AmpliSeq - Breast Cancer Cell Panel

Published: 3 November 2021| Version 1 | DOI: 10.17632/wk3yz9kc4x.1
Contributor:
Rebecca Lane

Description

Cell culture for the purposes of extracellular vesicle (EV) production requires reducing or removing the presence of FBS in the culture medium. This is because FBS is itself a rich source of EVs, which are physically indistinguishable from those produced by the cultured cells. Two alternate approaches have emerged to address this. The first is to culture cells in a complete FBS-supplemented medium, then incubate for a short period of time (~ 24 hours) in a serum free medium from which EVs may be isolated. The second is to supplement cells with FBS depleted of bovine EVs by overnight ultracentrifugation. It is well-known that serum deprivation impacts cell characteristics, but to date, no such study had sought to examine the effects of this in the EV production context. This study aimed to address this by conducting whole transcriptomic targeted sequencing (AmpliSeq) analysis of a panel of breast cancer cells cultured under EV production conditions. Specifically, eight cell lines (n=3 each) were cultured in complete medium (>10% FBS), a defined low serum medium with 1% EV-depleted FBS (Low Serum Medium) or cells cultured in complete medium then deprived of serum for 24 hours prior to collection (serum starvation). We hypothesised that serum deprivation would induce transcriptomic alterations to the cells, and that the specific nature of the gene expression changes would vary depending on patterns of ER, PR and HER2 expression. The data supported the hypothesis with differential expression analysis suggesting that distinct sets of genes were up- and down- regulated in ER+, HER2+ and triple negative breast cancer (TNBC) cells under each condition. The data also suggested that distinct sets of genes were modulated by the two methods of EV production. Importantly, however, the analysis verified that these transcriptomic changes did not appear to alter disease-relevant characteristics, with cells retaining their phenotypic traits regardless of culture method. This study provides an important validation of two commonly used methods for in vitro EV production.

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Institutions

University of Queensland

Categories

Breast Cancer, RNA Sequencing, Cell Culture, Cancer Cell Line

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