Name: Reproducible 3D Glioblastoma Migration Assay with Magnetic Nanoparticle Mediated Spheroid Localization Under Hypoxic Conditions (Video) | JoVE Journal
Uploaded: 2026-05-12T13:10:00.000+00:00
Duration: 7 min 55 s
Description: Learn a reproducible 3D glioblastoma migration assay using magnetic nanoparticles for spheroid localization under hypoxic conditions. This method enables precise imaging and comparison of invasive potential between patient-derived tumors.

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This work was supported by Research Ireland (Science Foundation Ireland) under grant code 20391A01 and the Health Research Board under grant code ERA-TRANSCAN-2022-002. Cells were kindly provided by our collaborators in the Dirks Lab, SickKids, Toronto and The Erasmus Medical Center (EMC), Rotterdam.

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Nothing to disclose

Modelling the invasive dynamics of highly aggressive tumors such as glioblastoma is critical for improving our understanding of disease processes and identifying more effective treatment strategies. The complex, adaptive, and plastic nature of glioblastoma cell migration necessitates preclinical models that can accurately capture the dynamic behaviors of individual glioma cells as they migrate from the central tumor mass. Importantly, modelling these processes across patient derived samples from different stages of disease enables researchers to better address the challenge of tumor heterogeneity and identify any stage specific changes in invasion dynamics.
Given this complexity, it is reasonable to assume that preclinical models should possess an equivalent level of sophistication. Here, we describe a refined method for generating uniformly sized patient derived gliomaspheres co-cultured with iron oxide nanoparticles. This approach allows controlled localization of gliomaspheres at the center of each well, enabling automated live cell imaging of invasion dynamics. Several studies have highlighted significant variability in spheroid size, shape, and growth kinetics when using conventional 3D culture methods, Sayde and colleagues demonstrated that spheroids can exhibit wide diameter distributions between those derived from unsorted versus sorted cancer stem cell populations, reflecting inherent variability in spheroid formation15,16. This heterogeneity has also been identified as a major source of reproducibility issues in spheroid based drug screening assays, further emphasizing the importance of generating homogeneous spheroid populations12. These findings stress the necessity of standardizing spheroid formation conditions prior to conducting functional analyses, challenges which this method directly addresses. By standardizing both spheroid size and position, this method significantly enhances reproducibility and minimizes variability typically associated with 3D cell culture systems, where uneven spheroid formation can affect downstream analysis.
During the initial development of this assay, we observed substantial variability in spheroid localization across replicates, requiring the use of a large number of wells per experiment but also many biological replicates to obtain statistically significant results. This not only substantially increased workload but also generated a significant amount of unusable data, reduced assay efficiency and reproducibility. The resulting variability therefore reflected loss of quantifiable data rather than differences in invasion behavior. By centralizing gliomaspheres within each well, magnetic localization ensures that images from all replicates can be consistently segmented and analyzed, even where biological differences in invasion dynamics are observed between samples.
The incorporation of iron oxide nanoparticles introduces a gentle magnetic force that consistently centralizes gliomaspheres across all wells, greatly improving the performance of the image processing pipeline, data processing, and post migration analyses, without affecting gliomasphere biology or viability8. A key advantage of this system is its compatibility with post migration multiplexed analysis, for example, Figure 6 represents analysis of the visualization of the glioblastoma stem cell marker CD44, which enables evaluation of stem like cell expression following migration and treatment across patient samples. This capability highlights the platforms potential for integrative studies linking invasive phenotypes to molecular features, an important consideration given the phenotypic plasticity of glioblastoma stem like cells in response to microenvironmental cues and their ability to evade treatment promoting inevitable tumor recurrence.
While this assay greatly improves the reproducibility of visualizing single spheroid invasion dynamics, there remains some critical limitations that must be considered for future assay optimization. Matrigel is a widely used model of basement membrane, however it is a complex, undefined extracellular matrix (ECM) substitute derived from murine sarcomas, which does not fully recapitulate the human brain microenvironment17. Future optimization of the assay could incorporate brain specific ECM models to better mimic the brain tumor microenvironment, which we believe is an imperative optimization step of in vitro brain invasion models18,19. While Matrigel remains a widely adopted model of ECM, it represents a non-brain specific extracellular matrix derived from murine sarcoma. As such, it does not fully recapitulate the biochemical and mechanical properties of the human brain microenvironment. Importantly, the magnetic localization strategy described here is matrix independent and could be readily adapted to brain specific ECM systems in future studies to further enhance physiological relevance.
Other considerations of the assay are the potential requirements of training a new WEKA segmentation model for each cell line due to sample heterogeneity and variability in invasion morphology. Additionally, both seeding density and nanoparticle concentration should be optimized for each cell line and spheroid type to maintain uniformity and viability prior to performing high throughput migration assays. Regardless of these limitations, this platform provides a scalable and adaptable framework for studying patient specific glioblastoma invasion and treatment response. Its compatibility with live cell imaging, automated segmentation pipelines, and multiplexed post assay analysis opens opportunities for high content screening of novel therapeutics, combination therapies, and mechanistic studies of invasion from highly heterogenous samples of patient tumors. Furthermore, extending this assay to co culture systems, such as resident brain cells such as astrocytes, neurons and microglia or models of blood brain barrier interactions yield additional insights into tumor stroma interactions that drive glioblastoma progression and recurrence.
Several technical steps were identified during assay development as critical determinants of reproducibility and overall assay performance. Standardizing the seeding density of cell lines prior to the assay ensures that initial gliomasphere size is consistent, this enables spheroid growth dynamics to be comparable across cell lines, treatments and conditions. Without this standardization step, the initial spheroid sizes will add a significant level of variability, which may then reflect variable responses of gliomaspheres to experimental conditions. Precise central localization of gliomaspheres within each well is a key requirement for accurate automated image acquisition and downstream segmentation. Off center spheroids resulted in substantial imaging noise and prevents the use of the WEKA segmentation as a means to segment the images into clearly defined regions. By ensuring all gliomaspheres are localized in the center, the same WEKA classifier can be used multiple times as noise is substantially reduced and image quality dramatically improves. A representation of this is shown in Figure 1. The incorporation of magnetic iron oxide nanoparticles is therefore a method of ensuring central location of gliomaspheres, minimizing positional variability and substantially improving image processing and comparability across replicates. Lastly, segmentation model training within the WEKA pipeline represents an additional critical standardization step. Due to inter-patient heterogeneity and differences in invasion morphology, a single segmentation model may not generalize across all samples. Retraining or refining classification parameters for each cell line ensures accurate probability maps of invasive areas and prevents quantification errors.
Overall, this assay provides a reproducible and scalable platform for studying the dynamic invasive behaviors of patient derived glioma samples in real time. Its compatibility with multiplexed imaging and downstream molecular analyses makes it a powerful and versatile tool for both basic and translational glioma research which will greatly enhance the translational relevance of pre-clinical glioblastoma models.

