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. Author manuscript; available in PMC: 2024 Jan 22.
Published in final edited form as: Methods Mol Biol. 2021;2269:37–47. doi: 10.1007/978-1-0716-1225-5_3

In Vitro Methods for the Study of Glioblastoma Stem-Like Cell Radiosensitivity

Joseph H McAbee 1,2,3, Charlotte Degorre-Kerbaul 1, Philip J Tofilon 1
PMCID: PMC10802913  NIHMSID: NIHMS1955894  PMID: 33687670

Abstract

Ionizing radiation is a critical component of glioblastoma (GBM) therapy. Recent data have implicated glioblastoma stem-like cells (GSCs) as determinants of GBM development, maintenance, and treatment response. Understanding the response of GSCs to radiation should thus provide insight into the development of improved GBM treatment strategies. Towards this end, in vitro techniques for the analysis of GSC radiosensitivity are an essential starting point. One such method, the clonogenic survival assay has been adapted to assessing the intrinsic radiosensitivity of GSCs and is described here. As an alternative method, the limiting dilution assay is presented for defining the radiosensitivity of GSC lines that do not form colonies or only grow as neurospheres. In addition to these cellular strategies, we describe γH2AX foci analysis, which provides a surrogate marker for radiosensitivity at the molecular level. Taken together, the in vitro methods presented here provide tools for defining intrinsic radiosensitivity of GSCs and for testing agents that may enhance GBM radioresponse.

Keywords: Glioblastoma, In vitro radiosensitivity, Clonogenic survival, Limiting dilution assay, γH2AX

1. Introduction

Radiotherapy remains a primary treatment modality for glioblastomas (GBMs) significantly contributing to the prolongation of patient survival [1]. However, whereas many GBMs initially respond, they essentially all recur; even in combination with surgery and chemotherapy, the median survival of patients with GBM continues to be dismal with the majority succumbing to disease within 2 years of diagnosis [2]. Although GBM cells are generally considered to be highly migratory and invasive [3], local recurrence is overwhelmingly within the initial treatment volume, which indicates that GBM cells in situ are extremely radioresistant [4]. Defining the processes and molecules responsible for this radioresistance should provide a rational basis for designing target-based strategies that enhance GBM radiosensitivity and therapeutic response. Towards this goal, a model system that accurately simulates the biology of GBMs is a critical requirement. Whereas the biology of long-established glioma cell lines has little in common with these brain tumors in situ [5], GBM stem-like cells (GSCs) are a clonogenic subpopulation thought to be critical to the development, maintenance, and treatment response of GBMs [6-8]. For in vitro experimentation, GSCs are initially isolated from human GBM surgical specimens as neurospheres and grown in neural basal medium containing epidermal growth factor and basic fibroblast growth factor, that is, stem cell growth medium. As a model system for investigating GBM radioresistance, we previously determined the in vitro radiosensitivity of GSCs using a clonogenic assay [9], the gold standard for defining intrinsic radiosensitivity.

The in vitro clonogenic survival assay measures the consequences of radiation on the proliferative potential of individual cells; the survival curves generated from this assay reflect the two principal mechanisms of radiation-induced cell death: apoptosis and mitotic catastrophe [10]. Of note, as for cells isolated from most human solid tumors, irradiation of GSCs induces a minimal level of apoptosis with the majority of cells undergoing mitotic death [9]. Critical to the clonogenic assay is the ability of cells to grow as an attached monolayer and to form distinct colonies when seeded sparsely (i.e., at clonogenic density). Initial attempts at defining GSC radiosensitivity by clonogenic analysis used culture media containing 10% FBS [8]. However, although facilitating colony formation, the presence of FBS creates a differentiation-inducing environment resulting in the loss of stem cell characteristics [5, 11], which complicates data interpretation. An alternative approach is to seed GSCs disaggregated from neurospheres onto standard tissue culture plates coated with poly-L-lysine (PLL) containing stem cell growth medium and maintained at 5% O2 [9, 12]. Under these conditions, the GSCs grow as adherent colonies and, in contrast to growth in medium containing FBS, maintain their stem-like cell characteristics [9, 11]. This method allows for the generation of radiation clonogenic survival curves that can be used to compare the radiosensitivity of GSC lines as well as to evaluate potential radiosensitizers.

