What is cancer immunotherapy?
Cancer immunotherapy consists of multiple approaches that focus on harnessing and enhancing the innate powers of the immune system to fight cancer. While traditional small molecule chemotherapy continues to play a critical role in cancer treatment, immunotherapy is rapidly gaining traction; in 2014 immunotherapies constituted ~50% of the overall oncology pharmacopeia. Cancer immunotherapies can be divided into three major categories: (1) cytokines/immunomodulation agents, (2) monoclonal antibodies, and (3) cell-based therapies. Though monoclonal antibodies currently represent the largest class of commercialized cancer immunotherapies, cell-based therapies are rapidly making headway. This class of patient-specific therapies involve collecting immune cells from a cancer patient, engineering them (via genetic manipulation or peptide/adjuvant stimulation) to recognize and kill cancer cells, growing large numbers of these and reintroducing them into the same patient.
What are the different ways the immune system can be harnessed to target tumors?
As shown in the figure below, immune cell-mediated tumor cell killing can involve the components of both the innate and adaptive immune systems including: (1) natural killer (NK) cells, (2) cytotoxic T cells (MHC-dependent), (3) antibodies secreted by B lymphocytes, (4) engineered antibodies such as bispecific antibodies and bispecific T cell engagers (BiTEs), (5) genetically engineered T cells targeting specific tumor antigens (e.g., CAR-T; MHC-independent), and (6) macrophage-mediated phagocytosis.
What assays are used to study immune cell killing?
Many in vitro assays have been developed to screen and evaluate the efficacy of immune cell-mediated killing. The most common of these is the release assay where effector cell-mediated disruption of the target cell membrane results in leakage of its cytoplasmic contents into the culture medium. Endogenous biomolecules (such as lactate dehydrogenase) or previously added exogenous labels (such as the radioisotopes 51Cr or 111In) that leak into the media are then measured as an indirect readout of the damage caused by effector cells. Alternative endpoint methods include flow cytometry, ELISA-based granzyme measurement, and morphometric analyses by microscopy. Although traditional end-point assays still serve as important tools for evaluating the efficacy of immune cell-mediated killing, there is a critical need for more efficient, homogeneous, automated assays (requiring less hands-on time) that aren’t hampered by the artifacts and hassle of using labels and that can provide quantitative results.
What is the xCELLigence RTCA assay principle?
ACEA’s xCELLigence® Real-Time Cell Analysis (RTCA) instruments utilize gold microelectrodes embedded in the bottom of microtiter wells to non-invasively monitor cell status including cell number, shape/size and attachment. The major distinguishing features of this technology include enhanced sensitivity, the preclusion of labels and, importantly, kinetic measurement of cell health/behavior.
Step 1: Adherent target cells (i.e. tumor cells) are first seeded in the wells of an electronic microtiter plate (E-Plate®). Adhesion of cells to the gold microelectrodes impedes the flow of electric current between electrodes. This impedance value, plotted as a unitless parameter called “Cell Index”, increases as cells proliferate and then plateaus as cells approach 100% confluence.
Step 2: When added subsequently, non-adherent effector cells (i.e. immune cells) in suspension do not cause impedance changes in and of themselves (due to lack of adherence to the gold microelectrodes).
Step 3: If effector cells induce the destruction of the target adherent tumor cells, the corresponding cytolytic activity can be sensitively and precisely detected. The continuous acquisition of impedance data for each well of an E-Plate enables the generation of real-time killing curves for multiple conditions simultaneously.
Key benefits of using xCELLigence to monitor immune cell-mediated killing:
- Label-Free: Allowing for more physiological assay conditions; labeling or secondary assays aren’t required.
- Real-Time: Quantitative monitoring of both fast (hours) and slow (days) killing kinetics.
- Sensitive: Capable of evaluating low effector cell to target cell ratios that are physiologically relevant.
- Simple Workflow: Requires only the addition of effector cells to target cells (in the presence or absence of antibodies); homogeneous assay without additional sample handling.
- Automatic Data Plotting: RTCA software enables facile data display and objective analysis, precluding the subjective data vetting that is common to imaging-based assays.