Table of Contents

• High-Throughput Screening in Cancer Research
• Microplate Assays for Cancer Research
• Unlock the potential of microplate readers in cancer research

High-Throughput Screening in Cancer Research

High-throughput screening (HTS) has revolutionized the field of cancer research by enabling the rapid and systematic evaluation of compounds for their potential as anticancer agents. This approach represents a pivotal step in the early stages of cancer drug discovery.

HTS involves the methodical evaluation of thousands to millions of compounds from libraries to identify potential candidates that interact with specific biological targets in a predicted manner. These candidates, often referred to as "leads" or "hits," serve as the basis for further optimization and development of potential anticancer agents.

Moreover, the recent advancements in personalized cancer treatment have been driven by the development of targeted drugs. These treatments can leverage innovations in organoid technology, which involves the culture of organoids with self-renewal capacity, self-organization, and long-term proliferation while mimicking many characteristics of primary tissues. Patient-derived organoid (PDO) libraries have emerged as living biobanks, enabling in-depth analyses of tissue function, development, tumor initiation, and cancer pathobiology. PDOs are versatile and can be utilized for sequencing analyses, drug screening, targeted therapy testing, tumor microenvironment studies, and genetic engineering applications.

In the field of cancer research, these instruments are commonly employed to assess the effects of compounds on cancer cell lines, study the expression of specific genes or proteins, and evaluate various cellular responses. They play a pivotal role in identifying potential anticancer agents and advancing our understanding of cancer biology through large-scale, systematic screening efforts.

Robotics and liquid handling devices are crucial for the automation of HTS. These technologies enable the precise dispensing of compounds, cells, and reagents into multi-well plates, ensuring consistency and minimizing human error.

In summary, the integration of microplate readers, 3D biology, robotics, liquid handling devices, and dedicated software has revolutionized cancer research by expediting the discovery of potential anticancer compounds. While HTS is not a substitute for the entire drug discovery process, it plays a critical role in lead identification and enhances our understanding of cancer biology.

Microplate Assays for Cancer Research

Microplate assays have emerged as valuable tools, offering high-throughput capabilities and the ability to analyze numerous samples simultaneously. These assays allow researchers to investigate various aspects of cancer biology, from cell viability and cytotoxicity to protein-protein interactions and enzyme activity. In this article, we will explore some of the key microplate assays used in cancer research:

Cell viability assays

Cell viability assays are used to determine the number of viable and proliferating cells in a sample. They are crucial for assessing the impact of different treatments on cancer cells, helping researchers evaluate the potential of anticancer agents. Common methods include using dyes that are taken up by living cells, and the resulting signal is measured by a microplate reader.

• The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay is largely used in research to assess cell viability and proliferation with colorimetric readouts. Yellow MTT dye is reduced by metabolically active cells to form purple formazan product (Figure 1). This is solubilized and the absorbance intensity is measure on a microplate reader at 590 nm. MTT is quite inexpensive and relatively fast as it can give results within four hours. In the market there are other microplate assays that work similarly, for example yellow XTT (2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide) is reduced by metabolically active cells to orange formazan (absorbance read, OD 450-500 nm) or resazurin dye (alamarBlue) is reduced to resorufin (fluorescence read, ex 560 / em 590).
  
  Read application note: Assess cell viability and proliferation with colorimetric readouts

Figure 1. Schematic of the MTT assay mechanism

• The ATP (adenosine triphosphate) assay is a homogeneous, stable luminescence assay for determining the number of viable cells in culture. The method is based on quantitation of the ATP present in the well, an indicator of metabolically active cells (Figure 2). Several companies sell ready-to-use compounds that need to be added 1:1 in the well with the samples, and after a very short incubation, the sample can be read on a microplate reader equipped with luminescence detection mode. As mentioned above, several tests in cancer research are performed on 3D models. Performing viability assay on spheroids can be challenging due to the difficulty of ensuring the reagent reaches the core of the spheroid. Nevertheless, ATP-based viability assays specific for 3D cultures are now available, making it easier to rapidly assess drug response in both 2D and 3D model systems.
  
  Learn more about ATP assays:
  • Measure cancer cell viability using a homogeneous, stable luminescence assay
  • Rapidly assess drug response in 2D and 3D breast cancer model systems with a luminescent viability assay

Figure 2. Schematic of the ATP-based luciferin assay mechanism

• The EarlyTox™ Live/Dead Assay from Molecular Devices contains two markers for live or dead cells: calcein AM and ethidium homodimer-III (EthD-III). The nonfluorescent calcein AM permeates the intact cell membrane and is converted into fluorescent calcein by intracellular esterases in live cells. EthD-III is non-fluorescent and impermeant to an intact plasma membrane. When cell membrane integrity is compromised (by cell death), EthD-III enters cells and binds to nucleic acids, resulting in bright red fluorescence in dead cells (Figure 4).
  
