Preliminary Study On Molecular Mechanisms Of Perineural Invasion Of Adenoid Cystic Carcinoma With An In Vitro Coculture Model

Adenoid cystic carcinoma (ACC) is a common malignant tumor in salivary glands. ACC has the propensity for perineural invasion (PNI), which could cause pain and paralysis, occult extension beyond apparently clear resection margins and result in local recurrence. Although PNI has been found for more than a century, there has been little progress in the understanding of molecular mechanisms behind this entity, which partly because of lacking effective research models for the complex interaction between nerve and tumor cell. We described a PNI in vitro model by which we could research on the propensity of PNI. We also investigated the gene change in the ACC cell-nerve interaction by Gene Chips. Resently, axonal guidance molecules Eph receptors were found not only regulating axonal growth in embryo development but also expressing outside nerve system. In adult, Eph receptors could regulate the oncogenesis and vascularigenesis in many malignant tumors. We postulate the Eph receptors could also regulate the axonal growth in tumors. This research focused mainly on the expression and function of EphA2 receptor and ephrin A1 ligand to find a possible relationship between EphA2/ephrin A1 and PNI in ACC.PartⅠConstruction of PNI in vitro model of adenoid cystic carcinomaObjective:To construct an in vitro PNI model of ACC. Methods:Mouse dorsal root ganglia (DRG) and human ACC cell lines (ACC-2 and ACC-M) were co-cultured in Matrigel matrix. The interaction between cancer cells and nerves were observed through contrast phase microscope and identified by immunofluorescence double staining. Results:In the in vitro model, neurites outgrowth from DRG to ACC cells was observed at day 1. After the neurites contacting with the ACC cells at day 3, ACC-M cells migrated along the neurites and infiltraed to the DRG at last, just as the PNI in vivo. The neurites without contacting with ACC cells could keep outgrowing. The ACC cell clony aera was significantly larger in the ACC cell-DRG coculture than that in control group. Conclusion:The PNI in vitro model could simulate the process of perineural invasion. This study showed the utility of ACC cell-DRG coculture model to studying molecular mechanism of perineural in ACC.PartⅡImpact of neural microenvironment on invasion, survival and gene expression of ACC cellsObjective:This study aimed to analyse the relationship between PNI and clinico-pathological factors in ACC and the proliferation and apoptotic changes between ACC tumor cells invasion the nerve and far away from the nerve, to investigate the changing of adhesion, invasion and survival ability of ACC cells in the PNI in vitro model, and to analyse the gene changes in the PNI in vitro model by GeneChips. Methods:With immunohistochemical staining of S-100 in 32 ACC tissue sections, the incidence and types of PNI were examined to analyse the relationship between PNI and clinicopathological factors in ACC. By immune stain of Ki-67 and TUNEL assay, the proliferation index and apoptotic index were recorded. Then the proliferating and apoptotic changes between ACC tumor cells around the nerve and far away the nerve were analysed. The changing of adhesion, invasion and survival ability of ACC cells in the PNI in vitro model was measured by proliferation index, apoptotic index, MTT assay and Transwell assay. The gene changing in the PNI in vitro model was detected by GeneChips. Results:PNI was identified in 25 of 32 ACC cases (78.1%). The tubular type had a lower incidence of PNI. The incidence of PNI was higher in solid and cribriform pattern of ACC. However, there was no significant relationship between PNI and the other clinicopathological factors. Increasing of proliferation and decreasing of apoptosis were found in ACC cells around nerve contrasting with ACC cells far away nerve (p<0.05). The mean proliferation index was significantly higher in ACC cells in the PNI model, while the apoptotic activity determined by apoptotic index was significantly decreased (p<0.05). The invasion ability of ACC cells in coculture was significantly increased (p<0.05). However, the condition medium could enhance the adhesion ability of ACC cells to the brain tissues non-significantly. MTT assay showed an increase of growth of ACC cells with condition medium. By GeneChips, we found 369 genes up-regulated and 920 genes down-regulated in the co-culture model. Conclusion:The occurrence PNI was correlated with pathological pattern of ACC. ACC cells obtained increased ability of invasion and survival when interacting with nerves. The gene changing of ACC cell-nerve interaction might involve in the process of PNI.PartⅢStudy on the roles of EphA2/ephrinAl in perineural invasion of ACCObjective:To investigate the expression and function of EphA2 and ephrin A1 in ACC and the relationship with PNI. Methods:32 ACC tissue sections were immunohistochemically stained with EphA2 and ephrin A1 antibody to analyse relashionship between expression of EphA2/ephrin A1 and clinicopathological factors. Western blot and qRT-PCR were performed to analyse the expression of EphA2 and ephrin A1 both in protein and mRNA level. Immunoprecipitation was applied to examine the phosphorylation status of EphA2 receptors. The biological behaviors of ACC cells and ACC cell-nerve interaction were also examined when EphA2 activity was changed under EphA2-Fc and ephrin A1-Fc stimulation. Results:EphA2 was overexpressed in 23 ACC cases (93.8%) and ephrin A1 was overexpressed in 31 (96.9%). The overexpression of EphA2 and ephrinAl were significantly correlated with the incidence of PNI. EphA2 and ephrinAl were also upregulated in protein and mRNA level, with the expression in ACC-M much higher than ACC-2. In ACC tissue and ACC cells, the phosphorylation of EphA2 receptor was very low. Increasing the EphA2 receptor activity by ephrinAl-Fc could reduce the proliferation and invasion activity. Blocking the Eph signal by EphA2-Fc could inhibit the nerve cell moving towards ACC cells and the neurites outgrowth to ACC cells. Conclusion: EphA2/ephrinAl signal could affect the proliferation and invasion of ACC cells and take a part in PNI process.