A neurodevelopmental epigenetic programme mediated by SMARCD3–DAB1–Reelin signalling is hijacked to promote medulloblastoma metastasis

Cell lines and cell culture

MED8A (provided by M. D. Taylor, The Hospital for Sick Children, Toronto, Canada) and D556 (provided by D. D. Bigner, Duke University Medical Center, Durham, NC, USA) were cultured in DMEM supplemented with 20% FBS (Sigma-Aldrich, F2442); D425 and D458 (provided by S. Agnihotri, UPMC Children’s Hospital of Pittsburgh, Pittsburgh, PA, USA) were cultured in IMEM supplemented with 20% FBS; and D341 (purchased from American Type Culture Collection (ATCC, HTB-187)) was cultured in EMEM supplemented with 20% FBS. The hcNSCs provided by E. H. Raabe (Johns Hopkins University School of Medicine, Baltimore, MD, USA) were cultured in NSC medium as previously described22,49. 293T packaging cells from ATCC (CRL-3216) were cultured in DMEM with 10% FBS.

Mice and animal housing

Animal experiments were performed with the approval of the University of Pittsburgh Animal Care and Use Committee (protocol number 21049271). Female and male ICR SCID mice aged 4–6 weeks were purchased from Taconic Biosciences (model ICRS-F/ICRS-M). C57BL/B6 mice aged 4–6 weeks were purchased from The Jackson Laboratory (strain 000664) and were bred and maintained at the CHP Rangos Research Center under pathogen-free conditions. All mice were housed under a 12-h light–dark cycle, a temperature range of 21–23 °C and relative humidity of 55 ± 10%.

Orthotopic MB mouse models

For orthotopic xenograft MB models, SCID mice were anaesthetized with an intraperitoneal injection of ketamine–xylazine solution (1.75 ml of 100 mg ml–1 ketamine and 0.25 ml of 100 mg ml–1 xylazine in 8 ml sterile water) at a dosage of 100 μl per 20 g body weight, then placed into a stereotactic apparatus equipped with a z-axis (Kopf). A small hole was bored in the skull 2.0 mm posterior and 2.0 mm lateral to the lambada using a dental drill. Cells (1 × 105) infected with luciferase-ZsGreen (Addgene, 39196) lentivirus in 3 μl DPBS were injected into the right cerebellum 2.5 mm below the surface of the brain using a 10 μl Hamilton syringe with an unbevelled 30-gauge needle. For virus-induced spontaneous MB models, postnatal C57BL/B6 mice were used for the stereotactic injection of lentiviruses into the cerebellum as described above. Animals were monitored for tumour development by assessing neurological function and signs (for example, hunchback, seizure and posterior paralysis). For in vivo BLI, mice were given intraperitoneal injections of 150 μg per g d-luciferin (GoldBio, LUCK-100) and anaesthetized with 2.5% isoflurane in an induction chamber. At 10 min after injection, animals were imaged using Perkin Elmer lumina IVIS S5 systems. In vivo MRI brain imaging was carried out using a Bruker BioSpec 70/30 USR spectrometer operating at 7-Tesla field strength with the following parameters: field of view of 3.0 × 2.0 cm; acquisition matrix of 384 × 256; acquisition slice thickness of 0.60 mm; repetition time/echo time = 2,177 ms/14 ms. Mice with neurological deficits or moribund appearance were euthanized. Brains were removed after transcardial perfusion with 4% paraformaldehyde (PFA) and then fixed in 4% PFA for paraffin embedding or making OCT frozen tissue blocks.

Treatment

Dasatinib (MedChemExpress, HY-10181) was dissolved in DMSO (75 mg ml–1, 37.5 mg ml–1 or 0 mg ml–1) and 25-fold diluted with 50 mmol per litre sodium acetate buffer (pH 4.6; Sigma, S7899). Mice were treated with dasatinib at a dose of 5 μl per g body weight through oral gavage into the stomach using curved feeding needles (Kent Scientific, FNC-20-1.5-2).

