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Defining tumor-associated vascular heterogeneity in pediatric high-grade and diffuse midline gliomas

Defining tumor-associated vascular heterogeneity in pediatric high-grade and diffuse midline gliomas The blood–brain barrier (BBB) plays important roles in brain tumor pathogenesis and treatment response, yet our understanding of its function and heterogeneity within or across brain tumor types remains poorly characterized. Here we analyze the neurovascular unit (NVU) of pediatric high-grade glioma (pHGG) and diffuse midline glioma (DMG) using patient derived xenografts and natively forming glioma mouse models. We show tumor-associated vas- cular differences between these glioma subtypes, and parallels between PDX and mouse model systems, with DMG models maintaining a more normal vascular architecture, BBB function and endothelial transcriptional program rela- tive to pHGG models. Unlike prior work in angiogenic brain tumors, we find that expression of secreted Wnt antago - nists do not alter the tumor-associated vascular phenotype in DMG tumor models. Together, these findings highlight vascular heterogeneity between pHGG and DMG and differences in their response to alterations in developmental BBB signals that may participate in driving these pathological differences. Keywords: Pediatric high-grade glioma, Diffuse midline glioma, Blood brain barrier, Endothelial cells, Neurovascular unit, Diffuse intrinsic pontine glioma, H3K27M, Wnt signaling Introduction across adult and pediatric brain tumor entities [3, 4]. Our The blood–brain barrier (BBB) is a specialized vascular understanding of intra- and inter-tumoral BBB heteroge- structure within the brain formed by the neurovascular neity continues to improve with advancements in defin - unit (NVU) which consists of endothelial cells, pericytes, ing molecular subgroups of human brain tumors, and the astrocytes and neurons [1]. While essential for normal development of accurate patient-derived xenograft (PDX) brain function and homeostasis, the BBB poses a prob- and genetically engineered mouse models (GEMMs) that lem for treating CNS related diseases since the majority faithfully recapitulate features of primary human brain of drugs and small molecules display limited brain pen- tumors. etration [2]. BBB function was historically considered Pediatric high-grade gliomas (pHGGs) are among disrupted in brain tumors based on studies using aggres- the most common childhood brain tumors and can be sive adult glioma models that do not accurately reflect divided into multiple subgroups based different fea - the diversity and pathological heterogeneity identified tures including histology, location, mutation status and molecular profile [5 –8]. One of the most lethal pHGG types are H3K27M mutant diffuse midline glio - mas (DMGs), which encompass midline and brainstem *Correspondence: timothy.phoenix@uc.edu Division of Pharmaceutical Sciences, James L. Winkle College gliomas that harbor H3K27M mutations [7, 9]. Treat- of Pharmacy, University of Cincinnati, Cincinnati, OH, USA ment options remain limited for DMG patients, and no Full list of author information is available at the end of the article © The Author(s) 2021. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. The Creative Commons Public Domain Dedication waiver (http:// creat iveco mmons. org/ publi cdoma in/ zero/1. 0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Wei et al. acta neuropathol commun (2021) 9:142 Page 2 of 18 chemotherapy or targeted therapy has demonstrated IUE mouse models significant survival benefits thus far [8 , 10]. One pro- All IUE related mouse work was done according to insti- posed reason for the failure of systemically delivered tutional and IACUC review boards (University of Cincin- therapies is that DMGs maintains a more intact BBB nati). The IUE procedure to generate pediatric high-grade compared to other non-brainstem tumors, as clinicians glioma and diffuse midline glioma mouse models was have noted that DMGs frequently display little to no performed as previously described [18]. Briefly, IUE contrast enhancement on magnetic resonance imag- pHGG mouse models were created by lateral ventricle ing (MRI) [11, 12]. Yet a detailed characterization or injection of PdgfraD842V + H3.3G34R + DNP53 + Pbase direct comparison of tumor-associated vasculature in plasmids (all at 1  µg/µL). IUE DMG mouse model th cortical pHGGs and DMGs has not been systematically were created by 4 ventricle injection of Pdg- performed. fraD842V + H3.3K27M + DNP53 + Pbase plasmids. Here, employing patient derived xenografts (PDX) Control BS or CTX IUE conditions were created th and in utero electroporation (IUE) based mouse mod-by 4 or lateral ventricle injections respectively of els of cortical pHGG and brainstem DMG, we perform H3.3WT + DNP53 + Pbase plasmids. Survival curves a histological and molecular analysis of tumor-associ- were generated in GraphPad Prism using Log-rank Man- ated vasculature. We show that cortical pHGGs induce tle-Cox statistical tests. changes traditionally associated with glioblastoma vas- culature [13], including abnormal vessel morphology Patient derived xenograft pHGG and DMG models and BBB disruption, while brainstem DMGs maintain a and primary tumor samples similar vascular content and architecture to that of the Pediatric HGG and DIPG/DMG patient derived xeno- normal brainstem. Additional transcriptomics studies graft (PDX) samples are from Dr. Esther Hulleman’s support our phenotypic analyses and suggest that DMG group at VU University Medical Center, Amsterdam, endothelial cells are maintained in a stable and mature The Netherlands. All PDX experiments were carried state. While expression of Wnt-antagonists in other out at the VU University Medical Center in accordance aggressive pediatric brain tumors, such as medulloblas- with the Declaration of Helsinki and national and insti- toma [14, 15] and adult glioblastoma induces BBB dis- tutional guidelines. 5- to 6-week old NOD/SCID/Il2rg-/- ruption [16, 17], we find that their expression does not mice (Jackson) were intracranially injected with 500,000 alter the BBB or tumor-associated vascular phenotype cells of each of the following primary cultures: VUMC- in DMG models. Our findings provide direct evidence DIPG-F [19], VUMC-DIPG-G, VUMC-HGG-11 [20], of tumor-associated vascular differences between DMG VUMC-HGG-14 [21]. The stereotactic coordinates that and pHGG tumors and underscore the need to con- were used to inject into the pons (VUMC-DIPG-F and sider intra- and inter-tumor heterogeneity in vascular -G) were 0.8  mm laterally, 1  mm caudally, and 4.5  mm content and BBB function when developing therapeutic ventrally from the lambda. The stereotactic coordinates strategies that target tumor cells or their vasculature. that were used to inject into the striatum (VUMC- HGG-11 and -14) were 2  mm laterally, 1  mm cranially, and 3 mm ventrally from the bregma. Cells were injected Materials and methods in an injection volume of 5 μL at a flow rate of 1 μL/ Plasmid constructs minute to minimize the neurological side effects of the DNA plasmids were construct as we previously procedure. Primary tumor-tissue was obtained through described [18]. PBCAG- H3.3G34R-Ires-eGFP was surgical resection (pHGG) or via a brain autopsy study generated by InFusion (Takara) PCR site directed (DIPG/DMG) in the Amsterdam UMC (Amsterdam, the mutagenesis from an H3F3A template plasmid Netherlands), in accordance with the declaration of Hel- (Addgene #42,632) and insertion into EcoR1 linearized sinki and approved by the institutional review board of PBCAG-Ires-eGFP. PBCAG-Dkk1-Ires-eGFP was gen- Amsterdam UMC, location VUmc (METc VUmc, study erated by PCR amplification of Dkk1 and insertion by number: VUMC2009/237) and the Scientific Committee InFusion ligation into EcoR1 linearized PBCAG-Ires- of the Dutch Childhood Oncology Group (DCOG). eGFP. PBCAG-Fzd8-CRD-IgG was created by PCR amplification of Fzd8-CRD-IgG (Addgene #16,689) Tissue collection, processing and immunostaining and insertion by InFusion ligation into EcoR1 line- Upon development of neurological symptoms, mice were arized PBCAG-Ires-eGFP. All plasmids were verified by deeply anesthetized before perfusion with cold DPBS fol- Sanger sequencing (CCHMC DNA sequencing core), lowed by 1% PFA. Brains were rapidly dissected in cold and plasmid stocks prepared by using NuceloBond Xtra DPBS and then fixed in 100% MeOH at 4  °C overnight. Maxi EF endotoxin-free kits (Machery-Nagel). W ei et al. acta neuropathol commun (2021) 9:142 Page 3 of 18 Samples were rehydrated at 4  °C in DPBS for 4  h before Magnetic cell sorting (MACS) embedding in 3% low-melt agarose gel (IBI scientific, Endothelial cells (ECs) were purified using magnetic #B70051). 150 um thick free-floating sections were cut sorting using MACS (Miltenyl Biotec) according to the using a Leica vibratome. Floating sections were incubated manufacturer’s protocol. All antibodies, buffers and in blocking solution (PBS + 0.5% Triton x-100 + 10% nor- equipment were purchased from Miltenyl Biotec. Briefly, mal donkey serum) at RT for 30  min before incubating tumor or normal tissues were micro dissected under a with primary antibodies at 4  °C overnight. The next day fluorescent stereoscope, and 3–4 tumors or control tis - sections were washed in DPBS, transferred to blocking sue samples were pooled together to minimize vari- solution containing the appropriate secondary antibod- ability between individual tumors. Samples were then ies (1:500) and incubated at room temperature for 2  h. dissociated by incubation in a Collagenase IV cocktail Finally, sections were incubated in Hoechst (1:1000 in for 45 min at 37 °C. Collagenase IV dissociation solution PBS) for 10 min before final DPBS washing and mounting was made by mixing 32 mg collagenase IV (Worthington, onto slides (Fisher, Superfrost), and coverslipped (Pro- #LS004209), 10  mg Deoxyribonuclease I (Worthington, long Gold Antifade, ThermoFisher). Primary antibodies #LS002007), 20 mg Soybean trypsin inhibitor (Worthing- used in this study include: eGFP (Aves, #GFP1020), CD31 ton, #LS003587) with 10 mL DPBS. Following incubation (BD Biosciences, #550,274), Glut1 (Millipore, #07–1401), and trituration, cell suspensions were passed through Collagen IV (BioRad, #161,115), Desmin (Cell Signal- 40  μm mesh cell strainers. Myelin and cellular debris ing, #5332), human Vimentin (eBioscience, #11–9897- were removed using debris removal solution (Milte- 82), Claudin-5 (Thermofisher, #352,588), Plvap (BD nyl Biotec #130–109-398), and red blood cells (RBCs) Biosciences, #550,274), Ter119 (Invitrogen #14–5921-82) removed by incubation in ACK lysing buffer (Thermo and Hoechst (ThermoFisher). Corresponding secondary Fisher, #A10492-01). Cell pellets were resuspended in 90 antibodies used were all purchased from Jackson Immu- μL PEB buffer / 10 million cells. PEB buffer was prepared noResearch. Images were acquired on a confocal micro- by diluting MACS BSA stock solution (#130–091-376) scope (Nikon A1), and image analysis was performed in 1:20 with autoMACS rinsing solution (#130–091-222). NIH Image J. Statistical analyses of these data were per- Cells were blocked by adding 10  μl of FcR blocking rea- formed in GraphPad prism as described in the manu- gent and incubated with 15 μL of CD45 microbeads script. P-values of ≤ 0.05 were considered statistically (#130–052-301) at 4  °C for 15  min, then applied to MS significant. column (#130–042-201) against a magnetic separator For histology, mouse brain tissue was fixed in 10% for - followed by two washes with PEB buffer. CD45-negative malin overnight and transferred to 70% ethanol before flow through cells were then collected and labeled with paraffin embedding. 