Clinical trial design and oversight
A phase 2 open-label study was conducted at the NIH (NCT03571191). This investigator sponsored study was supported by Amgen, Inc; study design, conduct, and analyses were performed by the investigators. Subjects received denosumab for 6-months at a dose of 120 mg every 4 weeks, with loading doses on weeks 2 and 3 26. Percutaneous FD lesion biopsies were performed at baseline and 6-months in 6 of 8 total adult subjects (Supplemental Fig. 1). Biopsies were deferred in 2 subjects; 1 due to the presence of isolated craniofacial FD, and 1 due to intercurrent illness unrelated to study intervention. Biopsy sites were chosen jointly by the investigators and surgeon to ensure a minimally invasive procedure, and were performed in the interventional radiology suite at the NIH Clinical Center using core needles under CT guidance. The same sites were biopsied pre- and post denosumab treatment.
In vivo and ex vivo models of Fibrous Dysplasia
Mice expressing the GαsR201C variant were generated as described 8. At 10 weeks of age, expression of GαsR201C was induced in the limbs by switching to doxycycline-supplemented food (100 ppm Purina Mod LabDiet 5001, PMI Nutrition International, Saint Louis, MO). Six mice received anti-RANKL antibody (6 mg/Kg, BE0191, Bioxcell Lebanon, NH) and 6 received rat IgG2A isotype control, as well as 6 littermate controls not carrying tet- GsαR201C (6 mg/Kg, BE0089, Bioxcell) by subcutaneous injections on days 28, 30, 35, 42, 49 and 56 after induction. Plasma was collected weekly, and on day 58 mice were euthanized and tissue was collected. On day 58 all mice were euthanized, and FD or control tissue was extracted from the distal ulna and radius, cleaned from muscle and tendons, and snap-frozen for RNA analysis. Mice were then perfused with PBS and Z-fix fixative, and both hindlimbs were extracted for histology and µCT analysis.
GαsR201C expression was induced in ex vivo bone marrow cultures 27 and then treated with anti-RANKL antibody. MC3T3-E1 clone 4 and 14 cells were cultured as previously described 28. Additional methods are included in the Supplementary appendix.
Mouse plasma measurements
120 µL of mouse blood was collected weekly via retro-orbital eye collection. At the time of euthanasia, 500–1000 µL was obtained from the vena cava. Blood was stored in heparinized vials and plasma was obtained. TRAP5b, CT-X and P1NP was measured using IDS ELISA kits SB-TR103, AC-06F1 and AC-33F1, respectively.
Mouse X-rays and microCT
Mice were anesthetized with 2–5% of isoflurane and X-ray images of the hind limbs were obtained on a Faxitron Ultrafocus system (Hologic, Marlborough MA). A semi-quantitative score was developed and validated to quantify the disease burden (Table S1). Three independent examiners blindly evaluated X-rays of both hindlimbs for each mouse and timepoint in an independent fashion.
Right hindlimbs were dissected and scanned using a Scanco µCT 50 at 10 µm, 70 kVp, 80 µA, and 900 ms integration time (Scanco, Wangen-Brüttisellen, Switzerland). Reconstructed images were analyzed with Analyze 14 (AnalyzeDirect, Overland Park KS) and calibrated against hydroxyapatite phantoms with known densities. The volume of interest (VOI) was defined as the distal tibia sector between 500 µm below the fibula insertion point and 200 µm above the intermedium. The Smart Trace tool was used to outline the distal tibia every 50 µm in the sagittal direction and then Propagate Objects was used to connect the 2D tracings, semi-automatically segmenting the VOI.
Following segmentation, the average bone mineral density (BMD) in milligrams of hydroxyapatite per cubic centimeter (mg HA/cm3) was obtained for the entire VOI. Distal tibiae were further analyzed by obtaining the number of voxels per BMD unit and binned into three categories: soft tissue (< 350 mg HA/cm3), partially mineralized tissue (350–600 mg HA/cm3), and mineralized tissue (> 600 mg HA/cm3).
