We would like to thank the funding sources for this work: the National Institutes of Health National Cancer Institute (R37 CA222563 to J.M.), the Coulter Foundation (J.M.), and Virginia Tech ICTAS-CEH (J.M. &#38; J.H.). The GSCs used in this assay were derived by Jakub Godlewski, Ph.D. (Harvard Medical School).

This protocol was developed in compliance with the guidelines set forth by the Virginia Tech Institutional Biosafety Committee.
NOTE: Perform all procedures in a BSL-2 cell culture cabinet unless otherwise specified. Please consult the institution&#39;s biosafety committee for guidance on the use of human cells.
1. Calculations for gel preparation
<ol>
	<li>Calculate the number of conditions needed for the experiment. 
	NOTE: A &#34;condition&#34; is defined by the contents of the solution (e.g., cell concentrations, collagen concentrations, etc.), not externally applied variables, like the fluid pressure head used in this example.</li>
	<li>Calculate the total volume of gel needed for each condition solution. 
	NOTE: The volume required for 12-well gels that use 12 mm diameter tissue culture inserts is 100-150 &#181;L per insert. At least three technical replicates are recommended when quantifying invasion. Additionally, to account for volume loss from pipetting error, add the volume of an additional replicate to each condition solution.</li>
	<li>Calculate the&#160;total volume of gel solution required for the experiment from step 1.2.</li>
	<li>Calculate the total volume of 3 mg/mL collagen solution required for the experiment. Add at least 10% to this volume to account for volume loss when pipetting. 
	Total volume 3 mg/mL collagen = 0.4&#160;&#215; Total volume of gel solution</li>
	<li>Calculate the volumes of high-concentration rat tail collagen I, sterile 1 N NaOH, sterile 10x PBS, and sterile water for each component in the 3 mg/mL collagen solution at the given final concentrations (cf): collagen I cf = 3 mg/mL, NaOH cf = 2.3% v/v of the collagen I volume, and PBS cf =1x. Dilute the solutions in sterile ultrapure (18.2 M&#937;&#183;cm) water.</li>
	<li>Calculate the volumes of 3 mg/mL collagen solution, 1% w/v&#160;methacrylated hyaluronic acid, and the photoinitiator, 17 mg/mL lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP), and cell culture media for each of the condition solutions at the given final concentrations (cf): collagen cf = 1.2 mg/mL, hyaluronic acid cf = 0.4% w/v, LAP cf = 2% v/v of the hyaluronic acid volume. Dilute the solutions in cell culture media containing cells.</li>
	<li>Calculate the number of cells for each condition solution at the desired ratio (e.g., 100,000 GSCs per 100 uL hydrogel at a ratio of 4:1:1 GSCs:astrocytes:microglia).</li>
</ol>
2. Preparing materials
<ol>
	<li>In the week prior to the experiment, plate GSCs, astrocytes, and microglia. 
	NOTE: Plate to yield approximately double the number of cells than what is needed for gels to account for both passaging and the loss of cells during the procedure.</li>
	<li>Prepare a working solution of the photoinitiator, LAP, at 17 mg/mL in sterile 1x PBS. Protect it from light.</li>
	<li>Reconstitute the methacrylated hyaluronic acid in 1x PBS per the manufacturer&#39;s instructions. Seal the lid and rock the suspension gently for 1 h, or until dissolved, at 4 &#176;C. 
	NOTE: The methacrylated hyaluronic acid used in this protocol is stable for approximately 1 month at 4 &#176;C as specified by the manufacturer. Protect it from light.</li>
	<li>Set up a UV lamp using a collimated light-emitting diode (LED) with a nominal wavelength of 385 nm at an intensity of 50 mW/cm2 at the surface of the gel. Place the lamp in the cell culture cabinet. 
	CAUTION: UV light can damage the eyes. Wear protective eyewear, and do not look directly into the lamp. 
	NOTE: Conduct separate experiments to ensure that viability or the functional outcome being measured is not significantly impacted by 385 nm light exposure at 50 mW/cm2 over the exposure period (45 s).</li>
	<li>Prepare media (astrobasal media + 0.5% v/v N-2 + 1% v/v B-27 without vitamin A) for the experiment. 
	NOTE: Combine media components and filter through a 0.22 &#181;m pore vacuum system to ensure sterility. Avoid freeze-thaw cycles of the growth factors (N-2 and B-27) to prevent degradation.</li>
	<li>Prepare 1 N NaOH in a fume hood with the splash guard in place. 
	CAUTION: 1 N NaOH is highly corrosive. Handle with care, wear eye protection, or use a splash guard, and keep the solution under a fume hood.</li>
	<li>Prepare sterile ultrapure (18.2 M&#937;&#183;cm) water.</li>
</ol>
3. Passaging cells and labeling GSCs
<ol>
	<li>Passage astrocytes, microglia, and GSCs according to the manufacturer&#39;s instructions.</li>
	<li>Label the GSCs with Hoechst 33342 following the manufacturer&#8217;s recommendation. 
	CAUTION: Hoescht 33342 can cause serious eye irritation and can induce DNA damage. Handle with care.</li>
	<li>Centrifuge all cells at 200 x g for 5 min at RT.</li>
	<li>Gently dump the supernatant and remove excess media with a pipette.</li>
	<li>Resuspend the GSCs in 1x PBS.</li>
	<li>Centrifuge the GSCs at 200 x g for 5 min.</li>
	<li>Resuspend all cells in the media (astrobasal media + 0.5% N-2 v/v + 1% v/v B-27) for the experiment.</li>
	<li>Count all the cells.</li>
	<li>Add the absolute number of cells needed for each condition solution into one vial per condition solution. Place these at 37 &#176;C, 5% CO2 until use.</li>
</ol>
4. Preparing hydrogels
NOTE: Perform the steps after opening the methacrylated hyaluronic acid and photoinitiator in the dark until the gels have been photo-crosslinked.
<ol>
	<li>Place the 8-11 mg/mL (high concentration) collagen, 1 N NaOH, sterile ultrapure (18.2 M&#937;&#183;cm) water, sterile 10x PBS, one microcentrifuge tube (for the 3 mg/mL collagen solution), methacrylated hyaluronic acid, and the photoinitiator on ice. 
	NOTE: All materials that come into contact with the collagen solution must be kept on ice to prevent premature crosslinking.</li>
	<li>Place the tissue culture inserts in the plate and label the plate.</li>
	<li>Using the calculated values, prepare the 3 mg/mL collagen solution by combining high-concentration collagen, 1 N NaOH, ultrapure (18.2 M&#937;&#183;cm) water, and 10x PBS. Add components dropwise and keep the pipette tip submerged when mixing to prevent bubble formation. 
	NOTE: If bubbles do form, centrifugation or tapping the tube on a hard surface often removes the bubbles. Furthermore, media containing phenol red will be yellow if the pH is too acidic; more NaOH should be added in this case.</li>
	<li>Centrifuge cells at 200 x g for 5 min at RT.</li>
	<li>Gently remove excess media from the top of each cell condition solution, leaving the volume of media required for the condition. Place these solutions on ice. 
	NOTE: Do not transfer the cells out of this vial to avoid loss of cells. Instead, add all gel condition components to this vial, then mix gently and distribute the gels in the plate.</li>
	<li>Add the calculated volume of 3 mg/mL collagen solution for a final concentration of 1.2 mg/mL to each cell condition solution from step 4.5.</li>
	<li>Add the calculated volume of 1% w/v methacrylated hyaluronic acid for a final concentration of 0.4% w/v&#160;to each cell condition solution from step 4.5.</li>
	<li>Add the calculated volume of 17 mg/mL LAP to each cell condition solution from step 4.5. 
	NOTE: Add this solution just prior to distributing gels in the plate to maximize viability; LAP in liquid form is cytotoxic.</li>
	<li>One at a time, mix each condition tube and add 100-150 &#181;L to each tissue culture insert. Keep the pipette tip submerged to avoid bubble formation.</li>
	<li>Turn on the 385 nm UV lamp (set up at 50 mW/cm2) at a constant current.</li>
	<li>Photo-crosslink each gel, one at a time, by exposing each well to the UV light for 45 s each.</li>
	<li>Crosslink the collagen by placing the plate at 37 &#176;C for 30-45 min.</li>
	<li>Apply the fluid pressure head to gels using astrobasal + 0.5% N-2 v/v + 1% v/v B-27.
	<ol>
		<li>Net zero flow (static): Add 700 &#181;L of media in the well-plate and 100 &#181;L in the tissue culture insert.</li>
		<li>Net flow: Add 100 &#181;L of media in the well-plate and 700 &#181;L in the tissue culture insert. 
		NOTE: Use capillary action to add 100 &#181;L underneath the tissue culture insert; this volume will not fill the well plate.</li>
	</ol>
	</li>
	<li>Place the plate at 37 &#176;C, 5% CO2, for at least 18 h or up to 5 days. 
	NOTE: Systems can incubate for up to 5 days depending on the needs of the assay, but an 18-24 h timepoint is sufficient for initial invasion, viability, or proliferation experiments.</li>
</ol>
5. Fixing cells for invasion analysis