Glioblastoma remains one of the most significant challenges in neuro oncology. This disease is driven by extensive inter and intra patient heterogeneity, robust evasion of response to standard of care, resistance to apoptotic induction, and remarkable plasticity in response to environmental conditions1 . More recently, the complexity of the tumor microenvironment (TME) has gained considerable attention, particularly the dynamic nature of glioma stem like cells (GSCs), which can switch phenotype and cell state in response to microenvironmental changes2,3. This phenotypic plasticity promotes tumor progression and contributes to inevitable recurrence despite aggressive frontline standard of care treatment. Surgical resection remains the most effective intervention for improving patient prognosis, however, due to the highly infiltrative and microscopic invasive nature of glioblastoma, complete tumor removal is not achievable4. Modelling the invasive dynamics of patient samples across various disease stages and in tumor specific conditions will facilitate a deeper understanding into the dynamic and plastic ability of glioblastoma migration and therefore help to identify effective novel treatment approaches5. Therefore, developing physiologically relevant models that capture patient specific invasion and migration dynamics is essential for enhancing pre-clinical research and progressing these objectives. Moreover, such models are critical for evaluating the efficacy of novel anti-tumor therapies, which could in theory be developed for use in combination with surgery to prevent residual microscopic invasion and ultimately reduce recurrence risk. The complexity of glioblastoma justifies the need for advanced complex preclinical models that can recapitulate key tumor architectural features. Patient derived three-dimensional models have gained increased attention for their ability to preserve cell to cell interactions, maintain gradients of nutrients, oxygen, and therapeutic agents, and replicate the structural integrity of native tumors6,7. However, visualizing these 3D models presents technical challenges, including spheroid positioning, imaging variability, and assay reproducibility.
Here, we describe a patient specific 3D glioblastoma migration model performed under hypoxic conditions, enabling the investigation of migratory dynamics across samples derived from various patient tumor subtypes and disease stages, including treatment naive and recurrent tumor samples. This platform allows assessment of differential responses to therapeutic agents and supports multiplex post migration analysis through image segmentation, enabling quantitative evaluation of migration dynamics, spheroid growth and protein expression. The incorporation of magnetic iron oxide nanoparticles allows consistent centralization of spheroids within each well by applying a gentle magnetic force8. Spheroids cultured in standard multiwell plates often exhibit a tendency to drift toward the well edges, a behavior believed to be influenced by the formation of liquid menisci and peripheral evaporation, especially in wells at the plate perimeter9,10,11. These phenomena introduce imaging artifacts and background noise, particularly during transmitted light microscopy which can complicate time lapse imaging of invasion dynamics. Unlike 2D monolayer cultures, where regions of uniform cell density can be selected for imaging, single spheroid invasion assays require precise and consistent positioning of the entire spheroid and migration area within the imaging field. This adds an additional challenge, as drift or irregular positioning within the well can compromise data quality and reproducibility. Therefore, consistently centralizing spheroids within multiwell plates minimizes these challenges and improves not only imaging quality but experimental consistency, image processing pipelines and post migration analyses. Additionally, initiating spheroid cultures at uniform cell densities in these specialized spheroid culture plates promotes reproducible spheroid formation, reducing the variability in morphology, growth rate, and structural integrity10,11. In practical terms, typical gliomaspheres used in these assays range from 150&#8211;300 &#181;m in diameter, forming reproducibly under standardized cell densities. Cultures are maintained under hypoxic conditions (1%&#8211;2% O2).
The platform described here supports moderate to high throughput formats, such as 96&#8209;well plates, enabling multiple replicates per patient sample, and is scalable for drug screening or multi-condition assays12 .Optimizing spheroid positioning and culture conditions is therefore essential to enhance the robustness and clinical relevance of 3D preclinical models used to study glioma invasive potential and other dynamic cellular processes.

