Optimization of in vitro model for analysis of tumor cell migration dynamics

Kravchenko, A. O.; Kosach, V. R.; Shkarina, Kateryna; Zaiets, I. V.; Tykhonkova, I. O.; Хоруженко, А. И.

doi:10.7124/bc.000a00

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“…First, spheroids were passed through a sterile nylon mesh (Spectrum, USA) with a pore diameter of 100 μm to remove large cell aggregates. The second step of the filtration was carried out using sterile filter mesh (Spectrum, USA) with a pore diameter of 30 μm for the single cell elimination [27].…”

Section: Methodsmentioning

confidence: 99%

Sensitivity of MCF-7 cells with differential expression of S6K1 isoforms to the regulatory impact of fibroblasts

et al. 2020

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To investigate the involvement of mTOR/S6K1 cell signaling network, with focus on S6K, in response of tumor cells to regulatory impact of fibroblasts. Methods. Cell culture, including co-cultivation of fibroblasts and tumor cells, immunofluorescence analysis, Western blot analysis, assessment of cell migration by scratch test, and transformation of multicellular spheroid in monolayer cell colony. Results. The present work showed the positive effect of stromal cells on the phosphorylation level of the components of mTOR/S6K1 signaling cascade: p85S6K1, p70S6K1 and mTOR in human breast adenocarcinoma MCF-7 cells. To determine, which of the S6K1 isoforms, p85S6K1, p70S6K1 or p60S6K1, is the most sensitive to the extracellular environment, the stable MCF-7 cell lines with edited expression of S6K1 isoforms were used. It was found that selective expression of p60S6K1 led to the changes in morphological features of tumor cells under both two-and three-dimensional culture conditions. These cells also exhibited high levels of focal adhesion kinase (FAK) phosphorylation and large protein content of Zo-1, CD29, CD44 compatible with their high migration potential in the scratch test. Besides, the cells, selectively expressing p60S6K1, were resistant to fibroblastproducing factors and rapamycin. It was also demonstrated that fibroblasts increased tumor cell motility in scratch test and spheroid outspreading assay under co-cultivation conditions in paracrine manner, whereas the direct contact of tumor spheroids with the fibroblast monolayer significantly reduced the velocity of spheroid outspreading. Conclusions. The data obtained indicate that not only the differential expression of S6K1 isoforms in MCF-7 cells but also their ratio are important signaling parameters determining cell survival and response to microenvironment factors.

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Section: Methodsmentioning

confidence: 99%