NOTE: Do not allow the tissue culture insert membrane to dry out, and do not apply the solutions directly to the membrane to avoid detachment of cells from the membrane.
<ol>
	<li>Aspirate media from the tissue culture inserts and well-plate.</li>
	<li>Suction the gels from the tissue culture inserts with a P1000 tip. 
	NOTE: Gels can be retained for imaging by leaving the gels in the well during this step and following the alternative fixation protocol in the note of step 5.4.</li>
	<li>Gently wash the well-plate well with 1x PBS so that each tissue culture insert membrane is covered.</li>
	<li>Add 4% v/v formalin in PBS to the well-plate so that each tissue culture insert membrane is covered and incubate for 15-20 min at RT. 
	CAUTION: Formalin is a suspected carcinogen and can cause serious eye damage. Use formalin in a fume hood with the splash guard in place. 
	NOTE: This incubation length will only fix cells on the tissue culture insert membrane. If gels are kept for imaging, fix gels for 1 h at 4 &#176;C, gently rocking, and add enough formalin to the tissue culture insert to permeate the gel. After fixing, thoroughly wash gels with PBS for at least 20 min.</li>
	<li>Wash each well with 1x PBS. Afterward, add an additional 1x PBS to each well-plate well and tissue culture insert.</li>
	<li>Use a cotton swab to remove gel fragments by wiping the upper side of each tissue culture insert membrane. Return any lost 1x PBS to each well after wiping to avoid drying out the membranes.</li>
	<li>Wash each well&#160;and tissue culture insert in 1x PBS. 
	NOTE: The plate can be sealed and placed at 4 &#176;C if needed, but image membranes as soon as possible to avoid loss of fluorescence.</li>
</ol>
6. Imaging and quantification
<ol>
	<li>On the DAPI channel, image each membrane. If possible, take a tiled fluorescence image of the entire well. If this is not possible, at 20x, image the center of the well and four regions in a cross formation that intersects the center point. 
	NOTE: Exclude the edges of the well to avoid gel fragments and do not overlap regions of interest (ROIs).</li>
	<li>Count the number of cells in each image.</li>
	<li>Calculate the percent of seeded GSCs that invaded for each well: 
	<img alt="Equation 1" src="/files/ftp_upload/66604/66604eq01.jpg" style="margin: auto;" /></li>
	<li>Calculate the mean and standard deviation of percent invasion across technical replicates.</li>
</ol>
7. Alternative endpoint: flow cytometry
NOTE: In addition to or instead of analyzing invasion, unfixed gels may be degraded&#160;and the cells harvested for flow cytometry or other endpoint analyses. An example protocol analyzing cell viability (fixable live/dead stain), proliferation (Ki67 antibody), and stemness (CD71 antibody) is provided. Other markers of interest can be substituted in this protocol after validating staining efficacy. In particular, a wide variety of markers exist that target GSCs (reviewed in25). This protocol begins after step 4.14.
<ol>
	<li>Degrade the hydrogels. 
	NOTE: A 2x concentration of collagenase and dispase solutions are prepared first with experimental media (astrobasal + 0.5% v/v N-2 + 1% v/v B-27 without vitamin A), which is then added into an equal volume of hydrogel to reach a final concentration of 1x. As different cells have different responses to enzymatic degradation, checking cell viability and yield (Figure 2) is strongly recommended. The range of the final collagenase concentration should be 0.2-0.4 mg/mL, and the range of final dispase concentration should be 0.01-0.04 mg/mL; in this example, 0.3 mg/mL of collagenase and 0.02 mg/mL of dispase is used to degrade the hydrogels.
	<ol>
		<li>Prepare 2x collagenase and dispase solutions (gel degradation media solution). Dilute stock collagenase to 0.6 mg/mL and stock dispase to 0.04 mg/mL with experimental media (astrobasal + 0.5% N-2 v/v + 1% v/v B-27 without vitamin A).</li>
		<li>Remove the gels from the tissue culture inserts and transfer them into a V-bottom 96-well plate.</li>
		<li>Add the equivalent volume of gel degradation media solution per hydrogel to each well. For example, add 100 &#181;L of gel degradation media solution to 100 &#181;L of hydrogel.</li>
		<li>Incubate the plate on a shaker at 300-500 RPM (orbit: 2 mm) at 37 &#176;C for 15-30 min. 
		NOTE: Try the minimum interval, 15 min incubation, if cells are sensitive to enzymatic degradation.</li>
		<li>Pipette each hydrogel up and down 30-50 times to break up the hydrogels. 
		NOTE: Hydrogels will be easy to pipette once they are completely degraded. If gels cannot be easily pipetted, the gels must be incubated on a shaker at 300-500 RPM (orbit: 2 mm)&#160;at 37 &#176;C for another 15 min. Then, pipette each hydrogel up and down 30-50 times to break up the gels. &#8203;Importantly, the incubation time for degrading hydrogels should not exceed 1 h; this could lead to low cell viability.</li>
		<li>Centrifuge the plate at 625 x g for 1 min at 4 &#176;C, and discard the supernatant. 
		NOTE: The supernatant may be removed by either pipetting or by quickly inverting the plate over a waste tray.</li>
		<li>Resuspend the cells with 50 &#181;L of 1x PBS and pool three technical replicates into one well to yield a single biological replicate.</li>
		<li>Centrifuge the plate at 625 x g for 1 min at 4 &#176;C, and discard the supernatant to continue onto flow cytometry.</li>
	</ol>
	</li>
	<li>Block non-specific binding.
	<ol>
		<li>Wash the cells with 200 &#181;L of 1x PBS/well, centrifuge the plate at 625 x g for 1 min at 4 &#176;C, and discard the supernatant. Perform all of the following steps on ice unless otherwise specified.</li>
		<li>Resuspend the cells with 200 &#181;L of blocking solution (1x PBS with 10% v/v FBS) and incubate the plate for 15 min at RT.</li>
		<li>Wash the cells with 200 &#181;L of 1x PBS/well, centrifuge the plate at 625 x g for 1 min at 4 &#176;C, and discard the supernatant.</li>
	</ol>
	</li>
	<li>Label cells with a live/dead stain.
	<ol>
		<li>Test the cell viability using a fixable live/dead near-infrared stain. Dilute the live/dead stain with 1x PBS to 1:1000.</li>
		<li>Resuspend the cells in one well with 200 &#181;L of 70% ethanol for a negative control.</li>
		<li>Resuspend the cells in the remaining wells with 200 &#181;L of 1x PBS.</li>
		<li>Incubate the plate for 5-10 min on ice, centrifuge the plate at 625 x g for 1 min at 4 &#176;C, and discard the supernatant.</li>
		<li>Add 50 &#181;L of live/dead solution to the respective wells and incubate the plate on ice for 15 min.</li>
		<li>Centrifuge the plate at 625 x g for 1 min at 4 &#176;C and discard the supernatant.</li>
		<li>Wash the cells twice with 200 &#181;L of flow buffer (HBSS with 2% w/v BSA) per well.</li>
		<li>Centrifuge the plate at 625 x g for 1 min at 4 &#176;C and discard the supernatant.</li>
	</ol>
	</li>
	<li>Perform surface immunostaining for cellular markers of interest (CD71 antibody).
	<ol>
		<li>Dilute the CD71 antibody with flow buffer (HBSS with 2% w/v BSA) at 1:50.</li>
		<li>Add 50 &#181;L of antibody solution to the respective wells and incubate the plate on ice for 15 min.</li>
		<li>Centrifuge the plate at 625 x g for 1 min at 4 &#176;C and discard the supernatant.</li>
		<li>Wash the cells once with 200 &#181;L of flow buffer (HBSS with 2% w/v BSA) per well.</li>
		<li>Centrifuge the plate at 625 x g for 1 min at 4 &#176;C and discard the supernatant.</li>
	</ol>
	</li>
	<li>Perform intracellular immunostaining for cellular markers of interest (Ki67 antibody).
	<ol>
		<li>Use the Foxp3 transcription factor staining buffer set to fix and permeabilize the cells. Add 100 &#181;L of Foxp3 fixation/permeabilization working solution to each well. Resuspend the cell pellets by pipetting up and down.</li>
		<li>Incubate the plate on ice or at RT for 30-60 min in the dark.</li>
		<li>Centrifuge the plate at 625 x g for 1 min at 4 &#176;C and discard the supernatant.</li>
		<li>Wash the cells twice with 200 &#181;L of 1x permeabilization buffer per well.</li>
		<li>Centrifuge the plate at 625 x g for 1 min at 4 &#176;C and discard the supernatant.</li>
		<li>Add 100 &#181;L of blocking solution (1x PBS with 2% v/v FBS) for 15 min at RT.</li>
		<li>Centrifuge the plate at 625 x g for 1 min at 4 &#176;C and discard the supernatant.</li>
		<li>Dilute the Ki67 antibody with blocking solution (1x PBS with 2% v/v FBS) at 1:25.</li>
		<li>Add 50 &#181;L of 1:25 Ki67 antibody solution to the respective wells and incubate the plate for 30 min at RT in the dark.</li>
		<li>Centrifuge the plate at 625 x g for 1 min at 4 &#176;C and discard the supernatant.</li>
		<li>Wash the cells twice with 200 &#181;L of 1x permeabilization buffer per well.</li>
		<li>Resuspend the cells with 200 &#181;L of flow buffer.</li>
	</ol>
	</li>
	<li>Process the samples using a flow cytometer. An example gating strategy is detailed in Cornelison et al. 202212.</li>
</ol>

