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Multi-omics analysis defines 5-fluorouracil drug resistance in 3D HeLa carcinoma cell model
Lin Wang1, Xueting Wang1, Tong Wang1
1State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai, People's Republic of China.
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.
Insights
Researchers developed a 3D multicellular tumor spheroid model to study cervical cancer drug resistance. This model revealed that 5-fluorouracil resistance in HeLa cells is linked to altered metabolism, endoplasmic reticulum function, and extracellular matrix changes.
Area of Science:
- Oncology
- Biotechnology
- Cell Biology
Background:
- Cervical cancer poses a global health challenge, with drug resistance limiting treatment efficacy.
- Efficient in vitro tumor models are crucial for improving drug screening and clinical applications.
- Multicellular tumor spheroids (MTSs) offer a transitional model between 2D cultures and in vivo xenografts, recapitulating tumor traits.
Purpose of the Study:
- To establish a rapid, reproducible, and scalable in vitro model for generating uniform multicellular tumor spheroids (MTSs).
- To investigate the molecular mechanisms underlying 5-fluorouracil (5-FU) resistance in HeLa cervical cancer cells within a 3D MTS model.
- To compare chemoresistance and molecular profiles between 2D monolayer cultures and 3D MTSs.
Main Methods:
- Development of a high-throughput liquid overlay method for rapid MTS generation.
- Cytotoxicity assays to assess 5-FU resistance in 2D vs. 3D models.
- Multi-omics analysis (proteomics, metabolomics, transcriptomics) to explore molecular differences.
- Analysis of protein synthesis, endoplasmic reticulum (ER) homeostasis, and extracellular matrix (ECM) protein expression.
Main Results:
- A reproducible protocol for generating uniform 3D MTSs was established.
- HeLa cells in 3D MTSs exhibited significantly enhanced 5-FU resistance (resistance index of 5.72) compared to 2D cultures.
- 3D MTSs showed decreased mitochondrial function and TCA cycle activity, with a metabolic shift towards glycolysis.
- Upregulation of heat shock proteins indicated enhanced protein folding and ER homeostasis in 3D MTSs.
- Increased expression of ECM proteins (laminin, collagen) in 3D MTSs formed a physical barrier to drug penetration.
Conclusions:
- The study successfully developed a scalable and reproducible 3D MTS model for cervical cancer drug screening.
- Glycolytic metabolism, ER homeostasis, and ECM protein expression are critical factors contributing to 5-FU chemoresistance in HeLa cells.
- The 3D MTS model provides valuable insights into tumor biology and drug resistance mechanisms relevant to clinical applications.
