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Quantitative investigation of MDA-MB-231 breast cancer cell motility: dependence on epidermal growth factor concentration and its gradient.

ABSTRACT: Enhanced cell motility is one of the primary features of cancer. Accumulated evidence demonstrates that Epidermal Growth Factor Receptor (EGFR) mediated pathways play an important role in breast cancer cell proliferation and migration. We have quantified the MDA-MB-231 breast cancer cell migration in response to the stimulation of EGFR pathways with their ligand EGF to determine how the cell motility of MDA-MB-231 cells depends on the ligand concentration and gradient. Analysis at the single cell level combined with mathematical modeling and the ability to vary the ligand concentration and gradients locally using microfluidic devices allowed us to separate the unique contributions of ligand concentration and ligand gradient to cell motility. We tracked the motility of 6600 cells individually using time lapse imaging under varying EGF stimulation conditions. Trajectory analysis of the tracked cells using non-linear multivariate regression models showed that: (i) cell migration of MDA-MB-231 breast cancer cells depends on the ligand gradient but not on the ligand concentration. This observation was valid for both the total (direction independent) and directed (along gradient direction) cell velocities. Although the dependence of the directed motility on ligand gradient is to be expected, the dependence of the total velocity solely on ligand gradient was an unexpected novel observation. (ii) Enhancement of the motilities of individual cells in a population upon exposure to the ligand was highly heterogeneous, and only a very small percentage of cells responded strongly to the external stimuli. Separating out the non-responding cells using quantitative analysis of individual cell motilities enabled us to establish that enhanced motility of the responding cells indeed increases monotonically with increasing EGF gradient. (iii) A large proportion of cells in a population were unresponsive to ligand stimulation, and their presence introduced considerable random intrinsic variability to the observations. This indicated that studying cell motilities at the individual cell level is necessary to better capture the biological reality and that population averaging methods should be avoided. Studying motilities at the individual cell level is particularly important to understand the biological processes that are possibly driven by the action of a small portion of cells in a population, such as metastasis. We discuss the implications of our results on the total and chemotactic movement of cancer cells in the tumor microenvironment.

SUBMITTER: Islam T

PROVIDER: S-EPMC5624528 | biostudies-literature | 2017 Sep

REPOSITORIES: biostudies-literature

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Quantitative investigation of MDA-MB-231 breast cancer cell motility: dependence on epidermal growth factor concentration and its gradient.

Islam Tanzila T Resat Haluk H

Molecular bioSystems 20170901 10

Enhanced cell motility is one of the primary features of cancer. Accumulated evidence demonstrates that Epidermal Growth Factor Receptor (EGFR) mediated pathways play an important role in breast cancer cell proliferation and migration. We have quantified the MDA-MB-231 breast cancer cell migration in response to the stimulation of EGFR pathways with their ligand EGF to determine how the cell motility of MDA-MB-231 cells depends on the ligand concentration and gradient. Analysis at the single cel ...[more]

PMID: 28799616

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Project description:Since bone metastatic breast cancer is an incurable disease, causing significant morbidity and mortality, understanding of the underlying molecular mechanisms would be highly valuable. Here, we describe in vitro and in vivo evidence for the importance of serine biosynthesis in the metastasis of breast cancer to bone. We first characterized the bone metastatic propensity of the MDA-MB-231(SA) cell line variant as compared to the parental MDA-MB-231 cells by radiographic and histological observations in the inoculated mice. Genome-wide gene expression profiling of this isogenic cell line pair revealed that all the three genes involved in the L-serine biosynthesis pathway, phosphoglycerate dehydrogenase (PHGDH), phosphoserine aminotransferase 1 (PSAT1), and phosphoserine phosphatase (PSPH) were upregulated in the highly metastatic variant. This pathway is the primary endogenous source for L-serine in mammalian tissues. Consistently, we observed that the proliferation of MDA-MB-231(SA) cells in serine-free conditions was dependent on PSAT1 expression. In addition, we observed that L-serine is essential for the formation of bone resorbing human osteoclasts and may thus contribute to the vicious cycle of osteolytic bone metastasis. High expression of PHGDH and PSAT1 in primary breast cancer was significantly associated with decreased relapse-free and overall survival of patients and malignant phenotypic features of breast cancer. In conclusion, high expression of serine biosynthesis genes in metastatic breast cancer cells and the stimulating effect of L-serine on osteoclastogenesis and cancer cell proliferation indicate a functionally critical role for serine biosynthesis in bone metastatic breast cancer and thereby an opportunity for targeted therapeutic interventions. Parental MDA-MB-231 cells and MDA-MB-231(SA) cells were cultured in cell culture flasks. RNA was isolated in order to compare the gene expression profiles of these cell variants. Total of two samples. No replicates.

Project description:Breast cancer remains a leading cause of death in women worldwide. Although breast cancer therapies have greatly advanced in recent years, many patients still develop tumour recurrence and metastasis, and eventually succumb to the disease due to chemoresistance. Citral has been reported to show cytotoxic effect on various cancer cell lines. However, the potential of citral to specifically target the drug resistant breast cancer cells has not yet been tested, which was the focus of our current study.The cytotoxic activity of citral was first tested on MDA-MB-231 cells in vitro by MTT assay. Subsequently, spheroids of MDA-MB-231 breast cancer cells were developed and treated with citral at different concentrations. Doxorubicin, cisplatin and tamoxifen were used as positive controls to evaluate the drug resistance phenotype of MDA-MB-231 spheroids. In addition, apoptosis study was performed using AnnexinV/7AAD flowcytometry. Aldefluor assay was also carried out to examine whether citral could inhibit the ALDH-positive population, while the potential mechanism of the effect of citral was carried out by using quantitative real time- PCR followed by western blotting analysis.Citral was able to inhibit the growth of the MDA-MB-231 spheroids when compared to a monolayer culture of MDA-MB-231 cells at a lower IC50 value. To confirm the inhibition of spheroid self-renewal capacity, the primary spheroids were then cultured to additional passages in the absence of citral. A significant reduction in the number of secondary spheroids were formed, suggesting the reduction of self-renewal capacity of these aldehyde dehydrogenase positive (ALDH+) drug resistant spheroids. Moreover, the AnnexinV/7AAD results demonstrated that citral induced both early and late apoptotic changes in a dose-dependent manner compared to the vehicle control. Furthermore, citral treated spheroids showed lower cell renewal capacity compared to the vehicle control spheroids in the mammosphere formation assay. Gene expression studies using quantitative real time PCR and Western blotting assays showed that citral was able to suppress the self-renewal capacity of spheroids and downregulate the Wnt/?-catenin pathway.The results suggest that citral could be a potential new agent which can eliminate drug-resistant breast cancer cells in a spheroid model via inducing apoptosis.

Dataset Information

Quantitative investigation of MDA-MB-231 breast cancer cell motility: dependence on epidermal growth factor concentration and its gradient.

Publications

Quantitative investigation of MDA-MB-231 breast cancer cell motility: dependence on epidermal growth factor concentration and its gradient.

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