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A novel 3D co-culture model augmented with single-cell sequencing to study the response of breast cancer cells to cisplatin

ABSTRACT: Three-dimensional (3D) cell direct co-culture is an essential method for cancer research, which can overcome the limitations of traditional research based on cell lines. Single-cell RNA sequencing can detect the cell heterogeneity of direct co-culture of 3D cells at the single-cell level,However, there is still lack verification. The purpose of this study is to verify the superiority of single-cell RNA sequencing combined with 3D cell direct co-culture in breast cancer research and prove that it can become a powerful solution for cancer cell culture in vitro.In this study ,we found that the different cell culture modes showed very different responses to cisplatin intervention. In MCF-7 alone culture mode, there was a large difference in the transcriptome of MCF-7 after cisplatin intervention compared with the group not given drug intervention; while in direct co-culture mode, there was also a difference between the cisplatin intervention group and the control group, but this difference was relatively small. To further seek evidence that the direct co-culture mode is closer to the real environment in vivo, we analyzed the differential genes in the direct co-culture mode, and the enriched signaling pathways were similar to the results in the CancerSEA database, and the enriched signaling pathways included apoptosis, DNA damage, hypoxia, and metastasis. Conclusions: Single-cell sequencing can facilitate direct co-culture of 3D cells in tumor research, a model that more closely resembles the real pathophysiological environment in vivo.

ORGANISM(S): Homo sapiens

PROVIDER: GSE205243 | GEO | 2022/06/04

REPOSITORIES: GEO

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Project description:Autologous stem cell therapy has potential for biologic treatment of disc degeneration. Due to ease of harvest and abundance, adipose-derived mesenchymal stem cells (AD-MSC) are readily available. Our objectives were: 1) To develop/validate methods to harvest AD-MSC and direct them to a disc-like phenotype by three-dimensional (3D) culture and TGF-Ã3 exposure; 2) To perform gene expression profiling for human AD-MSC and annulus cells in 3D culture; 3) To test whether disc cell-AD-MSC co-culture could augment proteoglycan production. Stem cell plasticity offers potential for future biologic therapies for disc degeneration. Data indicated that human AD-MSC can successfully be manipulated in 3D culture to express gene products important in the disc ECM (types I and II collagen, chondroitin sulfate, keratin sulfate, decorin), and that co-culture of annulus cells with AD-MSC enhances proteoglycan production. Studies defined gene expression patterns of AD-MSC and human annulus cells in 3D culture, important as we explore the potential of MSC in biologic therapies for disc degeneration. Experiment Overall Design: AD-MSC were extracted from human adipose tissue, and characterized as stem cells using accepted criteria (direction into osteoblasts or chondrocytes; and cell surface marker criteria). Three AD-MSC cultures were grown in 3D with or without TGF-Ã3 for 2-3 weeks. Disc Tissue samples were obtained from surgical disc procedures performed on patients with herniated discs and degenerative disc disease. Seven disc cultures were also grown in 3-D for 2 weeks. RNA was harvested according to instructions with the Trizol isolation method, checked for quality using the 2100 Bioanalyzer (Agilent Technologies, Inc., Santa Clara, CA USA), reverse-transcribed to double-stranded cDNA, subjected to two rounds of transcription, and hybridized to the DNA microarray in the Affymetrix Fluidics Station 400. Affymetrix human U133 X3P arrays were used. Using Genesifter, gene expression in AD-MSC with and witout TGF-B3 was compared to annulus cells. Gene expression of stem cells with TGF-Ã3 was also compared to stem cells grown in the absence of TGF-Ã3.

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A novel 3D co-culture model augmented with single-cell sequencing to study the response of breast cancer cells to cisplatin

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