Ex vivo time-lapse imaging

Mouse brains were collected after perfusion with ice-cold HBSS and then embedded in 4% low-melting agarose diluted in HBSS. The 300 µm sagittal slices were obtained using a Leica VT1000S vibratome with a speed of 0.1 mm s–1 and an amplitude of 1 mm. Slices were cultured on 0.4 µm culture inserts placed on MatTek glass-bottom dishes with the slice culture medium.

After 3 h in a cell culture incubator, the MatTek glass-bottom dishes were moved to a confocal microscope chamber with humidity and 5% CO2. A Zeiss LSM 719 confocal microscope was used to obtain acquisitions every 6 min with a z-stack.

The MTrackJ plugin in ImageJ (Fiji 1.53C) was used for analysing the videos to obtain the position of one cell at a specific time point \(p_n\), instantaneous distance travelled \(d_n = d\left( } \right)\), total distance travelled \(d_} = \mathop \limits_^ d_n\), net distance travelled \(d_} = d\left( \right)\), instantaneous trajectory time \(\Delta t\) and total trajectory time \(t_} = \left( \right)\Delta t\). The instantaneous speed \(s_n = d_n/\Delta t\), average speed \(s_a = d_}/t_}\), velocity \(v = d_}/t_}\) and directionality \(D = d_}/d_}\) were calculated using the calculated variables in MTrackJ.

Lentivirus production and transduction of target cells

The expression vectors were generated by cloning the respective open reading frame into a pLenti6.3 vector using the Gateway Cloning system. The lentiviral CRISPR–Cas9 vectors were generated by ligating the oligonucleotides of sgRNA sequences (Supplementary Table 5) into lentiCRISPRv2-Blast (Addgene, 83480) or lentiGuide-Puro (Addgene, 52963) and then validated by Sanger DNA sequencing. Gene expression was validated by RT–qPCR (primers listed in Supplementary Table 6) or immunoblotting in lentivirus-infected target cells. For enhancer deletion, genomic DNA was extracted (New England BioLabs, T3010S) and amplified by PCR (ApexBio, K1025), then gel purified (Qiagen, 28704) and sequencing validated. Genotyping PCR primers are listed in Supplementary Table 7. Lentiviruses were produced in 293T cells with a packaging system (pCMVR8.74, pMD2.G, pRSV-Rev) per the vendor’s instruction.

MTS assay and BrdU assays

For the MTS assay, 5,000 cells were seeded into a 96-well plate with 150 μl medium. Then, 30 μl of the combined MTS–PMS solution (Promega, G5430) was pipetted into each well. After 2 h of incubation, 100 μl medium out of the total 150 μl medium was pipetted into a new 96-well plate, and absorbance at 490 nm was measured using a BioTech Synergy HTX. For the bromodeoxyuridine (BrdU) assay, cells were incubated overnight at 4 °C with anti-BrdU antibody after being incubated in medium containing BrdU for 1 h, fixation, HCl incubation and blocking. A fluorescence-conjugated antibody was used to visualize the anti-BrdU-labelled cells.

Scratch wound-healing assay and Transwell assay

Cells were seeded into a 12-well plate and allowed to reach 95% confluence. Wounds were made with pipette tips, and images were captured at specific time points and analysed using ImageJ. For time-lapse imaging, cells were seeded on MatTek dishes and wounds were made with pipette tips at 95% confluency. The MatTek dishes were moved to a confocal microscope chamber with humidity and 5% CO2. A Zeiss LSM 719 confocal microscope was used for imaging acquisition as described for ex vivo brain slices.

Transwell assays were performed in Falcon 24-well insert systems (8.0 μm pore sizes). After 6 h of starvation, cells were seeded in Transwell inserts at 1 × 106 cells per well in medium without FBS or with dasatinib, and the inserts were transferred into medium containing FBS or dasatinib. After 24 h or 48 h of incubation, cells were fixed and stained using a Hema 3 stain set (Fisher Scientific, 22-122911) or directly stained with calcein AM (BD Biosciences, 564061).

