The NSCLC cell line A549 was purchased from the ATCC collection. Furthermore, cells in monolayer culture not only have higher sensitivity to anticancer drugs upon treatment but they become quiescent and decrease proliferation rate and increase apoptosis more quickly than 3D models or in vivo tumors. Only 26% of cells in 3D reached the same concentration of drugs as the cells treated in 2D dishes because the synthesis of ECM components was more intense in 3D cultures. The monolayer culture of the human breast cancer cell line MCF-7 showed 2.6-fold higher accumulation of doxorubicin, paclitaxel, and tamoxifen compared to three-dimensional cultivation on porous biodegradable polymeric microparticles. It has been shown by us and others that the cells grown on 2D surfaces demonstrate higher drug sensitivity. So far, the traditional in vitro screening of drug candidates in cell-based assays has been based on adherent cultures (2D assays), but several studies reported the application of cancer multicellular spheroids for screening and target identification. Three-dimensional spheroids mimic the organoid of a solid tumor, while 2D culture methods fail to represent different tissue areas within tumors, such as proliferating, quiescent and necrotic core zones. Investigation of tumor spheroids revealed, that the core of the spheroid was similar to in vivo conditions. Various extracellular matrix (ECM) components and their homologues (collagen, gelatin, etc.) are used to facilitate the adhesion of cells to the carriers. Therefore, 3D cultures of NSCLC cells bearing more putative cancer targets should be used in drug screening as the preferred technique rather than the Petri-dish.Ĭurrently, there is a wide variety of techniques for three-dimensional cell culture methods: specially designed incubators, tubes, microcarriers and growing matrices. Multidimensional single cell proteome profiling revealed that 3D cultures represent a transition from 2D to in vivo conditions by intermediate marker expression of TRA-1-60, TMEM45A, pan-keratin, CD326, MCT4, Gal-3, CD66, GLUT1, and CD274. In 3D systems CA9, CD24, and EGFR showed higher expression than in vivo. The described twelve markers convincingly highlighted a unique pattern reflecting intra-tumor heterogeneity of 3D samples and in vivo A549 lung cancer cells. Additionally, TRA-1-60, pan-keratins, CD326, Galectin-3, and CD274, markers with known clinical significance have been investigated at single cell resolution. Gene expression analysis enabled the selection of markers that were overexpressed: TMEM45A, SLC16A3, CD66, SLC2A1, CA9, CD24, or repressed: EGFR either in vivo or in long-term 3D cultures. Proliferation, viability, and cell cycle phase distribution has been investigated. Here, we characterize lung cancer markers using single cell mass cytometry to compare different in vitro cell culturing methods: two-dimensional (2D), carrier-free, or bead-based 3D culturing with in vivo xenografts. Single cell genomics and proteomics with the combination of innovative three-dimensional (3D) cell culture techniques can open new avenues toward the understanding of intra-tumor heterogeneity.
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