5  μm thick sections were prepared CD31 microbeads (#130–097-418) and passed through on a microtome (Lecia), and processed for hematoxylin– a new MS column against a magnetic separator followed eosin (H&E) staining. For immunohistochemistry of pri- by two washes with PEB buffer. CD45-negative/CD31- mary patient samples 5  µm thick paraffin sections were positive ECs retained within the MS column were eluted prepared and stained using standardized techniques. in PEB buffer by expelling with the provided column Primary antibodies used include anti-CD31 (Dako, plunger. Purified cell numbers were determined using an #M0823), anti-Cldn5 (Invitrogen, #34–1600) and anti- automated cell counter (Thermo Fisher) and then pro - Glut1 (Millipore, #07–1401). Stains were developed with cessed for total RNA isolation. secondary HRP and DAB immunoreactivity secondary kits (Dako) and counterstained with Hematoxylin before mounting. RNA preparation, real‑time quantitative PCR, whole transcriptome sequencing and analysis Total RNA was isolated from freshly isolated samples TMR dextran BBB permeability assay using the NucleoSpin Plus RNA kit (Macherey–Nagel) 10-kDa Tetramethylrhodamine (TMR)-dextran (Ther - as previously described [18]. cDNA was synthesized moFisher # D1817) was dissolved in sterile DPBS at a using SuperScript Vilo cDNA synthesis kit (Thermo concentration of 10  mg/ml. TMR dextran was adminis- Fisher, #11,754,050) according to manufacture proto- tered as previously described elsewhere [22]. Following col. Real-time PCR was performed using a Bio-Rad CFX circulation of the dextran tracer brains were harvested qPCR system. The fold increase was determined using in ice-cold DPBS and fixed in 4% PFA overnight. Fixed the 2 − ΔΔCT method. Gapdh was used as endogenous brains were washed in DPBS the next day, followed by control to normalize mRNA expression level. The fol - incubation in 30% sucrose for 48 h at 4˚C before embed- lowing primers were purchased from IDT. Gapdh, Fwd: ding in tissue freezing media. 50  μm thick free-floating AGG TCG GTG TGA ACG GAT TTG, Rvs: TGT AGA sections were cut using a Leica cryostat. Wei et al. acta neuropathol commun (2021) 9:142 Page 4 of 18 CCA TGT AGT TGA GGT CA; Cd31, Fwd,: ACG CTG using iGEAK and gProfiler using default settings [24]. GTG CTC TAT GCA AG, Rvs: TCA GTT GCT GCC Transcription factor protein–protein interaction net- CAT TCA TCA; Tie2, Fwd: GAG TCA GCT TGC TCC works were generated using Enrichr [25]. All RNA- TTT ATG G, Rvs: AGA CAC AAG AGG TAG GGA seq files are deposited in Gene Expression Omnibus as ATT GA; Vegfr2, Fwd: TTT GGC AAA TAC AAC CCT GSE179372. TCA GA, Rvs: GCA GAA GAT ACT GTC ACC ACC; NeuN, Fwd: ATC GTA GAG GGA CGG AAA ATT GA, Results Rvs: GTT CCC AGG CTT CTT ATT GGT C; Cd68, pHGG and DMG patient derived xenografts display Fwd: TGT CTG ATC TTG CTA GGA CCG, Rvs: GAG tumor‑associated vascular differences. AGT AAC GGC CTT TTT GTG A; Dkk1, Fwd: CTC The presence of contrast enhancement (CE) in mag - ATC AAT TCC AAC GCG ATC A, Rvs: GCC CTC ATA netic resonance imaging (MRI), indicating gadolinium GAG AAC TCC CG. RNA-sequencing was performed as leakage outside of blood vessels, is a common feature in previously described [18]. RNA quality control was per- most glioblastomas [13]. Yet prior radiological studies formed on a bioanalyzer (BioRad) to ensure the quality have noted DMG patients tend to display limited to no of each sample submitted. For isolation of polyA RNA, CE, suggesting the maintenance of a mostly intact BBB a NEBNext Poly(A) mRNA Magnetic Isolation Module [11, 12, 26]. To investigate potential vascular differences (New England BioLabs) was used for polyA RNA puri- between DMG and pHGG tumors, we examined availa- fication with a total of 1  μg good quality total RNA as ble orthotopic PDX models [19–21]. Staining PDX tumor input. The SMARTer Apollo NGS library prep system samples with a human specific tumor cell marker (hVi - (Takara) was used for automated polyA RNA isolation. mentin) and pan-endothelial marker (CD31) revealed For RNA sequencing library preparation, the library for minimal changes in the vasculature phenotype of DMG RNA-seq was prepared by using the NEBNext Ultra II PDXs (Fig.  1a, b). In contrast pHGG PDXs displayed Directional RNA Library Prep Kit (New England Bio- decreased blood vessel density, vascular branchpoints Labs). After indexing via PCR enrichment (8 cycles), the and enlarged lumens (Fig. 1b, d, e). Staining for the tight amplified libraries together with the negative control junction marker Claudin5 (Cldn5) and BBB associated were cleaned up for quality control analysis. To study dif- glucose transporter Glut1 (also known as Slc2a1) found ferential gene expression, individually indexed and com- both expressed in pHGG and DMG PDX vasculature, patible libraries were proportionally pooled (~ 25 million although pHGG PDXs displayed small but significant reads per sample in general) for clustering in the cBot decreases in Glut1-expressing vessels, along with regions system (Illumina). Libraries at the final concentration of of disorganized Cldn5 tight junctions in vessels (Fig.  1c, 15  pM were clustered onto a single-read flow cell using f). In contrast, DMG PDX blood vessels showed no the IlluminaTruSeq SR Cluster Kit v3, and sequenced to noticeable differences in the expression or organization 51 bp using theTruSeq SBS Kit v3 on the Illumina HiSeq of Glut1 and Cldn5 compared to normal brain regions system. Sequence reads were aligned to the reference (Fig.  1c). In parts of pHGG tumors that contained genome using the TopHat aligner and reads aligning to necrotic regions there was a transition of Glut1 stain- each known transcript were counted using Bioconduc- ing from vascular to non-vascular cells (i.e., tumor cells tor packages for next-generation sequencing data analy- or associated macrophages), likely indicating a metabolic sis. The differential expression analysis between different response to hypoxic conditions [27] (Additional file  1: sample types was performed using the negative binomial Fig. S1). Additional staining of primary human DMG statistical model of read counts as implemented in the and pHGG samples for CD31, Cldn5 and Glut1 showed edgeR Bioconductor package. Transcriptional profiles similar patterns to PDX models, providing further evi- were interrogated with iGEAK (Interactive Gene Expres- dence for vascular differences between these tumors sion Analysis Kit for microarray and RNA-seq data), an (Additional file  1: Fig. S2). This data, together with prior R (v3.3.2) and JavaScript based open-source desktop radiological studies in patients and PDX models [12, 19, application [23]. Functional enrichment of differentially 26, 28], suggests DMGs maintain a relatively intact BBB expressed gene lists between conditions was performed compared to cortical pHGGs. (See figure on next page.) Fig. 1 Tumor associated vascular differences in orthotopic patient derived xenograft pHGG and DMG models. Representative immunofluorescent images of pHGG, DMG PDX samples and normal brain labeled with a hVimentin and Hoechst, b CD31 and c Claudin-5 and Glut1 and Hoechst. Scale bar = 20 μm. Quantification of d CD31-positive blood vessel density in the normal brain (n = 5), DMG PDX (n = 5) and pHGG PDX (n = 3), e branchpoints and f % Glut1-positive blood vessels in DMG PDX (n = 5) and pHGG PDX (n = 3). Data are presented as mean ± SEM *P < 0.05, **p < 0.001, ***p < 0.0001, unpaired t-test with Mann–Whitney posthoc comparison W ei et al. acta neuropathol commun (2021) 9:142 Page 5 of 18 Fig. 1 (See legend on previous page.) Wei et al. acta neuropathol commun (2021) 9:142 Page 6 of 18 Cortical pHGG and brainstem DMG mouse models vessels with diameters ranging from 2 μm to 40 μm, and recapitulate tumor‑associated vascular differences bordering rim regions with a mixture of abnormal and To further examine vascular differences between pHGG normal vessel phenotypes. (Additional file  1: Fig. S3). and DMGs we employed recently developed glioma u Th s, de novo IUE mouse models of brainstem DMG mouse models created by in utero electroporation (IUE) and supratentorial pHGG recapitulate the architectural [18, 29]. DMG mouse models were made by brainstem differences found in PDX tumor-associated vascular targeted IUE of Piggybac DNA plasmids expressing Pdg- networks, providing an accurate system to study tumor- fraD842V, DNp53 and H3.3K27M. To generate corti- blood vessel interactions. cal pHGG mouse models, we replaced H3.3K27M with a plasmid expressing H3.3G34R, combine with Pdg- pHGG and DMG mouse models display differences fraD842V and DNp53 expressing Piggybac DNA plas- in vascular integrity and BBB function mids. Both H3K27M DMG and H3G34R pHGG IUEs To gain additional insight into these vascular differ - resulted in the generation of fully penetrant gliomas ences we examined the vascular permeability of control in successfully electroporated offspring. H3K27M IUE and IUE tumor models by circulation of a fluorescently DMGs formed at a significantly shorter latency com - labeled dextran tracer (10  kDa TMR-Dextran). No pared to H3G34R IUE pHGGs (median survival for extravascular leakage was found in control brainstem and IUE DMG model (n = 12) was 30  days and 79  days for cortical samples, or in IUE DMG tumors (Fig. 4a, b). On HGG (n = 19) respectively; Log-rank Mantel-Cox test; the other hand, IUE pHGG tumors displayed extravas- P < 0.0001) (Fig. 2a). This is in agreement with prior stud - cular dextran leakage, suggesting an increased level ies demonstrating the ability of H3K27M mutations to of vascular permeability (Fig.  4a, b). This was also sup - accelerate glioma formation [18, 30, 31], and with recent ported by the presence of extravascular red blood cells in data showing that the H3G34R mutation does not sig- IUE pHGG, but not IUE DMG tumors, as visualized by nificantly alter the formation or latency of PdgfraD842V TER119 staining (Fig. 4c). expressing gliomas [32]. Cortical pHGG mouse models Interactions between endothelial cells and neighbor- displayed histological features of glioblastoma, includ- ing cell types which make up the neurovascular unit, ing pseudopalisading necrosis and microvascular prolif- (pericytes, astrocytes and neurons), are essential for eration, while brainstem DMG models displayed features instructing and maintaining blood–brain barrier func- of grade III (3) high-grade gliomas (Fig.  2c, d). Control tion and vascular integrity. Immunostaining control cortical and brainstem IUEs (DNp53 + H3.3WT) did not and IUE glioma mouse models with Desmin, a marker effectively drive gliomagenesis, with only one tumor aris - of pericyte and smooth muscle cells, revealed no ing out of all samples (n = 23) (Fig. 2a). change in the vascular pericyte coverage in IUE DMG Upon collecting tumor samples, we noted areas of mac- tumors, which maintained the same extent of pericyte roscopic hemorrhage in all the cortical pHGGs, a feature coverage as the normal brainstem (Fig.  5a, d). IUE not found in brainstem DMG models (Fig.  2b). To com- pHGG tumors displayed a significant decrease in peri- pare tumor-associated vasculature within these models cyte investment compared to normal cortex and IUE we stained normal control, IUE DMG and IUE pHGG DMGs (Fig. 5a, d). Changes in the extracellular matrix tumors with the pan-endothelial marker CD31 and quan- protein Collagen IV (ColIV) further demonstrated dif- tified blood vessel density, diameter and branchpoints. ferences between IUE pHGG and DMG tumors, with Relative to normal cortical vessels, IUE pHGGs displayed IUE pHGG tumors showing decreased ColIV basement significantly enlarged and dilated vessels (Fig.  3a, b), membrane staining, indicating changes in the neuro- reduced overall vascular density (Fig.  3a, c) and reduced vascular unit compared to normal brain and IUE DMG vessel branching (Fig.  3a, d). While there was no signifi - tumors (Fig.  5b, e). We also performed co-immuno- cant difference between normal brainstem and IUE DMG labeling for Glut1 and Plasmalemma Vesicle Associ- blood vessels, comparison of IUE pHGG and DMG ves- ated Protein (Plvap), a protein involved in endothelial sels revealed pHGG tumors displayed a mean vascular fenestrae diaphragms, caveolae and trans-endothelial diameter approximately two times larger than that of channels [33–35]. Plvap expression was not detected DMG tumors, and reduced vessel density and branching in Glut1-positive blood vessels in control brain