Suniti nib treatment completely inhibited vascularization if the treatment was started before tumors were vascularized. Discussion In the present work, A 07 GFP Insider Treasures
Nilotinib Uncovered and R 18 GFP mela noma xenografts grown in dorsal window chambers were used as preclinical models. It has previously been shown that intradermal A 07 and R 18 xenografts retain several characteristic features of the original patient tumors, including histological appearance, angiogenic potential, and vessel density. Moreover, intradermal A 07 and R 18 xenografts have been shown to differ substantially in angiogenic potential, vessel density, growth rate, and oxygenation status. These differences were maintained when A 07 GFP and R 18 GFP tumors were grown in dorsal window chambers despite the transfection with GFP, the confinement of tumor growth by the chamber preparations, the small size of the tumors, and the elevated temperature during tumor growth.
Consequently, A 07 GFP and R 18 GFP tumors grown in dorsal window chambers should be ap propriate models for investigating the effect of sunitinib treatment on tumor vasculature and oxygenation. Tumors were treated with two different doses of suni tinib in the current study. Both sunitinib doses have been shown to result in sufficient plasma concentra tions in athymic mice to inhibit VEGFR and PDGFR phosphorylation in xenografts of human melanoma, human glioma, and human colon carcinoma. Lower sunitinib doses have been shown to result in insufficient plasma concentrations and no inhibition of VEGFR and PDGFR phosphorylation in the same xenograft models.
Moreover, the higher sunitinib dose has been shown to reduce ves sel density and improve vascular function in human glioma xenografts. The two sunitinib doses should therefore be well suited to evaluate the effect vessels with plasma only. The function of tumor vascula ture was assessed by using a novel first pass imaging method which involves recording movies of the dynamic distribution of a fluorescent vascular tracer after an intravenous bolus injection . From the recorded first pass imaging movies, BST images and cor responding BST frequency distributions were produced. We have previously shown that the BST assay is highly reproducible, sufficiently sensitive to detect gradients in BST along vessel segments, and sufficiently sensitive to indentify the majority of tumor vessels.
It has previously been shown that A 07 and R 18 cells express and secrete VEGF A and interleukin 8, and that the angiogenic activity can be significantly reduced by inhibiting VEGF A in both xenograft lines. The secretion rate of VEGF A and IL 8 has been shown to be higher for A 07 cells than for R 18 cells and, in addition, A 07 cells have been shown to express and secrete basic fibroblast growth factor whereas R 18 cells do not secrete this factor.
Sunitinib treatment did not affect BST To investigate the effect of sunitinib treatment on the function Major Tactics Over RKI-1447 Unveiled of tumor vasculature, first pass imaging movies were recorded, and BST images and BST frequency dis tributions were produced. BST was not affected by suni tinib treatment. This is shown qualitatively in Figure 6, which shows representative BST images and the corre sponding BST frequency distributions from day 2 and 4 of an untreated A 07 GFP tumor, an A 07 GFP tumor treated with sunitinib, an un treated R 18 GFP tumor, and an R 18 GFP tumor treated with sunitinib. The black re gion in the sunitinib treated A 07 GFP tumor reflects an avascular region which contains hypoxic tumor tissue.
Figure 7 shows BST for all tumors included in the short term treatment experiments, illus trating that untreated and sunitinib treated tumors did not differ in BST on either day 2 or 4, regardless of whether A 07 GFP tumors were treated with 20 mg kg day sunitinib, A 07 GFP tumors were treated with 40 mg kg day sunitinib, or R 18 GFP tumors were treated with 40 mg kg day sunitinib. Sunitinib treatment induced hypoxia To investigate the effect of sunitinib treatment on tumor hypoxia, tumors were resected and submitted to histo logical examination immediately after the last intravital microscopy imaging session. Untreated A 07 GFP tumors did not show hypoxic regions whereas sunitinib treated A 07 GFP tumors showed multiple hypoxic regions. The hypoxic regions co localized with avascular regions or regions with very low vessel density, and were found in both central and peripherial parts of sunitinib treated A 07 GFP tumors.
3 out of 6 untreated R 18 GFP tumors and 7 out of 8 sunitinib treated R 18 GFP tumors showed scat tered hypoxic regions. R 18 GFP tumors did not show avascular regions, and the hypoxic regions reflected low overall vessel densities. The hypoxic area fractions were significantly higher in sunitinib treated A 07 GFP tumors than in untreated A 07 GFP tumors, and a non significant trend towards higher hypoxic area fractions in sunitinib treated R 18 GFP tumors com pared to untreated R 18 GFP tumors was observed. Prolonged sunitinib treatment reduced tumor growth In a separate experiment, A 07 GFP tumors were treated with 40 mg kg day sunitinib or vehicle for 8 days.
By day 6, untreated tumors grew close to the window chamber boundaries, and mice bearing untreated tumors were sacri ficed to avoid growth restriction by the chamber prepara tions. Sunitinib treated tumors were significantly smaller, and were allowed to grow for two more days. The prolonged sunitinib treatment further reduced vessel densities, and increased interstitial distances, but did not affect BST. Our ex perimental model did not allow evaluation of longer treat ment periods.
