We would like to thank Dr Vasiliki Economopoulos for her in-house script to measure the fuluorescence signal in ImageJ. We would also want to thank the CITIUS (University of Seville) and all their personnel for their support during the demonstration. This work was supported by the Spanish FEDER I + D + i-USE, US-1264152 from University of Seville, and the Ministerio de Ciencia, Innovaci&#243;n y Universidades PID2021-126090OA-I00

The four different mushroom variants described in this protocol were obtained from a commercial source (see Table of Materials).
1. Isolation of fungal &#946;-glucans
<ol>
	<li>Extraction and isolation of soluble mushroom polysaccharides 
	NOTE: Soluble mushroom polysaccharides (SMPs) were obtained according to the procedure schematically shown in Figure 1.
	<ol>
		<li>Gently rinse fresh P. ostreatus, P. djamor, H. erinaceus,&#160;and G. lucidum fruiting bodies (about 2,000 g/mushroom) in distilled water five times.</li>
		<li>Dry the fruit bodies at 50 &#177; 2 &#176;C in a conventional air-drying oven until a constant weight is reached (~24 h).</li>
		<li>Ground the dried mushrooms in a blade mill, obtaining about 200 g of powder from each mushroom.</li>
		<li>Suspend 100 g of mushroom powder (MP) (P. ostreatus, P. djamor, H. erinaceus, and G. lucidum) in 1,000 mL of H2Od. Then, autoclave at 121 &#176;C for 15 min, and finally leave at room temperature for 30 min.</li>
		<li>Centrifuge the resulting suspension at 6,000 x g for 10 min at 4 &#176;C.</li>
		<li>Dry the precipitate containing insoluble mushroom polysaccharides (IMPs) at 50 &#177; 2 &#176;C in an air-drying oven for 24 h.</li>
		<li>Discard the precipitate and keep the supernatant. Concentrate the supernatant 10 times in a rotary evaporator.</li>
		<li>Precipitate the concentrate containing SMPs with ethanol (80% final concentration) at 4 &#176;C overnight.</li>
		<li>Centrifuge the ethanol suspension at 6,000 x g for 15 min at 4 &#176;C, retain the pellet (precipitate), and discard the supernatant with a pipette.</li>
		<li>Wash the precipitate three times with 80% ethanol before dissolving it in H2Od (10% w/v). Adjust the pH to 6.5/7 and the temperature to 37 &#176;C, and treat with 2 U and 4 U of &#945;-amylase and glucoamylase, respectively, to solubilize &#945;-glucans following the manufacturer&#39;s instructions (see Table of Materials).</li>
		<li>After the treatment with &#945;-amylase/glucoamylase, adjust the pH and temperature to 8.0 and 50 &#176;C, respectively, and treat with alcalase (2.5 U/g of protein) (see Table of Materials) to solubilize the proteins. 
		NOTE: This sequential enzymatic treatment removes most &#945;-glucans and proteins that co-precipitate with &#946;-glucans in the ethanol precipitation.</li>
		<li>After hydrolysis, centrifuge the hydrolysate at 6,000 x g for 15 min at 4 &#176;C, and the clean supernatant concentrate five times in a rotary evaporator. Precipitate again with 80% ethanol.</li>
		<li>Solubilize the resulting precipitate in H2Od and dialyze in distilled water for 24 h using cellulose tubing membranes (12,000 Da cut-off membranes; see Table of Materials) to remove low molecular weight molecules. Recover the water-soluble portion and freeze-dry it to produce soluble &#946;-glucans (S&#946;Gs).</li>
	</ol>
	</li>
	<li>Sugar and protein measurement
	<ol>
		<li>Measure the total sugar content of each fraction by the phenol-sulfuric acid method, using glucose as standard8. 
		NOTE: Quantitation of &#946;-glucan content may also be done by using the &#946;-glucan assay kit (mushroom and yeast; see Table of Materials), based on enzymatic hydrolysis and the activity of oxidoreductases: namely exo-1,3-&#946;-glucanase, glucose oxidase, &#946;-glucosidase, and peroxidase, with the subsequent formation of the quinoneimine. Follow the manufacturer&#39;s instructions, with slight modifications.</li>
		<li>Use 18 MH2SO4 instead of 12 MH2SO4.</li>
		<li>Evaluate the content of the total glucans and &#945;-glucans separately.</li>
