Fig. 4.

BCN057 mitigates radiation toxicity in intestinal organoids derived from mice and human tissue. a Microscopic images (phase contrast) of intestinal organoids along with b a histogram of budding crypt/total crypt ratio demonstrating that BCN057 treatment improves organoid growth compared with irradiated (IR) controls (2 Gy, *p < 0.006; 4 Gy, *p < 0.008; 6 Gy, *p < 0.003; 8 Gy, *p < 0.002). Images at 10× (indicated with arrow) and 40× demonstrated the presence of a budding crypt in irradiated organoids treated with BCN057. c Confocal microscopic images of organoids developed from Lgr5-EGFP-CRE-ERT2; R26- ACTB-tdTomato-EGFP mice demonstrated that BCN057 treatment increases the presence of Lgr5-positive cells (green) in budding crypts compared with irradiated controls. tdTomato is constitutively expressed in these mice as membrane-bound protein, and therefore allows better visualization of cellular morphology. d Microscopic image (phase contrast) of organoids developed from human non-malignant colon demonstrating the loss of crypt domain exposed to irradiation (8 Gy). Both bright field and hematoxylin and eosin (H&E) staining demonstrated complete loss of budding crypt at 72 h post-irradiation. However, BCN057 treatment at 1 h post-radiation rescued the organoids from radiation toxicity and accelerated crypt-villus budding. Note the presence of a budding crypt-like structure in the BCN057-treated group. Ki67 staining demonstrated positive staining in the budding crypt-like structure in the BCN057-treated organoids indicating cell proliferation in this group. However, irradiated untreated organoids did not show any Ki67-positive budding crypt-like structure. e Ratio of budding crypts to total number of crypts in human colonic organoids is increased with BCN057 treatment in irradiated organoids compared with untreated irradiated controls (2 Gy, *p < 0.009; 4 Gy, *p < 0.006; 6 Gy, *p < 0.001; 8 Gy, *p < 0.003; unpaired t test, two-tailed). f qPCR analysis demonstrated that BCN057 treatment increases the mRNA levels of Wnt target genes in epithelial cells isolated from irradiated human colonic organoids. Data are the average of three human subjects