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Longitudinal Morphological and Physiological Monitoring of Three-dimensional Tumor Spheroids Using Optical Coherence Tomography

Tumor spheroids have been developed as a three-dimensional (3D) cell culture model in cancer research and anti-cancer drug discovery. However, currently, ...

Nexcelom Bioscience LLC

Oncologists have investigated the effect of protein or chemical-based compounds on cancer cells to identify potential drug candidates. Traditionally, the growth inhibitory and cytotoxic effects of the drugs are first measured in 2D in vitro models, and then further tested in 3D xenograft in vivo models. Although the drug candidates can demonstrate promising inhibitory or cytotoxicity results in a 2D environment, similar effects may not be observed under a 3D environment. In this work, we developed an image-based high-throughput screening method for 3D tumor spheroids using the Celigo image cytometer. First, optimal seeding density for tumor spheroid formation was determined by investigating the cell seeding density of U87MG, a human glioblastoma cell line. Next, the dose-response effects of 17-AAG with respect to spheroid size and viability were measured to determine the IC value. Finally, the developed high-throughput method was used to measure the dose response of four drugs (17-AAG, paclitaxel, TMZ, and doxorubicin) with respect to the spheroid size and viability. Each experiment was performed simultaneously in the 2D model for comparison. This detection method allowed for a more efficient process to identify highly qualified drug candidates, which may reduce the overall time required to bring a drug to clinical trial.

High-Throughput 3D Tumor Spheroid Screening Method for Cancer Drug Discovery Using Celigo Image Cytometry.

To ensure cell-based assays are performed properly, both cell concentration and viability have to be determined so that the data can be normalized to generate meaningful and comparable results. Cell-based assays performed in immuno-oncology, toxicology, or bioprocessing research often require measuring of multiple samples and conditions, thus the current automated cell counter that uses single disposable counting slides is not practical for high-throughput screening assays. In the recent years, a plate-based image cytometry system has been developed for high-throughput biomolecular screening assays. In this work, we demonstrate a high-throughput AO/PI-based cell concentration and viability method using the Celigo image cytometer. First, we validate the method by comparing directly to Cellometer automated cell counter. Next, cell concentration dynamic range, viability dynamic range, and consistency are determined. The high-throughput AO/PI method described here allows for 96-well to 384-well plate samples to be analyzed in less than 7 min, which greatly reduces the time required for the single sample-based automated cell counter. In addition, this method can improve the efficiency for high-throughput screening assays, where multiple cell counts and viability measurements are needed prior to performing assays such as flow cytometry, ELISA, or simply plating cells for cell culture. 

A high-throughput AO/PI-based cell concentration and viability detection method using the Celigo image cytometry.

The ability to accurately measure cell viability is important for any cell-based assay. Traditionally, viability measurements have been performed using the trypan blue exclusion method on a hemacytometer, which allows researchers to visually distinguish viable from nonviable cells. While the trypan blue method can work for cell lines or primary cells that have been rigorously purified, in more complex samples such as PBMCs, bone marrow, whole blood, or any sample with low viability, this method can lead to errors. In recent years, advances in optics and fluorescent dyes have led to the development of automated benchtop image-based cell counters for rapid cell concentration and viability measurement. In this work, we demonstrate the use of image-based cytometry for cell viability detection using single-, dual-, or multi-stain techniques. Single-staining methods using nucleic acid stains such as EB, PI, 7-AAD, DAPI, SYTOX Green, and SYTOX Red, and enzymatic stains such as CFDA and Calcein AM, were performed. Dual-staining methods using AO/PI, CFDA/PI, Calcein AM/PI, Hoechst/PI, Hoechst/DRAQ7, and DRAQ5/DAPI that enumerate viable and nonviable cells were also performed. Finally, Hoechst/Calcein AM/PI was used for a multi-staining method. Fluorescent viability staining allows exclusion of cellular debris and nonnucleated cells from analysis, which can eliminate the need to perform purification steps during sample preparation and improve efficiency. Image cytometers increase speed and throughput, capture images for visual confirmation of results, and can greatly simplify cell count and viability measurements.

Assessment of Cell Viability with Single-, Dual-, and Multi-Staining Methods Using Image Cytometry.