RNA extraction and sequencing
Mouse and human samples were snap-frozen, pulverized using an automated dry pulverizer (CP02 cryoPREP, Covaris, Woburn MA) and immediately transferred to Trizol (Thermofisher). Phenol-chloroform RNA extraction was carried out. cDNA synthesis, library construction and sequencing were performing by Novogene (University of California, Davis, CA). Briefly, messenger RNA was purified from total RNA using poly-T oligo-attached magnetic beads. After fragmentation, the first strand cDNA was synthesized using random hexamer primers followed by the second strand cDNA synthesis, end repair, A-tailing, adapter ligation, size selection, amplification, and purification. The library was checked with Qubit and real-time PCR for quantification and bioanalyzer for size distribution detection. Quantified libraries were pooled and sequenced on an Illumina NovaSeq 6000 sequencer, according to effective library concentration and data amount using 150 x 150 paired-end mode. After sequencing, the base-called demultiplexed (fastq) read qualities were determined using FastQC (v0.11.2) 29, aligned to the GENCODE v32 human genome (GRCh38.v32) form human samples and GENCODE M23 mouse genome (GRCm38.M23) for mouse samples and gene counts generated using STAR (v2.7.3a) 30. Post-alignment qualities were generated with Picard tools. An expression matrix of raw gene counts was generated using R 31 and filtered to remove low counts genes (defined as those with less than 5 reads in at least one sample). The filtered expression matrix was used to generate a list of differentially expressed genes between the sample groups using DESeq2 32 and analyzed with principal component analysis (PCA). Reads corresponding to locations chr20:909,365 and chr20:58,909,366 were analyzed in order to detect and quantify GNAS p.R201C (C > T) and pR201H (G > A) substitutions respectively. An FD tissue genetic signature comprising 276 genes was derived from comparing WT versus Gnas R201C expressing mice that were more than ± 2 log2 fold change and were significantly (adjusted p-value ≤ 0.05) differentially expressed between both groups. This gene signature was converted to human genes based on homology scores using the Biomart service, which reduced it to 202 genes. Likewise, a list of genes positively correlated with the proliferation marker PCNA in healthy human tissues was compiled from Venet et al 9. A custom .gmt file was compiled from the FD, meta PCNA signatures and MSigDB GO-derived genesets corresponding to osteoclast and osteoblast differentiation and activity biological processes. For osteoblasts, the geneset GO:0001649 “osteoblast differentiation” list was used. For osteoclasts, we combined two genesets: GO:0030316 “osteoclast differentiation” and GO:0045453 “bone resorption”. The gene set variation analysis (GSVA) 33 method was used to compute enrichment scores for each sample against these selected genesets using the Poisson kernel and the resulting matrix was submitted to unsupervised clustering using the euclidean metric. Selected gene expression heatmaps were generated from log transformed TMM-normalized gene counts using the heatmap.2 R function.
Bone marrow explant and cell culture
Tibiae and femurs were dissected from uninduced mice or wild-type littermates and bone marrow cells (BMSCs) were pooled, plated and cultured as previously described 34. Cultures below passage 4 were used for the experiments. For GαsR201C expression induction in BMSCs subset of the explants, cells were plated at ~ 40% confluency in 6-well plates as well as in 24-well plates, and angiogenesis µ-slides (cat no. 81506 Ibidi, Firchburg, WI) and treated with 5µM doxycycline (Sigma, # D9891-5G). During induction, media were refreshed daily. Neutralizing RANKL antibody (BE0191, Bioxcell) was added at 1µg/mL. MC3T3-E1 clone 4 and 14 cells were cultured as previously described28.
The human BMSCs cultured used for BaseScope optimization were cultured as previously described 11.
Histology and cell culture staining
Tissue and culture preparation
Dissected mouse hindlimbs and human baseline and post-treatment bone biopsies were fixed in Z-fix (Anatech, USA) overnight at 4°C. Samples for paraffin embedding (PE) were decalcified in 0.25 M EDTA at 4°C. Samples were then embedded and sectioned into 5 µm sections. Slides were stored in 4°C or -20°C until staining. Sections from all PE samples were deparaffinized in xylene and rehydrated using a graded ethanol series for subsequent staining. Tissue morphology was assessed by staining sections for H&E.
Tissue samples for cryosectioning were shipped to Bonebase (at UConn Health, Farmington, CT) in formalin and in wet ice, embedded and cryosectioned following the methods in Dyment et al 35.
Cell cultures were fixed with warm freshly prepared 4% formaldehyde in PBS (Sigma, F1268) at 37◦C.
