Exposure of Patient-Derived Mesenchymal Stromal Cells To

Author Manuscript Published OnlineFirst on July 8, 2020; DOI: 10.1158/1541-7786.MCR-20-0091 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

1 Research Article

2 Exposure of patient-derived mesenchymal stromal cells to TGFB1 supports fibrosis 3 induction in a pediatric acute megakaryoblastic leukemia model 4 Theresa Hack1, Stefanie Bertram2, Helen Blair3, Verena Börger4, Guntram Büsche5, Lora 5 Denson1, Enrico Fruth1, Bernd Giebel4, Olaf Heidenreich3,6, Ludger Klein-Hitpass7, 6 Laxmikanth Kollipara8, Stephanie Sendker1, Albert Sickmann8,9,10, Christiane Walter1, Nils 7 von Neuhoff1, Helmut Hanenberg1,11, Dirk Reinhardt1, Markus Schneider1,* and Mareike 8 Rasche1,*

9 1 Department of Pediatric Hematology and Oncology, University Children’s Hospital Essen, 10 Essen, Germany 11 2 Department of Pathology, University Hospital Essen, Essen, Germany 12 3 Newcastle University, Wolfson Childhood Cancer Research Centre, Translation and Clinical 13 Research Institute, Newcastle upon Tyne, United Kingdom 14 4 Institute for Transfusion Medicine, University Hospital Essen, Essen, Germany 15 5 Department of Pathology, Hannover Medical School, Hannover, Germany 16 6 Princess Máxima Center for Pediatric Oncology, Utrecht, The Netherlands 17 7 Department of Cell Biology, University Hospital Essen, Essen, Germany 18 8 Leibniz-Institut für Analytische Wissenschaften – ISAS – e.V., Dortmund, Germany 19 9 Department of Chemistry, College of Physical Sciences, University of Aberdeen, Aberdeen, 20 Scotland, United Kingdom 21 10 Medizinische Fakultät, Medizinische Proteom-Center (MPC), Ruhr-Universität Bochum, 22 Bochum, Germany 23 11 Department of Otorhinolaryngology and Head/Neck Surgery, Heinrich Heine University, 24 Düsseldorf, Germany 25 * These authors contributed equally to this work.

26 Keywords: leukemic bone marrow niche, myeloid leukemia in children with Down 27 syndrome (ML-DS), acute megakaryoblastic leukemia (AMKL), TGFB1, bone marrow 28 fibrosis

29 Running title: TGFB1 supports fibrosis in pediatric AMKL

30 Funding: G.B. was supported by the Cluster of Excellence “Rebirth, humanized animal

31 models”, funded by the Deutsche Forschungsgemeinschaft, Germany. T.H. was funded by

32 the Boehringer Ingelheim Fonds with a travel grant. D.R. was funded by the Essener

33 Elterninitiative zur Unterstützung krebskranker Kinder e.V.. S.S. was funded by the

34 Essener Ausbildungsprogramm "Labor und Wissenschaft" für den aerztlichen Nachwuchs.

35 Correspondence to: Markus Schneider, Department of Pediatric Hematology and

36 Oncology, University Children’s Hospital Essen, 45147 Essen, Germany, email:

37 [email protected], phone: +49-201-723-1056, fax: +49-201-723-5591;

Downloaded from mcr.aacrjournals.org on October 1, 2021. © 2020 American Association for Cancer Research. Author Manuscript Published OnlineFirst on July 8, 2020; DOI: 10.1158/1541-7786.MCR-20-0091 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

38 Mareike Rasche, Department of Pediatric Hematology and Oncology, University Children’s

39 Hospital Essen, 45147 Essen, Germany, email: [email protected], phone:

40 +49-201-723-1051, fax: +49-201-723-5591

41 Conflicts of Interest: The authors declare no conflict of interest regarding this study. The 42 funders had no role in the design of the study; in the collection, analyses, or interpretation 43 of data; in the writing of the manuscript, or in the decision to publish the results.

44 Abstract counts: 241

45 Word counts: 5004

46 Number of figures and tables: 5

48 Abstract: Bone marrow fibrosis (BMF) is a rare complication in acute leukemia. In 49 pediatrics, it predominantly occurs in acute megakaryoblastic leukemia (AMKL) and here 50 especially in patients with trisomy 21, called myeloid leukemia in Down syndrome (ML- 51 DS). Defects in mesenchymal stromal cells (MSCs) and cytokines specifically released by 52 the myeloid blasts are thought to be the main drivers of fibrosis in the bone marrow niche 53 (BMN). In order to model the BMN of pediatric AMKL patients in mice, we first established 54 MSCs from pediatric patients with AMKL (n=5) and ML-DS (n=9). Healthy donor (HD) 55 control MSCs (n=6) were generated from unaffected children and adolescents ≤18 years 56 of age. Steady-state analyses of the MSCs revealed that patient-derived MSCs exhibited 57 decreased adipogenic differentiation potential and enrichment of proliferation-associated 58 genes. Importantly, TGFB1 exposure in vitro promoted early profibrotic changes in all three 59 MSC entities. To study BMF induction for longer periods of time, we created an in vivo 60 humanized artificial BMN subcutaneously in immunodeficient NSG mice, using a mixture of 61 MSCs, HUVECs and Matrigel. Injection of AMKL blasts as producers of TGFB1 into this 62 BMN after eight weeks induced fibrosis grade I/II in a dose-dependent fashion over a time 63 period of four weeks. Thus, our study developed a humanized mouse model that will be 64 instrumental to specifically examine leukemogenesis and therapeutic targets for AMKL 65 blasts in future. 66 Implications: TGFB1 supports fibrosis induction in a pediatric acute megakaryoblastic 67 leukemia model generated with patient-derived mesenchymal stromal cells.

68 Introduction

69 Acute megakaryoblastic leukemia (AMKL) is diagnosed in approximately 10% of 70 pediatric acute myeloid leukemia (AML) patients [1]. Here, AMKL occurs in two different 71 settings: myeloid leukemia in children with Down syndrome (ML-DS) and also in patients 72 with euploid genomes [1-3]. ML-DS is preceded by a transient abnormal myelopoiesis 73 (TAM) in infants, accompanied by GATA1s mutation in blasts and liver fibrosis. Despite 74 excellent event-free and overall survival, patients with ML-DS still have an increased risk of 75 severe chemotherapy-associated complications [2-5]. In contrast, pediatric AMKL in 76 patients without DS is a heterogeneous disease and usually arises in children until the age 77 of three years with mostly poor outcome [1]. 78 The physiological bone marrow niche (BMN) is a complex network consisting of 79 extracellular matrix (ECM) proteins, non-hematopoietic cells as well as soluble factors. 80 Mesenchymal stromal cells (MSCs) as multipotent progenitor cells are important members 81 of the BMN, which contribute to these physiological functions and can differentiate into 82 other cells in the BMN such as osteoblasts and adipocytes [6]. However, in context of 83 myeloid disorders such as AMKL [1, 7], myeloproliferative neoplasm (MPN), hairy cell

