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A Photopolymerizable Hyaluronic Acid-Collagen Model of the Invasive Glioma Microenvironment with Interstitial Flow
In This Article
Summary
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.
Abstract
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.
Introduction
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 tu....
Protocol
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's biosafety committee for guidance on the use of human cells.
1. Calculations for gel preparation
- Calculate the number of conditions needed for the experiment.
NOTE: A "condition.......
Representative Results
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 G62
Discussion
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, o.......
Acknowledgements
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. & J.H.). The GSCs used in this assay were derived by Jakub Godlewski, Ph.D. (Harvard Medical School).
....Materials
| Name | Company | Catalog Number | Comments |
| 12 Well Tissue Culture Plate, Sterile | Celltreat Scientific Products | 229112 | |
| 250 mL Filter System, PES Filter Material, 0.22 µm, 50 mm, Sterile | DOT Scientific | 667706 | |
| 385 nm, 1650 mW (Min) Mounted LED, 1700 mA | Thorlabs | M385LP1-C1 | |
| 75cm2 Tissue Culture Flask - Vent Cap, Sterile | Celltreat Scientific Products | 229341 | |
| 8.0 μm Cell Culture Plate Insert 12 mm Diameter | Millicell | PI8P01250 | |
| Absolute Ethanol, 200 proof, Molecular Biology Grade | Thermo Fisher Scientific | T038181000CS | Ethanol for flow cytometry dead cell control. |
| Astrocyte Medium (Astrofull) | ScienCell Research Laboratories | 1801 | Contains astrobasal, FBS, and penicillin/streptomycin. |
| B-27 Supplement (50x), minus vitamin A (Gibco) | Thermo Fisher Scientific | 12587010 | |
| BSA (MACS) | Miltenyi Biotec | 130-091-376 | |
| CD71 antibody (eBioscience, Invitrogen) | Fisher Scientific | 25-0719-41 | |
| Cell Counting Chambered Slides | Nexcelom Bioscience | CHT4-PD100-002 | |
| Cell Scrapers | Biologix USA | 70-1250 | |
| Cellometer K2 Fluorescent Cell Counter (Nexelcom Bioscience) | VWR | NEXCCMK2-SK150-FCS | |
| Centrifuge - Low-Speed | Eppendorf | 5702 R | Centrifuge for cell culture. |
| Clear Polystyrene 96-Well Microplates, Corning | Fisher Scientific | 07-200-108 | V-bottom plates for flow cytometry staining. |
| CO2 Incubator, 150L, Heracell 150i (Thermo Scientific) | Thermo Fisher Scientific | 50116047 | |
| Collagen I, High Concentration, Rat Tail | Corning | 354249 | |
| Collagen I, Rat Tail | Corning | 354236 | "Low" concentration for coating adherent flasks. |
| Collagenase (CAS# 9001-12-1) | United States Biological | C7511-30 | |
| Collimation Adapter for Olympus BX & IX, AR Coating: 350 - 700 nm | Thorlabs | COP1-A | |
| Cotton Swabs, Q-tips Precision Tips | Amazon | B01KCJB3R2 | |
| Dispase (CAS# 9001-92-7) | United States Biological | D3760 | |
| DMEM, high glucose (Gibco) | Thermo Fisher Scientific | 11330032 | |
| EVOS FL | Invitrogen | AMF4300 | |
| Fetal Bovine Serum (Gibco) | Thermo Fisher Scientific | 26140079 | For microglia culture. |
| Formalin solution, neutral buffered, 10% (Sigma-Aldrich) | Millipore Sigma | HT501128 | |
| Foxp3 / Transcription Factor Staining Buffer Set (eBioscience, Invitrogen) | Thermo Fisher Scientific | 00-5523-00 | |
| Glioma stem cells | n/a | n/a | Can be patient derived or commercial glioma stem cell lines. |
| Guava easyCyte HT System | Millipore Sigma | 0500-4008 | Flow cytometer. |
| HBSS (Sigma-Aldrich) | Millipore Sigma | H6648 | |
| HEPES (1 M) (Gibco) | Thermo Fisher Scientific | 15630080 | |
| High-Power 1-Channel LED Driver with Pulse Modulation, 10.0 A Max, 50.0 V Max | Thorlabs | DC2200 | Interface for UV Lamp. |
| Hoechst 33342 Solution (20 mM) (Thermo Scientific) | Thermo Fisher Scientific | 62249 | |
| Human Astrocytes | ScienCell Research Laboratories | 1800 | Primary astrocytes derived from the cerebral cortex. |
| Human EGF Recombinant Protein (Gibco) | Thermo Fisher Scientific | PHG0311 | |
| Human FGF-basic (FGF-2/bFGF) (aa 10-155) Recombinant Protein (Gibco) | Thermo Fisher Scientific | PHG0021 | |
| Immortalized Human Microglia - hTERT | Applied Biological Materials | T0251 | |
| Incu-mixer MP Heated Microplate Vortexer, 2 position | Benchmark Scientific | H6002 | |
| Ki67 REAfinity, PerCP-Vio 700 antibody | Miltenyi Biotec | 130-120-420 | |
| LED UV Curing Meter | Gigahertz-Optik | X1-RCH-116 | Optometer to measure UV light intensity. |
| Lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) | Advanced Biomatrix | 5269-100MG | Photoinitiator. |
| LIVE/DEAD Fixable Near-IR (Invitrogen) | Thermo Fisher Scientific | L24975 | |
| Methacrylated hyaluronic acid (photoHA) | Advanced Biomatrix | 5212-100MG | |
| Microcentrifuge, Sorvall ST8R (Thermo Scientific) | Fisher Scientific | 75-997-203 | Centrifuge for flow cytometry staining. |
| N-2 Supplement (100X) (Gibco) | Thermo Fisher Scientific | 17502048 | |
| Neurobasal-A Medium (Gibco) | Thermo Fisher Scientific | 10888022 | |
| PBS (10X), pH 7.4 without Ca & Mg | Quality Biological | 119-069-101 | |
| Sodium hydroxide, pellets ACS (CAS# 1310-73-2) | VWR | 97064-476 | |
| Synergy Ultrapure Water Purification System (MilliporeSigma) | Fisher Scientific | SYNS0HFUS | |
| Trypsin-EDTA (0.25%), phenol red (Gibco) | Thermo Fisher Scientific | 25200056 | |
| ViaStain AOPI Staining Solution | Nexcelom Bioscience | CS2-0106 |
References
- Ostrom, Q. T., et al. CBTRUS statistical report: Primary brain and other central nervous system tumors diagnosed in the United States in 2016-2020. Neuro Oncol. 25, 1-99 (2023).
- Stupp, R., et al.
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