Immunoblotting (IB), IHC and immunofluorescence (IF)

For IB, cells were collected, washed with PBS, lysed in RIPA buffer (Millipore, 20-188) with protease and phosphatase inhibitor mini-tablet (Thermo Fisher, A32961) and centrifuged at 10,000g at 4 °C for 15 min. Protein lysates were subjected to SDS–PAGE on a 12% gradient polyacrylamide gel, transferred onto polyvinylidene fluoride membranes, which were incubated with the indicated primary antibodies, washed and probed with horseradish peroxidase (HRP)-conjugated secondary antibodies. For IHC staining, brain sections were incubated with the indicated primary antibodies overnight at 4 °C after deparaffinization, rehydration, antigen retrieval (Vector Laboratories, H3300), quenching of endogenous peroxidase and blocking. The sections were incubated with HRP-conjugated horse anti-rabbit IgG polymer (Vector Laboratories, MP-7401) for 1 h, and then diaminobenzidine using DAB substrate (Vector Laboratories, SK-4105) for 1–15 min at room temperature, followed by haematoxylin staining. Images were acquired using a Nikon Eclipse E800 and scanned using DigiPath’s digital pathology scanner. For IF staining, mouse brains were isolated and fixed in 4% PFA overnight and then processed for OCT frozen tissue blocks. OCT frozen brain sections were thawed at room temperature for 30 min, rinsed and rehydrated with PBS 3 times. After blocking with PBS buffer containing 10% FBS, 1% BSA and 0.3% Triton, the sections were incubated with the indicated primary antibodies overnight at 4 °C following species-appropriate secondary antibodies coupled to AlexaFluor dyes (594 or 647, Invitrogen) for 1 h at room temperature. Vectashield with DAPI (Vector Laboratories, H-1500) was used to mount coverslips. Images were acquired using a Leica DMI8 microscope and analysed using ImageJ. Information about antibodies used for these assays are described in Supplementary Table 8.

Metastasis evaluation by direct fluorescence

The presence or absence of metastatic deposits was observed under a direct fluorescence stereoscope (Leica M165FC). Images were acquired with consistent exposure settings during the experiments. The spines of mice were defined as positive if a single metastatic deposit was observable.

Flow cytometry and FACS

Mice showing neurological signs of late-stage brain tumours or deemed end point were killed, and blood was collected through cardiac exsanguination under deep general anaesthesia. The collected blood (500–900 µl) was swiftly prepared for flow cytometry using RBC lysis buffer (Invitrogen, 00-4333), and cells were suspended in ice-cold PBS with 1% BSA and 2 mM EDTA. After incubation with propidium iodide (Thermo Fisher, P3566) in the dark, the stained cells were analysed using a BD Fortessa analyser. FACS was performed using a BD FACSAria cell sorter. Data were analysed using FlowJo software (v.10.6.1).

RNA isolation, RT–qPCR and RNA-seq

RNA was isolated using a RNeasy Plus Mini kit (Qiagen, 74134) and then used for first-strand cDNA synthesis (Invitrogen, 28025-013). RT–qPCR was performed using PowerUp SYBR Green master mix (Applied Biosystems, A25742). The relative expression of genes was normalized using ribosomal protein L39 (RPL39) as a housekeeping gene.

For RNA-seq, sequencing libraries were generated using a NEBNext Ultra RNA Library Prep kit for Illumina following the manufacturer’s recommendations, and index codes were added to attribute sequences to each sample. After cluster generation, the library preparations were sequenced using a NovaSeq 6000 platform, and paired-end reads were generated. Reads were aligned using Hisat2 (v.2.1.0) against the hg38 genome and transcriptome. After initial mapping, the aligned reads were filtered out if their best placements were only mapped to unique genomic coordinates. The statistical environment R was used to perform all the statistical analyses and graph plots.