84 leukemia and myelodysplastic syndrome [8], MSCs are key for the pathological changes 85 that occur, as they can transform to contractile myofibroblast with high α-smooth muscle 86 actin (αSMA) expression, extensive secretion of ECM proteins and increased stromal 87 fibers [9, 10]. These changes ultimately lead to bone marrow fibrosis (BMF), which 88 histologically is defined by decreased or absent adipocytes [11], an increase in bone 89 marrow stromal fibers [10] and in late stage occurrence of osteosclerosis [12]. As most 90 disorders with increased bone marrow stromal fibers are associated with abnormalities of 91 the number or function of megakaryocytes [10], it is not surprising that the histological 92 pictures of AMKL in adults with consecutive development of BMF is indistinguishable to the 93 changes observed in the BMN in the pediatric AMKL patients with and without trisomy 21 94 [7]. 95 A major factor for the pathological changes that occur in the leukemic BMN in AMKL 96 are increased concentrations of TGFB1, which is predominantly secreted by 97 megakaryocytes and platelets [13] and also AMKL blasts [8, 14]. TGFB1 is one of the 98 strongest inducer of collagen synthesis and considered as key driver of BMF [13, 15, 16]. 99 Several reports support the profibrotic role of TGFB1 in AMKL [17, 18], but it remains 100 unclear whether the secretion of TGFB1 from leukemic megakaryoblasts may be sufficient 101 to induce the BMF by itself, leading to a self-reinforcing leukemic BMN, or if additional 102 factors such as pathological changes in non-hematopoietic cells e.g. MSCs are essential 103 prerequisite for in the induction of BMF [19, 20]. 104 We therefore decided to use our unique resources in pediatric leukemia to address the 105 question whether specific changes in the MSCs derived from children and adolescents 106 with AMKL (with or without trisomy 21) exist that are necessary for the BMF observed in 107 pediatric and also adult AMKL or whether the simple overexpression of TGFB1 by AMKL 108 blasts is already sufficient for the BMF to occur. To this end, we analyzed MSCs derived 109 from pediatric AMKL with (ML-DS) or without Down syndrome or from healthy donors 110 (HDs) ≤18 years in vitro under different conditions or in vivo by generating a humanized 111 artificial BMN in immunodeficient mice. We further tested which pathological changes can 112 be induced in the humanized BMN in vivo by TGFB1. 113 Elucidating the interactions between leukemic blasts and the MSCs derived from 114 AMKL patients without or with Morbus Down in an in vitro or in vivo model systems will 115 ultimately be instrumental to identify potentially druggable target genes to improve the 116 outcome of not just AMKL but also other hematological conditions with BMF.

117 Materials and Methods

118 Cell lines, patients and healthy donor samples

119 In order to mimic a leukemic BMN with marrow fibrosis as occurring in the context of 120 AMKL in children and adolescents (<18 years of age) either being affected or not by 121 Morbus Down, patients diagnosed with either ML-DS or AMKL according with 2016 122 revision of the World Health Organization classification of myeloid neoplasms and acute 123 leukemia were enrolled in this study [2, 3]. After informed consensus of the patients and/or 124 their legal guardian, we isolated multipotent MSCs from bone marrow aspirates of ML-DS 125 (n=9) and AMKL patients (n=5) according to established protocols [21]. We also isolated 126 MSCs from HD (n=6) ≤18 years of age (Supplementary Table S1). MSCs were generated 127 from human BM mononuclear cells (MNCs), isolated from BM aspirates using Ficoll- 128 PaqueTM Plus (GE Healthcare) [21]. MSCs were expanded in DMEM with GlutaMax 129 (ThermoFisher), 10% (v/v) human platelet lysate (PL), 1% penicillin/streptomycin at 37°C, 2 130 5% CO2 and high humidity in Nunc™ EasyFill™ Cell Factory™ Systeme 2528 cm 131 (ThermoFisher) until passage (P) 3 and cryoconserved. MSCs experiments were 132 performed with P3, cells were pre-cultured until a density of 80-90% in T-75 tissue culture 133 flasks. After ≤2 passages, the isolated cells were analyzed for the typical hallmarks of 134 MSCs according to the ISCT minimal criteria for defining multipotent MSCs [22]. 135 The study was approved by the Ethics Committee including the approval for material 136 of healthy donors and patients (local ethic committee of the University Duisburg- Essen 137 (AZ: 16-7069-BO), local ethic committee of the Hannover Medical School (AZ: 1381-2012) 138 and local ethic committee of the University Muenster (AZ: 3 V Creutzig). The ML-DS cell 139 line CMK (DSMZ no. ACC392) was obtained from the DSMZ and cultured in RPMI 1640 140 (Lonza), 10% (v/v) fetal bovine serum and 1% penicillin/streptomycin. Human umbilical 141 vein endothelial cells (HUVECs) were purchased from PromoCell and cultured in EBM-2 142 (Lonza). Cell authentication was conducted using the GenePrint® 10 System (Promega) 143 with analysis on a 3500 Genetic Analyzer (ThermoFisher). Cells were tested periodically 144 and confirmed as Mycoplasma free by the PCR-based method (Myco-For: 5’- 145 gggagcaaacaggattagataccct-3’, Myco-Rev: 5’-tgcaccatctgtcactctgttaacctc-3’ and Myco- 146 Rev2: 5’-tgcaccatctgtcactccgttaacctc-3’ ).

147 Immunophenotyping

148 The immunophenotypic characterization of MSCs followed the criteria of the 149 International Society of Cellular Therapy (ISCT) [22]. To control for background 150 fluorescence and determine the threshold for positive expression, unstained MSCs were 151 acquired and used as negative control. Expression of individual markers was recorded, as 152 percentage of positive cells after subtracting the baseline auto-fluorescence levels (list of 153 antibodies, see Supplementary Material and Methods). Data were analyzed with Kaluza 154 software 2.1 (Beckman Coulter).

155 Adipogenic differentiation

156 MSCs were seeded in three independent experiments in 24-well plates and cultured 157 for 16 days in MesenCultTM Adipogenic Differentiation Medium (STEMCELL Technologies) 158 according to the manufacturer’s instructions. For treatment, MSCs were treated with 10 159 ng/ml TGFB1. Fat drops of adipocytes were stained with Oil Red O (Sigma-Aldrich). 160 Images were taken from the stained cell layer and analyzed for the percentage/amount of 161 positively stained areas, followed by quantification using Image J program [23]. Each 162 experiment was separately quantified.

163 Osteogenic differentiation

164 MSCs were seeded in three independent experiments in 24-well plates and cultured 165 for 16 days in OsteoMAX XFTM Differentiation Medium (Sigma-Aldrich) according to the 166 manufacturer’s instructions. For treatment with TGFB1, MSCs were treated with 10 ng/ml 167 TGFB1. Calcium depots of osteoblasts were stained with Alizarin Red S (Sigma-Aldrich). 168 To evaluate the osteogenic differentiation, stained cells were captured by optical 169 microscopy (4x magnification) and quantified by using Image J (see details in 170 Supplementary Material and Methods). Each experiment was separately quantified.

171 Proliferation assay

172 CellTiter Glo® Luminescence Assay (Promega) was performed in triplicates and 173 repeated two times with 1x104 MSCs per well in a 96-well plate. The cells were measured 174 after 24 hours according to the manufacturer’s instructions.

175 Colony forming unit fibroblast assay

176 Colony forming unit-fibroblast assay (CFU-F) was performed in 6-well plates with 200 177 cells in triplicates and repeated two times according to the manufacturer’s instructions of 178 MesenCult™-ACF Plus Culture Kit (STEMCELL Technologies). After 9 days, colonies 179 were fixed, stained with Giemsa. Colonies made up of more than 40 cells were scored as 180 CFU and were counted.

