Watching Cancer Immunotherapy Work in Real Time

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The human immune system is equipped with the tools to recognize, attack, and eliminate cancerous cells, but too often cancer cells find ways to evade this immunosurveillance by shutting down key immune cells, especially within the tumor microenvironment. To outmaneuver tumor escape mechanisms, researchers have been developing strategies to enhance the immune system and neutralize cancer cell defenses.

One such strategy, called adoptive T-cell transfer, involves extracting T cells from an individual, enhancing and growing them ex vivo in the lab through cell and genetic engineering, followed by reinfusion. By equipping the T cells with receptors that target cancer antigens, researchers can invigorate the body’s search-and-destroy mechanisms to take out cancer cells. Once the cells have been modified ex vivo and prior to injecting them into the individual, it’s critical to analyze their potency and selectivity. The Agilent xCELLigence Real-Time Cell Analysis (RTCA) system is a valuable tool for evaluating the cell-killing ability of these enhanced immune cells against cancer cells in real time (Figure 1). By measuring the immune-cell-mediated killing of cancer cells accurately, reproducibly, and in real time, the xCELLigence system helps researchers design safe and efficacious cell therapies.

Agilent xCELLigence RTCA MP
Figure 1. The Agilent xCELLigence RTCA MP instrument monitors immune cell killing in a label-free, real-time manner. The MP (multiple plate) model can host up to six 96-well E-Plates. The instrument is placed in a standard CO2 cell culture incubator and interfaces via a cable with analysis and control units that are housed outside the incubator.

“Agilent’s Cell Analysis division is focused on helping researchers harness the power of the immune system to develop effective immunotherapies. With the xCELLigence RTCA technology, the complex interaction between immune cells and tumor cells can be continuously monitored in real time, allowing researchers to rapidly test the efficacy of different immunotherapy approaches,” said Xiaobo Wang, general manager, ACEA Biosciences, part of Agilent.

Cancers caused by HPV present an ideal target for this strategy. Tumor formation depends on antigens unique to the virus, and healthy cells don’t produce those proteins. Researchers in the lab of Stephen Schoenberger, PhD, at the La Jolla Institute are working to engineer T cells against HPV antigens.

Tumor-infiltrating lymphocytes (TILs) are immune cells that naturally react to tumor antigens and can surround and penetrate solid tumors. The researchers isolated TILs that reacted to HPV antigen E6 from an individual with an HPV-related cancer, head and neck squamous cell carcinoma (HNSCC). They sequenced the T-cell receptor specific to the E6 antigen and, using a lentiviral vector, introduced it into T cells. The xCELLigence RTCA instrument was used to test the selectivity and potency of the engineered T cells.

“We’re measuring the ability of a T cell to kill a target cell,” explained Schoenberger. “That’s something that people in my line of work have been trying to measure since the 1960s.” Previous methods involved labeling the cells with radioactive tags and inferring cell death by the radioactivity released into the media. Avoiding radioactivity is one benefit of the xCELLigence system, Schoenberger said. Another is flexibility in target cell selection.

“Not every tumor cell that expresses an antigen grows well in vitro, and some of them don’t load well with these radioactive labels,” he pointed out. “The Agilent xCELLigence system gives us much more flexibility in the range of target cells that we can use in the assay.”

This project depended on being able to use a very rare human tumor cell line that grows in culture and expresses HPV E6 antigen.

The xCELLigence system detects cell death by a cellular impedance assay, which measures the flow of electrical current through a cell culture. Agilent’s patented 96-well microtiter E-plates are coated with gold biosensors that monitor changes in cell number, cell size, and cell attachment. The cells act as insulators, so the more cells that are attached to the biosensors, the more they impede the current. The nonperturbing nature of the readout allows for dynamic monitoring of tumor cells and their interaction with immune cells.

Only the cancer cells adhere to the sensors, while the effector T cells float free. By detecting the decrease in impedance generated by the killing of the target cells by immune cells, the instrument can quantify the cell-killing effects of the T cells in real time.

By testing four sets of conditions, the researchers were able to compare how well the engineered cells eliminated the cancer. (Figure 2). To assess tumor cell growth, they plated cancer cells alone, with no treatment, to see what unchecked growth looked like. They also tested the killing ability of unmodified T cells, and as a positive control, they tested the chemotherapy drug cisplatin. In the fourth condition, they assessed  the immune cells specifically engineered to produce T-cell receptors against the HPV antigen E6. Using the xCELLigence RTCA system, they produced real-time, precise, and reproducible measurements of the cancer cells’ response.

Dynamic monitoring of a cancer line
Figure 2. Dynamic monitoring of a cancer line to assess the capacity of the TCR to specifically kill tumor lines expressing the HPV E6 protein at 10:1 effector to target ratio. The green curve shows the natural growth of the tumor cells with no added effector cells. The red curve demonstrates that the chemotherapeutic, cisPt, can kill these lines within 48 hours. To assess nonspecific (alloreactivity) background killing by the CD8 T cells, the growth of CD8 T cells that were stimulated and expanded but not subjected to CRISPR or LV editing are represented by the purple curve. The neoantigen-engineered CD8 T cells (blue line) shows superior killing capacity than even chemotherapy at very early time points.
©Schoenberger Lab 2020 Reprinted with Permission

“The quantitative and real-time nature of the impedance data made it easy to compare the potency between treatments,” wrote Martin Naradikian, PhD, a postdoctoral researcher in the Schoenberger lab. The data revealed that the T cells engineered to hunt down E6 performed far better than the unmodified cells and showed killing ability equivalent to cisplatin at some time points.

The researchers also tested various ratios of effector cells to target cells, ranging from 20:1 to 1:1. The exquisite sensitivity of the xCELLigence RTCA enabled the testing of effector cells at low, physiologically relevant concentrations. For this project, they took readings every 15 minutes, but the xCELLigence system allows the user to define the interval between measurements, from seconds to hours.

With its simple and high-throughput workflow, the xCELLigence RTCA system easily allowed for direct comparison of the different samples (Figure 3). The simple workflow lets users monitor target cell killing continuously and auto-matically over a period of hours or days, noninvasively, with no dyes or labels. The xCELLigence RTCA instrument can read an entire 96-well plate in 15 seconds.  With the capability to read up to six plates independently, the xCELLigence RTCA MP (multiple plate) instrument also cuts down on scheduling conflicts, allowing researchers to run multiple experiments at the same time.

Agilent Cancer IMT Workflow
Figure 3. Agilent xCELLigence RTCA technology offers a high-throughput and easy workflow.

Agilent continues to add new features to improve productivity and versatility. “While this approach is simple and easy to use, we learned that our users would also like to visually validate immune and tumor cell activity in real time, which would typically require a separate experiment,” said ACEA’s Xiaobo Wang. “To simplify their workflow, we recently released the xCELLigence RTCA eSight, which combines the power of live cell imaging with real-time impedance-based readings. This allows researchers to obtain highly reproducible dual measurements with the convenience of one single set up.”

The researchers in Schoenberger’ s lab who used the xCELLigence eSight appreciated the ability to visualize the cells. “You can do light and fluorescence imaging of your target cells, you can watch them disappear, or move, or whatever you’re interested in,” said Schoenberger. “Seeing is believing.”

 

To learn more about this application, download the Agilent application note “Real-Time Specificity and Potency Assessment of Human Papilloma Virus Specific Engineered T Cells”.

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Not for use in diagnostic procedures.

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