Patient and healthy volunteer-derived BMSCs cultures were pelleted at 4C, 300G, 5 mins, embedded in OCT media, frozen and cryosectioned for Gαs mRNA detection optimization and validation.
TRAP and ALP enzymatic detection
Mouse cryosections were stained for TRAP and ALP enzymatic activity at Bonebase following previously described methods 35. Mouse culture TRAP staining was performed with Cosmo Bio LTD TRAP Staining Kit (Cat no. PMC-AK04F-COS, Cosmo Carlsbad, CA). Human PE sections were incubated with TRAP staining solution (Wako, Cat No 294-67001) for 25 min at RT, then counterstained with Methyl Green and mounted using EcoMount.
Immunohistochemistry
PE sections were deparaffinized in xylene and rehydrated using a graded ethanol series. Endogenous peroxidase activity was blocked using 3% H2O2 in methanol. For Runx2 and Mcm2, antigen retrieval was performed using Uni-Trieve (Innovex Biosciences, Cat No NB325) for 30 min at 55°C or 45 min at 70°C, respectively. Non-specific binding was blocked using goat or rabbit serum (Vector, Cat No PK-6105 and PK-6101) as appropriate. Next, both human and mouse sections were incubated with rabbit anti-Runx2 (1:400, Abcam, Cat No ab192259), rabbit anti-Mcm2 (1:200, Abcam, Cat No 108935), human sections were incubated with rabbit anti-osteocalcin (1:200, Proteintech, Cat No 23418-1-AP), and mouse sections were incubated with goat anti-sclerostin (1:100, R&D Systems, AF1589) overnight at 4°C. Rabbit and goat isotype control antibodies were used at a similar concentration (BioLegend, Cat No 910801; R&D Systems, Cat No AB-108-C, respectively) and no significant unspecific staining was observed (not shown). Sections were then incubated with corresponding goat anti-rabbit or rabbit anti-goat biotinylated secondary antibodies (Vector, Cat No PK-6105 and PK-6101) for 45 min at RT, followed by VECTASTAIN Elite ABC Reagent (Vector, Cat No PK-6100) for 30 min at RT. Finally, staining was developed using DAB-EASY tablets (Acros Organics, Thermo Fisher Scientific, Cat No AC328005000) until desired stain intensity was achieved. Samples were counterstained with Methyl Green (Vector, H-3402-500) and mounted using EcoMount (Biocare Medical, Cat No EM897L).
In vitro fluorescence and immunofluorescence
Mouse marrow explant cultures were stained with antibodies against Tsg101 (NOVUS, 4A10), Runx2 (Abcam, 76956), Ki-67 (CST, D3B5) and Rank (Abcam, 13918). Mouse-Alexa555 (CST, 4409) and Rabbit-Alexa647 (CST, 4414) were used as fluorescent secondary antibodies. Osteoclast fusion was evaluated by fluorescence microscopy as previously described 36 using phallodin-Alexa488 and Hoechst (Invitrogen, cat no. H3570 and A30106 respectively) to label actin cytoskeleton and nuclei. For all stains, cells were permeabilized 10 min in PBS with 0.1% Triton X100 in PBS and 5% FBS (IF Buffer) was used to suppress non-specific binding. Then the cells were incubated with primary antibodies overnight in IF Buffer. After washes in IF Buffer, we placed the cells for 2h at room temperature in IF Buffer with secondary antibodies. Images were captured on a Zeiss LSM 800 airyscan, confocal microscope using a C-Apochromat 63x/1.2 water immersion objective.
mRNA in situ hybridization
For human SOST mRNA hybridization, sections were deparaffinized and incubated with RNAscope Hydrogen Peroxide (Advanced Cell Diagnostics [ACD], Cat No 322335) for 10 min at RT. Target retrieval was performed using ACD Custom Pretreatment Reagent (ACD, Cat No 300040) for 45 min at 40°C. Probes were hybridized as instructed and consisted of dapB (bacterial gene, negative control), PPIB (housekeeping gene), and SOST (sclerostin). The remaining hybridization steps were performed using RNAscope 2.5 HD Detection Reagents- RED (ACD, Cat No 322360) with the exception of AMP5 incubation which was extended to 60 min. Sections were counterstained with 50% Gil’s Hematoxylin I (Sigma-Aldrich, Cat No GHS132) and mounted with EcoMount (Biocare Medical, Cat No EM897L).