181 Gene expression analysis using microarrays

182 MSCs were trypsinized and lysed with RLT buffer (Qiagen) containing 0.1% β 183 Mercaptoethanol. RNA extraction with DNase digestion was performed with RNeasy Mini 184 Kit (Qiagen). For microarray analyses, we used the Affymetrix GeneChip platform 185 employing the WT Plus assay for sample preparation and microarray hybridization. 200 ng 186 total RNA was used for the human Clariom S microarrays (Affymetrix) (Supplementary

187 Material and Methods for details). GeneChip data have been submitted to GEO 188 (GSE138861).

189 Quantitative RT-PCR after TGFB1 treatment

190 MSCs were treated for 48 hours with 10 ng/ml TGFB1. Total RNA from MSCs were 191 isolated as described above accept from DNase digestion. cDNA was synthesized using 192 SuperScriptTM VILOTM cDNA synthesis kit (ThermoFisher). Quantitative real time 193 polymerase chain reaction (qRT-PCR) was carried out using the StepOnePlusTM 194 (ThermoFisher) and commercial TaqMan Gene Expression Assays (Supplementary Table 195 S4; ThermoFisher). Relative PPARG and IBSP quantification of untreated MSCs was 196 calculated using 2ΔCT values with the house keeping genes GAPDH and TUB1A1. All 197 samples were set relative to one HD. Relative quantification between treated to untreated 198 was calculated using the -ΔΔCT values with the house keeping genes.

199 In vivo artificial bone marrow niche formation

200 NOD.Cg-PrkdcscidIl2rgtm1WjL/SzJ (NSG) mice were purchased from the Jackson 201 Laboratory. Mice were housed in specific pathogen-free conditions in the animal facility of 202 University Hospital Essen. All mouse experiments were performed with the approval of the 203 local ethics committee (LANUV) for animal use under the permission A1503/15 (Date of 204 approval 07/2015). 205 MSCs (1x106) were mixed with HUVECs (1x106) in 0.1 ml Matrigel (Corning) and 0.1 206 ml OsteoMAX XFTM Differentiation Medium (Sigma-Aldrich) immediately before 207 subcutaneously injections into the both flanks of immunodeficient NSG mouse (12-20 208 weeks old) [24, 25]. After 8 weeks, CMK cells (1x106 or 5x106) were injected into the 209 formed artificial bone marrow niche (Ossicle-like structure). The mice were sacrificed after 210 12 weeks in total. Ossicle-like structure were harvested, fixed in 4% neutral buffered 211 Formalin (Morphisto) and embedded in paraffin. Sections of 3 μm were produced. 212 Histological standard stains were performed: Hematoxylin and eosin (HE staining kit; 213 Morphisto), Masson Goldner (MG) Trichrom (staining kit; Morphisto) light-green was 214 replaced with aniline blue and the silver impregnation method, following Gomori’s 215 technique. For immunohistochemical staining the following primary antibodies were used 216 (Supplementary Table S5). 217 For antigen retrieval the sections were first incubated with EDTA pH 9, accept from 218 sections for alkaline phosphatase (ALP) staining. Sections for ALP were first incubated 219 with sodium citrate buffer (pH 6) and were further processed with the ZytoChem Plus 220 (HRP) Polymer Kit (Zytomed Systems) according to the manufacturer’s instructions.

221 Slides were scanned with the digital whole slide scanner Aperio AT2 (400 slide 222 capacity; lens setting 40x) by the central facility of the Westdeutsche Biobank Essen, using 223 the image management system Aperio Image Scope (Version 12.2.3.8013). To evaluate 224 the size of ossicle-like structure, we marked the edge of every ossicle-like structure by 225 hand and calculated the area in mm2. The histopathological analysis of the generated 226 ossicle-like structure was performed by an experienced pathologist determining the 227 obtained tissue as humanized BMN. In addition the evaluation of the fibrosis grade was 228 performed in accordance with the European Consensus on Grading Bone Marrow criteria 229 on the basis of the fibrotic fibers, identified in the silver stains, as hallmark for BMF [12].

230 Statistical analysis

231 Data were analyzed using GraphPad Prism Software 8. For comparison between the 232 groups of MSCs, significance was determined with the unpaired two-sided student’s t-test 233 with equal variances except for PPARG qPCR data, here we performed an unpaired 234 single-sided student’s t-test, because we expected only a down regulation. For comparison 235 between treated vs. untreated, significance was determined with the paired two-sided 236 student’s t-test with equal variances. Values of experimental data are expressed as mean 237 of biological replicates. P-values (*P<0.05; **P<0.01; ***P<0.001) were considered to be 238 statistically significant.

239 Results

240 Immunophenotyping demonstrates interindividual heterogeneity, but no disease- 241 specific expression patterns. 242 Initial characterization of the required criteria for MSCs, adherence to plastic tissue, 243 immunophenotypic profiling of required marker by flow cytometric analysis and 244 differentiation under standard in vitro differentiation conditions [22], revealed homogenous 245 features and thus confirmed that the patient-derived MSCs fulfilled the minimal ISCT 246 criteria for MSCs on several levels (see Figure S1). 247 In order to extensively characterize the MSCs from the different sources, we stained 248 the cells with a panel of 30 antibodies including classical surface markers, 5 integrins and 249 6 tetraspanins by flow cytometry. Although subtle individual differences between the 250 different MSCs were detectable, the extended phenotypic analysis of the cell surface 251 antigens failed to identify major differences between the ML-DS-, AMKL- and HD-derived 252 MSCs (Supplementary Table S2), thus suggesting that distinct functional properties of 253 these MSCs might only be apparent, if the cells are challenged by other means. As TGFB1 254 has been reported to play an important role in formation of the BMF in AMKL, we also 255 checked whether the inactive form of TGFB1, LAP TGFB1, is differentially expressed on

256 the MSCs from the three different sources. The results however demonstrated that TGFB1 257 is not expressed in any of the patient- or HD-derived MSCs. 258 259 Disease-derived MSCs exhibit a decreased adipogenic differentiation potential 260 Next, we compared the dynamics of the differentiation potential of the three different 261 MSC types. The HD-, ML-DS- and AMKL-derived MSCs all showed an osteogenic 262 differentiation potential (Figure 1A), that was comparable between the three MSC entities 263 (Figure 1B). Accordingly, the expression levels of IBSP, an early differentiation marker of 264 osteogenesis [26], were similar in the non-differentiated MSCs of all three entities (Figure 265 1C). 266 In contrast, the adipogenic differentiation of ML-DS- and AMKL-derived MSCs was 267 significantly decreased when compared to the differentiation of HD-derived MSCs. 268 Specifically, the adipogenically differentiated HD-derived MSCs showed clear formation of 269 fat drops, whereas the fat drops were largely missing in the ML-DS-derived MSCs (Figure 270 1D). Quantification revealed 3 to 11% stained area of fat drops in HD-derived MSCs 271 (Figure 1E). ML-DS-derived MSCs displayed only a stained area of 0 to 6% and a 4-fold 272 reduction in ML-DS-derived MSCs (*P<0.05), while AMKL-derived MSCs only showed on 273 average a 2-fold reduction in the adipogenic differentiation potential. In support of this 274 pathological differentiation potential, the expression of PPARG, the master regulator of 275 adipogenesis [27], was already significantly decreased in non-differentiated patient-derived 276 MSCs (Figure 1F). Thus, the MSCs of ML-DS and AMKL patients showed a decreased 277 adipogenic differentiation potential which seems to be intrinsically determined already in 278 the earliest passage by a lower expression of PPARG. 279 280 Gene expression profiling indicates a higher proliferation activity in disease-derived 281 MSCs 282 We next analyzed whether gene expression profiles would allow to readily distinguish 283 between the patient-derived and the HD-derived MSCs. To this end, mRNA was extracted 284 from non-differentiated early passage MSCs of 8 ML-DS and 5 AMKL patients as well as 5 285 HDs and then analyzed on the Affymetrix GeneChip platform. Unsupervised hierarchical 286 clustering analysis revealed heterogeneity but no consistent clustering of the individual 287 samples within their disease entities existed (Supplementary Figure S2). 288 Analysis of the expression profiles with the PartekGS gene set enrichment analysis 289 (GSEA) software using standard filter settings revealed that the hallmark gene sets 'E2F- 290 targets' and 'G2M-checkpoint' were significantly enriched in AMKL- and ML-DS-derived 291 MSCs (p<0.01; Table 1, Supplementary Figure S3) when compared to the HD-derived 292 MSCs gene expression profiles, thus suggesting that higher proliferation activities were