Replicating Glioblastoma Invasion with Hyaluronan-Collagen I Hydrogel System

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The authors have no relevant conflicts of interest to disclose.

Assembling the TME model includes six fundamental steps: 1) passaging cells and separating the cells between like conditions, 2) assembling a concentrated collagen solution for all conditions, 3) combining the gel components (cells, collagen, methacrylated hyaluronic acid, and the photoinitiator) for each condition 4) plating gels, 5) crosslinking by UV exposure and heat, and 6) adding the fluid pressure head. After 18 h or more, the gels can be harvested for flow cytometry, RNA extraction, protein extraction, imaging, or other downstream applications, while the tissue culture insert membranes can be fixed to quantify invasion. Thus, besides preparing cells before the day of the experiment, the full process, including seeding the model and downstream applications, typically takes no more than 2-4 days to complete.
The TME model can be adapted to represent different microenvironments and cancers. Namely, the cell types and their ratios can be modified, or to achieve different flow rates or stiffnesses, matrix composition can be altered. Extending the time gels are photocrosslinked under the UV light or collagen crosslinked at 37 &#176;C will increase stiffness. The media used should be adapted for any new cells added to the model. Furthermore, cells can be added on top of the gel in the flow media to more accurately model cellular infiltration for immune cell models. However, cellular viability and relevant factors like maintenance of stemness or the extent of reactive gliosis should be assessed before establishing any new parameters. Reactive gliosis can be assessed by GFAP and CD68 expression as described12 while stemness can be measured using various GSC markers25, though it should be noted that there is increasing evidence that both reactive gliosis and stemness are transient states represented by a preponderance of markers; thus experimentally relevant markers should be selected26,27. In the TME model that we have described, CD68 and GFAP expression are relatively low in isolated glia, but it is often either increased or decreased in the presence of different GSC lines, and the expression of CD68 and GFAP in glia negatively correlates with CD71+ stemness in GSCs12.
Numerous models exist that recapitulate aspects of brain microenvironments and generally fall under one or more of the three categories: microtissue models that autologously secrete matrix (i.e., spheroids and organoids in liquid or solid matrices), premade matrices (scaffolds), and microfluidic devices; these were reviewed recently by Tate and Munson (2019)28. The major advantages of the model described in this paper over these other categories are that this model 1) recapitulates the brain tumor matrix, which is otherwise absent in 2D cultures or liquid cell suspensions 2) operates autonomous of tubing, pumps, and other electronics typically associated with microfluidic models, 3) can be set up with materials that do not require in-house production, unlike some scaffolds and microfluidic devices29, and 4) utilizes a brain-based, nonproprietary hyaluronic acid matrix that is definable rather than matrix extracts like Matrigel30, minimizing confounding factors. Hyaluronic acid is abundant in GBMs31 and regulates flow-enhanced glioma invasion via CD449. More generally, the influence of ECM composition, biophysical forces, and the nature of the 3D environment (e.g., hypoxia and reduced penetration of therapeutics) on proliferation, metabolism, stemness, and differentiation, response to therapeutics, and invasiveness is significant and has been extensively reviewed32,33,34,35,36.
Limitations of this model include the fact that it cannot yet replicate complex tissue architecture like vasculature or neuronal patterning. Furthermore, imaging often requires expensive imaging equipment (i.e., confocal microscopes) due to light scattering by the hydrogel. Additionally, the UV light intensity and the duration of UV exposure for crosslinking varies widely in published literature37,38,39,40 and can be adjusted to optimize cell viability and function, if needed. In particular, high-intensity UV light has been shown to reduce the cellular viability of sensitive cell types due to reactive oxygen species (ROS) production38. If initial cellular viability experiments are suboptimal, protocols can be employed to reduce the effect of ROS on viability38. However, any adjustments will change the physical properties of the gel and, potentially, the flow rate.
The TME model is most directly useful for modeling fluid flow in the invasive tumor microenvironment in glioblastoma. However, the model can be adapted to represent different microenvironments, regardless of whether a flow is included or the cancer type, insofar as cells are viable and continue to represent the physiology of the respective disease. In particular, recent studies have highlighted that neurons are a driving force of glioblastoma invasion41,42; thus, including neurons in the TME model may be of interest for studying their effect on GSC invasion but would require further model optimization beyond the scope of this protocol. Alternatively, versions of the model without GSCs but including glia and other relevant cell types like neurons are useful for modeling outcomes in non-neoplastic diseases in the brain28. Importantly, soluble candidate therapeutics can easily be tested with the model before pursuing in vivo work.

