| The "seed"(selected cancer cells with metastatic capacity), defined by Paget in his "seed and soil " theory, can grow only on congenial "soil" (the microenvironment suitable for its growth in specific organs and tissues)and form metastasis. Skeleton is one of the most common metastasis target organs for breast cancer. Osteopontin, as a protein in extracelluar matrix, is tightly associated with the affinity for the tumor metastasis to specific organs. Osteopontin can induce breast epithelial cell migration and invasion. Previous researches on osteopontin demonstrated that it is highly expressed in malignant tumor tissues, such as colon cancer, gastric cancer, breast cancer, prostate cancer, liver cancer, lung caner and malignant melanoma. Furthermore, it is positively correlated with tumor metastasis. Treatment targeting on OPN or its receptors may represent a particularly suitable focus for therapeutic intervention. And this protein will probably be a predicting molecular for tumor metastasis and prognosis.Chapter 1 Study of the expression of osteopontin in breast cancer and its correlation with metastasisObjective To explore the expression level of osteopontin in breast cancer, to analysis the relation between osteopontin and metastasis of breast cancer, to investigate the feasibility of establishing mathematical model for predicting metastasis of breast cancer.Methods According to the following clinical data, the patients were divided into 4 groups: Group A(breast cancer with distant metastasis, 40 samples), Group B(breast cancer without distant metastasis, 30samples), Group C(breast fibroma, 16 samples), Group D(normal breast, 9 samples). The expression level of OPN in breast tissue was detected by immunohistochemical S-P method. The relationship between Osteopontin and metastasis of breast cancer were analyzed by single-factorial evaluation. Furtherly, factors correlated with breast cancer metastasis were screened using multifactorial logistic regression and the mathematical prediction model were established. Fresh tissues from breast cancer patients without metastasis,breast cancer patients with bone metastasis and their involved lymph nodes were respectively collected, and the normal tissues 5cm distant from cancer tissue as control. The mRNA expression level of osteopontin was measured by RT-PCR methods. And the difference between each group was compared.Results The positive rate in Group A and Group B was 85.0% and 56.7%, respectively. And the positive rate in the whole patients with breast cancer was 72.9% (51/70), including 26 cases (37.1%) with high expression levels. The positive rate in Group C and Group D was 18.8% and 33.3%, respectively. And no case with high expression levels was found. Compared with normal breast tissues and breast fibroma tissues, the positive rate in breast cancer tissues was significantly higher (P＜0.01). The positive rate of osteopontin expression in the breast cancer patients with metastasis was higher than that in the breast cancer patients without metastasis (P＜0.05). Four factors——systemic treatment,ER status,involved lymph nodes and TNM staging——associated with breast cancer metastasis were screened using multivariate Logistic regression analysis. On this basis, the equation predicting the status of metastasis in breast cancer was established. Its sensitivity and specificity was 72.5% and 70.0%, respectively. Its overall accuracy was 71.4% The expression of OPN mRNA was highest in breast cancer tissues with bone metastasis and in metastatic lymph nodes, lower in breast cancer tissues without bone metastasis and lowest in breast tissues adjacent to breast cancer and in normal tissues.Conclusions The expression level of osteopontin in breast cancer was higher than that of OPN in normal breast and fibroma, which indicate that it is potentially a target for therapy in breast cancer. Single-factorial analysis demonstrated osteopontin is positively correlated with tumor metastasis. However, osteopontin is not selected as an independent factor associated with breast cancer metastasis using multivariate logistic regression analysis, which suggests that osteopontin may effect on metastasis by polygenic combination.Chapter 2 Effects of the anti-osteopontin antibody on biological characterization of human highly metastatic breast cancer cell line MDA-MB-231Objective To study the Effects of the anti-OPN antibody on biological characterization of the breast cancer cell line MDA-MB-231 with high potential for metastasis.Methods Taken established human breast cancer cell line MDA-MB-231 in vitro as research object, the expression level of OPN mRNA and protein was detected by RT-PCR method and Western blotting method, respectively, using human breast cancer cell line MCF-7 as control. The breast cancer cell line MDA-MB-231 was treated with or without the anti-OPN antibody. Plotted dose-effects curve and cell growth curve, and monolayer