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MECHANISMS REGULATING GROWTH PROGRESSION OF SALIVARY GLAND NEOPLASMS USING MUCOEPIDERMOID CARCENOMA PRIMARY CELLS

2017-04-17 14:57:18

Georgetown University April 17 2017


Ahmad Mohmad Alamri



Abstract


Acquiring patient-derived primary cancer cells is a means towards individualized cancer treatment and a critical step for precision medicine. Strategies that allow efficient isolation of primary cells that enable genetic and chemosensitivity studies reflective of the original cancer are critical. The first goal of this project was to study and compare culture conditions (conditional reprograming culture (CRC) with a rho kinase inhibitor (Y-27632) and irradiated J2 feeder cells and EpiCultâ„¢ (STEMCELL Tech) (EpiC)) for establishment efficiency, differentiation, allograft generation, and transcriptome characterization, of triple negative primary mammary non cancer and cancer cells isolated from genetically engineered mouse models with different Brca1 gene dosages (100%, 50%, 0%). The second goal was to establish and propagate primary epithelial cells from malignant and benign human salivary gland neoplasms using CRC and to perform transcriptome and exome analyses on different tumor regions. A low-grade sublingual salivary mucoepidermoid carcinoma (MEC) was used as an exemplar for further analyses including comparison of growth parameters under different culture conditions (non-CR 2D, 3D, and xenograft), RNAseq and exomeseq analyses, and chemosensitivity assays of AKT inhibitors MK2206 and GSK690693 in 2 and 3D culture formats. Addressing the first goal, transcriptome comparison of CRC and EpiC cultures revealed 1700 differentially iv expressed genes by passage 20. Trp53 gene family up-regulation and increased expression of epithelial differentiation genes was observed in CRC while in EpiC, expression of epithelial-mesenchymal-transition genes was
up-regulated. The number of differentially expressed genes was diminished by short-term passaging. For the second goal a primary cell biobank was established from benign and malignant salivary gland tissue. No driver mutations were identified form exome sequencing. MEC primary cells grew under different conditions and developed into xenografts. RNAseq revealed upregulation of AKT pathway genes. The primary cells were sensitive to MK2206 but not GSK690603. In conclusion, CRC technology was an efficient means of generating primary cancer cell cultures and results from genetic and chemosensitivity studies demonstrated the reproducibility of this approach, even when cells from different areas of a tumor were taken. AKT pathway is upregulated in MEC primary cells and could be a promising candidate for targeted therapies.




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