293 present in early passage MSCs from both disease entities when compared to HD-derived 294 MSCs. In addition, the 'mitotic spindle' gene set, also associated with cellular proliferation, 295 was significantly higher expressed in ML-DS-derived MSCs (p<0.01, Table 1, 296 Supplementary Figure S3). 297 It is known that the interferon response is constitutively hyperactive/activated in DS 298 cells, most likely due to the fact that interferon alpha receptor is located on chromosome 299 21 [28]. We therefore interpreted the findings that genes of the 'Interferon-alpha response' 300 gene set were significantly enriched in ML-DS-derived MSCs but not in AMKL-derived 301 MSCs (Table 1) as confirmation for the validity of our analyses. 302 In order to verify whether the increased expression levels for proliferation-associated 303 gene sets in patient-derived MSCs is reflected by increased proliferation of these cells 304 compared to HD-derived MSCs, we analyzed the proliferation activity of the cells after 24 305 hours and after 9 days, respectively. As shown in Figure 2A and 2B, neither the short-term 306 proliferation analysis by a commercial assay (Promega) nor the long-term CFU-F assay 307 revealed any differences between the three types of MSCs. 308 309 TGFB1 impairs the osteogenic and adipogenic differentiation of MSCs and 310 stimulates fibrosis 311 TGFB1 is secreted in large quantities by the leukemic megakaryoblasts [10, 17] and is 312 considered to be a key driver for the BMF that often develops in patients with AMKL [10, 313 15]. Therefore, we added 10 ng/ml TGFB1 during the adipogenic and osteogenic 314 differentiation of MSCs from HDs (n=5), ML-DS (n=7) and AMKL (n=5) donors, 315 respectively. As shown in Figure 3A, the addition of TGFB1 clearly reduced the osteogenic 316 differentiation in 12 different primary MSC lines from ML-DS and AMKL patients and also 317 in the 5 MSC lines from HDs (HD: *P<0.05, ML-DS: **P<0.01, AMKL: **P<0.01). In 318 addition, trying to differentiate the primary MSCs to adipogenesis is completely abolished 319 in all MSC lines that, in the absence of TGFB1, were still capable of differentiation into 320 adipocytes (Figure 3B): all MSCs derived from HDs, one MSC line from a patient with ML- 321 DS and 3 MSC lines from patients with AMKL. 322 Next, we analyzed whether the effects of TGFB1 exposure on the expression levels of 323 genes that are associated with either adipogenesis or osteogenesis can already be 324 detected early in MSCs of all three entities. As shown in Figure 3C, treatment with TGFB1 325 led to a significantly reduced expression of adipogenic (PPARG) [27] and osteogenic 326 (IBSP) [26] hallmark genes (***P<0.001) in standard culture conditions without exogenous 327 stimulation of differentiation (Figure 3B). In addition, the expression of genes that are 328 associated with fibrosis (ACTA2, COL1A1 and COL3A1) [9, 29] were significantly 329 increased after TGFB1 treatment (***P<0.001).

330 In summary, solely the exposure of primary MSCs from either ML-DS or AMKL 331 patients and also MSCs from HDs to TGFB1 clearly inhibited their differentiation towards 332 osteoblasts and adipocytes. Already after 48 hours, TGFB1 had induced a fibrotic gene 333 expression profile in MSCs, regardless of their origin, that is characterized for BMF, 334 decreased adipogenic differentiation and increased fibrosis marker expression. 335

336 Development of a robust in vivo model to study the leukemic BMN 337 Finally, in order to better understand the complex interaction between leukemic blasts 338 and MSCs in the leukemic BMN over time, we established an in vivo model for a leukemic 339 BMN. Our model was based on the protocols from Chen et al. and Reinisch et al. to 340 generate so called human ossicles in vivo in mice [24, 25]. 341 As only very limited cellular material was available from our pediatric patients, we had 342 to initially expand the MSCs further. In addition, we used HUVECs instead of more 343 immature endothelial colony forming cells (ECFCs) as a modification to the protocol, which 344 was already applied by multiple groups (reviewed in [30]). We mixed the patient and HD- 345 derived MSCs with HUVECs and Matrigel and then subcutaneously injected the mixtures 346 into both hind limb flanks of NSG mice. 8 weeks later, we performed direct injection of the 347 human leukemic megakaryoblast cell line CMK as TGFB1 source [31] into the ossicle-like 348 structure. After 12 weeks in total, the mice were sacrificed and the uninjected and injected 349 ossicle-like structures were harvested for further analyses (Figure 4A). 350 Overall, 40 ossicle-like structures were generated using MSCs from the different 351 entities, 16 of which were injected with the CMK leukemic megakaryoblasts. The 352 histological stains HE as well as MG allowed to identify the normal structures in the 353 ossicle-like structures including capillaries and erythrocytes (Figure 4B) and the injected 354 MSCs were visualized in the tissues using the VIM stain. Using a human CD31 antibody 355 revealed that only a minor proportion of the endothelial cells were stained, suggesting that 356 also murine cells contributed to the endothelium in these artificial structures. Importantly, 357 the positive staining of cells for COL1 and ALP indicated that at least early stages of bone 358 formation were present in these ossicle-like structures, as these two proteins are accepted 359 as early differentiation markers for osteogenesis [26, 32, 33]. Histologically, the origin of 360 the MSCs did not influence the formation and morphology of the ossicle-like structures 361 (data not shown). We therefore concluded that injection of MSCs-HUVECs-Matrigel 362 mixtures was able to generate an artificial mostly human BMN in NSG mice in vivo, 363 independent of the MSCs origin. Successful engraftment of leukemic megakaryoblasts in 364 the ossicle-like structures after 8 weeks led to a dramatic increase in the size of the 365 organoids, mainly due to the growth of the leukemic cells, as visualized by human CD45 366 staining (Figure 4C). 11

367 Finally, to study the effect of the interaction between the leukemic megakaryoblasts 368 and the BMN, we injected one or five million CMK cells 8 weeks after starting the ossicle 369 formation into the structures palpable under the skin in the flanks of the mice. Injected 370 animals were sacrificed 4 weeks later without any treatment and the ossicle-like structures 371 were harvested and histologically analyzed. When the sizes (surface areas of the tissue 372 sections) of untreated ossicle-like structures were compared to those injected with 373 leukemic megakaryoblasts (Figure 4D), ossicle-like structures with leukemic 374 megakaryoblasts showed significant increases in size (+CMK (1x106): ***P<0.001, +CMK 375 (5x106): ***P<0.001). Again, no differences in the sizes of the ossicle-like structures were 376 noted for the entity of MSCs that were used in these experiments (data not shown). 377 378 Leukemic megakaryoblastic cells induced BMF in the generated leukemic BMN 379 As the leukemic BMN of AMKL is characterized by a varying degree of BMF, we 380 histologically analyzed the ossicle-like structures according to the European Consensus on 381 Grading Bone Marrow [12]. In order to evaluate to which degree the injected leukemic 382 megakaryoblasts, as the local source of TGFB1 [31], had induced BMF in the ossicle-like 383 structures, we used the silver staining to reveal reticulin fibers in the humanized BMN. As 384 shown in Figure 4E and F, only ossicle-like structures with injected leukemic 385 megakaryoblasts displayed fibrotic fibers with grade I and II fibrosis. Ossicle-like structures 386 injected with 1x106 CMK cells developed grade I fibrosis in 70% of cases. All ossicle-like 387 structures injected with 5x106 CMK cells developed fibrosis (grade I in 67% and grade II 388 fibrosis in 33%). Importantly, the induction of fibrosis by the leukemic megakaryoblasts 389 occurred in all MSC lines, independent of their origins (Supplementary Table S3, data not 390 shown).

