Over the years significant efforts have been made to adapt Vero cells to suspension in order to engineer high-throughput and scalable vaccine production platforms, however, those efforts were limited by hurdles such as low cell viability and long doubling time. In order to design more efficient strategies for successful adaptation to suspension cultures maintaining acceptable cell viability and doubling time, it is necessary to better understand the genetic and phenotypic changes triggered by the adaptation to the suspension state. Thus, we propose in this paper a comparative functional genomics analysis of adherent and suspension Vero cells from the gene to the protein interaction network level.
Indeed, correlations between differential expression analysis (Figure 2, 3), metabolic pathway enrichment analyses (Figure 4), gene set enrichment analyses (Figure 5) and network topology analyses (Tables 1, 2) highlight key events such as the upregulation of immune response, exocytosis and vacuole pathways which are involved in the storage of waste and other exocytosis molecules.
Furthermore, several events of checks-and-balances were observed across the different analysis methods used (Table 3): lipids metabolism and lipid pathways are also upregulated via pathway enrichment while the metabolic analysis revealed a downregulation of CPT1 and palmitate associated metabolic reactions which regulate fatty acid oxidation in mitochondria and whose upregulation impairs glucose homeostasis [18]. In parallel, the gluconeogenesis metabolic pathway is upregulated while the glycolytic pathway is downregulated thus hindering ATP generation. That resulting stress is met with not only a downregulation of the proline metabolic pathway which constitute a checkpoint that is reported to promote proline accumulation during stress [19], but also an upregulation of asparagine metabolism which promotes the cell adaptation to nutrient depravation and/or hypoxia [15] thus promoting cell survival.
Surprisingly, as observed in HEK293 cells adapted to suspension[20], cellular component organization pathways associated with cell adhesion such as actin filament-based process, regulation of cell adhesion, apical part of cell, plasma membrane bounded cell projection morphogenesis and extracellular matrix are upregulated via pathway enrichment analysis, while NTA showed an upregulation of the cell adhesion PPI network with CDH18 as its central gene and which can be considered as possible target for engineering in order to improve the cells adaptation to suspension. This upregulation of cell adhesion related genes could be due to the cells’ attempt to restore the attachment to culture surfaces and surrounding cells which could explain the aggregates that are often observed in Vero cells suspension cultures and the cell rings that form on the suspension culture dishes.
On the other hand, the adherens junction pathway, which regulates cell-cell adhesion and is essential for viability (via the control of cell proliferation, polarity, shape, motility and survival) [21], is downregulated alongside the aspartate metabolic pathway, the mitochondrial 1-carbon metabolic pathway, the MYC and E2F targets pathway, which could explain the low cell density observed during Vero cells adaptation to suspension.
Moreover, the pathways related to cell division, mitotic cell cycle phase transition are downregulated via pathway enrichment, which is confirmed by the downregulation of the folate metabolic pathway which leads to cell cycle arrest at G0/G1 [22] and the downregulation of FYN which is central to the networks associated with the control of cell growth, thus, providing some insight in the origin of the long doubling time observed in Vero cells adapted to suspension culture. Nonetheless, Vero cells attempt to balance that effect on doubling time via the upregulation of the glycine, threonine and serine pathway which associated with an unrestrained cell cycle progression [14], and the upregulation of RHOU, ESR1 which are central to the anatomical structure morphogenesis pathway and more precisely cell proliferation and antiapoptotic regulations.
Lastly the epithelial to mesenchymal transition (EMT) pathway is downregulated in Vero cells adapted to suspension, thus highlighting the fact that the adaptation to suspension is not associated with EMT as previously shown with HEK293 cells adapted to suspension [20].
To conclude, we present in this paper key genes pathways at play during the adaptation of Vero cells to suspension and their complex checks-and-balances which could assist in the successful adaptation of Vero cells to suspension. Indeed, those key genes, notably associated with cell adhesion or hindering cell viability or doubling time could be potentially targeted via gene editing as new strategies for the adaptation to suspension, but also the observed competitions between the regulation of competing pathways can be studied more in detail via targeted perturbations using gene editing tools such as CRISPR [23].