| Craniopharyngioma is an intracranial extra-axial epithelial tumor of sellar or parasellar region, which grows along the cranial pharyngeal tube..Craniopharyngioma is one of most common intracranial congenital tumor which accounts for6-13%of all intracranial tumors and constitutes60%tumors of the sellar region in children. Since the first description of craniopharyngioma as a distinct tumor in the pituitary region by Austrian neuropathologist Jakob Erdheim in1904; many extensive and in-depth study on craniopharyngioma has been conducted. As yet, more than100years later, expert consensus on the origin, pathological features and biological characteristics of this formidable neoplasm is still lacking due to the lack of the tumor research platform and technical conditions.Craniopharyngiomas are benign tumors which are classified into, adamantinomatous variant and squamous papillary variant by WHO. The adamantinomatous craniopharyngioma often form finger-like protrution invades the surrounding brain tissue, leading to difficult to total resection of the tumor, postoperative recurrence and worse clinical prognosis compared to the latter variant. The results of several single-center studies have shown that the prognosis of adamantinomatous craniopharyngioma is worse than the squamous papillary variant, but the mechanism is not clear. Although many scholars had undertaken extensive research on the adamantinomatous craniopharyngioma in the organizational level, the progress of basic research of adamantinomatous craniopharyngioma is slow due to the lack of stable in vitro and in vivo models. Several scholars cultured the adamantinomatous craniopharyngioma in vitro and used in functional experiments, however, characteristics of tumor cells was not mentioned in the study. At the same time, few in vivo xenograft model of adamantinomatous craniopharyngioma are reported. The contemporary understanding of the biological characteristics of this tumor can be further expanded by establishment of the adamantinomatous craniopharyngioma experimental model.We previously found that the formation of inflammatory adhesions of adamantinomatous craniopharyngioma with the surrounding normal brain tissue is closely related to tumor total resection, postoperative recurrence, prognosis of patients in follow up; the spontaneous rupture of craniopharyngioma cysts and cyst fluid spillage during surgery can lead to aseptic meningitis; histologically, intense fibrillary gliosis and infiltration of inflammatory cells are frequently present in the surrounding parenchyma. These observations suggest that tumor cells may produce certain inflammatory cytokines that might play an important role in tumor progression and prognosis of patients. Mori et al has demonstrated IL-6as an important inducer of craniopharyngioma associated inflammation affecting the prognosis of patients, however the elucidation of the role of IL-6in craniopharyngioma in vitro is not yet reported due to the lack of technical conditions.IL-6is multifunctional cytokine that contributes to the regulation of hemopoiesis, immunity and inflammatory response, also contributes to regulate physiological function of central nervous and cardiovascular system, and associates with cell survival, proliferation and wound healing. High expression of IL-6can be found in colorectal cancer, breast cancer and other epithelial tumors, as well as glioma and other central nervous system tumors, which is closely related with prognosis. Tumor cells or mesenchymal cells in tumor microenvironment can secrected IL6, through autocrine or paracrine secretion. IL-6after binding to its receptors forms a complex to activate the abnormal signaling pathways, contributing to the promotion of tumorigenesis, tumor angiogenesis, tumor migration and invasion. However, the role of IL-6in adamantinomatous craniopharyngioma is still not clear. We contemplate that the IL-6secretion by adamantinomatous craniopharyngioma or mesenchymal cells in tumor microenvironment through either autocrine or paracrine secretion might promote characteristics change in tumor cells and thus affect the prognosis of this patients?In this study, we established the adamantinomatous craniopharyngioma finite cell line by primary culture in vitro as the necessity of the scientific reserch of craniopharyngioma and further investigate the biological characters of the tumor cells in order to elucidate the role of IL-6in craniopharyngioma. Additionally, we established the xenotransplanted tumor model of human adamantinomatous craniopharyngioma by implanting the adamantinomatous craniopharyngioma cells into the nude mice subcutaneously. The antibody array technology was used to investigate the expression of inflammatory cytokines in the different inflammation grade of the adamantinomatous craniopharyngioma tissue; which was followed by the stimulation of the adamantinomatous craniopharyngioma in vitro by IL-6to investigate the role of IL6on biological characters of tumor cells. Based on the findings of this experiment the possible mechanism of IL-6on craniopharyngioma cells is discussed. The aim of our study is to elucidate the relationship between inflammation and craniopharyngioma that might aid in development of clinical treatment for craniopharyngioma. Chapter I Section I Cell culture and characterization of adamantinomatous craniopharyngiomaObjective:To investigate an efficient method of primary adamantinomatous craniopharyngioma cells culture and observe the behaviour of adamantinomatous craniopharyngioma cells in vitro.Methods:The specimen of adamantinomatous craniopharyngioma were treated with trypsin and cultured in vitro, and purified by filter membrane. The cell morphology was observed by inverted microscope and hematoxylin eosin staining. The expression of Pan-CK was examined by immunocytochemistry. The MTT assay and flow cytometry were employed to study the cell proliferation and cell cycle.Results:The success rate of primary culture and passage culture is88.9%and81.3%. Human adamantinomatous craniopharyngioma cells showed adherent growth in serum-containing medium. The tumor cells were large with abundant cytoplasm were large and characteristically demonstrated assemebled growth. A