391 Discussion

392 This study was designed to investigate the interaction of leukemic cells and the BMN 393 in pediatric patients with AMKL using cells from children and adolescents with or without 394 Morbus Down. To this end, we characterized and compared bone marrow-derived MSCs 395 which were isolated at the time of diagnosis from pediatric AMKL and ML-DS patients. 396 MSCs are one of the main cell types forming the BMN and have been implemented of at 397 least contributing to the pathological changes in the BMN that often occur in AMKL and 398 other myeloid malignancies, namely BMF [34, 35]. Importantly, we also were able to 399 compare these AMKL patient-derived MSCs with MSCs from healthy pediatric donors ≤18 400 years of age. Although the HDs were older (mean age 12.5 years) compared to our ML-DS 401 (mean age 9 months) and AMKL (mean age 27 months) patients, no clear differences in 402 the immunophenotype between the MSCs of the AMKL and ML-DS patients or HDs were 12

403 apparent, despite using a rather large panel of 30 monoclonal antibodies in flow cytometry 404 to analyze MSCs in early passages. Therefore, although possible [36, 37], we do not 405 consider that the ages ot the MSC donors introduced a relevant bias in our experiments. In 406 addition the osteogenic differentiation potential, was comparable between the patient- and 407 the HD-derived MSCs. In contrast, using differentiation into the adipocytes as readout, we 408 demonstrated a clearly diminished differentiation potential in the primary MSC lines 409 derived from AMKL and ML-DS patients compared to the HD-derived MSCs. We were also 410 able to support these observations by the finding that expression of the master regulator 411 gene of adipogenesis PPARG was already down-regulated in non-differentiated MSCs 412 derived from both types of patients. It is noteworthy, that this clearly impaired adipogenesis 413 in the patient-derive MSCs was the only difference that unequivocally allowed to 414 distinguish between disease-associated and healthy donor MSCs, despite the remarkable 415 interindividual heterogeneity of the primary cells in all experiments. 416 Although MSCs from pediatric patients have already been described [38], our analysis 417 is the first characterization of MSCs in pediatric AMKL and ML-DS in the literature. We 418 therefore could only compare our findings to those reported for MSCs characterizations in 419 adult AML patients. In this analysis, it became quickly evident that the findings in MSCs 420 from adults are very heterogenous discussing either a decreased [39, 40], a normal [41, 421 42] or an increased [41, 42] adipogenic differentiation potential. A similar heterogeneity 422 was also reported for the immunophenotypes and the osteogenic differentiation potential . . 423 In addition, comparing the findings in our MSC study and to reports in the literature is 424 rather inadequate due to three main reasons. Firstly, almost all studies use a mixture of 425 MSCs from a variety of leukemic subtypes and none purely concentrated on AMKL 426 patients, as this type of AML is very rare in adults. Secondly, most MSC studies in the 427 literature use MSCs derived from heavily (pre-)treated and/or relapsed adult AML patients 428 [39-42], while we obtained our MSCs from patients at diagnosis prior to any treatment. 429 Thirdly, different culture conditions in the studies most likely influenced the results and 430 makes any comparison between different studies more problematic: e.g. the use of platelet 431 lysates instead of animal serum already changes the basic characteristics of the MSCs 432 [43, 44]. 433 In order to detect more subtle differences between the three MSC entities, we next 434 performed unsupervised gene expression analysis on 18 MSC lines, however, this 435 analysis did not allow to define clusters for the MSCs from AMKL and ML-DS patients or 436 the HDs. Analysis of hallmark gene sets revealed that a slight but significant increase in 437 proliferation-associated genes exists in the patient-derived MSCs at early passage, but the 438 differences were too small to be detectable in vitro without introducing differentiation of the 439 cells.

440 Overall, our in vitro model is consistent with the clinical picture of the leukemic, fibrotic 441 BMN in AMKL and supports TGFB1 as main driver of BMF. Here, it should be noted that 442 the effects of TGFB1 on MSCs can also be concentration-dependent which adds another 443 level of complexity: Lieb et al. 2004- demonstrated in vitro that high TGFB1 concentration 444 (10 ng/ml) decrease osteogenic differentiation in MSCs, while low dose (0.1-1 ng/ml) 445 stimulates osteogenic differentiation [45].In addition, Battula et al. and Hanoun et al. 446 reported the induction of an osteoblastic niche in adult AML [40, 46], while Frisch et al. 447 described a severe functional inhibition of osteoblastic cells in a murine AML model [47]. 448 The pathological changes in the BMN therefore seem to depend not only on the amount of 449 TGFB1, but also on other factors including PDGF and EGF secreted by the individual 450 blasts of each patient [15, 16]. 451 In adult patients, several stages of BMF occur over time [12]. As most cell culture 452 experiments with primary MSCs are rather arbitrary and too short, we decided to create an 453 artificial in vivo model for a humanized leukemic BMN. Based on the initial work by Chen et 454 al. and Reinisch et al. [24, 25] and essential improvements by others (reviewed in [30]) we 455 injected early passage primary patient- and HD-derived MSCs mixed with HUVECs and 456 Matrigel subcutaneously on both flanks of immunodeficient NSG mice. Importantly, early 457 passage HUVECs implanted with Matrigel can already form some sort of a human 458 vascular network when subcutaneously injected, when MSCs are co-implanted with the 459 HUVECs, also perivascular functions of the MSCs are present in this newly formed tissue 460 [30]. We also demonstrated early stages of bone formation in our artificial BMN, as proven 461 by the positive staining for the early osteogenic differentiation markers ALP and COL1. 462 Therefore, we would like to propose the term “ossicle-like structure” for our artificial BMN 463 and thereby intend to clearly distinguish our in vivo model from the “classical” ossicle as 464 described by Reinisch et al. [25]. 465 Our Ossicle-like structures containing human MSCs formed an artificial BMN over a 466 time period of 8-12 weeks. 24 of these ossicle-like structures were analyzed histologically 467 and never demonstrated any histological sign of fibrosis. However, injection of leukemic 468 megakaryoblasts producing TGFB1 induced fibrotic fibers and grade I and II fibrosis 469 according to the European Consensus on Grading Bone Marrow [12] within 4 weeks, 470 regardless of the origin of MSCs. When increasing the number of injected AMKL blasts five 471 times, the grade of developing fibrosis was remarkably increased, probably by the local 472 increase of TGFB1 [45]. Thus, a humanized leukemic BMN with primary MSCs from 473 pediatric patients can be formed in vivo in immunodeficient mice from the sparse material 474 obtained from the patients prior to any treatment, thus allowing long-term studies to 475 specifically target leukemic blasts from pediatric as well as adult AMKL patients in the 476 BMN.

477 In addition, the results obtained in our in vivo and also in vitro model strongly support 478 the dominant role of TGFB1 in fibrosis induction in the BMN. Although we detected some 479 abnormalities in the patient-derived MSC lines, the massive of TGFB1 - either added in 480 culture or produced by the CMK cell line - induced the identical histological changes in 481 vitro and in vivo in MSCs from HDs, thereby identifying the TGFB1 production of AMKL 482 blasts as paramount for the induction of BMF in our models. It remains to be determined 483 whether disease-derived MSCs will even be necessary to model the fibrotic BMN of AMKL 484 patients. 485 The next steps for our model in vivo will be to evaluate whether the BMF completely 486 disappears when the elevated levels of TGFB1 are normalized, as has been demonstrated 487 in a rat model [48]. We are also aware that the mechanisms leading to fibrosis and 488 ultimately osteosclerosis are more complex and should be further analyzed: e.g. additional 489 cytokines as PDGF or the activation of the JAK/STAT pathway might also be critical for the 490 progression of the disease and maybe can be separately targeted, as reported in MPN- 491 induced BMF [49, 50]. We hope that our described in vivo mouse model can be used in 492 future to answer some of these open questions and to examine novel combinations of 493 potential therapeutic targets for our patients.

