Abstract
Simple SummaryThe research field of 3D cell cultivation in hydrogels is continuously growing. To be able to analyze the reaction of melanoma cells to 3D cultivation in alginate hydrogel on a molecular level, whole transcriptome sequencing was performed. Intriguingly, we could not only unravel differences between the gene regulation in 2D and 3D cultures but could also correlate the culture switch to the physiological process of tumor plasticity based on the observed patterns. Thereby, the role of EGR1 in controlling tumor plasticity and progression in melanoma was revealed. We conclude that the combination of cell culture models using biomaterials and whole transcriptome analysis leads to a deeper molecular understanding of cancer cells, herewith defining new therapeutic targets.Alginate hydrogels have been used as a biomaterial for 3D culturing for several years. Here, gene expression patterns in melanoma cells cultivated in 3D alginate are compared to 2D cultures. It is well-known that 2D cell culture is not resembling the complex in vivo situation well. However, the use of very intricate 3D models does not allow performing high-throughput screening and analysis is highly complex. 3D cell culture strategies in hydrogels will better mimic the in vivo situation while they maintain feasibility for large-scale analysis. As alginate is an easy-to-use material and due to its favorable properties, it is commonly applied as a bioink component in the growing field of cell encapsulation and biofabrication. Yet, only a little information about the transcriptome in 3D cultures in hydrogels like alginate is available. In this study, changes in the transcriptome based on RNA-Seq data by cultivating melanoma cells in 3D alginate are analyzed and reveal marked changes compared to cells cultured on usual 2D tissue culture plastic. Deregulated genes represent valuable cues to signaling pathways and molecules affected by the culture method. Using this as a model system for tumor cell plasticity and heterogeneity, EGR1 is determined to play an important role in melanoma progression.
Highlights
Cell culture models have contributed decisively to our contemporary knowledge of cell biology in healthy and diseased organisms
As Kruppel-like factor 2 (KLF2) is a transcription factor known to mediate the tumor suppressor gene p53, we looked at CDKN1A, a known direct target of p53, and found it to be significantly induced in 3D alginate
With 10 out of 11 most significantly induced genes in 3D alginate being significantly induced in melanoma cells compared to Normal human epithelial melanocytes (NHEMs), we revealed a strong correlation of genes upregulated1i3nof32D3 alginate with genes induced during melanoma development (Figure 4A)
Summary
Cell culture models have contributed decisively to our contemporary knowledge of cell biology in healthy and diseased organisms. By cultivating cells encapsulated in hydrogels, the physiological conditions including proliferation rates, metabolism, cell morphology, and extracellular matrix (ECM) production can be represented more accurately than on flat surfaces [1,2,3,4]. This is relevant for biomedical applications like tissue engineering, and the detailed investigation of cellular effects in cancer research and drug development. One major advantage of 3D culture models compared to in vivo animal models is their rather low complexity, making them highly definable and allowing researchers to selectively modulate the system and analyze molecular aspects in an isolated manner. Animal models and patient data are the means of choice to substantiate and confirm the in vitro results (reviewed in [5])
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