		<li>Measure the resulting &#946;-glucan values as the difference between the total glucan and &#945;-glucan (triplicate) values following the Kjeldahl protocol. In certain cases, protein content can be determined by the Lowry method, using albumin to plot the calibration curve11,12.</li>
	</ol>
	</li>
	<li>Ultraviolet absorption spectroscopy analysis
	<ol>
		<li>Obtain the S&#946;G ultraviolet (UV) spectra using a UV-visible spectrophotometer (see Table of Materials) by scanning the samples in the 200-400 nm region (Figure 2).</li>
		<li>Prepare 1.0 mg/mL of each S&#946;G in H2Od, transfer the solution to a quartz cuvette, and scan at room temperature.</li>
	</ol>
	</li>
	<li>Molecular weight distribution analysis
	<ol>
		<li>Estimate the homoegeneity of S&#946;Gs and molecular weight of polymers by size exclusion chromatography (SEC) using a high-performance liquid chromatography (HPLC) system (see Table of Materials) equipped with a refractive index detector and an ultra-hydrogel linear gel-filtration column (300 mm x 7.8 mm; Figure 3).</li>
		<li>Perform the assay at 40 &#176;C using deionized water as eluent at a flow rate of 0.5 mL/min-1 and dextrans (110, 80, and 50 kDa) as standards (see Table of Materials). Collect a 5 mL fraction.</li>
	</ol>
	</li>
	<li>Fourier-transform infrared (FTIR) analysis
	<ol>
		<li>Record the infrared spectra (Figure 4) on an FTIR spectrometer in the range of 4000-500 cm-1. The samples should be previously mixed with KBr to form films (standard FTIR procedure; see manufacturer&#39;s instructions and Table of Materials).</li>
	</ol>
	</li>
	<li>Molecular composition analysis
	<ol>
		<li>Estimate the molecular compositions of S&#946;Gs by high-performance thin-layer chromatography (HPTLC) as well as gas chromatography coupled to mass spectrometry (GC-MS), following standard procedures12.</li>
	</ol>
	</li>
</ol>
2. In vitro study of &#946;-glucan-induced microglia stimulation
<ol>
	<li>Cell culture of mouse glioblastoma and microglia cells in 8-well chamber slides 
	NOTE: This protocol is specific for GL261 (glioblastoma) and BV2 (microglia) cell lines. However, with slight modifications, these steps could potentially be used to study other cancer and immune cell lines.
	<ol>
		<li>Prepare Dulbecco&#39;s modified Eagle&#39;s medium (DMEM) complete medium modified with L-glutamin, 4.5 g/L D-glucose, and without pyruvate. Add 10% of fetal bovine serum (FBS) and 1% penicillin/streptomycin (see Table of Materials). Pre-warm the material in a water bath at 37 &#176;C for 15 min.</li>
		<li>Thaw frozen BV2 and GL261 aliquots into a water bath (37 &#176;C) for 2 min, and just before they completely thaw, carry them into a laminar flow hood and plate the cells into two different sterile T25 flasks (one for each cell line).</li>
		<li>Incubate the T25 flasks at 37 &#176;C, 5% CO2 until the culture is confluent. 
		NOTE: Depending on the freezing conditions and the time under cryopreservation, the time until confluence may vary. These cell lines usually require between 3 to 5 days to reach confluency in a T75 flask.</li>
		<li>After the BV2 cell culture becomes confluent, transfer it into 8-well chamber slides 0.6 x 106 cells/well. Keep the 8-well chamber slides in the incubator for 24 h.</li>
		<li>Once the microglia cells are plated into the 8-well chamber slides, repeat the same protocol with the GL261 cells.</li>
	</ol>
	</li>
	<li>Activation of microglia with &#946;-glucans
	<ol>
		<li>Coat the BV2 cells with four different &#946;-glucans (P. ostreatus, P. djamor, G. lucidum, and H. erinaceus) at a 0.2 mg/mL concentration for 72 h. One experimental condition must remain untreated (normal medium), acting as the control group.</li>
		<li>Collect the supernatant with a pipette after 72 h and pass the remaining volume through a 0.20 &#181;m syringe filter. Then, freeze the supernatant at -80 &#176;C for at least 24 h.</li>
	</ol>
	</li>
	<li>Treatment of GL261 with pre-activated microglia-conditioned medium
	<ol>