  Read application note: Measuring cell health on the SpectraMax® iD3 reader with cell viability assays

Cytotoxicity assays

In cancer research it is crucial to understand how cells respond to various treatments, including potential anticancer agents. These microplate assays can help researchers differentiate between apoptotic (programmed cell death) and necrotic (uncontrolled cell death) processes.

• A variety of assays have been designed to detect and measure different cell death processes, including apoptosis and necrosis. In one such assay from Promega, the binding of two annexin V fusion proteins containing complementary subunits of NanoBiT® luciferase to exposed phosphatidylserine on the outer leaflet of cell membranes during the apoptotic process enables the generation of a luminescent signal. Upon loss of membrane integrity, a DNA-binding dye enters the cell and generates a green fluorescent signal (Figure 3).

Figure 3. In healthy cells the Annexin V-LgBiT and Annexin V-SmBiT (NanoBiT) fusion proteins are far apart, and no luminescence is generated. The profluorescent DNA dye does not bind to DNA, and fluorescent signal is also absent (A). In early apoptosis, luminescence remains low until PS exposure brings the Annexin V fusion proteins close together, forming a functional luciferase, while the profluorescent DNA dye remains outside the cells. Luminescence is generated, and fluorescence remains absent (B). During secondary necrosis, fluorescent signal is generated upon loss of membrane integrity during late-stage apoptosis, when the DNA dye can enter the cell. Both luminescence and fluorescence are generated in this case (C).

• In the LDH (Lactate Dehydrogenase) assay, LDH is released into the cell culture medium when cell membranes are disrupted, as in the case of dead or dying cells.
• Caspase activity is measured using a fluorogenic substrate that emits fluorescence upon cleavage by active caspases. For example, the EarlyTox Caspase-3/7 R110 Assay provides a single-step, homogenous assay that is specifically designed for microplate readers. The fluorogenic substrate (Ac-DEVD)2-R110 contains two DEVD consensus target sequences and is completely hydrolyzed in cell lysate by the enzymes in two successive steps. Hydrolysis of both DEVD peptides releases the green-fluorescent dye rhodamine 110 (R110), resulting in a substantial fluorescence increase, with excitation at 490 nm and emission at 520 nm (Figure 4). Similarly, the The EarlyTox Caspase-3/7 NucView™ 488 Assay enables detection of apoptosis in intact cell populations through use of NucView 488 Caspase-3 substrate. The advantage of this microplate assay is that it has been optimized for use with both microplate readers and imaging systems. This assay kit, used together with SpectraMax microplate readers or the SpectraMax MiniMax 300 Imaging Cytometer, provides users with a flexible approach to the quantitation of apoptosis. Apoptotic cells can be measured directly as a function of total fluorescence per well, or by imaging and counting individual cells, with comparable results for both methods. Finally, we have shown that the EarlyTox Caspase-3/7 NucView™ 488 Assay also works very well on spheroids. Specifically, we assessed apoptosis in magnetically bioprinted HepG2 spheroids (Figure 4).

Figure 4. Schematic of different options that can be pursued when performing toxicity studies with the EarlyTox Cell Viability Kits .

Reporter gene assays

These microplate assays involve introducing reporter genes into cancer or other cell types to monitor the activity of specific genes or signaling pathways. The reporter gene's expression can be quantified using microplate readers, providing insights into the regulation of cancer-related genes and pathways. One of the most widely used is the luciferase reporter assay, which enables the study of gene expression at the transcriptional level. It is popular because it is relatively inexpensive and gives rapid, quantitative measurements. Firefly luciferase is widely used as a reporter to study gene regulation and function. It is a very sensitive reporter due to the lack of any endogenous luciferase activity in mammalian cells or tissue. Firefly luciferase catalyzes ATP-dependent oxidation of D-luciferin with the resulting emission of light (Figure 1A). Luciferase from the sea pansy Renilla reniformis is often used in multiplexed luciferase assays as a second reporter for normalizing transfection efficiency and for studying gene regulation and function. Renilla luciferase catalyzes coelenterazine oxidation by oxygen to produce light (Figure 1B). Dual luciferase assays enable the measurement of both firefly and Renilla luciferase activity in a single sample, with firefly acting as the experimental reporter and Renilla the control (Figure 5).

Figure 5. Chemical reactions catalyzed by firefly luciferase (A) and Renilla luciferase (B).

