Modic type 1 changes (MC1) are painful vertebral bone marrow lesions frequently found in patients suffering from chronic low-back pain. Marrow fibrosis is a hallmark of MC1. Bone marrow stromal cells (BMSCs) are key players in other fibrotic bone marrow pathologies, yet their role in MC1 is unknown. The present study aimed to characterise MC1 BMSCs and hypothesised a pro-fibrotic role of BMSCs in MC1. BMSCs were isolated from patients undergoing lumbar spinal fusion from MC1 and adjacent control vertebrae. Frequency of colony-forming unit fibroblast (CFU-F), expression of stem cell surface markers, differentiation capacity, transcriptome, matrix adhesion, cell contractility as well as expression of pro-collagen type I alpha 1, α-smooth muscle actin, integrins and focal adhesion kinase (FAK) were compared. More CFU-F and increased expression of C-X-C-motif-chemokine 12 were found in MC1 BMSCs, possibly indicating overrepresentation of a perisinusoidal BMSC population. RNA sequencing analysis showed enrichment in extracellular matrix proteins and fibrosis-related signalling genes. Increases in pro-collagen type I alpha 1 expression, cell adhesion, cell contractility and phosphorylation of FAK provided further evidence for their pro-fibrotic phenotype. Moreover, a leptin receptor high expressing (LEPRhigh) BMSC population was identified that differentiated under transforming growth factor beta 1 stimulation into myofibroblasts in MC1 but not in control BMSCs. In conclusion, pro-fibrotic changes in MC1 BMSCs and a LEPRhigh MC1 BMSC subpopulation susceptible to myofibroblast differentiation were found. Fibrosis is a hallmark of MC1 and a potential therapeutic target. A causal link between the pro-fibrotic phenotype and clinical characteristics needs to be demonstrated.

Background Vertebral endplate signal intensity changes visualized by magnetic resonance imaging termed Modic changes (MC) are highly prevalent in low back pain patients. Interconvertibility between the three MC subtypes (MC1, MC2, MC3) suggests different pathological stages. Histologically, granulation tissue, fibrosis, and bone marrow edema are signs of inflammation in MC1 and MC2. However, different inflammatory infiltrates and amount of fatty marrow suggest distinct inflammatory processes in MC2. Aims The aims of this study were to investigate (i) the degree of bony (BEP) and cartilage endplate (CEP) degeneration in MC2, (ii) to identify inflammatory MC2 pathomechanisms, and (iii) to show that these marrow changes correlate with severity of endplate degeneration. Methods Pairs of axial biopsies (n = 58) spanning the entire vertebral body including both CEPs were collected from human cadaveric vertebrae with MC2. From one biopsy, the bone marrow directly adjacent to the CEP was analyzed with mass spectrometry. Differentially expressed proteins (DEPs) between MC2 and control were identified and bioinformatic enrichment analysis was performed. The other biopsy was processed for paraffin histology and BEP/CEP degenerations were scored. Endplate scores were correlated with DEPs. Results Endplates from MC2 were significantly more degenerated. Proteomic analysis revealed an activated complement system, increased expression of extracellular matrix proteins, angiogenic, and neurogenic factors in MC2 marrow. Endplate scores correlated with upregulated complement and neurogenic proteins. Discussion The inflammatory pathomechanisms in MC2 comprises activation of the complement system. Concurrent inflammation, fibrosis, angiogenesis, and neurogenesis indicate that MC2 is a chronic inflammation. Correlation of endplate damage with complement and neurogenic proteins suggest that complement system activation and neoinnervation may be linked to endplate damage. The endplate-near marrow is the pathomechanistic site, because MC2 occur at locations with more endplate degeneration. Conclusion MC2 are fibroinflammatory changes with complement system involvement which occur adjacent to damaged endplates.