Glioblastoma is a devastating disease, marked by short survival1 that is extended only moderately by clinically-available treatments2,3. This impediment to effective therapy is largely driven by the highly infiltrative nature of chemoresistant glioma stem cells (GSCs) that are inaccessible to resection and seed recurrent tumors4. Concurrently, GSCs are highly plastic, responding to diverse stimuli in the tumor microenvironment (TME) in order to survive and invade5,6. Specifically, the densely populated tumor and leaky vasculature produce a steep pressure differential at the tumor border, increasing interstitial fluid flow (IFF) in distinct regions that correlate with GSC invasion7. This increased IFF influences8,9,10 and often enhances8,9 GSC invasion via molecular pathways that are amenable to pharmacological inhibition. However, this process is confounded by the influence of glia in the TME on glioma invasion; indeed, glia have been described to modulate glioma invasion in numerous contexts11, and preliminary evidence in our lab indicates a similar influence of glia on IFF-enhanced glioma invasion12. Thus, our lab has developed a tunable, 3D TME model that replicates these invasive tumor border regions by incorporating both IFF and glia-GSC interactions to quantify GSC invasion and other outcomes (i.e., surface or intracellular protein expression, RNA expression, etc.) as influenced by IFF, glia, and/or candidate therapeutics12(Figure 1).
The TME model is similar to other tissue culture insert-based interstitial fluid flow assays (i.e., static/flow assays) that have been widely published8,9,10,13,14,15,16,17,18. The main distinctions of this model are the incorporation of a nonproprietary and tunable collagen-hyaluronic acid matrix, inclusion of astrocytes and microglia into the matrix at ratios representative of patient TMEs12, and the system is entirely enclosed in a well-plate, lacking external tubing, reservoirs, or pumps. Hyaluronic acid is among the principal components of the brain extracellular matrix19, but it is not sufficient to permit fluid flow; thus, collagen I is included in the mixture. Crosslinking occurs in two steps: a free-radical-initiated chain growth polymerization whereby upon exposure to ultraviolet (UV) light, free radicals are produced from the photoinitiator and generate chemical crosslinks between methacrylated hyaluronic acid molecules20, and neutralization of acid-preserved collagen and exposure to heat to form collagen fibers21,22,23.
Various parameters that were optimized for this assay have been described in previously published work in thiol-modified hyaluronan-collagen hydrogels8,12. Specifically, the cells (primary human GSCs24, primary human cortical astrocytes, and&#160;immortalized human microglia) used for this assay are at least 60% viable for 3 days in gels composed of astrobasal media supplemented with GSC growth factors, 0.5% v/v N-2 and 1% v/v B-27 without vitamin A12. In cases where the gels must be degraded (e.g., for protein extraction or flow cytometry), collagenase and dispase are used and maintain sufficient viability (Figure 2).
To quantify invasion, GSCs must be fluorescently labeled before they are combined with glia in the matrix. This may be achieved by labeling with Hoechst 33342 (as described below), cell trackers12, genetic modification, or transduction. Regardless, the viability of cells labeled with new markers should be measured before incorporating them into the TME model. Alternatively, if invasion will not be quantified but the gels will be harvested for protein extractions, RNA extractions, or other outcomes, cells often do not need to be fluorescently labeled during the gel preparation process. In that case, gels can be either formalin-fixed for imaging, degraded and cells lysed for RNA extraction, or degraded using proteases for flow cytometry and subsequently lysed with protein lysis buffers for Western blots or other protein work.
Here, we present a protocol for incorporating human astrocytes, microglia, and patient-derived GSCs at a patient-defined ratio, 4:1:112, into a 1.2 mg/mL collagen I, 4 mg/mL hyaluronan matrix under static of flow conditions. The specific parameters used for an assay (i.e., cellular ratios, stiffness, cell types, etc.) can be altered to represent different contexts if viability is adequate.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/66604/66604fig01.jpg" /> 
Figure 1: Diagram of the TME model consisting of GSCs, astrocytes, and microglia in a hydrogel under a fluid pressure head. Cells are embedded in a collagen-hyaluronic acid hydrogel inside a tissue culture insert. Gravitational force drives the net downward flow. Pores in the tissue culture insert membrane allow cells invading downward to attach&#160;to the bottom surface of the tissue culture insert, and these cells can be fixed, imaged, and quantified. Created with BioRender.com. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66604/66604fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/66604/66604fig02.jpg" /> 
Figure 2: Cellular viability following hydrogel degradation. G34s (GSCs), astrocytes, and microglia were seeded in the TME model at 4:1:1 and incubated for 21 h at 37 &#176;C. Gels were removed and degraded using 0.3 mg/mL collagenase + 0.02 mg/mL dispase for a total of either 30, 45, or 60 min, pipetting to mix every 15 min (30 min condition) or at 30 min then every 15 min (45 min and 60 min conditions). After the gels were degraded, the cells were stained with acridine orange (all cells) and propidium iodide (dead cells) and counted using an automated cell counter. Viability is reported for all cells within the TME model. Cells maintained at least 70% viability over 30-60 min. Data is presented as mean &#177; SEM; n = 3 biological replicates. