Patients and samples

In total, 18 specimens were obtained from patients (female, age range from 31 to 77 years) with radiation enteritis (RE) who had received radiation therapy with a total dose of 50–98 Gy for cervical cancer, were diagnosed with RE on the basis of pelvic radiotherapy history radiological and colonoscopic features as described previously [26], and underwent surgical resection of the affected segment of intestine. Then, seven paired RE specimens were obtained from resected RE lesions and paired adjacent non-injured intestinal tissues. Specimens were snap-frozen or formalin-fixed for further use immediately after acquisition.

Animal study

All animals were raised in specific pathogen-free conditions and maintained in a controlled temperature environment with a 12 h light/dark cycle. The radiation-induced intestinal injury mouse model was established using female C57BL/6J mice aged 8–10 weeks purchased from GemPharmatech Co., Ltd. (Nanjing, China). After anesthesia, mice received abdominal irradiation with a single dose of 10.5 Gy containing the gastrointestinal tract using a RS2000 X-ray Irradiator (Rad Source Technologies, Inc., Boca Raton, FL, USA), and the rest of the body including the thorax, head and neck, and extremities were shielded. Mice in the normal control group only received sham irradiation after anesthesia. Mice were euthanized 4 weeks after irradiation and small intestinal tissues were removed for histological examination.

For AAV treatment experiment, the mice were intraperitoneally injected with AAV-Wnt4 (AAV-CMV-Wnt4-FLAG-GdGreen, OBiO Technology Co., Ltd, Shanghai, China) or AAV-vector (AAV-CMV-GdGreen, OBiO Technology Co., Ltd, Shanghai, China) at a dose of 2 × 1011 vg/mouse on the next day after 10.5 Gy of abdominal irradiation. At 4 weeks after irradiation, the mice were sacrificed and the small intestine tissues were collected for further examination.

We defined symmetric and asymmetric crypt fission in mouse small intestinal tissue with reference to previous report [27]. Symmetric crypt fission: bifurcated crypt showing two daughter crypts of the same length, diameter, and shape connected by a single crypt lumen. Asymmetric crypt fission: branched crypt showing two or more daughter crypts with varying diameters, lengths, and shapes connected by a single crypt lumen.

Mouse crypt isolation and organoid culture

Mouse crypt isolation and organoid culture were performed as described previously [28]. Briefly, mouse small intestine was removed and opened longitudinally. After gently scraping the surface feces and mucus, the intestinal tissue was cut into 2-mm pieces and washed with cold phosphate buffered saline (PBS). Tissues were incubated in 5 mM ethylenediaminetetraacetic acid (EDTA) at 4 °C for 30 min. After incubation, the supernatant was removed, and the tissue pieces were resuspended in ice-cold D-PBS and vigorously triturated using a 25-ml pipette. The separated crypts in the supernatant were filtered through a 70-μm filter into a new tube [precoated with 1% bovine serum albumin (BSA)] and then centrifuged at 500 g for 5 min. The pellet crypts were then resuspended in Matrigel (BD Biosciences, San Jose, CA, USA) and transferred to 24-well cell culture plate. Mouse IntestiCult™ Organoid Growth Medium (Stemcell Technologies) was added after the Matrigel was polymerized and changed every 2 days.

Lentivirus transfection

For the preparation of lentiviral particles,Wnt4-overexpressing plasmid (pEZ-Lv242-Wnt4, GeneCopoeia, Maryland, USA) or the control plasmid (pEZ-Lv242-vector, GeneCopoeia, Maryland, USA) were transfected in HEK 293FT cells together with lentiviral packaging vectors using PEI transfection solution. Supernatants containing virus were collected 48 and 72 h after transfection and centrifuged at 50,000 g for 90 min. The viral pellets were then resuspended in 500 μl of organoid culture medium supplemented with 10 μg/ml polybrene.