Modic type 1 changes (MC1) are vertebral bone marrow lesions and associate with low back pain. Increased serum C-reactive protein (CRP) has inconsistently been associated with MC1. We aimed to provide evidence for a role of CRP in the tissue pathophysiology of MC1 bone marrow. From thirteen MC1 patients undergoing spinal fusion at MC1 levels, vertebral bone marrow aspirates from MC1 and intra-patient control bone marrow were taken. Bone marrow CRP, IL-1, and IL-6 were measured with enzyme-linked immunosorbent assays; lactate dehydrogenase (LDH) was measured with a colorimetric assay. CRP, IL-1, and IL-6 were compared between MC1 and control bone marrow. Bone marrow CRP was correlated with blood CRP and with bone marrow IL-1, IL-6, and LDH. CRP expression by marrow cells was measured with PCR. Increased CRP in MC1 bone marrow (mean difference: +0.22 mg CRP/g protein, 95% CI [-0.04, 0.47], p=0.088) correlated with blood CRP (r=0.69, p=0.018), with bone marrow IL-1 (=0.52, p=0.029) and IL-6 (=0.51, p=0.031). Marrow cells did not express CRP. Increased LDH in MC1 bone marrow (143.1%, 95% CI [110.7%, 175.4%], p=0.014) indicated necrosis. A blood CRP threshold of 3.2 mg/L detected with 100% accuracy increased CRP in MC1 bone marrow. In conclusion, the association of CRP with inflammatory and necrotic changes in MC1 bone marrow provides evidence for a pathophysiological role of CRP in MC1 bone marrow.

BackgroundModic type 1 changes (MC1) are painful vertebral bone marrow lesions often found in patients with chronic low back pain. Damage of the endplate associates with MC1, is a risk factor for MC1 progression, and associates with pain. Recent evidence suggests a role of MC bone marrow neutrophils in endplate damage.ObjectivesThe aim of this study was 1) to show that MC1 bone marrow neutrophils are activated and 2) to show that activated blood neutrophils degrade cartilage endplates (CEPs).MethodsFrom MC1 patients undergoing lumbar spinal fusion, MC1 and intra-patient control bone marrow neutrophils (CD45+CD66b+) were isolated from bone marrow aspirates by cell sorting. Bulk RNA sequencing was performed (n = 5 + 5), differentially expressed genes (DEGs) were identified, and enrichment analysis was performed. In addition, neutrophil activation was assessed as CD66b expression by flow cytometry (n = 3 + 3). To assess the effect of activated neutrophils on CEP degradation, a neutrophil-mediated CEP damage model was established. Blood neutrophils were isolated from one donor and exposed to lumbar CEPs. A lumbar CEP was collected from six patients undergoing spinal fusion. From each CEP, three punch biopsies (ø=4-mm) were taken and halved. The six CEP biopsy halves per patient (1A,1B; 2A, 2B; 3A, 3B) were exposed for 18 h at 37 °C to: 1A) 0.75 U/ml collagenase P (positive control) and 1B) Hanks balanced salt solution (negative control); 2A) conditioned medium from 25 mio/ml activated neutrophils (100 nM PMA, 3 h, 37 °C) and 2B) conditioned medium from 25 mio/ml non-activated neutrophils; 3A) conditioned medium from 12.5 mio/ml activated neutrophils (100 nM PMA, 3 h, 37 °C) and 3B) conditioned medium from 12.5 mio/ml non-activated neutrophils. Exposure supernatant and CEP tissues were assayed for sulphated glycosaminoglycans (sGAG) and hydroxyproline (as measure for total collagen). Release of sGAG and hydroxyproline from condition 1A, 2A, and 3A was normalized to condition 1B, 2B and 3B, respectively. Relative release was tested against null hypothesis (µ0 = 100%) using a one sample t-test.Results185 genes were differentially expressed between MC1 and control vertebral bone marrow neutrophils. Enrichment analysis revealed an activated pro-inflammatory transcriptome (Figure 1a). Flow cytometric analysis confirmed neutrophil activation on protein level (measured as % of CD66high) in MC1 (control: 43.1% ± 15.7%, MC1: 54.1% ± 16.7%, p = 0.018) (Figure 1b).Exposure of CEP tissues to conditioned medium from activated neutrophils significantly increased the release of sGAG from the CEP tissues in a dose-dependent manner (25 mio/ml: 380.1% ± 177%, p = 0.012; 12.5 mio/ml: 123.7% ± 22.3%, p = 0.048, positive control: 545.0 % ± 302.8 %, p = 0.016) (Figure 1c) but there was no significant hydroxyproline release (not shown).Our data shows that neutrophils in MC1 bone marrow are activated and activated blood neutrophils degrade CEPs. Hence, neutrophils in MC1 bone marrow might promote and exacerbate CEP damage. Therefore, MC1 are not just reactive changes but can drive CEP damage that enhances a vicious inflammatory crosstalk with the adjacent disc.Figure 1.(a) Top 5 upregulated biological processes in MC1 bone marrow neutrophils (b) Representative flow cytometry image c) Relative sGAG released from CEP tissues. * P < 0.05.ConclusionWe identified neutrophils as a potentially important player in MC1. Therefore, the present findings could have implications for treatment strategies to mitigate CEP damage in MC1.REFERENCES:NIL.Acknowledgements:We thank the FOREUM Foundation for supporting this project.Disclosure of InterestsIrina Heggli: None declared, Mohamed Habib: None declared, Justin Scheer: None declared, Nick Herger: None declared, Tamara Mengis: None declared, Christoph Laux: None declared, Florian Wanivenhaus: None declared, Jose Miguel Spirig: None declared, Michael Betz: None declared, Mazda Farshad: None declared, Oliver Distler Speakers bureau: Bayer, Boehringer Ingelheim, Janssen, Medscape, Consultant of: 4P-Pharma, Abbvie, Acceleron, Alcimed, Altavant Siences, Amgen, AnaMar, Arxx, AstraZeneca, Baecon, Blade, Bayer, Boehringer Ingelheim, Corbus, CSL Behring, Galapagos, Glenmark, Horizon, Inventiva, Kymera, Lupin, Miltenyi Biotec, Mitsubishi Tanabe, MSD, Novartis, Pfizer, Prometheus, Redxpharna, Roivant, Sanofi and Topadur, Grant/research support from: Kymera, Mitsubishi Tanabe, Boehringer Ingelheim, Aaron Fields: None declared, Stefan Dudli: None declared.

