Meningeal-tumor interactions define distinct modes of leptomeningeal colonization in medulloblastoma

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Ryan, Amy C. Gross, Annika M. Albrecht, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8042018/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 01 Apr, 2026 Read the published version in Acta Neuropathologica Communications → Version 1 posted 9 You are reading this latest preprint version Abstract Medulloblastoma, the most common malignant brain tumor in children, shows a pronounced tendency to spread to the leptomeninges. Leptomeningeal metastasis accounts for nearly all medulloblastoma-related deaths, yet the cellular and molecular mechanisms driving this process remain poorly understood. Progress has been hindered by limited access to patient samples, the fragile anatomy of the leptomeninges, and the lack of robust preclinical models. Here, we developed an in vitro model that captures key features of the early leptomeningeal niche. We demonstrate that human meningeal cells promote medulloblastoma survival and proliferation under nutrient-deprived conditions both in vitro and in vivo . Using this system, we uncovered mechanisms that govern distinct patterns of leptomeningeal colonization in vivo . This physiologically informed and experimentally validated model offers a tractable platform for elucidating the molecular basis of leptomeningeal colonization and for identifying therapeutic vulnerabilities that can prevent medulloblastoma dissemination. Medulloblastoma leptomeningeal metastasis adhesion co-culture models Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Nearly all deaths from medulloblastoma, the most common malignant brain tumor of childhood, are due to leptomeningeal metastasis [ 1 – 5 ]. The biological mechanisms that enable tumor cells to survive and expand within the unique leptomeningeal compartment remain poorly defined[ 6 ]. Consequently, therapeutic strategies capable of eradicating disseminated tumor cells from the leptomeninges are lacking, particularly for patients who relapse after standard-of-care therapy[ 7 ]. The leptomeninges, comprising the pia mater, subarachnoid space (filled with cerebrospinal fluid - CSF), and arachnoid mater, were once viewed primarily as structural coverings to the brain and spinal cord but are now recognized as dynamic microenvironments that actively participate in central nervous system health and disease, including cancer[ 8 – 11 ]. Within the CSF, medulloblastoma cells exist in at least two states: free-floating within the fluid or adherent to the leptomeningeal surface of the brain and spinal cord. Adherent lesions may form diffuse, laminar coatings or discrete nodules along the leptomeninges, yet the mechanisms governing these distinct metastatic patterns remain unknown[ 12 , 13 ]. This knowledge gap underscores the need to understand how interactions between tumor cells and leptomeningeal stromal elements influence colonization, survival, and progression. Advances in leptomeningeal metastasis biology have been hindered by several experimental and clinical barriers[ 14 ]. Surgical access to leptomeningeal lesions is limited due to their diffuse nature and lack of therapeutic benefit from resection, precluding comprehensive molecular analysis of matched primary and metastatic samples. In the few available cases, metastatic lesions exhibit marked biological divergence from their primary tumors, underscoring the need to study the metastatic niche directly[ 15 ]. Moreover, while mouse models have been invaluable for identifying molecular drivers of medulloblastoma initiation, endpoints are reached due to the rapid growth of cerebellar tumors that cannot be surgically resected, not leptomeningeal metastasis[ 16 ]. Lastly, conventional in vitro models fail to replicate the cellular composition and nutrient-deprived microenvironment of the CSF, limiting their translational relevance. Organotypic co-culture systems have proven valuable for modeling tumor–host interactions that occur during metastatic dissemination to lung and bone marrow in other solid tumor contexts[ 17 – 20 ]. Leptomeningeal stromal-tumor co-cultures have been applied in studies of adult cancers, but have not been well characterized for medulloblastoma; moreover, previous in vitro findings have not been validated within the in vivo leptomeningeal niche[ 21 , 22 ]. To enable reductionist studies of leptomeningeal biology in medulloblastoma, we developed and validated an in vitro model that recapitulates key features of the leptomeningeal niche. By co-culturing established and patient-proximal medulloblastoma cell lines with primary human leptomeningeal fibroblasts, we demonstrate that meningeal cells promote tumor cell survival and proliferation under nutrient-deprived conditions. We further identify medulloblastoma models that reproducibly form laminar or nodular growth patterns in vitro and in vivo , revealing a cell-biological mechanism that explains this morphological divergence. This physiologically informed, experimentally validated model enables the dissection of the molecular determinants of leptomeningeal colonization and the identification of therapeutic vulnerabilities to prevent medulloblastoma dissemination. Materials and Methods Cell Culture Cell lines utilized in this manuscript were routinely authenticated with short tandem repeat profiling and Mycoplasma testing (Laboratory Corporation of America). Cells used for experiments that were grown in suspension were routinely replaced within two months after being revived from liquid nitrogen storage. Primary cells used for experiments were used within 10 passages after being revived from liquid nitrogen storage. Medulloblastoma cell lines (and their molecular sub-group) used in this study include: D425 (Group 3), MED-411FH (Group 3), MED-2112FH (Group 3), and CHLA-01-MED (Group 4). D425 Med (D425) cells (RRID: CVCL_1275) were derived from a primary medulloblastoma tumor resection at Duke University Medical Center and obtained from the laboratory of Dr. Jeffery Leonard[ 23 , 24 ]. Med-411FH (MED411) cells and Med-2112FH (MED2112) cells are cell lines derived from early passage patient-derived xenografts developed by the laboratory of Dr. Jim Olson and distributed by the Brain Tumor Resource Lab at Seattle Children’s Hospital[ 25 ]. CHLA-01-MED was obtained from American Type Culture Collection as described previously[ 26 ]. Human meningeal cells (HMC) (ScienCell Research Laboratories, Cat. #1400) are primary leptomeningeal cells derived from human donors. We have used > 5 distinct lots with similar results. D425 and HMC cells were cultured in DMEM (Corning, 10-013-CV) supplemented with 10% FBS (Atlanta Biologicals, S11150H). MED411, MED2112, and CHLA-01-MED cells were cultured in NeuroCult NS-A Basal Medium (Human) (Stemcell technologies, Cat. #05750) supplemented with NeuroCult NS-A Proliferation Kit (Human) (Stemcell technologies, Cat. #05751), human epidermal growth factor (20 ng/mL, ThermoFisher, AF-100-15-500UG) and human fibroblast growth factor-basic (20ng/mL, ThermoFisher, 100-18B-50UG). HMC survival optimization HMC were grown to confluence in 24-well plates. Media was exchanged for either basal growth media, serial dilutions of growth media with artificial CSF (ACSF, Tocris, Cat. No. 3525), ACSF + 60mg/dL glucose, or DMEM/F12 (Gibco, Cat. #21041025). After 72 hours, cell viability was assessed using alamarBlue (ThermoFisher, A50100), and cells were photographed under light-phase microscopy. Medulloblastoma-leptomeningeal fibroblast co-culture Human medulloblastoma cells and meningeal cells were co-cultured to generate an in vitro model that captures key interactions between tumor cells and the surrounding stroma of leptomeningeal metastasis within a reductionist setting. HMCs were seeded onto 0.4 µm transwell filters or 1.5# glass coverslips (Electron Microscopy Sciences, 72204-04) in 6- or 24-well dishes and cultured with DMEM supplemented with 10% FBS. After HMCs formed a confluent monolayer (48–72 hours), the base media was removed and replaced with serum-free, growth-factor free DMEM/F12 containing medulloblastoma cells. For transwell experiments, DMEM/F12 was placed in the top and bottom wells. Cell-free extracellular matrix HMC were grown to confluence on 1.5# coverslips for 72 hours. Cell-free HMC-derived matrix was generated as previously described [ 27 ]. Medulloblastoma cells were plated onto coated coverslips for 48–72 hours in their base media before processing for immunofluorescence. Animal Studies All animal procedures were approved by the Institutional Animal Care and Use Committee of the Abigail Wexner Research Institute at Nationwide Children’s Hospital (IACUC protocol AR15-00022). CB17-SCID mice [CB17SC scid−/−; RRID: IMSR_TAC:cb17sc] were used for all in vivo experiments. Subcutaneous Tumor Model : Mice were anesthetized and injected subcutaneously in the flank with 5 × 10⁵ medulloblastoma cells, with or without 1 × 10⁶ HMCs. Tumor growth was monitored biweekly via caliper measurements. Mice were euthanized, and tumors were harvested upon a total tumor volume of 2000 mm³. Spontaneous Leptomeningeal Metastasis Model : Mice were anesthetized with isoflurane and placed in a stereotaxic apparatus (Kopf Instruments, Model 940). Intracerebellar injection of 8 × 10⁴ medulloblastoma cells in 3 µL of Neurocult media was performed at 2mm lateral, 2mm posterior, and 2.5 mm deep relative to lambda. Post-operative care included analgesia and daily monitoring. At neurological endpoint (rapid weight loss and an enhanced body condition score of less than 9, and/or neurological clinical signs including head tilts, seizures, and/or log rolling), mice were euthanized and processed for histology. Experimental Leptomeningeal Metastasis Model : Medulloblastoma cells (2.5 × 10⁴ in 3 µL Neurocult media) were injected into the cisterna magna under aseptic conditions. Post-operative care and endpoint monitoring mirrored the spontaneous model. Histology Mice were euthanized at defined endpoints, sternotomy was performed, and mice were perfused with 20 ml of 0.9% saline injection (BD PosiFlush, cat #306546) followed by 30 ml of neutral buffered formalin (NBF) (Sigma-Aldrich, HT5012-1CS) through the left ventricle. Heads and spinal columns were cleaned of connective tissue and placed in NBF at 4°C for 48 hours. Brains were dissected from skull and spines were decalcified in 10% EDTA (Elabscience, E-IR-R112) for 10 days at room temperature, and then processed for paraffin embedding (FFPE). For standard histological observation, FFPE sections were counterstained with hematoxylin and eosin via conventional methods. Immunohistochemistry and Immunofluorescence Unstained 4µm sections were deparaffinized with xylene and rehydrated through ethanol series. Heat-mediated antigen retrieval was performed with Tris-EDTA buffer (TE; pH 9) with 0.2% v/v Tween 20 (Fisher Bioreagents, BP337-100). Cells growing on #1.5 glass coverslips or 0.4µm filters were fixed in