		<li>Once the GL261 is 80% confluent within the 8-well chamber slides, add &#946;-glucan-treated microglial medium (step 2.2.2) at a final volume concentration of 25% for 72 h (total volume: 250 &#181;l/well).</li>
		<li>Remove the medium after 72 h incubation and discard it.</li>
		<li>Wash the cells with phosphate buffer saline (PBS; pH 7.4, 0.1 M) three times for 5 minutes.</li>
		<li>Fix the cells by adding 200 &#181;L of 4% paraformaldehyde (PFA) at 4 &#176;C for 15 min. 
		NOTE: Depending on the different antibodies that might be used for immunofluorescence, fixation methods may differ from typical 4% PFA. Alcohol-based fixatives may be more efficient in preserving certain epitopes.</li>
	</ol>
	</li>
	<li>Immunofluorescence study
	<ol>
		<li>Wash the samples with PBS with triton X (PBST; 0.01%) for 10 min three times.</li>
		<li>Remove the PBST and add bovine serum albumin (BSA) blocking buffer 10% in PBST (Table 1) for 30 min at room temperature.</li>
		<li>Remove the blocking buffer and add 200 &#181;L per well of PBS containing the primary antibodies mixture (1:500 rat Ki-67 monoclonal antibody and 1:500 rabbit cleaved caspase-3 antibody; see Table of Materials). Incubate overnight at 4 &#176;C.</li>
		<li>After 24 h of incubation at 4 &#176;C, leave the samples at room temperature for 30 min.</li>
		<li>Wash the wells three times for 10 min with PBS on a shaker (low speed).</li>
		<li>Remove the PBS and replace with 200 &#181;L per well of PBS containing the mixture of secondary antibodies (1:200 donkey anti-rat IgG (H + L) highly cross-adsorbed secondary antibody, Alexa Fluor 488 and 1:200 donkey anti-rabbit IgG (H + L) highly cross-adsorbed secondary antibody, Alexa Fluor 647; see Table of Materials) for 45 min at room temperature in the dark.</li>
		<li>Wash the samples with PBS for 10 min on a multipurpose shaker.</li>
		<li>Remove the PBS and add 200 &#181;L per well of 4&#8242;,6-diamidino-2-phenylindole (DAPI) diluted in PBS (1:5,000) for 1 min.</li>
		<li>Remove DAPI (see Table of materials) and wash the cells for 5 min in PBS.</li>
		<li>Remove the well frame and add 50 &#181;L of PBS:glycerol (1:1) on each well and cover with a coverslip.</li>
		<li>Seal the slides with nail polish.</li>
		<li>Acquire images at 20x using a confocal microscope system (see Table of materials).</li>
	</ol>
	</li>
</ol>
3. Quantification and analysis of tumor cell proliferation and apoptosis
NOTE: In order to measure the potential effect of the different &#946;-glucans on tumor cell proliferation and apoptosis, an in-house script was used in ImageJ software to quantify the number of positive pixels of Ki67 (proliferation) and cCasp3 (apoptosis)13.
<ol>
	<li>Open ImageJ. Click on the Plugins button. Click on Coloc2, a plugin previously installed in the plugin folder, and finally select the image to analyze11. 
	NOTE: This plugin was available following previous contact with Dr. Vasiliki Economopoulos (veconom@uwo.ca). Instead of the script, both ImageJ and Fiji software have different tools for colocalization analysis (see Table of materials), with similar properties.</li>
	<li>Set thresholds according to the control (untreated, DMEM only) conditions. Click on the OK button. 
	NOTE: In order to avoid background and intensity discrepancies, all images must be taken under the same conditions. Preferably, imaging sessions should be performed on the same day, and microscope parameters unaltered across images.</li>
	<li>Ensure that the resulting images of the colocalized pixels and a summary window providing the percentage or raw number of pixels above the threshold pop up. Normalize the results with respect to the control (untreated) conditions. 
	NOTE: All data are given as mean &#177; SEM. Statistical analysis was performed using graphing and analysis software (see Table of Materials). A one-way ANOVA with Tukey&#39;s multiple comparison test was used. Errors are represented as standard error of the mean (s.e.m.) (*p &#60; 0.05, **p &#60; 0.01).</li>
</ol>

