Establishment and characterization of a cell line from human adenoid cystic carcinoma of the lacrimal glands and a nude mouse transplantable model.

Using tissue block culture techniques, we established a new human tumor cell line derived from adenoid cystic carcinoma of the lacrimal glands (LACC-1). The LACC-1 cell line was successfully subcultured for more than 100 passages during the last two years. The outgrowth of cells was observed by day 5 after seeding, and then the cells were generated slowly. The first passage proceeded by day 32, and the classical epithelioid cell colonies formed by day 69 after inoculation. After eight passages, homogeneous epithelioid tumor cells appeared when we combined continuous passage, mechanical scraping, repeated adherence, and dissociation methods to remove the fibroblast cells. LACC-1 cells appeared as a histologically solid pattern and continuous passage culture. The population doubling time was approximately 37.1 h. LACC-1 cells appeared as an epithelioid monolayer culture on the cell culture flask and presented with a cobblestone-like appearance when they reached confluency. The nucleus was large and round with many abnormal mitoses. The nucleoplasm ratio was high. Multinucleated tumor giant cells appeared. LACC-1 cells showed a tendency to have overlapping growth without contact inhibition when the cell density continued to increase. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) showed that the LACC-1 cells were malignant tumor cells that were poorly differentiated. The surface of the LACC-1 cells exhibited affluent microvilli, protuberances and filopodia under SEM. The no. 84 generation LACC-1 cell line was inoculated subcutaneously into the subaxillary of nude mice and the tumorigenic potential was evident. The formation rate of the transplanted tumors was 100% at day 7 after inoculation. This finding showed that the LACC-1 cell line was malignant with tumorigenic ability. The xenograft tumors retained the same histological characteristics of a solid pattern as the LACC-1 original tumor after inoculation for 49 days. Under TEM observation, the xenograft tumor cells had the same ultrastructure as the LACC-1 cells. Immunohistochemical examination revealed the similarity of both cytoskeletal proteins (e.g., cytokeratin, vimentin, desmin and α-SMA) and S-100 expression in the original tumor, LACC-1 cells and xenograft tumors. Immunoreactivity of these proteins was gradually decreased in these three tissues. Reverse transcription-polymerase chain reaction demonstrated that the xenograft tumors originated from the human. Based on these results, the LACC-1 cell line provides a useful model for studying the biological characteristics of human ACC of the lacrimal glands.

[Isolation and identification of cancer stem cells from human LACC cell line].

OBJECTIVE: To find ways to isolate and culture lacrimal adenoid cystic carcinoma (LACC) stem cells from human LACC cell line and to identify their biological properties. METHODS: Experimental research. LACC cells were digested, centrifuged and suspended in specific serum free medium (SFM) to get LACC 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 assay was used to determine the proliferation and chemotherapeutic drug resistance of stem cell spheres. The expressions of cell surface markers CD44⁺/CD24⁻ were detected by flow cytometry. Stem cell spheres differentiation were induced by dropping serum medium into SFM and the morphologic changes were observed. The IC50 and UV optical density of two kinds of cells were tested by Student's t-test. RESULTS: About (0.92 ± 0.02) % cells were clonogenic in LACC cell line. They could survive and proliferate to form free-floating cell spheres in SFM, which could stably passaged. The IC50 values of cancer stem cells and LACC cell line were 22.53 mg/L and 11.06 mg/L (t = 37.94, P < 0.05), which suggested that cancer stem cells were more resistant to cis-platinum than LACC cell line. In flow cytometry assay, 84.25% stem cell spheres were CD44⁺/CD24⁻ cells, comparing with 23.77% in LACC cell line. Stem cell spheres could differentiate into normal adenoid cystic carcinoma cells in medium with serum. CONCLUSION: LACC stem cell spheres, containing a large number of cancer stem cells, could be obtained by serum free suspension culture. This provide us an ideal model system for cancer stem cell research.

[Heterotransplantation of human lacrimal adenoid cystic carcinoma cell line into nude mouse].

OBJECTIVE: To investigate characterization of human lacrimal adenoid cystic carcinoma line LACC and establish a model of human LACC. METHODS: Experimental research. Human LACC cells were injected into subcutaneous tissue of nude mice. The length (L) and width (W) of the tumor were measured every day after injection of LACC cells, and the tumor volume was calculated as L×W(2)×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, 49 days after injection of LACC cells. Histologic examination, immunohistochemical analysis for Keratin,Vimentin,α-SMA, S-100, Desmin, PCR analysis for human ß-actin and electron microscopy were performed. Whether tumor metastasis to lungs, livers and lymph nodes of oxter were observed by hematoxylin-cosin staining method. RESULTS: Heterotransplanted tumors were observed in all 10 mice after injection of LACC cells. The results of HE staining and transmission electron microscope indicated that heterotransplanted tumor possesses typical histological characterization of epithelial carcinoma. The results of immunohistochemistry 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 ß-actin, indicating their human origin. Histologic examination show that tumors didn't metastasize to lung, liver and lymph node. CONCLUSIONS: Human LACC cell line possesses characterization of malignant carcinoma and also possesses 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 is relatively easy, quick, 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.

[Primary culture and morphological characteristics of human lacrimal adenoid cystic carcinoma cells].

OBJECTIVE: To explore a method of primary culturing human adenoid cystic carcinoma cells of lacrimal gland. METHODS: Experiment research. Tumor tissue was obtained from the surgical material of a patient diagnosed as lacrimal adenoid cystic carcinoma in Tianjin Medical University Eye Hospital during May 16th to June 1st.We gained primary cells via tissue culture techniques. Mixed cells were removed through several ways.Observed cell morphological characteristics by phase contrast microscope, scanning electron microscope and transmission electron microscope. Cyto-immunochemical staining was applied to detect the expressions of vimentin, desmin, S-100, cytokeratin, pan and CD34. Their expressions were also detected in the tumor tissue except CD34. Made cell growth curve and calculated cell doubling time. RESULTS: The outgrowth of cells was observed by day 5 after seeding tissues, and then cells generated slowly. The first passage proceeded by day 32, and the classical epithelioid cell colonies was observed by day 69 after inoculation. Purified cells of human lacrimal adenoid cystic carcinoma were obtained after the removal of mixed cells through several ways, which have been successfully subcultured for more than 100 passages. The 25th passage LACC cells appeared to be typically epithelioid cells, they showed contact inhibition as the density high enough.SEM and TEM showed the 25th passage LACC cells were malignant tumor cells poorly differentiated. They showed positive reaction with vimentin, cytokeratin (pan) as well as S-100, but negative reaction with desmin and CD34, which were consistent with the tumor tissue. The cell growth curve turned like a sigmoid one, and the cell doubling time was 37.1 h. CONCLUSIONS: We gained purified LACC cells, and understood the morphological characteristics, laying the foundation for the establishment of a human lacrimal adenoid cystic carcinoma cell line.

[The application of serum free medium in the research of isolating cancer stem cells].

The isolation and identification of cancer stem cells are the key points in exploring characteristics of cancer stem cells at present. Several species of cancer stem cells, for instance, retinoblastoma tumor stem cells, cancer stem cells of melanoma of choroid, breast cancer stem cells, lung cancer stem cells, colon cancer stem cells, etc., have been isolated and cultured successfully by serum free medium while their biological functions and characteristics are acquired. This review focus on the application of serum free medium in the research of isolating cancer stem cells, both ocular and general, in terms of providing foundation for further research on ocular cancer stem cells.