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66604/66604fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table><tbody><tr><td>12 Well Tissue Culture Plate, Sterile</td><td>Celltreat Scientific Products&amp;nbsp;</td><td>229112</td><td></td></tr><tr><td>250 mL Filter&amp;nbsp; System, PES Filter Material, 0.22 &amp;micro;m, 50 mm, Sterile</td><td>DOT Scientific</td><td>667706</td><td></td></tr><tr><td>385 nm, 1650 mW (Min) Mounted LED, 1700 mA&amp;nbsp;</td><td>Thorlabs&amp;nbsp;</td><td>M385LP1-C1</td><td></td></tr><tr><td>75cm&lt;sup&gt;2&lt;/sup&gt; Tissue Culture Flask - Vent Cap, Sterile</td><td>Celltreat Scientific Products</td><td>229341</td><td></td></tr><tr><td>8.0 &amp;mu;m Cell Culture Plate Insert 12 mm Diameter</td><td>Millicell</td><td>PI8P01250</td><td></td></tr><tr><td>Absolute Ethanol, 200 proof, Molecular Biology Grade</td><td>Thermo Fisher Scientific</td><td>T038181000CS</td><td>Ethanol for flow cytometry dead cell control.</td></tr><tr><td>Astrocyte Medium (Astrofull)</td><td>ScienCell Research Laboratories</td><td>1801</td><td>Contains astrobasal, FBS, and penicillin/streptomycin.&amp;nbsp;</td></tr><tr><td>B-27 Supplement (50x), minus vitamin A (Gibco)</td><td>Thermo Fisher Scientific</td><td>12587010</td><td></td></tr><tr><td>BSA (MACS)</td><td>Miltenyi Biotec&amp;nbsp;</td><td>130-091-376</td><td></td></tr><tr><td>CD71 antibody (eBioscience, Invitrogen)</td><td>Fisher Scientific&amp;nbsp;</td><td>25-0719-41</td><td></td></tr><tr><td>Cell Counting Chambered Slides</td><td>Nexcelom Bioscience</td><td>CHT4-PD100-002</td><td></td></tr><tr><td>Cell Scrapers</td><td>Biologix USA&amp;nbsp;</td><td>70-1250</td><td></td></tr><tr><td>Cellometer K2 Fluorescent Cell Counter (Nexelcom Bioscience)</td><td>VWR&amp;nbsp;</td><td>NEXCCMK2-SK150-FCS</td><td></td></tr><tr><td>Centrifuge - Low-Speed</td><td>Eppendorf&amp;nbsp;</td><td>5702 R</td><td>Centrifuge for cell culture.</td></tr><tr><td>Clear Polystyrene 96-Well Microplates, Corning</td><td>Fisher Scientific</td><td>07-200-108</td><td>V-bottom plates for flow cytometry staining.</td></tr><tr><td>CO&lt;sub&gt;2&lt;/sub&gt;&amp;nbsp;Incubator, 150L, Heracell 150i (Thermo Scientific)</td><td>Thermo Fisher Scientific</td><td>50116047</td><td></td></tr><tr><td>Collagen I, High Concentration, Rat Tail</td><td>Corning</td><td>354249</td><td></td></tr><tr><td>Collagen I, Rat Tail</td><td>Corning</td><td>354236</td><td>&quot;Low&quot; concentration for coating adherent flasks.</td></tr><tr><td>Collagenase (CAS# 9001-12-1)</td><td>United States Biological</td><td>C7511-30</td><td></td></tr><tr><td>Collimation Adapter for Olympus BX &amp;amp; IX, AR Coating: 350 - 700 nm</td><td>Thorlabs</td><td>COP1-A</td><td></td></tr><tr><td>Cotton Swabs, Q-tips Precision Tips&amp;nbsp;</td><td>Amazon</td><td>B01KCJB3R2</td><td></td></tr><tr><td>Dispase (CAS# 9001-92-7)&amp;nbsp;</td><td>United States Biological</td><td>D3760</td><td></td></tr><tr><td>DMEM, high glucose (Gibco)</td><td>Thermo Fisher Scientific</td><td>11330032</td><td></td></tr><tr><td>EVOS FL</td><td>Invitrogen</td><td>AMF4300</td><td></td></tr><tr><td>Fetal Bovine Serum (Gibco)</td><td>Thermo Fisher Scientific</td><td>26140079</td><td>For microglia culture.</td></tr><tr><td>Formalin solution, neutral buffered, 10% (Sigma-Aldrich)&amp;nbsp;</td><td>Millipore Sigma</td><td>HT501128</td><td></td></tr><tr><td>Foxp3 / Transcription Factor Staining Buffer Set (eBioscience, Invitrogen)</td><td>Thermo Fisher Scientific</td><td>00-5523-00</td><td></td></tr><tr><td>Glioma stem cells</td><td>n/a</td><td>n/a</td><td>Can be patient derived or commercial glioma stem cell lines.&amp;nbsp;</td></tr><tr><td>Guava easyCyte HT System</td><td>Millipore Sigma</td><td>0500-4008</td><td>Flow cytometer.</td></tr><tr><td>HBSS (Sigma-Aldrich)&amp;nbsp;</td><td>Millipore Sigma</td><td>H6648</td><td></td></tr><tr><td>HEPES (1 M) (Gibco)</td><td>Thermo Fisher Scientific</td><td>15630080</td><td></td></tr><tr><td>High-Power 1-Channel LED Driver with Pulse Modulation, 10.0 A Max, 50.0 V Max&amp;nbsp;</td><td>Thorlabs</td><td>DC2200</td><td>Interface for UV Lamp.</td></tr><tr><td>Hoechst 33342 Solution (20 mM) (Thermo Scientific)</td><td>Thermo Fisher Scientific</td><td>62249</td><td></td></tr><tr><td>Human Astrocytes</td><td>ScienCell Research Laboratories</td><td>1800</td><td>Primary astrocytes derived from the cerebral cortex.</td></tr><tr><td>Human EGF Recombinant Protein (Gibco)</td><td>Thermo Fisher Scientific</td><td>PHG0311</td><td></td></tr><tr><td>Human FGF-basic (FGF-2/bFGF) (aa 10-155) Recombinant Protein (Gibco)</td><td>Thermo Fisher Scientific</td><td>PHG0021</td><td></td></tr><tr><td>Immortalized Human Microglia - hTERT</td><td>Applied Biological Materials&amp;nbsp;</td><td>T0251</td><td></td></tr><tr><td>Incu-mixer MP Heated Microplate Vortexer, 2 position</td><td>Benchmark Scientific&amp;nbsp;</td><td>H6002</td><td></td></tr><tr><td>Ki67 REAfinity, PerCP-Vio 700 antibody</td><td>Miltenyi Biotec</td><td>130-120-420</td><td></td></tr><tr><td>LED UV Curing Meter</td><td>Gigahertz-Optik</td><td>X1-RCH-116</td><td>Optometer to measure UV light intensity.</td></tr><tr><td>Lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP)&amp;nbsp;</td><td>Advanced Biomatrix</td><td>5269-100MG&amp;nbsp;</td><td>Photoinitiator.</td></tr><tr><td>LIVE/DEAD Fixable Near-IR (Invitrogen)&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>L24975</td><td></td></tr><tr><td>Methacrylated hyaluronic acid (photoHA)</td><td>Advanced Biomatrix</td><td>5212-100MG</td><td></td></tr><tr><td>Microcentrifuge, Sorvall ST8R (Thermo Scientific)</td><td>Fisher Scientific</td><td>75-997-203</td><td>Centrifuge for flow cytometry staining.</td></tr><tr><td>N-2 Supplement (100X) (Gibco)</td><td>Thermo Fisher Scientific</td><td>17502048</td><td></td></tr><tr><td>Neurobasal-A Medium (Gibco)</td><td>Thermo Fisher Scientific</td><td>10888022</td><td></td></tr><tr><td>PBS (10X), pH 7.4 without Ca &amp;amp; Mg</td><td>Quality Biological</td><td>119-069-101</td><td></td></tr><tr><td>Sodium hydroxide, pellets ACS (CAS# 1310-73-2)</td><td>VWR&amp;nbsp;</td><td>97064-476</td><td></td></tr><tr><td>Trypsin-EDTA (0.25%), phenol red (Gibco)</td><td>Thermo Fisher Scientific</td><td>25200056</td><td></td></tr><tr><td>ViaStain AOPI Staining Solution</td><td>Nexcelom Bioscience</td><td>CS2-0106</td><td></td></tr></tbody></table>

