Establishment of a gastrointestinal epithelial organoid assay to assess bowel toxicity in response to chemoradiation treatment

Introduction
Chemoradiation is the most common non-surgical treatment for MIBC. However, chemoradiation is associated with acute and late intestinal toxicity, as irradiation (IR) of the surrounding small bowel and rectum is virtually inevitable. In the search for new, less toxic radiosensitisers, it is important to have an assay to determine normal intestinal toxicity. To meet this need, we have investigated whether gastrointestinal toxicity can be predicted ex vivo through the use of organoids. Particularly, we determined a range of doses that are safe to the gastrointestinal tract in order to study bowel toxicity to chemoradiation. The ultimate aim is to develop an assay that determines organoid response to irradiation/radiosensitiser combinations, which can recapitulate the in vivo normal tissue toxicity assay from animals.
Methodology
Crypts were isolated from the small intestine of mice and cultured in organoid medium containing growth factors. For subculture, organoids cultured on day 7 were passaged. Undissociated organoids were cryopreserved by resuspension in freezing medium. One month after cryopreservation, organoids were thawed with or without Rho kinase inhibitor and subjected to different seeding conditions. The protocol for passaging was modified to achieve optimal organoid formation efficiency. Efficiency of organoid fragment seeding was quantified based on variable Matrigel concentrations and densities of organoid fragment seeding. The assay was tested by irradiating organoids at doses between 0.5 Gy to 6 Gy and treating organoids with variable concentrations of radiosensitisers gemcitabine and panobinostat. Irradiation and drugs were combined to assess whether the organoid response to chemoradiotherapy treatment recapitulated in vivo results.
Results
Organoid cryopreservation techniques were refined such that a collection of murine intestinal organoids has been established (n=32). Organoids were successfully cryopreserved and had good freeze-thaw efficiency with 86% survival. Recovery and subsequent growth of cryopreserved organoids was significantly improved by adding Rho-kinase inhibitor to the freezing medium and after thawing procedures. During assay development, seeding 50 organoid fragments per well in a 24-well plate was found to result in the optimum organoid density that allowed for adequate growth and imaging. More favourable organoid formation efficiencies were seen using Matrigel concentrations 1.5 times that of the organoid fragment-suspension medium volume, compared to a one-to-one ratio of medium and Matrigel (p=0.03).
When testing the assay, organoid growth and budding was not affected by radiation doses lower than 2 Gy. For organoids that received 2 Gy radiation, budding was halted and there was premature disaggregation (10-15%), where epithelial cells had shed into the lumen, by day 3 post-irradiation. Administration of doses higher than 2 Gy resulted in significant disaggregation (>80%) as soon as 24 hours after irradiation.
For organoids exposed to gemcitabine and panobinostat, significant disaggregation began at doses of 25 nM and 50 nM respectively. Decreased organoid growth was seen when combining the drugs with radiation that was indicated by a decrease in organoid size.
Conclusion
The development of this assay could present a promising modality for assessing the outcome of chemoradiation combinations on the small intestine. This assay is being adapted for use in patientderived intestinal organoids.

Advisor: Kiltie, A