regions of nearly two and five times smaller than that of DMG or IUE DMG tumors, but could be found in some IUE tumors respectively (Fig.  3a–d). IUE pHGG tumors do pHGG vessels (Fig.  5c, f ). While expression of Plvap contain diverse intra-tumoral vascular features, with could be sporadically found in pHGG blood vessels, its core regions showing tortuous and chaotic angiogenic expression was not accompanied by the loss of Glut1, W ei et al. acta neuropathol commun (2021) 9:142 Page 7 of 18 Fig. 2 Generation of IUE pHGG and DMG mouse models. a Kaplan–Meier survival curves for IUE conditions: control brainstem (H3.3WT + DNp53, n = 11), control cortex (H3.3WT + DNp53, n = 12), IUE DMG (PdgfraD842V + DNp53 + H3.3K27M, n = 12) and IUE pHGG (PdgfraD842V + DNp53 + H3.3G34R, n = 19). ***p < 0.0001, Log-rank Mantel-Cox test. b Representative whole brain brightfield and GFP images depicting the regional location of IUE DMG and pHGG mouse models. Arrowheads point towards GFP-positive tumor regions. Scale bar = 1 mm. c H&E staining of IUE pHGG and IUE DMG sections. d High magnification inset images of H&E stained sections. Scale bars = 500 µm (top panels) and 50 µm (bottom panels) as previously described in medulloblastomas [14]. state of BBB functionality. These data show that DMG This would suggest at least the partial maintenance of blood vessels maintain numerous attributes of the nor- a signaling program that regulates Glut1 expression mal NVU, while many of these elements are altered or in endothelial cells, rendering a hybrid or fluctuating partially disrupted in cortical pHGGs. Wei et al. acta neuropathol commun (2021) 9:142 Page 8 of 18 Fig. 3 IUE pHGG and DMG mouse models recapitulate PDX tumor-associated vascular differences. a Representative immunofluorescent z-stack projection images of CD31-positive blood vessels in each experimental condition. Scale bar = 20 μm. Quantification of b CD31-positive blood vessel diameter, c density and d branch points. Control cortex and brainstem groups (n = 3), IUE DMG (n = 6) and IUE pHGG (n = 4). Data are presented as mean ± SEM. ***p < 0.0001; unpaired t-test with Mann–Whitney posthoc comparison Endothelial transcriptomes highlight differences ECs. Further separation between normal brain ECs and and similarities between pHGG, DMG and normal brain IUE DMG EC groups was evident, with a second branch endothelial signaling programs point dividing these groups (Fig.  6a). A similar pattern We next purified endothelial cells and vessel fragments emerged by principal component analysis (PCA), with from normal brain regions (cortex, cerebellum and samples from each group clustering in the same gen- brainstem) and IUE DMG and pHGG tumors to exam- eral region, and IUE pHGG ECs segregating the furthest ine molecular differences by whole-transcriptome analy - away from normal brain ECs (Additional file  1: Fig. S5). sis. Using antibody labeled magnetic beads to isolate Comparison of differentially expressed genes (FC > 4, adj. Cd31 + / Cd45- endothelial cells and vascular fragments, p-val < 0.05) between IUE DMG EC and IUE pHGG EC comparison of positive and negatively sorted popula- identified over-represented gene sets related to immune tions from normal brain samples showed positive enrich- system interactions (adaptive immune system, MHC ment for endothelial (Cd31, Tie2, Vegfr2) and pericyte class II antigen presentation), extracellular matrix regula- (Pdgfrb) genes, and negative enrichment for microglia tion (ECM organization, ECM degradation), and vascular (Cd68) and neuronal (NeuN, Tub3) genes (Additional interactions (cell surface interactions at the vascular wall, file  1: Fig. S4). Following RNA-seq, hierarchical cluster- platelet activation signaling and aggregation) (Fig.  6b, ing by Pearson’s correlation of all samples revealed two c). Further analysis of differentially up-regulated genes main branches separating IUE pHGG EC from nor- (FC > 2, adj. p-val < 0.05) in IUE pHGG ECs displayed mal brain region ECs (BS, CB and CTX) and IUE DMG enrichment in pathways related to immune response W ei et al. acta neuropathol commun (2021) 9:142 Page 9 of 18 Fig. 4 pHGG and DMG mouse models display differences in BBB function. a Representative whole brain images of brightfield and fluorescent TMR-dextran signal in control and IUE tumor conditions. Scale bar = 1 mm. b Representative immunofluorescent z-stack projection images of TMR-dextran and CD31 labeled sections. Scale bar = 20 μm. c Representative immunofluorescent z-stack projection images of control and IUE tumor conditions labeled with CD31 and Ter119 depicting the retention or extravascular leakage of red blood cells within samples. Scale bar = 20 μm Expression of secreted Wnt‑antagonists does not alter related pathways, while those up-regulated in IUE DMG DMG vascular phenotype ECs included extracellular matrix organization, SLC- DMGs are invasive brain tumors, and our vascular anal- mediated transmembrane transport, and signaling path- yses indicate minimal disruption to established blood ways (Hippo, Wnt) associated with BBB function [36–39] vessels within brain regions harboring tumor cells. This (Fig. 6d, Additional file  2: Table 1). Thus, beside preserv - is corroborated by the expression pattern of genes asso- ing their morphology and blood–brain barrier function, ciated with endothelial tip or stalk cell identity [40, 41]. IUE DMG blood vessels appear to maintain transcrip- IUE DMG ECs display increased expression of stalk cell tional programs that closely align with normal brain genes, while IUE pHGG ECs show higher expression of endothelium. Wei et al. acta neuropathol commun (2021) 9:142 Page 10 of 18 Fig. 5 Mural cell coverage and BBB associated marker expression differences between pHGG and DMG mouse models. Representative immunofluorescent z-stack projection images of a Desmin and CD31, b Collagen IV and CD31, and c Plvap and Glut1 in each experimental condition. Scale bar = 20 μm. d, e Quantification of CD31-positive vessel coverage by desmin and collagen IV respectively. f Quantification of Plvap-positive area in Glut1-positive blood vessels in each condition. Data are presented as mean ± SEM. n = 3 for all conditions. *p < 0.05, **p < 0.001, ***p < 0.0001; unpaired t-test with Mann Whitney posthoc comparison W ei et al. acta neuropathol commun (2021) 9:142 Page 11 of 18 tip cell genes (Fig.  7a). Further supporting the idea of a vasculature, which recapitulate findings in biopsy and more mature and stable vascular state in DMGs, analy- autopsy derived patient specimens. While variations in sis of transcription factor protein–protein interaction glioma BBB function have been appreciated within the (PPI) networks identified Sox17, a transcription factor field, including regional differences in glioma mouse (TF) that is highly expressed in mature brain endothelial models [44], a detailed comparison that catalogs and cells [42], as the most significantly enriched TF in DMG validates these differences between malignant gliomas ECs (Fig. 7b). Sox17 is a positive inducer of Wnt signaling has not been carried out. We show that the vascular net- [42], and together with Ctnnb1, which was also enriched work within DMGs remains mostly intact with respect in DMG ECs (Fig. 7b), may promote stability through the to blood vessel morphology, BBB function and tran- maintenance of proper Wnt signaling levels. Within IUE scriptional programs, while cortical pHGGs display both pHGG ECs many enriched TFs in the PPI were associ- phenotypic and transcriptional changes related to disor- ated with immune responses. These included Stat3, ganized angiogenesis, inflammation and BBB dysfunction which was the most significantly enriched TF, and inter - (Fig. 8). Moreover, DMG tumors display limited sensitiv- feron response factors (IRF3/6) and NOD2, all of which ity to the expression of secreted Wnt antagonists, which can drive downstream signaling related to immune sys- have been shown to drive BBB dysfunction in glioblas- tem activity. toma and medulloblastoma [14, 16], suggesting heteroge- Canonical Wnt-signaling is essential for blood–brain neity in the response of tumor-associated blood vessels to barrier formation in the developing brain [36, 37]. In extrinsic signals in the tumor microenvironment. addition, prior work in medulloblastoma and adult glio- In pathological conditions, including brain tumors, blastoma have shown that inhibition of endothelial the BBB presents a conundrum for treatment strategies. Wnt-signaling, by either expression of secreted Wnt- On one hand, the BBB is commonly cited as an impor- antagonists such as Dkk1 and Wif1 [14, 16], or genetic tant factor in brain tumor treatment resistance since the deletion of Wnt signaling components in endothelial majority of drugs and small molecules display limited cells [17], results in tumor vascular abnormalities and BBB penetration [3]. On the other hand, poor perfu- blood–brain barrier dysfunction. To test whether Wnt sion in abnormal and “dead end” vascular structures that antagonists could alter the vascular phenotype of DMG lack BBB function can impede drug delivery [45]. Stud- tumor models we expressed the Wnt receptor antagonist ies examining angiogenesis and BBB specification dur - Dkk1, or a secreted version of the Fzd8 receptor (Fzd8- ing normal CNS development have identified endothelial CRD-IgG) [43] in our IUE DMG mouse model (Addi- Wnt signaling as an essential regulator [36, 37]. The vas - tional file1: Fig. S6). IUE DMG tumors expressing empty cular phenotype of Wnt mutants shares many common vector control or the secreted Wnt antagonist (Dkk1 or features with that in glioblastoma, including chaotic Fzd8-CRD-IgG) developed tumors with similar laten- architecture, hemorrhaging, the formation of glomeruli cies, and analysis of vascular content and supporting structures and a lack of BBB functionality [36, 37, 46–49], components, such as ECM proteins, did not identify any suggesting a direct link between Wnt signaling disruption significant changes induced by secreted Wnt antagonist and brain tumor vascular abnormalities. Indeed, com- (Fig. 7c). Our data suggests that differences in the angio - pared to normal brain and DMG ECs, pHGG ECs show genic state of tumor-associated vasculature will influ - a modestly decreased Wnt-signaling at the transcriptome ence how they respond to other external cues, adding an level, indicating that alterations in this essential BBB additional layer of complexity to interactions within the signaling pathway likely participate in generating pHGG tumor microenvironment. vascular abnormalities. Despite these changes, pHGG blood vessels retain some features of the BBB, as most Discussion endothelial cells express Glut1, and only a small sub- Our analyses across pHGG and DMG implant based set co-express Plvap, a component of fenestrated pores PDX and native forming IUE mouse models reveal phe- [35]. This could be due to residual levels of Wnt signal - notypic and molecular differences in tumor-associated ing within gliomas, as BBB-specific Wnt-ligands (Wnt7a, (See figure on next page.) Fig. 6 pHGG and DMG endothelial transcriptomes highlight heterogeneity of tumor-associated and normal brain endothelial signaling programs. a Hierarchical clustering of Pearson’s correlation plot visualizing the correlation values between samples. Scale bar represents the range of the correlation coefficients displayed. b Heatmap of the top 25 most significant (adj. p-value) differentially expressed genes between IUE DMG EC and IUE pHGG EC. c Gene sets enriched by over-representation analysis of differentially expressed genes (FC > 4, adj. p < 0.05) between IUE pHGG EC and IUE DMG ECs. d Gene sets enriched by over-representation analysis of differentially expressed genes (FC > 2, adj. p < 0.05) up-regulated in IUE pHGG EC or IUE DMG ECs Wei et al. acta neuropathol commun (2021) 9:142 Page 12 of 18 Fig. 6 (See legend on previous page.) W ei et al. acta neuropathol commun (2021) 9:142 Page 13 of 18 Wnt7b and Norrin) are expressed by glial lineage cell- tumor cells, which is dependent on Stat3 activation types, including oligodendrocyte progenitors