Interleukin-33 Potentiates TGF-ß Signaling to Regulate Intestinal Stem Cell Regeneration After Radiation Injury.

Epithelial regeneration is critical for barrier maintenance and organ function after intestinal radiation injury. Accumulating evidence indicates that the interleukin family members play critical roles in intestinal stem-cell-mediated epithelial regeneration. However, little is known about the relationship between interleukin 33 (IL-33)/ST2 axis and intestinal regeneration after radiation injury. We demonstrate here that IL-33 expression significantly increased after radiation treatment. Deficiency of IL-33/ST2 promotes intestinal epithelial regeneration, resulting in a reduction of mortality during radiation-induced intestine injury. Using ex vivo organoid cultures, we show that recombinant IL-33 promotes intestinal stem cell differentiation. Mechanistically, the effects of IL-33 were mediated by activation of transforming growth factor-ß signaling. Our findings reveal a fundamental mechanism by which IL-33 is able to regulate the intestinal crypt regeneration after tissue damage.

SIRT1 inhibitors mitigate radiation-induced GI syndrome by enhancing intestinal-stem-cell survival.

High-dose radiation exposure induces gastrointestinal (GI) stem cell death, resulting in denudation of the intestinal mucosa and lethality from GI syndrome, for which there is currently no effective therapy. Studying an intestinal organoid-based functional model, we found that Sirtuin1(SIRT1) inhibition through genetic knockout or pharmacologic inhibition significantly improved mouse and human intestinal organoid survival after irradiation. Remarkably, mice administered with two doseages of SIRT1 inhibitors at 24 and 96 h after lethal irradiation promoted Lgr5+ intestinal stem cell and crypt recovery, with improved mouse survival (88.89% of mice in the treated group vs. 0% of mice in the control group). Moreover, our data revealed that SIRT1 inhibition increased p53 acetylation, resulting in the stabilization of p53 and likely contributing to the survival of intestinal epithelial cells post-radiation. These results demonstrate that SIRT1 inhibitors are effective clinical countermeasures to mitigate GI toxicity from potentially lethal radiation exposure.

Bach2 Deficiency Promotes Intestinal Epithelial Regeneration by Accelerating DNA Repair in Intestinal Stem Cells.

Epithelial regeneration is critical for barrier maintenance and organ function after intestinal injury, although the repair mechanisms are unclear. Here, we found that Bach2 deficiency promotes intestinal epithelial cell proliferation during homeostasis. Moreover, genetic inactivation of Bach2 in mouse intestinal epithelium facilitated crypt regeneration after irradiation, resulting in a reduction in mortality. RNA-sequencing analysis of isolated crypts revealed that Bach2 deficiency altered the expression of numerous genes, including those regulating double-strand break repair. Mechanistic characterizations indicated that Bach2 deletion facilitated DNA repair in intestinal crypt cells, as evidenced by faster resolution of γ-H2AX and 53BP1 foci in Bach2-/- crypt cells, compared with Bach2+/+ control. Together, our studies highlight that Bach2 deficiency promotes intestinal regeneration by accelerating DNA repair in intestinal stem cells after radiation damage.

Organoid modelling identifies that DACH1 functions as a tumour promoter in colorectal cancer by modulating BMP signalling.

BACKGROUND: Dachshund homologue 1 (DACH1) is highly expressed in LGR5+ intestinal stem cells and colorectal tumours. However, the roles of DACH1 in intestinal cell stemness and colorectal tumorigenesis remain largely undefined. METHODS: We used immunohistochemistry, western blotting and quantitative real-time PCR to analyse DACH1 expression in colorectal cancer (CRC) samples. CRISPR/Cas9 gene editing and lentiviral vector-mediated overexpression and shRNA-mediated knockdown of DACH1 were utilized to modulate DACH1 expression in cell lines and organoids. An intestinal organoid-based functional model was analysed, and cancer cell colony formation, sphere formation assays and murine xenotransplants were performed to reveal the role of DACH1 in CRC cell proliferation, stemness and tumorigenesis. Immunofluorescence, co-immunoprecipitation, RNA interference and microarray data analyses were conducted to demonstrate the association between DACH1 and the bone morphogenetic protein (BMP) signalling pathway. FINDINGS: DACH1 is specifically expressed in discrete crypt base cells, and increased DACH1 expression was found in all stages of CRC. Moreover, the high expression of DACH1 independently predicted poor prognosis. In colon cancer cells, shRNA-mediated suppression of DACH1 inhibited cell growth in vitro and in vivo. By studying the intestinal organoid-based functional model, we found that depletion of DACH1 reduced the organoid formation efficiency and tumour organoid size. DACH1 overexpression stimulated both colonsphere formation and tumour organoid formation in the context of dysregulated BMP signalling. Mechanistic characterizations indicated that overexpression of DACH1 affects a subset of stem cell signature genes implicated in stem cell proliferation and maintenance through the suppression of BMP signalling via SMAD4. INTERPRETATION: Together, our study highlights DACH1 as an integral regulator of BMP signalling during intestinal tumorigenesis, and DACH1 could be a potential prognostic marker and therapeutic target for colorectal cancer patients.

