Abstract
We investigated how varying the composition of cell culture formulations and growing cancer cells at different densities might affect tumor cell genotype. Specifically, we compared gene expression profiles generated by human MDA-MB-231 breast cancer cells cultured in different media [minimum essential medium (MEM), Dulbecco’s modified Eagle’s medium (DMEM), or Roswell Park Memorial Institute (RPMI)-1640 medium] containing different concentrations of fetal bovine serum (FBS) or different sera (equine or bovine) that were grown at different cell densities. More than 2,000 genes were differentially modulated by at least a 2-fold difference when MDA-MB-231 cancer cells were 90% confluent and compared with cultures that were 50% confluent. Altering the concentration of serum produced an even more pronounced effect on MDA-MB-231 cancer cell gene expression in that 2,981 genes were differentially expressed in a comparison between cells cultured in 0.1% FBS and same cell density cultures that were maintained in 10% FBS. A comparison between MDA-MB-231 cancer cells that were 90% confluent in MEM, DMEM, or RPMI-1640 media, all containing 10% FBS, resulted in 8,925 differentially expressed genes. Moreover, one-quarter (25.6%) of genes from our genome-wide expression analysis were expressed at significantly different levels by cells grown in MEM, DMEM, or RPMI-1640 media. Genes associated with epithelial-mesenchymal transition (EMT) were among the genes that were differentially modulated by cells grown in different cell culture formulations and these genes were verified at the protein level. Collectively, these results underscore the importance of accurate reporting and maintenance of uniform culture conditions to ensure reproducible results.
Highlights
Recent reports have sought to raise awareness of the growing number of cancer research studies whose findings cannot be independently reproduced [1]
The cells were grown as monolayers in MEM supplemented with 10% fetal bovine serum (FBS) and harvested for analysis when they were 50 or 90% confluent in culture dishes
1,100 genes were significantly upregulated in cancer cells that were 90% confluent when compared to cells that were 50% confluent
Summary
Recent reports have sought to raise awareness of the growing number of cancer research studies whose findings cannot be independently reproduced [1]. A similar assessment of 67 projects (47 of which were oncology studies) by researchers from Bayer HealthCare (Leverkusen, Germany) revealed that only one‐quarter of the published data could be reproduced [2]. Several reasons have been proposed to explain the high rate of contrasting results among different laboratories, including investigator bias, inappropriate statistical analysis of results, and insufficient sample size [1,2]. Investigator bias is a broad category that includes manipulation of the analysis and selective reporting of data [3]. It is well known that the smaller the experimental sample size, the less likely the research findings are to be true [3]. There are no comprehensive analyses of the effects of cell culture modifications on the cancer cell transcriptome
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
