Abstract
Cervical cancer is a serious health problem in women around the globe. However, the use of clinical drug is seriously dampened by the development of drug resistance. Efficient in vitro tumor model is essential to improve the efficiency of drug screening and the accuracy of clinical application. Multicellular tumor spheroids (MTSs) can in a way recapitulates tumor traits in vivo, thereby representing a powerful transitional model between 2D monolayer culture and xenograft. In this study, based on the liquid overlay method, a protocol for rapid generation of the MTSs with uniform size and high reproducibility in a high-throughput manner was established. As expected, the cytotoxicity results showed that there was enhanced 5-fluorouracil (5-FU) resistance of HeLa carcinoma cells in 3D MTSs than 2D monolayer culture with a resistance index of 5.72. In order to obtain a holistic view of the molecular mechanisms that drive 5-FU resistance in 3D HeLa carcinoma cells, a multi-omics study was applied to discover hidden biological regularities. It was observed that in the 3D MTSs mitochondrial function-related proteins and the metabolites of the tricarboxylic acid cycle (TCA cycle) were significantly decreased, and the cellular metabolism was shifted towards glycolysis. The differences in the protein synthesis, processing, and transportation between 2D monolayer cultures and 3D MTSs were significant, mainly in the heat shock protein family, with the up-regulation of protein folding function in endoplasmic reticulum (ER), which promoted the maintenance of ER homeostasis in the 3D MTSs. In addition, at the transcript and protein level, the expression of extracellular matrix (ECM) proteins (e.g., laminin and collagen) were up-regulated in the 3D MTSs, which enhanced the physical barrier of drug penetration. Summarizing, this study formulates a rapid, scalable and reproducible in vitro model of 3D MTS for drug screening purposes, and the findings establish a critical role of glycolytic metabolism, ER hemostasis and ECM proteins expression profiling in tumor chemoresistance of HeLa carcinoma cells towards 5-FU.Graphical
Highlights
Cancer is the leading cause of death among people under the age of 70 in most countries
By recording and observing Multicellular tumor spheroids (MTSs) every day, we found that the growth of MTSs reached the plateau on the 6th day, on which the MTSs were chosen for the follow-up experiments
Through comparative proteome and metabolome analysis (Figs. 5a-d, 6a, b), we found that in 3D MTSs the biological processes related to the mitochondrial function were significantly down-regulated, and the intermediates involved in TCA cycle were decreased in 3D MTSs, but there were no significant differences in the enzymes and intermediate metabolites of the glycolytic pathway
Summary
Cancer is the leading cause of death among people under the age of 70 in most countries. To satisfy rapid growth and proliferation, tumor cells require a large amount of nutrients to meet their material and energy needs (Zhu and Thompson 2019). Tumor cells mainly rely on aerobic glycolysis ( known as the Warburg effect) for rapid energy and precursor supply, which is accompanied by the production of large amounts of lactate (Vander Heiden et al 2009). The nucleotide metabolic pathway of tumor cells is highly efficient and nucleotide imbalance further induces tumor-associated mutations (Aird and Zhang 2015). Based on the complex genetic diversities and epigenetic differences of tumor cells, there are great differences between the individual differences of different patients and the tissue heterogeneity of the same patient are huge, which seriously hinders the effective treatment of tumors (Hensley et al 2016; Singh et al 2010)
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