NBF for 10 minutes at room temperature. Following three rinses in 1x Phosphate Buffered Saline (PBS) (Corning, 21-040-CM), coverslip or histological sections were then incubated for at least 1 hour in blocking solution consisting of PBS, 0.2% triton100 (v/v) (Sigma, X-100-100ml), and 3% bovine serum albumin (w/v) (Sigma, A-7888) at room temperature. Coverslips or histological sections were then incubated overnight in the following primary antibodies (diluted in blocking solution): hamster anti-Podoplanin (Developmental Studies Hybridoma Bank(DSHB), 8.1.1), rabbit anti-Fibronectin (Abcam, ab2413), rabbit anti-Collagen IV (Abcam, ab6586), rabbit anti-Synaptophysin (Abcam, ab32127), rabbit anti-GFAP (Abcam, ab68428), rabbit anti-CD31 (Abcam, ab182981), rabbit anti-pan laminin (Novus Biologicals, NB300-144), rabbit anti- Cleaved Caspase 3 (Cell Signaling Technologies-CST, 9664), rabbit anti- phospho-Histone Ser10 (CST, 53348), goat anti-CD45 (R&D Systems, AF114), goat anti-OTX2 (R&D Systems, AF1979), sheep anti-S100A6 (R&D Systems, AF4584), or mouse anti-Vimentin (Abnova, SRL33). Following three rinses in PBS, sections or coverslips were incubated with appropriate AlexaFluor-labeled secondary antibodies (Invitrogen), DAPI (Invitrogen, D1306) and, where indicated, Phalloidin AlexaFluor 488, 568 or 647 (Invitrogen), or Wheat Germ Agglutinin (WGA) AlexaFluor 647 (Invitrogen, W11261) diluted in blocking solution for 1 hour at room temperature. Following PBS rinses, coverslips and tissue sections were mounted in Fluoromount G (Invitrogen, 00495802). Microscopy and image analysis Confocal microscopy images were obtained using a Crest-X-Light V3 spinning disk confocal on a Nikon Ti2-E inverted microscope, equipped with either a Hamamatsu ORCA Fusion camera or a Zeiss 800 laser scanning microscope, and featuring 4X, 20X, and 40X air objectives. Raw ND2 or czi files were imported into ImageJ for post-image processing and quantitation. All manipulation of images (brightness/contrast) were done uniformly to images. Mitotic (pH3+) and apoptotic (CC3+) were quantified by creating binary images with a manual threshold, then subjecting the binary images to the Analyze Particles function (size > 10µm 2 ). Index was generated by dividing the cells (pH3 + or CC3+) by the total number of medulloblastoma cells (OTX2+). Spreading of medulloblastoma cells in co-culture was quantified by generating a binary image of OTX2 image and processed using the Skeletonize function. Skeletonized images were then quantified with Analyze Skeleton function. Percent spreading on HMC-derived ECM was quantified manually using the Cell Counter Plugin. H/E images were obtained with Aperio FL ScanScope digital slide scanner with 20X objective, and metastasis length was quantified using Aperio ImageScope Software (V12.4.3). Statistical analysis All statistical analyses for in vitro assays and animal studies were performed using GraphPad Prism v10. The number of biological replicates (independent experiments or animals) is specified in the figure legends, along with the appropriate statistical tests and P values. Data are presented as the mean with error bars that represent the standard error of the mean. Results Pial fibroblast-medulloblastoma adhesion within the early metastatic niche To guide the design of physiologically relevant in vitro models, we first generated models of the early leptomeningeal metastatic niche in vivo . Among medulloblastoma subgroups, Group 3 and Group 4 tumors most frequently disseminate to the leptomeninges[ 3 ]. Because Group 4 models are not widely available, we orthotopically injected three human Group 3 medulloblastoma cell lines (D425, MED411, and MED2112) into the cerebella of immunocompromised mice to assess spontaneous metastasis (Fig. 1 A). At humane endpoint, brains and spines were examined histologically for evidence of dissemination. Although all models formed robust primary tumors with comparable survival (Fig. 1 B), small leptomeningeal lesions were detected only in D425 and MED411 (Fig. 1 C). These findings highlight the potential limitations of orthotopic xenografts for studying leptomeningeal metastasis, as rapid primary tumor growth restricts the development of marked leptomeningeal disease. We next defined the stromal architecture of the leptomeningeal niche in D425 spontaneous metastasis lesions. The cellular aspects of the leptomeninges include pial and arachnoid fibroblasts, penetrating blood vessels, a subpial basement membrane, tissue-resident and recruited immune cells, and the glial limitans [ 8 , 9 , 28 – 30 ]. Multiparameter immunohistochemistry identified pial and arachnoid fibroblasts (Podoplanin), endothelial cells (CD31), basement membrane (laminin), glial limitans (GFAP), and immune cells (CD45). Tumor cells, identified by OTX2 expression (not shown for clarity), were consistently juxtaposed to pial fibroblasts, suggesting a preferential tumor–fibroblast interaction during early dissemination (Fig. 1 D-G). Based on these observations and recently published data[ 31 ], we selected leptomeningeal fibroblasts as the primary stromal component for developing an in vitro co-culture model of the early leptomeningeal niche. Human leptomeningeal fibroblasts support medulloblastoma survival and growth Leptomeningeal fibroblasts can be isolated from human donors and cultured in vitro [ 32 ]. To enhance the physiological relevance by mimicking the nutrient-deprived state of the CSF, we first defined the minimal media conditions capable of supporting primary human leptomeningeal fibroblasts (HMC). Standard HMC media (DMEM + 10% FBS) was diluted with increasing concentrations of artificial CSF (aCSF) containing physiologic glucose or replaced with serum- and growth factor–free DMEM/F12 ( Supplemental Fig. 1A ). While HMCs failed to survive in aCSF, confluent monolayers were maintained in DMEM/F12 ( Supplemental Fig. 1B–C ). Under these conditions, HMCs retained expression of pial markers including fibronectin, collagen IV, laminin, S100A6, and vimentin ( Supplemental Fig. 1D ). Thus, HMCs can be maintained under serum- and growth factor–free conditions that approximate the nutrient limitations of CSF. Supplemental Fig. 1 Optimization of HMC growth under serum-free and growth-factor-free conditions. A) Schematic depicting the dilution conditions followed to minimize growth factor and serum conditions while maximizing HMC proliferation and survival. B) Quantification of cellular viability of HMCs under different nutrient conditions. The plot represents results for n = 3 experiments. C) Representative phase contrast images of HMCs under the different nutrient conditions. D) Representative immunofluorescence images of HMCs demonstrating that they retain expression of common pial markers under serum- and growth-factor-free conditions. Scale bar = 50µm. Using these optimized conditions, we next tested whether HMCs promote medulloblastoma cell survival and proliferation in short-term co-culture (Fig. 2 A). Under nutrient-deprived conditions, D425 and MED411 cells exhibited reduced proliferation and increased apoptosis, as shown by phospho-H3 (S10) and cleaved caspase-3 (CC3) staining (Fig. 2 B–E). Co-culture with HMCs restored proliferation and reduced apoptosis to near steady-state levels, indicating that leptomeningeal fibroblasts provide trophic support under nutrient-limited conditions. To assess whether HMCs enhance tumorigenicity in vivo , we performed dual flank injections in immunocompromised mice, injecting tumor cells alone on one side and tumor + HMCs on the opposite side (Fig. 2 F). Co-injection with HMCs increased tumor establishment ( D425 : 55.5% tumor take rate in single injection vs 88.9% tumor take rate in co-injection, MED411 : 66.6% tumor take rate in single injection vs 100% tumor take rate in co-injection) and accelerated growth in both D425 and MED411 models (Fig. 2 G). Notably, HMC injection alone did not result in tumorigenesis, suggest the difference is not attributable to expansion of meningeal cells. Together, these data demonstrate that leptomeningeal fibroblasts actively promote medulloblastoma survival and proliferation both in vitro and in vivo . Distinct laminar and nodular morphologies reflect divergent modes of leptomeningeal colonization Medulloblastoma metastatic lesions adopt diffuse laminar or distinct nodular phenotypes in patients, though the mechanisms underlying these morphologies have not been elucidated due to lack of preclinical models. Having validated short-term co-culture feasibility, we next examined whether extended co-culture recapitulates the morphologic diversity of leptomeningeal metastases. A panel of medulloblastoma neurosphere lines—including Group 3 models (D425, MED411, MED2112) and one Group 4 model (CHLA-01-MED)—was co-cultured with HMCs under nutrient-deprived conditions for 7 days. Two reproducible and morphologically distinct growth patterns emerged: a laminar phenotype (D425, MED411) in which tumor cells disseminated from neurospheres and spread across the fibroblast monolayer, and a nodular phenotype (MED2112, CHLA-01-MED) characterized by compact, spherical aggregates with limited dispersion (Fig. 3 A). To generate a quantitative metric of laminar versus nodular growth, OTX2 + tumor cell masks were converted to skeletonized representations, enabling measurement of branch number and extent as indicators of surface spreading (Fig. 3 B-C). Laminar models (D425 and MED411) demonstrated significantly higher branching than nodular models (MED2112 and CHLA-01-MED), indicative of a more diffuse growth pattern in vitro . We next sought to determine whether in vitro behaviors mirrored in vivo dissemination patterns. Because leptomeningeal disease progression is prevented by rapid primary tumor growth in spontaneous models (Fig. 1 ), we hypothesized that direct injection of tumor cells into CSF would allow for leptomeningeal tumor progression and evaluation of growth patterns between different models. Previous works have seeded medulloblastoma cells into CSF through injection of tumor cells into lateral ventricle or cisterna magna[ 31 , 33 , 34 ]. Medulloblastoma cells are thought to enter CSF through either direct invasion into 4th ventricle or following hematogenous dissemination[ 35 ]. Because the CSF within the 4th ventricle circulates directly into the cisterna magna, we chose intracisternal injections to inoculate D425, MED411, or MED2112 cells directly into the CSF. The resulting metastatic lesions recapitulated the in vitro patterns: laminar models (D425 and MED411) exhibited extensive surface coating of the intracranial and spinal meninges, whereas the nodular model MED2112 formed discrete, localized deposits (Fig. 3 D). We quantified lesion length as a surrogate measure of laminar versus nodular growth and found that laminar lesions extended significantly farther along the pial surface than nodular lesions (Fig. 3 E). We acknowledge the limitations of using quantitative metrics such as length to capture categorical morphologic