Cayman’s cellular metabolism screening services will generate a full profile of mitochondrial function and cellular bioenergetics for preclinical compounds. We provide a progressive set of primary screens that determine mitochondrial function in intact cells and functional assays to screen potential effectors of the electron transport chain, mitochondrial inner membrane permeability, and mitochondrial ATP production or reactive oxygen species generation. We sat down with one of our scientists, David Hoffman, Ph.D., a mitochondrial biochemist and expert in the design of biochemically relevant experimental models, to discuss the work he is doing to assess mitochondrial respiration, glycolysis, and function.
My degree is in biochemistry with a primary focus on the mechanics of mitochondrial function and ROS production under tightly controlled oxygen concentrations. Since then, I’ve focused on broadening my knowledge of cellular metabolism and mitochondrial biochemistry as it pertains to different organismal systems, cell types, and disease states. I started working in this area in 2004.
We use an Agilent Seahorse XFe96—an instrument I’ve been using since my graduate work—which really helps to streamline our process. With this instrument, we can measure the key indicators of cellular metabolism—mitochondrial oxygen consumption and extracellular acidification—simultaneously in a 96-well microchamber plate. Responses to substrates, inhibitors, and other compounds can be detected through the 4-port injection system and automated mixing at programmable intervals. All of this is captured in real time using the XFe96 system software. To maintain experimental integrity, methods are optimized for each system and everything is standardized in order to ensure reproducibility.
The tumor microenvironment possesses a unique metabolic environment consisting of low oxygen concentrations, low pH, and high concentrations of fatty acids. We can stimulate and culture immune cells and cancer cells under hypoxic conditions utilizing our BioSpa™ 8 Automated Incubator, which among other things, functions as an atmospherically controlled incubator attached to the Cytation™ 5 Cell Imaging Multi-Mode Plate Reader from BioTek. From there, we can assess metabolic function using the XFe96, or measure cellular ROS generation, mitochondrial membrane potential, and apoptotic markers through high-content imaging using the Cytation™ 5. The poster I presented at the 2019 meeting of the American Association for Cancer Research is a good example of the variety of cell-based assays we have at our disposal for cellular metabolism in the tumor microenvironment.
For mitochondrial toxicity, the first thing we’d look at would be oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) on the XFe96 at a concentration 10-fold higher than the Cmax. Based on our findings, we could then de-risk compounds or classify them as inhibitors or uncouplers. Next, we would look at changes in metabolic phenotype and fuel preferences. For the inhibitors, we could further narrow down the mechanism of toxicity by focusing on the individual components of the electron transport chain (ETC) through the use of our MitoCheck® assays—which we developed exclusively to assess the activity of Complexes I-IV of the ETC and the mitochondrial ATP synthase.
Previously there was no great commercial resource for targeting the individual complexes of the ETC. The assays available at the time were time-consuming and reliant on antibodies for detection. Our assays are tailored specifically to each complex in the ETC, using distinct sets of substrates and inhibitors. We also created the MitoCheck® Mitochondrial (Tissue) Isolation Kit, which enables the measurement of ETC activity in intact mitochondria removed from isolated tissue samples to provide a more simple and biochemically relevant experimental model system that is capable of phosphorylating ATP. Examples of how these assays can be used in conjunction with determining mitochondrial phenotypes can be viewed in the links below.
For the XFe96 experiments, we use Agilent’s Cell Normalization Software, which is compatible with the BioTek Cytation™ 5 Cell Imaging Multi-Mode Plate Reader. This allows us to automate the cell count based on Hoechst staining, and incorporate that with the OCR data from the Seahorse to report OCR/cell. For other experiments, such as the MitoCheck® ETC activity assays, we normalize based on protein concentration or protein/unit of citrate synthase. We designed the MitoCheck® Citrate Synthase Activity Assay Kit to accomplish this task.
Cellular metabolism is dynamic. Some cell types are able to compensate for compound-induced mitochondrial dysfunction by upregulating other metabolic pathways. This often goes unnoticed until there is a high metabolic demand and can often be overlooked in a cell viability assay.
The proton ionophore FCCP has long been used to deplete the electrochemical proton gradient, uncoupling oxidative phosphorylation from the ETC. Mitochondria compensate for this change by increasing oxygen consumption to restore the diminished ATP generation. Uncouplers are a valuable tool to study bioenergetics. However, if the concentration of uncoupler is excessive, cytotoxicity will occur, and cellular oxygen consumption will plummet. Thus, for each new cell line tested, careful titration of the uncoupler is always good practice to minimize toxicity problems. In general, FCCP is effective in a very narrow range of concentrations, which can be limiting in certain experimental settings, especially if this concentration is not high enough to dissipate the proton gradient. Furthermore, FCCP is not specific to mitochondria and demonstrates ionophore activity on plasma membranes in addition to mitochondrial membranes, which can generate off-target effects such as disruption of the microtubule cytoskeleton, inhibition of lysosome activity, or activation of ion channels.
We’ve shown that a second generation ionophore, BAM15, can be used as an uncoupler at a wider effective concentration range compared to FCCP and—at proper titration—circumvents the cell toxicity issue. In contrast to FCCP, BAM15 is highly selective for the inner mitochondrial membrane and does not depolarize the plasma membrane at high concentrations. This data can be viewed in our application note: Characterizing a Better Uncoupler.
Screening customers are always concerned with maintaining reproducibility across higher throughput assay runs. We are currently working to integrate the XFe96 with Agilent’s AssayMap Bravo robotic liquid handling platform, which will provide our service customers with even greater integrity in assay reproducibility regardless of throughput.
Written reports are provided detailing the experiments conducted, results and summary, and possible next steps. We usually schedule a call with clients following delivery of a report to discuss results in detail and answer any questions.
We have a very talented group of scientists at Cayman with a very wide range of expertise. For example, I’ve recently become very interested in the tumor microenvironment. It’s incredibly unique from a metabolic standpoint, but my knowledge of tumor-associated immune cells and their impact on the microenvironment is, for lack of a better term, not a personal strength. Luckily for me, our Immunology Services group does have this expertise, and can be brought into the project planning process. I feel like this is what really makes Cayman Services stand out. If I have a question pertaining to immunology, cell biology, enzymology, structural biology, medicinal chemistry, or mass spectrometry, the diversity in background and technical skills within Cayman, combined with the intimate knowledge of the project brought by our clients, allows us to talk through project design, review results, and plan next steps as a true team that’s invested in the outcomes. It’s not uncommon for a project to start with the assessment of cellular metabolism and bioenergetic responses to a particular compound, and then progress to consultation with other resident experts to run in vitro tox assays, optimize drug design & synthesis, and perform additional compound profiling and bioanalysis.
Learn more about our service capabilities at www.caymanchem.com/services.