Cell viability has a critical impact on product quantity and quality during the biomanufacturing of therapeutic proteins. An advanced understanding of changes in the cellular and conditioned media ...proteomes upon cell stress and death is therefore needed for improved bioprocess control. Here, a high pH/low pH reversed phase data independent 2D‐LC‐MSE discovery proteomics platform was applied to study the cellular and conditioned media proteomes of CHO‐K1 apoptosis and necrosis models where cell death was induced by staurosporine exposure or aeration shear in a benchtop bioreactor, respectively. Functional classification of gene ontology terms related to molecular functions, biological processes, and cellular components revealed both cell death independent and specific features. In addition, label free quantitation using the Hi3 approach resulted in a comprehensive shortlist of 23 potential cell viability marker proteins with highest abundance and a significant increase in the conditioned media upon induction of cell death, including proteins related to cellular stress response, signal mediation, cytoskeletal organization, cell differentiation, cell interaction as well as metabolic and proteolytic enzymes which are interesting candidates for translating into targeted analysis platforms for monitoring bioprocessing response and increasing process control.
A 2D‐LC‐MSE discovery proteomics platform was applied to study the cellular and conditioned media proteomes of CHO‐K1 apoptosis and necrosis models where cell death was induced by staurosporine exposure or aeration shear in a benchtop bioreactor. The comparison of the proteomics profiles allowed for the identification and functional classification of proteins specific and independent of individual cell death types as well as the shortlisting of 23 potential cell viability marker proteins which could be used for improved bioprocessing control.
MiR-7 acts as a tumour suppressor in many cancers and abrogates proliferation of CHO cells in culture. In this study we demonstrate that miR-7 targets key regulators of the G1 to S phase transition, ...including Skp2 and Psme3, to promote increased levels of p27(KIP) and temporary growth arrest of CHO cells in the G1 phase. Simultaneously, the down-regulation of DNA repair-specific proteins via miR-7 including Rad54L, and pro-apoptotic regulators such as p53, combined with the up-regulation of anti-apoptotic factors like p-Akt, promoted cell survival while arrested in G1. Thus miR-7 can co-ordinate the levels of multiple genes and proteins to influence G1 to S phase transition and the apoptotic response in order to maintain cellular homeostasis. This work provides further mechanistic insight into the role of miR-7 as a regulator of cell growth in times of cellular stress.
RNA sequencing (RNASeq) has been widely used to associate alterations in Chinese hamster ovary (CHO) cell gene expression with bioprocess phenotypes; however, alternative messenger RNA (mRNA) ...splicing, has thus far, received little attention. In this study, we utilized RNASeq for transcriptomic analysis of a monoclonal antibody (mAb) producing CHO K1 cell line subjected to a temperature shift. More than 2,465 instances of differential splicing were observed 24 hr after the reduction of cell culture temperature. A total of 1,197 of these alternative splicing events were identified in genes where no changes in abundance were detected by standard differential expression analysis. Ten examples of alternative splicing were selected for independent validation using quantitative polymerase chain reaction in the mAb‐producing CHO K1 cell line used for RNASeq and a further two CHO K1 cell lines. This analysis provided evidence that exon skipping and mutually exclusive splicing events occur in genes linked to the cellular response to changes in temperature and mitochondrial function. While further work is required to determine the impact of these changes in mRNA sequence on cellular phenotype, this study demonstrates that alternative splicing analysis can be utilized to gain a deeper understanding of post‐transcriptional regulation in CHO cells during biopharmaceutical production.
RNA sequencing (RNASeq) has been widely used to associate alterations in Chinese hamster ovary (CHO) cell gene expression with bioprocess phenotypes; however, alternative messenger RNA (mRNA) splicing, has thus far, received little attention. The authors demonstrate that the CHO cell response to subphysiological bioreactor temperature not only results in the widespread alteration of gene expression but also has an equally dramatic effect on mRNA splicing.
Lapatinib has clinical efficacy in the treatment of trastuzumab-refractory HER2-positive breast cancer. However, a significant proportion of patients develop progressive disease due to acquired ...resistance to the drug. Induction of apoptotic cell death is a key mechanism of action of lapatinib in HER2-positive breast cancer cells.
