Three-dimensional (3D) tumor models have been increasingly used to investigate and characterize cancer drug compounds. The ability to perform high-throughput screening of 3D multicellular tumor spheroids (MCTS) can highly improve the efficiency and cost-effectiveness of discovering potential cancer drug candidates. Previously, the Celigo Image Cytometer has demonstrated a novel method for high-throughput screening of 3D multicellular tumor spheroids. In this work, we employed the Celigo Image Cytometer to examine the effects of 14 cancer drug compounds on 3D MCTS of the glioblastoma cell line U87MG in 384-well plates. Using parameters such as MCTS diameter and invasion area, growth and invasion were monitored for 9 and 3 d, respectively. Furthermore, fluorescent staining with calcein AM, propidium iodide, Hoechst 33342, and caspase 3/7 was performed at day 9 posttreatment to measure viability and apoptosis. Using the kinetic and endpoint data generated, we created a novel multiparametric drug-scoring system for 3D MCTS that can be used to identify and classify potential drug candidates earlier in the drug discovery process. Furthermore, the combination of quantitative and qualitative image data can be used to delineate differences between drugs that induce cytotoxic and cytostatic effects. The 3D MCTS-based multiparametric scoring method described here can provide an alternative screening method to better qualify tested drug compounds.

Thalidomide notoriously caused severe birth defects, particularly to the limbs, in those exposed in utero following maternal use of the drug to treat morning sickness. How the drug caused these birth defects remains unclear. Many theories have been proposed including actions on the forming blood vessels. However, thalidomide survivors also have altered nerve patterns and the drug is known for its neurotoxic actions in adults following prolonged use. We have previously shown that CPS49, an anti-angiogenic analog of thalidomide, causes a range of limb malformations in a time-sensitive manner in chicken embryos. Here we investigated whether CPS49 also is neurotoxic and whether effects on nerve development impact upon limb development. We found that CPS49 is neurotoxic, just like thalidomide, and can cause some neuronal loss late developing chicken limbs, but only when the limb is already innervated. However, CPS49 exposure does not cause defects in limb size when added to late developing chicken limbs. In contrast, in early limb buds which are not innervated, CPS49 exposure affects limb area significantly. To investigate in more detail the role of neurotoxicity and its impact on chicken limb development we inhibited nerve innervation at a range of developmental timepoints through using β-bungarotoxin. We found that neuronal inhibition or ablation before, during or after limb outgrowth and innervation does not result in obvious limb cartilage patterning or number changes. We conclude that while CPS49 is neurotoxic, given the late innervation of the developing limb, and that neuronal inhibition/ablation throughout limb development does not cause similar limb patterning anomalies to those seen in thalidomide survivors, nerve defects are not the primary underlying cause of the severe limb patterning defects induced by CPS49/thalidomide.

There have been 1600 FDA approved drugs since their inception in 1938. Currently it costs approximately 883 million dollars to bring a drug candidate to the market, and often times the potential candidate fails at the beginning of the clinical phase. Therefore, it is important to develop drug screening methods that are more clinically relevant or predictable. The current 2D methods for cancer drug discovery have had some difficulty in identifying potential drug candidates that can be used for clinical testing. To overcome this challenge, there has been an increase in research of 3D tissue culture that facilitated the development of new in-vitro tumor model assays. Traditional 3D spheroid analysis method relied heavily on visual observation using standard microscopy, which is time-consuming and has high operator variations. In the recent years, high-throughput image-based cytometers, such as Celigo, have demonstrated the ability to perform bright-field and fluorescence cell-based assays. Celigo imaging cytometer can be employed to rapidly analyze and characterize 3D tumor spheroids, which can be used to generate both quantitative and qualitative results. In this work, we demonstrate a high-throughput 3D tumor spheroid screening method using the Celigo imaging cytometer to screen the effects of 14 drug compounds (NIH/NCAT) on U87MG spheroid size, matrigel invasion, and tumor spheroid viability. First, a dose response experiment is performed to screen the growth inhibitory effects of the drug compounds. In addition to direct spheroid size analysis, dose inhibitory responses of tumor invasion into the matrigel are also examined. Finally, the use of specific fluorescent dyes such as Calcein AM, PI, and Caspase 3/7 were used to screen drug induced cytotoxicity on the tumor spheroids. The results showed that Celigo imaging cytometer can quickly generate accurate growth inhibitory data to identify potential drug candidates. Furthermore, tumor invasion were clearly observed and quantified in the captured images, as well as fluorescent analysis of tumor spheroid viability. By utilizing the 3D spheroid screening method, researchers can rapidly characterize and quantify drug effects on tumor spheroids in a high-throughput format, which can improve the efficiency of identifying more qualified cancer drug candidates.