Eight- to 12-week-old female C57BL/6 mice (Envigo, Harlan, The Netherlands) and female p16-3MR mice (kindly provided by Marco Demaria) were bred in the central animal facility of the University Medical Center Groningen. The mice were maintained under conventional conditions. Animal experimental procedures were approved by the Central Committee Animal Experimentation of the Dutch government and the Institute Animal Welfare Body at the University Medical Center Groningen.
Salivary glands were fixed with 4% formaldehyde and embedded into paraffin. Five micrometer paraffin sections were dewaxed and boiled for 8 min with pre-heated antigen retrieval buffer. Subsequently, sections were incubated with the following primary antibodies: mouse anti-γH2AX (Merck, 06-636, Darmstadt, Germany), mouse anti-BCL-2 (Dako, M0887, Glostrup, Denmark), mouse anti-p16 (CINtec® Histology Kit, 9517, Mannheim, Germany), or rabbit anti-Aquaporin5 antibody (Alomone labs, AQP-005, Jerusalem, Israel). Visualization for bright field microscopy was accomplished by adding specific secondary biotin carrying antibodies, biothynlated rabbit anti-mouse (Dako, E0413), or biothynlated Swine anti-rabbit (Dako, E0431) at 1:300 dilution. Nuclear counterstaining was performed with hematoxylin. The percentage of area positive for AQP5 staining in salivary gland tissue was quantified using ImageJ on five representative fields at ×10 magnification on three mice per group.
Isolation of mouse salivary gland cells and organoid culture
Murine submandibular salivary glands were dissected from 8- to 12-week-old female p16-3MR mice. Salivary gland cells were isolated and cultured to form organoids as described previously22. In short, salivary glands were mechanically and enzymatically dissociated and cultured in DMEM-12 (Gibco/Invitrogen, 11320-074, Bleiswijk, The Netherlands) medium consisting of 1% penicillin/streptomycin (Gibco, NY, USA), glutamax (2 mM; ThermoFischer Scientific, 35050038, Paisley, UK), EGF (20 ng/ml; Sigma-Aldrich, E9644, Zwijndrecht, The Netherlands), FGF2 (20 ng/ml; peprotech, 100-18B, NJ, USA), N2 (1×; Gibco, 17502-048), insulin (10 μg/ml; Sigma-Aldrich, I6634), and dexamethasone (1 μM; Sigma-Aldrich, d4902), here called minimal medium. After 3 days, primary spheres were dissociated into single cells, seeded in Matrigel and cultured in minimal medium supplemented with Y-27632 (10 μM; Abcam, ab120129, Cambridge, UK), 10% R-spondin1 conditioned medium (provided by C. Kuo), and 50% Wnt3a conditioned medium to form organoids. After 7 days organoids were passaged by dissociation into single cells and cultured as described above.
Organoids were collected 7 days after (sham) irradiation, fixed and stained overnight with X-Gal solution according to the manufacturer’s instructions (Merck Millipore, KAA002RF, MA, USA). Senescent cells were identified as blue-stained cells under light microscopy. The percentage of cells positive for SA-β-gal staining in salivary gland tissue was quantified using ImageJ on three representative fields at ×20 magnification on three mice per group.
Renilla luciferase assay
The p16-3MR gene-reporter system used in this study was as previously described23. Briefly, p16-3MR mice carry a three molecular reporter protein (Luciferase-mRFP-HSVtk fusion protein), which is regulated by the p16 promoter (Fig. 2d). The luciferase protein can be measured using the Renilla luciferase assay. Therefore, p16-3MR mice can be used to track radiation-induced senescence in 3D cultured organoids in vitro. Organoids derived from the salivary glands of six p16-3MR mice were collected and dissociated into single cells. The Renilla luciferase assay system was used according to the manufacturer′s protocol. In total, 100,000 cells were lysed with 100 μl of 1× Renilla luciferase assay lysis buffer. For each reaction, 20 μl of cell lysate was added to a well of a 96-well plate (Greiner Bio-one, 655075, Frickenhausen, Germany). Each sample was analyzed in triplicate.