In situ hybridization of Gαs p.R201 variants mRNA was achieved using the novel Advanced Cell Diagnostics [ACD] Basescope duplex system with custom-made probes and following manufacturer protocols. To optimize and validate the method, human bone marrow stromal cells (BMSCs) primary cultures containing GNAS p.R201C, GNAS pR201H and WT variants were cultured, pelleted by centrifugation and embedded in OCT cryosectioning media. Sections of 5 µm were assessed for RNA hybridization of two candidate probes for each GNAS p.R201C, GNAS pR201H and WT variant in collaboration with ACD team. Images of hybridization with the probes validated for further studies are shown in Fig S4A.
Briefly, human sections were incubated with RNAscope Hydrogen Peroxide (ACD, Cat No 322335) for 10 min at RT, retrieved with ACD Custom Pretreatment Reagent (ACD, Cat No 300040) for 60 min at 40°C. Positive controls PPIB (high-expression housekeeping gene) and POLR2A (low-expression housekeeping gene) and negative control dapB (bacterian gene) probes, and custom-designed probes for wild-type GNAS (ACD, Cat No 1061061-C1) as well as the two most common mutations in FD: GNASR201C (c.601C > T, ACD, Cat No 1061041-C2) and GNASR201H (c.602G > A, ACD, Cat No 1061051-C2). BaseScope Duplex Detection Reagent Kit (ACD, Cat No 323810); AMP7 and AMP11 times were increased to 45 min and 60 min, respectively, to increase staining intensity. Sections were counterstained using 50% Gil’s Hematoxylin I and mounted with VectaMount Permanent Mounting Medium (Vector, Cat No H-5000). Staining of the positive and negative control probes are shown in Fig S4B-G.
Microscopy imaging
Chromogenically stained PE sections were scanned using a NanoZoomer S60 Digital slide scanner (Hamamatsu, Cat No C13210-01) at 400X magnification.
Fluorescent cryogenic sections were scanned using a Axioscan 7 fluorescence scanner (Zeiss, Oberkochen, Germany) 35 with filters appropriate for the detection of DAPI (Em 460nm, Ab 350nm), Elf97 TRAP (Em 550nm, Ab 375nm) and ALP (Em 605nm, Ab 545nm). Scanned slides were manually aligned and converted to Adobe Photoshop .psd files.
Cell immunofluorescence images were captured on a Zeiss LSM 800 airyscan, confocal microscope using a C-Apochromat 63x/1.2 water immersion objective. For osteoclast fusion assay, 8 randomly selected fields of view were imaged using Alexa488, Hoechst and phase contrast compatible filter sets (BioTek) on a Lionheart FX microscope using a 10x/0.3 NA Plan Fluorite WD objective lense (BioTek) using Gen3.10 software (BioTek).
Microscopy quantification
For bone content and cellularity, images from H&E-stained PE sections were analyzed using a custom script in ImageJ (NIH). For human sections, complete biopsy sections were analyzed. For mice, 1–3 fields of 0.6 by 0.4 mm of randomly selected FD lesional tissue were analyzed per sample. Non-bone or non-FD tissue were removed using Adobe Photoshop 2021 when necessary (Adobe, San Jose, CA). On image J, pixel to area calibration was performed and mineralized and fibrous tissue areas were manually traced, and nuclei were selected in these areas using a semi-automatic color threshold selection followed by a watershed separation plugin. Nuclei number and mineralized and fibrotic areas were calculated using the “analyze particles” function. Total nuclei number obtained with this method was used as denominator to calculate the ratio of positive cells for TRAP activity, Rank, and Mcm2 in consecutive sections form the human biopsies.
For mouse TRAP and ALP analysis in mouse FD cryosections, multiplexed fluorescence 1 by 1 mm images of the distal tibiae were opened in Adobe Photoshop and color levels were adjusted and exported as single-layer tiff files corresponding to TRAP, ALP or DAPI for image quantification. Images were then analyzed with QuPath version 0.3.2, an open-source software for digital pathology analysis 37. After calibrating the pixel to area ratio of the images, cell nuclei were identified by DAPI staining and counted using the “cell detection” function. TRAP and ALP-stained areas were traced using the “pixel classification” function in which the area of fluorescence (µm2) is calculated based on a pixel color intensity threshold. Average TRAP and ALP stain per cell was calculated as the ratio of µm2 TRAP or ALP positive area and number of nuclei in each quantified image.