The SpectraMax DuoLuc™ Reporter Assay Kit enables sensitive quantitation of both firefly and Renilla luciferases in mammalian cells. Serial injection of two optimized detection reagents allows the luciferases to be assayed in the same microplate well. For example, we have shown how the DuoLuc reporter assay and SpectraMax iD5 Multi-Mode Microplate Reader are used to detect nuclear factor-κB (NF-κB) activation in a mammalian cell model. NF-κB is very important for the regulation of inflammation, immunity, proliferation, differentiation and apoptosis.

Learn more about the DuoLuc reporter assay:

• Monitor NF-κB activation with a sensitive dual luciferase reporter assay on the SpectraMax iD5
• Detect dual luciferase expression on the FlexStation 3 microplate reader

Enzyme activity assays

Enzymes, such as kinases and phosphatases, play a crucial role in cancer signaling pathways. Enzyme activity assays involve measuring the activity of these enzymes using substrates that produce a detectable signal. Microplate readers quantify the enzymatic reaction, allowing researchers to assess the impact of different treatments on enzyme activity.

• IMAP® Technology from Molecular Devices enables rapid, homogeneous, and non-radioactive assay of kinases, phosphatases, and phosphodiesterases (Figure 7) and is suited for both assay development and high-throughput screening. IMAP assays are based on binding of phosphate to immobilized metal coordination complexes on nanoparticles. When IMAP binding entities bind to phosphorylated substrate, molecular motion of the peptide is altered, and fluorescence polarization (FP) for the fluorescent label attached to the peptide increases (Figure 6, left). In a TR-FRET version of the assay, the inclusion of a terbium (Tb) donor enables a fluorescent energy transfer to occur when phosphorylated substrate is present (Figure x, right). This microplate assay is detected in a time-resolved fluorescence mode, which virtually eliminates fluorescence interference from assay components or compounds in a screen. TR-FRET also offers flexibility in substrate size and concentration.
  
  Learn more about IMAP assays:
  • IMAP FP kinase assays on the SpectraMax M5 Multi-Mode Microplate Reader
  • IMAP phosphodiesterase assays on SpectraMax Multi-Mode Microplate Readers

Figure 6. IMAP FP and TR-FRET phosphodiesterase assay principle.

• THUNDER™ is a versatile and cost-effective cell-based assay platform developed by Bioauxilium Research for measuring individual phosphorylated and total endogenous proteins. Specifically, the THUNDER™ Phospho-ERK1/2 (T202/Y204) assay kit is a homogeneous TR-FRET sandwich immunoassay with a simple workflow consisting of three steps that can indirectly report the higher or lower activity of the tyrosine kinase receptor, located upstream from the MAPK/ERK cell signaling pathway (Figure 8).
  
  Read application note: Cell-based measurement of ERK1/2 phosphorylation with THUNDER TR-FRET assay

Figure 8 THUNDER TR-FRET sandwich immunoassay. Binding of both the Eu-Ab1 and FR-Ab2 to the analyte enables a transfer of energy from the Europium chelate to the acceptor fluorophore, resulting in signal at 665 nm that is detected using a microplate reader with time-resolved detection mode.

Protein-protein interaction assays

Researchers use microplate readers to detect and quantify interactions between specific proteins in cancer pathways. These interactions can reveal the roles of these proteins in cancer development and progression. Drug treatments are often designed with the aim to disrupt certain protein-protein interaction. Thus, it is important to have microplate assays that facilitate the study of interactions between specific proteins.

• BRET (bioluminescence resonance energy transfer) is a technique for measuring protein-protein or protein-ligand interactions that involves a bioluminescent donor and a fluorescent acceptor. When donor and acceptor are closer than 10 nm to each other, the donor excites the acceptor, which then emits fluorescence. By tagging one protein of interest with the donor and its binding partner with the acceptor, one can measure protein interactions by using a microplate reader to detect light emitted by the donor and acceptor (Figure 9).
  
  Learn more about NanoBRET technology:
  • Measure p53-MDM2 protein interaction with NanoBRET technology
  • NanoBRET Technology on the SpectraMax i3x Multi-Mode Microplate Reader
  • Assessing Reader Capability for NanoBRET™ Technology: Two Simple and Effective Assays

Figure 9. NanoBRET assay. When a NanoLuc-Protein A fusion (energy donor) interacts with a fluorescently labeled HaloTag-Protein B fusion (energy acceptor), donor and acceptor are brought close together and energy is transferred.

