Dynamic, Large-Scale Profiling of Transcription Factor Activity from Live Cells in 3D Culture

Abstract

BackgroundExtracellular activation of signal transduction pathways and their downstream target transcription factors (TFs) are critical regulators of cellular processes and tissue development. The intracellular signaling network is complex, and techniques that quantify the activities of numerous pathways and connect their activities to the resulting phenotype would identify the signals and mechanisms regulating tissue development. The ability to investigate tissue development should capture the dynamic pathway activity and requires an environment that supports cellular organization into structures that mimic in vivo phenotypes. Taken together, our objective was to develop cellular arrays for dynamic, large-scale quantification of TF activity as cells organized into spherical structures within 3D culture.Methodology/Principal FindingsTF-specific and normalization reporter constructs were delivered in parallel to a cellular array containing a well-established breast cancer cell line cultured in Matrigel. Bioluminescence imaging provided a rapid, non-invasive, and sensitive method to quantify luciferase levels, and was applied repeatedly on each sample to monitor dynamic activity. Arrays measuring 28 TFs identified up to 19 active, with 13 factors changing significantly over time. Stimulation of cells with β-estradiol or activin A resulted in differential TF activity profiles evolving from initial stimulation of the ligand. Many TFs changed as expected based on previous reports, yet arrays were able to replicate these results in a single experiment. Additionally, arrays identified TFs that had not previously been linked with activin A.Conclusions/SignificanceThis system provides a method for large-scale, non-invasive, and dynamic quantification of signaling pathway activity as cells organize into structures. The arrays may find utility for investigating mechanisms regulating normal and abnormal tissue growth, biomaterial design, or as a platform for screening therapeutics.

Inhibition of CDK-mediated phosphorylation of Smad3 results in decreased oncogenesis in triple negative breast cancer cells

“…To further examine the impact of CDKi on EMT related events we used a novel transcription factor (TF) reporter cell array 21,22,31 and found that CDK2i treatment of TNBC cells led to an early increase in activity of b-catenin, Smad3, Snail, Sp1, Twist, YY1, and Zeb1 (Fig. 4).…”

“…21,22 After treating MDA-MD-231 cells with control or CDK2i, activities of 11 TFs associated with oncogenesis were measured at several time points. Factors selected for study were based on association with proliferation, apoptosis, and metastasis ( Table 2).…”

“…To form an array, MDA-MB-231 cells were transduced with lentivirus encoding TA-FLuc or one of the TF reporter genes, as previously described, 21,22,31 (25,000 PP/cell), by centrifugation, resuspension in Matrigel (BD Biosciences), and seeding into wells of a black 384-well plate (Greiner BioSciences, Monroe, NC). Cells were treated with 0.1% DMSO control or 240 nM CDK2i at indicated time points.…”

“…One mM d-luciferin (Perkin Elmer, Waltham, MA) was added to wells and plates were incubated at 37 C for 30 minutes, followed by imaging with In Vivo Imaging System (Caliper). Data were analyzed using R. 54 Statistical methodology 22 was updated accordingly: data below the background that contained non-infected cells were removed (confidence interval [CI] D 99.5%) and missing data were replaced with the average value of the same treated wells. Subsequently, data were further normalized by the average bioluminescence intensity of the control wells, TA-Fluc, for the same time and treatment and logarithmically transformed (in based 2).…”

Lentiviral Arrays for Live-cell Dynamic Monitoring of Gene and Pathway Activity During Stem Cell Differentiation

“…[1][2][3][4][5][6] However, due to low transfection efficiencies, acquiring stably transfected cells requires long-term drug selection limiting the application of this approach to cell lines as opposed to primary cells. Also, the transient nature of transfection makes it difficult to follow cells for a long time (days to weeks) as required for stem cell differentiation.…”