Glioblastoma disrupts the ependymal wall and extracellular matrix structures of the subventricular zone

Patient-derived GBM BTIC line GBM1A, originally established as line 020913 [40] and extensively characterized by our collaborators, was cultured as neurospheres in Dulbecco's modified Eagle's medium/F-12 medium supplemented with EGF and FGF (20 ng/mL each). To localize cells in vivo, cells were transduced with a lentivirus for GFP-luciferase (GFP-luc; RediFect™ Red-FLuc-GFP, Perkin Elmer CLS960003) and sorted using fluorescence-activated cell sorting for GFP.

Animal experiments were approved by the Mayo Clinic Institutional Animal Care and Use Committee. Mice were housed in an AAALAC-accredited facility abiding by all federal and local regulations. Male immunosuppressed athymic nude mice (J:NU; Jackson Laboratory strain 007850) were maintained at Mayo Clinic Jacksonville with ad libitum access to food and water and a 12-h light–dark cycle. Animals were injected with GBM1A GFP-luc + BTICs for experiments at 6 weeks of age.

Mice were anesthetized with isoflurane inhalation and placed into a stereotactic frame. 3.5 × 10⁵ GBM1A GFP-luc + BTICs were injected in 2 μL of sterile PBS at a rate of 0.5 μL/minute. Animals were randomly assigned to one of three groups; LV-proximal vehicle injection (PBS), LV-distal GBM, and LV-proximal GBM (n = 20 per group total). LV-proximal and LV-distal surgical sites were established in the following coordinates in mm relative to bregma as previously described [39]; LV-proximal: AP: 1.0, L: 1.2, D: 2.3; LV-distal: AP: 1.0, L:2.1, D: 2.3. Mice were maintained for 4 weeks following tumor implantation for immunohistological analysis. Mice were then anesthetized with ketamine-xylazine and perfused with 0.9% saline followed by 4% paraformaldehyde (PFA). Brains were extracted and postfixed in 4% PFA overnight, then stored in PBS with 0.1% sodium azide at 4ºC. Animals used for wholemount experiments were perfused with 0.9% saline and brains were immediately processed as described below. Animals used for transmission electron microscopy analysis were perfused by 0.9% saline followed by 2% PFA/2.5% glutaraldehyde, then processed.

The lateral wall of the LV was dissected out of the brain hemisphere ipsilateral to injection as described previously [41]. After dissection of the LV, wholemounts (n = 3 per group) were fixed overnight in 4% PFA. The next day, wholemounts were permeabilized by incubation in 0.1% Triton in PBS (PBS-TX), blocked for 1 h at room temperature in 10% normal donkey serum in PBS-TX, then incubated with primary antibodies at various concentrations (Table 1) for 3 days at 4 °C diluted in blocking solution. The wholemounts were then washed with PBS-TX and incubated with secondary antibodies in blocking solution at a concentration of 1:500 overnight at 4 °C protected from light. Wholemounts were then washed with PBS, counterstained with DAPI, and mounted on glass slides before imaging.

Animals (n = 3 per group) were perfused with 0.9% saline and tissue was fixed by immersion with 2% PFA/2.5% glutaraldehyde in 0.1 M phosphate buffer for 1 h. Samples were then post-fixed in 1% osmium tetroxide (Electron Microscopy Sciences) for 45 min at 4 °C. Samples were washed with deionized water and partially dehydrated in increasing concentrations of ethanol up to 100%. Subsequently, critical point drying and sputtering with gold/palladium alloy was performed at the Central Service for Experimental Research of the University of Valencia.

Brains (n = 5 per group) were sectioned into 50 μm coronal sections using a Precisionary Compresstome VF-300-0Z vibrating microtome. Sections were permeabilized and blocked as described above. Sections were incubated with primary antibodies (Table 1) diluted in blocking solution overnight at 4 °C. The next day sections were washed and incubated with secondary antibodies (1:500 dilution) in the dark for 1 h at room temperature in blocking solution. Sections were then washed with PBS and counterstained with DAPI as a nuclear marker. For lipid droplet labeling, sections were permeabilized with 0.1% saponin and incubated with HCS LipidTOX Red Neutral Lipid Stain (Invitrogen H34476) diluted 1:100 in PBS for 1 h before mounting on glass slides.