For lentiviral transduction of organoids, organoids were harvested by mechanically disrupting the Matrigel, and then digested with trypsin for 3 min at 37 °C. After removal of trypsin by centrifugation, the organoids were mixed with the virus solution and centrifuged on 32 °C at 600 g for 1 h in a 48-well plate to enhance transduction efficacy. The organoid-virus mixture was incubated in a culture incubator for 1 h at 37 °C, and then resuspended with 500 μl of organoid culture medium and centrifuged for 5 min at 500 g to pellet organoids. The organoid pellet was then embedded in 50 μl of ice-cold matrigel and cultured in 0.5 ml of mouse IntestiCult™ Organoid Growth Medium.

siRNA transfection

siRNA transfection of organoids was performed according to previous study [29]. The siRNA transfection solution was prepared by mixing Wnt4 siRNA (RiboBio Co. Ltd, Guangzhou, China) or control siRNA (RiboBio Co. Ltd, Guangzhou, China) with RNAiMAX (Invitrogen) in DMEM (Gibco) containing 10% FBS (Gibco), and was added directly to organoids after 2 days of culture in a 24-well plate. The medium was replaced with fresh organoid culture medium 48 h after transfection.

Histology, immunohistochemistry (IHC), and immunofluorescence (IF)

Human and mouse intestinal tissues were fixed with 4% paraformaldehyde and paraffin embed. Sections were stained with hematoxylin and eosin (HE).

IHC staining was carried out using SP-9000 SPlink Detection Kit (ZSGB-Bio, Beijing, PR China) following the manufacturer’s protocol. The following primary antibodies were used: anti-WNT4 (1:100, 14371-1-AP, Proteintech group), anti-Ki67 (1:300, ab16667, Abcam) and anti-lysozyme (1:300, ab108508-40, Abcam) antibodies.

For IF staining of tissue sections, permeabilization was performed with PBS containing 0.1% Triton X-100. After blocking with 1% BSA, the sections were incubated with primary antibodies at 4 °C overnight, washed and subsequently incubated with fluorescence-conjugated secondary antibodies Alexa Fluor 488 (1:1000, A-32723, Invitrogen) or Alexa Fluor 594 (1:1000, A-11037, Invitrogen) at room temperature for 1 h. Nuclei were counterstained with DAPI (Beyotime, Beijing, China). Confocal laser scanning was performed using a Leica TCS-SP8 confocal microscope (Mannheim, Baden-Wuerttemberg, Germany). The primary antibodies utilized in this study were anti-EphB3 (1:300, AF432-SP, R&D systems), anti-lysozyme (1:300, ab108508-40, Abcam), anti-WNT4 (1:100, 14371-1-AP, Proteintech group), anti-Flag (1:200, F1804, Sigma), and anti-ROR2 (1:8, Ror2, DSHB) antibodies.

For IF staining of organoids, organoids isolated from Matrigel were fixed with 4% paraformaldehyde at 37 °C for 45 min, permeabilized using 1% Triton-X100 for 1 h, and then incubated with blocking buffer (3% normal goat serum, 1% BSA and 0.2% Triton-X100 in PBS) for 1 h at room temperature. The following primary antibodies were used to incubate overnight with the organoids in Working Buffer (0.3% normal goat serum, 0.1% BSA, 0.2% Triton-X100 in PBS): anti-lysozyme (1:200, ab108508-40, Abcam), anti-Ki67 (1:200, ab16667, Abcam), anti-WNT4 (1:50, 14371-1-AP, Proteintech group), and anti-ROR2 (1:4, Ror2, DSHB) antibodies. After washing, AlexaFluor-conjugated secondary antibodies (1:1000, Invitrogen) along with Phalloidin (1:100, PF00003, Proteintech group) were applied overnight in Working Buffer. Nuclei were counterstained with DAPI (1:5000, Beyotime, Beijing, China) or Hoechst 33342 (1:5000, Beyotime, Beijing, China). After mounting with ProLong Gold antifade (Meilunbio), images were taken with Lecia TCS-SP8 or STELLARIS 8 FALCON confocal microscopes (Mannheim, Baden-Wuerttemberg, Germany).

Time series of live imaging of organoid culture

Live imaging of organoids was captured using Operetta CLS HCS (PerkinElmer, USA). Imaging processing was conducted using the Harmony 4.8 software (PerkinElmer, USA).