<table><tbody><tr><td>96 WELL BIOPRINTING KIT, CLEAR</td><td>Greiner</td><td>655840</td><td></td></tr><tr><td>Accutase</td><td>Invitrogen</td><td>00-4555-56</td><td>n/a</td></tr><tr><td>B-27 Supplement (50X), serum&amp;nbsp;free without vitamin A</td><td>Gibco</td><td>12587010</td><td>10%</td></tr><tr><td>Basic Fibroblast Growth Factor, Human&amp;nbsp;(bFGF)</td><td>PeproTech</td><td>100-18B</td><td>20&amp;nbsp;ng/ml</td></tr><tr><td>Celldiscoverer&amp;nbsp;7</td><td>Zeiss</td><td>-</td><td></td></tr><tr><td>Corning Spheroid Microplates</td><td>Corning</td><td>CLS4515</td><td></td></tr><tr><td>Corning Matrigel Matrix</td><td>Corning</td><td>354234</td><td>1:100 final dilution</td></tr><tr><td>Countess Automated Cell Counter</td><td>Invitrogen</td><td>-</td><td></td></tr><tr><td>DMEM-F12&amp;nbsp;(500ml)</td><td>Thermofischer</td><td>11320-074</td><td>n/a</td></tr><tr><td>Epidermal Growth Factor Human (EGF)</td><td>PeproTech</td><td>AF-100-15</td><td>20&amp;nbsp;ng/ml</td></tr><tr><td>FITC Mouse Anti-Human CD44</td><td>BD Pharmingen</td><td>555478</td><td>1:100</td></tr><tr><td>Greiner CELLSTAR 96 Well Polystyrene Cell Culture Microplates with F-Bottom/Chimney Well, &amp;micro;Clear, Black, TC, Sterile, With Lid</td><td>Greiner</td><td>655090</td><td></td></tr><tr><td>Heparine</td><td>Thermofischer</td><td>A16198</td><td>5&amp;micro;g/ml</td></tr><tr><td>Hoechst 33258</td><td>&amp;nbsp;Sigma Aldrich (Merck)</td><td>861405</td><td>1&amp;micro;g/ml</td></tr><tr><td>NanoShuttle-PL</td><td>Greiner</td><td>657841</td><td>1.5&amp;nbsp;&amp;micro;l/10,000 cells</td></tr><tr><td>Penicillin - Streptomycin</td><td>Merck</td><td>P4333</td><td>1%</td></tr></tbody></table>