small amount of bubble like structure was seen in the cytoplasm. The tumor cells demonstrated slow growth, the growth rate of cells in1st-5th day is statistically significant difference (F=190.748..P<0.0001). The cells were diploid and depicted irregular polygonal and epithelioid form. The tumor cells in G0-G1,46.8±3.61%; in G2-M,37.3±1.51%; in S,15.9±2.77%.Conclusion:The finite adamantinomatous craniopharyngioma cell line could be cultured in a serum medium and isolated, cultured and expanded effectively by the membrane sub-filtering techinque, which was beneficial for the follow-up experiments. The adamantinomatous craniopharyngioma cells demonstrated the Pan-CK positive phenotype, slow growth mainly in the G0-G1phase with no aneuploid cells. Chapter I Section IIEstablishment of subcutaneous xenotransplanted tumor model of human adamantinomatous craniopharyngioma in nude miceObjective:Establishment of subcutaneous xenotransplanted tumor model of human adamantinomatous craniopharyngioma in mude mice on the basis of the primary adamantinomatous craniopharyngioma cells culture previously.Methods:Craniopharyngioma cells were primarily cultured by trypsin digestion. The purified cells in third passage were then implanted subcutaneously into the nude mice. The specimens were studied by microscope to ensure the characteristics of the xenotransplanted tumor.Results:The subcutaneous xenotransplanted tumor can be established by implanting the adamantinomatous craniopharyngioma cells into the nude mice subcutaneously. Adamantinomatous craniopharyngioma cells can survive in the BALB/c nude mice. The xenograft can be found on8days after implantation. The tumor formation rate at the back was37.5%. The disorderly growth of the squamous epithelial tumor cells can be found under microscope in subcutaneous tissue of nude mice.Conclusion:The subcutaneously xenotransplanted tumor model of human adamantinomatous craniopharyngioma in nude mice was successfully established and it might be beneficial to condunct further studies on this tumor. However, the model is not stable, and the rate of tumor formation is too low. More research is needed to optimize the conditions of the xenotransplanted tumor establishment. Chapter II Section I Epithelial-mesenchymal transition and clinico-pathological correlation in craniopharyngiomaObjective:To assess the immunophenotypic changes associated with epithelial mesenchymal transition (EMT) in craniopharyngioma, especially at the tumour invasive front, and correlate the findings with clinicopathological features and patient outcomes.Methods:42craniopharyngiomas were, subjected to the detection of EMT makers (vimentin, E-cadherin and β-catenin) by immunohistochemistry. The relationships between expression of these markers and various clinico-pathological indicators and clinical outcomes of these tumors were analyzed.Results:There was statistically significant difference in the expression of vimentin and E-cadherin between adamantinomatous and papillary variants in whole tumor and at the tumor invasive front (χ2=11.181, P=0.004,χ2=6.931,P=0.031;χ2=17.393, P<0.001, χ2=8.501, P=0.014). There was statistically significant difference in the cytoplasmic or nuclear accumulation of β-catenin between the two variants above at the tumor invasive front (χ2=19.765, P<0.001). The expression of vimentin and E-cadherin but β-catenin in whole-tumour sections were associated with tumor recurrence, postoperative weight and hypothalamic disturbances (χ2=23.834, P<0.001, r=0.643, P<0.001, r=0.637, P<0.001; χ2=16.526, P<0.001, r=-0.579, P<0.001, r=-0.714,P<0.001), and the expression of vimentin and E-cadherin/β-catenin at the tumor invasive front were colligated with tumor recurrence, postoperative weight and hypothalamic disturbances (χ2=19.782, P<0.001, r=0.642, P<0.001, r=0.573, P=0.001;χ2=11.475, P=0.003, r=-0.54, P<0.001, r=-0.626, P<0.001;χ2=16.572, P<0.001, r=-0.54, P<0.001, r=-0.506, P=0.001).Conclusions:Our study exemplifies, for the first time, the potential prognostic implications of vimentin, E-cadherin and β-catenin expression in craniopharyngiomas. Epithelial-mesenchymal transition may represent a crucial mechanism in the progression of craniopharyngiomas. Chapter ⅡSection Ⅱ The effection of Interleukin-6on the migration characteristics of adamantinomatous craniopharyngioma cells in vitroObjective:To elucidate the effect and mechanism of the IL-6on the migration characteristics of adamantinomatous craniopharyngioma cells in vitro..Methods; We demonstrated the inflammation and clinico-pathological correlation in craniopharyngioma previously. In this study, antibody array assay was used to assess the expression of different inflammatory molecules in adamantinomatous craniopharyngioma tissues at different inflammatory levels. Further, the effect and mechanism of IL-6on adamantinomatous craniopharyngioma cell migration in vitro was investigated.Results:The inflammation of craniopharyngioma was associated with pathological classification (χ2=6.603, P=0.037), the rate of total resection (χ2=8.188, P=0.017), calcification (γ=0.326, P=0.022) and postoperative HSS (y=0.376, P=0.008). The expression of IL-6and other inflammatory factors varied in tumor tissue at different level of inflammatory exposure. Expressions of IL6-IL6Ra-GP130were detected by immunohistochemistry and soluble IL-6R in the cystic fluid and supernatant by craniopharyngioma cells and fibroblasts were determined by ELISA. Migration in adamantinomatous craniopharyngioma was promoted by IL-6treatment in a dose-dependent manner (F=112.390, P﹤0.0001; F=126.654, P﹤0.0001). The expression of Vimentin was increased and E-cadherin was decreaseed by IL-6treatment.Conclusion:The inflammation in craniopharyngioma was significantly associated with pathological classification. Additionally this inflammation was associated with the rate of total resection, calcification and postoperative HSS. Antibody arrays demonstrated a significant change in cytokine profiles in patients with different degrees of inflammation. IL-6might promot migration in vitro via classic-and trans-signaling pathways by inducing EMT in adamantinomatous craniopharyngioma. |
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