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There are no competing interests to declare.

This work describes the use of well-established techniques to successfully isolate, purify, and characterize the content of S&#946;Gs from four different fungi. The results showed how after hot water extraction of SMPs, obtained from P. ostreatus, P. djamor, G. lucidum, and H. erinaceus,&#160;followed by hydrolytic treatment with &#945;-amylase, glucosidase, and protease, the content of &#945;-glucan and protein were reduced, thus significantly enriching the amount of pure S&#946;Gs.</...

Despite the advent of novel achievements in the field of neuro-oncology, the life expectancy of glioblastoma patients remains meager. Gold-standard therapies against brain tumors are based on the amalgamation of surgery, radiotherapy, and chemotherapy. However, in the last decade, immunotherapy has emerged as a powerful strategy to treat different types of cancer1. Thus, the possibility of harnessing the body&#39;s immune response against tumor cells has recently become the fourth pillar of oncology.
It has long been known that one of the biggest challenges in the field is to overcome the strong immunosuppression found within the tumor microenvironment2. Particularly, in the case of glioblastoma, one of the most common and aggressive forms of brain cancer, unraveling key pathways that orchestrate such pro-tumoral scenarios and finding novel compounds that could counteract the depressing response of the immune system might pave the way for future therapies against this incurable disease.
The brain possesses its own immune system cells, and the most relevant cell type are microglia. These cells have been proven to have a rather complex behavior across different central diseases3. In the case of primary brain tumors (e.g., glioblastoma), these cells are shifted toward an anti-inflammatory phenotype that supports tumor cells to colonize the brain parenchyma3. Numerous publications have enhanced the major role of these cells during tumor progression. One of the main reasons for this is that glioma-associated microglia and infiltrated macrophages (GAMs) account for one-third of the total tumor mass, thus suggesting the unequivocal influence of their activation states during brain tumor progression4,5.
In that vein, fungal &#946;-glucans have been described as potent molecules triggering effective immune responses, including phagocytosis and pro-inflammatory factors production, leading to the elimination of pernicious agents6,7,8,9,10. Fungal &#946;-glucans have generally been studied using extracts from different mushroom parts. However, the attribution of specific effects requires its purification to avoid ambiguities and to be able to understand the mechanism of action of such molecules as immunomodulatory agents8.
In this work, soluble &#946;-glucans are purified from the fruiting body of four different mushrooms, regularly employed as edible (Pleurotus ostreatus and Pleurotus djamor) and as medicinal (Ganoderma lucidum and Hericium erinaceus) mushrooms. In particular, these four mushrooms have great use in the food and pharmaceutical industry and were produced within an environmentally friendly circular economy in a commercial enterprise (see Table of Materials).
In order to lay the foundation for the future use of fungal &#946;-glucans in brain cancer therapies, well-defined purification strategies and preclinical studies delving into their putative interaction with immune system cells are essential to evaluate their potential role as anti-tumor mediators. This work describes the numerous steps of isolation and purification needed to retrieve the soluble &#946;-glucans contained within the fruiting bodies of the selected mushroom. Once successfully purified, microglia cells are activated to enhance their inflammatory phenotype. Mouse glioblastoma cells (GL261) are coated with a different microglia-conditioned medium, previously treated with these extracts, and then its effect on tumor cells&#39; behavior is evaluated. Interestingly, pilot studies from our lab (data not shown) have uncovered how pro-inflammatory microglia may slow tumor cell migration and invasion properties not only in glioblastoma cells but also in other cancer cell lines. This multidisciplinary work may provide a useful tool for oncology researchers to test promising compounds able to boost the immune response in many different types of tumors.

