such cell adhesion molecules found up - regulated in suspension cell lines may thus be appropriate cell line engineering targets for improved bioprocess performance of suspension cell lines. further evaluation of the differentially expressed genes between adherent and suspension hek293 progeny cell lines, based on metabolic gene set analysis, highlighted changes in biosynthesis of aromatic amino acids and pathways related to lipids and / or cholesterol metabolic processes ( fig. 4b ). these metabolic changes could be a result of different growth media compositions used for the cultivation of either adherent or suspension cells that may imply different concentrations of for instance amino acids, glucose or serum. when reducing the number of differentially expressed genes to those that consistently showed differential expression between adherent and suspension cells in pairwise comparisons of all cell lines, the cholesterol and sterol biosynthesis and metabolism pathway were found to be most significantly different between the cell types ( fig. 5b ). moreover, five of the consistently up - regulated genes in suspension hek293 compared to adherent encode enzymes that have either direct roles in the cholesterol biosynthesis pathway ( msmo1, hmgcs1 and idi1 ), or proteins that are associated with cholesterol metabolism by various processes ( npc1l1 and insig1 ). as suspension cell lines are cultivated under serum free conditions, the increased expression of genes associated with for instance cholesterol in suspension cell lines may be a result of a lower cholesterol content in the medium. however, as cholesterol is a major component of the cell membrane and has an important function for membrane structure and cell signaling, the differential expression of genes associated with cholesterol synthesis and metabolism may also be of importance for the different morphologies between adherent and suspension hek293 cells. indeed, previous studies have shown that cholesterol plays a critical role in regulating the formation of cell - to - cell interactions in endothelial cells and that depletion of cholesterol reduces cell adhesion and increases endothelial cell stiffness. increased cell surface stiffness has been reported for hek293 cells in suspension compared to adherent state as a result of up - regulation and re - organization of the actin cytoskeleton. this may partly be a result of altered cholesterol levels in the cell membrane since cholesterol is a regulator of the actin cytoskeleton and cholesterol depletion has been shown to induce actin polymerization. interestingly, the insulin - induced gene 1 protein ( insig1 ), which was up - regulated in suspension compared to adherent hek293, is a negative regulator of cholesterol synthesis and important for cholesterol homeostasis and knockout of insig1 has previously been shown to result in cholesterol accumulation. notably, a lower cholesterol biosynthesis in suspension cell lines compared to the original hek293 strain was indeed predicted using ipa ( supplementary fig. s7 ). it should however be noted that this prediction does not take into consideration the effect of insig1, instead the predicted reduction in cholesterol biosynthesis in suspension cells compared to the hek293 cell line is a result of down - regulation of sc5d ( lathosterol oxidase ). from a bioprocess perspective, differences in intracellular cholesterol synthesis and metabolism may also be of interest with regards to the secretory capacity of a cell line since previous findings has shown that cholesterol is essential for er to golgi transport within the secretory pathway and that secreted productivity of cho cells increases upon elevated intracellular cholesterol levels, through silencing of insig1, possibly due to increasing the volume of the golgi compartment. it would therefore be of interest to gain further knowledge about the cholesterol content and distribution within hek293 cell lines and potentially evaluate if this pathway can be targeted for enhanced bioproductivity without having a deleterious impact on suspension growth or cell morphology. four of the 38 genes ( id1, smad7, txnip and lox ) that were consistently differentially expressed between adherent and suspension hek293 have previously been annotate d to play a role in epithelial to mesenchymal transition ( emt ), the event where stationary epithelial cells lose their cell – cell adhesion and change into motile and invasive mesenchymal cells. however, when evaluating the expression of common markers for mesenchymal and endothelial phenotypes as well as predicting the outcome of the emt pathway using ipa, the parental hek293 strain showed the most mesenchymal - like phenotype whereas suspension cell lines were predicted to have reduced transition from epithelial to mesenchymal phenotype compared with hek293 ( supplementary fig. s8 ). these results indicate that the suspension adaptation of hek293 lineages does not follow the emt pathway. altogether nine of the 38 identified genes ( lox, id1, adamts1, zic1, kcnma1, dhrs3, rarg, col4a6 and arrdc4 ), with differential expression between all adherent and suspension comparisons of hek293 ( fig. 5a ), were shown to have significantly different expression between adherent and suspension cells also in an extended validation of the genes in a set of 63 human cell lines from the hpa database ( fig. 6c ). five of these genes ( lox, id1, zic1, dhrs3 and rarg ) showed a consistent expression profile ( same direction of up - or downregulation ) between adherent and suspension cells compared to the results presented in fig. 5b, suggesting a key role of these genes in the morphologies of adherent and suspension human cell lines. in support of this hypothesis, up - regulation of id1, as found in adherent cells compared to suspension cell lines, has been associated with the mesenchymal - to - epithelial transition. moreover, id1 silencing has also been shown to significantly reduce adhesion of neural stem cells and conversely, increased id1 expression in epithelial cells has been related to increased adhesion. in addition, lysyl oxidase ( lox ), an enzyme responsible for the covalent cross - linking between elastin and collagen in the extracellular matrix, has been shown to be important for cell – matrix adhesion formation, supporting the adherent phenotype of adherent cells but is also associated with cell invasion and induction of emt. besides the emt - related genes, the additional three genes ( rarg, zic1 and dhrs3 ), consistently up - regulated in adherent cells compared to suspension cell lines, have previously been associated with increased cell adhesion through the retinoic acid signaling pathway. in line with this, retinol metabolism was found to be down-regulate d in suspension cells in the metabolic gene set analysis ( fig. 4b ). our study has outlined the genomic and transcriptomic variations between six industrially relevant hek293 cell lines, in an attempt to improve the understanding of their respective differences in phenotype. we report a selective pressure to develop certain expression profiles during the evolution and continuous cultivation, evidenced by the numerous genes and pathways detailed here. the key common changes between hek293 and its progeny cell lines involve in particular cell membrane proteins and processes related to cell adhesion, motility and the organization of various cellular components such as the cytoskeleton and extracellular matrix. in addition, changes associated with differences between adherent and suspension cell growth in particularly involve changes in cell adhesion protein expression, cholesterol metabolism and a set of six key genes ( rarg, id1, zic1, lox and dhrs3 ) with potentially key roles in the differentiation between the two groups. these results could be of importance when pursuing further cell line engineering or bioprocess optimization of these and other human cell lines.