Immune-cell recognition and killing of unwanted target cells, such as emergent tumor cells, is a critical component of the human host defense mechanism. Antibody-dependent cell-mediated cytotoxicity (ADCC) and T cell killing are two mechanisms of cell-mediated immune response. Each of these processes involves the stimulation of immune cell sub-populations, such as natural killer (NK) cells or cytotoxic T lymphocytes (CTL), which then actively lyse target cells. Understanding the interplay between immune and cancer cells and restoring and promoting the immune system’s capacity to fight and eliminate tumors ("cancer immunotherapy" or "immuno-oncology") is an exciting and promising research field.
An integrated solution for real-time visualization and automated analysis of immune cell-mediated killing of tumor cells – all within your tissue culture incubator. Ideal for:
Time-lapse movies acquired using IncuCyte® live-cell analysis system showing a T cell sub population of PBMCs, activated with anti-CD3 antibody and IL-2, killing target SK-OV-3 tumor cells. Labeling of the SK-OV-3 cancer cells with the IncuCyte® NucLight Red Live-Cell Labeling reagent enables direct counting of viable tumor cell numbers over-time. Addition of the IncuCyte® Caspase 3/7 apoptosis reagent enables simultaneous detection of cells undergoing apoptosis (green fluorescence).
McCormack E, Adams KJ, Hassan NJ, Kotian A, Lissin NM, Sami M, et al. Bi-specific TCR-anti CD3 redirected T-cell targeting of NY-ESO-1- and LAGE-1-positive tumors. Cancer Immunol Immunother, 62(4):773–85, 2013
Adams, KJ et al. The use of induced pluripotent stem (iPS) cells for the safety testing of enhanced affinity TCR-transduced T cells. Cancer Research, AACR; April 5-9, San Diego, CA, 2014
“Redirected killing by ImmTACs can be missed in short-term killing assays such as 51Cr-release and even the extended LDH-release assay. The adaptation and use of the IncuCyte™ technology is therefore of critical importance for assessing ImmTAC potency and specificity under the strictest possible conditions”.
Adams, K. Redirected T cell activity by high affinity TCR-Anti-CD3 Bispecific candidate therapeutics. PhD Dissertation, Cardiff University (2013)
Visualize immune cell/tumor cell interplay using IncuCyte immune cell killing assays. (1) Physical contact between a small cytotoxic T cell and a larger labeled tumor cell (red). T lymphocyte division. (2) Tumor cells under attack from a cytotoxic T lymphocyte: The "kiss of death". (3) Tumor cell cytoplasmic granulation immediately followed by caspase 3/7 labeling (green), nuclear condensation and cell death. (4) Tumor cell mitosis: One cell becomes two.
Visualize and quantify immune cell interactions with tumor cells within a mixed culture using IncuCyte FabFluor-488. A549 CytoLight Red tumor cells were mixed with either pre-activated or non-activated PBMCs in the presence of IncuCyte FabFluor-488-α-CD45 and IncuCyte Opti-Green to label the total lymphocyte population. Images at 2 hours post PBMC addition, show interactions between CD45+ cells (green) and A549 cells (red). Quantification of the interaction (overlay, yellow mask in images) reveals a marked increase in the interaction of activated effector cells with the target cells indicating cell engagement for immune killing of tumor cells (as shown in the bar graphs). This analysis was performed using the Cell-By-Cell Analysis Software Module.
Measure tumor cell death and proliferation (optional) in real time using IncuCyte immune cell killing assays. User-friendly IncuCyte® software enables direct image-based detection of apoptotic tumor cells (green objects, outlined in yellow) and real-time counting of live tumor cells (IncuCyte® NucLight Red labeled nuclei, outlined in blue). Kinetic readouts reveal the time dependence of treatment effects.
Measure non-adherent tumor cell death and proliferation and immune cell proliferation using Cell-By-Cell Analysis in real time. Ramos NucLight Red cells were co-cultured with non-activated and pre-activated (CD3/IL-2) PBMCs in the presence of Annexin V Green Apoptosis Reagent. Using Non-Adherent Cell-By-Cell Analysis Software, images were acquired at 20X every 2 hours. Populations classified with red fluorescence to identify target and effector cell populations. Target cell proliferation, target cell apoptotic index, and effector cell proliferation were calculated and graphed over time for increasing target to effector cell ratios.
ADCC: Trastuzumab (Herceptin®) induced immune cell killing of SKOV-3 ovarian cancer cells in immune-cell killing spheroid model: HER2-positive SKOV-3 NucLight Red spheroids (2.5K/well) were seeded with PBMCs (6.25K/well) and treated with Herceptin (mAb targeting HER2 receptors). Herceptin induced inhibition of SKOV-3 spheroid growth.
ADCC: Trastuzumab (Herceptin) induced immune cell killing of SKOV-3 ovarian cancer cells. PBMCs were co-cultured with either SKOV-3 NucLight Red (HER2-positive) or A549 NucLight Red (HER2-negative) tumor cells. Time courses of trastuzumab-induced concentration-dependent inhibition of tumor cell proliferation in SKOV-3 (A) but not A549 (B) cells. Concentration-response curve to trastuzumab for inhibition of proliferation in SKOV-3 cells (C). Examples of unprocessed (top) and masked images (bottom) in the presence and absence of antibody (72h post addition, D).
Common methods used to assess immune cell killing of cancer cells are often end-point, require cell lifting (e.g., flow cytometry) or measure indirect read outs of tumor cell viability (e.g., LDH, GAPDH release assays) or immune cell activation (ELISpot). All are generally non-image based. The table below shows the capabilities and challenges of some common approaches.
|IncuCyte™ immune cell killing assays||Flow cytometry||51Cr release assay||GAPDH/LDH-release assay||DELFIA® TRF Assays||ELISpot||ADCC reporter bioassay (Promega)|
|Real-time cell visualization|
|Direct measurement of tumor cell death|
|Tumor cell viability measurements|
|Assess long term killing >24h|
|Flexible choice of target and effector cells|
|ADCC and T cell killing formats|
|Mix and read, no wash|
|Cells can be used for further analysis|
|No labeling antibodies|
|Product name||Product Data Sheet||Safety Data Sheet||Qty||Cat. No.|
|IncuCyte® Caspase-3/7 Green Apoptosis Assay Reagent||1||4440|
|IncuCyte® Caspase-3/7 Red Apoptosis Assay Reagent||1||4704|
|IncuCyte® NucLight Red Lentivirus (EF-1α, Puro)||1||4476|
|IncuCyte® NucLight Green Lentivirus (EF-1α, Puro)||1||4475|
|IncuCyte® NucLight Red Lentivirus (EF-1α, Bleo)||1||4478|
|IncuCyte® NucLight Green Lentivirus (EF-1α, Bleo)||1||4477|
|IncuCyte® CytoLight Rapid Green Reagent – for cell labeling||1||4705|
|IncuCyte® CytoLight Rapid Red Reagent – for cell labeling||1||4706|
|IncuCyte® Mouse IgG1 FabFluor-488 Antibody Labeling Reagent||1 vial (50 µg)||4745|
|IncuCyte® Mouse IgG2a FabFluor-488 Antibody Labeling Reagent||1 vial (50 µg)||4743|
|IncuCyte® Mouse IgG2b FabFluor-488 Antibody Labeling Reagent||1 vial (50 µg)||4744|