Patient-Derived Organoids Predict Chemoradiation Responses of Locally Advanced Rectal Cancer.

Accumulating evidence indicates that patient-derived organoids (PDOs) can predict drug responses in the clinic, but the ability of PDOs to predict responses to chemoradiation in cancer patients remains an open question. Here we generate a living organoid biobank from patients with locally advanced rectal cancer (LARC) treated with neoadjuvant chemoradiation (NACR) enrolled in a phase III clinical trial. Our co-clinical trial data confirm that rectal cancer organoids (RCOs) closely recapitulate the pathophysiology and genetic changes of corresponding tumors. Chemoradiation responses in patients are highly matched to RCO responses, with 84.43% accuracy, 78.01% sensitivity, and 91.97% specificity. These data imply that PDOs predict LARC patient responses in the clinic and may represent a companion diagnostic tool in rectal cancer treatment.

Inhibition of SIRT1 promotes taste bud stem cell survival and mitigates radiation-induced oral mucositis in mice.

Taste loss is one of the debilitating complications in radiation-induced oral mucositis (RIOM), as occurs in head and neck cancer patients undergoing radiotherapy. We report here a radio-mitigation effect of Sirtuin 1 (SIRT1) inhibitors in taste bud organoids and a mouse model of radiation-induced taste bud injury. The organoids, developed from circumvallate (CV) papilla, were irradiated with single dose of X-rays and inhibitors of SIRT1 or SIRT2 were added 24 h later. The survival was evaluated by measuring the number and size of regenerated organoids after irradiation (IR). Oral mucositis (OM) was induced by IR of the oral region of Lgr5-lacZ transgenic mice. The surviving Lgr5+ taste bud stem cells were identified after lacZ-staining and the mucosal ulceration on tongue dorsal surface was determined by histological methods. Results showed that SIRT1 inhibitors (nicotinamide, EX527, salermide and sirtinol), but not SIRT2 inhibitors, significantly improve taste bud organoid survival after IR. Remarkably, administration of nicotinamide (NAM), a recognized inhibitor of SIRT1 to mice 24 h after IR promotes the survival of Lgr5+ taste bud stem cells, resulting in alleviated tongue mucositis. In conclusion, SIRT1 inhibitors promote Lgr5+ taste bud stem cell survival and mitigate RIOM in mice. These observations have important implications for efforts to develop therapeutic strategies against taste dysfunction and mucosal ulceration in RIOM.

Activated B Lymphocyte Inhibited the Osteoblastogenesis of Bone Mesenchymal Stem Cells by Notch Signaling.

Estrogen is very important to the differentiation of B lymphocytes; B lymphopoiesis induced by OVX was supposedly involved in osteoporosis. But the effects of B lymphocytes on the osteogenic differentiation of bone mesenchymal stem cells (BMSCs) are not clear. In this study, we detected bone quality and bone loss in a trabecular bone by electronic universal material testing machine and microcomputed tomography (micro-CT) in OVX and splenectomized-ovariectomy (SPX-OVX) rats. Additionally, changes in lymphocytes (B lymphocyte, CD4+ and CD8+ T lymphocytes, and macrophages) in the bone marrow were analyzed by flow cytometry. The osteogenesis of BMSCs cocultured with normal and LPS-pretreated B lymphocytes was detected by BCIP/NBT and Alizarin red S staining. Measurement of the Notch2, Notch4, Hey1, Hey2, Hes1, and runt-related transcription factor 2 (Runx2) expression in BMSCs cocultured with B lymphocytes was done using real-time PCR. The effects of dexamethasone and DAPT (inhibitor of Notch signaling) on osteogenesis of BMSCs were detected by BCIP/NBT, Alizarin red S staining, and real-time PCR. Osteoporosis happened in OVX rats, more serious in SPX-OVX rats, B lymphocytes increased in OVX rats, and sharply higher in SPX-OVX rats. Osteoporosis did not happen in SPX rats which is still companied with a high increase of B lymphocytes. LPS-pretreated B lymphocytes suppressed the osteogenesis of BMSCs, but the normal B lymphocytes could not. The LPS-pretreated B lymphocytes upregulated the expression of Notch4, Hes1, and Hey2 and downregulated the expression of Runx2 in BMSCs. Dexamethasone and DAPT could downregulate the high expression of Notch4, Hes1, Hey2 and upregulate the low expression of Runx2 in BMSCs which cocultured with LPS treated B lymphocytes, the inhibited ALP and Alizarin red staining in BMSCs which cocultured with LPS treated B lymphocytes also partly restored.