Project description:Cancer-associated fibroblasts (CAFs) have been recognized as important contributors to cancer development and progression. However, opposing evidence has been published whether CAFs, in addition to epigenetic, also undergo somatic genetic alterations and whether these changes contribute to carcinogenesis and tumour progression. We combined multiparameter DNA flow cytometry, flow-sorting and 6K SNP-arrays to study DNA aneuploidy, % S-phase, loss of heterozygosity (LOH) and copy number alterations (CNAs) to study somatic genetic alterations in cervical cancer-associated stromal cell fractions (n = 58) from formalin-fixed, paraffin-embedded (FFPE) samples. Tissue sections were examined for the presence of CAFs. Microsatellite analysis was used to study LOH. By flow cytometry we found no proof for DNA aneuploidy in the vimentin-positive stromal cell fractions of any samples (CV G0G1 population 3.7% +/- 1.2; S-phase 1.4% +/- 1.8). The genotype concordance between the stromal cells and the paired normal endometrium samples was > 99.9%. No evidence for CNAs or LOH was found in the stromal cell fractions. In contrast, high frequencies of DNA content abnormalities (43/57), a significant higher S-phase (14.6% +/- 8.5)(p = 0.0001) and substantial numbers of CNAs and LOH were identified in the keratin-positive epithelial cell fractions (CV G0G1 population 4.1% +/- 1.0). Smooth muscle actin and vimentin immunohistochemistry verified the presence of CAFs in all cases tested. LOH hot-spots on chromosomes 3p, 4p and 6p were confirmed by microsatellite analysis but the stromal cell fractions showed retention of heterozygosity only. From our study we conclude that stromal cell fractions from cervical carcinomas are DNA diploid, have a genotype undistinguishable from patient-matched normal tissue and are genetically stable. Stromal genetic changes do not seem to play a role during cervical carcinogenesis and progression. In addition, the stromal cell fraction of cervical carcinomas can be used as reference allowing large retrospective studies of archival FFPE tissues for which no normal reference tissue is available. Paired experiment, Endometrial (non-tumor) cells vs stromal cells from cervical tumors. Biological replicates: 58 patients. From 5 tumors also the tumor fraction was profiled.

Project description:Recurrent non-medullary thyroid carcinoma (NMTC) is a rare disease. We initially characterized 27 recurrent NMTC: 13 papillary thyroid cancers (PTC), 10 oncocytic follicular carcinomas (FTC-OV), and 4 non-oncocytic follicular carcinomas (FTC). A validation cohort composed of benign and malignant (both recurrent and non-recurrent) thyroid tumours was subsequently analysed (n = 20). Methods Data from genome-wide SNP arrays and flow cytometry were combined to determine the chromosomal dosage (allelic state) in these tumours, including mutation analysis of components of PIK3CA/AKT and MAPK pathways. Results All FTC-OVs showed a very distinct pattern of genomic alterations. Ten out of 10 FTC-OV cases showed near-haploidisation with or without subsequent genome endoreduplication. Near-haploidisation was seen in 5/10 as extensive chromosome-wide monosomy (allelic state [A]) with near-haploid DNA indices and retention of especially chromosome 7 (seen as a heterozygous allelic state [AB]). In the remaining 5/10 chromosomal allelic states AA with near diploid DNA indices were seen with allelic state AABB of chromosome 7, suggesting endoreduplication after preceding haploidisation. The latter was supported by the presence of both near-haploid and endoreduplicated tumour fractions in some of the cases. Results were confirmed using FISH analysis. Relatively to FTC-OV limited numbers of genomic alterations were identified in other types of recurrent NMTC studied, except for chromosome 22q which showed alterations in 6 of 13 PTCs. Only two HRAS, but no mutations of EGFR or BRAF were found in FTC-OV. The validation cohort showed two additional tumours with the distinct pattern of genomic alterations (both with oncocytic features and recurrent). Conclusions We demonstrate that recurrent FTC-OV is frequently characterised by genome-wide DNA haploidisation, heterozygous retention of chromosome 7, and endoreduplication of a near-haploid genome. Whether normal gene dosage on especially chromosome 7 (containing EGFR, BRAF, cMET) is crucial for FTC-OV tumour survival is an important topic for future research. 28 thyroid tumors from 27 patients were profiled by SNP array. Comparisons between different types were made.

Project description:Hui2014 - Age-related changes in articular cartilage This model is described in the article: Oxidative changes and signalling pathways are pivotal in initiating age-related changes in articular cartilage Wang Hui1, David A Young1, Andrew D Rowan1, Xin Xu2, Tim E Cawston1, Carole J Proctor1,3 Annals of the Rheumatic Diseases Abstract: Objective: To use a computational approach to investigate the cellular and extracellular matrix changes that occur with age in the knee joints of mice. Methods: Knee joints from an inbred C57/BL1/6 (ICRFa) mouse colony were harvested at 3–30?months of age. Sections were stained with H&E, Safranin-O, Picro-sirius red and antibodies to matrix metalloproteinase-13 (MMP-13), nitrotyrosine, LC-3B, Bcl-2, and cleaved type II collagen used for immunohistochemistry. Based on this and other data from the literature, a computer simulation model was built using the Systems Biology Markup Language using an iterative approach of data analysis and modelling. Individual parameters were subsequently altered to assess their effect on the model. Results: A progressive loss of cartilage matrix occurred with age. Nitrotyrosine, MMP-13 and anaplastic lymphoma kinase (ALK1) staining in cartilage increased with age with a concomitant decrease in LC-3B and Bcl-2. Stochastic simulations from the computational model showed a good agreement with these data, once transforming growth factor-? signalling via ALK1/ALK5 receptors was included. Oxidative stress and the interleukin 1 pathway were identified as key factors in driving the cartilage breakdown associated with ageing. Conclusions: A progressive loss of cartilage matrix and cellularity occurs with age. This is accompanied with increased levels of oxidative stress, apoptosis and MMP-13 and a decrease in chondrocyte autophagy. These changes explain the marked predisposition of joints to develop osteoarthritis with age. Computational modelling provides useful insights into the underlying mechanisms involved in age-related changes in musculoskeletal tissues. This model is hosted on BioModels Database and identified by: BIOMD0000000560. To cite BioModels Database, please use: BioModels Database: An enhanced, curated and annotated resource for published quantitative kinetic models. To the extent possible under law, all copyright and related or neighbouring rights to this encoded model have been dedicated to the public domain worldwide. Please refer to CC0 Public Domain Dedication for more information.

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