The Cultivation And Identification Of Human Lacrimal Gland Adenoid Cystic Carcinoma Cancer Stem Cells

Objective 1. Cultivate Human Adenoid Cystic Carcinoma cells line of the Lacrimal Gland for the extraction of Cancer Stem Cells. 2. Separate and cultivate Cancer Stem Cells of Human Lacrimal gland Adenoid Cystic Carcinoma. 3. Study and identify the Cancer Stem Cells’ related charactaristics of Human Adenoid Cystic Carcinoma of the Lacrimal gland. Methods 1. Part 1 : The extraction and cultivation of Cancer Stem Cells.The Human Adenoid Cystic Carcinoma cells line of the Lacrimal Gland were cultivated in normal serum medium.Choose the cells that grew well according to the curve of cell growth, and pancreatic enzyme to digestive adherent cells into single-cell suspension,and then vaccinate them into serum-free medium in a certain density. Replace the medium regularly,observe the morphological changes and obtain the Cancer Stem Cells sphere for next study. 2. Part 2 :Identification of the Cancer Stem Cells of the Human Adenoid Cystic Carcinoma.Ganther the stably-grewed lacrimal gland adenoid cystic carcinoma Cancer Stem Cells spheres cultivated in Serum-free suspension culture. Using microscope to observe the morphological changes, flow cytometry instrument for detecting the expression of classical stem cell markers CD133 and ABCG2 and transwell chamber detecting the cancer stem cell aggressivity at different periods.Also multi-directional differentiation potential of the cancer stem cells such as differentiation to vascular endothelial cells were tested using the endothelial cells medium. Vitro xenotransplantation experiment were implemented to test the differences of tumorigenic force between two kinds of cells.Results 1. LACC cells grew adeherently to plates in serum containg medium, forming paving stone samples. Differented from the normal tumor cells which growed in a paved stones style,when transfered to serum-free medium,these LACC cells gradually began floating in the culture,and gradually gathered into cells microspheres with close connection among cells.The cells microspheres became bigger and much regular as time going on,.and can form satisfactory microspheres after 10~12 days with beaded or grapes-like appearance. The Cancer Stem Cells microspheres kept a stable state of clone through continuous subculture. 2. The collected CSCs microspheres showed related features as stem cell: 1) Phase contrast microscope observation showed the normal LACC cells grew adherently with a paving stone style, while the microspheres cultivated in serum free medium presented specific morphological feature as grapes,sharply different to the normal tumor cells. 2) Flow Cytometry experiments showed that the expression ratio of stem cells related marker of CD133 in LACC-CSCs was(35.67±6.86) %, significantly different to LACC with(0.46±0.48) %,(t = 8.867, P < 0.05). Similarly,the expression ratio of stem cell marker of ABCG2 in LACC-CSCs was(39.99±4.54)%, significantly different to LACC with(6.75±1.34)%,(t=-9.932,P<0.05).It reminded us that LACC-CSCs expressed the stem cells related markers, different to the normal tumor cells. 3) In vitro experiment of Matrigel invasion, LACC-CSCs went through the matrigel basement membrane averagely(32.60±8.79)/HP contrary to LACC of average(10.20 + 2.77)/HP after 24 hours,showing statistically significance(t= 5.433, P< 0.05) between the two groups.After training for 48 hours,the difference between two groups was still obviously(t=5.779,P<0.05)with LACC-CSCs average(62.60±4.83)/HP to LACC(44±5.34)/HP. LACC-CSCs had stronger organization migration that may cause severe tissue damage than LACC. 4) When induced by serum medium containing VEGF and bFGF in an environment of low oxygen, LACC-CSCs grew adherent gradually and cells morphological changed after continous induction to long spindle cells. When cultured into three-dimensional matrix structure they formed vessel samples, and expressed vascular endothelial markers CD31 and CD34. LACC-CSCs had the ability of directional differentiation as the stem cells. 5) In the transplanted tumor in vitro experiment,we observed tumors in both LACC and LACC-CSCs groups,while the last group showed a stronger tumorigenic with shorter tumor formation time and larger tumors. Conclusions 1. The CSCs of the Human Adenoid Cystic Carcinoma of Lacrimal Gland can be obtained through the serum-free culture,and was expected to collect more by enhancing the culture conditions. 2. The collected CSCs microspheres showed related features as stem cells with the following characteristics: strong aggressivity,tumorigenic force,directional differentiation potential under special environment.This experiment will establish the foundation of further study and treatment trategies targetd to Cancer Stem Cells.