Cancer spheroids and tumoroids are versatile, three-dimensional (3D) in vitro models generated from cancer cells and tumor tissues. Because these 3D cancer models more closely resemble complex tumor biology and cellular interactions observed in vivo, scientists are using them to evaluate candidate immune cell products and accelerate preclinical drug development for urgently needed cellular immunotherapies in solid tumor applications. The simple and sensitive assays of the Maestro Z accurately measure tumor growth and immune cell killing of 3D cancer spheroid models.
Accurately measuring cell potency is critical for developing and improving immunotherapies. Cancer spheroids placed in a CytoView-Z plate can be noninvasively monitored to track real-time growth and assess the potency of therapeutic candidates.
A cancer spheroid composed of HER2-expressing ovarian cancer cells in a CytoView-Z plate. Growth of spheroids of different starting sizes are monitored over 50 hours. The addition of HER2-targeted CAR T cells at different effector:target ratios at 24 hours shows a dose-dependent decrease in spheroid size.
When it comes to measuring the effects of cell-based immunotherapies on solid tumors, 2D monolayers may not tell the full story. Download our new app note to discover how next-generation in vitro tumor models on Axion's Maestro Z platform can accelerate immunotherapy discovery—with no labels, dyes, or complicated steps.
Solid tumors are characterized by a harsh tumor microenvironment, which can substantially impact interactions between tumors and immune therapies like chimeric antigen receptor (CAR) T-cell therapy. While two-dimensional (2D) cell cultures remain an indispensable platform for high-throughput drug screening and toxicity testing in oncology, 3D cancer spheroids offer an in vitro model closer to the complex tumor biology. A comparison of CAR T cell-mediated cytolysis in 3D in vitro models and 2D monolayers using the Maestro Z found a decreased potency of HER-2 specific CAR T cells in the 3D tumor model- much as solid tumors demonstrate increased resistance to treatment in vivo.
CAR T cell-mediated cytolysis of cancer spheroids is lower than the cytolysis of matching E:T ratios in monolayer cultures over time (A), resulting in a higher EC50 for the spheroid cultures (B).
Cancer spheroids are formed over four days in ulta-low attachment U-bottom plates. Individual spheroids are then transferred into each well of a CytoView-Z plate. One day post spheroid plating, CAR T cell suspension is added at desired effector to target (E:T) ratios. Changes in spheroid size are tracked label-free and in real time with the Impedance Module software.
Additional cancer spheroid culture and recording protocol is available.
The advantages of measuring immune cell-mediated cell killing on the Maestro Z, ZHT, Pro, and Edge platforms:
Continuous cell monitoring – Up to 384 simultaneous live recordings from your spheroids. Now you can track immune cell-mediated cell killing in real time, even when you are out of the lab.
Analyze cell activity label-free – Perform noninvasive electrical measurements from the cultured cell population, circumventing the use of dyes/reporters that can perturb your cell model and confound results.
Precise assay environment – No need for an additional cell culture incubator, saving valuable lab space and money. The smart environmental chamber finely controls heat and CO₂ while rejecting electrical noise and mechanical vibrations.
See your cells – Sometimes you just want to look at your cells under a microscope. The Cytoview-Z 96-well plates have a viewing window in each well which allows cell visualization.
Probe cell models in the same plate they were cultured in – Other higher throughput platforms (e.g. flow cytometry) often require cell samples to be transferred into a single-cell suspension before testing. In the case of adherent cells this is not ideal since they exist as a functional network of interlinked cells.
Smartphone App for your assay – You can't always be in the lab. But changes in immune cell-mediated cell killing seldom occur at convenient time points. The Maestro Z App allows you to see live results and system status on your smartphone.
It’s easy – With effortless assay setup and intuitive analysis software designed for quick export of figures and results, you can now focus on the science.