a photopolymerizable hyaluronic acid-collagen model of the invasive glioma microenvironment with interstitial flow

Glioblastoma recurrence is a major hindrance to treatment success and is driven by the invasion of glioma stem cells (GSCs) into healthy tissue that are inaccessible to surgical resection and are resistant to existing chemotherapies. Tissue-level fluid movement, or interstitial fluid flow (IFF), regulates GSC invasion in a manner dependent on the tumor microenvironment (TME), highlighting the need for model systems that incorporate both IFF and the TME. We present an accessible method for replicating the invasive TME in glioblastoma: a hyaluronan-collagen I hydrogel composed of human GSCs, astrocytes, and microglia seeded in a tissue culture insert. Elevated IFF can be represented by applying a fluid pressure head to the hydrogel. Additionally, this model can be tuned to replicate inter- or intra-patient differences in cellular ratios, flow rates, or matrix stiffnesses. Invasion can be quantified, while gels can be harvested for a variety of outcomes, including GSC invasion, flow cytometry, protein or RNA extraction, or imaging.

Representative data for invasion (Figure 3), viability, and the expression of Ki67 and CD71 via flow cytometry (Figure 4) are provided for GSC lines as previously published for a thio-modified hyaluronan-collagen hydrogel12. The presence of astrocytes and microglia within the TME model has a differential effect on GSC invasion dependent on the cell line (Figure 4). Specifically, the GSCs G44 and G6224 were seeded in the TME model with and without astrocytes and microglia and exposed to a fluid pressure head (flow) or net zero flow (static).
The presence versus absence of astrocytes and microglia within the TME model was associated with a reduction in G44 invasion within the static condition (Figure 3; p &#60; 0.05). In contrast, neither flow nor the presence of astrocytes and microglia significantly affected G62 invasion. These differing effects of the tumor microenvironment and flow on&#160;invasion have been described for additional cell lines12 and should be investigated for new cell lines that are incorporated into the model system.
To analyze the viability, Ki67+ proliferation, and CD71+ stemness of GSCs in the TME model, the GSC line G3424 was seeded with fluorescent-dye labeled astrocytes and microglia, and static or flow was applied. Then, the gels were degraded to isolate the cells for flow cytometry. Cells were stained with fixable live/dead, then fixed and labeled with Ki67 or CD71 antibodies12.&#160;Figure 4A depicts the general gating protocol for live CD71+, Ki67+ G34s as differentiated from astrocytes and microglia. G34 proliferation (Ki67) was significantly increased in flow relative to static when alone in the gels (p &#60; 0.001) and in the presence versus absence of glia within the static condition (p &#60; 0.05) (Figure 4B). Though statistically nonsignificant, G34 trended toward increased CD71 expression during flow when alone in the gels; this trend was not observed in the presence of glia (Figure 4C). This method may be used to quantify other markers of interest for each cell type within the TME model.
Notably, due to the variability introduced by multiple factors that can potentially influence glioma invasion (gel stiffness, reactive gliosis, phenotypic changes due to selection and passaging, etc.), it is often helpful to report the effect size, and it is necessary to generate technical replicates and conduct a power analysis for biological replicates before assaying new cell lines. Generally, n = 3-6 biological replicates are acceptable. To minimize variability and maximize power, conditions should all be run within the same experiment, including comparisons between different GSC lines, when possible.
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/66604/66604fig03v2.jpg" /> 
Figure 3: Differential influence of the tumor microenvironment on flow-stimulated invasion across two GSC lines. Fluorescently labeled GSCs (G44 and G62) were seeded in hydrogels alone (-A/M) or with both astrocytes and microglia (+A/M). A fluid pressure head was applied to &#34;flow&#34; conditions; zero net fluid flow was applied to &#34;static&#34; conditions. Invaded GSCs were fixed on the tissue culture insert membrane, counted, and averaged between technical replicates. The average invasion was plotted for each biological replicate. G44 invasion was significantly reduced by the presence of A/M within the static condition (p &#60; 0.05). In contrast, neither flow nor the presence of A/M influenced invasion in G62. The proportion of seeded GSCs that invaded is plotted as mean &#177; SEM; n = 3 biological replicates; *p &#60; 0.05 for a matched 2-way ANOVA followed by Tukey&#39;s multiple comparisons. Data is reformatted and reprinted with permission from Cornelison et al. 202212. The statistical analysis was revised to include only the cell lines of interest. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66604/66604fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/66604/66604fig04.jpg" /> 
Figure 4: Measuring viability, proliferation (Ki67), and stemness (CD71) in GSCs within the TME model. GSCs were seeded with or without fluorescent-dye labeled astrocytes (green) and microglia (deep red) in the TME model and exposed to net zero fluid flow (static) or net flow (flow). Gels were degraded and stained with live/dead near-infrared dye and with Ki67 or CD71 antibodies for flow cytometry. Isotype controls are shown in gray. (A) Cell types were gated, and viable cells quantified via live/dead near-infrared stain. (B) Ki67-proliferation in G34s is significantly increased in flow relative to static when alone in the hydrogel (p &#60; 0.001) and in the presence of astrocytes and microglia (A/M) within the static condition (p &#60; 0.05). (C) In contrast, though statistically nonsignificant, G34 stemness, represented by CD71 expression, is maintained in the static conditions by including astrocytes and microglia in the TME model. Data is presented as mean &#177; SEM; n = 3 biological replicates. *p &#60; 0.05 and ***p &#60; 0.0001 for a two-way ANOVA followed by Tukey&#39;s multiple comparisons. The figure is reformatted and reprinted with permission from Cornelison et al. 202212; additional data is included demonstrating the static conditions in panels B and C. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66604/66604fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Author Spotlight: Patient-Informed 3D Model for Studying Glioblastoma Invasion via Interstitial Fluid Flow

We present a method for replicating the glioma tumor microenvironment at the invasive front that incorporates interstitial fluid flow. This model is a hyaluronan-collagen I hydrogel in a tissue culture insert where a fluid pressure head can be applied. Invasion can be quantified, and cells can be isolated or lysed.

A Photopolymerizable Hyaluronic Acid-Collagen Model of the Invasive Glioma Microenvironment with Interstitial Flow

Glioblastoma recurrence is a major hindrance to treatment success and is driven by the invasion of glioma stem cells (GSCs) into healthy tissue that are ...

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744 Views.  Fralin Biomedical Research Institute at Virginia Tech Carilion. We present a method for replicating the glioma tumor microenvironment at the invasive front that incorporates interstitial fluid flow. This model is a hyaluronan-collagen I hydrogel in a tissue culture insert where a fluid pressure head can be applied. Invasion can be quantified, and cells can be isolated or lysed. 

Video: Author Spotlight: Patient-Informed 3D Model for Studying Glioblastoma Invasion via Interstitial Fluid Flow

Micropatterned Surfaces to Study Hyaluronic Acid Interactions with Cancer Cells

Cancer invasion and progression involves a motile cell phenotype, which is under complex regulation by growth factors/cytokines and extracellular matrix (ECM) components within the tumor microenvironment. Hyaluronic acid (HA) is one stromal ECM component that is known to facilitate tumor progression by enhancing invasion, growth, and angiogenesis1. Interaction of HA with its cell surface receptor CD44 induces signaling events that promote tumor cell growth, survival, and migration, thereby increasing metastatic spread2-3. HA is an anionic, nonsulfated glycosaminoglycan composed of repeating units of D-glucuronic acid and D-N-acetylglucosamine. Due to the presence of carboxyl and hydroxyl groups on repeating disaccharide units, native HA is largely hydrophilic and amenable to chemical modifications that introduce sulfate groups for photoreative immobilization 4-5. Previous studies involving the immobilizations of HA onto surfaces utilize the bioresistant behavior of HA and its sulfated derivative to control cell adhesion onto surfaces6-7. In these studies cell adhesion preferentially occurs on non-HA patterned regions.
To analyze cellular interactions with exogenous HA, we have developed patterned functionalized surfaces that enable a controllable study and high-resolution visualization of cancer cell interactions with HA. We utilized microcontact printing (uCP) to define discrete patterned regions of HA on glass surfaces. A "tethering" approach that applies carbodiimide linking chemistry to immobilize HA was used 8. Glass surfaces were microcontact printed with an aminosilane and reacted with a HA solution of optimized ratios of EDC and NHS to enable HA immobilization in patterned arrays. Incorporating carbodiimide chemistry with mCP enabled the immobilization of HA to defined regions, creating surfaces suitable for in vitro applications. Both colon cancer cells and breast cancer cells implicitly interacted with the HA micropatterned surfaces. Cancer cell adhesion occurred within 24 hours with proliferation by 48 hours. Using HA micropatterned surfaces, we demonstrated that cancer cell adhesion occurs through the HA receptor CD44. Furthermore, HA patterned surfaces were compatible with scanning electron microscopy (SEM) and allowed high resolution imaging of cancer cell adhesive protrusions and spreading on HA patterns to analyze cancer cell motility on exogenous HA.

