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Abstract
BackgroundRadiation-induced rectal epithelial damage is a very common side effect of pelvic radiotherapy and often compromise the life quality and treatment outcome in patients with pelvic malignancies. Unlike small bowel and colon, effect of radiation in rectal stem cells has not been explored extensively. Here we demonstrate that Lgr5-positive rectal stem cells are radiosensitive and organoid-based transplantation of rectal stem cells mitigates radiation damage in rectum.MethodsC57Bl6 male mice (JAX) at 24 h were exposed to pelvic irradiation (PIR) to determine the radiation effect in pelvic epithelium. Effect of PIR on Lgr5-positive rectal stem cells (RSCs) was determined in Lgr5-EGFP-Cre-ERT2 mice exposed to PIR. Effect of PIR or clinically relevant fractionated PIR on regenerative response of Lgr5-positive RSCs was examined by lineage tracing assay using Lgr5-eGFP-IRES-CreERT2; Rosa26-CAG-tdTomato mice with tamoxifen administration to activate Cre recombinase and thereby marking the ISC and their respective progeny. Ex vivo three-dimensional organoid cultures were developed from Lgr5-EGFP-Cre-ERT2 mice. Organoid growth was determined by quantifying the budding crypt/total crypt ratio. Organoids from Lgr5-EGFP-ires-CreERT2-TdT mice were transplanted in C57Bl6 male mice exposed to PIR. Engraftment and repopulation of Lgr5-positive RSCs were determined after tamoxifen administration to activate Cre recombinase in recipient mice. Statistical analysis was performed using Log-rank (Mantel-Cox) test and paired two-tail t test.ResultExposure to pelvic irradiation significantly damaged rectal epithelium with the loss of Lgr5+ve rectal stem cells. Radiosensitivity of rectal epithelium was also observed with exposure to clinically relevant fractionated pelvic irradiation. Regenerative capacity of Lgr5+ve rectal stem cells was compromised in response to fractionated pelvic irradiation. Ex vivo organoid study demonstrated that Lgr5+ve rectal stem cells are sensitive to both single and fractionated radiation. Organoid-based transplantation of Lgr5+ve rectal stem cells promotes repair and regeneration of rectal epithelium.ConclusionLgr5-positive rectal stem cells are radiosensitive and contribute to radiation-induced rectal epithelial toxicity. Transplantation of Lgr5-positive rectal stem cells mitigates radiation-induced rectal injury and promotes repair and regeneration process in rectum.
Highlights
Rectal injury is a major limiting factor for definitive chemo-radiation therapy of pelvic malignancies, such as prostate cancer, bladder cancer, or ovarian cancer, collectively 20–30% of all malignancies [1, 2]
Rectal stem cells in organoid is sensitive to irradiation To determine the radiosensitivity of RSCs, we have developed organoids from Lgr5-eGFP-IRES-CreERT2 mice and exposed to irradiation
Microscopic image with × 10 and × 40 magnification demonstrated loss of budding crypt in irradiated organoids. c Survival assay (ATP uptake assay) demonstrated significant reduction of organoid survival in a dose-dependent manner. d Confocal microscopic images of organoids developed from Lgr5-EGFP-CRE-ERT2; R26- ACTB-tdTomato-EGFP mice demonstrated loss of Lgr5+ve cells (Green) in budding crypt from irradiated organoids compared to un-irradiated control. tdTomato is constitutively expressed in these mice as membranebound protein allows better visualization of cellular morphology. e Histogram demonstrating significant decrease in Lgr5 +ve cells in irradiated rectal organoids compared to un-irradiated control (p < 0.0004)
Summary
Rectal injury is a major limiting factor for definitive chemo-radiation therapy of pelvic malignancies, such as prostate cancer, bladder cancer, or ovarian cancer, collectively 20–30% of all malignancies [1, 2]. Radiation proctitis is a common and debilitating consequence of radiation therapy-induced damage to the rectal tissue characterized by acute mucosal loss, inflammation, followed by progressive tissue scarring leading to organ fibrosis. Previous research on radiation-induced toxicity in small bowel demonstrated that radiation-induced loss of intestinal stem cell is the major cause of mucosal damage [4,5,6]. Unlike radiationinduced small bowel toxicity majority of the reports on radiation-induced rectal injury described radiation-induced proctitis/fibrosis [7, 8]. Radiation-induced rectal epithelial damage is a very common side effect of pelvic radiotherapy and often compromise the life quality and treatment outcome in patients with pelvic malignancies. Unlike small bowel and colon, effect of radiation in rectal stem cells has not been explored extensively. We demonstrate that Lgr5-positive rectal stem cells are radiosensitive and organoid-based transplantation of rectal stem cells mitigates radiation damage in rectum
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