We used QuPath point tool in PE scans to manually label and count cells positive for TRAP, Rank (multinuclear osteoclasts or mononuclear precursors, Mcm2 and, sclerostin-stained (either by RNA hybridization in humans or immunohistochemistry in mice) and non-stained osteocytes. For ACD Basescope™ quantification, we used this tool in 400x images to label hematoxylin-stained nuclei in proximity of detected GαsR201C or GαsR201H mRNA molecules (green dots), quantified as mutant cells, regardless of the neighboring presence of wildtype Gαs mRNA molecules (red dots). Nuclei only associated to wildtype Gαs mRNA molecules where quantified as wildtype cells. Positive controls PPIB (high-expression housekeeping gene) and POLR2A (low-expression housekeeping gene) and negative control dapB (bacterian gene) probes were used.
Osteoclast fusion efficiency was evaluated as the number of fusion events between osteoclasts with obvious ruffled boarders and ≥ 3 nuclei in phalloidin-Alexa488/Hoechst images, as described previously 38. Since regardless of the sequence of fusion events, the number of cell-to-cell fusion events required to generate syncytium with N nuclei is always equal to N-1, we calculated the fusion number index as Σ (Ni − 1) = Ntotal − Nsyn, where Ni = the number of nuclei in individual syncytia and Nsyn = the total number of syncytia.
RUNX2 and OCN stains in human samples were quantified by a scoring method, since the variable intensity of staining made impossible a quantification based on a cell positivity color threshold. For the secreted factor OCN, stain was normally dispersed and not associated to individual cells, so a collection of blinded 100x microscopy images were studied and assigned to 5 scores based on their staining level (being 0 no stain and 5 maximum stain). Then two images of each stain level were provided to the readers for training purposes. For RUNX2, descriptive scores were developed and shown in table S2. All assessments that required human evaluation were examined by at least 3 trained readers in a blinded fashion.
In vitro bone resorption assay
Bone resorption was evaluated using bone resorption assay kit from Cosmo Bio USA according to the manufacturer’s instructions. In short, fluoresceinamine-labeled chondroitin sulfate was used to label 24-well, calcium phosphate-coated plates. Media were collected at 4–5 days post induction and fluorescence of the media was evaluated as recommended by the manufacturer.
Enrichment and quantification of Extracellular Vesicle (EV) Fractions
Ex vivo cultures were cultured in complete culture media supplemented with FBS depleted of EVs (via ultracentrifugation at 150,000xg for > 2 hours). 24 hours later, conditioned media was collected, and cells/large cell debris were depleted via centrifugation (15 mins at 4,000xg). Next, an EV fraction was enriched via ultracentrifugation (150,000xg for 1.5 hours). Alternatively, exoEasy Maxi Kit (Qiagen, Hilden Germany) was used to isolate EVs. EV-enriched fractions were evaluated via Western Blot using anti-Tsg101 (cat no. 4A10, NOVUS, Louis MO) and anti-RANK (cat no. 13918 Abcam, Cambridge UK) antibodies. Tsg101 and Rank bands staining was quantified using densitometry via ImageJ.
Statistical analyses
Data are expressed as individual datapoints and mean ± SEM for all values. Results were tested 375 for normality using Shapiro-Wilk test and analyses were performed using non-paired t-tests or Mann-Whitney tests. For longitudinal measures, paired two tailed t-tests compared baseline to timepoint values. Data are reported as average and standard deviation when shown, unless otherwise indicated, and analyses were conducted on GraphPad Prism 8.0.2 by LFdC. Culture experiments were evaluated by two tailed t-tests paired by donor mouse, and non-paired t-tests for MC3T3-E1 experiments by JW.
Study approval
Clinical trial NCT03571191 was approved by the NIH Investigational Review Board, and informed consent was obtained from all subjects. The study was monitored by a data safety and monitoring committee organized by the National Institute of Dental and Craniofacial Research. Mouse experiments were conducted under a protocol approved by the NIH/NIDCR Animal Care and Use committee (ASP 19–897).