494 Author Contributions: T.H., M.S. and M.R. designed the study, T.H., S.B., H.B., V.B., 495 E.F., B.G., O.H., L.K.H., L.K., A.S., C.W., H.H., M.S. and M.R. collected and interpreted 496 data. T.H., L.K.H., L.K., H.H., M.S. and M.R. wrote the draft of the manuscript; and all 497 authors gave final approval of manuscript.

498 Acknowledgments: L.K. and A.S. acknowledge the support by the Ministerium für Kultur 499 und Wissenschaft des Landes Nordrhein-Westfalen, the Regierende Bürgermeister von 500 Berlin - inkl. Wissenschaft und Forschung, and the Bundesministerium für Bildung und 501 Forschung. The Westdeutsche Biobank Essen supported the work by digitalization of the 502 slides with immunohistological staining’s. We also thank Michael Möllmann and Nicole 503 Schuster for their assistance with the animal experiments.

504 References

505 1. Schweitzer J, Zimmermann M, Rasche M, von Neuhoff C, Creutzig U, Dworzak M, 506 Reinhardt D, Klusmann J-H. Improved outcome of pediatric patients with acute 507 megakaryoblastic leukemia in the AML-BFM 04 trial. Annals of hematology. 2015; 508 94: 1327-36. 509 2. Arber DA, Orazi A, Hasserjian R, Thiele J, Borowitz MJ, Le Beau MM, Bloomfield 510 CD, Cazzola M, Vardiman JW. The 2016 revision to the World Health Organization 511 classification of myeloid neoplasms and acute leukemia. Blood. 2016; 127: 2391- 512 405.

513 3. Swerdlow SH, Campo E, Harris NL, Pileri SA. (2017). WHO Classification of 514 Tumours of Haematopoietic and Lymphoid Tissues: International Agency for 515 Research on Cancer). 516 4. Klusmann JH, Creutzig U, Zimmermann M, Dworzak M, Jorch N, Langebrake C, 517 Pekrun A, Macakova-Reinhardt K, Reinhardt D. Treatment and prognostic impact 518 of transient leukemia in neonates with Down syndrome. Blood. 2008; 111: 2991-8. 519 5. Creutzig U, Reinhardt D, Diekamp S, Dworzak M, Stary J, Zimmermann M. AML 520 patients with Down syndrome have a high cure rate with AML-BFM therapy with 521 reduced dose intensity. Leukemia. 2005; 19: 1355. 522 6. Ehninger A, Trumpp A. The bone marrow stem cell niche grows up: mesenchymal 523 stem cells and macrophages move in. J Exp Med. 2011; 208: 421-8. 524 7. Pagano L, Pulsoni A, Vignetti M, Mele L, Fianchi L, Petti M, Mirto S, Falcucci P, 525 Fazi P, Broccia G. Acute megakaryoblastic leukemia: experience of GIMEMA trials. 526 Leukemia. 2002; 16: 1622. 527 8. Zahr AA, Salama ME, Carreau N, Tremblay D, Verstovsek S, Mesa R, Hoffman R, 528 Mascarenhas J. Bone marrow fibrosis in myelofibrosis: pathogenesis, prognosis 529 and targeted strategies. Haematologica. 2016; 101: 660-71. 530 9. Schmitt-Gräff A, Skalli O, Gabbiani G. α-smooth muscle actin is expressed in a 531 subset of bone marrow stromal cells in normal and pathological conditions. 532 Virchows Archiv B. 1989; 57: 291. 533 10. Kuter D, Bain B, Mufti G, Bagg A, Hasserjian R. Bone marrow fibrosis: 534 pathophysiology and clinical significance of increased bone marrow stromal fibres. 535 Br J Haematol. 2007; 139: 351-62. 536 11. Rozman C, Cervantes F, Rozman M, MERCADER JM, Montserrat E. Magnetic 537 resonance imaging in myelofibrosis and essential thrombocythaemia: contribution 538 to differential diagnosis. British journal of haematology. 1999; 104: 574-80. 539 12. Thiele J, Kvasnicka HM, Facchetti F, Franco V, van der Walt J, Orazi A. European 540 consensus on grading bone marrow fibrosis and assessment of cellularity. 541 Haematologica. 2005; 90: 1128-32. 542 13. Cowley SA, Groopman JE, Avraham H. Effects of transforming growth factor beta 543 on megakaryocytic cell fusion and endomitosis. Int J Cell Cloning. 1992; 10: 223- 544 31. 545 14. Hasselbalch HC. The role of cytokines in the initiation and progression of 546 myelofibrosis. Cytokine Growth Factor Rev. 2013; 24: 133-45. 547 15. Roberts AB, Sporn MB, Assoian RK, Smith JM, Roche NS, Wakefield LM, Heine 548 UI, Liotta LA, Falanga V, Kehrl JH, et al. Transforming growth factor type beta: 549 rapid induction of fibrosis and angiogenesis in vivo and stimulation of collagen 550 formation in vitro. Proc Natl Acad Sci U S A. 1986; 83: 4167-71. 551 16. Kimura A, Katoh O, Hyodo H, Kuramoto A. Transforming growth factor-beta 552 regulates growth as well as collagen and fibronectin synthesis of human marrow 553 fibroblasts. Br J Haematol. 1989; 72: 486-91. 554 17. Terui T, Niitsu Y, Mahara K, Fujisaki Y, Urushizaki Y, Mogi Y, Kohgo Y, Watanabe 555 N, Ogura M, Saito H. The production of transforming growth factor-beta in acute

556 megakaryoblastic leukemia and its possible implications in myelofibrosis. Blood. 557 1990; 75: 1540-8. 558 18. Reilly J, Barnett D, Dolan G, Forrest P, Eastham J, Smith A. Characterization of an 559 acute micromegakaryocytic leukaemia: evidence for the pathogenesis of 560 myelofibrosis. British journal of haematology. 1993; 83: 58-62. 561 19. Schepers K, Pietras EM, Reynaud D, Flach J, Binnewies M, Garg T, Wagers AJ, 562 Hsiao EC, Passegue E. Myeloproliferative neoplasia remodels the endosteal bone 563 marrow niche into a self-reinforcing leukemic niche. Cell Stem Cell. 2013; 13: 285- 564 99. 565 20. Kumar B, Garcia M, Weng L, Jung X, Murakami J, Hu X, McDonald T, Lin A, 566 Kumar A, DiGiusto D. Acute myeloid leukemia transforms the bone marrow niche 567 into a leukemia-permissive microenvironment through exosome secretion. 568 Leukemia. 2018; 32: 575. 569 21. Digirolamo CM, Stokes D, Colter D, Phinney DG, Class R, Prockop DJ. 570 Propagation and senescence of human marrow stromal cells in culture: a simple 571 colony-forming assay identifies samples with the greatest potential to propagate 572 and differentiate. Br J Haematol. 1999; 107: 275-81. 573 22. Dominici M, Le Blanc K, Mueller I, Slaper-Cortenbach I, Marini F, Krause D, Deans 574 R, Keating A, Prockop D, Horwitz E. Minimal criteria for defining multipotent 575 mesenchymal stromal cells. The International Society for Cellular Therapy position 576 statement. Cytotherapy. 2006; 8: 315-7. 577 23. Cai R, Nakamoto T, Hoshiba T, Kawazoe N, Chen G. Control of simultaneous 578 osteogenic and adipogenic differentiation of mesenchymal stem cells. J Stem Cell 579 Res Ther. 2014; 4: 1000223. 580 24. Chen Y, Jacamo R, Shi Y, Wang R, Battula V, Konoplev S, Strunk D, Hofmann N, 581 Reinisch A, Konopleva M, Andreeff M. Human extramedullary bone marrow in 582 mice: a novel in vivo model of genetically controlled hematopoietic 583 microenvironment. Blood. 2012; 119: 4971-80. 584 25. Reinisch A, Thomas D, Corces M, Zhang X, Gratzinger D, Hong W, Schallmoser K, 585 Strunk D, Majeti R. A humanized bone marrow ossicle xenotransplantation model 586 enables improved engraftment of healthy and leukemic human hematopoietic cells. 587 Nat Med. 2016; 22: 812-21. 588 26. Tiaden AN, Breiden M, Mirsaidi A, Weber FA, Bahrenberg G, Glanz S, Cinelli P, 589 Ehrmann M, Richards PJ. Human serine protease HTRA1 positively regulates 590 osteogenesis of human bone marrowǦderived mesenchymal stem cells and 591 mineralization of differentiating boneǦforming cells through the modulation of 592 extracellular matrix protein. Stem Cells. 2012; 30: 2271-82. 593 27. Lefterova MI, Haakonsson AK, Lazar MA, Mandrup S. PPARγ and the global map 594 of adipogenesis and beyond. Trends in Endocrinology & Metabolism. 2014; 25: 595 293-302. 596 28. Sullivan KD, Lewis HC, Hill AA, Pandey A, Jackson LP, Cabral JM, Smith KP, 597 Liggett LA, Gomez EB, Galbraith MD. Trisomy 21 consistently activates the 598 interferon response. Elife. 2016; 5: e16220.

