Live Organoid Cyclic Imaging

Introduction:: Organoids are becoming increasingly relevant in biology and medicine for their physiological complexity and accuracy in modeling human disease. To fully assess their biological profile while preserving their spatial information, spatiotemporal imaging tools are warranted. While previously-developed imaging techniques such as 4D live imaging and light-sheet imaging have yielded important clinical insights, these technologies lack the combination of multiplexed and longitudinal analysis. To address these challenges, we implemented a click chemistry-based ultra-fast cyclic imaging method for the multiplex imaging and longitudinal monitoring of living organoids. Our technology exploits bioorthogonal click chemistry to quench fluorescent signals from the surface and intracellular of labeled cells across multiple cycles, allowing for longitudinal profiling of many markers.


Materials and Methods:: The technology is based on the idea of rapidly and effectively quenching antibody-conjugated fluorescent dyes of varying wavelengths without obstructing native biochemical processes. More specifically, the technology take advantage of the tetrazine/trans-cyclooctene (Tz/TCO) reaction's remarkable ultrafast kinetics and bioorthogonal nature. This process is achieved using a modular linker that connects the fluorochromes and antibodies with an embedded TCO molecule (antibody-TCO-fluorophores) for clicking with Tz conjugated to a quencher. Based on our previous work, the appropriate quencher that was chosen for our application was the black hole quencher (BHQ3). Tz-BHQ3 has been reported to accelerate the interaction between the Tz-TCO click reaction, achieving immediate (seconds) and highly efficient ( >95%) quenching of the dye's fluorescence. This prevents time-consuming cleavage or bleaching steps and permits rapid cellular analysis. To validate this click chemistry-based ultra-fast cyclic imaging method on organoids, we chose patient-derived glioblastoma organoids as our model system.




Results, Conclusions, and Discussions:: We applied our bioorthogonal click-based chemistry technology to profile organoids, allowing for the analysis of multiple proteins and longitudinal monitoring over an extended timeframe. More specifically, we screened for specific and nonspecific glioblastoma markers in human brain organoids while monitoring their viability. Through this multiplex analysis, we are able to observe the relative co-localization and the relative abundance of these glioblastoma markers. To demonstrate the combination of multiplexing and longitudinal monitoring in the human brain organoids, we investigate the effectiveness of chimeric antigen receptor (CAR)-T cell based immunotherapy in live patient-derived glioblastoma organoid models over the course of 5-days. Overall, we anticipate that the findings and applications of this work can be broadly translated into investigating physiological developments in other organoid systems.


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