<table><tbody><tr><td>8-well chamber slides</td><td>Thermo Fisher, USA</td><td>171080</td><td></td></tr><tr><td>Air-drying oven</td><td>J.P. Selecta S.A., Spain</td><td>2000210</td><td></td></tr><tr><td>Albumin</td><td>Sigma-Aldrich, St. Louis</td><td>A7030</td><td></td></tr><tr><td>Alcalase</td><td>Novozymes, Denmark</td><td>protease</td><td></td></tr><tr><td>Alexa Fluor 488</td><td>Thermofisher, USA</td><td>A32731</td><td></td></tr><tr><td>Alexa Fluor 647</td><td>Thermofisher, USA</td><td>A32728</td><td></td></tr><tr><td>Blade mill</td><td>Retsch, Germany</td><td>&amp;nbsp;SM100</td><td></td></tr><tr><td>Bovine Serum Albumin</td><td>MERK, Germany</td><td>A9418</td><td></td></tr><tr><td>Cellulose tubing membrane</td><td>Sigma-Aldrich, St. Louis</td><td>D9402</td><td></td></tr><tr><td>Centrifuge</td><td>MERK, Germany</td><td>Eppendorf, 5810R</td><td></td></tr><tr><td>Colocalisation pluggins</td><td>ImageJ</td><td></td><td>(https://imagej.net/imaging/colocalization-analysis&amp;nbsp;)</td></tr><tr><td>DAPI</td><td>MERK, Germany</td><td>28718-90-3</td><td></td></tr><tr><td>Dextrans</td><td>Pharmacosmos, Holbalk, Denmark</td><td>Dextran 410, 80, 50</td><td></td></tr><tr><td>Dulbecco&amp;acute;s modified Eagle&amp;acute;s medium, Gluta MAXTM</td><td>Gibco, Life Technologies, Carlsbad, CA, USA</td><td>10564011</td><td></td></tr><tr><td>Extenda (&amp;alpha;- Amylase/Glucoamylase)</td><td>Novozymes, Denmark</td><td></td><td></td></tr><tr><td>Fetal bovine serum</td><td>Gibco, Life Technologies, Carlsbad, CA, USA</td><td>A4736301</td><td></td></tr><tr><td>FT-IR spectromete</td><td>Bruker-Vertex, Switzerland</td><td>VERTEX 70v</td><td></td></tr><tr><td>Graphing and analysis software</td><td>GraphPad Prism (GraphPad Software, Inc.)</td><td></td><td></td></tr><tr><td>H&lt;sub&gt;2&lt;/sub&gt;SO&lt;sub&gt;4&lt;/sub&gt;</td><td></td><td></td><td></td></tr><tr><td>HPLC system</td><td>Waters Corp, Milford, MA, USA</td><td>Waters 2695 HPLC</td><td></td></tr><tr><td>Incubator</td><td>Eppedorf</td><td>Galaxy 170S</td><td></td></tr><tr><td>Mass Spectometer</td><td>Q Exactive GC, Thermo Scientific</td><td>725500</td><td></td></tr><tr><td>Paraformaldehyde</td><td>MERK, Germany</td><td>P6148</td><td></td></tr><tr><td>Penicillin/streptomycin</td><td>Sigma-Aldrich, St. Louis</td><td>P4458</td><td></td></tr><tr><td>pH meter</td><td>Crison, Barcelona, Spain</td><td>Basic 20</td><td></td></tr><tr><td>Phosphate-buffered saline</td><td>Gibco, Life Technologies, Carlsbad, CA, USA</td><td>1010-015</td><td></td></tr><tr><td>Rabbit Cleaved Caspase-3 (Asp175) Antibody</td><td>Abcam, UK</td><td>ab243998</td><td></td></tr><tr><td>Rat Ki-67 Monoclonal</td><td>Thermofisher, USA</td><td>MA5-14520</td><td></td></tr><tr><td>Rotary evaporator</td><td>B&amp;uuml;chi Ib&amp;eacute;rica S.L.U., Spain</td><td>El Rotavapor R-100</td><td></td></tr><tr><td>Ultra-hydrogel linear gel-filtration column (300 mm x 7.8 mm)</td><td>Waters Corp, Milford, MA, USA</td><td>WAT011545</td><td></td></tr><tr><td>UV-Visible spectrophotometer</td><td>Amersham Bioscience, UK</td><td>Ultrospec 2100 pro</td><td></td></tr><tr><td>VectaMount</td><td>Vector Laboratories, C.A, USA</td><td>H-5000-60</td><td></td></tr><tr><td>Water bath</td><td>J.P. Selecta S.A., Spain</td><td></td><td></td></tr><tr><td>Zeiss LSM 7 DUO Confocal Microscope System.</td><td>Zeiss, Germany</td><td></td><td></td></tr><tr><td>&amp;beta;-glucan Assay Kit</td><td>Megazyme, Bray, Co. Wicklow, Ireland</td><td>K-BGLU</td><td></td></tr><tr><td>&amp;beta;-glucans</td><td>Setas y Hongos del Sur, S.L.</td><td></td><td>Supplied the four variants of mushrooms</td></tr></tbody></table>

isolation and purification of fungal &#946;-glucan as an immunotherapy strategy for glioblastoma

One of the biggest challenges in developing effective therapies against glioblastoma is overcoming the strong immune suppression within the tumor microenvironment. Immunotherapy has emerged as an effective strategy to turn the immune system response against tumor cells. Glioma-associated macrophages and microglia (GAMs) are major drivers of such anti-inflammatory scenarios. Therefore, enhancing the anti-cancerous response in GAMs may represent a potential co-adjuvant therapy to treat glioblastoma patients. In that vein, fungal &#946;-glucan molecules have long been known as potent immune modulators. Their ability to stimulate the innate immune activity and improve treatment response has been described. Those modulating features are partly attributed to their ability to bind to pattern recognition receptors, which, interestingly, are greatly expressed in GAMs. Thus, this work is focused on the isolation, purification, and subsequent use of fungal &#946;-glucans to enhance the tumoricidal response of microglia against glioblastoma cells. The mouse glioblastoma (GL261) and microglia (BV-2) cell lines are used to test the immunomodulatory properties of four different fungal &#946;-glucans extracted from mushrooms heavily used in the current biopharmaceutical industry: Pleurotus ostreatus, Pleurotus djamor, Hericium erinaceus,&#160;and Ganoderma lucidum. To test these compounds, co-stimulation assays were performed to measure the effect of a pre-activated microglia-conditioned medium on the proliferation and apoptosis activation in glioblastoma cells.