colony formation test were used to evaluated cell proliferation. The migration and invasion potential of cancer cells with different treatments was analyzed by the wound healing assay and matrigel invasion activities, respectively. MTT assay was used to assess the inhibitory effect of co-treatment with doxorubicin and the anti-OPN antibody on cell proliferation.Results The high level of osteopontin expression in human breast cancer cell line MDA-MB-231 was validated by RT-PCR and Western blotting analysis, compared with breast cancer cell line MCF-7. The dose-effects curve demonstrated that the inhibiting proliferation effect of the anti-OPN antibody was dose-dependent. The cell growth curve of human breast cancer cell line MDA-MB-231 manifested the antibody had effects on cell growth, made cell growth to be slow and prolonged the double-time of cancer cell. Monolayer colony formation test exhibited the number of forming colony decreased in the breast cancer cells treated with the antibody. Would-healing assay showed the invasion capability of the breast cancer cells treated with the antibody significantly weakened. Matrigel invasion activities in vitro revealed the number of penetrated cells decreased in the breast cancer cells treated with the antibody. The effect of doxorubicin on inhibiting cell proliferation of breast cancer cells was enhanced by co-treatment with antibody in the range of 0.1-1μg/ml. And the 50% inhibitory concentration(IC50) of doxorubicin was reduced to 52% when combined with the anti-OPN antibody compared to the treatment with doxorubicin alone.Conclusions The osteopontin mRNA and protein expression in human breast cancer cell line MDA-MB-231 was high levels. The cell growth curve and Monolayer colony formation test both demonstrated the inhibitory effect of anti-OPN antibodies on the proliferation of human breast cancer cell line MDA-MB-231. The dose-effects curve showed that the inhibitory effects with dose-dependent relation. Would-healing assay and matrigel invasion activities testified that the inhibitory effects of migration and invasion capability of the breast cancer cells treated with the antibody. The anti-OPN antibody could augment the effect of doxorubicin on inhibiting cell proliferation, exhibiting a synergistic effect.Chapter 3 Establishment of animal model with breast cancer metastasis to bone and treatment with an antibody against OsteopontinObjective To explore the treatment of breast cancer metastasis to bone in vivo with the anti-OPN antibody.Methods 24 female BALB/C-nu/nu nude mice were inoculated into left cardiac ventricle with MDA-MB-231 cell suspension to establish bone metastasis model of breast cancer in vivo. Mice were divided into four treatment groups(n=6/group) according to different treatment: Group A (treatment with high dose of the anti-OPN antibody), Group B(treatment with low dose of the anti-OPN antibody), Group C(treatment with non- specific antibody), Group D(treatment with normal saline). The mice were followed for the observation of general condition, such as vitality and body weight changes. Animals were radiographed for the changes of fore and hind limb long bone at the end of week 3 and week 5, respectively. Long bones were removed from mice at the time of killing and carried on histopathologic examination. The levels of MMP-2 and MMP-9 expression were measured by immunohistochemical method.Results The body weight of mice had been weakly loss in 72 hours after inoculation and then recovered. Until the day 25 after inoculation, mice treatment with normal saline and non-specific antibody began to be weight loss, emaciated and sluggish. There was no similar phenomenon in mice treatment with anti-OPN antibodies. X-ray findings included defect of the cortex, spot-like destruction, moutheaten-like destruction, massive osteolytic destruction, and periosteal reaction, swelling of soft tissue and pathological fracture. The results showed that for Group C and Group D, the osteolytic bone lesion occurred earlier than that of Group A and Group B. At later stage, Group C and Group D had more severe osteolytic bone lesion than Group A and Group B. The metastatic lesions of breast cancer cells in heart injection model was were diagnosed by histopathologic examination. Except mice with failed model, the incidence of breast cancer metastasis to bone in Group A and Group B was 20.0 %(1/5) and 33.3 %(2/6), which was significantly lower than that of Group C and Group D (4/4 and 5/5). The MMP-2 and MMP-9 expression levels of Group A and Group B decreased, compared with Group C and Group D.Conclusions A xenograft model for breast cancer in nude mice was successfully established. Initial study in vivo indicated that treatment with the anti-OPN antibody could inhibit human breast cancer cells MDA-MB-231 metastasizing to bone and lower the expression levels of MMP-2 and MMP-9.
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