Single-cell microarray enables high-throughput evaluation of DNA double-strand breaks and DNA repair inhibitors

Abstract

A key modality of non-surgical cancer management is DNA damaging therapy that causes DNA double-strand breaks that are preferentially toxic to rapidly dividing cancer cells. Double-strand break repair capacity is recognized as an important mechanism in drug resistance and is therefore a potential target for adjuvant chemotherapy. Additionally, spontaneous and environmentally induced DSBs are known to promote cancer, making DSB evaluation important as a tool in epidemiology, clinical evaluation and in the development of novel pharmaceuticals. Currently available assays to detect double-strand breaks are limited in throughput and specificity and offer minimal information concerning the kinetics of repair. Here, we present the CometChip, a 96-well platform that enables assessment of double-strand break levels and repair capacity of multiple cell types and conditions in parallel and integrates with standard high-throughput screening and analysis technologies. We demonstrate the ability to detect multiple genetic deficiencies in double-strand break repair and evaluate a set of clinically relevant chemical inhibitors of one of the major double-strand break repair pathways, non-homologous end-joining. While other high-throughput repair assays measure residual damage or indirect markers of damage, the CometChip detects physical double-strand breaks, providing direct measurement of damage induction and repair capacity, which may be useful in developing and implementing treatment strategies with reduced side effects.

CometChip: A High-throughput 96-Well Platform for Measuring DNA Damage in Microarrayed Human Cells

“…The ability to process 96 samples on the same platform not only reduces labor and time, but also reduces experimental noises and greatly enhances reproducibility. Inter sample variation is significantly lower than the traditional slide-to-slide variation, which can be 2 fold higher than the variation observed with this chip 6,7 . Reduced noise also leads to improved sensitivity, enabling detection of more subtle differences among samples.…”

“…Second, the microwells can be made with variable sizes to accommodate cell types of different sizes. Under circumstances that multiple cells are captured in a single microwell, multi-cell comets are selfcalibrated and yield consistent results compared to single cell comets 6,7 . Another benefit of patterning cells in a microarray is that all comets are then at the same focal plane with fixed positions, reducing noise due to unfocused comets and facilitating automated imaging and analysis.…”

“…Together, these limitations contribute to its relatively low acceptance in large-scale studies. Through integration of engineering technologies and principles, the CometChip brings a solution to the problems of throughput and inconsistencies that are associated with the traditional comet assay 6,7 . Briefly, to create the chip, a mold is microfabricated using photolithography to create microposts with diameters as small as that of a single cell.…”

“…We have demonstrated in previous publications the detection window of both versions. 6,7 Although the comet assay has been routinely used for basic research on DNA damage and repair and genotoxicity testing 1,2,[8][9][10][11][12][13][14][15] , the assay has been reported having poor reproducibility due to sample-to-sample, person-to-person and lab-to-lab variability. Additionally, the assay is low throughput, in part because image acquisition and data analysis are laborious.…”

Droplet-microarray on superhydrophobic–superhydrophilic patterns for high-throughput live cell screenings

“…It has raised the rate of scientific discoveries by enabling multiple parallel live cell experiments for the simultaneous evaluation of the effects of multiple factors on the phenotype of cells 1, 2 . Phenotypic cell-based screenings based on genetic perturbation of cells via RNA interference or gene over-expression are routine in fundamental science when investigating fundamental biological processes like cell signaling 3 , cell cycle control 4 , DNA repair machinery 5 , cell differentiation 6,7 , apoptosis 8,9 etc. HTS of live cells is widely employed in the pharmaceutical industry in fields such as drug discovery, antibody production and the toxicity testing of new drug candidates and bioactive compounds.…”