Tissue cultures of immortalized human cells, also known as established cell lines, are broadly accessible and cost‐efficient tools for biomedical research. We here review potential genetic sources of systematic error in cell line experiments due to clonal evolution in vitro. In particular, the authors highlight alterations in telomere function over prolonged culture and population bottlenecks, respectively, as two commonly overlooked phenomena that can result in significant alterations in cell line genotypes over just one or a few passages in vitro. These alterations may include changes in mutation status of oncogenes and large scale chromosomal imbalances. We introduce a simple list of factors to be avoided in order to reduce the risk of data misinterpretation due to clonal evolution, including unacknowledged in vitro selection pressures, prolonged culture per se, harsh population size reductions, experiments at early phases after establishment, and the employment of cell lines not sufficiently analyzed by high resolution genetic techniques.

The relationship between the three-dimensional (3D) nuclear telomere architecture and specific genetic alterations in papillary thyroid carcinoma (PTC), in particular in cancer stem-like cells (CSLCs), has not yet been investigated. We isolated thyrospheres containing CSLCs from B-CPAP, K1, and TPC-1 PTC-derived cell lines, representative of tumors with different genetic backgrounds within the newly identified BRAF V600E -like PTC subgroup, and used immortalized normal human thyrocytes (Nthy-ori 3.1) as control. We performed quantitative fluorescence in situ hybridization, 3D imaging, and 3D telomere analysis using TeloView software to examine telomere dysfunction in both parental and thyrosphere cells. Among the 3D telomere profile, a wide heterogeneity was observed, except for telomere intensity. Our findings indicate that CSLCs of each cell line had longer telomeres than parental cells, according to telomere intensity values, which correlate with telomere length. Indeed, the thyrosphere cells had lower numbers of lower-intensity telomeres (≤5,000 arbitrary fluorescent units, a.u.), compared with parental cancer cells, as well as parental control cells, (p < 0.0001). The B-CPAP thyrospheres showed a decreased number of higher intensity telomeres (>17,000 a.u.) than K1 and TPC-1 cells, as well as control cells (p < 0.0001). By selecting PTC-derived cell lines with different genetic backgrounds characteristic of BRAF V600E -like PTC subgroups, we demonstrate that thyrosphere cells with BRAF V600E and TP53 mutations show shorter telomeres than those harboring RET/PTC or BRAF V600E and wild-type TP53.Hence, our data reveal a trend towards a decrease in telomere shortening in CSLCs, representing the early cancer-promoting subpopulation, as opposed to parental cells representing the tumor bulk cells. K E Y W O R D S 3D quantitative nuclear telomere analysis, cancer stem-like cells, papillary thyroid carcinoma, quantitative fluorescence in situ hybridization 1 | INTRODUCTION Papillary thyroid carcinoma (PTC) is the most common thyroid malignancy, and it accounts for 85-90% of differentiated thyroid tumors (DeLellis, Lloyd, Heitz & Eng, 2004). Recently, it has been reported that, at the genomic level, PTC consists of two highly distinct classes reflecting the tumor histology: the BRAF V600E -like nodules, predominantly characterized by BRAF V600E mutations and Rearranged during Transfection (RET) kinase fusions, which show the true papillary architecture; and RAS-like nodules, with RAS mutations, J Cell Physiol. 2019;234:5175-5185. wileyonlinelibrary.com/journal/jcp