Quantitative proteomic analysis of Carboplatin resistance in 2D and 3D cultured high-grade serous ovarian cancer cells

Drug discovery and development has historically been performed on cells cultured as flat monolayers that do not mimic an in-vivo environment. Thus, there is currently a growing interest in 3D cell culture systems for drug screening and discovery that are more likely to provide physiologically reliable information. Here, we present the first quantitative profiling of carboplatin-induced proteome comparing 2D and 3D models of High-grade Serous Ovarian Cancer (HGSOC) cells. With over 300,"000 cases and approximately 200,"000 deaths per year, ovarian cancer is the leading cause of death from gynaecological malignancies and high-grade serous ovarian cancer (HGSOC) is the most common form of ovarian cancer.

We investigated the carboplatin effects on the proteome of two2 sets of commercially available and biologically-related cell lines with different sensitivity to carboplatin: UWB1/UWB1-BRCA and PEO1/PEO4 that were isolated from the same patient before and after developing chemoresistance. Cells were cultured as 3D spheroids in a basement membrane (Cultrex) and as 2D monolayers for comparison, half the replicateswith or without were exposed exposure to sublethal micromolar concentrations (twice the IC50) of carboplatin. Liquid chromatography coupled to tandem mass spectrometry (data-dependent acquisition mode) enabled protein quantitation based on a bottom-up approach relying on labelling of trypsin-digested proteins with isobaric mass tags (TMT) for direct comparison across samples and replicates. Isobaric labelling-based LC-MS analysis with Real Time Search MS3 acquisition enabled the relative quantification of We identified more thanover 6500 proteins across each all samples and achieveddemonstrating deep proteome coverage with high accuracy and precision. Using a 3-way ANOVA statistical analysis, we further identified more than 900 differentially regulated proteins differentially expressed (-log10 ANOVA value > 1.30) and were able to compare proteinwith variations in among the experimental factors groupings based on : 1)encompassing carboplatin response, culture dimension and carboplatin treatment. resistant vs sensitive phenotype 2) 2D vs 3D and 3) treated vs non-treated criteria.

We found important significant changes differences in proteins expression levels involved inof proteins involved in DNA damage responses as well as in DNA packing (nucleosome) and regulation. H1-4 histone linker and isoforms (H1-3, H1-5) were downregulated (p value ≤ 0.05) in sensitive and treated 3D models, while being upregulated (p value ≤ 0.05) in resistant and treated 3D models;, while opposite results were found in 2D models (p value ≤ 0.05). Overall, our work highlights identifies major differences among 2D and 3D cultured samples and highlights the need testifies of the crucialness of implementing near patient ovarian cancer models that are closer to tumour structure characteristics for drug discovery.