reproducible 3d glioblastoma migration assay with magnetic nanoparticle mediated spheroid localization under hypoxic conditions

Reproducible 3D Glioblastoma Migration Assay with Magnetic Nanoparticle Mediated Spheroid Localization Under Hypoxic Conditions

We describe a reproducible 3D migration assay to model the migratory and invasive potential of patient-derived glioblastoma gliomaspheres from newly diagnosed and recurrent tumors under clinically relevant hypoxic conditions. Uniform sized gliomaspheres are transferred onto a thin layer of extracellular matrix and co-cultured with magnetic iron oxide nanoparticles, which enable their centralized localization within culture wells via placement onto a magnetic plate holder. The inclusion of magnetic iron oxide nanoparticles on seeding facilitates precise, localized imaging of individual patient derived gliomaspheres via a gentle magnetic force and optimizes automated image processing pipelines by reducing positional variability. This assay supports detailed study of glioblastoma migratory behavior in a physiologically relevant microenvironment and allows direct comparison of invasive potential and migratory behavior between newly diagnosed and recurrent patient derived gliomaspheres. The method is compatible with live cell imaging and multiplexed analysis, offering a scalable platform for preclinical investigation of glioblastoma migration, invasive potential, and therapeutic response.

Using the described 3D migration assay under hypoxic conditions, gliomaspheres from recurrent patient tumors were treated with either vehicle or Drug X, and invasion dynamics were monitored every h for 72 h. Figure 5 describes the WEKA segmentation analysis of invasion dynamics, revealing that Drug X significantly reduces the invasive potential of recurrent gliomaspheres compared to vehicle controls under clinically relevant hypoxic conditions.
To further assess the impact of Drug X on stem cell characteristics post migration, gliomaspheres were fixed and immunostained for the expression and localization of the glioblastoma stem cell marker CD44, as shown in Figure 6. This multiplexed analysis facilitates visualization of migration dynamics and stem cell marker expression, providing insights into how Drug X modulates both invasive potential and stem-like properties of recurrent glioblastoma models under clinically relevant hypoxic conditions.
<img alt="Gliomasphere migration analysis: graph, bar chart, microscopy images, drug effect on tumor cell movement." class="xfigimg" src="/files/ftp_upload/70365/70365fig5.jpg" data-altupdatedat="1782792046000" data-alt="Figure 5"> 
Figure 5: Optimized migration assay highlights the efficacy of novel Drug X in preventing recurrent gliomasphere migration. Patient derived recurrent gliomaspheres treated with vehicle or Drug X for 72 h under hypoxic conditions. Invasion dynamics were captured every h for 72 h. (A) Representative transmitted light images of invasion dynamics at 0 h and 72 h in top panel - Vehicle treated and bottom panel - Drug X treated. (B) Representative probability maps of invasion area used in the segmentation analysis. (C) Recurrent gliomaspheres treated with Drug X displayed significantly less invasive potential vs vehicle treated gliomaspheres. Quantification of the invasive area at 72 h revealed significantly decreased invasion in Drug X treated gliomaspheres, represented as total area covered at the time of fixation. Data represent invasion area over time from n=3 independent replicates. Statistical significance was assessed using an unpaired t-test; p &lt; 0.005 is indicated by **. <a href="https://www.jove.com/files/ftp_upload/70365/70365fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Fluorescence microscopy, CD44 expression, Hoechst stain, drug impact on cell morphology, diagram." class="xfigimg" src="/files/ftp_upload/70365/70365fig6.jpg" data-altupdatedat="1782792046000" data-alt="Figure 6"> 
Figure 6: Migration and stem cell marker expression following treatment. Gliomaspheres described in Figure 5 were fixed post migration and immunostained using an antibody targeting the glioblastoma stem cell marker CD44 following treatment with either vehicle or Drug X. This figure demonstrates the type of multiplexed post migration analysis that can be performed using standard immunostaining techniques. Images show CD44-FITC (green) and Hoechst (nuclei, blue). Scale bar = 200 µm. <a href="https://www.jove.com/files/ftp_upload/70365/70365fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplementary File 1: Resaving multiscene CZI files from the microscope as a single scenes as TIF files.<a href="https://www.jove.com/files/ftp_upload/70365/1._Export_Multiscene_as_Tiff_v02.ijm" target="_blank">Please click here to download this file.</a>
Supplementary File 2: Generating probability maps using a Weka model.<a href="https://www.jove.com/files/ftp_upload/70365/2._WEKA_segmentation_v02.ijm" target="_blank">Please click here to download this file.</a>
Supplementary File 3: Segmentation analysis without threshold. <a href="https://www.jove.com/files/ftp_upload/70365/3._Segmentation_analysis_v01.ijm" target="_blank">Please click here to download this file.</a>
Supplementary File 4: Segmentation analysis with threshold. <a href="https://www.jove.com/files/ftp_upload/70365/3._Segmentation_analysis_v02.ijm" target="_blank">Please click here to download this file.</a>