and astro- [61]. Additionally, increased cytokine expression caused cytes [50–53]. It could also be due to variability in Vegf by interactions between microglia and glioma cells can signaling, as Vegf ligands are required for the formation activate endothelial Jak / Stat3 signaling, resulting in of fenestrated blood vessels in the choroid plexus [54]. increased vascular permeability in  vitro [62]. Together, While Wnt signaling is essential for sprouting angiogen- this can lead to increased endothelial expression of leu- esis into the developing CNS and BBB formation [36, 37, kocyte adhesion molecules, which are associated with 49], how it interfaces with traditional pro-angiogenic fac- BBB dysfunction and inflammation [63, 64]. DMGs tors like Vegf in development and pathological conditions tend to display low T-cell infiltration compared to other remains an open area of investigation. Another possibil- gliomas [65]. Whether differences in the tumor micro - ity is that only certain mechanisms employed by the BBB environment and pHGG and DMG vascular properties are altered in pHGGs. While Glut1 expression is a BBB directly or indirectly influence the differential recruit - associated marker in CNS vasculature, its expression can ment of infiltrating immune cells into tumors will be of be maintained in the presence of other NVU alterations particular interest to further delineate. that impact BBB function, such pericyte loss [55, 56]. We find that DMG vessels are not particularly sensi - Pericyte coverage has been shown to directly mediate tive to the expression of secreted Wnt antagonists, which transcytosis rates [55–57], and in healthy brain, recep- have previously been shown to drive BBB dysfunction tor mediated transcytosis allows the selective crossing of in glioblastoma [16] and medulloblastoma [14, 15]. This plasma proteins, which switches to a more general tran- finding, taken together with our data showing DMGs scytosis mechanism with aging and pericyte loss [58]. contain a stable network of blood vessels in a mature Decreased pericyte coverage in pHGGs could have a par- endothelial transcriptional state, lead us to postulate that ticular impact on transcytosis and could in part explain differences in the angiogenic state of brain tumors plays why Glut1 expression is maintained in regions that dis- a role in their responsiveness to fluctuations in Wnt sig - play vascular permeability. nals. Canonical Wnt signaling by specific ligands (Wnt7a, Beside our immuno-staining characterization that Wnt7b, Ndp) and co-receptor complexes (Fzd4, Gpr124, demonstrates DMG blood vessels retain normal morpho- Reck, Lrp5/6) is essential for BBB induction and matura- logical features and BBB function, we find that DMG ECs tion in the developing brain [36, 37, 46, 47, 66–69]. Yet, maintain a transcriptional program similar to that of nor- inhibition or deletion of these Wnt ligand or receptor mal brain ECs. Examination of endothelial tip and stalk components in the mature brain under normal homeo- cell gene expression reveals increased expression of stalk static conditions does not impact vascular integrity or cell genes in DMG ECs, and elevated expression of angi- BBB function [17, 42]. Levels of endothelial Wnt signal- ogenic tip cell genes in pHGG ECs. Moreover, PPI net- ing in the brain change over the course of brain devel- work analysis identified Sox17 as the most significantly opment and maturation. Previous studies have shown enriched differentially expressed transcription factor in canonical Wnt signaling, using the BAT (beta-catenin DMG ECs. Sox17 expression is highest in more mature activated reporter) LacZ reporter mouse, decreases in brain endothelial cells [42], suggesting that tumor-asso- brain endothelial cells as they mature [42, 70]. Moreover, ciated blood vessels in DMGs mainly consist of existing expression of Apcdd1, an inhibitor of the canonical Wnt mature vasculature, and not newly created vessels that pathway, increases with age, ensuring the proper level of develop in a highly organized fashion. Transcription fac- Wnt signaling required for proper angiogenesis and BBB tors enriched in pHGG EC PPI networks were mainly development [71]. Understanding how these differences related to inflammatory mediated pathways, agreeing in Wnt signaling during vascular development and mat- with the general immune-related signatures identified uration apply to brain tumors will be important to con- when comparing to normal or DMG ECs. Stat3 is a criti- sider not only for DMGs, but also for pHGGs and adult cal mediator of immune related responses in gliomas [59, glioblastomas, since they also contain regions of tumor- 60]. Interactions between microglia and glioma tumor associated vasculature that are not engaged in active cells can promote a mesenchymal cell state in glioma angiogenesis. (See figure on next page.) Fig. 7 Expression of secreted Wnt-antagonists does not alter DMG vascular phenotype. a Heatmap of endothelial stalk and tip cell associated gene expression in IUE pHGG and DMG ECs. b Transcription factor protein–protein interaction networks enriched in IUE pHGG or DMG ECs. c Representative immunofluorescent z-stack projection images of CD31 labeled or CD31 and Collagen IV labeled blood vessels in IUE DMG control, Dkk1, or Fzd8-CRD-IgG tumors. Scale bar = 20 μm Wei et al. acta neuropathol commun (2021) 9:142 Page 14 of 18 Fig. 7 (See legend on previous page.) W ei et al. acta neuropathol commun (2021) 9:142 Page 15 of 18 Fig. 8 Summary of pHGG and DMG models and vascular phenotypes associated with each tumor type. Patient derived xenografts and in utero electroporation based cortical pHGG and brainstem DMG models were utilized to investigate in vivo vascular phenotypes. Across model systems, DMG tumor-associated blood vessels consistently displayed vascular phenotypes, BBB function and transcriptional programs similar to normal brain endothelium. pHGG tumor-associated blood vessels were associated with abnormal vascular phenotypes, BBB dysfunction, and transcriptional changes In summary, we present a detailed analysis of pHGG several other mechanisms of BBB opening, including and DMG tumor-associated vascular profiles, highlight - transcytosis, have been described after ultrasound treat- ing blood vessel heterogeneity and differences between ment [74]. Other methods to circumvent the BBB are these deadly brain tumors. Additionally, our data shows convection-enhanced delivery (CED), in which drugs are DMGs respond differently to variations in Wnt signal - directly infused into the parenchyma or tumor under a ing levels, pointing out a need to further understand how hydrostatic pressure gradient [75], the use of nanopar- canonical Wnt signaling and Vegf signaling interplay to ticles, or intranasal/intra-arterial delivery [76]. A bet- regulate angiogenesis and BBB specification both in nor - ter understanding of the tumor vasculature can help to mal CNS development and in pathological settings. As decide which method to use in certain tumor types. current outcomes for most malignant gliomas are dismal, Together, the present work provides new insights that regardless of their vascular phenotype, there is a critical emphasize the need to consider vascular heterogeneity need for new and improved therapeutic strategies [8]. among brain tumors in the development of new thera- For example, strategies to “normalize” leaky and tortur- peutic strategies. ous blood vessels within brain tumors could be accom- plished by stabilizing endothelial Wnt signaling. This Abbreviations could provide the benefit of promoting normal vascu - BBB: Blood brain barrier; DMG: Diffuse midline glioma; PHGG: Pediatric high- lar growth and BBB function within tumors, enhancing grade glioma; GEMMs: Genetically engineered mouse models; NVU: Neuro- vascular unit; CNS: Central nervous system; MRI: Magnetic resonance imaging; the perfusion and vascularity of brain tumors even bet- CE: Contrast enhancement; IUE: In utero electroporation; PDX: Patient derived ter than current anti-Vegf therapies. Recent strategies xenograft; BS: Brainstem; CTX: Cortex; CB: Cerebellum; H&E: Hematoxylin and have leveraged receptor mediated transcytosis to deliver eosin; qPCR: Quantitative real-time polymerase chain reaction; TMR: Tetra- methylrhodamine; ECs: Endothelial cells; Cldn5: Claudin5; Plvap: Plasmalemma cargo into the brain [72, 73]. By defining the expression vesicle associated protein; TF: Transcription factor; COLIV: Collagen IV; MACS: of receptors in endothelial cells across the normal brain Magnetic cell sorting; FUS: Focused ultrasound; CED: Convection enhanced and brain tumor types, one can develop approaches to delivery. target the delivery of new therapies into brain tumors that have been traditionally hard to penetrate. Transcy- Supplementary Information tosis can also be upregulated using (microbubble-medi- The online version contains supplementary material available at https:// doi. org/ 10. 1186/ s40478- 021- 01243-1. ated) focused ultrasound (FUS). This is a non-invasive method that temporarily opens the BBB in a targeted Additional file 1 contains data related to supplementary figures 1–6. location. Although drug delivery by FUS is thought to This includes vascular stains in (Fig S1) PDX and (Fig S2) primary pHGG mainly function through mechanical stimulation of the and DMG samples, examples of intratumoral heterogeneity in IUE pHGG blood vessels and consequent opening of tight-junctions, mouse models (Fig S3), validation of endothelial purity following magnetic Wei et al. acta neuropathol commun (2021) 9:142 Page 16 of 18 Netherlands. Amsterdam Leukodystrophy Center, Amsterdam UMC, Amster- cell sorting (Fig S4), PCA of endothelial samples (Fig S5), and validation of dam, The Netherlands. Dkk1 expression in IUE pHGG overexpression models (Fig S6). Received: 2 July 2021 Accepted: 10 August 2021 Additional file 2 contains data related to transcriptional analysis of endothelial samples from normal brain regions, IUE pHGG and IUE DMG mouse models. Tab1: DEGs comparing DMG vs. pHGG ECs; Tab2: DEGs comparing BS vs. DMG ECs; Tab3: DEGs comparing Ctx vs. pHGG ECs; Tab 4: Pathways enriched in pHGG ECs compared to DMG ECs; Tab5: Pathways enriched in DMG ECs compared to pHGG ECs. References 1. Daneman R, Prat A (2015) The blood-brain barrier. Cold Spring Harb Perspect Biol 7:a020412. https:// doi. org/ 10. 1101/ cshpe rspect. a0204 12 Acknowledgements 2. Muldoon LL, Soussain C, Jahnke K, Johanson C, Siegal T, Smith QR, Hall We would like to thank members of the pathology core at CCHMC, and the WA, Hynynen K, Senter PD, Peereboom DM et al (2007) Chemotherapy Genomics, Epigenomics, and Sequencing Core at UC (supported in part by delivery issues in central nervous system malignancy: a reality check. J CEG grant NIEHS P30-ES006096) for their assistance. Clin Oncol 25:2295–2305. https:// doi. org/ 10. 1200/ JCO. 2006. 09. 9861 3. Heffron TP (2016) Small molecule kinase inhibitors for the treatment of Authors’ contributions brain cancer. J Med Chem. https:// doi. org/ 10. 1021/ acs. jmedc hem. 6b006 XW performed the majority of experiments and data analysis and contributed to the writing and editing of the manuscript. MHM performed experiments 4. Sarkaria JN, Hu LS, Parney IF, Pafundi DH, Brinkmann DH, Laack NN, Gian- related to PDX models and contributed to the writing and editing of the nini C, Burns TC, Kizilbash SH, Laramy JK et al (2018) Is the blood-brain manuscript. MB and MB performed experiments related to primary pHGG and barrier really disrupted in all glioblastomas? A critical assessment of DMG human samples. EH supervised MHM and contributed to the writing existing clinical data. Neuro Oncol 20:184–191. https:// doi. org/ 10. 1093/ and editing of the manuscript. TNP conceived the project, supervised XW, neuonc/ nox175 assisted with planning of experiments and oversaw writing and editing of the 5. Jones C, Baker SJ (2014) Unique genetic and epigenetic mechanisms manuscript. 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Defining tumor-associated vascular heterogeneity in pediatric high-grade and diffuse midline gliomas