features, mirroring the challenges faced clinically in assessing leptomeningeal disease[ 36 , 37 ]. Collectively, the combination of in vitro and in vivo findings provide preclinical evidence for biologically distinct modes of leptomeningeal colonization. Laminar lesions disrupt the fibroblast layer and adhere to the subpial basement membrane We performed long-term live-cell imaging of HMC–tumor co-cultures to directly visualize tumor–fibroblast interactions. Both laminar and nodular medulloblastoma cells adhered rapidly to fibroblasts within hours of plating ( Supplemental Videos 1–4 ). However, laminar cells progressively displaced the fibroblast monolayer and spread along the underlying substrate, whereas nodular cells remained compact and adherent to pial cells. Fixed co-cultures imaged after 5 days confirmed these distinct morphologies at high resolution (Fig. 4 A–E). In vivo immunohistochemistry of D425, MED411, and MED2112 leptomeningeal lesions corroborated these findings: laminar tumor cells penetrated the pial fibroblast layer to adhere directly to the laminin-positive subpial basement membrane. We did not observe invasion through the basement membrane or disruption of the glial limitans, consistent with clinical observations that parenchymal extension of disseminated disease is rare in medulloblastoma. In contrast, nodular lesions remained juxtaposed to intact pial fibroblasts (Fig. 4 F–H). Thus, laminar and nodular lesions represent distinct modes of tumor–meningeal interaction, differing in their degree of fibroblast displacement and engagement with the subpial basement membrane. Adhesion to meningeal extracellular matrix distinguishes laminar from nodular cells We next sought to determine how medulloblastoma cells interact with extracellular matrix (ECM) derived from leptomeningeal fibroblasts given the differential patterns of spread observed in previous experiments. To directly quantify medulloblastoma behavior on pial ECM, we generated cell-free HMC ECM by culturing HMC on coverslips and performing a decellularization method described previously[ 27 ] (Fig. 5 A–C). Decellularized ECM retains both the structure and biological properties of leptomeningeal-deposited ECM, even in the absence of leptomeningeal fibroblasts (noted by the absence of nuclear DAPI staining). D425, MED411, MED2112, and CHLA-01-MED were plated on decellularized HMC-derived ECM for 24 hours. Laminar cells (D425 and MED411) adopted an elongated morphology with filopodia-like projections characteristic of spreading and migration. In contrast, nodular cells (MED2112 and CHLA-01-MED) retained cortical actin organization and failed to spread on HMC-derived ECM (Fig. 5 D-F). Lastly, nodular cells exhibit significantly increased apoptosis when cultured on HMC-derived extracellular matrix, indicating reduced fitness and impaired adaptation to ECM-dependent growth conditions (Fig. 5 G). Discussion Leptomeningeal metastasis remains the principal cause of treatment failure and death in children with medulloblastoma, yet its underlying biology has remained elusive. Here, we developed an in vitro model that faithfully mirrors the morphologic diversity of leptomeningeal metastasis observed in vivo . Using this system, we identify two distinct modes of leptomeningeal colonization—laminar and nodular—that arise from differential tumor–stroma adhesion mechanisms. Our findings build upon previous iterations of in vitro meningeal and ECM medulloblastoma models by providing in vivo validation[ 22 , 38 , 39 ]. Future studies will define the molecular mechanisms that distinguish laminar and nodular phenotypes and determine whether cellular plasticity exists between these states. Integrating in vitro co-culture systems with both spontaneous and experimental leptomeningeal metastasis models establishes a versatile platform for delineating the stage-specific roles of metastatic mediators; whether they govern invasion and dissemination from the primary site, colonization of the leptomeninges, or subsequent progression within the leptomeningeal niche [ 34 , 40 – 42 ]. Additionally, understanding the pathways that regulate these phenotypic transitions may have translational significance, given differential therapeutic sensitivities between clinical phenotypes [ 12 , 43 ]. The model described here provides a tractable platform to investigate therapeutic response as a function of leptomeningeal colonization architecture. The leptomeningeal niche as a trophic microenvironment A recent landmark paper from the Taylor group identified medulloblastoma-induced changes to the leptomeningeal environment during colonization and established the importance of tumor-host interactions in leptomeningeal metastasis[ 31 ]. Our findings agree with those observations by providing direct evidence that human meningeal cells provide trophic support to medulloblastoma cells. Co-culture of medulloblastoma cells with HMC rescued tumor cell proliferation and suppressed apoptosis under nutrient-deprived conditions, demonstrating that fibroblast-derived cues compensate for the metabolic limitations of CSF. Similar trophic effects have been described in lung and bone marrow metastases, suggesting that stromal support is a conserved feature of the metastatic niche across organ systems[ 44 ]. By defining leptomeningeal fibroblasts as key mediators of this process, our study establishes a cellular foundation for dissecting paracrine and adhesion-dependent interactions that sustain leptomeningeal metastasis. Modeling implications and future directions Traditional xenograft models are limited in their ability to capture the temporal or spatial features of leptomeningeal dissemination due to rapid intracerebellar tumor growth and limited sampling of metastatic lesions. The co-culture system described here complements these in vivo models by offering a reductionist platform for mechanistic dissection and therapeutic testing. Integration of single-cell and spatial transcriptomic profiling will enable mapping of tumor–stroma signaling networks and identification of context-specific dependencies. In parallel, the model provides an experimental framework for evaluating compounds that disrupt adhesion-dependent survival. Future studies will delineate the mechanisms that distinguish cell–cell from cell–matrix adhesion in nodular and laminar metastases, as disrupting these interactions may represent context-specific therapeutic vulnerabilities within the leptomeningeal niche. While our model captures key aspects of the leptomeningeal niche, it lacks other microenvironmental components, such as immune or vascular cells, that may modulate colonization dynamics and progression[ 31 , 35 , 45 , 46 ]. Incorporating these elements in future iterations will further enhance physiologic relevance. The models used in this study were predominantly Group 3 medulloblastoma lines with MYC amplification, reflecting both their aggressive and metastatic phenotype and their availability [ 47 ]. Future work will extend these studies to WNT-, SHH-activated, and non-MYC–amplified Group 3/4 tumors, ideally using patient-derived xenografts or primary human specimens, to capture the full molecular spectrum of medulloblastoma leptomeningeal metastasis. Ultimately, coupling these advanced models with clinical and genomic data from patient-derived metastasis samples will enable a more complete understanding of metastatic evolution in medulloblastoma. Conclusions Our study delineates modes by which medulloblastoma colonizes the leptomeninges. We identify differential interactions with fibroblast-derived extracellular matrix as a key determinant in influencing metastatic phenotype. This framework establishes the leptomeningeal niche as both a driver of metastatic persistence and a therapeutic vulnerability, offering new avenues for targeting the most lethal stage of pediatric medulloblastoma. Declarations Ethics approval and consent to participate This study used pre-established and patient-derived human cell lines that are commercially available (Brain Tumor Resource Lab, ATCC, Sigma-Aldrich and ScienCell). According to institutional and national guidelines, research involving such cell lines does not require ethics committee approval because the material is anonymized and information identifying the donor is not accessible. Consent for publication De-identified MR images were obtained after approval from Nationwide Children’s Hospital Institutional Review Board. Consent was not deemed to be necessary. Competing interests LJG is supported by Pelotonia on a Pelotonia Graduate Scholar Fellowship. Funding This work was funded by CancerFree KIDS (Wolf Pack Research Grant, JBR), St. Baldrick’s Foundation Scholar Award (JBR), Alex’s Lemonade Stand Foundation Young Investigator Award (JBR), Hyundai Hope On Wheels, Bridge2K (JBR), and Nationwide Children’s Hospital (JBR). LJG is supported by Pelotonia on a Pelotonia Graduate Scholar fellowship. RDR is supported by Alex’s Lemonade Stand Crazy 8 Award, NIH R01CA260178, NIH CTSA Grant UL1TR002733, CancerFree KIDS Foundation, and Nationwide Children’s Director Strategic Development Fund. Acknowledgments We are indebted to Dr. Robert Wechsler-Reya, Elizabeth Thompson, and Daphne Koubourli for their technical expertise in in vivo modeling. We are grateful to members of the laboratory of Dr. Ryan Roberts for valuable discussions and critical reading of this manuscript. We also acknowledge the Histopathology and Microscopy Core facilities at Nationwide Children’s Hospital Abigail Wexner Research Institute for their excellent technical expertise. The authors acknowledge the use of ChatGPT5, solely for text editing and clarity. All schematics in this manuscript were generated using BioRender. Authors’ information (optional) Not applicable. Author Contribution LJG- design of work, acquisition, analysis, and interpretation of data, draft of manuscriptLR- design of work, acquisition, analysis, and interpretation of data, reviewed manuscriptAG-acquisition, analysis, and interpretation of data, reviewed manuscriptAA-acquisition of data, reviewed manuscriptJW-acquisition of data, reviewed manuscript JL-acquisition of data, critical materials, review and revision of manuscriptRR-interpretation of data, review and revision of manuscript JR-conception and design of work, acquisition, analysis, and interpretation of data, review and revision of manuscript Acknowledgement We are indebted to Dr. Robert Wechsler-Reya, Elizabeth Thompson, and Daphne Koubourli for their technical expertise in in vivo modeling. We are grateful to members of the laboratory of Dr. Ryan Roberts for valuable discussions and critical reading of this manuscript. We also acknowledge the Histopathology and Microscopy Core facilities at Nationwide Children’s Hospital Abigail Wexner Research Institute for their excellent technical expertise. The authors acknowledge the use of ChatGPT5, solely for text editing and clarity. All schematics in this manuscript were generated using BioRender. Data Availability All data supporting the findings of this study are available within the paper and its Supplementary Information. References Hill RM et al (2020) Time, pattern, and outcome of medulloblastoma relapse and their association with tumour biology at diagnosis and therapy: a multicentre cohort study. Lancet Child Adolesc Health 4(12):865–874 Michalski JM et al (2021) Children's Oncology Group Phase III Trial of Reduced-Dose and Reduced-Volume Radiotherapy With Chemotherapy for Newly Diagnosed Average-Risk Medulloblastoma. 