Quantification of Cytokine-Induced Cell Death in Human Colonic Organoids Using Live Fluorescence Microscopy

The inflammatory cytokines interferon-gamma and TNF-have existed for hundreds of millions of years and are critical for effective host defense against intercellular pathogens. Our research group is trying to understand how these cytokines work to kill cells, such as epithelial cells in the gut and cancer cells. Commonly used systems to study intestinal epithelial cell death mechanisms include mouse models and immortalized cancer cell lines.

Human test organoids are advantageous as they're generated directly from patient biopsies and retain many physiological and morphological characteristics of the parent tissue. This means they have increased translational value. Organ research has issues with experiments reproducibility, and has several technical challenges compared to traditional cell culture.

The current techniques for measuring organoid cell death also have limitations. Some are only semi-quantitative, do not measure cell death directly, cannot measure single organoid responses, or require expensive equipment and complex protocols. Our protocol for quantitative analysis of organoid cell death is straightforward, robust, and inexpensive.

We used our protocol to measure single organoid responses to cytotoxic cytokine combinations, but it can be easily adapted to study any type of perturbagen. This method is useful for research on cell death, epithelial barrier function, or mucosal immunology. Recently we have reported that interferon-gamma and TNF-synergize to induce inflammatory cell death in gut epithelial cells and colon cancer cells.

They do this via the JAK1/2-STAT1 signaling pathway. In future work, we wish to understand what type of cell death this is and how JAK1 and JAK2 kill the cells.