A Study Of Intratumoral Distribution Difference For Tumor Angiogenesis And Microvessel Density Within Osteosarcoma

Objective: To study the distribution difference of VEGF and microvessel density (MVD) within human osteosarcoma specimens as well as within xenografts of human osteosarcoma cell line MG63 in subcutis of nude mice; To analyze the relationship of the VEGF expression and MVD in different region of the tumor; To provide experimental reference to the definition of site clinically about where the specimen should be drawn, and to the clinical and basic research about the angiogenesis of osteosarcoma.Methods: 1 Three intact osteosarcoma specimens were selected to establish three-dimensional coordinate system in which tumor center were taken as the original point, and 1 cm was taken as basic unit on the vertical axis, the sagittal axis, the coronal axis to obtain space coordinates. Tissue masses were divided respectively according to space coordinates, and were imbedded in paraffin after fixation in 10% formalin. Then tissue masses were used to make tissue chips on which each point diameter 1.0mm was ordered a partition for 0.5mm. Each tumor produced a tissue chip. Each point on the chips represented a tissue mass. Paraffin section was cut for thickness of 5μm. VEGF protein expression and MVD of human osteosarcoma were examined by VEGF polyclonal antibody and CD34 monoclonal antibody by immunohistochemical staining method. Then VEGF expression quantitative values (positive goals integrated optical density, IOD) and MVD were calculated by MoticMed6.0 imaging processing software. Referring to the definition of rim and center of musculoskeletal neoplasm by MA and Meng, all the points on each tissue chip was classified into three groups. The radiation-shape lines were connected from the center (O) of Intratumoral maximal internal tangent circles to the random points A~G in the surface of the tumors. And each line was divided into three equal segments by the medial 1/3 points and the lateral 1/3 points. Linked the medial 1/3 points and the lateral 1/3 points respectively and formed two cocenric circles whose shapes were similar to the tumor, by which tumor was divided into three parts. The medial part (Inner layer) was the center of the tumor; the lateral part (Outer layer) was the edge of the tumor; the intermediate part (Intermediate layer) was the junctional zone of above two parts. According to the preceding method, points of each tumor were selected for three groups: Inner layer, Intermediate layer, Outer layer. 2 Established subcutaneous model of osteosarcoma-bearing nude mice: 0.2ml cell suspension (about 100 million each mouse) of osteosarcoma cell line MG-63 from ATCC was inoculated in subcutaneous layer of 20 nude mice' left axillary posterior region respectively. Osteosarcoma all appeared in a week. After 2 weeks, xenografts were taken out of nude mice. 12 xenografts which had equal size (about 1.0*1.0*1.0 cm~3) were used for the cutting of the frozen section (thickness for 8μm). VEGF protein expression and MVD of xenografts were examined by VEGF polyclonal antibody and CD34 monoclonal antibody through immunohistochemical staining method. 2-4 continuous slices were cut at intervals of 1mm in each xenografs, and all the slices were labeled clearly by sequence. Each slice marked by VEGF polyclonal antibody and CD34 monoclonal antibody moved around the center of the slice in left-right and up-down direction under light microscope. When moving for 1mm, a space coordinate point was defined. Every coordinate point representing different position of the slice was taken pictures to calculate MVD, VEGF IOD. All the coordinate points in a xenografs were representative of different space position. The points full of necrotic tissue were excluded. Referring to the definition of rim and center of musculoskeletal neoplasm by MA and Meng, all the points of each xenograft was classified into two groups. Center of xenograft was taken as the original point; circular region of the diameter 5mm divided the tumor into two parts: The medial part (Inner layer), the lateral part (Outer layer). Inner layer was the center of the tumor; Outer layer was the edge of the tumor. So points of each tumor were selected for two groups: Inner layer, Outer layer. Then VEGF IOD and MVD were calculated by MoticMed6.0 imaging processing software.Results: 1 The results of 3 osteosarcoma were consistent. There were no difference between Outer layer and Intermediate layer with regard to VEGF protein expression and MVD distribution within tumors (P>0.05), but there were significant difference between Intermediate layer, Outer layer and Inner layer (P<0.01). Edge of human osteosarcoma had a higher VEGF protein expression and denser microvessel than the center. There was significant positive correlation between the expression intensity of VEGF and MVD in the Inner layer, Intermediate layer, Outer layer of osteosarcomas. 2 The analytic results of xenografts were as follows: there was significant difference statistically between Outer layer and Inner layer (P<0.01). Edge of xenografts had a higher VEGF protein expression and denser microvessel than the center. There was significant positive correlation between the expression intensity of VEGF and MVD in the Inner layer and Outer layer.Conclusions: Difference existed in the different region of the human osteosarcomas and xenografts of human osteosarcoma in nude mice. When there were specimens needing pathological examination, we should draw the materials from the edge of the specimens to benefit analysis, even if necrosis did not exist in the center. When investigating the angiogenesis of osteosacoma, we should indicate the specimen where it was obtained to avoid the experimental deviation due to intratumaral distribution difference of microvessel.