However, not all GSCs are amenable to the clonogenic assay. Some GSC lines only proliferate when grown in suspension as neurospheres. Other GSCs proliferate as monolayer cultures when tissue culture plates are coated with PLL or, as an alternative substrate that enhances attachment, polyornithine/laminin but do not form the distinct colonies necessary for clonogenic survival analysis. In these situations, the limiting dilution assay (LDA) may be applicable. Similar to clonogenic assays, the LDA determines the effects of radiation on cell proliferation and/or the ability to grow into neurospheres, which can be especially applicable to GSCs. The LDA typically involves seeding a serial dilution of cells into rows of a 96-well plate (see below), before or after irradiation [13, 14]. After allowing 2–3 weeks for proliferation, the wells are scored as positive or negative and survival curves constructed (see below). While it may be possible to determine surviving fraction based on neurospheres per well, it must be cautioned that at the time of irradiation wells do not contain single cells. In contrast to the clonogenic assay, which involves irradiation of single cells as they are attached in monolayer culture, irradiation of wells containing multiple cells growing in suspension can result in aggregation, which could influence neurosphere counts and complicate data interpretation. Finally, short-term cellular assays based on cell proliferation at 2–3 days after irradiation are not appropriate for assessing radiosensitivity of cells that undergo mitotic death (e.g., those isolated from solid tumors, normal fibroblasts). Whereas this type of assay may be applicable to chemotherapy, it does not account for the transient cell cycle delay that occurs after irradiation.

In addition to the cellular assays described above, at the molecular level, analysis of γH2AX foci provides insight into GSC radiosensitivity. γH2AX foci correspond to radiation-induced DNA double-strand breaks (DSBs) and their dispersal correlates with DSB repair [15, 16]. Because DSBs are the critical lesion in radiation-induced cell death, γH2AX foci can serve as a surrogate measure of radiosensitivity [17, 18]. Moreover, given that a shared target of many radiosensitizing agents involves some aspect of the DSB repair process, quantifying γH2AX foci dispersal as a function of time after irradiation can be useful in identifying drugs that enhance radiation-induced cell death. Because foci can be evaluated in 1–2 days after irradiation and because their measure is amenable to high-throughput technology [19], γH2AX foci analysis provides an advantage as a screening approach for GSC radiosensitizers.

2. Materials

2.1. Cell Culture

  1. Stem cell medium: DMEM/F12, 2% B27 supplement without vitamin A, 50 ng/mL each of human EGF and FGF.

  2. TrypLE Express.

  3. Defined Trypsin Inhibitor.

  4. Phosphate-buffered saline (PBS).

  5. 40 μm cell strainers.

  6. Beckman coulter cell counter and isotonic diluent (or similar cell counting platform).

  7. 37 °C, 5% CO2, 5% O2 Incubator (see Note 1).

  8. Light microscope.

  9. Centrifuge.

2.2. Clonogenic Survival Assay

  1. 0.1% Poly-l-Lysine in H2O.

  2. 6-well plates.

  3. 0.5% Crystal violet solution: 2% Crystal violet diluted in methanol.

  4. Stereomicroscope.

2.3. Limiting Dilution Assay

  1. Flat bottom 96-well plates.

  2. Multichannel pipette.

  3. Light microscope.

  4. ELDA: Extreme Limiting Dilution Analysis online software (http://bioinf.wehi.edu.au/software/elda/).

2.4. γH2AX Staining

  1. Poly-l-Ornithine: 1:500 in PBS.

  2. Laminin: 1:500 in PBS.

  3. 2-Chamber slides (see Note 2).

  4. Fixative: 10% neutral buffered formalin.

  5. PBS-T: PBS with 0.5% Tween 20.

  6. Permeabilization buffer: PBS with 0.2% Triton.

  7. Blocking buffer: PBS-T, 5% Goat serum, 1% BSA.

  8. Primary antibody: anti-phospho-Histone H2AX Ser 139 (Millipore; 1:1000 concentration).

  9. Secondary antibody: Alexa Fluor 488 goat anti-mouse IgG (Molecular Probes; 1:1000 concentration; or use secondary antibody of choice).

  10. ProLong Gold Antifade with DAPI.

  11. Fluorescent microscope.

  12. Plate rocking device.

  13. Image analysis software (e.g., ImageJ).

2.5. Ionizing Radiation Source

  1. XRAD320 (Precision X-ray, Inc.) or similar source of ionizing radiation.

3. Methods

3.1. Clonogenic Survival Assay

  1. All steps should be performed under sterile conditions until the final steps of staining and analysis. Once made or opened, reagents should be stored at 4 °C unless otherwise specified by manufacturer.