We present a 3D glioblastoma migration model using patient derived magnetic gliomaspheres generated from newly diagnosed and recurrent tumors. This platform allows visualization of patient specific migration dynamics and assessment of therapeutic efficacy. The assay can be multiplexed post migration to evaluate protein localization and expression patterns.

We describe a reproducible 3D migration assay to model the migratory and invasive potential of patient-derived glioblastoma gliomaspheres from newly ...

We focused on visually capturing glioblastoma tumor migration, allowing the anti-invasive properties of therapies to be reliably and reproducibly examined. The incredible heterogeneity of glioblastoma is difficult to model. This protocol captures individual glioblastoma tumor invasiveness in a reproducible manner.To begin obtain the gliomaspheres in a culture flask and keep all the reagents ready under sterile conditions. Using a 10-milliliter serological pipette and pipette aid, collect the gliomaspheres from the flask. Transfer the gliomaspheres into a 15-milliliter conical tube.Centrifuge at 300g for three minutes, and discard the supernatant without disturbing the cell pellet. Resuspend the pellet in one-milliliter Accutase at room temperature. Use a P-1000 pipette to agitate the solution every two to three minutes and incubate for a total of 10 to 15 minutes.After incubation, add an equal volume of gliomasphere media to the cell suspension to deactivate the Accutase. Centrifuge the single cell suspension at 300g for three minutes at 37 degrees Celsius. Then, resuspend the cell pellet in three to five milliliters of complete gliomasphere medium, depending on the pellet size.Note the exact volume used for subsequent cell counting. Take a 10-microliter aliquot of the single cell suspension and gently mix it with 10 microliters of trypan blue in a 1.5-milliliter microcentrifuge tube. Load 10 microliters of this mixture into an automated cell counter.Prepare a master mix of the glioma cells in the gliomasphere medium containing one microgram per milliliter propidium iodide and 0.05 microliters iron oxide nanoparticles. Seed 500 single glioma cells per well from this master mix into an ultra-low attachment 96-well spheroid microplate. Incubate the plate for 48 hours at 37 degrees Celsius, 5%carbon dioxide, and 21%oxygen in a humidified cell culture incubator to allow gliomasphere formation.To prepare the Matrigel, dilute the Matrigel extracellular matrix stock 1:10 in ice cold complete neurosphere medium after 48 hours of gliomasphere formation. Perform a second 1:10 dilution to obtain the Matrigel working solution. Keep the diluted Matrigel on ice.Pre-chill the imaging plate and store it on ice. Dispense 50 microliters of the Matrigel working solution into each well of a 96-well micro clear flat bottom plate. Incubate the plate at 37 degrees Celsius for at least one hour.Then, remove 30 microliters from each well, leaving 20 microliters of polymerized Matrigel coating the bottom of each well. Spray the magnetic plate with 70%ethanol and wipe dry under sterile conditions. Place the Matrigel-coated microplate onto a magnetic 96-well plate drive.Pipette 80 microliters of medium containing the single gliomasphere from each well. Gently dispense it onto the Matrigel-coated plate. Treat the spheroids according to the experimental design.Now, transfer the plate with the magnetic drive to the imaging system for live imaging. Configure a programmable inverted live cell microscope to acquire live cell images. Set the camera to 2x2 Binning.Gently load the multi-well plate without the magnetic drive into the microscope. Adjust the microscope stage, acceleration, and speed to 10%If applicable, select the plate material in the software so the optics are adjusted to match. Adjust the magnification to encompass the entire invasive field of view.Bring a gliomasphere into focus and select the 5X objective and 0.5X tube lens to yield 2.5X magnification. Check both the phase gradient contrast, transmitted light, and fluorescence channels. Adjust the exposure time and light intensity to avoid overexposure.Assign a position that includes the x, y, and z-coordinates. Use hardware auto-focusing to ensure consistent imaging of the same focal plane. Gliomaspheres derived from newly-diagnosed and recurrent patient tumors exhibited dual phenotypes and random cell-to-cell associations.Transmitted light images of gliomaspheres cultured for 48 hours showed a dark, speckled appearance in gliomaspheres containing iron oxide nanoparticles. Gliomaspheres cultured without nanoparticles showed random localization, whereas those cultured with nanoparticles showed consistent central localization. Representative transmitted light images showed invasion dynamics at zero hours and 72 hours in vehicle-treated gliomaspheres and Drug X-treated gliomaspheres.Quantification of the invasive area at 72 hours, measured as the total area covered at the time of fixation, revealed significantly decreased invasion in Drug X-treated gliomaspheres compared with vehicle-treated controls. This protocol enables the scalable, high-throughput analysis of glioblastoma tumor-specific migration changes in response to various conditions and our therapies. Optimizing iron oxide concentration ensures minimal use and controlled steroid positioning, greatly enhancing assay consistency, accuracy, and overall performance.Future studies can develop more representative brain ECM matrices to better mimic glioblastoma migration, and thereby improving translational relevance in in vitro systems.