599 29. Shehata AM, Kandel SH, Rizk SH, Khalifa KA, Fouad AS. Collagen I and collagen 600 III expression in fibrotic bone marrow. Menoufia Medical Journal. 2016; 29: 360. 601 30. Abarrategi A, Mian SA, Passaro D, Rouault-Pierre K, Grey W, Bonnet D. Modeling 602 the human bone marrow niche in mice: From host bone marrow engraftment to 603 bioengineering approaches. Journal of Experimental Medicine. 2018; 215: 729-43. 604 31. Hattori H, Matsuzaki A, Suminoe A, Ihara K, Nakayama H, Hara T. High expression 605 of plateletǦderived growth factor and transforming growth factorǦβ1 in blast cells 606 from patients with Down Syndrome suffering from transient myeloproliferative 607 disorder and organ fibrosis. British journal of haematology. 2001; 115: 472-5. 608 32. Granéli C, Thorfve A, Ruetschi U, Brisby H, Thomsen P, Lindahl A, Karlsson C. 609 Novel markers of osteogenic and adipogenic differentiation of human bone marrow 610 stromal cells identified using a quantitative proteomics approach. Stem cell 611 research. 2014; 12: 153-65. 612 33. Köllmer M, Buhrman JS, Zhang Y, Gemeinhart RA. Markers are shared between 613 adipogenic and osteogenic differentiated mesenchymal stem cells. Journal of 614 developmental biology and tissue engineering. 2013; 5: 18. 615 34. Schneider R, Mullally A, Dugourd A, Peisker F, Hoogenboezem R, Van Strien P, 616 Bindels E, Heckl D, Busche G, Fleck D, Muller-Newen G, Wongboonsin J, Ventura 617 Ferreira M, et al. Gli1+ Mesenchymal Stromal Cells Are a Key Driver of Bone 618 Marrow Fibrosis and an Important Cellular Therapeutic Target. Cell Stem Cell. 619 2017; 20: 785-800 e8. 620 35. Decker M, Martinez-Morentin L, Wang G, Lee Y, Liu Q, Leslie J, Ding L. Leptin- 621 receptor-expressing bone marrow stromal cells are myofibroblasts in primary 622 myelofibrosis. Nature cell biology. 2017; 19: 677. 623 36. Mareschi K, Ferrero I, Rustichelli D, Aschero S, Gammaitoni L, Aglietta M, Madon 624 E, Fagioli F. Expansion of mesenchymal stem cells isolated from pediatric and 625 adult donor bone marrow. J Cell Biochem. 2006; 97: 744-54. 626 37. Palomares Cabeza V, Hoogduijn MJ, Kraaijeveld R, Franquesa M, Witte-Bouma J, 627 Wolvius EB, Farrell E, Brama PAJ. Pediatric Mesenchymal Stem Cells Exhibit 628 Immunomodulatory Properties Toward Allogeneic T and B Cells Under 629 Inflammatory Conditions. Front Bioeng Biotechnol. 2019; 7: 142. 630 38. Knuth CA, Kiernan CH, Palomares Cabeza V, Lehmann J, Witte-Bouma J, Ten 631 Berge D, Brama PA, Wolvius EB, Strabbing EM, Koudstaal MJ, Narcisi R, Farrell E. 632 Isolating Pediatric Mesenchymal Stem Cells with Enhanced Expansion and 633 Differentiation Capabilities. Tissue Eng Part C Methods. 2018; 24: 313-21. 634 39. Boyd AL, Reid JC, Salci KR, Aslostovar L, Benoit YD, Shapovalova Z, Nakanishi 635 M, Porras DP, Almakadi M, Campbell CJ. Acute myeloid leukaemia disrupts 636 endogenous myelo-erythropoiesis by compromising the adipocyte bone marrow 637 niche. Nature cell biology. 2017; 19: 1336. 638 40. Battula VL, Le PM, Sun JC, Nguyen K, Yuan B, Zhou X, Sonnylal S, McQueen T, 639 Ruvolo V, Michel KA. AML-induced osteogenic differentiation in mesenchymal 640 stromal cells supports leukemia growth. JCI insight. 2017; 2.

641 41. Corradi G, Baldazzi C, Očadlíková D, Marconi G, Parisi S, Testoni N, Finelli C, 642 Cavo M, Curti A, Ciciarello M. Mesenchymal stromal cells from myelodysplastic 643 and acute myeloid leukemia patients display in vitro reduced proliferative potential 644 and similar capacity to support leukemia cell survival. Stem cell research & therapy. 645 2018; 9: 271. 646 42. Azadniv M, Myers JR, McMurray HR, Guo N, Rock P, Coppage ML, Ashton J, 647 Becker MW, Calvi LM, Liesveld JL. Bone marrow mesenchymal stromal cells from 648 acute myelogenous leukemia patients demonstrate adipogenic differentiation 649 propensity with implications for leukemia cell support. Leukemia. 2019. 650 43. Astori G, Amati E, Bambi F, Bernardi M, Chieregato K, Schafer R, Sella S, 651 Rodeghiero F. Platelet lysate as a substitute for animal serum for the ex-vivo 652 expansion of mesenchymal stem/stromal cells: present and future. Stem Cell Res 653 Ther. 2016; 7: 93. 654 44. Ren J, Ward D, Chen S, Tran K, Jin P, Sabatino M, Robey PG, Stroncek DF. 655 Comparison of human bone marrow stromal cells cultured in human platelet growth 656 factors and fetal bovine serum. J Transl Med. 2018; 16: 65. 657 45. Lieb E, Vogel T, Milz S, Dauner M, Schulz M. Effects of transforming growth factor 658 β1 on bonelike tissue formation in three-dimensional cell culture. II: osteoblastic 659 differentiation. Tissue engineering. 2004; 10: 1414-25. 660 46. Hanoun M, Zhang D, Mizoguchi T, Pinho S, Pierce H, Kunisaki Y, Lacombe J, 661 Armstrong S, Duhrsen U, Frenette P. Acute myelogenous leukemia-induced 662 sympathetic neuropathy promotes malignancy in an altered hematopoietic stem cell 663 niche. Cell Stem Cell. 2014; 15: 365-75. 664 47. Frisch BJ, Ashton JM, Xing L, Becker MW, Jordan CT, Calvi LM. Functional 665 inhibition of osteoblastic cells in an in vivo mouse model of myeloid leukemia. 666 Blood. 2012; 119: 540-50. 667 48. Yanagida M, Ide Y, Imai A, Toriyama M, Aoki T, Harada K, Izumi H, Uzumaki H, 668 Kusaka M, Tokiwa T. The role of transforming growth factorǦβ in PEGǦrHuMGDFǦ 669 induced reversible myelofibrosis in rats. British journal of haematology. 1997; 99: 670 739-45. 671 49. Friedman SL, Sheppard D, Duffield JS, Violette S. Therapy for fibrotic diseases: 672 nearing the starting line. Science translational medicine. 2013; 5: 167sr1-sr1. 673 50. Mead AJ, Mullally A. Myeloproliferative neoplasm stem cells. Blood. 2017; 129: 674 1607-16.

