the results presented here indeed show that they are highly similar both on a genomic and transcriptomic level and confirm the previously reported findings that standard propagation of hek293 cell lines does not alter the genomic profile to a large extent. furthermore, based on the hierarchical clustering, the adherent progeny cell lines showed a higher degree of divergence from each other compared to suspension cells. this may be a result of the independent transformation and isolation of the 293t and 293e lineages by the stable integration of different viral genes in different labs. the relatively low expression level of ebna1 observed in 293e cells may be an effect of cultivating cells in the absence of geneticin in this study, in order to minimize differences in cultivation conditions between cell lines. interestingly, a truncated version of the large t antigen was also found to be expressed in 293e cells, which has to our knowledge not been reported previously ( fig. 1e and supplementary fig. s2 ). this sequence was likely derived from the prsvneo plasmid that was co - transfected with pcmv - ebna1 during the isolation of the 293e c18 clone. the overall comparison of the genomic and transcriptomics profiles of hek293 cell lines suggests that the parental hek293 strain has the highest divergence amongst the cell lines. common changes in gene copy number gain or loss ( fig. 2 and supplementary fig. s3 ) and consensus differential expression alterations ( fig. 3 ) were observed amongst all progeny cell lines when compared to hek293. for instance, a common dense pattern of copy number gain and loss for progeny cell lines was observed on chromosome 13 and 18 ( fig. 2a ). such patterns, found across all or several lineages of hek293 isolated independently by different methods, suggests a selective advantage for altered copy numbers of such loci in regard to the phenotypes of the cell lines. on chromosome 13, several genes associated with the cytoskeleton ( bora, mzt1, pibf1, dach1 and klhl1 ) had a copy number gain in all progeny cell lines ( supplementary table s3 ). indeed, consensus gene expression changes associated with cytoskeleton organization were observed between all progeny hek293 cell lines and hek293 ( fig. 3a ). in addition, bora, mzt1 and dach1 were found amongst the 329 genes commonly differentially expressed between all adherent and suspension cell lines ( supplementary table s3 ). moreover, amongst the five genes with high impact snvs found in all progeny cell lines compared to hek293, one gene ( sgcd ) encodes the cytoskeletal protein delta - sarcoglycan. within the gained region of chromosome 18 common to all progeny cell lines except 293e, there are several cell adhesion molecules ( dsc1, dsc2, dsc3, dsg1, dsg2, dsg3 and dsg4 ), which may render this region prone to gene copy number variation during cell line development. the observed enrichment of cell adhesion go - terms ( go: 0, 007, 156 and go: 0, 098, 742 ) amongst genes with common high / moderate impact snps unique to progeny cell lines compared to hek293 cells also supported common genomic alterations in progeny cell lines involved in cell adhesion. combined with the observed down - regulation of the entire cell adhesion gene set in progeny cell lines compared to hek293 ( fig. 3a ), results highlight changes in cytoskeleton and cell adhesion during continuous cultivation and cell line development of hek293 cells. such traits associated with cell adhesion, cell motility and extracellular matrix organization may result from selective pressure, through inefficiency of detaching the most adherent cells, during single cell cloning. indeed, the adherent progeny cell lines 293e and 293t are easier to detach by trypsinization compared to the parental hek293, potentially through altered expression of cell adhesion, cytoskeletal and cell membrane proteins in accordance with the observed consensus changes between progeny cell lines and the parental hek293. moreover, specific genomic regions of more local gain or loss of specific genes were observed, including a loss of fumarate hydratase ( fh ) gene copies. the loss of fh copies was previously observed for hek293 by lin and coworkers and was suggested to play an important part in the transformed phenotype of the cell line. in line with this, our results showed that several of the hek293 progeny cell lines ( 293e, 293 - f and freestyle 293 - f ) were found to only maintain half the number of fh gene copies compared to the original hek293 ( supplementary fig. s4 ), supporting an advantageous loss of the fh gene in the hek293 cell lineages. furthermore, a conserved pattern of substantial gain ( > 1. 5 log2 - fold change ) of the tle4 gene and surrounding loci, was observed for all progeny cells except 293e ( supplementary fig. s4 ). interestingly, tle4 has previously been reported to have both a tumor suppressor function and to be associated with promoting tumor growth in different studies of different cancers. moreover, a significant loss of the adam3a pseudogene ( < - 1 log2 - fold change ), which has previously been associated with different cancers, was observed in all progeny cell lines except 293 - h compared to hek293 ( supplementary fig. s4 ). the specific gain of tle4 and / or loss of adam3a loci and their association with tumor development, suggest important functions of these genes in the evolution of hek293 cell lines, potentially through effects on proliferation and / or evasion of normal cell senescence of progeny cell lines. in bioproduction processes for pharmaceutical proteins, suspension cell lines enable large - scale cultivation in bioreactors, which is required in order to meet the demands for marketed drugs. however, the adaptation of cells from adherent to suspension growth and the differential cultivation procedures between adherent and suspension cells induces phenotypic changes to the cell lines. in order to develop a deeper understanding of such changes, we evaluated differences in gene expression levels between adherent and suspension progeny hek293 cells. consensus differential expression results were found related to up - regulation of genes associated with cell component organization such as membrane, cytoskeleton and cell junction in suspension compared to adherent cells ( fig. 5a ). noteworthy, cell adhesion was found up - regulated in suspension compared to adherent cells. amongst the most significant differentially expressed genes ( adjusted p - value < 0. 01 ) in the cell adhesion gene set ( supplementary table s7 ), several members of the cadherin superfamily, including many different protocadherins ( pcdh ), desmoglein 2 ( dsg2 ) and desmocollin 2 ( dsc2 ) were found significantly up - regulated in suspension cells compared to adherent cells. this family of genes are involved in the formation of adherence junctions between cells. notably, dsg2 and dsc2 are located in the region on chromosome 18 that had gained genomic copies in all progeny cell lines except 293e compared to the parental strain ( fig. 2a ). moreover, four protocadherin members showed the highest fold - change of up - regulated genes in suspension cells ( supplementary table s7 ). the higher expression of such cell adhesion molecules in suspension cell lines compared to adherent progeny hek293 cells may be explained by the loss of culture dish support to grow on in case of suspension cells. upon disrup tion of adhesion interactions with other cells and extracellular matrix, a natural cellular response may be to increase or maintain the expression of adhesion molecules in an attempt to restore such connections. the ability of the suspension cell lines to form cell aggregates during suspension cultivation and the ease of the cells to attach to culture dish surfaces upon cultivation without shaking, can be speculated to support these findings.