Sunitinib Main Industry Secrets For BMS-345541 Disclosed treated tumors had significantly longer segment lengths than untreated tumors, whereas sunitinib treatment did not affect vessel tortuosities. The sunitinib induced effect on segment lengths was more pronounced for A 07 GFP tumors than for R 18 GFP tumors. Sunitinib treatment increased median vessel diameter but inhibited vessel diameter increase in remaining vessels sunitinib treated tumors did not differ from untreated tumors in size regardless of whether A 07 GFP tumors were treated with 20 mg kg day sunitinib, A 07 GFP tumors were treated with 40 mg Sunitinib treated A 07 GFP tumors showed higher me dian vessel diameters than untreated A 07 GFP tumors, whereas sunitinib treated R 18 GFP tumors did not dif fer from untreated R 18 GFP tumors in median vessel diameter.
To investigate changes in vessel diameters of remaining vessels, the diameter was mea sured in the same vessels on subsequent days. Ves sels in untreateted tumors showed an increase in vessel diameter from day 1 to day 2, and from day 1 to day 4. Vessels in A 07 GFP tumors treated with 20 mg kg day sunitinib showed similar changes in vessel diameter, whereas A 07 GFP and R 18 GFP tumors treated with 40 mg kg day sunitinib showed lower increases in vessel diameters than untreated tumors. Consequently, the increase in median vessel diameter did not reflect increases in the vessel diameters of remaining vessels, but rather a selective pruning of small vessels.
Immunohistochemical investigations showed that sunitinib treatment did not affect vascular basement membrane and pericyte coverage and confirmed that sunitinib treatment reduced vessel density Histological sections of A 07 GFP and R 18 GFP window chamber tumors were stained for CD31, collagen IV, or SMA to visualize endothelial cells, vascular basement membrane, and pericytes. Vessels in A 07 GFP tumors showed vascular basement membrane and were covered with pericytes. Long bands with positive SMA staining originating in CD31 positive vessel walls were observed in A 07 GFP tumors, revealing large net works of pericytes in these tumors. In contrast, most ves sels in R 18 GFP tumors did not show vascular basement membrane, and pericytes were only found adjacent to microvessels. Sunitinib treated tumors did not differ from untreated tumors in vascular basement membrane or pericyte coverage and, consequently, the dif ferences in vessel maturation between A 07 GFP and R 18 GFP tumors were observed regardless of whether un treated or sunitinib treated tumors were considered. Immunohistochemical preparations stained for CD31 con firmed that sunitinib treatment significantly reduced vessel density.
Immunohistochemistry was done by using a peroxidase based indirect staining method. An anti pimonidazole rabbit polyclonal antibody, an anti CD31 rabbit polyclonal antibody, an anti collagen IV rabbit polyclonal antibody, Nilotinib or an anti SMA rabbit polyclonal antibody was used as primary antibody. Diaminobenzidine was used as chromogen, and hematoxylin was used for counterstaining. Hypoxic area fractions were determined by image analysis. Number of microvessel profiles per mm2 of tumor tissue was scored manually and used as parameter for micro vascular density. Statistical analysis Statistical comparisons of data were carried out by the Students t test when the data complied with the condi tions of normality and equal variance. Under other con ditions, comparisons were done by nonparametric analysis using the Mann Whitney rank sum test.
The Kolmogorov Smirnov method was used to test for nor mality, and the Levenes test was used to test for equal variance. Probability values of P 0. 05, determined from two sided tests, were considered significant. The statis tical analysis was performed by using the SigmaStat stat istical software. Results Short term sunitinib treatment did not affect tumor growth Mice were divided in groups with matched tumor size to receive sunitinib treatment or no treatment. Sunitinib treatment was started 6 or 12 days after tumor initiation. At these time points, defined as day 0, A 07 GFP and R 18 GFP tumors were of similar size, and had developed vascular net works. During the short treatment period, kg day sunitinib, or R 18 GFP tumors were treated with 40 mg kg day sunitinib.
Sunitinib treatment reduced vessel density To investigate effects of sunitinib treatment on vascular morphology, mice treated with sunitinib or vehicle were submitted to intravital microscopy. Sunitinib treatment reduced vessel density in both A 07 GFP and R 18 GFP tumors. This is shown qualitatively in Figure 3 which shows representative intravital microscopy images of an untreated A 07 GFP tumor, an A 07 GFP tumor treated with sunitinib, an untreated R 18 GFP tumor, and an R 18 GFP tumor treated with sunitinib. To quantify these qualitative observations, vascular masks were produced and vessel densities and interstitial distances were calcu lated. Sunitinib treated tumors had significantly lower vessel densities and significantly higher interstitial dis tances than untreated tumors .
The sunitinib induced reduction in vessel densities was more pronounced for A 07 GFP tumors than for R 18 GFP tumors. For sunitinib treated A 07 GFP tumors, the density of small vessels decreased more than the density of large vessels, implying that sunitinib treat ment selectively pruned small vessels. For sunitinib treated R 18 GFP tumors, the densities of small and large vessels were similarly reduced.
