Applications of High-Throughput Clonogenic Survival Assays in High-LET Particle Microbeams

Georgantzoglou, Antonios; Merchant, Michael J; Jeynes, Jonathan C. G.; Mayhead, Natalie; Punia, Natasha; Butler, Rachel E.; Jena, R.

doi:10.3389/fonc.2015.00305

Cited by 4 publications

(2 citation statements)

References 53 publications

(70 reference statements)

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“…This experiment was stopped at 36 h because the stain had started to fade. The use of an alternative stain with a longer lifetime, or a bright field microscope without the use of stains, could increase observation time indefinitely [ 23 ]. The fact that all the observed cell division occurred in the last 12 h of the experiment also demonstrates that the AMOEBA system maintained a level of osmolality that allowed cell division to occur throughout the whole time period.…”

Section: Discussionmentioning

confidence: 99%

Automated microbeam observation environment for biological analysis—Custom portable environmental control applied to a vertical microbeam system

England

Bigelow

Merchant

et al. 2017

Sensors and Actuators B: Chemical

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Vertical Microbeams (VMB) are used to irradiate individual cells with low MeV energy ions. The irradiation of cells using VMBs requires cells to be removed from an incubator; this can cause physiological changes to cells because of the lower CO2 concentration, temperature and relative humidity outside of the incubator. Consequently, for experiments where cells require irradiation and observation for extended time periods, it is important to provide a controlled environment. The highly customised nature of the microscopes used on VMB systems means that there are no commercially available environmentally controlled microscope systems for VMB systems. The Automated Microbeam Observation Environment for Biological Analysis (AMOEBA) is a highly flexible modular environmental control system used to create incubator conditions on the end of a VMB. The AMOEBA takes advantage of the recent “maker” movement to create an open source control system that can be easily configured by the user to fit their control needs even beyond VMB applications. When applied to the task of controlling cell medium temperature, CO2 concentration and relative humidity on VMBs it creates a stable environment that allows cells to multiply on the end of a VMB over a period of 36 h, providing a low-cost (costing less than $2700 to build), customisable alternative to commercial time-lapse microscopy systems. AMOEBA adds the potential of VMBs to explore the long-term effects of radiation on single cells opening up new research areas for VMBs.

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Section: Discussionmentioning

confidence: 99%

Automated microbeam observation environment for biological analysis—Custom portable environmental control applied to a vertical microbeam system

England

Bigelow

Merchant

et al. 2017

Sensors and Actuators B: Chemical

Self Cite

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“…Several recent papers describe methodologies that approximate clonongic assays in HTS formats by miniaturizing to small well formats and utilizing automated image analysis to score fixed and stained colonies (7)(8)(9)(10)(11). The closest approximation of a clonogenic HTS among this body of work is a 'High Content' screen of Non Small Cell Lung Cancer cells in 96-well plates (10).…”

Section: Introductionmentioning

confidence: 99%

A high throughput screen with a clonogenic endpoint to identify radiation modulators of cancer

Gomes

Frederick

Jacobsen

et al. 2022

Preprint

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Clonogenic assays evaluate the ability of single cells to proliferate and form colonies. This process approximates the regrowth and recurrence of tumors after treatment with radiation or chemotherapy, and thereby provides a drug discovery platform for compounds that block this process. However, because of their labor-intensive and cumbersome nature, adapting canonical clonogenic assays for high throughput screening (HTS) has been challenging. We overcame these barriers by developing an integrated system that automates cell- and liquid-handling, irradiation, dosimetry, drug administration, and incubation. Further, we developed a fluorescent live-cell based automated colony scoring methodology that identifies and counts colonies precisely based upon actual nuclei number rather than colony area, thereby eliminating errors in colony counts caused by radiation induced changes in colony morphology. We identified 13 cell lines from 7 cancer types, where radiation is a standard treatment module, that exhibit identical radiation and chemoradiation response regardless of well format and are amenable to miniaturization into small-well HTS formats. We performed pilot screens through a 1584 compound NCI Diversity Set library using two cell lines representing different cancer indications. Radiation modulators identified in the pilot screens were validated in traditional clonogenic assays, providing proof-of-concept for the screen. The integrated methodology, hereafter 'clonogenic HTS', exhibits excellent robustness (Z' values >0.5) and shows high reproducibility (>95%). We propose that clonogenic HTS we developed can function as a drug discovery platform to identify compounds that inhibit tumor regrowth following radiation therapy, to identify new efficacious pair-wise combinations of known oncologic therapies, or to identify novel modulators of approved therapies.

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A High Throughput Screen with a Clonogenic Endpoint to Identify Radiation Modulators of Cancer

et al. 2022

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Clonogenic assays evaluate the ability of single cells to proliferate and form colonies. This process approximates the regrowth and recurrence of tumors after treatment with radiation or chemotherapy, and thereby provides a drug discovery platform for compounds that block this process. However, because of their labor-intensive and cumbersome nature, adapting canonical clonogenic assays for high throughput screening (HTS) has been challenging. We overcame these barriers by developing an integrated system that automates cell- and liquid-handling, irradiation, dosimetry, drug administration, and incubation. Further, we developed a fluorescent live-cell based automated colony scoring methodology that identifies and counts colonies precisely based upon actual nuclei number rather than colony area, thereby eliminating errors in colony counts caused by radiation induced changes in colony morphology. We identified 13 cell lines from 7 cancer types, where radiation is a standard treatment module, that exhibit identical radiation and chemoradiation response regardless of well format and are amenable to miniaturization into small-well HTS formats. We performed pilot screens through a 1,584 compound NCI Diversity Set library using two cell lines representing different cancer indications. Radiation modulators identified in the pilot screens were validated in traditional clonogenic assays, providing proof-of-concept for the screen. The integrated methodology, hereafter “clonogenic HTS”, exhibits excellent robustness (Z' values > 0.5) and shows high reproducibility (>95%). We propose that clonogenic HTS we developed can function as a drug discovery platform to identify compounds that inhibit tumor regrowth following radiation therapy, to identify new efficacious pair-wise combinations of known oncologic therapies, or to identify novel modulators of approved therapies.

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Applications of High-Throughput Clonogenic Survival Assays in High-LET Particle Microbeams

Cited by 4 publications

References 53 publications

Automated microbeam observation environment for biological analysis—Custom portable environmental control applied to a vertical microbeam system

Automated microbeam observation environment for biological analysis—Custom portable environmental control applied to a vertical microbeam system

A high throughput screen with a clonogenic endpoint to identify radiation modulators of cancer

A High Throughput Screen with a Clonogenic Endpoint to Identify Radiation Modulators of Cancer

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