• TR-FRET assays address protein-protein interactions, nuclear receptor assays, receptor dimerization, ligand binding, receptor internalization, enzyme assays, and many more. They involve the use of two fluorophores, a donor and an acceptor, to label proteins or other biomolecules of interest. Proximity between two biomolecules enables fluorescence resonance energy transfer (FRET) to occur from the donor to the acceptor fluorophore. TR-FRET uses long-lifetime fluorescent lanthanide cryptates as donors, minimizing short-lived background fluorescence from compounds and other materials. The detection of time-resolved fluorescence resonance energy transfer (TR-FRET) with a microplate reader provides a readout of biomolecular binding. (See also IMAP TR-FRET, above.)
• Fluorescence polarization (FP) is widely used to monitor binding events in solution. It can be used to assess protein-antibody binding, DNA hybridization, enzyme activity, and it is well suitable for high-throughput screening. A small, fluorescently labeled molecule (tracer) emits mostly depolarized light when excited with plane-polarized light, because the tracer tumbles rapidly during the time between excitation and emission. However, when the tracer binds a much larger molecule, it rotates more slowly, and the emitted light remains largely polarized. The amount of polarized/depolarized light will give a quantification of the interaction between the protein and the tracer. (See also IMAP FP, above.)
  
  Read application note: Establishing and optimizing a fluorescence polarization assay

ELISA (Enzyme-Linked Immunosorbent Assay)

ELISAs are widely used for the quantification of proteins in cancer research. Microplate readers measure the signals generated in these assays to determine the concentration of specific proteins, such as cytokines or growth factors, which are often relevant to cancer biology

Learn more about ELISAs by downloading our eBook, ELISA explained: from basics to practical application.

AlphaScreen & AlphaLISA

AlphaLisa is a bead-based, homogeneous assay for studying molecular interactions in a microplate format. Compared to traditional ELISA methods, which have several wash steps that can damage cell monolayers and are very time consuming, AlphaLISA do not require washing steps, minimizing cell loss, resulting in faster and more accurate results.

Read application note: AlphaLISA Screen on the SpectraMax Paradigm Multi-Mode Microplate Detection Platform

TR-FRET (including HTRF)

HTRF is a versatile technology developed for detecting biomolecular interactions. It combines fluorescence resonance energy transfer (FRET) technology with time-resolved (TR) measurement of fluorescence, allowing elimination of short-lived background fluorescence.

Read application note: HTRF Human TNFα Assay on SpectraMax Paradigm Multi-Mode Microplate Reader

The assay uses donor and acceptor fluorophores. When donor and acceptor are close enough to each other, excitation of the donor by an energy source (e.g., a flash lamp) triggers an energy transfer to the acceptor, which in turn emits specific fluorescence at a given wavelength. HTRF is also widely used in substitution of classic ELISA as it provides a simple, no wash strategy to detect and quantify proteins in as little as two hours. Moreover, it has a wide detectable range, and it is scalable with long signal stability.

Read application note: Normalize HTRF cytokine assays to cell viability

ROS (Reactive Oxygen Species) Assays

ROS are associated with oxidative stress and cancer development. Microplate readers can quantify ROS levels, helping researchers understand the oxidative state of cancer cells and how it may contribute to cancer progression.

Read application note: Measuring reactive oxygen species with SpectraMax microplate readers

Cell cycle analysis

Cell cycle analysis is an important study often necessary in cancer research. Usually, it is performed through flow cytometry. Nevertheless, the SpectraMax® i3x Multi-Mode Microplate Reader equipped with the SpectraMax® MiniMax™ 300 Imaging Cytometer can be used to image and analyze images of FUCCI (fluorescent ubiquitination-based cell-cycle indicator) spheroids. FUCCI spheroids have been developed to study cancer cell cycle progression as they allow the identification of cells in various phases of the cell cycle. The FUCCI technology is based on the overexpression of two modified cell cycle-dependent proteins, geminin and Cdt1, each respectively fused to a green fluorophore (AmCyan for geminin) and a red fluorophore (mCherry for Cdt1). Cdt1 and geminin levels fluctuate differentially throughout the cell cycle: Cdt1 levels peak in G1 phase; while geminin levels rise in late S, G2 and M phase. This results in the nucleus of FUCCI-expressing cells appearing red in G1 phase and green in late S, G2 and M phase (Figure 10). Although this assay is not indicated for HTS screenings, it is an important microplate assay that can be implemented once compound leads have been selected for further and deeper characterization.

Read application note: Acquire and analyze images of FUCCI spheroids on the SpectraMax MiniMax cytometer

Figure 10. FUCCI cell cycle analysis. mCherry-Cdt and AmCyan-geminin fluorescently labeled cell cycle proteins are expressed or degraded during different phases of the cell cycle such that cells appear red during G1 phase and green during S, G2, and M phases. Cellular imaging can be used to monitor the cell cycle under various experimental conditions.

Unlock the potential of microplate readers in cancer research

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