O-phosphate (CA-4-P), which was chosen as a model VDA. Mouse fibrosarcoma cell lines that are capable of expressing all vascular endothelial growth factor (VEGF) isoforms (control) or only single isoforms of VEGF (VEGF120, VEGF164, or VEGF188) were developed under endogenous VEGF promoter control. Once tumors were established, VEGF isoform expression did not affect growth or blood flow rate. However, VEGF188 was uniquely associated with tumor vascular maturity, resistance to hemorrhage, and resistance to CA-4-P. Pericyte staining was much greater in VEGF188 and control tumors than in VEGF120 and VEGF164 tumors. Vascular volume was highest in VEGF120 and control tumors (CD31 staining) but total vascular length was highest in VEGF188 tumors, reflecting very narrow vessels forming complex vascular networks. I.v. administered 40 kDa FITC-dextran leaked slowly from the vasculature of VEGF188 tumors compared with VEGF120 tumors. Intravital microscopy measurements of vascular length and RBC velocity showed that CA-4-P produced significantly more vascular damage in VEGF120 and VEGF164 tumors than in VEGF188 and control tumors. Importantly, this translated into a similar differential in therapeutic response, as determined by tumor growth delay. Results imply differences in signaling pathways between VEGF isoforms and suggest that VEGF isoforms might be useful in vascular-disrupting cancer therapy to predict tumor susceptibility to VDAs.

Topics: Angiogenesis Inhibitors; Animals; Cell Line, Tumor; Enzyme-Linked Immunosorbent Assay; Female; Fibrosarcoma; Immunohistochemistry; Mice; Mice, SCID; Neovascularization, Pathologic; Protein Isoforms; RNA, Messenger; Stilbenes; Vascular Endothelial Growth Factor A

5,6-Dimethylxanthenone-4-acetic acid (DMXAA) and combretastatin A4 phosphate (CA-4-P) markedly inhibit tumor blood flow in mice and are both currently in clinical trial. One of the challenges in clinical evaluation of antivascular agents is the monitoring of tumor blood flow inhibition in individual patients. This study investigates, using mouse models, whether a new marker for tissue hypoxia, (99m)technetium-labeled 2,2'-(1,4-diaminobutane)bis(2-methyl-3-butanone) dioxime (99mTc-labeled HL-91; Prognox)] has potential for the scintigraphic monitoring of tumor response to antivascular agents. Determination of radioactivity in dissected tissues 3 h after DMXAA (80 micromol/kg) or CA-4-P (227 micromol/kg) was injected indicated that both drugs inhibited blood flow (86RbCl uptake; 84 and 87%, respectively) and increased 99mTc-labeled HL-91 levels (350 and 300%, respectively) selectively in murine RIF-1 tumors. Planar imaging of 99mTc-labeled HL-91 3 h after DMXAA injection showed a dose-dependent increase in tumor levels above a threshold of 50 micromol/kg; this same threshold was observed for the inhibition of tumor blood flow (determined using Hoechst 33342). DMXAA also inhibited blood flow--and increased 99mTc-labeled HL-91 uptake--in MDAH-MCa-4 mouse mammary carcinomas and in NZMN10 human melanoma xenografts. Whether 99mTc-labeled HL-91 might also be useful as a biomarker for tumor cell killing was investigated by clonogenic assay of surviving cells 15 h after imaging 99mTc-labeled HL-91 in RIF-1 tumors. Log cell kill in individual tumors showed a statistically significant linear correlation (P < 0.001) with 99mTc-labeled HL-91 uptake after 60 micromol/kg (r2 = 0.79) and 70 micromol/kg (r2 = 0.44) but not at 80 micromol/kg DMXAA. The lack of correlation at high doses presumably reflects the insensitivity of the tumor-averaged 99mTc-labeled HL-91 signal to small regions in which tumor blood flow is preserved (which will limit log cell kill). The results indicate the potential of 99mTc-labeled HL-91 for the noninvasive imaging of tumor blood flow inhibition by antivascular drugs in humans.

Topics: Angiogenesis Inhibitors; Animals; Antineoplastic Agents; Biomarkers, Tumor; Cell Hypoxia; Fibrosarcoma; Humans; Mammary Neoplasms, Experimental; Melanoma; Mice; Mice, Inbred C3H; Neoplasm Transplantation; Neoplasms, Experimental; Organotechnetium Compounds; Oximes; Radionuclide Imaging; Radiopharmaceuticals; Stilbenes; Xanthenes; Xanthones