  2. Add 1 mL poly-l-lysine (PLL) to each well of a 6-well plate. Rock plates gently to ensure that all well surfaces are coated. Allow poly-l-lysine to stand for 1 h to overnight in 37 °C incubator.

  3. Remove PLL from wells and wash twice with sterile PBS. PLL can be utilized up to three times before being discarded. Aspirate final PBS wash and let wells dry in 37 °C incubator for several hours. Drying overnight is preferred.

  4. As glioblastoma stem-like cells are typically maintained as neurospheres, spin down neurospheres at 138 × g for 3 min at room temperature. To limit the amount of cell damage, the level of centrifugation should be kept to a minimum, i.e., just enough to pellet the neurospheres, which will depend on the size of the spheres. Aspirate supernatant and resuspend in 1 mL TryplE express. After appropriate incubation time (30 s to 5 min) at room temperature, add 2 mL Defined Trypsin Inhibitor and 4 mL sterile PBS (see Note 3).

  5. Pipette up and down until neurospheres have been disaggregated and are no longer visible in pipette (see Note 4). Filter cell suspension through a sterile 40 μm cell strainer; ensure a single cell suspension using microscope.

  6. Spin cells down at 215 × g for 5 min at room temperature. Aspirate supernatant and resuspend in 5 mL sterile PBS. Count cells with Coulter counter or hemocytometer.

  7. Spin cells down for a final time at 215 × g for 5 min, aspirate supernatant, and resuspend in stem cell media at 1 × 105 cells/mL in a 50 mL conical tube. Perform serial 1:10 dilutions in stem cell media until desired dilution is reached.

  8. Add appropriate number of cells to PLL-coated plates with a final volume of 2 mL of stem cell media per 9.5 cm2 well (see Note 5). Incubate plates overnight at 37 °C, 5% CO2, and 5% O2 to allow cells to attach prior to radiation treatment (see Note 6).

  9. After allowing time for cell attachment and recovery, plates are irradiated using an X-ray or other ionizing radiation source. Radiation should be delivered prior to the first doubling (for GSCs this is typically 24 h), which ensures that single cells and not doublets are irradiated. The radiation dose range typically delivered to GSCs is 1–6 Gy using at least 3 doses to allow for the generation of a survival curve. After irradiation, plates are returned to a 37 °C, 5% CO2, and 5% O2 incubator and fed up to twice per week by adding 1 mL of stem cell medium (see Note 7).

  10. In general, the maximum number of GSC colonies form in 14–21 days, which should be detectable under a stereomicroscope. Gently decant media into a waste container; 0.5% crystal violet is then added by dripping stain along the wall of the well, which should prevent colony detachment. After allowing crystal violet to stain cells for up to 5 min at room temperature, rinse plates by immersion in a large bowl of water (do not expose colonies directly to running water). Properly dispose of crystal violet. At this point, colonies should be clearly visible.

  11. Count number of colonies per well with a stereomicroscope and colony counting pen and record. Colonies are defined as containing at least 25 cells. After irradiation cell death (permanent loss of proliferative activity) can occur after 1 or more divisions. For this reason and because the goal of the clonogenic assay is to define “surviving” cells, it is important to set the minimum number of cells per colony at 25, which corresponds to 4–5 divisions.

  12. Using these results, a radiation survival curve can be constructed. Towards this end, the surviving fraction (SF) for each dose of radiation is calculated by determining the plating efficiency (PE) (number of colonies divided by the number of cells seeded × 100), which is then divided by the PE determined for the untreated control sample. Survival curves are constructed by plotting the surviving fraction versus radiation dose. The control (0 Gy) SF is set at 1.0 on the y-axis with the remaining SF data points plotted on a log scale (decreasing from 1.0); the x-axis corresponds to the radiation dose and is plotted on a linear scale (a semi-log plot).

3.2. Limiting Dilution Assay (See Note 8)

  1. Under a sterile hood, collect neurospheres in suspension by centrifugation at 138 × g for 3 min at room temperature. As in the clonogenic assay, use the minimum amount of centrifugation that will pellet the neurospheres.

  2. Perform steps 4–6 in Subheading 3.1 as outlined above to obtain single cell suspension. Perform serial dilutions to obtain a suspension with 200 cells/100 μL (see Note 9).

  3. Fill every well of a 96-well plate with 100 μL of stem cell medium. It is important to avoid the production of bubbles during the entire experiment.