Background:Modic type 1 changes (MC1) are vertebral bone marrow lesions associated with non-specific low back pain (LBP). The pathophysiology of MC1 includes inflammation, fibrosis, and high bone turnover. Bone marrow stromal cells (BMSCs) are key regulators of these processes: BMSCs contribute to inflammation by regulating myelopoiesis/osteoclastogenesis; BMSCs can differentiate into osteoblasts contributing to high bone turnover, and BMSCs can differentiate into pro-fibrotic myofibroblasts.Objectives:To identify dysregulated biological processes in MC1 BMSCs contributing to the pathobiology of MC1.Methods:Bone marrow aspirates were obtained from LBP patients with MC1 undergoing lumbar spinal fusion. Aspirates were taken prior to screw insertion. From each patient, a MC1 and a healthy control (HC) aspirate from the adjacent vertebral body was collected. BMSCs were isolated by plastic adherence and expanded. RNA from BMSCs passage 3 was sequenced (n=3 + 3) (Illumina Novaseq) and gene ontology of significantly dysregulated genes (p-value < 0.05) was analyzed. Specificity and rate of BMSC matrix adhesion were quantified: BMSCs (n=8 + 8) were seeded on fibronectin-coated, collagen-I-coated, and non-coated plastic dishes. BMSC adhesion was evaluated from 15min to 30min (Δ 30min - 15min). Percentage of adherent cells of MC1 and HC BMSC was compared with paired t-test. In order to identify integrins responsible for dysregulated cell-matrix adhesion, gene expression of 15 relevant integrins was measured by quantitative real-time PCR (qPCR). Normalized expressions were compared between MC1 and HC BMSC with paired t-test. Integrin β1 protein level was semi quantitatively analyzed by Western Blot (n = 5 + 5) and normalized to β-Actin expression.Results:By RNA sequencing, 154 genes were differentially expressed between MC1 and HC BMSCs (p-value ≤ 0.01; log2-ratio ≥ 0.5). Gene ontology enrichment analysis revealed an overrepresentation of the biological process “cell-matrix adhesion” among all significantly regulated genes (p-value < 9.3e-13). A change in cell adhesion was corroborated with adhesion assay. Binding (Δ 30min - 15min) to collagen I (MC1 + 16%, HC +10%, p-value = 0.10), fibronectin (MC1 + 17%, HC +6%, p-value = 0.03), and non-coated surface (MC1 + 46%, HC +35%, p-value = 0.05) was increased in MC1 (Figure 1). Integrin gene expression analysis revealed significant upregulation of integrin beta-1 gene (ITGB1) in MC1 vs. HC (fold change = 1.24, p-value = 0.047), whereas there was no significant difference between the other integrins tested. On protein level, integrin β1 was upregulated in MC1 in four out of five patients (Figure 2).Figure 1.Adhesion assay.Figure 2.Western Blot analysis.Conclusion:Adhesion of BMSCs to matrix and integrin β1 expression are increased in MC1. Integrin β1 is essential for cell-matrix adhesion and an important contributor to the initiation and progression of tissue fibrosis, a hallmark of MC1. Therefore, BMSCs and integrin β1 might be relevant novel targets for the treatment of MC1.Disclosure of Interests: :Irina Heggli: None declared, Susanne Epprecht: None declared, Tamara Mengis: None declared, Astrid Juengel: None declared, Michael Betz: None declared, Jose Spirig: None declared, Florian Wanivenhaus: None declared, Florian Brunner: None declared, Mazda Farshad: None declared, Oliver Distler Grant/research support from: Grants/Research support from Actelion, Bayer, Boehringer Ingelheim, Competitive Drug Development International Ltd. and Mitsubishi Tanabe; he also holds the issued Patent on mir-29 for the treatment of systemic sclerosis (US8247389, EP2331143)., Consultant of: Consultancy fees from Actelion, Acceleron Pharma, AnaMar, Bayer, Baecon Discovery, Blade Therapeutics, Boehringer, CSL Behring, Catenion, ChemomAb, Curzion Pharmaceuticals, Ergonex, Galapagos NV, GSK, Glenmark Pharmaceuticals, Inventiva, Italfarmaco, iQvia, medac, Medscape, Mitsubishi Tanabe Pharma, MSD, Roche, Sanofi and UCB, Speakers bureau: Speaker fees from Actelion, Bayer, Boehringer Ingelheim, Medscape, Pfizer and Roche, Stefan Dudli: None declared