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Abstract

The blood–brain barrier (BBB) plays important roles in brain tumor pathogenesis and treatment response, yet our understanding of its function and heterogeneity within or across brain tumor types remains poorly characterized. Here we analyze the neurovascular unit (NVU) of pediatric high-grade glioma (pHGG) and diffuse midline glioma (DMG) using patient derived xenografts and natively forming glioma mouse models. We show tumor-associated vas- cular differences between these glioma subtypes, and parallels between PDX and mouse model systems, with DMG models maintaining a more normal vascular architecture, BBB function and endothelial transcriptional program rela- tive to pHGG models. Unlike prior work in angiogenic brain tumors, we find that expression of secreted Wnt antago - nists do not alter the tumor-associated vascular phenotype in DMG tumor models. Together, these findings highlight vascular heterogeneity between pHGG and DMG and differences in their response to alterations in developmental BBB signals that may participate in driving these pathological differences. Keywords: Pediatric high-grade glioma, Diffuse midline glioma, Blood brain barrier, Endothelial cells, Neurovascular unit, Diffuse intrinsic pontine glioma, H3K27M, Wnt signaling Introduction across adult and pediatric brain tumor entities [3, 4]. Our The blood–brain barrier (BBB) is a specialized vascular understanding of intra- and inter-tumoral BBB heteroge- structure within the brain formed by the neurovascular neity continues to improve with advancements in defin - unit (NVU) which consists of endothelial cells, pericytes, ing molecular subgroups of human brain tumors, and the astrocytes and neurons [1]. While essential for normal development of accurate patient-derived xenograft (PDX) brain function and homeostasis, the BBB poses a prob- and genetically engineered mouse models (GEMMs) that lem for treating CNS related diseases since the majority faithfully recapitulate features of primary human brain of drugs and small molecules display limited brain pen- tumors. etration [2]. BBB function was historically considered Pediatric high-grade gliomas (pHGGs) are among disrupted in brain tumors based on studies using aggres- the most common childhood brain tumors and can be sive adult glioma models that do not accurately reflect divided into multiple subgroups based different fea - the diversity and pathological heterogeneity identified tures including histology, location, mutation status and molecular profile [5 –8]. One of the most lethal pHGG types are H3K27M mutant diffuse midline glio - mas (DMGs), which encompass midline and brainstem *Correspondence: timothy.phoenix@uc.edu Division of Pharmaceutical Sciences, James L. Winkle College gliomas that harbor H3K27M mutations [7, 9]. Treat- of Pharmacy, University of Cincinnati, Cincinnati, OH, USA ment options remain limited for DMG patients, and no Full list of author information is available at the end of the article © The Author(s) 2021. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. The Creative Commons Public Domain Dedication waiver (http:// creat iveco mmons. org/ publi cdoma in/ zero/1. 0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Wei et al. acta neuropathol commun (2021) 9:142 Page 2 of 18 chemotherapy or targeted therapy has demonstrated IUE mouse models significant survival benefits thus far [8 , 10]. One pro- All IUE related mouse work was done according to insti- posed reason for the failure of systemically delivered tutional and IACUC review boards (University of Cincin- therapies is that DMGs maintains a more intact BBB nati). The IUE procedure to generate pediatric high-grade compared to other non-brainstem tumors, as clinicians glioma and diffuse midline glioma mouse models was have noted that DMGs frequently display little to no performed as previously described [18]. Briefly, IUE contrast enhancement on magnetic resonance imag- pHGG mouse models were created by lateral ventricle ing (MRI) [11, 12]. Yet a detailed characterization or injection of PdgfraD842V + H3.3G34R + DNP53 + Pbase direct comparison of tumor-associated vasculature in plasmids (all at 1  µg/µL). IUE DMG mouse model th cortical pHGGs and DMGs has not been systematically were created by 4 ventricle injection of Pdg- performed. fraD842V + H3.3K27M + DNP53 + Pbase plasmids. Here, employing patient derived xenografts (PDX) Control BS or CTX IUE conditions were created th and in utero electroporation (IUE) based mouse mod-by 4 or lateral ventricle injections respectively of els of cortical pHGG and brainstem DMG, we perform H3.3WT + DNP53 + Pbase plasmids. Survival curves a histological and molecular analysis of tumor-associ- were generated in GraphPad Prism using Log-rank Man- ated vasculature. We show that cortical pHGGs induce tle-Cox statistical tests. changes traditionally associated with glioblastoma vas- culature [13], including abnormal vessel morphology Patient derived xenograft pHGG and DMG models and BBB disruption, while brainstem DMGs maintain a and primary tumor samples similar vascular content and architecture to that of the Pediatric HGG and DIPG/DMG patient derived xeno- normal brainstem. Additional transcriptomics studies graft (PDX) samples are from Dr. Esther Hulleman’s support our phenotypic analyses and suggest that DMG group at VU University Medical Center, Amsterdam, endothelial cells are maintained in a stable and mature The Netherlands. All PDX experiments were carried state. While expression of Wnt-antagonists in other out at the VU University Medical Center in accordance aggressive pediatric brain tumors, such as medulloblas- with the Declaration of Helsinki and national and insti- toma [14, 15] and adult glioblastoma induces BBB dis- tutional guidelines. 5- to 6-week old NOD/SCID/Il2rg-/- ruption [16, 17], we find that their expression does not mice (Jackson) were intracranially injected with 500,000 alter the BBB or tumor-associated vascular phenotype cells of each of the following primary cultures: VUMC- in DMG models. Our findings provide direct evidence DIPG-F [19], VUMC-DIPG-G, VUMC-HGG-11 [20], of tumor-associated vascular differences between DMG VUMC-HGG-14 [21]. The stereotactic coordinates that and pHGG tumors and underscore the need to con- were used to inject into the pons (VUMC-DIPG-F and sider intra- and inter-tumor heterogeneity in vascular -G) were 0.8  mm laterally, 1  mm caudally, and 4.5  mm content and BBB function when developing therapeutic ventrally from the lambda. The stereotactic coordinates strategies that target tumor cells or their vasculature. that were used to inject into the striatum (VUMC- HGG-11 and -14) were 2  mm laterally, 1  mm cranially, and 3 mm ventrally from the bregma. Cells were injected Materials and methods in an injection volume of 5 μL at a flow rate of 1 μL/ Plasmid constructs minute to minimize the neurological side effects of the DNA plasmids were construct as we previously procedure. Primary tumor-tissue was obtained through described [18]. PBCAG- H3.3G34R-Ires-eGFP was surgical resection (pHGG) or via a brain autopsy study generated by InFusion (Takara) PCR site directed (DIPG/DMG) in the Amsterdam UMC (Amsterdam, the mutagenesis from an H3F3A template plasmid Netherlands), in accordance with the declaration of Hel- (Addgene #42,632) and insertion into EcoR1 linearized sinki and approved by the institutional review board of PBCAG-Ires-eGFP. PBCAG-Dkk1-Ires-eGFP was gen- Amsterdam UMC, location VUmc (METc VUmc, study erated by PCR amplification of Dkk1 and insertion by number: VUMC2009/237) and the Scientific Committee InFusion ligation into EcoR1 linearized PBCAG-Ires- of the Dutch Childhood Oncology Group (DCOG). eGFP. PBCAG-Fzd8-CRD-IgG was created by PCR amplification of Fzd8-CRD-IgG (Addgene #16,689) Tissue collection, processing and immunostaining and insertion by InFusion ligation into EcoR1 line- Upon development of neurological symptoms, mice were arized PBCAG-Ires-eGFP. All plasmids were verified by deeply anesthetized before perfusion with cold DPBS fol- Sanger sequencing (CCHMC DNA sequencing core), lowed by 1% PFA. Brains were rapidly dissected in cold and plasmid stocks prepared by using NuceloBond Xtra DPBS and then fixed in 100% MeOH at 4  °C overnight. Maxi EF endotoxin-free kits (Machery-Nagel). W ei et al. acta neuropathol commun (2021) 9:142 Page 3 of 18 Samples were rehydrated at 4  °C in DPBS for 4  h before Magnetic cell sorting (MACS) embedding in 3% low-melt agarose gel (IBI scientific, Endothelial cells (ECs) were purified using magnetic #B70051). 150 um thick free-floating sections were cut sorting using MACS (Miltenyl Biotec) according to the using a Leica vibratome. Floating sections were incubated manufacturer’s protocol. All antibodies, buffers and in blocking solution (PBS + 0.5% Triton x-100 + 10% nor- equipment were purchased from Miltenyl Biotec. Briefly, mal donkey serum) at RT for 30  min before incubating tumor or normal tissues were micro dissected under a with primary antibodies at 4  °C overnight. The next day fluorescent stereoscope, and 3–4 tumors or control tis - sections were washed in DPBS, transferred to blocking sue samples were pooled together to minimize vari- solution containing the appropriate secondary antibod- ability between individual tumors. Samples were then ies (1:500) and incubated at room temperature for 2  h. dissociated by incubation in a Collagenase IV cocktail Finally, sections were incubated in Hoechst (1:1000 in for 45 min at 37 °C. Collagenase IV dissociation solution PBS) for 10 min before final DPBS washing and mounting was made by mixing 32 mg collagenase IV (Worthington, onto slides (Fisher, Superfrost), and coverslipped (Pro- #LS004209), 10  mg Deoxyribonuclease I (Worthington, long Gold Antifade, ThermoFisher). Primary antibodies #LS002007), 20 mg Soybean trypsin inhibitor (Worthing- used in this study include: eGFP (Aves, #GFP1020), CD31 ton, #LS003587) with 10 mL DPBS. Following incubation (BD Biosciences, #550,274), Glut1 (Millipore, #07–1401), and trituration, cell suspensions were passed through Collagen IV (BioRad, #161,115), Desmin (Cell Signal- 40  μm mesh cell strainers. Myelin and cellular debris ing, #5332), human Vimentin (eBioscience, #11–9897- were removed using debris removal solution (Milte- 82), Claudin-5 (Thermofisher, #352,588), Plvap (BD nyl Biotec #130–109-398), and red blood cells (RBCs) Biosciences, #550,274), Ter119 (Invitrogen #14–5921-82) removed by incubation in ACK lysing buffer (Thermo and Hoechst (ThermoFisher). Corresponding secondary Fisher, #A10492-01). Cell pellets were resuspended in 90 antibodies used were all purchased from Jackson Immu- μL PEB buffer / 10 million cells. PEB buffer was prepared noResearch. Images were acquired on a confocal micro- by diluting MACS BSA stock solution (#130–091-376) scope (Nikon A1), and image analysis was performed in 1:20 with autoMACS rinsing solution (#130–091-222). NIH Image J. Statistical analyses of these data were per- Cells were blocked by adding 10  μl of FcR blocking rea- formed in GraphPad prism as described in the manu- gent and incubated with 15 μL of CD45 microbeads script. P-values of ≤ 0.05 were considered statistically (#130–052-301) at 4  °C for 15  min, then applied to MS significant. column (#130–042-201) against a magnetic separator For histology, mouse brain tissue was fixed in 10% for - followed by two washes with PEB buffer. CD45-negative malin overnight and transferred to 70% ethanol before flow through cells were then collected and labeled with paraffin embedding. 