  4. Add 100 μL of cell dilution made above (200 cells/100 μL) to all the wells in row A for a final volume of 200 μL.

  5. With the multichannel pipette, thoroughly mix solutions in row A and remove 100 μL from row A to transfer to row B. For the purposes of this example, the highest cell concentration will be 100 cells/well (After removing 100 μL from row A, row A is left with 100 cells).

  6. Thoroughly mix solutions in row B and remove 100 μL to transfer cells to row C. Continue in a similar fashion through row H (leave 200 μL media in row H, while all other wells will have 100 μL) to obtain the example range: Row A (100 cells), B (50), C (25), D (12), E (6), F (3), G (1), H (0).

  7. With a light microscope visually verify that wells A-G contain cell solutions composed of single cells and that there are no more than three cells in row H. Once verified, return plates to 37 °C incubator with 5% O2.

  8. Twenty-four hours after seeding cells, irradiate individual plates with a range of radiation doses, typically 2–8 Gy, and return each to incubator.

  9. At 14–21 days post-irradiation, remove plates from the incubator and examine under a light microscope. Determine and record the number of positive wells (wells that contain one or more spheres of approximately 30 cells) and/or the number of spheres per well.

  10. With the recorded number of positive wells, use ELDA: Extreme Limiting Dilution Analysis online software to calculate the cancer cell initiating frequency and significance (http://bioinf.wehi.edu.au/software/elda/) [20].

3.3. γH2AX Foci

  1. Add 1 mL poly-L-ornithine (PO) to each chamber of 2-chambered slides. Rock slides gently to ensure that all chamber surfaces are coated. Allow PO to stand for at least 4 h to overnight in 37 °C incubator.

  2. Remove PO from chambers and wash twice with sterile PBS. PO can be utilized up to three times before being discarded. Aspirate final PBS wash and add laminin in PBS to chamber and incubate at 37 °C for at least 4 h to overnight.

  3. Perform steps 3–6 in Subheading 3.1 as outlined above to generate a single GSC suspension. The number of cells seeded per chamber should be sufficient to generate an evenly distributed monolayer without overcrowding. Incubate cells on chamber slides until cells reach approximately 70% confluency. If cultures become confluent, it will be difficult to accurately visualize individual cells.

  4. When cells reach appropriate density, slides can be irradiated. The dose range is typically 1–3 Gy; for dispersal studies, which correlate with repair of radiation-induced DNA double-strand breaks (DSBs), typically, slides receive 2 Gy and are fixed at 1, 6, 16, and 24 h post-irradiation (see Note 10). However, IR dose and time points can vary based on cell line and study variables.

  5. For collection, chambers are washed with PBS prior to fixing cells with 10% neutral buffered formalin for 10 min at room temperature.

  6. Remove fixative and wash cells three times with PBS. After fixation, slides can be stored in the last PBS wash at 4 °C for 7–10 days prior to staining.

  7. For staining, permeabilize cell membranes with 0.2% Triton/PBS for 10 min at room temperature. After permeabilization, rinse with PBS-T once.

  8. To prevent nonspecific binding of primary antibody, add blocking buffer for 1 h at room temperature with shaking.

  9. After blocking, add the primary antibody, anti-phospho-histone H2AX Ser 129, at 1:1000 concentration in blocking buffer. Incubate for 2 h at room temperature or overnight at 4 °C. If utilizing a small volume of buffer with antibody, place slides on a plate rocking device.

  10. When primary antibody incubation is complete, wash cells three times with PBS-T for 5–10 min per wash at room temperature.

  11. Immediately add secondary antibody, Alexa Fluor 488 goat anti-mouse IgG (or secondary of choice), at 1:1000 concentration in blocking buffer. Incubate for 2 h at room temperature with shaking.

  12. Again, wash cells three times with PBS-T for 5–10 min per wash. Remove final PBS-T wash and remove chamber from slide.

  13. Apply 1 drop of ProLong Gold Antifade with DAPI (stains nuclei) to each chamber area and cover with glass cover slip. Allow to dry for 1 h to overnight with applied weight on top of slide to remove air bubbles. Slides can be stored for several weeks at 4 °C prior to imaging.

  14. Image on fluorescent microscope at 40–63 ×.

  15. Utilize ImageJ to analyze TIF image files to count number of foci per nuclei. Alternatively, foci can be counted manually in each nuclei. Count at least 25–50 nuclei per condition and time point.

4. Notes

  1. Standard incubators use ambient oxygen levels (air) of approximately 20%. However, in the GBM in situ environment oxygen levels are generally 5% or less. We have found that culturing GSCs in an incubator that maintains an oxygen level of 3–5%, which better simulates the GBM microenvironment, enhances their stem cell-like characteristics, including their clonogenicity, as compared to cells maintained at ambient oxygen levels [12].