We examined alterations in regulation of the intrinsic and extrinsic apoptosis pathways in cell line models of acquired lapatinib resistance both in vitro and in patient samples from the NCT01485926 clinical trial, and investigated potential strategies to exploit alterations in apoptosis signalling to overcome lapatinib resistance in HER2-positive breast cancer.
In this study, we examined two cell lines models of acquired lapatinib resistance (SKBR3-L and HCC1954-L) and showed that lapatinib does not induce apoptosis in these cells. We identified alterations in members of the BCL-2 family of proteins, in particular MCL-1 and BAX, which may play a role in resistance to lapatinib. We tested the therapeutic inhibitor obatoclax, which targets MCL-1. Both SKBR3-L and HCC1954-L cells showed greater sensitivity to obatoclax-induced apoptosis than parental cells. Interestingly, we also found that the development of acquired resistance to lapatinib resulted in acquired sensitivity to TRAIL in SKBR3-L cells. Sensitivity to TRAIL in the SKBR3-L cells was associated with reduced phosphorylation of AKT, increased expression of FOXO3a and decreased expression of c-FLIP. In SKBR3-L cells, TRAIL treatment caused activation of caspase 8, caspase 9 and caspase 3/7. In a second resistant model, HCC1954-L cells, p-AKT levels were not decreased and these cells did not show enhanced sensitivity to TRAIL. Furthermore, combining obatoclax with TRAIL improved response in SKBR3-L cells but not in HCC1954-L cells.
Our findings highlight the possibility of targeting altered apoptotic signalling to overcome acquired lapatinib resistance, and identify potential novel treatment strategies, with potential biomarkers, for HER2-positive breast cancer that is resistant to HER2 targeted therapies.
Background
The use of intraoperative MRI (iMRI) during treatment of gliomas may increase extent of resection (EOR), decrease need for early reoperation, and increase progression-free and overall ...survival, but has not been fully validated, particularly in the pediatric population.
Objective
To assess the accuracy of iMRI to identify residual tumor in pediatric patients with glioma and determine the effect of iMRI on decisions for resection, complication rates, and other outcomes.
Methods
We retrospectively analyzed a multicenter database of pediatric patients (age ≤ 18 years) who underwent resection of pathologically confirmed gliomas.
Results
We identified 314 patients (mean age 9.7 ± 4.6 years) with mean follow-up of 48.3 ± 33.6 months (range 0.03–182.07 months) who underwent surgery with iMRI. There were 201 (64.0%) WHO grade I tumors, 57 (18.2%) grade II, 24 (7.6%) grade III, 9 (2.9%) grade IV, and 23 (7.3%) not classified. Among 280 patients who underwent resection using iMRI, 131 (46.8%) had some residual tumor and underwent additional resection after the first iMRI. Of the 33 tissue specimens sent for pathological analysis after iMRI, 29 (87.9%) showed positive tumor pathology. Gross total resection was identified in 156 patients (55.7%), but this was limited by 69 (24.6%) patients with unknown EOR.
Conclusions
Analysis of the largest multicenter database of pediatric gliomas resected using iMRI demonstrated additional tumor resection in a substantial portion of cases. However, determining the impact of iMRI on EOR and outcomes remains challenging because iMRI use varies among providers nationally. Continued refinement of iMRI techniques for use in pediatric patients with glioma may improve outcomes.
The use of microRNAs (miRNAs) for improving the efficiency of recombinant protein production by CHO cells is gaining considerable interest for their ability to regulate entire molecular networks. ...Differential miRNA expression profiling and large-scale transient screening have been the prerequisite for the selection of miRNA candidates for stable manipulation, reported in CHO cells expressing a range of recombinant products. We selected a potent and well characterised tumour suppressor miRNA, miR-34a, as a high priority candidate for CHO cell engineering based on the conservation of both its sequence and function across species and cell type. Ectopic expression of miR-34a retained its functional conservation in CHO-SEAP cells by inhibiting growth by 90 % in addition to decreasing the viable cell population by 30 % when compared to controls. When the miR-34 family was stably depleted using a miRNA sponge decoy vector, the overall product yield was enhanced by ~2-fold in both fed-batch and small scale clonal batch cultures, despite having a negative impact on cell growth. These findings further strengthen the utility of miRNAs as engineering tools to modify and improve CHO cell performance.