Impedance-based cell analysis is a well-established technique for monitoring the presence, morphology, and behavior of cells in culture. Impedance describes the obstruction to alternating current flow. To measure impedance, small electrical currents are delivered to electrodes embedded in a cell culture substrate. The opposition to current flow from one electrode to another defines the impedance of the cell-electrode interface. When cells are present and attached to the substrate, they block these electrical currents and are detected as an increase in impedance.
Impedance is sensitive to many aspects of cell behavior: attachment, spreading, shape, cell-cell connections (e.g. tight junctions), and death. Even small transient changes, such as swelling or signaling, are detectable by impedance. Because impedance is noninvasive and label free, the dynamics of these changes can be monitored in real time over minutes, hours, or even days without disturbing the biology.
Interdigitated electrodes embedded in the cell culture substrate at the bottom of each well detect small changes in the impedance of current flow caused by cell presence, attachment, and behavior.
In the example below, the electrodes are initially uncovered before cells are added. The electrical current passes easily and the impedance is low. When cells begin to attach and cover the electrodes, less electrical current passes and the impedance is high. After dosing with a cytotoxic agent, cells die or detach, and the impedance decreases back towards baseline.
Impedance measures how much electrical signal (orange arrows) is blocked by the cell-electrode interface. Impedance increases as cells cover the electrode and decreases back to baseline due to cell death.
Many cell-based assays are endpoint assays, limited to a single snapshot in time. Repeating these assays at multiple time points can be labor intensive, time consuming, and costly. Key time points can be easily missed. Impedance-based cell analysis is nondestructive and label free, meaning that cellular dynamics can be monitored continuously.
The impedance assay can be used to characterize dynamic cell profiles, revealing how cells grow, attach, and interact over time. Each cell type exhibits a different cell profile, or “fingerprint”, of dynamic cell behavior. These profiles are sensitive to cell type, density, purity, and environmental factors. In this example, the Maestro Z impedance assay readily distinguished cell profiles across different cell densities and cell types.
(A, B) HeLa cells were seeded on a CytoView-Z plate at varying densities and the impedance was continuously monitored by the Maestro Z. Impedance scaled proportionally with cell density and readily distinguished different densities of the same cell type. (C) Maestro monitored the growth of three cell types, HeLa, A549, and Calu-3, and readily distinguishes their distinct cell profiles over time.
The Maestro Z impedance assay can also be used to capture the kinetics of cell responses to drugs or immune cell therapies. The kinetics, which cannot be captured by endpoint assays, often provide key insights into the efficacy of novel therapies. In the example below, the Maestro Z impedance assay was used to quantify the kinetics of cytotoxicity of chemotherapy agents.
A549 cells were dosed with dox, vehicle (DMSO), or tergazyme. Wells dosed with tergazyme showed an immediate decrease in impedance, reflecting complete cell death. Higher doses of dox resulted in a slower decrease in impedance and cell death. Cells dosed with 1 μM dox reached 50% cytolysis at 31 hrs.
Impedance varies with frequency, such that different frequencies reveal different aspects of cell biology. The small currents used to measure impedance will always take the path of least resistance. At low frequencies, such as 1 kHz, the impedance of the cell membrane is relatively high, forcing the current to flow under and between the cells. Low frequencies provide details about barrier integrity, the presence of gap junctions, and transepithelial or transendothelial resistance (TEER).
At high frequencies, such as 41.5 kHz, the impedance (and capacitive reactance) of the cell membrane is relatively low. Thus, most of the current couples capacitively through the cell membranes, providing information about the cell layer such as confluency and coverage.
In other words, low frequencies are sensitive to “what” cells are there, whereas high frequencies are sensitive to “how many” cells are there. The Maestro Z impedance assay uses multiple frequencies to provide the most information about the cells, simultaneously, continuously, and in real time.
Multiple frequencies were used to simultaneously and continuously monitor the coverage and barrier function (TEER) of Calu-3 and A549 cells. Coverage, measured as resistance at 41.5 kHz, increases over time for both cell types. TEER, measured at 1 kHz, reveals that only Calu-3 cells form a strong barrier, as they express tight junctions to block flow between neighboring cells.