A novel approach that allows the high-resolution analysis of cancer cell interactions with exogenous hyaluronic acid (HA) is described. Patterned surfaces are fabricated by combining carbodiimide chemistry and microcontact printing.

Cancer invasion and progression involves a motile cell phenotype, which is under complex regulation by growth factors/cytokines and extracellular matrix ...

Microfluidic Device for Recreating a Tumor Microenvironment in Vitro

We have developed a microfluidic device that mimics the delivery and systemic clearance of drugs to heterogeneous three-dimensional tumor tissues in vitro. Nutrients delivered by vasculature fail to reach all parts of tumors, giving rise to heterogeneous microenvironments consisting of viable, quiescent and necrotic cell types. Many cancer drugs fail to effectively penetrate and treat all types of cells because of this heterogeneity. Monolayers of cancer cells do not mimic this heterogeneity, making it difficult to test cancer drugs with a suitable in vitro model. Our microfluidic devices were fabricated out of PDMS using soft lithography. Multicellular tumor spheroids, formed by the hanging drop method, were inserted and constrained into rectangular chambers on the device and maintained with continuous medium perfusion on one side. The rectangular shape of chambers on the device created linear gradients within tissue. Fluorescent stains were used to quantify the variability in apoptosis within tissue. Tumors on the device were treated with the fluorescent chemotherapeutic drug doxorubicin, time-lapse microscopy was used to monitor its diffusion into tissue, and the effective diffusion coefficient was estimated. The hanging drop method allowed quick formation of uniform spheroids from several cancer cell lines. The device enabled growth of spheroids for up to 3 days. Cells in proximity of flowing medium were minimally apoptotic and those far from the channel were more apoptotic, thereby accurately mimicking regions in tumors adjacent to blood vessels. The estimated value of the doxorubicin diffusion coefficient agreed with a previously reported value in human breast cancer. Because the penetration and retention of drugs in solid tumors affects their efficacy, we believe that this device is an important tool in understanding the behavior of drugs, and developing new cancer therapeutics.

Microfluidic Device for Recreating a Tumor Microenvironment in Vitro

We present the procedure for fabrication and operation of a microfluidic device that recreates heterogeneous tumor microenvironments in vitro. The variability in apoptosis within tumor tissue was quantified using fluorescent stains and the effective diffusion coefficient of the chemotherapeutic drug doxorubicin into tumor tissue was evaluated.

We have developed a microfluidic device that mimics the delivery and systemic clearance of drugs to heterogeneous three-dimensional tumor tissues in ...

Three-dimensional Cell Culture Model for Measuring the Effects of Interstitial Fluid Flow on Tumor Cell Invasion

The growth and progression of most solid tumors depend on the initial transformation of the cancer cells and their response to stroma-associated signaling in the tumor microenvironment 1. Previously, research on the tumor microenvironment has focused primarily on tumor-stromal interactions 1-2. However, the tumor microenvironment also includes a variety of biophysical forces, whose effects remain poorly understood. These forces are biomechanical consequences of tumor growth that lead to changes in gene expression, cell division, differentiation and invasion3. Matrix density 4, stiffness 5-6, and structure 6-7, interstitial fluid pressure 8, and interstitial fluid flow 8 are all altered during cancer progression.
Interstitial fluid flow in particular is higher in tumors compared to normal tissues 8-10. The estimated interstitial fluid flow velocities were measured and found to be in the range of 0.1-3 &mu;m s-1, depending on tumor size and differentiation 9, 11. This is due to elevated interstitial fluid pressure caused by tumor-induced angiogenesis and increased vascular permeability 12. Interstitial fluid flow has been shown to increase invasion of cancer cells 13-14, vascular fibroblasts and smooth muscle cells 15. This invasion may be due to autologous chemotactic gradients created around cells in 3-D 16 or increased matrix metalloproteinase (MMP) expression 15, chemokine secretion and cell adhesion molecule expression 17. However, the mechanism by which cells sense fluid flow is not well understood. In addition to altering tumor cell behavior, interstitial fluid flow modulates the activity of other cells in the tumor microenvironment. It is associated with (a) driving differentiation of fibroblasts into tumor-promoting myofibroblasts 18, (b) transporting of antigens and other soluble factors to lymph nodes 19, and (c) modulating lymphatic endothelial cell morphogenesis 20.
The technique presented here imposes interstitial fluid flow on cells in vitro and quantifies its effects on invasion (Figure 1). This method has been published in multiple studies to measure the effects of fluid flow on stromal and cancer cell invasion 13-15, 17. By changing the matrix composition, cell type, and cell concentration, this method can be applied to other diseases and physiological systems to study the effects of interstitial flow on cellular processes such as invasion, differentiation, proliferation, and gene expression.

Interstitial fluid flow is elevated in solid tumors and can modulate tumor cell invasion. Here we describe a technique to apply interstitial fluid flow to cells embedded in a matrix and then measure its effects on cell invasion. This technique can be easily adapted to study other systems.

The growth and progression of most solid tumors depend on the initial transformation of the cancer cells and their response to stroma-associated signaling ...

Co-culture of Glioblastoma Stem-like Cells on Patterned Neurons to Study Migration and Cellular Interactions

Glioblastomas (GBMs), grade IV malignant gliomas, are one of the deadliest types of human cancer because of their aggressive characteristics. Despite significant advances in the genetics of these tumors, how GBM cells invade the healthy brain parenchyma is not well understood. Notably, it has been shown that GBM cells invade the peritumoral space via different routes; the main interest of this paper is the route along white matter tracts (WMTs). The interactions of tumor cells with the peritumoral nervous cell components are not well characterized. Herein, a method has been described that evaluates the impact of neurons on GBM cell invasion. This paper presents an advanced co-culture in vitro assay that mimics WMT invasion by analyzing the migration of GBM stem-like cells on neurons. The behavior of GBM cells in the presence of neurons is monitored by using an automated tracking procedure with open-source and free-access software. This method is useful for many applications, in particular, for functional and mechanistic studies as well as for analyzing the effects of pharmacological agents that can block GBM cell migration on neurons.

Here, we present an easy-to-use co-culture assay to analyze glioblastoma (GBM) migration on patterned neurons. We developed a macro in FiJi software for easy quantification of GBM cell migration on neurons, and observed that neurons modify GBM cell invasive capacity.

Glioblastomas (GBMs), grade IV malignant gliomas, are one of the deadliest types of human cancer because of their aggressive characteristics. Despite ...

Cancer Research

Glioblastoma Relapse Post-Resection Model for Therapeutic Hydrogel Investigations

Tumor recurrence is an important factor indicative of a poor prognosis in glioblastoma (GBM). Many studies are attempting to identify effective therapeutic strategies to prevent the recurrence of GBM after surgery. Bioresponsive therapeutic hydrogels capable of sustaining locally released drugs are frequently used for the local treatment of GBM after surgery. However, research is limited due to the lack of a suitable GBM relapse post-resection model. Here, a GBM relapse post-resection model was developed and applied in therapeutic hydrogel investigations. This model was constructed based on the orthotopic intracranial GBM model, which is widely used in studies on GBM. Subtotal resection was performed on the orthotopic intracranial GBM model mouse to mimic the clinical treatment. The residual tumor was used to indicate the size of the tumor growth. This model is easy to build, can better mimic the situation of GBM surgical resection, and can be applied in various studies on the local treatment of GBM relapse post-resection. As a result, the GBM relapse post-resection model provides a unique GBM recurrence model for effective local treatment studies of relapse post-resection.