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Supplementary Files D425HMCcocx.mp4 MED411HMCcocx.mp4 MED2112HMCcocx.mp4 CHLA01MEDHMCcocx.mp4 floatimage2.png Cite Share Download PDF Status: Published Journal Publication published 01 Apr, 2026 Read the published version in Acta Neuropathologica Communications → Version 1 posted Editorial decision: Revision requested 12 Dec, 2025 Reviews received at journal 11 Dec, 2025 Reviews received at journal 11 Dec, 2025 Reviewers agreed at journal 19 Nov, 2025 Reviewers agreed at journal 17 Nov, 2025 Reviewers invited by journal 17 Nov, 2025 Editor assigned by journal 15 Nov, 2025 Submission checks completed at journal 11 Nov, 2025 First submitted to journal 05 Nov, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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09:44:00","extension":"html","order_by":24,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":121421,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8042018/v1/0423ad5ae359db2278b60b26.html"},{"id":97652461,"identity":"e12cf6cf-81d8-4f39-b3ea-b30cc35d7716","added_by":"auto","created_at":"2025-12-08 06:44:07","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":900371,"visible":true,"origin":"","legend":"\u003cp\u003eThe early metastatic niche in medulloblastoma is composed of tumor cells and pial fibroblasts.\u003cstrong\u003e A) \u003c/strong\u003eSchematic representation for generating spontaneous leptomeningeal dissemination. \u003cstrong\u003eB) \u003c/strong\u003eKaplan-Meier survival curve depicting the probability of survival across different orthotopic medulloblastoma models. Logrank (Mantel-Cox) test: \u003cem\u003ep\u003c/em\u003e-value = 0.2198. \u003cstrong\u003eC) \u003c/strong\u003eRepresentative intracranial and spinal H/E for each medulloblastoma model (n=3/model). \u003cstrong\u003eD-G) \u003c/strong\u003eRepresentative images of D425 tumor cells in relation to cells in leptomeningeal niches (CD31=endothelial cells, PDPN=pial and arachnoid leptomeningeal fibroblasts, GFAP=astrocytes; glia limitans, Laminin=sub-pial basement membrane, CD45=immune cells). WGA=Wheat germ agglutinin, marks stroma, including pial and arachnoid layers. n=3. Scale bar= 50µm.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-8042018/v1/6cc8bbf2cdb81b7e2147eed6.png"},{"id":97652459,"identity":"ef26b0a8-3944-4ee5-9b0c-270e0ff4afe1","added_by":"auto","created_at":"2025-12-08 06:44:07","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":483097,"visible":true,"origin":"","legend":"\u003cp\u003eMeningeal cells promote medulloblastoma survival and growth\u003cstrong\u003e. A) \u003c/strong\u003eSchematic of co-culture development. \u003cstrong\u003eB-C)\u003c/strong\u003eRepresentative immunofluorescence images and quantitation of mitotic index (pH3+ cells/total tumor cells). One-way ANOVA with Dunnett’s multiple comparisons correction. n= 3 independent experiments, at least 300 cells/experiment. Scale bar=50µm. \u003cstrong\u003eD-E)\u003c/strong\u003eRepresentative images of cleaved caspase 3 (CC3) and quantitation of apoptotic index (CC3+ cells/total tumor cells). One-way ANOVA with Dunnett’s multiple comparisons correction. *p\u0026lt;0.05, **p\u0026lt;0.01, ***p\u0026lt;0.001, ****p\u0026lt;0.0001. n=3 independent experiments, at least 300 cells/experiment. Scale bar=50µm. \u003cstrong\u003eF)\u003c/strong\u003e Schematic of subcutaneous implantation of medulloblastoma cells (+human meningeal cells). \u003cstrong\u003eG) \u003c/strong\u003ePlot depicting tumor volume growth over time for each subcutaneously implanted medulloblastoma model. Quantification of % of total tumor volume taken up by the single injection vs co-injection tumor (tumor volume/total tumor volume X 100), n=10 mice/tumor line. One-tailed unpaired t-test. *p\u0026lt;0.05, **p\u0026lt;0.01, ***p\u0026lt;0.001, ****p\u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-8042018/v1/5341a0d57b7a0012bf842e74.png"},{"id":97674560,"identity":"34ba6cd5-a621-4941-abb1-d8e6f1984476","added_by":"auto","created_at":"2025-12-08 09:43:37","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":731325,"visible":true,"origin":"","legend":"\u003cp\u003eDistinct laminar and nodular growth patterns \u003cem\u003ein vitro\u003c/em\u003e mirror leptomeningeal colonization phenotypes \u003cem\u003ein vivo\u003c/em\u003e. \u003cstrong\u003eA)\u003c/strong\u003e Representative images of established co-cultures for a panel of medulloblastoma cell lines. Scale bar=50µm. \u003cstrong\u003eB)\u003c/strong\u003e Representative binary transformed images with skeletonization processing to quantify tumor cell spread. \u003cstrong\u003eC)\u003c/strong\u003e Quantification of number of branches in co-culture per medulloblastoma model. n=20 images per cell line from 3 independent experiments. Brown Forsythe and Welch’s ANOVA with Dunnett’s T3 multiple comparisons test. *p\u0026lt;0.05, **p\u0026lt;0.01, ***p\u0026lt;0.001, ****p\u0026lt;0.0001. \u003cstrong\u003eD)\u003c/strong\u003e Schematic representation of generation of experimental leptomeningeal dissemination in murine models. \u003cstrong\u003eE)\u003c/strong\u003e H\u0026amp;E of representative images of cranial and spinal leptomeningeal dissemination per medulloblastoma model from the experimental model of metastasis. n=3 mice/model. \u003cstrong\u003eF)\u003c/strong\u003e Quantification of metastatic lesion length for each experimental model. Brown Forsythe and Welch’s ANOVA with Dunnett’s T3 multiple comparisons test. *p\u0026lt;0.05, **p\u0026lt;0.01, ***p\u0026lt;0.001, ****p\u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-8042018/v1/c1db832698263aa147125c3e.png"},{"id":97652465,"identity":"31bfd038-3b2d-47b2-9c9b-48fd71598b0e","added_by":"auto","created_at":"2025-12-08 06:44:07","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":814522,"visible":true,"origin":"","legend":"\u003cp\u003eLaminar growing cells remodel the leptomeningeal pial layer during colonization. \u003cstrong\u003eA)\u003c/strong\u003e Representative image of HMC monolayer in single culture. \u003cstrong\u003eB-E)\u003c/strong\u003e Representative images of HMC layer in co-culture. \u003cstrong\u003eF-H) \u003c/strong\u003eIHC of spinal leptomeningeal metastases for three medulloblastoma experimental metastases. Arrows=sub-pial gap created by laminar spreading cells (T=tumor, P=pial layer, SP=sub-pial gap, BM= basement membrane). Scale bar=50µm.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-8042018/v1/d56d1007d5418535080e28ca.png"},{"id":97652467,"identity":"ec6f4aa2-8bce-40f5-b5d7-3e50f3bf328a","added_by":"auto","created_at":"2025-12-08 06:44:07","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1427526,"visible":true,"origin":"","legend":"\u003cp\u003eMedulloblastoma cells exhibit different patterns of spread on Human Meningeal Cell-derived extracellular matrix. \u003cstrong\u003eA)\u003c/strong\u003e Schematic for generating cell-free HMC-derived extracellular matrix (ECM). \u003cstrong\u003eB-C) \u003c/strong\u003eRepresentative immunofluorescence images of non-extracted \u003cstrong\u003e(B)\u003c/strong\u003eand extracted (cell-free) \u003cstrong\u003e(C)\u003c/strong\u003e matrix demonstrating that basement membrane proteins secreted by HMCs are maintained after cell lysis and ECM extraction (fibronectin, collagen IV and laminin). Scale bar=50µm. \u003cstrong\u003eD) \u003c/strong\u003eRepresentative images of medulloblastoma cells cultured on cell-free HMC-extracted ECM. Scale bar=50µm. \u003cstrong\u003eE) \u003c/strong\u003eQuantitation of % of cells spreading on HMC-derived ECM. n=3 independent experiments, 300 cells/experiment. Ordinary one-way ANOVA with Tukey’s multiple comparison testing. *p\u0026lt;0.05, **p\u0026lt;0.01, ***p\u0026lt;0.001, ****p\u0026lt;0.0001. \u003cstrong\u003eF) \u003c/strong\u003eRepresentative images of CC3 staining on cell-free HMC-extracted ECM. Scale bar=50µm. \u003cstrong\u003eG) \u003c/strong\u003eQuantitation of apoptotic index. n=4 independent experiments, 300 cells/experiment. Brown Forsythe and Welch’s ANOVA with Dunnett’s T3 multiple comparisons test. *p\u0026lt;0.05, **p\u0026lt;0.01, ***p\u0026lt;0.001, ****p\u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"floatimage6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8042018/v1/b8eccf7b4b4ef79c8b7eb27c.jpeg"},{"id":97652466,"identity":"73b9b724-d6a8-4269-914c-23387c328217","added_by":"auto","created_at":"2025-12-08 06:44:07","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":253473,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic depicting the proposed model for medulloblastoma leptomeningeal colonization: Medulloblastoma cells are recruited \u003cstrong\u003e(1)\u003c/strong\u003e and directly adhere to leptomeningeal pial cells \u003cstrong\u003e(2)\u003c/strong\u003e.\u003cstrong\u003e \u003c/strong\u003eLaminar disseminating cells disrupt the pial fibroblast layer \u003cstrong\u003e(3)\u003c/strong\u003e to expose and adhere to the underlying basement membrane\u003cstrong\u003e(4)\u003c/strong\u003e. Cell-matrix adhesion enables the spread of cells along the brain and spinal cord to form laminar, diffuse metastases. Nodular lesions remain adherent through cell-cell interactions between medulloblastoma cells and pial fibroblasts \u003cstrong\u003e(3’)\u003c/strong\u003e and expand outward \u0026nbsp;without diffuse spread along the leptomeninges \u003cstrong\u003e(4’)\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"floatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-8042018/v1/ca49bdb4e6af2ffb47077ca8.png"},{"id":106343978,"identity":"736b5de7-f7c6-4ae7-a687-bc8937f06605","added_by":"auto","created_at":"2026-04-07 16:11:23","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5192440,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8042018/v1/9cc59be7-e37b-441e-8ed8-b97be92d7ee6.pdf"},{"id":97652485,"identity":"8c2409ee-b117-487c-9b84-2c8edb38c49a","added_by":"auto","created_at":"2025-12-08 06:44:08","extension":"mp4","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":23731628,"visible":true,"origin":"","legend":"","description":"","filename":"D425HMCcocx.mp4","url":"https://assets-eu.researchsquare.com/files/rs-8042018/v1/be3fdaeb60f6fc88cc205edc.mp4"},{"id":97674809,"identity":"177124b5-30a0-4f0d-8e3a-e40d7aad4db8","added_by":"auto","created_at":"2025-12-08 