Discussion

Cancer immunotherapy offers the potential for specific eradication of tumour cells with few side effects and the development of immune memory that can prevent cancer recurrence. There are several pathways and cell types involved in immunologic driven tumour cell death but of most interest are the cytotoxic T cell and B cell driven mechanisms. Research in this area has traditionally been performed using various end point assays to look at total cell number or health e.g. 51chromium release assays or flow cytometery. Using these techniques the contribution of target and effector cell types to the final readout is often unclear and there is limited information around the temporal nature of events. With the use of kinetic live cell imaging on the IncuCyte ZOOM these biological events can be imaged and quantified in real time to provide data covering the complete response.

Using a co-culture model of tumour and effector cells in the IncuCyte ZOOM it is possible to follow tumour cell death, post T cell activation in real time. Target tumour cells e.g. A549 were seeded overnight in flat bottom 96 or 384 well plates. PBMC’s or isolated CD8+ T cells were then added to the wells in combination with various activators. The response was tracked by capturing images every 2 hours using a 10x objective over the next 4-7 days. The assay can be configured either using phase contrast imaging in combination with an apoptosis marker, caspase 3/7 reagent (Essen BioScience) or tracking of target cell proliferation using nuclear red labelled tumour cell lines (NucLight cell lines, Essen BioScience). Using caspase 3/7 reagent, dying cells are identified as fluorescent green objects that can be masked and counted by the IncuCyte ZOOM. Dying and labelled effector cells can be removed from this identified population with the use of size and shape exclusion filters. By using NucLight red labelled stable tumour cell lines the count of red objects provides a clean readout for the target tumour cell population in the well. Used in combination with caspase 3/7 reagent cell health can also be captured from the well. Either image capture method produces comparable results; however use of NucLight Red cell lines produces precise tumour population data. Data will be shown using activated PBMC’s and isolated CD8+ T cells in both 96 and 384 well plates to demonstrate the activation of effector T cells; temporal difference in target cell death due to altered effector/target cell ratios; quantification of effector T cell activation by different agents and demonstration of later stage pathway events related to T cell proliferation. A similar approach has been used to study tumour spheroid cell death following T cell activation.

Kinetic live cell imaging of T cell driven tumour cell death on the IncuCyte ZOOM can provide a wealth of in-depth information relating to either target or effector cell populations in isolation without the need for cell manipulation prior to assay. The system enables the generation of high definition images, data analysis of images and time lapse videos to demonstrate the event in real time which can greatly enhance the understanding of the biology involved.