(J-399) Intestinal enteroids as a platform to study intestinal repair post-radiation

Conventional radiation (CONV-RT) is a common cancer therapy that ablates tumorous tissue but is therapeutically limited by its detrimental effects on healthy tissue. CONV-RT is especially destructive of tissues that are highly proliferative, like the small intestinal epithelium, which results in radiation-induced enteritis that greatly reduces patient quality of life (Hauer-Jensen et al., 2014). An alternative radiation therapy known as FLASH radiation (FLASH-RT) has been shown to be equally as effective at targeting tumors as CONV-RT, while maintaining the integrity of intestinal crypts in murine models (Levy et al., 2020). Currently, there are two clinical reports of successful use of FLASH-RT for cancer patients (Bourhis et al., 2019; Mascia et al., 2023). Though promising, the underlying mechanisms behind the FLASH effect in human tissue remains unknown. Here, we leveraged intestinal enteroids, three-dimensional cell cultures that contain native intestinal cell types and recapitulate intestinal architecture, to study the human-specific response to FLASH-RT. While our data suggest that there is not a significant difference in tissue damage between radiation groups, FLASH-irradiated enteroids showed improved tissue repair and had a distinct transcriptional response compared to CONV-RT. These findings add to a growing body of work highlighting the exciting clinical implication for FLASH-RT in the clinic.

Intestinal enteroids were isolated from healthy murine and patient intestinal duodenum to isolate the most radio-sensitive region of the intestines. Primary cells were then embedded into a three-dimensional basal lamina matrix to form enteroids that mimic the native intestinal crypt, which houses the intestinal progenitor cells that are highly sensitive to radiation. The intestinal enteroids were radiated using either CONV-RT or FLASH-RT with a no radiation negative control.

First, we analyzed irradiated enteroids to determine differences in tissue repair across CONV-RT and FLASH-RT. We measured the extent of enteroid growth over time using a colony formation assay to compare the regenerative potential of intestinal tissue between radiation groups. Immunocytochemistry was used to observe structural differences in the regenerated enteroids using intestinal polarity markers (zonal occludens-1 and B-catenin). We quantified differences in enteroid growth and morphology by comparing the CONV-RT and FLASH-RT groups to non-irradiated enteroids.

We then looked at differences in DNA damage and repair following FLASH-RT and CONV-RT. Specifically, we measured the gene expression and protein localization of a key regulator of DNA repair, -H2AX. We also conducted RNA-sequencing to identify differences in gene expression across radiation groups at two distinct timepoints (1 hr and 96 hrs). These timepoints were selected to capture enteroid expression during the acute damage and proliferative phases of intestinal repair.

FLASH-RT displayed greater colony growth potential compared with CONV-RT. The FLASH-irradiated human cells were capable of reforming enteroids to a similar size as the no radiation control. In contrast, CONV-irradiated enteroids were consistently smaller and were less likely to be properly polarized compared to the FLASH-iradiated and no radiation groups.

We did not observe differences in -H2AX expression across CONV-RT and FLASH-RT. However, our transcriptomic analysis revealed distinct gene expression profiles across groups. A Principal Component Analysis (PCA) of our RNA-sequencing data resulted in two distinct clusters, consistent with the two timepoints following radiation, 1 hr and 96 hrs. We also observed that FLASH-irradiated enteroids clustered more closely to the no radiation control within both timepoints, suggesting that the gene expression of FLASH-irradiated enteroids was more like non-irradiated enteroids than CONV-RT. We also performed Gene Set Enrichment and GOTerm analyses to determine specific pathways that were enriched following radiation. The FLASH-RT enteroids demonstrated upregulation of genes associated with the WNT-family, cell-cell adhesion, and hypoxia response.

Our study validates human enteroids as a tool to investigate FLASH-RT. The differences in tissue repair between CONV-RT and FLASH-RT are consistent with what has been observed in murine models, suggesting this enteroid model offers a useful tool for future studies. While there were no significant differences in tissue damage between CONV-RT and FLASH-RT, our transcriptomic analysis suggests that there are differences in tissue repair across groups. Taken together, this study validates the use of intestinal enteroids as a platform to investigate human-specific mechanisms driving intestinal epithelium repair in future studies. Our results provide further evidence supporting continued pre-clinical and future clinical study of FLASH-RT.  

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