Quantitative real-time PCR
Cells were collected at designated time points. Total cellular RNA was extracted following the manufacturer’s instructions (Qiagen, RNeasy Mini Kit, Ref 74104, Hilden, Germany) to measure expression of cell cycle genes Cdkn2a (p16Ink4a), Cdkn1a (p21), and SASP genes (including Il6, Mcp1 Cxcl1), and the senescence transcriptome core signature Gdnf in mouse salivary gland organoid-derived cells (3–6 mice per group) and salivary gland tissue (6 mice per group). The primer sequences are listed in Supplementary Table 1. RNA reverse transcription was performed as described previously22. First-strand cDNA synthesis was performed by using 500 ng total RNA, 1 μl dNTP Mix (10 mM), 1 μl random primers (100 ng), 4 μl 5× First-stand Buffer, 2 μl DTT (0.1 M), 1 μl RNase OUTTM (40 units/μl), and 1 μl M-MLV RT (200 units), 20 μl in total for each reaction volume. To measure gene expression, the SYBR assay kit (Bio-Rad) was used. Briefly, 2.5 μl cDNA was mixed with 6.25 μl SYBR Green PCR Master Mix and 3.75 μl primers mix (20 μl forward primer, 20 μl reverse primer and 1160 μl dH2O, primer concentration of 100 nmol) for the genes of interest. qPCR conditions were as follows: 95 °C for 3 min, 39× (95 °C for 10 s, 55 °C for 10 s, and 72 °C for 30 s), 95 °C for 10 s, 65 °C for 5 s, 95 °C for 50 s. All reactions were run in triplicate on a BIO-RAD Real-Time PCR System. All the reagents mentioned above are from Invitrogen.
Salivary gland organoids derived from mice were harvested at designated time points and dissociated into single cells. For cell cycle analysis, after two washes with phosphate-buffered saline (PBS), cells were fixed with 70% ethanol, incubated overnight at 4 °C. Cells were collected by spinning them down for 5 min at 1000 r.p.m. at 4 °C, followed by washing with PBS. After two washes with PBS, cells were treated with 20 μl DNase free RNase A (stock concentration of 20 mg/ml) to remove residual RNA (Sigma-Aldrich) and incubated for 30 min at 37 °C. Four hundred microliters of propidium iodide solution (50 μg/ml) was added to cells and incubated for 1 h at room temperature. For CD24/CD29 population analysis, Pacific Blue™ anti-mouse CD24 (BioLegend, 101820, CA, USA) and FITC anti-rat CD29 (BD Biosciences, 555005, NJ, USA) antibody incubations were performed at room temperature for 15 min, followed by a wash step in PBS. Finally, propidium iodide solution (1 μg/ml) was added to the cells. Samples were analyzed using the XDP flow cytometry machine. Data were analyzed by FlowJo software.
SASP experiments with conditioned medium
Organoids cultured in WRY medium were (sham-) irradiated at day 5 (D5) in culture. Medium was collected at D12 and mixed with fresh medium in a 1:1 ratio, resulting in control (C50%) and IR (IR50%)-conditioned medium. 1 × 104 fresh single SGSCs released from passage 2 organoids were cultured with conditioned medium. Seven days later, Matrigel was dissolved using Dispase and organoid formation efficiency (OFE) was calculated as mentioned previously22.
In vitro irradiation and treatment with ganciclovir (GCV) and ABT263
The irradiation assay was performed as described previously24. In short, photon irradiation was performed using a Cesium-137 source with a dose rate of 0.59 Gy/min. All irradiation experiments were performed on 5-day-old organoids cultured in 12-well plates derived from at least three mice per group. Five-day-old organoids were irradiated with 7 Gy. Seven days later, 10 μg/ml GCV (Sigma-Aldrich, G2536) or 0.313 μM ABT263 (Selleckchem, Cat No. S1001, TX, USA,) were administrated to irradiated organoids and sham-irradiated control cells were incubated, while the same volume of vehicle solvent of GCV or ABT263 were added to the controls. Organoids were collected and dissociated into single cells, and reseeded to next passage at 1 × 104 cell density as mentioned previously. OFE was calculated 7 days later.
In vivo irradiation and treatment with ABT263
The irradiation experimental setup employed here was as described previously24. In short, the salivary glands of 2–3-month-old female C57BL/6 mice were (sham-) irradiated with a single dose of 15 Gy X-rays (Precision X-ray). Eight weeks after irradiation, mice were treated with vehicle (ethanol:polyethylene glycol 400:Phosal 50 PG at 10:30:60) or ABT263 (in ethanol:polyethylene glycol 400:Phosal 50 PG at 10:30:60) by oral gavage at 50 mg/kg per day for seven consecutive days for two cycles with a 2 week interval in between. Saliva production was measured as previously described25. Briefly, after stimulation with pilocarpine (2 mg/kg), saliva was collected for 15 min at baseline, 7, 13, and 22 weeks after irradiation and/or ABT263 treatment. This in vivo experiment was performed using eight mice per group.
GraphPad Software version 8 was used for all statistical analyses. Two-tailed Student’s t-test, Wilcoxon signed-rank test, and two-way ANOVA were used to estimate statistically significant differences between groups. Investigators were blinded to allocation during in vivo experiments and outcome assessments. All values were represented as means ± s.e.m. Numbers (n) for tested groups are stated in the figure legends. All p values were two-sided. P < 0.05 was considered to be statistically significant. All replicates in this study were samples from different mice.