Functional and genomic characterization of three novel cell lines derived from a metastatic gallbladder cancer tumor

BACKGROUND: Gallbladder cancer (GBC) is the most common tumor of the biliary tract. The incidence of GBC shows a large geographic variability, being particularly frequent in Native American populations. In Chile, GBC represents the second cause of cancer-related death among women. We describe here the establishment of three novel cell lines derived from the ascitic fluid of a Chilean GBC patient, who presented 46% European, 36% Mapuche, 12% Aymara and 6% African ancestry. RESULTS: After immunocytochemical staining of the primary cell culture, we isolated and comprehensively characterized three independent clones (PUC-GBC1, PUC-GBC2 and PUC-GBC3) by short tandem repeat DNA profiling and RNA sequencing as well as karyotype, doubling time, chemosensitivity, in vitro migration capability and in vivo tumorigenicity assay. Primary culture cells showed high expression of CK7, CK19, CA 19-9, MUC1 and MUC16, and negative expression of mesothelial markers. The three isolated clones displayed an epithelial phenotype and an abnormal structure and number of chromosomes. RNA sequencing confirmed the increased expression of cytokeratin and mucin genes, and also of TP53 and ERBB2 with some differences among the three cells lines, and revealed a novel exonic mutation in NF1. The PUC-GBC3 clone was the most aggressive according to histopathological features and the tumorigenic capacity in NSG mice. CONCLUSIONS: The first cell lines established from a Chilean GBC patient represent a new model for studying GBC in patients of Native American descent.

In vitro differentiation of human bone marrow mesenchy-mal stem cells into hepatocyte-like cells

Orthotropic liver transplantation [OLT] is the final procedure of both end stage and metabolic liver diseases. Hepatocyte transplantation is an alternative for OLT, but the sources of hepatocytes are limited. Bone marrow mesenchymal stem cells [BM-MSCs] can differentiate into hepatocyte-like cells and are a potential alternative source for hepatocytes. We aimed to investigate the differentiation potential of BM-MSCs into hepatocyte-like cells. Human BM-MSCs from a healthy donor were cultured and differentiated into hepatocyte-like cells. We investigated the expression of hepatocyte-specific markers in MSC-derived hepatocyte-like cells [MSC-HLC[s]] and evaluated their functionality using metabolic assays. MSC-HLCs expressed hepatocyte-specific markers at both mRNAand protein levels. In addition, the cells had the ability to uptake low density lipoprotein [LDL], clear ammonia, secrete albumin, and store glycogen. MSC-HLCs were transplanted into a familial hypercholesteromia patient. Human MSCs can be differentiated into partially functional hepatocyte-like cells. Thus, they could be a potential source for cell therapy in liver disorders

Fluctuation of circulating tumor cells in patients with lung cancer by real-time fluorescent quantitative-PCR approach before and after radiotherapy.

BACKGROUND AND AIMS: The failure to reduce the mortality of patients with solid tumours is mainly a result of the early dissemination of cancer cells to secondary site, which is usually missed by conventional diagnostic procedures used for tumour staging. The possibility to use easily accessible body fluids as a source for circulating tumour cells (CTCs) detection enables longitudinal observations of the disease. In the study, we evaluated the CTCs in lung cancer following locoregional radiation therapy. METHODS: Samples of 5 ml peripheral blood was taken from each lung cancer patients (n=15) both before and after the radiotherapy course. Meanwhile tumour size was determined by chest X-ray or computed tomography. Using cytokeratin 19(CK19) as marker, the blood samples were subjected to real time RT-PCR assay. All patients with lung cancer were treated with primary definitive and mediastinal radiotherapy. RESULTS: Compare to that of pre-treatment, the value of CK19 mRNA in peripheral blood after therapy decreased dramatically (5.0932+/-1.0628 vs. 4.2493+/-0.8323, t=3.192, P=0.007). The change of CK19 mRNA level before and after radiotherapy was closely related to the type (NSCLC vs. SCLC, 0.5389+/-0.9030 vs. 1.6826+/-0.9467, t=2.1465, P=0.051). Meanwhile, there appeared to be a close link between the grade (Well/Mod vs. Poor) and the change of CK19 mRNA (0.5024 vs. 1.5271, t=2.017, P=0.065). The change of CK19 mRNA level was related to variation of tumour burden during radiotherapy (r=0.0575, P=0.025). Of the 15 cases studied, 12 cases were positive before radiotherapy (12/15, 80%). The positive rate was 53% (8/15) after radiotherapy, meaning that four patients converted into negative after radiotherapy. CONCLUSIONS: The disseminated circulating cancer cells can be affected by radiotherapy; meanwhile further more systemic adjuvant treatment should be conducted. Due to concordance between molecular response and radiological remission, assessment of the therapeutic response might be possible by serial quantitative of CTCs.

Use of Rt-PCR in detecting disseminated cancer cells after incisional biopsy among oral squamous cell carcinoma patients.

PURPOSE: Tumour metastasis is the most clinically significant and enigmatic aspect of tumour behavior and is an unequivocal hallmark of malignancy. Until recent years little has been known about the transportation phase of vascular dissemination during biopsy, because of the technical difficulties in demonstrating circulating cancer cells. AIMS: This study examined whether cancer cell dissemination results from incisional biopsy in the peripheral blood by using Cytokeratin 19(CK-19) as the marker for Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR). In-house recipes without utilizing kits were employed to extract genomic and total RNA to make the procedure user friendly. MATERIALS AND METHODS: The study population consisted of n=10 patients who were clinically diagnosed for oral squamous cell carcinoma and who had not undergone any previous biopsies. 5 patients who were to undergo incisional biopsies for benign conditions served as controls. 5 ml of blood aspirates were collected before and within 15 minutes after incisional biopsy. CK-19 gene and a positive control gene beta actin were isolated to confirm the primers. Using the total RNA, RT-PCR was performed for beta actin and Ck 19 gene expression. RESULTS: Rt-PCR did show any expression for the CK-19 gene. CONCLUSION: In conclusion there was no evidence of dissemination of cancer cells in our study and the patients are on a regular follow up for the past one and half years. But larger sample size should be examined to make the procedure a diagnostic tool for cancer metastasis.