Successful purification of &#946;-glucans 
The mass of MP, SMPs, and S&#946;Gs obtained from fruiting bodies of P. ostreatus, P. djamor, G. lucidum, and H. erinaceus following the extraction and purification process is summarised in Table 1. The basic composition (total carbohydrates, &#946;-glucans, and protein) of MP, SMPs, and S&#946;Gs obtained from the fungi is depicted in Table 2. These results show how the protocol allowed ...

Watch this Scientific Journal Video about Isolation and Purification of Fungal Î²-Glucan as an Immunotherapy Strategy for Glioblastoma at JoVE.com

Isolation and Purification of Fungal &#946;-Glucan as an Immunotherapy Strategy for Glioblastoma

The present protocol describes the purification steps and subsequent studies of four different fungal &#946;-glucans as potential immunomodulatory molecules that enhance the anti-tumoral properties of microglia against glioblastoma cells.

One of the biggest challenges in developing effective therapies against glioblastoma is overcoming the strong immune suppression within the tumor ...

isolation-purification-fungal-glucan-as-an-immunotherapy-strategy-for

Research

JoVE Journal

Neuroscience

1.9K Views.  Universidad de Sevilla. Schimmel bèta-glucanen zijn belangrijke modulatoren van het immuunsysteem. Het verbeteren van de zuiverheids- en isolatieprotocollen is essentieel om hun potentiële rol als co-adjuvantia voor kankertherapie te bevestigen. Een hoge mate van vier verschillende bèta-glucanen werd bereikt met behulp van verschillende apparaten die vaak worden aangetroffen in moleculaire biologie en proteomische laboratoria.Dit protocol richt zich op het testen van de effecten van bèta-glucanen op micro...

Video: Isolation and Purification of Fungal β-Glucan as an Immunotherapy Strategy for Glioblastoma

Coculture Assays to Study Macrophage and Microglia Stimulation of Glioblastoma Invasion

Glioblastoma multiforme (grade IV glioma) is a very aggressive human cancer with a median survival of 1 year post diagnosis. Despite the increased understanding of the molecular events that give rise to glioblastomas, this cancer still remains highly refractory to conventional treatment. Surgical resection of high grade brain tumors is rarely complete due to the highly infiltrative nature of glioblastoma cells. Therapeutic approaches which attenuate glioblastoma cell invasion therefore is an attractive option. Our laboratory and others have shown that tumor associated macrophages and microglia (resident brain macrophages) strongly stimulate glioblastoma invasion. The protocol described in this paper is used to model glioblastoma-macrophage/microglia interaction using in vitro culture assays. This approach can greatly facilitate the development and/or discovery of drugs that disrupt the communication with the macrophages that enables this malignant behavior. We have established two robust coculture invasion assays where microglia/macrophages stimulate glioma cell invasion by 5 - 10 fold. Glioblastoma cells labelled with a fluorescent marker or constitutively expressing a fluorescent protein are plated without and with macrophages/microglia on matrix-coated polycarbonate chamber inserts or embedded in a three dimensional matrix. Cell invasion is assessed by using fluorescent microscopy to image and count only invasive cells on the underside of the filter. Using these assays, several pharmacological inhibitors (JNJ-28312141, PLX3397, Gefitinib, and Semapimod), have been identified which block macrophage/microglia stimulated glioblastoma invasion.

Understanding the malignant behavior of cancer requires creating accurate models of how tumor cells interact with components of the tumor microenvironment, such as macrophages. Here we describe two methods to study glioblastoma cell interaction with tumor associated macrophages and microglia where the effect on glioblastoma invasion is assessed.

Glioblastoma multiforme (grade IV glioma) is a very aggressive human cancer with a median survival of 1 year post diagnosis. Despite the increased ...

