Study Of VEGF Expression In Breast Cancer And Against VEGF Treatment On Breast Cancer Animal Model

English abstractStudy of VEGF expression in breast cancer and againstVEGF treatment on breast cancer animal modelZha xiaoming (student), Wu Zhengyan (tutor)ObjectiveIt is now widely recognized that angiogenesis is an essential step for solid tumor growth. Tumor microvessel density (MVD) has been shown to be an independent prognostic marker in several types of human tumors, including breast carcinoma. Vascular endothelial growth factor (VEGF) is a strong endothelial cell mitogen. Increased expression was shown to correlate with poor prognosis. In this study, VEGF expression was examined by immunohistochemical staining and molecular biological techniques, and compared with elinicopathologic factors, MVD and micrometastases in bone marrow. The significance of VEGF expression was studied in breast cancer. gainst-VEGF and its receptor KDR monoclonal antibodies were prepared, and the inhibitory effects of them on xenograft breast cancer MVF-7 cells on tumor nude mice were observed.Methods:1.The expressions of VEGF in paraffin-embedded sections and epithelial membrane antigen (EMA), keratins (KT) in bone marrow cells were studied by using immunohistochemical SABC staining in 30 breast cancer patients between April 1998 and Nov7l999. The exPression of VEGF the pathologica1 characteristic,bone marrow micrometastases and results of follow-up of thesepatients were comPared. Quantification of angiogenesis wasachieved by immunohistochemical staining of endothelial cellswith antibody to VIII-related antigen (RA).2. The exPression of VEGF mRNA in fresh tUmor samPles and' KT-19 mRNA in bone marrow cel1s were detected by reversetranscriptase-polyInerase chain reaction (RT~PCR) in 35 breastcancer patients from Ang 1999 to Dec l999. Meanwhile bonemarrow micrometastases by immunohistochemical staining usinganti-EMA and KT McAbs was compared with it by RT-PCR.3. Fusion-proteins of VEGF,,, and KDR I -IV were used -for'..immunizing BALB/c mice. The spleen cells from immunizedmice were fused with sp2/0 ce1ls and selective1y cultUre with HATmedium. ELISA method was used to select hybridomas. Theactivity of tWo McAbs against VEGF was measured by 'H-TdRincorporation on human umbi1ical vein endothe1ial ce1ls(mpEC).4. Human breast cancer MCF-7 cells were injected subcutaneously at2 X l0' in the right thorax region of 6 weeks old, female,BALB/C-nu/nU mice. These mice were randomly assigned to eachtreatment grouP after xenograt 5 days. AIl drugs wereadministered by intraPeritonea1 injection. The dosage of agentstested were as fOllows: anti-VEGF anibody (200ug, every other8day X l4), anti-KDR antibody (200ug,every other day X l4), 5-Fu (320ug (l6mg/kg) every other day X5) and saline (0.2m1every other day X l4) respectively Tumor volumes, calculated asthe product of length (L) and width (W), were monitored every 5days by a single observer All mice were sacrificed on day 35. Thetumor volumes and weight were measured. The expression ofVEGF MVD and micro-change of microvessel in t'Umor wereobserved.Results:l. Positive immunostaining for VEGF was observed by theimmunohistochemical staining technique in 20 out of 30 cases(66.7%), which was significantly associated with axillary lytnphnode metastasis, bone marrow micrometastasis and MVD. Therewas no correlative among positive VEGF and the presence ofestrogen receptof, tUmor size.2. The exPression of VEGF mRNA was observed by the RT-PCRtechnique in 29 out of 35 cases (82.86%), which was alsosignificantly associated with axillary lytnPh node metastasis, andbone marrow micrometastasis, no correlation with the presence ofestrogen receptor. But the expression of VEGF mRNA waspositive corre1ative with tumr size, which was differen frOm theresult by immunohistochemical staining. The result by RT-PCRwas more sensitive than that of immunohistochemical staining indetection of bone marrow micrometastasis.93.Two McAbs could...