Application of a fluorescence resonance energy transfer (FRET)‐based biosensor for detection of drug‐induced apoptosis in a 3D breast tumor model

Abstract

Two-dimensional (2D) cultures are commonly used for testing drug effects largely because of their easy maintenance. But they do not represent the spatial interactions of the cells within a tumor. Three-dimensional (3D) cultures can overcome those limitations thus mimicking the architecture of solid tumor. However, it is not easy to evaluate drug effects in 3D culture for a long time. This necessitates the development of a real-time and longitudinal analysis of 3D platforms. In this study, we transfected the plasmid DNA encoding the fluorescence resonance energy transfer (FRET)-based biosensor into human breast cancer cells and generated two cell lines of MCF7-C3 and MDA-MB-231-C3 (231-C3) cells. We used them to determine the activation of caspase-3, whereby healthy cells appear green and apoptotic cells appear blue by FRET imaging. As the caspase sensors can be constantly produced within the cells and quickly respond to caspase activation, we hypothesized that these sensor cells will allow longitudinal detection of apoptosis. MCF7-C3 and 231-C3 spheroids were generated and subjected to histological examination, gene expression studies, drug treatment, and FRET analyses. Our results demonstrated that MCF7-C3 cells formed tight 3D spheroids, and mimicked in vivo tumor architecture. The mRNA level of tumorigenic markers such as MMP-9, SOX2, and OCT4A were much higher in cells cultured in 3D than in 2D. Finally, upon treatment with paclitaxel, the FRET effect was reduced at the rim of MCF7-C3 spheroids in a dose and time-dependent manner demonstrating these sensor cells can be used to determine drug-induced apoptosis in a 3D set up. This study supports the possibility of developing a biosensor-based in vitro 3D breast tumor model for determination of anti-cancer drug penetration over a long course of time in a non-invasive manner.

High Shear Stresses under Exercise Condition Destroy Circulating Tumor Cells in a Microfluidic System

“…To investigate the effects of SS on inducing apoptosis in CTCs, we utilized engineered human breast cancer MDA-MB-231 cells (231-C3) that express apoptotic sensor for real-time detection of caspase activation22. Four SS conditions were selected in this experiment: 15, 30, 45, and 60 dynes/cm 2 covering the range of arterial SS from physiologically resting state19 to heavy exercise condition20.…”

“…Anderson Cancer Center, Houston, TX, USA. The fluorescence resonance energy transfer (FRET)-based caspase sensor C323 was transfected into MDA-MB-231 cells to generate a stable cell line named 231-C322. The 231-C3 cells can express a CFP-DEVD-YFP fusion protein, which can be cleaved by caspase-3 or caspase-7, thus serving as a live indicator of apoptosis.…”

“…the extremely low level of CTCs (1–5 cells/ml of patient’s blood sample7). Some of the breast cancer cells used in this study also stably expressed apoptotic sensor proteins which allow real-time detection of apoptosis182223. By combining the three technologies including the microfluidic circulatory system, metastatic cell lines, and apoptotic sensor, we were able to closely examine how high SS generated during intensive exercise destroys CTCs.…”

High expression of MnSOD promotes survival of circulating breast cancer cells and increases their resistance to doxorubicin

“…Recently, we generated two stable sensor 231-C3 and MCF7-C3 cell lines from metastatic breast cancer MDA-MB-231 and non-metastatic breast cancer MCF7 cells (Figure 1B) [17]. These sensor cells could change their color from green to blue under UV-irradiation-induced apoptosis due to the reduction of fluorescence resonance energy transfer (FRET) between the donor, cyan fluorescent protein (CFP), and the acceptor, yellow fluorescent protein (YFP) (Figure 1A-1C and Supplementary Video S1).…”

“…MCF7 and MDA-MB-231 cells were transfected with the sensor C3 plasmid using Lipofectamine 2000 (Invitrogen, USA). After selection with 500 μg/mL G418 (PAA, Germany), single-cell colonies expressing the C3 sensor were designated MCF7-C3 and 231-C3 cells (Figure 1B) [17]. …”