Analyses of scRNA-seq and sci-ATAC-seq3 data

For scRNA-seq analysis, genes not expressed in any cells had already been removed. Cells with fewer than 200 genes or more than 5,000 genes expressed or more than 10% mitochondrial genes expressed were removed using Seurat (v.3.2.3). Clusters generated using UMAP were assigned to cell types using known marker genes. For sci-ATAC-seq3, processed data were directly downloaded. Co-accessibility scores and Cicero gene activity scores were calculated using Cicero (v.1.6.2). Data were visualized using Sushi. Cellranger (v.5.0.1) was also used to analyse the scRNA-seq and sci-ATAC-seq3 data.

ATAC-seq

Approximately 100,000 nuclei were extracted from freshly collected MED8A cells with SMARCD3 WT or KO by incubating for 15 min on ice in lysis buffer (10 mM Tris-HCl, pH 7.5, 10 mM NaCl, 3 mM MgCl2, 0.1% NP-40, 0.1% Tween-20 and 0.01% digitonin). Samples were washed in 1 ml wash buffer (10 mM Tris-HCl, pH 7.5, 10 mM NaCl, 3 mM MgCl2 and 0.1% Tween-20) and centrifuged at 500g for 10 min at 4 °C. The supernatant was removed, and nuclei pellets were flash-frozen in liquid nitrogen and stored at −80 °C.

Nuclei were incubated in transposition reaction mix using 50 µl 2× tagmentation buffer (Diagenode) and 5 µl preloaded tagmentase (Diagenode) for 30 min in a thermomixer set to 37 °C at 1,000 r.p.m. Following transposition, DNA was isolated using a Qiagen MinElute Reaction Cleanup kit. Samples were PCR amplified using NEBNext High-Fidelity 2× PCR master mix and 25 µM of Nextera 70* and 25 µM Nextera 50* primers. Samples were amplified using five cycles of PCR, then quantified by qPCR and amplified using three additional cycles. Samples were run on 1.5% agarose gel, and 150–500 bp bands from each sample lane were extracted. Libraries were run on a Fragment Analyzer according to the manufacturer’s instructions (Agilent) to validate library quality. Libraries were pooled and sequenced on using an Illumina NextSeq500.

CUT&RUN

The CUT&RUN protocol was performed as previously described36,37,67 with the following modifications.

MED8A cells with SMARCD3 WT or KO were diluted to 1 million cells in PBS. Cells were centrifuged and resuspended in 1 ml cold nuclear extraction buffer (20 mM HEPES-KOH, pH 7.9, 10 mM KCl, 0.5 mM spermidine, 0.1% Triton X-100, 20% glycerol and freshly added protease inhibitors) and incubated for 10 min on ice. Nuclei were then centrifuged, and pellets were resuspended in 600 µl of nuclear extraction buffer. Concanavalin A beads (400 µl bead slurry per 1 million nuclei) were prepared in binding buffer (20 mM HEPES-KOH, pH 7.9, 10 mM KCl, 1 mM CaCl2 and 1 mM MnCl2) and washed twice in binding buffer before adding the nuclei and incubating for 10 min at 4 °C with rotation.

Following nuclei binding to concanavalin A beads, samples were pre-blocked for 5 min at room temperature using 1 ml blocking buffer (20 mM HEPES, pH 7.5, 150 mM NaCl, 0.5 mM spermidine, 0.1% BSA, 2 mM EDTA and freshly added protease inhibitors). Bound nuclei were washed in 1 ml wash buffer (20 mM HEPES, pH 7.5, 150 mM NaCl, 0.5 mM spermidine, 0.1% BSA and freshly added protease inhibitors). Following this wash, nuclei were resuspended in 2 ml wash buffer and aliquoted in 250 µl volumes to eight 1.5 ml tubes for the individual antibody reactions (125,000 nuclei per antibody sample for each cell line). Nuclei were incubated for 1 h at room temperature with rotation with the primary antibody in wash buffer to a final concentration of 1:100. Negative controls were included for each cell line, in which no primary antibody was added. Following incubation, samples were washed twice in 1 ml wash buffer.