5  μm thick sections were prepared CD31 microbeads (#130–097-418) and passed through on a microtome (Lecia), and processed for hematoxylin– a new MS column against a magnetic separator followed eosin (H&E) staining. For immunohistochemistry of pri- by two washes with PEB buffer. CD45-negative/CD31- mary patient samples 5  µm thick paraffin sections were positive ECs retained within the MS column were eluted prepared and stained using standardized techniques. in PEB buffer by expelling with the provided column Primary antibodies used include anti-CD31 (Dako, plunger. Purified cell numbers were determined using an #M0823), anti-Cldn5 (Invitrogen, #34–1600) and anti- automated cell counter (Thermo Fisher) and then pro - Glut1 (Millipore, #07–1401). Stains were developed with cessed for total RNA isolation. secondary HRP and DAB immunoreactivity secondary kits (Dako) and counterstained with Hematoxylin before mounting. RNA preparation, real‑time quantitative PCR, whole transcriptome sequencing and analysis Total RNA was isolated from freshly isolated samples TMR dextran BBB permeability assay using the NucleoSpin Plus RNA kit (Macherey–Nagel) 10-kDa Tetramethylrhodamine (TMR)-dextran (Ther - as previously described [18]. cDNA was synthesized moFisher # D1817) was dissolved in sterile DPBS at a using SuperScript Vilo cDNA synthesis kit (Thermo concentration of 10  mg/ml. TMR dextran was adminis- Fisher, #11,754,050) according to manufacture proto- tered as previously described elsewhere [22]. Following col. Real-time PCR was performed using a Bio-Rad CFX circulation of the dextran tracer brains were harvested qPCR system. The fold increase was determined using in ice-cold DPBS and fixed in 4% PFA overnight. Fixed the 2 − ΔΔCT method. Gapdh was used as endogenous brains were washed in DPBS the next day, followed by control to normalize mRNA expression level. The fol - incubation in 30% sucrose for 48 h at 4˚C before embed- lowing primers were purchased from IDT. Gapdh, Fwd: ding in tissue freezing media. 50  μm thick free-floating AGG TCG GTG TGA ACG GAT TTG, Rvs: TGT AGA sections were cut using a Leica cryostat. Wei et al. acta neuropathol commun (2021) 9:142 Page 4 of 18 CCA TGT AGT TGA GGT CA; Cd31, Fwd,: ACG CTG using iGEAK and gProfiler using default settings [24]. GTG CTC TAT GCA AG, Rvs: TCA GTT GCT GCC Transcription factor protein–protein interaction net- CAT TCA TCA; Tie2, Fwd: GAG TCA GCT TGC TCC works were generated using Enrichr [25]. All RNA- TTT ATG G, Rvs: AGA CAC AAG AGG TAG GGA seq files are deposited in Gene Expression Omnibus as ATT GA; Vegfr2, Fwd: TTT GGC AAA TAC AAC CCT GSE179372. TCA GA, Rvs: GCA GAA GAT ACT GTC ACC ACC; NeuN, Fwd: ATC GTA GAG GGA CGG AAA ATT GA, Results Rvs: GTT CCC AGG CTT CTT ATT GGT C; Cd68, pHGG and DMG patient derived xenografts display Fwd: TGT CTG ATC TTG CTA GGA CCG, Rvs: GAG tumor‑associated vascular differences. AGT AAC GGC CTT TTT GTG A; Dkk1, Fwd: CTC The presence of contrast enhancement (CE) in mag - ATC AAT TCC AAC GCG ATC A, Rvs: GCC CTC ATA netic resonance imaging (MRI), indicating gadolinium GAG AAC TCC CG. RNA-sequencing was performed as leakage outside of blood vessels, is a common feature in previously described [18]. RNA quality control was per- most glioblastomas [13]. Yet prior radiological studies formed on a bioanalyzer (BioRad) to ensure the quality have noted DMG patients tend to display limited to no of each sample submitted. For isolation of polyA RNA, CE, suggesting the maintenance of a mostly intact BBB a NEBNext Poly(A) mRNA Magnetic Isolation Module [11, 12, 26]. To investigate potential vascular differences (New England BioLabs) was used for polyA RNA puri- between DMG and pHGG tumors, we examined availa- fication with a total of 1  μg good quality total RNA as ble orthotopic PDX models [19–21]. Staining PDX tumor input. The SMARTer Apollo NGS library prep system samples with a human specific tumor cell marker (hVi - (Takara) was used for automated polyA RNA isolation. mentin) and pan-endothelial marker (CD31) revealed For RNA sequencing library preparation, the library for minimal changes in the vasculature phenotype of DMG RNA-seq was prepared by using the NEBNext Ultra II PDXs (Fig.  1a, b). In contrast pHGG PDXs displayed Directional RNA Library Prep Kit (New England Bio- decreased blood vessel density, vascular branchpoints Labs). After indexing via PCR enrichment (8 cycles), the and enlarged lumens (Fig. 1b, d, e). Staining for the tight amplified libraries together with the negative control junction marker Claudin5 (Cldn5) and BBB associated were cleaned up for quality control analysis. To study dif- glucose transporter Glut1 (also known as Slc2a1) found ferential gene expression, individually indexed and com- both expressed in pHGG and DMG PDX vasculature, patible libraries were proportionally pooled (~ 25 million although pHGG PDXs displayed small but significant reads per sample in general) for clustering in the cBot decreases in Glut1-expressing vessels, along with regions system (Illumina). Libraries at the final concentration of of disorganized Cldn5 tight junctions in vessels (Fig.  1c, 15  pM were clustered onto a single-read flow cell using f). In contrast, DMG PDX blood vessels showed no the IlluminaTruSeq SR Cluster Kit v3, and sequenced to noticeable differences in the expression or organization 51 bp using theTruSeq SBS Kit v3 on the Illumina HiSeq of Glut1 and Cldn5 compared to normal brain regions system. Sequence reads were aligned to the reference (Fig.  1c). In parts of pHGG tumors that contained genome using the TopHat aligner and reads aligning to necrotic regions there was a transition of Glut1 stain- each known transcript were counted using Bioconduc- ing from vascular to non-vascular cells (i.e., tumor cells tor packages for next-generation sequencing data analy- or associated macrophages), likely indicating a metabolic sis. The differential expression analysis between different response to hypoxic conditions [27] (Additional file  1: sample types was performed using the negative binomial Fig. S1). Additional staining of primary human DMG statistical model of read counts as implemented in the and pHGG samples for CD31, Cldn5 and Glut1 showed edgeR Bioconductor package. Transcriptional profiles similar patterns to PDX models, providing further evi- were interrogated with iGEAK (Interactive Gene Expres- dence for vascular differences between these tumors sion Analysis Kit for microarray and RNA-seq data), an (Additional file  1: Fig. S2). This data, together with prior R (v3.3.2) and JavaScript based open-source desktop radiological studies in patients and PDX models [12, 19, application [23]. Functional enrichment of differentially 26, 28], suggests DMGs maintain a relatively intact BBB expressed gene lists between conditions was performed compared to cortical pHGGs. (See figure on next page.) Fig. 1 Tumor associated vascular differences in orthotopic patient derived xenograft pHGG and DMG models. Representative immunofluorescent images of pHGG, DMG PDX samples and normal brain labeled with a hVimentin and Hoechst, b CD31 and c Claudin-5 and Glut1 and Hoechst. Scale bar = 20 μm. Quantification of d CD31-positive blood vessel density in the normal brain (n = 5), DMG PDX (n = 5) and pHGG PDX (n = 3), e branchpoints and f % Glut1-positive blood vessels in DMG PDX (n = 5) and pHGG PDX (n = 3). Data are presented as mean ± SEM *P < 0.05, **p < 0.001, ***p < 0.0001, unpaired t-test with Mann–Whitney posthoc comparison W ei et al. acta neuropathol commun (2021) 9:142 Page 5 of 18 Fig. 1 (See legend on previous page.) Wei et al. acta neuropathol commun (2021) 9:142 Page 6 of 18 Cortical pHGG and brainstem DMG mouse models vessels with diameters ranging from 2 μm to 40 μm, and recapitulate tumor‑associated vascular differences bordering rim regions with a mixture of abnormal and To further examine vascular differences between pHGG normal vessel phenotypes. (Additional file  1: Fig. S3). and DMGs we employed recently developed glioma u Th s, de novo IUE mouse models of brainstem DMG mouse models created by in utero electroporation (IUE) and supratentorial pHGG recapitulate the architectural [18, 29]. DMG mouse models were made by brainstem differences found in PDX tumor-associated vascular targeted IUE of Piggybac DNA plasmids expressing Pdg- networks, providing an accurate system to study tumor- fraD842V, DNp53 and H3.3K27M. To generate corti- blood vessel interactions. cal pHGG mouse models, we replaced H3.3K27M with a plasmid expressing H3.3G34R, combine with Pdg- pHGG and DMG mouse models display differences fraD842V and DNp53 expressing Piggybac DNA plas- in vascular integrity and BBB function mids. Both H3K27M DMG and H3G34R pHGG IUEs To gain additional insight into these vascular differ - resulted in the generation of fully penetrant gliomas ences we examined the vascular permeability of control in successfully electroporated offspring. H3K27M IUE and IUE tumor models by circulation of a fluorescently DMGs formed at a significantly shorter latency com - labeled dextran tracer (10  kDa TMR-Dextran). No pared to H3G34R IUE pHGGs (median survival for extravascular leakage was found in control brainstem and IUE DMG model (n = 12) was 30  days and 79  days for cortical samples, or in IUE DMG tumors (Fig. 4a, b). On HGG (n = 19) respectively; Log-rank Mantel-Cox test; the other hand, IUE pHGG tumors displayed extravas- P < 0.0001) (Fig. 2a). This is in agreement with prior stud - cular dextran leakage, suggesting an increased level ies demonstrating the ability of H3K27M mutations to of vascular permeability (Fig.  4a, b). This was also sup - accelerate glioma formation [18, 30, 31], and with recent ported by the presence of extravascular red blood cells in data showing that the H3G34R mutation does not sig- IUE pHGG, but not IUE DMG tumors, as visualized by nificantly alter the formation or latency of PdgfraD842V TER119 staining (Fig. 4c). expressing gliomas [32]. Cortical pHGG mouse models Interactions between endothelial cells and neighbor- displayed histological features of glioblastoma, includ- ing cell types which make up the neurovascular unit, ing pseudopalisading necrosis and microvascular prolif- (pericytes, astrocytes and neurons), are essential for eration, while brainstem DMG models displayed features instructing and maintaining blood–brain barrier func- of grade III (3) high-grade gliomas (Fig.  2c, d). Control tion and vascular integrity. Immunostaining control cortical and brainstem IUEs (DNp53 + H3.3WT) did not and IUE glioma mouse models with Desmin, a marker effectively drive gliomagenesis, with only one tumor aris - of pericyte and smooth muscle cells, revealed no ing out of all samples (n = 23) (Fig. 2a). change in the vascular pericyte coverage in IUE DMG Upon collecting tumor samples, we noted areas of mac- tumors, which maintained the same extent of pericyte roscopic hemorrhage in all the cortical pHGGs, a feature coverage as the normal brainstem (Fig.  5a, d). IUE not found in brainstem DMG models (Fig.  2b). To com- pHGG tumors displayed a significant decrease in peri- pare tumor-associated vasculature within these models cyte investment compared to normal cortex and IUE we stained normal control, IUE DMG and IUE pHGG DMGs (Fig. 5a, d). Changes in the extracellular matrix tumors with the pan-endothelial marker CD31 and quan- protein Collagen IV (ColIV) further demonstrated dif- tified blood vessel density, diameter and branchpoints. ferences between IUE pHGG and DMG tumors, with Relative to normal cortical vessels, IUE pHGGs displayed IUE pHGG tumors showing decreased ColIV basement significantly enlarged and dilated vessels (Fig.  3a, b), membrane staining, indicating changes in the neuro- reduced overall vascular density (Fig.  3a, c) and reduced vascular unit compared to normal brain and IUE DMG vessel branching (Fig.  3a, d). While there was no signifi - tumors (Fig.  5b, e). We also performed co-immuno- cant difference between normal brainstem and IUE DMG labeling for Glut1 and Plasmalemma Vesicle Associ- blood vessels, comparison of IUE pHGG and DMG ves- ated Protein (Plvap), a protein involved in endothelial sels revealed pHGG tumors displayed a mean vascular fenestrae diaphragms, caveolae and trans-endothelial diameter approximately two times larger than that of channels [33–35]. Plvap expression was not detected DMG tumors, and reduced vessel density and branching in Glut1-positive blood vessels in control brain regions of nearly two and five times smaller than that of DMG or IUE DMG tumors, but could be found in some IUE tumors respectively (Fig.  3a–d). IUE pHGG tumors do pHGG vessels (Fig.  5c, f ). While expression