Heterotransplantation Of Human Lacrima Adenoid Cystic Carcinoma Cell Line And Isolation And Identification Of Cancer Stem Cells From The Cell Line

Objective1.To investigate characterization of human lacrimal adenoid cystic carcinoma cell line LACC.2.Establish a model of huaman LACC.3.To isolate and cultivate lacrimal adenoid cystic carcinoma cancer stem cells from human lacrimal adenoid cystic carcinoma cell line LACC.and identify their biological properties.Methods1.Part1Human LACC cells were injected into subcutaneous tissue of nude mice The length (L) and width (W) of the tumour were measured every day after injection of LACC cells, and the tumour volume was calculated as L×W2×1/2.Two mice bearing LACC tumor were randomly killed, removed the tumors anatomy and their lungs.livers.and lymph nodes of oxter,14.28,35,42,49days after injection of LACC cells. Histologic examination,immunohistochemical analysis for Keratin, Vimentin, α-SMA, S-100, Desmin,PCR analysis for human B-actin and electron microscopy were performed. Whether tumor metastasis to lungs,livers and lymph nodes of oxter were observed by hematoxylin-eosin staining method.2.Part2Cells LACC were dissociated into single cancer cells, suspended in specific serum free medium (SFM) to acquire lacrimal adenoid cystic carcinoma cancer stem cell spheres. Limiting dilution analysis and monoclonal formation assay were performed to determine the proportion of cancer stem cells in LACC cell line.MTT assays were performed to detect stem cell spheres proliferating potentiality and characterize the resistance to chemotherapeutic drug. The expression of cell surface markers CD44+/CD24-was detected with flow cytometry.And observe the inducing differentiation and cytologic and morphologic characteristics of the stem cell spheres.Results1.Heterotransplanted tumors resulted in all10mice after injection of LACC cells. The results of HE staining and transmission electron microscope indicated that heterotransplanted tumor posses typically histological characterization of epithelial carcinoma. The results of immunohistochemistrv showed that Keratin. S-100,Vimentin, α-SMA expression in tumor tissue were positive,but Desmin expression were negative. RT-PCR analysis revealed that tumors expressed human β-actin,indicating their human origin. Histologic examination show that tumors didn’t metastasis to lung,liver and lymph node.2. About (0.92±0.015)%of LACC cells were clonogenic in LACC cell line.They can generate nonnadherent and serial propagating cancer stem cells in serum-free medium,which with stronger propagating capability. and more resistant to cisplatin than LACC cell line.By using a FACScan.there were84.25%CD4+/CD24-cells in stem cell spheres.but23.77%CD44+/CD24-in LACC cell line. Stem cell spheres could differentiate normal lacrimal adenoid cystic carcinoma in serum-supplemented medium condition.Conclusions1. Human LACC cell line posses characterization of malignant carcinoma and also posses characterization of adenoid cystic carcinoma.It is found that the heterotransplanted LACC tumor has histologic features similar to the original LACC of the patient.This model with lacrimal cystic adenoid carcinoma in nude mice was relatively easy,quickly,and especially with high successful rate.So it can be considered as an ideal animal model for study on lacrimal cystic adenoid carcinoma in vivo, and it should be useful to investigate the progression and develop a new therapectic approach for human lacrimal cystic adenoid carcinoma.2. Lacrimal adenoid cystic carcinoma cancer stem cell spheres can be obtained by serum free suspended culture.which is enriched with this kind of stem cells,provide an ideal model svstem for cancer stem cell research.