Samples were incubated with 2.4 µl in-house purified pA-MNase per sample in 250 µl wash buffer for 30 min at room temperature with rotation. Samples were pre-equilibrated to 0 °C in an ice water bath for 5 min before 3 mM CaCl2 was added to initiate MNase digestion. Following a 30 min digestion in an ice water bath, the digestion reaction was chelated using 2XRSTOP+ buffer (200 mM NaCl, 20 mM EDTA, 4 mM EGTA, 50 µg ml–1 RNase A, 40 µg ml–1 glycogen and 10 pg ml–1 MNase-digested Saccharomyces cerevisiae mononucleosomes added for a spike-in control). Samples were incubated at 37 °C for 20 min and centrifuged to separate and release fragments. Protein was digested with ProK, and DNA was purified using PCI extraction and ethanol precipitation.

DNA libraries were prepared using end-repair, adenylation and NEBNext stem-loop adapter ligation. Fragments were then purified using AMPure XP beads (Beckman Coulter) and amplified using 15 cycles of high-fidelity PCR. A final AMPure clean-up step was performed to purify the DNA fragments before sequencing. Samples were run on 1.5% agarose gels to validate library quality before sequencing. Libraries were pooled and sequenced using an Illumina NextSeq500.

Defining accessible sites

Reads in ATAC-seq and CUT&RUN were mapped to the hg38 reference genome using bowtie2 (v.2.3.5.1) with the options “—very-sensitive -X 2000” and “—very-sensitive -X 2000 –dovetail”, respectively. PCR duplicates were removed using sambamba.

For ATAC-seq, using MACS3 (v.3.0.0a6) call peak with the options “-f BEDPE -B -q 0.01”, reads with a fragment size between 1 and 100 or between 180 and 247 were used to define peaks of accessibility across all sites. ChIPseeker was used to annotate the peaks.

For CUT&RUN, using MACS3 call peak with the options “—broad –broad-cutoff 0.1”, reads with a fragment size between 150 and 500 were used to define peaks of histone-marker-binding sites. Inputs were used as a control for peak calling.

Peaks and alignments were visualized using IGV (V.2.6.3).

Pathology analysis of patient tumour samples

The tissue microarray MB slides (formalin-fixed paraffin-embedded) for IHC were provided by C. G. Eberhart (Johns Hopkins University School of Medicine, Baltimore, MD, USA), approved by the institutional review board (protocol number NA_00015113). The ten paired primary and metastatic MB MRI images and slides (formalin-fixed paraffin-embedded) for IHC from Xiangya Hospital were used and analysed with approval by the institutional review board (number 202110207). Informed consent was obtained for the biorepositories that provided the above study materials. The pathology analysis of MB samples was conducted by at least two experienced neuropathologists. The study was compliant with all ethical regulations.

Statistics and reproducibility

All the boxplots show the interquartile range (IQR), whiskers denote quartile 3 + 1.5× the IQR or quartile 1 – 1.5× the IQR. Data points that were more or less than the whiskers were considered outliers. Column bar plots show the mean with standard deviation. Statistical parameters, including the exact value of n, the definition of centre, dispersion, precision measures, statistical test and statistical significance, are reported in the figures and figure legends. Data were judged to be significant when P < 0.05. No statistical methods were used to predetermine the sample sizes, but our sample sizes were similar to those reported in the previous publications49,68. In the experiments with dasatinib treatment for mice bearing MB, tumour sizes were assessed, and then mice were grouped to minimize variations in tumour size among the groups. No randomization was performed for other experiments. The investigators were blinded to assess protein expression in IHC and IF experiments; other data collection and analyses were not performed blind to the conditions of the experiments. No animals or data were excluded except for low-quality cells during scRNA-seq analysis. GSVA (v.1.36.3) and IPA (v.01-16) were used to calculate the meta-proliferating cell nuclear antigen scores and pathway analysis scores, respectively. R (v.3.5.1) and GraphPad Prism (v.9.1.0) were used for statistical analyses.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

留言 (0)

沒有登入
gif