The development of three-dimensional (3D) multicellular tumor spheroid models for cancer drug discovery research has increased in the recent years. The use of 3D tumor spheroid models may be more representative of the complex in vivo tumor microenvironments in comparison to two-dimensional (2D) assays. Currently, viability of 3D multicellular tumor spheroids has been commonly measured on standard plate-readers using metabolic reagents such as CellTiter-Glo® for end point analysis. Alternatively, high content image cytometers have been used to measure drug effects on spheroid size and viability. Previously, we have demonstrated a novel end point drug screening method for 3D multicellular tumor spheroids using the Celigo Image Cytometer. To better characterize the cancer drug effects, it is important to also measure the kinetic cytotoxic and apoptotic effects on 3D multicellular tumor spheroids. In this work, we demonstrate the use of PI and caspase 3/7 stains to measure viability and apoptosis for 3D multicellular tumor spheroids in real-time. The method was first validated by staining different types of tumor spheroids with PI and caspase 3/7 and monitoring the fluorescent intensities for 16 and 21 days. Next, PI-stained and nonstained control tumor spheroids were digested into single cell suspension to directly measure viability in a 2D assay to determine the potential toxicity of PI. Finally, extensive data analysis was performed on correlating the time-dependent PI and caspase 3/7 fluorescent intensities to the spheroid size and necrotic core formation to determine an optimal starting time point for cancer drug testing. The ability to measure real-time viability and apoptosis is highly important for developing a proper 3D model for screening tumor spheroids, which can allow researchers to determine time-dependent drug effects that usually are not captured by end point assays. This would improve the current tumor spheroid analysis method to potentially better identify more qualified cancer drug candidates for drug discovery research. © 2017 International Society for Advancement of Cytometry.

Real-time viability and apoptosis kinetic detection method of 3D multicellular tumor spheroids using the Celigo Image Cytometer.

Three-dimensional (3D) tumor spheroid models have gained increased recognition as important tools in cancer research and anticancer drug development. However, currently available imaging approaches used in high-throughput screening drug discovery platforms, for example, bright-field, phase contrast, and fluorescence microscopies, are unable to resolve 3D structures deep inside (>50 μm) tumor spheroids. In this study, we established a label-free, noninvasive optical coherence tomography (OCT) imaging platform to characterize 3D morphologic and physiologic information of multicellular tumor spheroids (MCTS) growing from approximately 250 to 600 μm in height over 21 days. In particular, tumor spheroids of two cell lines, glioblastoma (U-87MG) and colorectal carcinoma (HCT116), exhibited distinctive evolutions in their geometric shapes at late growth stages. Volumes of MCTS were accurately quantified using a voxel-based approach without presumptions of their geometries. In contrast, conventional diameter-based volume calculations assuming perfect spherical shape resulted in large quantification errors. Furthermore, we successfully detected necrotic regions within these tumor spheroids based on increased intrinsic optical attenuation, suggesting a promising alternative of label-free viability tests in tumor spheroids. Therefore, OCT can serve as a promising imaging modality to characterize morphologic and physiologic features of MCTS, showing great potential for high-throughput drug screening. .

Optical Coherence Tomography Detects Necrotic Regions and Volumetrically Quantifies Multicellular Tumor Spheroids.

Three-dimensional tumor spheroid models have been increasingly used to investigate and characterize cancer drug compounds. Previously, the Celigo image cytometer has demonstrated its utility in a high-throughput screening manner for evaluating potential drug candidates in a 3D multicellular tumor spheroid (MCTS) primary screen. In addition, we have developed real-time kinetic caspase 3/7 apoptosis and propidium iodide viability 3D MCTS assays, both of which can be used in a secondary screen to better characterize the hit compounds. In this work, we monitored the kinetic apoptotic and cytotoxic effects of 14 compounds in 3D MCTS produced from the glioblastoma cell line U87MG in 384-well plates for 9 days. The kinetic results allowed the categorization of the effects from 14 drug compounds into early and late cytotoxic, apoptotic, cytostatic, and no effects. The real-time apoptosis and viability screening method can serve as an improved secondary screen to better understand the mechanism of action of these potential drug candidates identified from the primary screen, allowing one to identify a more qualified drug candidate and streamline the drug discovery process of research and development.

Sarah Kessel

Department of Technology R&D

Nexcelom Bioscience LLC

Longitudinal Morphological and Physiological Monitoring of Three-dimensional Tumor Spheroids Using Optical Coherence Tomography