Tumor vasculature was visualized by using transillumination and filters for green light, and tumor vascular networks were mapped by recording 1 4 single frames with Nilotinib a 4 objective lens resulting in a field of view of 3. 80 3. 80 mm2 and a pixel size of 3. 7 3. 7 um2. To study the function of tumor vasculature, first pass imaging movies were recorded after a 0. 2 mL bolus of 50 mg mL tetramethylrhodamine isothiocyanate labeled dextran with a molecular weight of 155 kDa was injected into the lateral tail vein. First pass imaging movies were recorded at a frame rate of 22. 3 frames per second by using a 2 objective lens, resulting in a time resolution of 44. 8 ms, a field of view of 5. 97 5. 97 mm2, and a pixel size of 7. 5 7. 5 um2. All recordings were stored and analyzed offline.
Tumor size was calculated from the number of pixels showing GFP fluorescence. Analysis of vascular morphology Two dimensional projected vascular masks were pro duced manually from transillumination images recorded with the 4 objective lens. Interstitial distance and vessel diameter were computed from the vascular masks. Vessel density was calculated from skeletons of vascular masks. Vessel density of small or large vessels was calculated from skeletons of vascular masks only showing vessels with a diameter smaller or larger than 15 um. The ana lysis of vascular morphology is illustrated for a represen tative tumor in Figure 1. This figure shows high resolution transillumination images, the vascular mask, the skeleton of the vascular mask, and color coded vessel diameter superimposed on the vascular mask.
Vessel segment length and vessel tortuosity were calculated from 50 randomly selected vessel segments. Vessel tor tuosity was defined as T 100% SL, where SL represents the segment length and S represents the shortest distance between the branching points, as illustrated in Figure 1F. Change in vessel diameter was assessed by manually measuring the diameter of the same vessel segments on subsequent days. Analysis of vascular function Two dimensional projected vascular masks were pro duced from the movies recorded with the 2 objective lens as described previously. Blood supply time images were produced by assigning a BST value to each pixel of the vascular masks. The BST of a pixel was defined as the time difference between the frame showing maximum fluorescence intensity in the pixel and the frame showing maximum fluorescence intensity in the main tumor supplying artery, as described in de tail previously. Immunohistochemical detection of tumor hypoxia, microvessels, vascular basement membrane, and pericytes The tumors were resected immediately after the last intra vital microscopy examinations and fixed in phosphate buffered 4% paraformaldehyde.
The animal experiments were approved by the Norwegian National Animal Research Authority and were done according to the Interdisciplinary Principles and Guidelines for the Use of Animals in Research, Marketing, and Education. Cells and multicellular spheroids A 07 and R 18 human melanoma cells were constitu tively transfected buy inhibitor with green fluorescence protein by lipofection. The transfected cells used in the present experiments were obtained from our frozen stock and grown as monolayers in RPMI 1640 supplemented with 13% bovine calf serum, 250 ug mL penicillin, 50 ug mL strepto mycin, and 700 ug mL or 2200 ug mL genetecin. Multicellular spheroids were produced by seeding approximately 106 cells in 30 mL medium in plas tic tissue culture flasks coated with a thin layer of 1% agar.
The flasks were agitated for 2 h using a tilting platform, and aggregates of approximately 50 um in diam eter were formed. The spheroids were then allowed to grow in coated culture flasks before implantation in win dow chambers. Cells and spheroid cultures were incubated at 37 C in a humidified atmosphere of 5% CO2 in air and subcultured twice a week. Anesthesia Window chamber implantation and intravital micros copy examinations were carried out with anesthetized mice. Fentanyl citrate, fluanisone, and mida zolam were administered intraperitoneally in doses of 0. 63 mg kg, 20 mg kg, and 10 mg kg, respectively. After surgery, the mice were given a single injection of buprenorphine intrape ritoneally in a dose of 0. 12 mg kg to relieve pain.
Window chamber preparations Window chambers were implanted into the dorsal skin fold as described previously. Briefly, the chamber consisted of two parallel frames, and after implantation, the frames sandwiched an extended double layer of skin. Before the chamber was implanted, a circular hole with a diameter of approximately 6. 0 mm was made in one of the skin layers. A plastic window with a diameter of 6. 0 mm was attached to the frame on the surgical side with a clip to provide visual access to the fascial side of the opposite skin layer. Tumors were initiated by implanting spheroids or tumor specimens with a dia meter of 200 to 400 um onto the exposed skin layer. Sunitinib treatment Sunitinb L malate was dissolved in hydrochloric acid, polysorbate 80, polyethylene Glycol 300, sodium hydro xide, and sterile water.
Mice were treated with 20 or 40 mg kg day sunitinib or vehicle for 4 or 8 days, by oral administration. Intravital microscopy Intravital microscopy was performed before initiation of sunitinib treatment, and 1, 2, and 4 days after the start of treatment, or before, and 2, 4, 6 and 8 days after the start of treatment. The mice were kept in a specially constructed holder that fixed the window chamber to the microscope stage during intravital microscopy. The body core temperature was kept at 37 to 38 C by using a hot air gen erator.