  2. Poly-l-ornithine/laminin coating of tissue culture plates or slides enhances attachment of GSCs and induces a proliferation pattern of an evenly distributed monolayer. This is in contrast to PLL, which also enhances attachment but results in a colony forming growth pattern when cells are seeded at a sparse (clonogenic) density. The uniform distribution of cells over the surface of the slide (the result of poly-l-ornithine/laminin coating) allows for the straightforward visualization of nuclei using standard fluorescent microscopy.

  3. Incubation times with TryplE will depend on the GSC line. To minimize cell injury, exposure to TrypIE should be kept as short as possible. In general, 30 s of TryplE exposure is sufficient for many lines when coupled with physical disaggregation techniques (see Note 4). If neurospheres remain intact after 30 s of TryplE and pipetting, neurospheres may be spun down again and incubated with fresh TryplE for a slightly longer duration.

  4. An effective method of physical disaggregation is to attach a sterile, unfiltered 200 μL pipette tip to a sterile 10 mL pipette and pipette the entire cell suspension up and down through the 200 μL tip until neurospheres are no longer visible.

  5. The number of cells plated per well depends on the PE of the GSC line. For example, if a GSC line has a PE of 0.20, then control plates seeded at 100 cells should yield approximately 20 colonies. The number of cells seeded for the irradiated plates should then be increased to account for the expected level of cell killing. That is, if 2 Gy is expected to result in an SF of 0.50, then increase the cells seeded to 200, which should yield approximately 20 colonies (Irradiated PE/Control PE = SF of 0.50).

  6. It is also possible to plate cells for clonogenic analysis after treating with radiation. In this case, individual samples (cells in monolayer or neurospheres in suspension) are irradiated at a designated dose and then disaggregated into single cell suspensions that are counted and subjected to the necessary dilutions to obtain the desired cell concentration, which are then seeded into PLL-coated plates. However, to reduce the variability that can result from the multiple individual cell counts and dilution procedures, we find that reproducibility is improved when cells are plated and then irradiated, a protocol that involves the disaggregation and counting of a single sample. Moreover, plating cells and then irradiating after a recovery period reduces the potential for the disaggregation process to artifactually influence cellular radioresponse.

  7. The clonogenic assay can be used to identify potential radiation modifying agents. This typically involves an agent delivered alone and combined with each dose of radiation. The critical calculation is to account for the cell death (decreased PE compared to control) induced by treatment with the agent alone. By normalizing for agent-induced cell killing both the radiation only and the radiation + agent survival curves start at an SF of 1.0 allowing for a direct comparison. The radiation only and radiation + agent survival curves can be compared according to a dose modifying factor (DMF). This is defined as the ratio of the radiation dose that results in a given surviving fraction (typically 0.10) to the dose of radiation in the combined treatment that results in the same surviving fraction. A DMF of greater than 1 indicates radiosensitization and less than 1 indicates radioprotection.

  8. This 96-well plate assay can be used for adherent cells that do not form colonies on standard or coated tissue culture plates, cells with a migratory phenotype that only form an evenly distributed monolayer, or cells that divide only when grown as neurospheres.

  9. The range of cell concentrations used is dependent on the frequency of initiating cells in the fraction and on the intrinsic radiosensitivity of the cells. Therefore, LDA will need to be optimized for each individual cell line. An initial LDA using a wide range of cell concentrations can be used to find the best range of cell dilutions. The range should include concentrations with 100% positive wells or sphere formation down to cell concentrations with no positive wells or sphere formation.

  10. γH2AX foci analysis can also be used to test potential radiosensitizing agents. Because DSB repair correlates with radiosensitivity, testing a drug or a genetic/epigenetic manipulation involves determining the time course of foci dispersal after irradiation. For GSCs, the radiation dose is typically 2 Gy and foci are determined at 1, 6, and 24 h post-irradiation with and without the agent. A significantly greater number of foci remaining at 24 h in the radiation + agent samples as compared to radiation only is suggestive of an inhibition of DSB repair and putatively radiosensitization. At each time point, it is important to have samples treated with agent alone to control for the possible induction of γH2AX foci by the agent. The detection of a radiosensitizing agents using γH2AX foci analysis should be verified using the clonogenic assay.

Acknowledgments

JHM is supported by the NIH OxCam and Gates Cambridge Scholarships.

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