09:44:18","extension":"mp4","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":20907703,"visible":true,"origin":"","legend":"","description":"","filename":"MED411HMCcocx.mp4","url":"https://assets-eu.researchsquare.com/files/rs-8042018/v1/2aa89e92884212bcbc1603ea.mp4"},{"id":97674840,"identity":"73152409-2586-4dc6-9233-ab2feec8a290","added_by":"auto","created_at":"2025-12-08 09:44:25","extension":"mp4","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":20138721,"visible":true,"origin":"","legend":"","description":"","filename":"MED2112HMCcocx.mp4","url":"https://assets-eu.researchsquare.com/files/rs-8042018/v1/82c207031832768b0bd6c0a1.mp4"},{"id":97652484,"identity":"f1f699a3-d9d8-42aa-8ddc-5566d6f9a5fd","added_by":"auto","created_at":"2025-12-08 06:44:08","extension":"mp4","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":17543500,"visible":true,"origin":"","legend":"","description":"","filename":"CHLA01MEDHMCcocx.mp4","url":"https://assets-eu.researchsquare.com/files/rs-8042018/v1/b7a16ac0c19c58301a31a61f.mp4"},{"id":97652471,"identity":"8631251f-34df-4b58-8ba7-1d38c9f05bf2","added_by":"auto","created_at":"2025-12-08 06:44:07","extension":"png","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":523193,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8042018/v1/59f9eb5c07c0e17a2e3f69e7.png"}],"financialInterests":"No competing interests reported.","formattedTitle":"Meningeal-tumor interactions define distinct modes of leptomeningeal colonization in medulloblastoma","fulltext":[{"header":"Introduction","content":"\u003cp\u003eNearly all deaths from medulloblastoma, the most common malignant brain tumor of childhood, are due to leptomeningeal metastasis [\u003cspan additionalcitationids=\"CR2 CR3 CR4\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. The biological mechanisms that enable tumor cells to survive and expand within the unique leptomeningeal compartment remain poorly defined[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Consequently, therapeutic strategies capable of eradicating disseminated tumor cells from the leptomeninges are lacking, particularly for patients who relapse after standard-of-care therapy[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe leptomeninges, comprising the pia mater, subarachnoid space (filled with cerebrospinal fluid - CSF), and arachnoid mater, were once viewed primarily as structural coverings to the brain and spinal cord but are now recognized as dynamic microenvironments that actively participate in central nervous system health and disease, including cancer[\u003cspan additionalcitationids=\"CR9 CR10\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Within the CSF, medulloblastoma cells exist in at least two states: free-floating within the fluid or adherent to the leptomeningeal surface of the brain and spinal cord. Adherent lesions may form diffuse, laminar coatings or discrete nodules along the leptomeninges, yet the mechanisms governing these distinct metastatic patterns remain unknown[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. This knowledge gap underscores the need to understand how interactions between tumor cells and leptomeningeal stromal elements influence colonization, survival, and progression.\u003c/p\u003e\u003cp\u003eAdvances in leptomeningeal metastasis biology have been hindered by several experimental and clinical barriers[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Surgical access to leptomeningeal lesions is limited due to their diffuse nature and lack of therapeutic benefit from resection, precluding comprehensive molecular analysis of matched primary and metastatic samples. In the few available cases, metastatic lesions exhibit marked biological divergence from their primary tumors, underscoring the need to study the metastatic niche directly[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Moreover, while mouse models have been invaluable for identifying molecular drivers of medulloblastoma initiation, endpoints are reached due to the rapid growth of cerebellar tumors that cannot be surgically resected, not leptomeningeal metastasis[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Lastly, conventional \u003cem\u003ein vitro\u003c/em\u003e models fail to replicate the cellular composition and nutrient-deprived microenvironment of the CSF, limiting their translational relevance.\u003c/p\u003e\u003cp\u003eOrganotypic co-culture systems have proven valuable for modeling tumor\u0026ndash;host interactions that occur during metastatic dissemination to lung and bone marrow in other solid tumor contexts[\u003cspan additionalcitationids=\"CR18 CR19\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Leptomeningeal stromal-tumor co-cultures have been applied in studies of adult cancers, but have not been well characterized for medulloblastoma; moreover, previous \u003cem\u003ein vitro\u003c/em\u003e findings have not been validated within the \u003cem\u003ein vivo\u003c/em\u003e leptomeningeal niche[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. To enable reductionist studies of leptomeningeal biology in medulloblastoma, we developed and validated an \u003cem\u003ein vitro\u003c/em\u003e model that recapitulates key features of the leptomeningeal niche. By co-culturing established and patient-proximal medulloblastoma cell lines with primary human leptomeningeal fibroblasts, we demonstrate that meningeal cells promote tumor cell survival and proliferation under nutrient-deprived conditions. We further identify medulloblastoma models that reproducibly form laminar or nodular growth patterns \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e, revealing a cell-biological mechanism that explains this morphological divergence. This physiologically informed, experimentally validated model enables the dissection of the molecular determinants of leptomeningeal colonization and the identification of therapeutic vulnerabilities to prevent medulloblastoma dissemination.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eCell Culture\u003c/h2\u003e\u003cp\u003eCell lines utilized in this manuscript were routinely authenticated with short tandem repeat profiling and \u003cem\u003eMycoplasma\u003c/em\u003e testing (Laboratory Corporation of America). Cells used for experiments that were grown in suspension were routinely replaced within two months after being revived from liquid nitrogen storage. Primary cells used for experiments were used within 10 passages after being revived from liquid nitrogen storage. Medulloblastoma cell lines (and their molecular sub-group) used in this study include: D425 (Group 3), MED-411FH (Group 3), MED-2112FH (Group 3), and CHLA-01-MED (Group 4). D425 Med (D425) cells (RRID: CVCL_1275) were derived from a primary medulloblastoma tumor resection at Duke University Medical Center and obtained from the laboratory of Dr. Jeffery Leonard[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Med-411FH (MED411) cells and Med-2112FH (MED2112) cells are cell lines derived from early passage patient-derived xenografts developed by the laboratory of Dr. Jim Olson and distributed by the Brain Tumor Resource Lab at Seattle Children\u0026rsquo;s Hospital[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. CHLA-01-MED was obtained from American Type Culture Collection as described previously[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Human meningeal cells (HMC) (ScienCell Research Laboratories, Cat. #1400) are primary leptomeningeal cells derived from human donors. We have used\u0026thinsp;\u0026gt;\u0026thinsp;5 distinct lots with similar results. D425 and HMC cells were cultured in DMEM (Corning, 10-013-CV) supplemented with 10% FBS (Atlanta Biologicals, S11150H). MED411, MED2112, and CHLA-01-MED cells were cultured in NeuroCult NS-A Basal Medium (Human) (Stemcell technologies, Cat. #05750) supplemented with NeuroCult NS-A Proliferation Kit (Human) (Stemcell technologies, Cat. #05751), human epidermal growth factor (20 ng/mL, ThermoFisher, AF-100-15-500UG) and human fibroblast growth factor-basic (20ng/mL, ThermoFisher, 100-18B-50UG).\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eHMC survival optimization\u003c/h3\u003e\n\u003cp\u003eHMC were grown to confluence in 24-well plates. Media was exchanged for either basal growth media, serial dilutions of growth media with artificial CSF (ACSF, Tocris, Cat. No. 3525), ACSF\u0026thinsp;+\u0026thinsp;60mg/dL glucose, or DMEM/F12 (Gibco, Cat. #21041025). After 72 hours, cell viability was assessed using alamarBlue (ThermoFisher, A50100), and cells were photographed under light-phase microscopy.\u003c/p\u003e\n\u003ch3\u003eMedulloblastoma-leptomeningeal fibroblast co-culture\u003c/h3\u003e\n\u003cp\u003eHuman medulloblastoma cells and meningeal cells were co-cultured to generate an \u003cem\u003ein vitro\u003c/em\u003e model that captures key interactions between tumor cells and the surrounding stroma of leptomeningeal metastasis within a reductionist setting. HMCs were seeded onto 0.4 \u0026micro;m transwell filters or 1.5# glass coverslips (Electron Microscopy Sciences, 72204-04) in 6- or 24-well dishes and cultured with DMEM supplemented with 10% FBS. After HMCs formed a confluent monolayer (48\u0026ndash;72 hours), the base media was removed and replaced with serum-free, growth-factor free DMEM/F12 containing medulloblastoma cells. For transwell experiments, DMEM/F12 was placed in the top and bottom wells.\u003c/p\u003e\n\u003ch3\u003eCell-free extracellular matrix\u003c/h3\u003e\n\u003cp\u003eHMC were grown to confluence on 1.5# coverslips for 72 hours. Cell-free HMC-derived matrix was generated as previously described [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Medulloblastoma cells were plated onto coated coverslips for 48\u0026ndash;72 hours in their base media before processing for immunofluorescence.\u003c/p\u003e\n\u003ch3\u003eAnimal Studies\u003c/h3\u003e\n\u003cp\u003e All animal procedures were approved by the Institutional Animal Care and Use Committee of the Abigail Wexner Research Institute at Nationwide Children\u0026rsquo;s Hospital (IACUC protocol AR15-00022). CB17-SCID mice [CB17SC scid\u0026minus;/\u0026minus;; RRID: IMSR_TAC:cb17sc] were used for all \u003cem\u003ein vivo\u003c/em\u003e experiments.\u003c/p\u003e\u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eSubcutaneous Tumor Model\u003c/span\u003e: Mice were anesthetized and injected subcutaneously in the flank with 5 \u0026times; 10⁵ medulloblastoma cells, with or without 1 \u0026times; 10⁶ HMCs. Tumor growth was monitored biweekly via caliper measurements. Mice were euthanized, and tumors were harvested upon a total tumor volume of 2000 mm\u0026sup3;.\u003c/p\u003e\u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eSpontaneous Leptomeningeal Metastasis Model\u003c/span\u003e: Mice were anesthetized with isoflurane and placed in a stereotaxic apparatus (Kopf Instruments, Model 940). Intracerebellar injection of 8 \u0026times; 10⁴ medulloblastoma cells in 3 \u0026micro;L of Neurocult media was performed at 2mm lateral, 2mm posterior, and 2.5 mm deep relative to lambda. Post-operative care included analgesia and daily monitoring. At neurological endpoint (rapid weight loss and an enhanced body condition score of less than 9, and/or neurological clinical signs including head tilts, seizures, and/or log rolling), mice were euthanized and processed for histology.