of Plvap contain diverse intra-tumoral vascular features, with could be sporadically found in pHGG blood vessels, its core regions showing tortuous and chaotic angiogenic expression was not accompanied by the loss of Glut1, W ei et al. acta neuropathol commun (2021) 9:142 Page 7 of 18 Fig. 2 Generation of IUE pHGG and DMG mouse models. a Kaplan–Meier survival curves for IUE conditions: control brainstem (H3.3WT + DNp53, n = 11), control cortex (H3.3WT + DNp53, n = 12), IUE DMG (PdgfraD842V + DNp53 + H3.3K27M, n = 12) and IUE pHGG (PdgfraD842V + DNp53 + H3.3G34R, n = 19). ***p < 0.0001, Log-rank Mantel-Cox test. b Representative whole brain brightfield and GFP images depicting the regional location of IUE DMG and pHGG mouse models. Arrowheads point towards GFP-positive tumor regions. Scale bar = 1 mm. c H&E staining of IUE pHGG and IUE DMG sections. d High magnification inset images of H&E stained sections. Scale bars = 500 µm (top panels) and 50 µm (bottom panels) as previously described in medulloblastomas [14]. state of BBB functionality. These data show that DMG This would suggest at least the partial maintenance of blood vessels maintain numerous attributes of the nor- a signaling program that regulates Glut1 expression mal NVU, while many of these elements are altered or in endothelial cells, rendering a hybrid or fluctuating partially disrupted in cortical pHGGs. Wei et al. acta neuropathol commun (2021) 9:142 Page 8 of 18 Fig. 3 IUE pHGG and DMG mouse models recapitulate PDX tumor-associated vascular differences. a Representative immunofluorescent z-stack projection images of CD31-positive blood vessels in each experimental condition. Scale bar = 20 μm. Quantification of b CD31-positive blood vessel diameter, c density and d branch points. Control cortex and brainstem groups (n = 3), IUE DMG (n = 6) and IUE pHGG (n = 4). Data are presented as mean ± SEM. ***p < 0.0001; unpaired t-test with Mann–Whitney posthoc comparison Endothelial transcriptomes highlight differences ECs. Further separation between normal brain ECs and and similarities between pHGG, DMG and normal brain IUE DMG EC groups was evident, with a second branch endothelial signaling programs point dividing these groups (Fig.  6a). A similar pattern We next purified endothelial cells and vessel fragments emerged by principal component analysis (PCA), with from normal brain regions (cortex, cerebellum and samples from each group clustering in the same gen- brainstem) and IUE DMG and pHGG tumors to exam- eral region, and IUE pHGG ECs segregating the furthest ine molecular differences by whole-transcriptome analy - away from normal brain ECs (Additional file  1: Fig. S5). sis. Using antibody labeled magnetic beads to isolate Comparison of differentially expressed genes (FC > 4, adj. Cd31 + / Cd45- endothelial cells and vascular fragments, p-val < 0.05) between IUE DMG EC and IUE pHGG EC comparison of positive and negatively sorted popula- identified over-represented gene sets related to immune tions from normal brain samples showed positive enrich- system interactions (adaptive immune system, MHC ment for endothelial (Cd31, Tie2, Vegfr2) and pericyte class II antigen presentation), extracellular matrix regula- (Pdgfrb) genes, and negative enrichment for microglia tion (ECM organization, ECM degradation), and vascular (Cd68) and neuronal (NeuN, Tub3) genes (Additional interactions (cell surface interactions at the vascular wall, file  1: Fig. S4). Following RNA-seq, hierarchical cluster- platelet activation signaling and aggregation) (Fig.  6b, ing by Pearson’s correlation of all samples revealed two c). Further analysis of differentially up-regulated genes main branches separating IUE pHGG EC from nor- (FC > 2, adj. p-val < 0.05) in IUE pHGG ECs displayed mal brain region ECs (BS, CB and CTX) and IUE DMG enrichment in pathways related to immune response W ei et al. acta neuropathol commun (2021) 9:142 Page 9 of 18 Fig. 4 pHGG and DMG mouse models display differences in BBB function. a Representative whole brain images of brightfield and fluorescent TMR-dextran signal in control and IUE tumor conditions. Scale bar = 1 mm. b Representative immunofluorescent z-stack projection images of TMR-dextran and CD31 labeled sections. Scale bar = 20 μm. c Representative immunofluorescent z-stack projection images of control and IUE tumor conditions labeled with CD31 and Ter119 depicting the retention or extravascular leakage of red blood cells within samples. Scale bar = 20 μm Expression of secreted Wnt‑antagonists does not alter related pathways, while those up-regulated in IUE DMG DMG vascular phenotype ECs included extracellular matrix organization, SLC- DMGs are invasive brain tumors, and our vascular anal- mediated transmembrane transport, and signaling path- yses indicate minimal disruption to established blood ways (Hippo, Wnt) associated with BBB function [36–39] vessels within brain regions harboring tumor cells. This (Fig. 6d, Additional file  2: Table 1). Thus, beside preserv - is corroborated by the expression pattern of genes asso- ing their morphology and blood–brain barrier function, ciated with endothelial tip or stalk cell identity [40, 41]. IUE DMG blood vessels appear to maintain transcrip- IUE DMG ECs display increased expression of stalk cell tional programs that closely align with normal brain genes, while IUE pHGG ECs show higher expression of endothelium. Wei et al. acta neuropathol commun (2021) 9:142 Page 10 of 18 Fig. 5 Mural cell coverage and BBB associated marker expression differences between pHGG and DMG mouse models. Representative immunofluorescent z-stack projection images of a Desmin and CD31, b Collagen IV and CD31, and c Plvap and Glut1 in each experimental condition. Scale bar = 20 μm. d, e Quantification of CD31-positive vessel coverage by desmin and collagen IV respectively. f Quantification of Plvap-positive area in Glut1-positive blood vessels in each condition. Data are presented as mean ± SEM. n = 3 for all conditions. *p < 0.05, **p < 0.001, ***p < 0.0001; unpaired t-test with Mann Whitney posthoc comparison W ei et al. acta neuropathol commun (2021) 9:142 Page 11 of 18 tip cell genes (Fig.  7a). Further supporting the idea of a vasculature, which recapitulate findings in biopsy and more mature and stable vascular state in DMGs, analy- autopsy derived patient specimens. While variations in sis of transcription factor protein–protein interaction glioma BBB function have been appreciated within the (PPI) networks identified Sox17, a transcription factor field, including regional differences in glioma mouse (TF) that is highly expressed in mature brain endothelial models [44], a detailed comparison that catalogs and cells [42], as the most significantly enriched TF in DMG validates these differences between malignant gliomas ECs (Fig. 7b). Sox17 is a positive inducer of Wnt signaling has not been carried out. We show that the vascular net- [42], and together with Ctnnb1, which was also enriched work within DMGs remains mostly intact with respect in DMG ECs (Fig. 7b), may promote stability through the to blood vessel morphology, BBB function and tran- maintenance of proper Wnt signaling levels. Within IUE scriptional programs, while cortical pHGGs display both pHGG ECs many enriched TFs in the PPI were associ- phenotypic and transcriptional changes related to disor- ated with immune responses. These included Stat3, ganized angiogenesis, inflammation and BBB dysfunction which was the most significantly enriched TF, and inter - (Fig. 8). Moreover, DMG tumors display limited sensitiv- feron response factors (IRF3/6) and NOD2, all of which ity to the expression of secreted Wnt antagonists, which can drive downstream signaling related to immune sys- have been shown to drive BBB dysfunction in glioblas- tem activity. toma and medulloblastoma [14, 16], suggesting heteroge- Canonical Wnt-signaling is essential for blood–brain neity in the response of tumor-associated blood vessels to barrier formation in the developing brain [36, 37]. In extrinsic signals in the tumor microenvironment. addition, prior work in medulloblastoma and adult glio- In pathological conditions, including brain tumors, blastoma have shown that inhibition of endothelial the BBB presents a conundrum for treatment strategies. Wnt-signaling, by either expression of secreted Wnt- On one hand, the BBB is commonly cited as an impor- antagonists such as Dkk1 and Wif1 [14, 16], or genetic tant factor in brain tumor treatment resistance since the deletion of Wnt signaling components in endothelial majority of drugs and small molecules display limited cells [17], results in tumor vascular abnormalities and BBB penetration [3]. On the other hand, poor perfu- blood–brain barrier dysfunction. To test whether Wnt sion in abnormal and “dead end” vascular structures that antagonists could alter the vascular phenotype of DMG lack BBB function can impede drug delivery [45]. Stud- tumor models we expressed the Wnt receptor antagonist ies examining angiogenesis and BBB specification dur - Dkk1, or a secreted version of the Fzd8 receptor (Fzd8- ing normal CNS development have identified endothelial CRD-IgG) [43] in our IUE DMG mouse model (Addi- Wnt signaling as an essential regulator [36, 37]. The vas - tional file1: Fig. S6). IUE DMG tumors expressing empty cular phenotype of Wnt mutants shares many common vector control or the secreted Wnt antagonist (Dkk1 or features with that in glioblastoma, including chaotic Fzd8-CRD-IgG) developed tumors with similar laten- architecture, hemorrhaging, the formation of glomeruli cies, and analysis of vascular content and supporting structures and a lack of BBB functionality [36, 37, 46–49], components, such as ECM proteins, did not identify any suggesting a direct link between Wnt signaling disruption significant changes induced by secreted Wnt antagonist and brain tumor vascular abnormalities. Indeed, com- (Fig. 7c). Our data suggests that differences in the angio - pared to normal brain and DMG ECs, pHGG ECs show genic state of tumor-associated vasculature will influ - a modestly decreased Wnt-signaling at the transcriptome ence how they respond to other external cues, adding an level, indicating that alterations in this essential BBB additional layer of complexity to interactions within the signaling pathway likely participate in generating pHGG tumor microenvironment. vascular abnormalities. Despite these changes, pHGG blood vessels retain some features of the BBB, as most Discussion endothelial cells express Glut1, and only a small sub- Our analyses across pHGG and DMG implant based set co-express Plvap, a component of fenestrated pores PDX and native forming IUE mouse models reveal phe- [35]. This could be due to residual levels of Wnt signal - notypic and molecular differences in tumor-associated ing within gliomas, as BBB-specific Wnt-ligands (Wnt7a, (See figure on next page.) Fig. 6 pHGG and DMG endothelial transcriptomes highlight heterogeneity of tumor-associated and normal brain endothelial signaling programs. a Hierarchical clustering of Pearson’s correlation plot visualizing the correlation values between samples. Scale bar represents the range of the correlation coefficients displayed. b Heatmap of the top 25 most significant (adj. p-value) differentially expressed genes between IUE DMG EC and IUE pHGG EC. c Gene sets enriched by over-representation analysis of differentially expressed genes (FC > 4, adj. p < 0.05) between IUE pHGG EC and IUE DMG ECs. d Gene sets enriched by over-representation analysis of differentially expressed genes (FC > 2, adj. p < 0.05) up-regulated in IUE pHGG EC or IUE DMG ECs Wei et al. acta neuropathol commun (2021) 9:142 Page 12 of 18 Fig. 6 (See legend on previous page.) W ei et al. acta neuropathol commun (2021) 9:142 Page 13 of 18 Wnt7b and Norrin) are expressed by glial lineage cell- tumor cells, which is dependent on Stat3 activation types, including oligodendrocyte progenitors and astro- [61]. Additionally, increased cytokine expression caused cytes [50–53]. It could also be due to variability in Vegf by interactions between microglia and glioma cells can signaling, as Vegf