BackgroundModic changes (MC) are painful vertebral bone marrow lesions and are often found in patients with chronic low back pain. The adjacent intervertebral disc (IVD) seems to play an important role: the rapidly degrading disc stands in an inflammatory cross-talk with the MC bone marrow and MC develop almost always simultaneously cranial and caudal to a degenerated IVDs. Few studies have investigated expression of inflammatory cytokines and proteases expressed by IVD cells adjacent to MC, however, how this affects the ECM degeneration has not been determined.ObjectivesThe aim of this study was to identify MC-specific mechanisms in degenerated discs. We hypothesize that the ‘Modic discs’ have a distinct disc matrix degradome.MethodsDegenerated lumbar IVDs from MC1 (n=26), MC2 (n=20) and non-MC (n=19) levels from gender and age matched patients undergoing spinal fusion surgery were collected. Degradome was measured with N-terminal amine isotopic labeling of substrates (TAILS) liquid chromatography tandem mass spectrometry (LC-MS/MS). TAILS allows to identify degraded proteins by detecting de novo N-terminal peptides. Sequence motifs were calculated using TwoSample Logo web application to identify significantly enriched amino acids around the cleavage site of the top 50 upregulated peptides. Since proteases have preferences for amino acid sequences, different sequence motifs can indicate activity of different proteases. Proteases were matched to cleavage sites of the top 50 enriched MC1 and MC2 peptides using TopFINDer database.ResultsMean degree of disc degeneration as measured by Pfirrmann grade was not significantly different between all groups (MC1: 3.8 ± 0.9, MC2: 4.2 ± 0.6, non-MC: 3.6 ± 0.8). A total of 487 (MC1), 419 (MC2), and 404 (non-MC) different protein fragments were detected of which 21.0%, 12.2% and 9.4% were unique to MC1, MC2, and non-MC, respectively (Figure 1a). Comparing MC1 to non-MC discs, the degradome was more complex with a variety of ECM fragments enriched (e.g. type II collagen, fibrinogen), while fragments of type I collagen, clusterin, and fibronectin were depleted (Figure 1b). In MC2 discs, mainly fibronectin and clusterin were enriched, while type I collagen was depleted (Figure 1b). Sequence motifs show unique cleavage preferences for each group (Figure 1c) indicating activity of different proteases in MC1 and MC2 discs. TopFinder identified neutrophil elastase as an important protease in all groups targeting cleavage of different proteins. In MC1 and MC2 IVDs kallikrein 3 as well as MMP9 and MMP2 are associated with found cleavage products. Finally, in MC1 TopFINDer uniquely identifies multiple fragments created by MMP2, MMP7, MMP12, MMP13 or cathepsin B.ConclusionMC1, MC2 and non-MC discs have different ECM degradomes that may be caused by the activity of different proteases. Differences in disc degeneration mechanisms and in the type and amount of bioactive ECM fragments explain why not all degenerating discs lead to adjacent MCs.REFERENCES:NIL.Acknowledgements:NIL.Disclosure of InterestsTamara Mengis: None declared, Irina Heggli: None declared, Nick Herger: None declared, Borbala Aradi-Vegh: None declared, Bernd Roschitzki: None declared, Jonas Grossmann: None declared, Florian Brunner: None declared, Roy Marcus: None declared, Mazda Farshad: None declared, Oliver Distler Speakers bureau: Bayer, Boehringer Ingelheim, Janssen, Medscape, Consultant of: 4P-Pharma, Abbvie, Acceleron, Alcimed, Altavant Siences, Amgen, AnaMar, Arxx, AstraZeneca, Baecon, Blade, Bayer, Boehringer Ingelheim, Corbus, CSL Behring, Galapagos, Glenmark, Horizon, Inventiva, Kymera, Lupin, Miltenyi Biotec, Mitsubishi Tanabe, MSD, Novartis, Pfizer, Prometheus, Redxpharna, Roivant, Sanofi and Topadur, Grant/research support from: BI, Kymera, Mitsubishi Tanabe, Stefan Dudli: None declared.