After 4 weeks of tumor growth, mice (n = 5 per group) were anesthetized and 1 µL of 0.2% 1,1'-Dioctadecyl-3,3,3',3'-Tetramethylindocarbocyanine (DiI) in 2% DMSO was injected into the contralateral ventricle from the original injection site using a stereotactic frame. Coordinates for the DiI injection site are the following mm from bregma: AP: − 0.5, L: − 0.7, D: 2.0. 24 h after injection, mice were perfused and brains were sectioned and processed for IHC.

Samples (n = 3 per group) were sectioned into 200 µm sections and post-fixed with 2% osmium tetroxide (Electron Microscopy Sciences) for 2 h. Sections were then washed in deionized water, and partially dehydrated in 70% ethanol. Afterwards, the samples were contrasted with 2% uranyl acetate (Electron Microscopy Sciences) in 70% ethanol for 2 h at 4 °C. The samples were further dehydrated and infiltrated in Durcupan ACM epoxy resin (Sigma) at room temperature overnight, and then at 60 °C for 72 h. Once the resin was cured, SVZ sections were selected and cut into ultrathin Sects. (60–80 nm) using an Ultracut UC7 ultramicrotome (Leica Biosystems). These sections were placed on Formvar-coated single-slot copper grids (Electron Microscopy Sciences) and stained with lead citrate.

LV wholemount and coronal IHC preparations were visualized using a Zeiss LSM880 confocal microscope. For coronal sections, the entirety of the coronal SVZ was imaged dorsal to ventral on sections containing tumor or injection site. Images were taken using 10X, 20X, 40X, or 63X objectives. SEM images were obtained on a Hitachi S4800 microscope. TEM images were obtained with a FEI Tecnai Spirit G2 biotwin microscope with a Xarosa (20 Megapixel resolution) digital camera using Radius image acquisition software (EMSIS GmbH, Münster, Germany). ZEN Blue (Zeiss), ImageJ (NIH), and Vision4D (arivis) were used for image processing and quantification.

Images of lipid droplets along the entirety of the coronal SVZ section (dorsal to ventral) were acquired on the TEM. The number of lipid droplets per millimeter of SVZ was recorded. Images were opened with ImageJ. Scale was calibrated and lipid droplet area was measured using the oval selection tool.

Images of fractones along the entirety of the coronal SVZ section (dorsal to ventral) were acquired using IHC and TEM. The IHC images were loaded into Vision4D software. The SVZ was manually selected and a fluorescence intensity threshold was set for laminin gamma-1 (LMγ1) and NS-HSPGs. Puncta expressing both markers above threshold were considered a single fractone. The number of fractones per millimeter of SVZ was recorded. TEM images were opened with ImageJ. Fractal structures with an electron dense layer surrounded by an electron lucent layer were defined as fractones [32]. Scale was calibrated and fractone area was measured using the freehand selection tool.

Confocal images of ependymal cell junctions and channels were taken of the entirety of the ipsilateral SVZ section at the coronal section of injection at 20X magnification. The images were loaded into Vision4D software. The SVZ was manually selected and a fluorescence intensity threshold was set for each channel type. Puncta expressing markers above threshold were considered a single channel. Channel formations above the fluorescence threshold were automatically counted and recorded for analysis.

All data is represented as the mean ± standard error of the mean unless otherwise indicated. Statistical analysis and graphical representation were performed using GraphPad Prism 9 ® software. Normal distribution of the data was evaluated using the Shapiro–Wilk normality test. For comparisons among three groups, analysis of variance (ANOVA) with Tukey’s post-hoc correction was performed. The level of significance was determined as p < 0.05.

The data that support the findings of this study are available from the corresponding author upon reasonable request.