Cancer Research

Evaluation of Biomarkers in Glioma by Immunohistochemistry on Paraffin-Embedded 3D Glioma Neurosphere Cultures

Analysis of protein expression in glioma is relevant for several aspects in the study of its pathology. Numerous proteins have been described as biomarkers with applications in diagnosis, prognosis, classification, state of tumor progression, and cell differentiation state. These analyses of biomarkers are also useful to characterize tumor neurospheres (NS) generated from glioma patients and glioma models. Tumor NS provide a valuable in vitro model to assess different features of the tumor from which they are derived and can more accurately mirror glioma biology. Here we describe a detailed method to analyze biomarkers in tumor NS using immunohistochemistry (IHC) on paraffin-embedded tumor NS.

Neurospheres grown as 3D cultures constitute a powerful tool to study glioma biology. Here we present a protocol to perform immunohistochemistry while maintaining the 3D structure of glioma neurospheres through paraffin embedding. This method enables the characterization of glioma neurosphere properties such as stemness and neural differentiation.

Analysis of protein expression in glioma is relevant for several aspects in the study of its pathology. Numerous proteins have been described as ...

Isolation and Expansion of Human Glioblastoma Multiforme Tumor Cells Using the Neurosphere Assay

Stem-like cells have been isolated in tumors such as breast, lung, colon, prostate and brain. A critical issue in all these tumors, especially in glioblastoma mutliforme (GBM), is to identify and isolate tumor initiating cell population(s) to investigate their role in tumor formation, progression, and recurrence. Understanding tumor initiating cell populations will provide clues to finding effective therapeutic approaches for these tumors. The neurosphere assay (NSA) due to its simplicity and reproducibility has been used as the method of choice for isolation and propagation of many of this tumor cells. This protocol demonstrates the neurosphere culture method to isolate and expand stem-like cells in surgically resected human GBM tumor tissue. The procedures include an initial chemical digestion and mechanical dissociation of tumor tissue, and subsequently plating the resulting single cell suspension in NSA culture. After 7-10 days, primary neurospheres of 150-200 &mu;m in diameter can be observed and are ready for further passaging and expansion.

This video protocol demonstrates the isolation and expansion of stem like cells from surgically resected human glioblastoma mutliforme (GBM) tumor tissue using the neurosphere assay culture method.

Stem-like cells have been isolated in tumors such as breast, lung, colon, prostate and brain. A critical issue in all these tumors, especially in ...

Optimization of High Grade Glioma Cell Culture from Surgical Specimens for Use in  Clinically Relevant Animal Models and 3D Immunochemistry

Glioblastomas, the most common and aggressive form of astrocytoma, are refractory to therapy, and molecularly heterogeneous. The ability to establish cell cultures that preserve the genomic profile of the parental tumors, for use in patient specific in vitro and in vivo models, has the potential to revolutionize the preclinical development of new treatments for glioblastoma tailored to the molecular characteristics of each tumor.
Starting with fresh high grade astrocytoma tumors dissociated into single cells, we use the neurosphere assay as an enrichment method for cells presenting cancer stem cell phenotype, including expression of neural stem cell markers, long term self-renewal in vitro, and the ability to form orthotopic xenograft tumors. This method has been previously proposed, and is now in use by several investigators. Based on our experience of dissociating and culturing 125 glioblastoma specimens, we arrived at the detailed protocol we present here, suitable for routine neurosphere culturing of high grade astrocytomas and large scale expansion of tumorigenic cells for preclinical studies. We report on the efficiency of successful long term cultures using this protocol&#160;and suggest affordable alternatives for culturing dissociated glioblastoma cells that fail to grow as neurospheres. We also describe in detail a protocol for preserving the neurospheres 3D architecture for immunohistochemistry. Cell cultures enriched in CSCs, capable of generating orthotopic xenograft models that preserve the molecular signatures and heterogeneity of GBMs, are becoming increasingly popular for the study of the biology of GBMs and for the improved design of preclinical testing of potential therapies.

Protocol for propagation of dissociated high grade glioma surgical specimens in serum-free neurosphere medium to select for cells with cancer stem cell phenotype. For specimens that fail to grow as neurospheres, an alternative protocol is suggested. A paraffin embedding technique for maintaining the 3D neurosphere architecture for immunocytochemistry is described.

Glioblastomas, the most common and aggressive form of astrocytoma, are refractory to therapy, and molecularly heterogeneous. The ability to establish cell ...

