Organoids, sophisticated mini-organs with distinct similarities to human tissues and organs, are increasingly being used to model disease and generate therapeutics [1]. Despite their advantages, it can be difficult to gain an accurate and complete representation of their biological profile using previously-developed imaging methods. In particular, live organoid imaging has been used, but it cannot allow for multiplexing of molecular biomarkers [2]. Meanwhile, fixed organoid imaging, though capable of multiplexing [3], prevents longitudinal monitoring. We sought to overcome these challenges by incorporating the tetrazine/trans-cyclooctene (Tz/TCO) bioorthogonal click chemistry reaction into immunostaining to allow for the detection and quenching of multiple fluorescent signals in a longitudinal manner. This technology was successfully characterized on live and fixed patient-derived glioblastoma organoids before multiplexed, cyclic imaging was used to create a nuanced glioblastoma proteomic profile.

Intracellular protein imaging in fixed organoids and surface protein imaging in live organoids were completed at multiple timepoints to evaluate the time dependency of this staining/quenching method. For fixed organoids, 24 h was recommended for complete staining, while 2 mins was recommended for complete quenching. For the live organoids, there was minimal difference in staining between the timepoints tested. A longer amount of time, 7 mins, was recommended to ensure complete quenching in these organoids.

The potential of this technology to monitor disease was validated by the cyclic imaging of multiple glioblastoma protein markers in fixed and live patient-derived glioblastoma organoids. In fixed organoids, CD9 and CD81 had the highest expression. Both of these surface proteins are common to multiple cancers but may have particular relevance in glioblastoma [4]. Documented glioblastoma-associated surface proteins (CD44, CD133, and EGFR) showed a lower expression than CD9 and CD81 but a higher expression than HER2, another common cancer biomarker. The intracellular protein biomarkers all had observable expression when targeted. Biomarkers in the live organoids had similar expression as in the fixed organoids, including elevated expression of CD9 and CD81.

In both fixed and live organoids, CD9 and CD81 were highly expressed, elucidating their further study as glioblastoma biomarkers. Since this system is restricted to staining and quenching protein biomarkers, technologies should be developed for multiplexed and longitudinal analysis of other molecular biomarkers, including nucleic acids. Given the ability of this method to profile proteomic changes in organoids over time, it may help determine the diagnosis and prognosis of many conditions. Additionally, it holds promise in aiding in the development of personalized therapies tailored to individual patients.

[1] Tang, XY. et al. (2022). “Human organoids in basic research and clinical applications.” Signal Transduction and Targeted Therapy 7(168).
[2] Rios, A. And Clevers, H. (2018). "Imaging organoids: a bright future ahead.” Nature Methods 15(1): 24—26 .  
[3] Saglam-Metiner, P. et al. (2023). “Spatio-temporal dynamics enhance cellular diversity, neuronal function and further maturation of human cerebral organoids.” Communications Biology 6(173).
[4] Jennrich, S. et al. (2022). “CD9- and CD81-positive extracellular vesicles provide a marker to monitor glioblastoma cell response to photon-based and proton-based radiotherapy.” Frontiers in Oncology 12.