We first evaluated the effect of tumor proximity to the LV on the integrity of the ependymal cell barrier of the SVZ. Patient-derived GBM BTICs modified to express GFP were implanted at locations proximal or distal to the LV (Additional file 1: Fig. S1). A vehicle injection of PBS at the LV-proximal injection site was also included to account for injection artifact (Fig. 1A). After 4 weeks, the LV lateral walls were extracted, and ependymal cell wall integrity was assessed through LV wholemount imaging. In the LV-proximal injection group, we identified single GFP + cells from LV-proximal tumors which extended single processes into the LV, interacting directly with the CSF in a manner reminiscent of NSCs (Additional file 4: Video S1). This result was corroborated through IHC on coronal sections, where single GFP + cells were again seen extending processes to the LV lumen and cells were seen invading into the LV (Fig. 1B). Additionally, using SEM we identified invading cells on the apical ependymal cell wall only in LV-proximal tumor animals, suggesting they belong to GBM cells (Fig. 1C). In the vehicle injection group, the ependymal wall remained intact as evaluated by immunofluorescent LV wholemount analysis. Ependymal cells were well-outlined with β-catenin and occasionally formed pinwheel formations with GFAP + centers (Fig. 1D top, Additional file 2: Fig. S2) and no GFP + cells are found within the tissue. In the LV-distal GBM group, the ependymal cell wall remained intact, but was accompanied by low levels of GFAP + astrocytic gliosis (Fig. 1D middle, Additional file 2: Fig. S2). In contrast, the LV-proximal tumor animals contained localized areas of extreme LV wall disruption, where ependymal cells were lost and GFP + GBM cells and several GFAP + astrocytes had direct contact with the LV lumen (Fig. 1D bottom, Additional file 2: Fig. S2). Interestingly, GFP + areas were located to the ipsilateral LV and were not observed in any of the surrounding ventricular spaces by IHC or the spinal canal by in vivo imaging signal, suggesting localized entry of GBM cells into the ipsilateral LV.

Given the disruption of the ependymal wall, we next assessed whether LV-proximal GBM cells therefore have direct access to CSF and CSF-contained components. Following 4 weeks of GBM growth, 1 µL of 0.2% DiI solution was injected into the contralateral LV from original injection and brains were analyzed for DiI fluorescence after 24 h. In the presence of LV-distal GBM, very few GFP + tumor cells were labeled by the DiI stain, and those labeled appeared to be migrating towards the LV (Fig. 1E, left). In contrast, LV-proximal GBM tumors occasionally had high numbers of LV-invading cells (Fig. 1E, right, arrow) and had increased numbers of GFP + tumor cells labeled with DiI (Fig. 1E, right), indicating direct contact of the tumors with CSF. This result proves that LV-proximal GBM tumors have access to the CSF compartment and strongly suggests that CSF-contained components are able to directly signal to LV-proximal GBM cells and vice versa.

After observing the physical disruption of the ependymal cell barrier and the accompanied invasion of GBM cells, we were interested to see if ependymal cilia would remain intact in the presence of LV-proximal GBM. SEM analysis of the ipsilateral LV wall was performed to examine the status of cilia. In the LV-proximal vehicle injected animals, cilia were abundant and long, with a uniform orientation (Fig. 2A). Cilia appeared similar in LV-distal GBM injected animals, with a slight loss of uniformity in the orientation of cilia (Fig. 2B). In the LV-proximal GBM injected animals, however, there was a stark reduction in the number of cilia (Fig. 2C). The cilia of this group also showed to be shorter and lack uniform directionality, suggesting alterations in CSF flow. Such a reduction in cilia, in both size and number, has clear implications for CSF regulation and flow throughout the ventricular system.

In previous works, reduction in cilia density has been tied to accumulation of LDs [42]. Therefore, we were next interested to investigate whether ependymal cells accumulated LDs in response to nearby GBM. To do so, we measured the incorporation of LDs in ependymal cells using both IHC and TEM. Using LipidTOX to fluorescently label neutral lipids in β-catenin-outlined ependymal cells, we identified an accumulation of ependymal LDs in LV-proximal GBM compared to vehicle-injected and LV-distal GBM groups (Fig. 3A). This was verified using TEM, where ciliated ependymal cells of the SVZ were shown to significantly accumulate round LDs of homogenous density in the LV-proximal GBM group compared to LV-distal GBM and vehicle-injected mice (mean ± SEM Vehicle: 13.95 ± 1.69 LDs/mm SVZ, LV-distal GBM: 27.34 ± 1.61 LDs/mm SVZ, LV-proximal GBM: 56.19 ± 7.67 LDs/mm SVZ, p < 0.01; Fig. 3B, C). Additionally, the average LD area was significantly increased both by the presence of GBM and increasing tumor proximity to the LV (mean ± SEM Vehicle: 4.062 ± 0.696 µm², LV-distal GBM: 5.266 ± 0.324 µm², LV-proximal GBM: 6.743 ± 0.328 µm², p < 0.01; Fig. 3D). The accumulation of both number and size of LDs in the SVZ of LV-proximal GBM mice indicates increased stress in these cells, potentially due to physical disruption of the LV wall by tumor cells.