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Cancer Research

Source: Guyon, J., et al. A 3D Spheroid Model for Glioblastoma. J. Vis. Exp. (2020)The video demonstrates the generation of a three-dimensional spheroid model of tumor cell invasion. Spheroids are formed from human brain tumor cells and embedded in a collagen matrix. The tumor cells at the spheroid periphery adopt an invasive phenotype, secreting proteases to degrade the collagen and advancing to invade the surrounding matrix.

Generation of a Three-Dimensional Spheroid Model of Tumor Cell Invasion

Source: Pericoli, G. et. al., Live-3D-Cell Immunocytochemistry Assays of Pediatric Diffuse Midline Glioma. J. Vis. Exp. (2021)This video demonstrates live cell imaging to observe pediatric diffuse midline glioma cell migration using immunohistochemical labeling with anti-CD44 antibodies.

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Three-Dimensional (3D) Tumor Spheroid Invasion Assay

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Laser Capture Microdissection of Glioma Subregions for Spatial and Molecular Characterization of Intratumoral Heterogeneity, Oncostreams, and Invasion

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A 3D Spheroid Model for Glioblastoma

The blood-brain barrier is a significant hurdle in the delivery of therapies for glioblastoma, a disease for which there is no cure. Here, we report an in vivo image-guided iron oxide therapeutic nano platform that can bypass this physiological barrier by virtue of size and accumulate in the tumor.

Nanoparticle Delivery of an Oligonucleotide Payload in a Glioblastoma Multiforme Animal Model

Here, we establish a metabolic profiling platform integrating three-dimensional (3D) glioma spheroids and imaging-based normalization via extracellular flux analysis. This approach enables more accurate assessment of tumor metabolic responses to drug treatments and provides insights into potential mechanisms of resistance.

Advancements in the Metabolic Profiling of Three-Dimensional Brain Tumor Spheroids for Drug Screening

Learn a reproducible 3D glioblastoma migration assay using magnetic nanoparticles for spheroid localization under hypoxic conditions. This method enables precise imaging and comparison of invasive potential between patient-derived tumors.

Reproducible 3D Glioblastoma Migration Assay with Magnetic Nanoparticle Mediated Spheroid Localization Under Hypoxic Conditions

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