ligands are required for the formation activate endothelial Jak / Stat3 signaling, resulting in of fenestrated blood vessels in the choroid plexus [54]. increased vascular permeability in  vitro [62]. Together, While Wnt signaling is essential for sprouting angiogen- this can lead to increased endothelial expression of leu- esis into the developing CNS and BBB formation [36, 37, kocyte adhesion molecules, which are associated with 49], how it interfaces with traditional pro-angiogenic fac- BBB dysfunction and inflammation [63, 64]. DMGs tors like Vegf in development and pathological conditions tend to display low T-cell infiltration compared to other remains an open area of investigation. Another possibil- gliomas [65]. Whether differences in the tumor micro - ity is that only certain mechanisms employed by the BBB environment and pHGG and DMG vascular properties are altered in pHGGs. While Glut1 expression is a BBB directly or indirectly influence the differential recruit - associated marker in CNS vasculature, its expression can ment of infiltrating immune cells into tumors will be of be maintained in the presence of other NVU alterations particular interest to further delineate. that impact BBB function, such pericyte loss [55, 56]. We find that DMG vessels are not particularly sensi - Pericyte coverage has been shown to directly mediate tive to the expression of secreted Wnt antagonists, which transcytosis rates [55–57], and in healthy brain, recep- have previously been shown to drive BBB dysfunction tor mediated transcytosis allows the selective crossing of in glioblastoma [16] and medulloblastoma [14, 15]. This plasma proteins, which switches to a more general tran- finding, taken together with our data showing DMGs scytosis mechanism with aging and pericyte loss [58]. contain a stable network of blood vessels in a mature Decreased pericyte coverage in pHGGs could have a par- endothelial transcriptional state, lead us to postulate that ticular impact on transcytosis and could in part explain differences in the angiogenic state of brain tumors plays why Glut1 expression is maintained in regions that dis- a role in their responsiveness to fluctuations in Wnt sig - play vascular permeability. nals. Canonical Wnt signaling by specific ligands (Wnt7a, Beside our immuno-staining characterization that Wnt7b, Ndp) and co-receptor complexes (Fzd4, Gpr124, demonstrates DMG blood vessels retain normal morpho- Reck, Lrp5/6) is essential for BBB induction and matura- logical features and BBB function, we find that DMG ECs tion in the developing brain [36, 37, 46, 47, 66–69]. Yet, maintain a transcriptional program similar to that of nor- inhibition or deletion of these Wnt ligand or receptor mal brain ECs. Examination of endothelial tip and stalk components in the mature brain under normal homeo- cell gene expression reveals increased expression of stalk static conditions does not impact vascular integrity or cell genes in DMG ECs, and elevated expression of angi- BBB function [17, 42]. Levels of endothelial Wnt signal- ogenic tip cell genes in pHGG ECs. Moreover, PPI net- ing in the brain change over the course of brain devel- work analysis identified Sox17 as the most significantly opment and maturation. Previous studies have shown enriched differentially expressed transcription factor in canonical Wnt signaling, using the BAT (beta-catenin DMG ECs. Sox17 expression is highest in more mature activated reporter) LacZ reporter mouse, decreases in brain endothelial cells [42], suggesting that tumor-asso- brain endothelial cells as they mature [42, 70]. Moreover, ciated blood vessels in DMGs mainly consist of existing expression of Apcdd1, an inhibitor of the canonical Wnt mature vasculature, and not newly created vessels that pathway, increases with age, ensuring the proper level of develop in a highly organized fashion. Transcription fac- Wnt signaling required for proper angiogenesis and BBB tors enriched in pHGG EC PPI networks were mainly development [71]. Understanding how these differences related to inflammatory mediated pathways, agreeing in Wnt signaling during vascular development and mat- with the general immune-related signatures identified uration apply to brain tumors will be important to con- when comparing to normal or DMG ECs. Stat3 is a criti- sider not only for DMGs, but also for pHGGs and adult cal mediator of immune related responses in gliomas [59, glioblastomas, since they also contain regions of tumor- 60]. Interactions between microglia and glioma tumor associated vasculature that are not engaged in active cells can promote a mesenchymal cell state in glioma angiogenesis. (See figure on next page.) Fig. 7 Expression of secreted Wnt-antagonists does not alter DMG vascular phenotype. a Heatmap of endothelial stalk and tip cell associated gene expression in IUE pHGG and DMG ECs. b Transcription factor protein–protein interaction networks enriched in IUE pHGG or DMG ECs. c Representative immunofluorescent z-stack projection images of CD31 labeled or CD31 and Collagen IV labeled blood vessels in IUE DMG control, Dkk1, or Fzd8-CRD-IgG tumors. Scale bar = 20 μm Wei et al. acta neuropathol commun (2021) 9:142 Page 14 of 18 Fig. 7 (See legend on previous page.) W ei et al. acta neuropathol commun (2021) 9:142 Page 15 of 18 Fig. 8 Summary of pHGG and DMG models and vascular phenotypes associated with each tumor type. Patient derived xenografts and in utero electroporation based cortical pHGG and brainstem DMG models were utilized to investigate in vivo vascular phenotypes. Across model systems, DMG tumor-associated blood vessels consistently displayed vascular phenotypes, BBB function and transcriptional programs similar to normal brain endothelium. pHGG tumor-associated blood vessels were associated with abnormal vascular phenotypes, BBB dysfunction, and transcriptional changes In summary, we present a detailed analysis of pHGG several other mechanisms of BBB opening, including and DMG tumor-associated vascular profiles, highlight - transcytosis, have been described after ultrasound treat- ing blood vessel heterogeneity and differences between ment [74]. Other methods to circumvent the BBB are these deadly brain tumors. Additionally, our data shows convection-enhanced delivery (CED), in which drugs are DMGs respond differently to variations in Wnt signal - directly infused into the parenchyma or tumor under a ing levels, pointing out a need to further understand how hydrostatic pressure gradient [75], the use of nanopar- canonical Wnt signaling and Vegf signaling interplay to ticles, or intranasal/intra-arterial delivery [76]. A bet- regulate angiogenesis and BBB specification both in nor - ter understanding of the tumor vasculature can help to mal CNS development and in pathological settings. As decide which method to use in certain tumor types. current outcomes for most malignant gliomas are dismal, Together, the present work provides new insights that regardless of their vascular phenotype, there is a critical emphasize the need to consider vascular heterogeneity need for new and improved therapeutic strategies [8]. among brain tumors in the development of new thera- For example, strategies to “normalize” leaky and tortur- peutic strategies. ous blood vessels within brain tumors could be accom- plished by stabilizing endothelial Wnt signaling. This Abbreviations could provide the benefit of promoting normal vascu - BBB: Blood brain barrier; DMG: Diffuse midline glioma; PHGG: Pediatric high- lar growth and BBB function within tumors, enhancing grade glioma; GEMMs: Genetically engineered mouse models; NVU: Neuro- vascular unit; CNS: Central nervous system; MRI: Magnetic resonance imaging; the perfusion and vascularity of brain tumors even bet- CE: Contrast enhancement; IUE: In utero electroporation; PDX: Patient derived ter than current anti-Vegf therapies. Recent strategies xenograft; BS: Brainstem; CTX: Cortex; CB: Cerebellum; H&E: Hematoxylin and have leveraged receptor mediated transcytosis to deliver eosin; qPCR: Quantitative real-time polymerase chain reaction; TMR: Tetra- methylrhodamine; ECs: Endothelial cells; Cldn5: Claudin5; Plvap: Plasmalemma cargo into the brain [72, 73]. By defining the expression vesicle associated protein; TF: Transcription factor; COLIV: Collagen IV; MACS: of receptors in endothelial cells across the normal brain Magnetic cell sorting; FUS: Focused ultrasound; CED: Convection enhanced and brain tumor types, one can develop approaches to delivery. target the delivery of new therapies into brain tumors that have been traditionally hard to penetrate. Transcy- Supplementary Information tosis can also be upregulated using (microbubble-medi- The online version contains supplementary material available at https:// doi. org/ 10. 1186/ s40478- 021- 01243-1. ated) focused ultrasound (FUS). This is a non-invasive method that temporarily opens the BBB in a targeted Additional file 1 contains data related to supplementary figures 1–6. location. Although drug delivery by FUS is thought to This includes vascular stains in (Fig S1) PDX and (Fig S2) primary pHGG mainly function through mechanical stimulation of the and DMG samples, examples of intratumoral heterogeneity in IUE pHGG blood vessels and consequent opening of tight-junctions, mouse models (Fig S3), validation of endothelial purity following magnetic Wei et al. acta neuropathol commun (2021) 9:142 Page 16 of 18 Netherlands. Amsterdam Leukodystrophy Center, Amsterdam UMC, Amster- cell sorting (Fig S4), PCA of endothelial samples (Fig S5), and validation of dam, The Netherlands. Dkk1 expression in IUE pHGG overexpression models (Fig S6). Received: 2 July 2021 Accepted: 10 August 2021 Additional file 2 contains data related to transcriptional analysis of endothelial samples from normal brain regions, IUE pHGG and IUE DMG mouse models. Tab1: DEGs comparing DMG vs. pHGG ECs; Tab2: DEGs comparing BS vs. DMG ECs; Tab3: DEGs comparing Ctx vs. pHGG ECs; Tab 4: Pathways enriched in pHGG ECs compared to DMG ECs; Tab5: Pathways enriched in DMG ECs compared to pHGG ECs. References 1. Daneman R, Prat A (2015) The blood-brain barrier. Cold Spring Harb Perspect Biol 7:a020412. https:// doi. org/ 10. 1101/ cshpe rspect. a0204 12 Acknowledgements 2. Muldoon LL, Soussain C, Jahnke K, Johanson C, Siegal T, Smith QR, Hall We would like to thank members of the pathology core at CCHMC, and the WA, Hynynen K, Senter PD, Peereboom DM et al (2007) Chemotherapy Genomics, Epigenomics, and Sequencing Core at UC (supported in part by delivery issues in central nervous system malignancy: a reality check. J CEG grant NIEHS P30-ES006096) for their assistance. Clin Oncol 25:2295–2305. https:// doi. org/ 10. 1200/ JCO. 2006. 09. 9861 3. Heffron TP (2016) Small molecule kinase inhibitors for the treatment of Authors’ contributions brain cancer. J Med Chem. https:// doi. org/ 10. 1021/ acs. jmedc hem. 6b006 XW performed the majority of experiments and data analysis and contributed to the writing and editing of the manuscript. MHM performed experiments 4. Sarkaria JN, Hu LS, Parney IF, Pafundi DH, Brinkmann DH, Laack NN, Gian- related to PDX models and contributed to the writing and editing of the nini C, Burns TC, Kizilbash SH, Laramy JK et al (2018) Is the blood-brain manuscript. MB and MB performed experiments related to primary pHGG and barrier really disrupted in all glioblastomas? A critical assessment of DMG human samples. EH supervised MHM and contributed to the writing existing clinical data. Neuro Oncol 20:184–191. https:// doi. org/ 10. 1093/ and editing of the manuscript. TNP conceived the project, supervised XW, neuonc/ nox175 assisted with planning of experiments and oversaw writing and editing of the 5. Jones C, Baker SJ (2014) Unique genetic and epigenetic mechanisms manuscript. 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Acta Neuropathologica Communications – Springer Journals

Published: Aug 23, 2021

Keywords: Pediatric high-grade glioma; Diffuse midline glioma; Blood brain barrier; Endothelial cells; Neurovascular unit; Diffuse intrinsic pontine glioma; H3K27M; Wnt signaling

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