Fractones, ECM structures derived from ependymal cells, have been previously shown to mediate signals from the LV to NSCs and have been hypothesized to play a role in GBM biology [12, 31, 33]. To determine whether the ependymal-derived fractone structures are disrupted in response to tumor proximity to the LV, we evaluated the number of LMγ1 + /NS-HSPG + puncta in the ependymal wall of the SVZ using IHC. Interestingly, we identified a significant increase in SVZ fractone density in LV-proximal GBM mice compared to vehicle and LV-distal GBM-injected mice (mean ± SEM Vehicle: 12,993 ± 1514 fractones/mm² SVZ, LV-distal GBM: 13,826 ± 1620 fractones/mm² SVZ, LV-proximal GBM: 18,644 ± 1388 fractones/mm² SVZ, p < 0.05; Fig. 4A, B). Given the increase in fractone density, we were also interested in seeing if the size of these structures was changed in response to nearby GBM. Conversely, we found by TEM analysis that the average area of fractone structures is decreased in LV-proximal GBM compared to vehicle injected mice (mean ± SEM Vehicle: 0.710 ± 0.109 µm², LV-distal GBM: 0.589 ± 0.097 µm², LV-proximal GBM: 0.253 ± 0.037 µm², p < 0.05; Fig. 4C, D).

We were also interested to see whether fractone localization is affected by the presence of a LV-proximal GBM tumor. Using TEM, we determined that in the vehicle and LV-distal GBM injected animals, fractones were localized to the base of ependymal cells as has been previously reported. In contrast, animals with LV-proximal GBM occasionally had displaced fractone localization, with fractones being directly adjacent to the LV (Additional file 3: Fig. S3). These data show that fractones may have increased access to CSF in the presence of LV-proximal GBM.

Disruptions in cell junctions between ependymal cells may contribute to compromised ependymal cell function and barrier integrity. In particular, functional Cx-43 junctions are important for reactivation of proliferation in the subependymal niche in response to injury [43, 44]. Given previous findings implicating decreased NSC proliferation in response to GBM [39, 45], we were interested in how these junctions may be altered by GBM proximity to the niche. To examine cell junction integrity in ependymal cells, we performed IHC for Cx-43 junctions in the SVZ. We identified a significant decrease in Cx-43 + junctions in the presence of LV-proximal GBM compared to other groups (mean ± SEM Vehicle: 40,132 ± 2287 Cx-43 + junctions/mm² SVZ, LV-distal GBM: 46,053 ± 2029 Cx-43 + junctions/mm² SVZ, LV-proximal GBM: 28,036 ± 3347 Cx-43 + junctions/mm² SVZ, p < 0.05; Fig. 5A, B). The notable decrease in these junctions may partially drive the decreased neurogenesis in the SVZ previously observed in response to LV-proximal GBM.

Next, we were interested to examine other important ependymal cell channels, including AQP4 channels. These channels are integral for maintaining the structural and functional integrity of ependymal cells [46] and appear to co-regulate Cx-43 + gap junctions. IHC against AQP4 revealed a significant decrease in these channels in the LV-proximal GBM group compared to LV-distal GBM and LV-proximal vehicle (mean ± SEM Vehicle: 29,912 ± 1925 AQP4 + junctions/mm² SVZ, LV-distal GBM: 30,561 ± 4682 junctions/mm² SVZ, LV-proximal GBM: 16,278 ± 2318 junctions/mm² SVZ, p < 0.05; Fig. 5C, D). The combined decrease of AQP4 channels and Cx-43 junctions suggests loss of ependymal cell barrier integrity with implications for CSF production and fluid exchange with the parenchyma.