\u003c/p\u003e\u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eExperimental Leptomeningeal Metastasis Model\u003c/span\u003e: Medulloblastoma cells (2.5 \u0026times; 10⁴ in 3 \u0026micro;L Neurocult media) were injected into the cisterna magna under aseptic conditions. Post-operative care and endpoint monitoring mirrored the spontaneous model.\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eHistology\u003c/h2\u003e\u003cp\u003eMice were euthanized at defined endpoints, sternotomy was performed, and mice were perfused with 20 ml of 0.9% saline injection (BD PosiFlush, cat #306546) followed by 30 ml of neutral buffered formalin (NBF) (Sigma-Aldrich, HT5012-1CS) through the left ventricle. Heads and spinal columns were cleaned of connective tissue and placed in NBF at 4\u0026deg;C for 48 hours. Brains were dissected from skull and spines were decalcified in 10% EDTA (Elabscience, E-IR-R112) for 10 days at room temperature, and then processed for paraffin embedding (FFPE). For standard histological observation, FFPE sections were counterstained with hematoxylin and eosin via conventional methods.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eImmunohistochemistry and Immunofluorescence\u003c/h3\u003e\n\u003cp\u003eUnstained 4\u0026micro;m sections were deparaffinized with xylene and rehydrated through ethanol series. Heat-mediated antigen retrieval was performed with Tris-EDTA buffer (TE; pH 9) with 0.2% v/v Tween 20 (Fisher Bioreagents, BP337-100).\u003c/p\u003e\u003cp\u003eCells growing on #1.5 glass coverslips or 0.4\u0026micro;m filters were fixed in NBF for 10 minutes at room temperature. Following three rinses in 1x Phosphate Buffered Saline (PBS) (Corning, 21-040-CM), coverslip or histological sections were then incubated for at least 1 hour in blocking solution consisting of PBS, 0.2% triton100 (v/v) (Sigma, X-100-100ml), and 3% bovine serum albumin (w/v) (Sigma, A-7888) at room temperature. Coverslips or histological sections were then incubated overnight in the following primary antibodies (diluted in blocking solution): hamster anti-Podoplanin (Developmental Studies Hybridoma Bank(DSHB), 8.1.1), rabbit anti-Fibronectin (Abcam, ab2413), rabbit anti-Collagen IV (Abcam, ab6586), rabbit anti-Synaptophysin (Abcam, ab32127), rabbit anti-GFAP (Abcam, ab68428), rabbit anti-CD31 (Abcam, ab182981), rabbit anti-pan laminin (Novus Biologicals, NB300-144), rabbit anti- Cleaved Caspase 3 (Cell Signaling Technologies-CST, 9664), rabbit anti- phospho-Histone Ser10 (CST, 53348), goat anti-CD45 (R\u0026amp;D Systems, AF114), goat anti-OTX2 (R\u0026amp;D Systems, AF1979), sheep anti-S100A6 (R\u0026amp;D Systems, AF4584), or mouse anti-Vimentin (Abnova, SRL33). Following three rinses in PBS, sections or coverslips were incubated with appropriate AlexaFluor-labeled secondary antibodies (Invitrogen), DAPI (Invitrogen, D1306) and, where indicated, Phalloidin AlexaFluor 488, 568 or 647 (Invitrogen), or Wheat Germ Agglutinin (WGA) AlexaFluor 647 (Invitrogen, W11261) diluted in blocking solution for 1 hour at room temperature. Following PBS rinses, coverslips and tissue sections were mounted in Fluoromount G (Invitrogen, 00495802).\u003c/p\u003e\n\u003ch3\u003eMicroscopy and image analysis\u003c/h3\u003e\n\u003cp\u003eConfocal microscopy images were obtained using a Crest-X-Light V3 spinning disk confocal on a Nikon Ti2-E inverted microscope, equipped with either a Hamamatsu ORCA Fusion camera or a Zeiss 800 laser scanning microscope, and featuring 4X, 20X, and 40X air objectives. Raw ND2 or czi files were imported into ImageJ for post-image processing and quantitation. All manipulation of images (brightness/contrast) were done uniformly to images. Mitotic (pH3+) and apoptotic (CC3+) were quantified by creating binary images with a manual threshold, then subjecting the binary images to the \u003cem\u003eAnalyze Particles\u003c/em\u003e function (size\u0026thinsp;\u0026gt;\u0026thinsp;10\u0026micro;m\u003csup\u003e2\u003c/sup\u003e). Index was generated by dividing the cells (pH3\u0026thinsp;+\u0026thinsp;or CC3+) by the total number of medulloblastoma cells (OTX2+). Spreading of medulloblastoma cells in co-culture was quantified by generating a binary image of OTX2 image and processed using the Skeletonize function. Skeletonized images were then quantified with \u003cem\u003eAnalyze Skeleton\u003c/em\u003e function. Percent spreading on HMC-derived ECM was quantified manually using the \u003cem\u003eCell Counter\u003c/em\u003e Plugin. H/E images were obtained with Aperio FL ScanScope digital slide scanner with 20X objective, and metastasis length was quantified using Aperio ImageScope Software (V12.4.3).\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eAll statistical analyses for \u003cem\u003ein vitro\u003c/em\u003e assays and animal studies were performed using GraphPad Prism v10. The number of biological replicates (independent experiments or animals) is specified in the figure legends, along with the appropriate statistical tests and \u003cem\u003eP\u003c/em\u003e values. Data are presented as the mean with error bars that represent the standard error of the mean.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003ePial fibroblast-medulloblastoma adhesion within the early metastatic niche\u003c/h2\u003e\u003cp\u003eTo guide the design of physiologically relevant \u003cem\u003ein vitro\u003c/em\u003e models, we first generated models of the early leptomeningeal metastatic niche \u003cem\u003ein vivo\u003c/em\u003e. Among medulloblastoma subgroups, Group 3 and Group 4 tumors most frequently disseminate to the leptomeninges[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Because Group 4 models are not widely available, we orthotopically injected three human Group 3 medulloblastoma cell lines (D425, MED411, and MED2112) into the cerebella of immunocompromised mice to assess spontaneous metastasis (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). At humane endpoint, brains and spines were examined histologically for evidence of dissemination. Although all models formed robust primary tumors with comparable survival (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB), small leptomeningeal lesions were detected only in D425 and MED411 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). These findings highlight the potential limitations of orthotopic xenografts for studying leptomeningeal metastasis, as rapid primary tumor growth restricts the development of marked leptomeningeal disease.\u003c/p\u003e\u003cp\u003eWe next defined the stromal architecture of the leptomeningeal niche in D425 spontaneous metastasis lesions. The cellular aspects of the leptomeninges include pial and arachnoid fibroblasts, penetrating blood vessels, a subpial basement membrane, tissue-resident and recruited immune cells, and the glial limitans [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan additionalcitationids=\"CR29\" citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Multiparameter immunohistochemistry identified pial and arachnoid fibroblasts (Podoplanin), endothelial cells (CD31), basement membrane (laminin), glial limitans (GFAP), and immune cells (CD45). Tumor cells, identified by OTX2 expression (not shown for clarity), were consistently juxtaposed to pial fibroblasts, suggesting a preferential tumor\u0026ndash;fibroblast interaction during early dissemination (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD-G). Based on these observations and recently published data[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e], we selected leptomeningeal fibroblasts as the primary stromal component for developing an \u003cem\u003ein vitro\u003c/em\u003e co-culture model of the early leptomeningeal niche.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eHuman leptomeningeal fibroblasts support medulloblastoma survival and growth\u003c/h2\u003e\u003cp\u003eLeptomeningeal fibroblasts can be isolated from human donors and cultured \u003cem\u003ein vitro\u003c/em\u003e [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. To enhance the physiological relevance by mimicking the nutrient-deprived state of the CSF, we first defined the minimal media conditions capable of supporting primary human leptomeningeal fibroblasts (HMC). Standard HMC media (DMEM\u0026thinsp;+\u0026thinsp;10% FBS) was diluted with increasing concentrations of artificial CSF (aCSF) containing physiologic glucose or replaced with serum- and growth factor\u0026ndash;free DMEM/F12 (\u003cb\u003eSupplemental Fig.\u0026nbsp;1A\u003c/b\u003e). While HMCs failed to survive in aCSF, confluent monolayers were maintained in DMEM/F12 (\u003cb\u003eSupplemental Fig.\u0026nbsp;1B\u0026ndash;C\u003c/b\u003e). Under these conditions, HMCs retained expression of pial markers including fibronectin, collagen IV, laminin, S100A6, and vimentin (\u003cb\u003eSupplemental Fig.\u0026nbsp;1D\u003c/b\u003e). Thus, HMCs can be maintained under serum- and growth factor\u0026ndash;free conditions that approximate the nutrient limitations of CSF.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eSupplemental Fig.\u0026nbsp;1\u003c/strong\u003e\u003cp\u003eOptimization of HMC growth under serum-free and growth-factor-free conditions. \u003cb\u003eA)\u003c/b\u003e Schematic depicting the dilution conditions followed to minimize growth factor and serum conditions while maximizing HMC proliferation and survival. \u003cb\u003eB)\u003c/b\u003e Quantification of cellular viability of HMCs under different nutrient conditions. The plot represents results for n\u0026thinsp;=\u0026thinsp;3 experiments. \u003cb\u003eC)\u003c/b\u003e Representative phase contrast images of HMCs under the different nutrient conditions. \u003cb\u003eD)\u003c/b\u003e Representative immunofluorescence images of HMCs demonstrating that they retain expression of common pial markers under serum- and growth-factor-free conditions. Scale bar =\u0026thinsp;50\u0026micro;m.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eUsing these optimized conditions, we next tested whether HMCs promote medulloblastoma cell survival and proliferation in short-term co-culture (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Under nutrient-deprived conditions, D425 and MED411 cells exhibited reduced proliferation and increased apoptosis, as shown by phospho-H3 (S10) and cleaved caspase-3 (CC3) staining (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB\u0026ndash;E). Co-culture with HMCs restored proliferation and reduced apoptosis to near steady-state levels, indicating that leptomeningeal fibroblasts provide trophic support under nutrient-limited conditions.