3D cell culture and identification Groups
In the process of 3D cell culture, we found that the cell clusters formed by the co-culture of the three types of cells are smoother and more regular, while the cell clusters formed on Matrigel by simple MCF-7 have different sizes and irregular shapes(Figure 1A-F). We performed scRNA-seq on the 3D culture cells(Figure 1G), and after quality control, approximately 7088 cells met quality control metrics and were analyzed. Unsupervised clustering using the Seurat package identified six clusters of cells (cluster 0–5, Figure1H, I). Cluster 0, 1, and 3 were classified as HUVEC, Cluster 2 and 4 were classified as MCF-7, and Cluster 5 was classified as HMF(Figure 2A-B). We also screened the highly expressed genes of each group of cells, among which the HUVEC highly expressed genes included C15orf48, PLAU, CCL20, PDZK1IP1, and MT2A(Figure 2C-D). The MCF-7 highly expressed genes include TXNIP, COX6C, CRABP2, CCN5, CLDN4, TFF1, KRT19(Figure 2G-H), and HMFs’ highly expressed genes include MMP2, COL6A1, SPARC, DCN, and COL1A1(Figure 2E-F).
MCF-7 response after cisplatin stimulation at single-cell levels.
The purpose of this study was to compare the level of the single-cell MCF-7 cisplatin difference in response to the co-culture cultured alone. First, our results show that the transcriptomes of the two different states are very different (Figure 3A, B), and the difference between the two groups after cisplatin intervention is more significant (Figure 3C, D). However, there are also differences between the cisplatin intervention group and the control group under the mode of cell co-culture, but this difference is relatively mild. The highly expressed genes in MCF-7 after cisplatin intervention include HSPA8, HSPA1A, HSPA1B, FOS, STC1, LOXL2, CCN5, and MT2A, and the low-expressed genes include EIF5B, GDF15, USP8, XBP1, KRT18, and KRT19(Figure 4A). Next, we have performed signal pathway enrichment analysis and GO analysis of differential genes. The enrichment signal pathway of differential genes includes Protein processing in the endoplasmic reticulum, Antigen processing and presentation, and Estrogen signaling pathway(Figure 4A). CancerSEA is a specialized database aimed at single-cell resolution decoding comprehensive cancer cells’ different functional states. Finally, we analyze the differential genes and single-cell sequencing databases to further analyze the functions of the differentially expressed genes. The signal pathways enriched by these differential genes include Apoptosis, DNA damage, Hypoxia, and Metastasis, among others(Figure 4D-E).