Cell line development aims to generate and select clones with desirable characteristics. One of the most important parameters for biopharmaceutical cell selection is cell-specific productivity (Qp) ...or the quantity of product produced per cell per day. Fluorescence-activated cell sorting (FACS) is a powerful, high-throughput technique that facilitates multiparametric characterization and isolation of individual cell clones from heterogeneous populations. Here, we describe a FACS-based method for section of high-producing CHO cell clones.
Our ability to study Chinese hamster ovary (CHO) cell biology has been revolutionised over the last decade following the development of next generation sequencing technology and publication of ...reference DNA sequences for CHO cells and the Chinese hamster. RNA sequencing has not only enabled the association of transcript expression with bioreactor conditions and desirable bioprocess phenotypes but played a key role in the characterisation of protein coding and small noncoding RNAs. The annotation of long noncoding RNAs, and therefore our understanding of their role in CHO cell biology, has been limited to date. In this manuscript, we use high‐resolution RNASeq data to more than double the number of annotated lncRNA transcripts for the CHO K1 genome. In addition, the utilisation of strand‐specific sequencing enabled the identification of more than 1,000 new antisense and divergent lncRNAs. The utility of monitoring lncRNA expression is demonstrated through an analysis of the transcriptomic response to a reduction of cell culture temperature and identification of simultaneous sense/antisense differential expression for the first time in CHO cells. To enable further studies of lncRNAs, the transcripts annotated in this study have been made available for the CHO cell biology community.
Long non‐coding RNAs (lncRNAs) are emerging as potential cell line engineering candidates to increase the efficiency of biopharmaceutical production in Chinese hamster ovary (CHO) cells. In this manuscript, the authors describe the utilisation of RNA sequencing (RNASeq) to significantly increase the number of annotated CHO cell lncRNA transcripts. In addition, the authors demonstrate that lncRNA expression is altered upon the reduction of cell culture temperature. This study is an important enabling step for wider utilisation of lncRNAs for CHO cell genetic engineering
In this study, we report an investigation of a panel of clonally‐derived Chinese hamster ovary (CHO) cell lines exhibiting variability in the proportion of full‐length IgG4 Fc‐fusion protein ...produced. The recombinant protein was found to be degraded during cell culture into four shorter “clipped” species (three of the four cleavage sites occurred at arginine residues) and preliminary analyses suggested that a host cell enzyme was responsible for proteolysis. To identify the specific enzyme responsible, RNA sequencing was used to identify gene expression differences between the cell lines with a “high” and “low” clipping phenotype. From this analysis, six protease‐encoding genes were found to be significantly upregulated in those cell lines yielding the lowest proportion of full‐length IgG4 Fc‐fusion protein. Four of these protease candidates were deprioritized after examination of their cleavage site specificity. The remaining enzymes, Adam19 and Furin, were found to be capable of cleavage at arginine residues, and inhibitors for both proteases were added to cell‐free media to determine if the product degradation could be reduced. While the Adam19 inhibitor had no impact, Furin inhibitor I (specific for the proprotein convertase family of enzymes) was found to result in a 33–39% increase in complete IgG4 Fc‐fusion protein when compared with untreated samples.
The authors report the application of next generation sequencing to investigate mRNA changes associated with increased proteolysis of an IgG4 Fc‐fusion protein in a panel of clonally derived CHO cell lines. Differential gene expression analysis identified six upregulated proteases and following comparison of known substrates Adam19 and Furin proprotein convertase were prioritized. Subsequent addition of Furin Inhibitor I to cell free media was found to reduce the level of IgG4 Fc‐fusion protein degradation by at least 33% in comparison to an uninhibited control after 96 hrs incubation.
Objectives
We used miRNA and proteomic profiling to understand intracellular pathways that contribute to high and low specific productivity (Qp) phenotypes in CHO clonally derived cell lines (CDCLs) ...from the same cell line generation project.
Results
Differentially expressed (DE) miRNAs were identified which are predicted to target several proteins associated with protein folding. MiR-200a was found to have a number of predicted targets associated with the unfolded protein response (UPR) which were shown to have decreased expression in high Qp CDCLs and have no detected change at the mRNA level. MiR-200a overexpression in a CHO CDCL was found to increase recombinant protein titer by 1.2 fold and Qp by 1.8 fold.
Conclusion
These results may suggest a role for miR-200a in post-transcriptional regulation of the UPR, presenting miR-200a as a potential target for engineering industrially attractive CHO cell phenotypes.
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