\u003c/p\u003e\u003cp\u003eTo assess whether HMCs enhance tumorigenicity \u003cem\u003ein vivo\u003c/em\u003e, we performed dual flank injections in immunocompromised mice, injecting tumor cells alone on one side and tumor\u0026thinsp;+\u0026thinsp;HMCs on the opposite side (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF). Co-injection with HMCs increased tumor establishment (\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eD425\u003c/span\u003e: 55.5% tumor take rate in single injection vs 88.9% tumor take rate in co-injection, \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eMED411\u003c/span\u003e: 66.6% tumor take rate in single injection vs 100% tumor take rate in co-injection) and accelerated growth in both D425 and MED411 models (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG). Notably, HMC injection alone did not result in tumorigenesis, suggest the difference is not attributable to expansion of meningeal cells. Together, these data demonstrate that leptomeningeal fibroblasts actively promote medulloblastoma survival and proliferation both \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003eDistinct laminar and nodular morphologies reflect divergent modes of leptomeningeal colonization\u003c/h2\u003e\u003cp\u003eMedulloblastoma metastatic lesions adopt diffuse laminar or distinct nodular phenotypes in patients, though the mechanisms underlying these morphologies have not been elucidated due to lack of preclinical models. Having validated short-term co-culture feasibility, we next examined whether extended co-culture recapitulates the morphologic diversity of leptomeningeal metastases. A panel of medulloblastoma neurosphere lines\u0026mdash;including Group 3 models (D425, MED411, MED2112) and one Group 4 model (CHLA-01-MED)\u0026mdash;was co-cultured with HMCs under nutrient-deprived conditions for 7 days. Two reproducible and morphologically distinct growth patterns emerged: a \u003cb\u003elaminar phenotype\u003c/b\u003e (D425, MED411) in which tumor cells disseminated from neurospheres and spread across the fibroblast monolayer, and a \u003cb\u003enodular phenotype\u003c/b\u003e (MED2112, CHLA-01-MED) characterized by compact, spherical aggregates with limited dispersion (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). To generate a quantitative metric of laminar versus nodular growth, OTX2\u0026thinsp;+\u0026thinsp;tumor cell masks were converted to skeletonized representations, enabling measurement of branch number and extent as indicators of surface spreading (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB-C). Laminar models (D425 and MED411) demonstrated significantly higher branching than nodular models (MED2112 and CHLA-01-MED), indicative of a more diffuse growth pattern \u003cem\u003ein vitro\u003c/em\u003e.\u003c/p\u003e\u003cp\u003eWe next sought to determine whether \u003cem\u003ein vitro\u003c/em\u003e behaviors mirrored \u003cem\u003ein vivo\u003c/em\u003e dissemination patterns. Because leptomeningeal disease progression is prevented by rapid primary tumor growth in spontaneous models (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), we hypothesized that direct injection of tumor cells into CSF would allow for leptomeningeal tumor progression and evaluation of growth patterns between different models. Previous works have seeded medulloblastoma cells into CSF through injection of tumor cells into lateral ventricle or cisterna magna[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Medulloblastoma cells are thought to enter CSF through either direct invasion into 4th ventricle or following hematogenous dissemination[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Because the CSF within the 4th ventricle circulates directly into the cisterna magna, we chose intracisternal injections to inoculate D425, MED411, or MED2112 cells directly into the CSF. The resulting metastatic lesions recapitulated the \u003cem\u003ein vitro\u003c/em\u003e patterns: laminar models (D425 and MED411) exhibited extensive surface coating of the intracranial and spinal meninges, whereas the nodular model MED2112 formed discrete, localized deposits (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). We quantified lesion length as a surrogate measure of laminar versus nodular growth and found that laminar lesions extended significantly farther along the pial surface than nodular lesions (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). We acknowledge the limitations of using quantitative metrics such as length to capture categorical morphologic features, mirroring the challenges faced clinically in assessing leptomeningeal disease[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Collectively, the combination of \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e findings provide preclinical evidence for biologically distinct modes of leptomeningeal colonization.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003eLaminar lesions disrupt the fibroblast layer and adhere to the subpial basement membrane\u003c/h2\u003e\u003cp\u003eWe performed long-term live-cell imaging of HMC\u0026ndash;tumor co-cultures to directly visualize tumor\u0026ndash;fibroblast interactions. Both laminar and nodular medulloblastoma cells adhered rapidly to fibroblasts within hours of plating (\u003cb\u003eSupplemental Videos 1\u0026ndash;4\u003c/b\u003e). However, laminar cells progressively displaced the fibroblast monolayer and spread along the underlying substrate, whereas nodular cells remained compact and adherent to pial cells. Fixed co-cultures imaged after 5 days confirmed these distinct morphologies at high resolution (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA\u0026ndash;E).\u003c/p\u003e\u003cp\u003e\u003cem\u003eIn vivo\u003c/em\u003e immunohistochemistry of D425, MED411, and MED2112 leptomeningeal lesions corroborated these findings: laminar tumor cells penetrated the pial fibroblast layer to adhere directly to the laminin-positive subpial basement membrane. We did not observe invasion through the basement membrane or disruption of the glial limitans, consistent with clinical observations that parenchymal extension of disseminated disease is rare in medulloblastoma. In contrast, nodular lesions remained juxtaposed to intact pial fibroblasts (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF\u0026ndash;H). Thus, laminar and nodular lesions represent distinct modes of tumor\u0026ndash;meningeal interaction, differing in their degree of fibroblast displacement and engagement with the subpial basement membrane.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003eAdhesion to meningeal extracellular matrix distinguishes laminar from nodular cells\u003c/h2\u003e\u003cp\u003eWe next sought to determine how medulloblastoma cells interact with extracellular matrix (ECM) derived from leptomeningeal fibroblasts given the differential patterns of spread observed in previous experiments. To directly quantify medulloblastoma behavior on pial ECM, we generated cell-free HMC ECM by culturing HMC on coverslips and performing a decellularization method described previously[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e] (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA\u0026ndash;C). Decellularized ECM retains both the structure and biological properties of leptomeningeal-deposited ECM, even in the absence of leptomeningeal fibroblasts (noted by the absence of nuclear DAPI staining). D425, MED411, MED2112, and CHLA-01-MED were plated on decellularized HMC-derived ECM for 24 hours. Laminar cells (D425 and MED411) adopted an elongated morphology with filopodia-like projections characteristic of spreading and migration. In contrast, nodular cells (MED2112 and CHLA-01-MED) retained cortical actin organization and failed to spread on HMC-derived ECM (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD-F). Lastly, nodular cells exhibit significantly increased apoptosis when cultured on HMC-derived extracellular matrix, indicating reduced fitness and impaired adaptation to ECM-dependent growth conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eLeptomeningeal metastasis remains the principal cause of treatment failure and death in children with medulloblastoma, yet its underlying biology has remained elusive. Here, we developed an \u003cem\u003ein vitro\u003c/em\u003e model that faithfully mirrors the morphologic diversity of leptomeningeal metastasis observed \u003cem\u003ein vivo\u003c/em\u003e. Using this system, we identify two distinct modes of leptomeningeal colonization\u0026mdash;laminar and nodular\u0026mdash;that arise from differential tumor\u0026ndash;stroma adhesion mechanisms. Our findings build upon previous iterations of \u003cem\u003ein vitro\u003c/em\u003e meningeal and ECM medulloblastoma models by providing \u003cem\u003ein vivo\u003c/em\u003e validation[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Future studies will define the molecular mechanisms that distinguish laminar and nodular phenotypes and determine whether cellular plasticity exists between these states. Integrating \u003cem\u003ein vitro\u003c/em\u003e co-culture systems with both spontaneous and experimental leptomeningeal metastasis models establishes a versatile platform for delineating the stage-specific roles of metastatic mediators; whether they govern invasion and dissemination from the primary site, colonization of the leptomeninges, or subsequent progression within the leptomeningeal niche [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan additionalcitationids=\"CR41\" citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Additionally, understanding the pathways that regulate these phenotypic transitions may have translational significance, given differential therapeutic sensitivities between clinical phenotypes [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. The model described here provides a tractable platform to investigate therapeutic response as a function of leptomeningeal colonization architecture.\u003c/p\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003eThe leptomeningeal niche as a trophic microenvironment\u003c/h2\u003e\u003cp\u003eA recent landmark paper from the Taylor group identified medulloblastoma-induced changes to the leptomeningeal environment during colonization and established the importance of tumor-host interactions in leptomeningeal metastasis[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Our findings agree with those observations by providing direct evidence that human meningeal cells provide trophic support to medulloblastoma cells. Co-culture of medulloblastoma cells with HMC rescued tumor cell proliferation and suppressed apoptosis under nutrient-deprived conditions, demonstrating that fibroblast-derived cues compensate for the metabolic limitations of CSF. Similar trophic effects have been described in lung and bone marrow metastases, suggesting that stromal support is a conserved feature of the metastatic niche across organ systems[\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. By defining leptomeningeal fibroblasts as key mediators of this process, our study establishes a cellular foundation for dissecting paracrine and adhesion-dependent interactions that sustain leptomeningeal metastasis.