675

676 Figure legends

677 Figure 1. Osteogenic and adipogenic differentiation potential of MSCs. (A) 678 Osteogenic differentiation after 16 days in differentiation and control medium. 679 Representative pictures of HD-, ML-DS- and AMKL-derived MSCs are shown. (B) 680 Quantitative analysis of osteogenic differentiation showed no differences in AMKL- 681 (n=5) and ML-DS-derived MSCs (n=7) compared to HD-derived MSCs (n=5). Data 682 points of each sample display the mean color saturation (8-bit). (C) No differential 683 gene expression in non-differentiated MSCs for the osteogenic marker IBSP 684 measured by qRT-PCR. (D) Adipogenic differentiation status of HD- and ML-DS- 685 derived MSCs after 16 days of culture are depicted. (E) The quantitative analysis 686 showed the decreased adipogenic potential of ML-DS- (n=7; *P<0.05) and AMKL- 687 (n=5) compared to the differentiation potential of HD-derived MSCs (n=5). Data 688 points of each sample are shown as the mean of Oil Red O positive stained area 689 relative to total area in percent. (F) The adipogenic marker PPARG showed a 690 decreased gene expression in non-differentiated ML-DS- (n=3, P*<0.05) and AMKL- 691 derived MSCs (n=3; P*<0.05) compared to HD-derived MSCs (n=3). Scale bars: 100 692 μm (Adipogenesis), 5 mm (Osteogenesis).

693 Figure 2. MSCs showed no differences in cell activity and colony 694 formation/clonogenicity. (A) 24-hour proliferation assay for HD- (n= 5), ML-DS- (n= 7) 695 and AMKL-derived MSCs (n= 5). (B) Colony formation assay: Formation of CFU-F 696 colonies per 200 primary MSC lines from HDs (n= 3), ML DS (n= 3) and AMKL patients 697 (n= 3).

698 Figure 3. Effects of TGFB1 exposure on MSCs derived from patients with ML- 699 DS or AMKL and from HDs. (A, B) Experiments were performed with HD- (n=5) 700 ML-DS- (n=7) and AMKL-derived MSCs (n=5). After 16 days in differentiation 701 medium, the cultures were stained with Alizarin Red S for osteogenic differentiation 702 and Oil Red O for adipogenic differentiation, respectively, and then analyzed. (A) 703 Osteogenic differentiation: Quantitative analysis showed a decreased osteogenic 704 potential with TGFB1 treatment compared to untreated MSCs (ctr) (HD: *P <0.05; 705 ML-DS and AMKL: **P<0.01). Data points of each sample display mean color 706 saturation (8-bit). (B) Adipogenic differentiation: Quantitative analysis showed a 707 decreased adipogenic potential with TGFB1 treatment compared to ctr (HD: 708 *P<0.05). Data points of each sample displayed the mean of Oil Red O positive 709 stained area in percent. (C) Analysis of non-differentiated MSCs (HD- n=3, ML-DS- 710 n=3, AMKL-derived MSCs n=3) after 48h culture with 10 ng/ml TGFB1 by qRT-PCR. 711 Heatmap visualization showed the gene expression pattern of treated non-

712 differentiated MSCs relative to untreated (log2 fold; -3 blue to 3 red). TGFB1 713 decreased PPARG and IBSP expression (***P<0.001) and induced fibrosis gene 714 expression (ACTA2, COL1A1, COL3A1; ***P<0.001).

715 Figure 4. Establishment of an in vivo model for a leukemic BMN through 716 growth of leukemic megakaryoblasts and fibrosis induction in a human ossicle 717 in NSG mice. (A) Experimental schematic and timeline. In vitro cultured MSCs 718 (1x106) and HUVECs (1x106) were mixed with Matrigel and subcutaneously injected 719 into NSG mice. After 8 weeks, CMK cells were injected into the developed artificial 720 niche (ossicle). After 12 weeks in total, the mice were sacrificed and the ossicle-like 721 structures harvested and analyzed. (B) Histological and immunohistological 722 characterization of the ossicle-like structures. Representative analysis of an ossicle 723 after 12 weeks post-transplantation without CMK cells is shown. (C) Representative 724 ossicle-like structures without and with injection of CMK cells is shown. 725 Immunostaining for human CD45. Middle column: low magnification 700 μm 726 (untreated) and 5 mm (+CMK). Right column: high magnification 70 μm. (D) Sizes of 727 ossicle-like structures: Injection of CMK cells (1x106 n=10, ***P<0.001; 5x106 n=6, 728 ***P<0.001) increased the sizes of the ossicle-like structures compared to the sizes 729 of uninjected ossicle-like structures (n=24). (E) Percentage of fibrosis-positive 730 ossicle-like structures. Only ossicle-like structures with CMK cells displayed fibrosis 731 of grade I and II (CMK cells 1x106: 70% grade I; 30% grade 0 (w/o fibrosis); CMK 732 cells 5x106: 67% grade I; 33% grade II). (F) Representative silver staining of grade 0 733 to II fibrosis. Grade 0: untreated ossicle; grade I: ossicle with injection of 1x106 CMK 734 cells); grade II: ossicle with 5x106 CMK cells. Scale bar: 200 μm.

735 Tables

736 Table 1. GSEA of hallmark gene sets revealed enrichment of proliferation- 737 related gene sets (E2F targets, G2M checkpoint, mitotic spindle) in ML-DS- 738 (n=8) and/or AMKL-derived MSCs (n=5) compared to HD-derived MSCs (n=5). 739 (SIZE) Size of the gene set; (NES) normalized enrichment score; (FDR) false 740 discovery rate; (FWER) family-wise error rate; (q) q-value; (p) p-value. Cut-off: NES 741 < (-) 2.0; FWER < 0.01.

Group GS name Size NES FDR (q) FWER (p)

ML-DS HALLMARK_E2F_TARGETS 185 2.87 <0.01 <0.01

ML-DS HALLMARK_G2M_CHECKPOINT 188 2.79 <0.01 <0.01

ML-DS HALLMARK_MITOTIC_SPINDLE 194 2.12 <0.01 <0.01

ML-DS HALLMARK_INTERFERON_ALPHA_RESPONSE 91 2.09 <0.01 <0.01

AMKL HALLMARK_E2F_TARGETS 185 3.08 <0.01 <0.01

AMKL HALLMARK_G2M_CHECKPOINT 188 2.80 <0.01 <0.01

742

Exposure of patient-derived mesenchymal stromal cells to TGFB1 supports fibrosis induction in a pediatric acute megakaryoblastic leukemia model

Theresa Hack, Stefanie Bertram, Helen Blair, et al.

Mol Cancer Res Published OnlineFirst July 8, 2020.

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