The present protocol establishes a glioblastoma (GBM) relapse post-resection model using microscopy to investigate the therapeutic effect of an injectable, bioresponsive hydrogel in vivo.

Tumor recurrence is an important factor indicative of a poor prognosis in glioblastoma (GBM). Many studies are attempting to identify effective ...

Hyaluronic-Acid Based Hydrogels for 3-Dimensional Culture of Patient-Derived Glioblastoma Cells

Glioblastoma (GBM) is the most common, yet most lethal, central nervous system cancer. In recent years, many studies have focused on how the extracellular matrix (ECM) of the unique brain environment, such as hyaluronic acid (HA), facilitates GBM progression and invasion. However, most in vitro culture models include GBM cells outside of the context of an ECM. Murine xenografts of GBM cells are used commonly as well. However, in vivo models make it difficult to isolate the contributions of individual features of the complex tumor microenvironment to tumor behavior. Here, we describe an HA hydrogel-based, three-dimensional (3D) culture platform that allows researchers to independently alter HA concentration and stiffness. High molecular weight HA and polyethylene glycol (PEG) comprise hydrogels, which are crosslinked via Michael-type addition in the presence of live cells. 3D hydrogel cultures of patient-derived GBM cells exhibit viability and proliferation rates as good as, or better than, when cultured as standard gliomaspheres. The hydrogel system also enables incorporation of ECM-mimetic peptides to isolate effects of specific cell-ECM interactions. Hydrogels are optically transparent so that live cells can be imaged in 3D culture. Finally, HA hydrogel cultures are compatible with standard techniques for molecular and cellular analyses, including PCR, Western blotting and cryosectioning followed by immunofluorescence staining.

Here, we present a protocol for three-dimensional culture of patient-derived glioblastoma cells within orthogonally tunable biomaterials designed to mimic the brain matrix. This approach provides an in vitro, experimental platform that maintains many characteristics of in vivo glioblastoma cells typically lost in culture.

Glioblastoma (GBM) is the most common, yet most lethal, central nervous system cancer. In recent years, many studies have focused on how the extracellular ...

Hydrogel Arrays Enable Increased Throughput for Screening Effects of Matrix Components and Therapeutics in 3D Tumor Models

Cell-matrix interactions mediate complex physiological processes through biochemical, mechanical, and geometrical cues, influencing pathological changes and therapeutic responses. Accounting for matrix effects earlier in the drug development pipeline is expected to increase the likelihood of clinical success of novel therapeutics. Biomaterial-based strategies recapitulating specific tissue microenvironments in 3D cell culture exist but integrating these with the 2D culture methods primarily used for drug screening has been challenging. Thus, the protocol presented here details the development of methods for 3D culture within miniaturized biomaterial matrices in a multi-well plate format to facilitate integration with existing drug screening pipelines and conventional assays for cell viability. Since the matrix features critical for preserving clinically relevant phenotypes in cultured cells are expected to be highly tissue- and disease-specific, combinatorial screening of matrix parameters will be necessary to identify appropriate conditions for specific applications. The methods described here use a miniaturized culture format to assess cancer cell responses to orthogonal variation of matrix mechanics and ligand presentation. Specifically, this study demonstrates the use of this platform to investigate the effects of matrix parameters on the responses of patient-derived glioblastoma (GBM) cells to chemotherapy.

The present protocol describes an experimental platform to assess the effects of mechanical and biochemical cues on chemotherapeutic responses of patient-derived glioblastoma cells in 3D matrix-mimetic cultures using a custom-made UV illumination device facilitating high-throughput photocrosslinking of hydrogels with tunable mechanical features.

Cell-matrix interactions mediate complex physiological processes through biochemical, mechanical, and geometrical cues, influencing pathological changes ...

Fabrication Followed by 3D Encapsulation of Tumor Spheroids in PEG Hydrogels

Tumor Spheroid Fabrication and Encapsulation in Polyethylene Glycol Hydrogels for Studying Spheroid-Matrix Interactions

Three-dimensional (3D) encapsulation of spheroids is crucial to adequately replicate the tumor microenvironment for optimal cell growth. Here, we designed an in vitro 3D glioblastoma model for spheroid encapsulation to mimic the tumor extracellular microenvironment. First, we formed square pyramidal microwell molds using polydimethylsiloxane. These microwell molds were then used to fabricate tumor spheroids with tightly controlled sizes from 50-500 &#956;m. Once spheroids were formed, they were harvested and encapsulated in polyethylene glycol (PEG)-based hydrogels. PEG hydrogels are a versatile platform for spheroid encapsulation, as hydrogel properties such as stiffness, degradability, and cell adhesiveness can be tuned independently. Here, we used a representative soft (~8 kPa) hydrogel to encapsulate glioblastoma spheroids. Finally, a method to stain and image spheroids was developed to obtain high-quality images via confocal microscopy. Due to the dense spheroid core and relatively sparse periphery, imaging can be difficult, but using a clearing solution and confocal optical sectioning helps alleviate these imaging difficulties. In summary, we show a method to fabricate uniform spheroids, encapsulate them in PEG hydrogels and perform confocal microscopy on the encapsulated spheroids to study spheroid growth and various cell-matrix interactions.

Author Spotlight: In Vitro Hydrogel Model for Glioblastoma Microenvironment Study

Here, we present a protocol that enables fast, robust, and cheap fabrication of tumor spheroids followed by hydrogel encapsulation. It is widely applicable as it does not require specialized equipment. It would be particularly useful for exploring spheroid-matrix interactions and building in vitro tissue physiology or pathology models.

Three-dimensional (3D) encapsulation of spheroids is crucial to adequately replicate the tumor microenvironment for optimal cell growth.  Here, we ...

Matrix-Based Tumor Invasion Model Using Microglia Glioblastoma Co-Culture

Source: Coniglio, S., et al.&#160;<a href="https://app-jove-com.remotexs.ntu.edu.sg/v/53990">Coculture assays to study macrophage and microglia stimulation of glioblastoma invasion.</a> J. Vis. Exp. (2016)
In this video, glioblastoma and microglia cells are mixed with a polymer matrix solution and transferred to a porous chamber insert where a gel forms, embedding the cells. Microglia stimulate the migration of glioblastoma cells through the porous insert, mimicking tumor cell invasion into surrounding tissue, Cells are then fixed and prepared for further analysis.

Source: Coniglio, S., et al.&#160;Coculture assays to study macrophage and microglia stimulation of glioblastoma invasion. J. Vis. Exp. (2016)
In this ...

JoVE Encyclopedia of Experiments

Neuroscience

Neuropathology

Tumors

A Hyaluronic Acid-Based Hydrogel for 3-Dimensional Culture of Glioblastoma Cells

Source: Xiao, W.,&#160;et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/58176">Hyaluronic-Acid Based Hydrogels for 3-Dimensional Culture of Patient-Derived Glioblastoma Cells.</a> J. Vis. Exp. (2018).
This video demonstrates how gliomaspheres, derived from brain tumors, are separated into single cells and encapsulated in a hyaluronic acid-based hydrogel. This process promotes cell-cell interactions, forming a structured, three-dimensional glioblastoma model.

Hydrożel na bazie kwasu hialuronowego do trójwymiarowej hodowli komórek glejaka

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Thiolation of Hyaluronic Acid
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Hydrożel na bazie kwasu hialuronowego do trójwymiarowej hodowli komórek glejaka