The splicing factor SF3B1 is recurrently mutated in various tumors, including pancreatic ductal adenocarcinoma (PDAC). The impact of the hotspot mutation SF3B1K700E on the PDAC pathogenesis, however, remains elusive. Here, we demonstrate that Sf3b1K700E alone is insufficient to induce malignant transformation of the murine pancreas, but increases aggressiveness of PDAC if it co-occurs together with mutated KRAS and p53. We further demonstrate that SF3B1K700E reduces epithelial–mesenchymal transition (EMT) and confers resistance to TGF-β1-induced cell death, and provide evidence that this phenotype is in part mediated through aberrant splicing of Map3k7. Taken together, our work suggests that SF3B1K700E acts as an oncogenic driver in PDAC through enhancing resistance to the tumor suppressive effects of TGF-β.

BackgroundVertebral bone marrow lesions known as Modic type 1 changes (MC1) are a major contributor to unspecific lower back pain. Pain may relate to the higher innervation of MC1 endplates. Blocking neo-innervation in MC1 could be a promising treatment approach for MC1. Bone marrow stromal cells (BMSC) can produce neurotrophic factors and are dysregulated in MC1.ObjectivesThe aim of the study was to identify if BMSC in MC1 support neo-innervation through release of neurotrophic factors.MethodsThe RNA sequencing data set (ENA PRJEB39993) of MC1 BMSC vs. intra-patient control vertebral BMSC (n=3 MC1 + 3 intra-patient control) was used to investigate enriched gene sets. From additional patients, BMSC were isolated from vertebral bone marrow aspirates (n=4 MC1 + 4 intra-patient control) and were co-cultured with the neuroblastoma cell line SH-SY5Y for 8 days. Neurite outgrowth from SH-SY5Y was quantified as a measure for neurotrophic activity using microscopy. Thirty neurotrophic cytokines were analyzed in the supernatant of the co-culture using C-Series Human Neuro Discovery Array C2.ResultsGene set enrichment analysis of MC1 vs. intra-patient control BMSC identified gene sets associated with BDNF signaling such as “BDNF TRKB signaling” (normalized enrichment score (NES) = 1.71, p<0.001) and “mBDNF and proBDNF regulation of GABA neurotransmission” (NES = 1.61, p<0.001) amongst the top enriched gene sets.SH-SY5Y cells co-cultured with MC1 BMSC compared to intra-patient control showed significantly increased neurite outgrowth after 4 days (p = 0.045), 6 days (p = 0.027), and 8 days (p = 0.031). (Fig. 1a).Cytokine array analysis revealed significantly more mature brain-derived neurotrophic factor (mBDNF) (p = 0.021) and ciliary neurotrophic factor (CNTF) (p = 0.030) in supernatant of MC1 vs. control BMSC (Fig. 1b).ConclusionNeurotrophic activity of MC1 BMSC is increased. This might be mediated by BDNF and CNTF. Therefore, BDNF and CNTF may represent interesting novel treatment approaches for MC1 that directly target pain mechanisms.REFERENCES:NIL.Acknowledgements:NIL.Disclosure of InterestsTamara Mengis: None declared, Irina Heggli: None declared, Nick Herger: None declared, Borbala Aradi-Vegh: None declared, Florian Brunner: None declared, Mazda Farshad: None declared, Oliver Distler Speakers bureau: Bayer, Boehringer Ingelheim, Janssen, Medscape, Consultant of: 4P-Pharma, Abbvie, Acceleron, Alcimed, Altavant Siences, Amgen, AnaMar, Arxx, AstraZeneca, Baecon, Blade, Bayer, Boehringer Ingelheim, Corbus, CSL Behring, Galapagos, Glenmark, Horizon, Inventiva, Kymera, Lupin, Miltenyi Biotec, Mitsubishi Tanabe, MSD, Novartis, Pfizer, Prometheus, Redxpharna, Roivant, Sanofi and Topadur, Grant/research support from: BI, Kymera, Mitsubishi Tanabe, Stefan Dudli: None declared.