3D image processing and analysis

Organoids were imaged with a Lecia STELLARIS 8 FALCON confocal microscope (Mannheim, Baden-Wuerttemberg, Germany) using 40× objective lens, and Z stacks were taken at 1 μm steps. Measurements of crypt length and Paneth cell distance were based on 3D projections of organoids using ImageJ software (National Institutes of Health, Bethesda, MD, USA). Symmetric fission in organoids was assessed according to previous report [12]. That is, a ratio of the length of the shorter to the length of the longer daughter crypt greater than 0.75 was considered a symmetric fission. The localization of Paneth cells was determined on the basis of staining for lysozyme, and the distance of each Paneth cell from the crypt base was measured to analyze their distribution.

RT-qPCR

Total RNA in tissues or organoids was isolated using Trizol reagent (Invitrogen, Carlsbad, CA). Reverse transcription was carried out using ReverTra Ace Master Mix (TOYOBO, Osaka, Japan). Real-time quantitative polymerase chain reaction (RT-qPCR) was conducted with FastStart Essential DNA Green Master (Roche, Basel, Switzerland) using RT-PCR systems (Applied LightCycler96 Roche). GAPDH or ACTB served as the internal control. The primer sequences applied in this study are listed in Additional file 1, Supplementary Table 1.

Immunoprecipitation (IP)

IP assay was performed according to our previous study [28]. Briefly, whole small intestinal tissues from normal C57BL/6J mice (8–10 weeks, female) were lysed using lysis buffer (150 mM NaCl, 10 mM HEPES, pH 7.4, 1% NP-40). Lysates were precleaned with 25 μl of protein G affinity gel (E3403, Sigma, St Louis, MO, USA) at 4 °C for 1 h. The supernatant was immunoprecipitated with 1 μg of anti-ROR2 antibody (Ror2, DSHB) or mouse immunoglobulin G (IgG) isotype control (MAB002, R&D systems) at 4 °C overnight, and then incubated with 30 μl of protein G gel at 4 °C for 1 h. The immunoprecipitated complex was washed with wash buffer (150 mM NaCl, 10 mM HEPES, pH 7.4, 0.1% NP-40) six times, and eluted with 2× sample buffer, and then subjected to western blot analysis.

In situ hybridization (ISH)

The probe for Wnt4 was designed and synthesized by Servicebio (Wuhan, China). The probe sequences were as follows: 5′-TGAGTTTCTCGCACGTCTCCTCTT-3′, 5′-TCCACAAAGGACTGTGAGAAGGCTAC-3′, 5′-GGCTTTAGATGTCTTGTTGCACGTGC-3′.

Small intestines from mice at 4 weeks post-irradiation (10.5 Gy) and the normal control mice were fixed in formalin, embedded in paraffin and sliced into 4 μm sections. After being dewaxed and rehydrated, the sections were repaired in a citric acid antigen retrieval solution (pH 6.0) at 90 °C for 48 min and digested in proteinase K (20 μg/ml) at 40 °C for 10 min. After blocking endogenous peroxidase with 3% methanol –H2O2, sections were prehybridized at 37 °C for 1 h and hybridized in a solution containing the probes overnight at 40 °C. Sections were washed in 2 × SSC for 10 min at 37 °C, in 1 × SSC at 37 °C for 2 × 5 min and in 0.5 × SSC at room temperature for 10 min. Sections were then blocked in normal rabbit serum at room temperature for 30 min and incubated with anti-DIG-HRP at 37 °C for 50 min. After washing for several times with PBS, color development was performed with DAB and nuclei were counterstained with hematoxylin. Signal intensities in the crypt and villus regions were quantified separately with four random 200× magnification fields per slide using ImageJ software.

Statistical analysis

Data analyses were performed using GraphPad Prism 8 (GraphPad, San Diego, CA, USA). Unpaired or paired two-sided Student’s t-tests were used to calculate differences between two groups. one-way or two-way analysis of variance (ANOVA) with Tukey’s multiple comparisons test were applied to compare multiple groups. Pearson correlation coefficient test was used to analyze the relationship between variables. Bars represent mean ± SD. P < 0.05 was considered significant. All experiments were carried out at least three times.