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\u003ch2\u003eModeling implications and future directions\u003c/h2\u003e\u003cp\u003eTraditional xenograft models are limited in their ability to capture the temporal or spatial features of leptomeningeal dissemination due to rapid intracerebellar tumor growth and limited sampling of metastatic lesions. The co-culture system described here complements these \u003cem\u003ein vivo\u003c/em\u003e models by offering a reductionist platform for mechanistic dissection and therapeutic testing. Integration of single-cell and spatial transcriptomic profiling will enable mapping of tumor\u0026ndash;stroma signaling networks and identification of context-specific dependencies. In parallel, the model provides an experimental framework for evaluating compounds that disrupt adhesion-dependent survival. Future studies will delineate the mechanisms that distinguish cell\u0026ndash;cell from cell\u0026ndash;matrix adhesion in nodular and laminar metastases, as disrupting these interactions may represent context-specific therapeutic vulnerabilities within the leptomeningeal niche.\u003c/p\u003e\u003cp\u003eWhile our model captures key aspects of the leptomeningeal niche, it lacks other microenvironmental components, such as immune or vascular cells, that may modulate colonization dynamics and progression[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. Incorporating these elements in future iterations will further enhance physiologic relevance. The models used in this study were predominantly Group 3 medulloblastoma lines with MYC amplification, reflecting both their aggressive and metastatic phenotype and their availability [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. Future work will extend these studies to WNT-, SHH-activated, and non-MYC\u0026ndash;amplified Group 3/4 tumors, ideally using patient-derived xenografts or primary human specimens, to capture the full molecular spectrum of medulloblastoma leptomeningeal metastasis. Ultimately, coupling these advanced models with clinical and genomic data from patient-derived metastasis samples will enable a more complete understanding of metastatic evolution in medulloblastoma.\u003c/p\u003e\u003c/div\u003e"},{"header":"Conclusions","content":"\u003cp\u003eOur study delineates modes by which medulloblastoma colonizes the leptomeninges. We identify differential interactions with fibroblast-derived extracellular matrix as a key determinant in influencing metastatic phenotype. This framework establishes the leptomeningeal niche as both a driver of metastatic persistence and a therapeutic vulnerability, offering new avenues for targeting the most lethal stage of pediatric medulloblastoma.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003cp\u003eThis study used pre-established and patient-derived human cell lines that are commercially available (Brain Tumor Resource Lab, ATCC, Sigma-Aldrich and ScienCell). According to institutional and national guidelines, research involving such cell lines does not require ethics committee approval because the material is anonymized and information identifying the donor is not accessible.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003ch2\u003eConsent for publication\u003c/h2\u003e\u003cp\u003eDe-identified MR images were obtained after approval from Nationwide Children\u0026rsquo;s Hospital Institutional Review Board. Consent was not deemed to be necessary.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003cp\u003eLJG is supported by Pelotonia on a Pelotonia Graduate Scholar Fellowship.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e\u003cp\u003eThis work was funded by CancerFree KIDS (Wolf Pack Research Grant, JBR), St. Baldrick\u0026rsquo;s Foundation Scholar Award (JBR), Alex\u0026rsquo;s Lemonade Stand Foundation Young Investigator Award (JBR), Hyundai Hope On Wheels, Bridge2K (JBR), and Nationwide Children\u0026rsquo;s Hospital (JBR). LJG is supported by Pelotonia on a Pelotonia Graduate Scholar fellowship. RDR is supported by Alex\u0026rsquo;s Lemonade Stand Crazy 8 Award, NIH R01CA260178, NIH CTSA Grant UL1TR002733, CancerFree KIDS Foundation, and Nationwide Children\u0026rsquo;s Director Strategic Development Fund.\u003c/p\u003e\u003cp\u003eAcknowledgments\u003c/p\u003e\u003cp\u003eWe are indebted to Dr. Robert Wechsler-Reya, Elizabeth Thompson, and Daphne Koubourli for their technical expertise in \u003cem\u003ein vivo\u003c/em\u003e modeling. We are grateful to members of the laboratory of Dr. Ryan Roberts for valuable discussions and critical reading of this manuscript. We also acknowledge the Histopathology and Microscopy Core facilities at Nationwide Children\u0026rsquo;s Hospital Abigail Wexner Research Institute for their excellent technical expertise. The authors acknowledge the use of ChatGPT5, solely for text editing and clarity. All schematics in this manuscript were generated using BioRender.\u003c/p\u003e\u003cp\u003eAuthors\u0026rsquo; information (optional)\u003c/p\u003e\u003cp\u003eNot applicable.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eLJG- design of work, acquisition, analysis, and interpretation of data, draft of manuscriptLR- design of work, acquisition, analysis, and interpretation of data, reviewed manuscriptAG-acquisition, analysis, and interpretation of data, reviewed manuscriptAA-acquisition of data, reviewed manuscriptJW-acquisition of data, reviewed manuscript JL-acquisition of data, critical materials, review and revision of manuscriptRR-interpretation of data, review and revision of manuscript JR-conception and design of work, acquisition, analysis, and interpretation of data, review and revision of manuscript\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eWe are indebted to Dr. Robert Wechsler-Reya, Elizabeth Thompson, and Daphne Koubourli for their technical expertise in in vivo modeling. We are grateful to members of the laboratory of Dr. Ryan Roberts for valuable discussions and critical reading of this manuscript. We also acknowledge the Histopathology and Microscopy Core facilities at Nationwide Children\u0026rsquo;s Hospital Abigail Wexner Research Institute for their excellent technical expertise. The authors acknowledge the use of ChatGPT5, solely for text editing and clarity. All schematics in this manuscript were generated using BioRender.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eAll data supporting the findings of this study are available within the paper and its Supplementary Information.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eHill RM et al (2020) Time, pattern, and outcome of medulloblastoma relapse and their association with tumour biology at diagnosis and therapy: a multicentre cohort study. 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J Biotechnol 236:10\u0026ndash;25\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":true,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"acta-neuropathologica-communications","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"anec","sideBox":"Learn more about [Acta Neuropathologica Communications](https://actaneurocomms.biomedcentral.com/)","snPcode":"40478","submissionUrl":"https://submission.springernature.com/new-submission/40478/3","title":"Acta Neuropathologica Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Medulloblastoma, leptomeningeal metastasis, adhesion, co-culture models","lastPublishedDoi":"10.21203/rs.3.rs-8042018/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8042018/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eMedulloblastoma, the most common malignant brain tumor in children, shows a pronounced tendency to spread to the leptomeninges. Leptomeningeal metastasis accounts for nearly all medulloblastoma-related deaths, yet the cellular and molecular mechanisms driving this process remain poorly understood. Progress has been hindered by limited access to patient samples, the fragile anatomy of the leptomeninges, and the lack of robust preclinical models.\u003c/p\u003e\u003cp\u003eHere, we developed an \u003cem\u003ein vitro\u003c/em\u003e model that captures key features of the early leptomeningeal niche. We demonstrate that human meningeal cells promote medulloblastoma survival and proliferation under nutrient-deprived conditions both \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e. Using this system, we uncovered mechanisms that govern distinct patterns of leptomeningeal colonization \u003cem\u003ein vivo\u003c/em\u003e. This physiologically informed and experimentally validated model offers a tractable platform for elucidating the molecular basis of leptomeningeal colonization and for identifying therapeutic vulnerabilities that can prevent medulloblastoma dissemination.\u003c/p\u003e","manuscriptTitle":"Meningeal-tumor interactions define distinct modes of leptomeningeal colonization in medulloblastoma","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-08 06:44:02","doi":"10.21203/rs.3.rs-8042018/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-12-12T14:29:29+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-12T01:05:41+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-11T20:43:41+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"74696458150603854063120678845806667460","date":"2025-11-19T16:56:28+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"185725473314781444909431194250283882544","date":"2025-11-17T15:45:20+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-11-17T15:39:53+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-11-15T19:10:42+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-11-11T06:59:00+00:00","index":"","fulltext":""},{"type":"submitted","content":"Acta Neuropathologica Communications","date":"2025-11-05T22:23:05+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"acta-neuropathologica-communications","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"anec","sideBox":"Learn more about [Acta Neuropathologica Communications](https://actaneurocomms.biomedcentral.com/)","snPcode":"40478","submissionUrl":"https://submission.springernature.com/new-submission/40478/3","title":"Acta Neuropathologica Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"c927c392-5a0a-486b-b0ce-c6f1dc81bff3","owner":[],"postedDate":"December 8th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-04-07T16:07:55+00:00","versionOfRecord":{"articleIdentity":"rs-8042018","link":"https://doi.org/10.1186/s40478-026-02253-7","journal":{"identity":"acta-neuropathologica-communications","isVorOnly":false,"title":"Acta Neuropathologica Communications"},"publishedOn":"2026-04-01 15:59:05","publishedOnDateReadable":"April 1st, 2026"},"versionCreatedAt":"2025-12-08 06:44:02","video":"","vorDoi":"10.1186/s40478-026-02253-7","vorDoiUrl":"https://doi.org/10.1186/s40478-026-02253-7","workflowStages":[]},"version":"v1","identity":"rs-8042018","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8042018","identity":"rs-8042018","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}