Medicine

Generation of CAR T Cells for Adoptive Therapy in the Context of Glioblastoma Standard of Care

Adoptive T cell immunotherapy offers a promising strategy for specifically targeting and eliminating malignant gliomas. T cells can be engineered ex vivo to express chimeric antigen receptors specific for glioma antigens (CAR T cells). The expansion and function of adoptively transferred CAR T cells can be potentiated by the lymphodepletive and tumoricidal effects of standard of care chemotherapy and radiotherapy. We describe a method for generating CAR T cells targeting EGFRvIII, a glioma-specific antigen, and evaluating their efficacy when combined with a murine model of glioblastoma standard of care. T cells are engineered by transduction with a retroviral vector containing the anti-EGFRvIII CAR gene. Tumor-bearing animals are subjected to host conditioning by a course of temozolomide and whole brain irradiation at dose regimens designed to model clinical standard of care. CAR T cells are then delivered intravenously to primed hosts. This method can be used to evaluate the antitumor efficacy of CAR T cells in the context of standard of care.

The lymphodepletive and immunomodulatory effects of chemotherapy and radiation standard of care can be leveraged to enhance the antitumor efficacy of T cell immunotherapy. We outline a method for generating EGFRvIII-specific chimeric antigen receptor (CAR) T cells and administering them in the context of glioblastoma standard of care.

Adoptive T cell immunotherapy offers a promising strategy for specifically targeting and eliminating malignant gliomas. T cells can be engineered ex vivo ...

Immunology and Infection

Isolation and Flow Cytometric Analysis of Glioma-infiltrating Peripheral Blood Mononuclear Cells

Our laboratory has recently demonstrated that natural killer (NK) cells are capable of eradicating orthotopically implanted mouse GL26 and rat CNS-1 malignant gliomas soon after intracranial engraftment if the cancer cells are rendered deficient in their expression of the &#946;-galactoside-binding lectin galectin-1 (gal-1). More recent work now shows that a population of Gr-1+/CD11b+ myeloid cells is critical to this effect. To better understand the mechanisms by which NK and myeloid cells cooperate to confer gal-1-deficient tumor rejection we have developed a comprehensive protocol for the isolation and analysis of glioma-infiltrating peripheral blood mononuclear cells (PBMC). The method is demonstrated here by comparing PBMC infiltration into the tumor microenvironment of gal-1-expressing GL26 gliomas with those rendered gal-1-deficient via shRNA knockdown. The protocol begins with a description of&#160;how to culture and prepare GL26 cells for inoculation into the syngeneic C57BL/6J mouse brain. It then explains the steps involved in the isolation and flow cytometric analysis of glioma-infiltrating PBMCs from the early brain tumor microenvironment. The method is adaptable to a number of in vivo experimental designs in which temporal data on immune infiltration into the brain is required. The method is sensitive and highly reproducible, as glioma-infiltrating PBMCs can be isolated from intracranial tumors as soon as 24 hr post-tumor engraftment with similar cell counts observed from time point matched tumors throughout independent experiments. A single experimentalist can perform the method from brain harvesting to flow cytometric analysis of glioma-infiltrating PBMCs in roughly 4-6 hr depending on the number of samples to be analyzed. Alternative glioma models and/or cell-specific detection antibodies may also be used at the experimentalists&#8217; discretion to assess the infiltration of several other immune cell types of interest without the need for alterations to the overall procedure.

Presented here is a straightforward method for the isolation and flow cytometric analysis of glioma-infiltrating peripheral blood mononuclear cells that yields time-dependent quantitative data on the number and activation status of immune cells entering the early brain tumor microenvironment.

Our laboratory has recently demonstrated that natural killer (NK) cells are capable of eradicating orthotopically implanted mouse GL26 and rat CNS-1 ...

Stereotactic Adoptive Transfer of Cytotoxic Immune Cells in Murine Models of Orthotopic Human Glioblastoma Multiforme Xenografts

Glioblastoma multiforme (GBM), the most frequent and aggressive primary brain cancer in adults, is generally associated with a poor prognosis, and scarce efficient therapies have been proposed over the last decade. Among the promising candidates for designing novel therapeutic strategies, cellular immunotherapies have been targeted to eliminate highly invasive and chemo-radioresistant tumor cells, likely involved in a rapid and fatal relapse of this cancer. Thus, administration(s) of allogeneic GBM-reactive immune cell effectors, such as human V&#978;9V&#948;2 T lymphocytes, in the vicinity of the tumor would represents a unique opportunity to deliver efficient and highly concentrated therapeutic agents directly into the site of brain malignancies. Here, we present a protocol for the preparation and the stereotaxic administration of allogeneic human lymphocytes in immunodeficient mice carrying orthotopic human primary brain tumors. This study provides a preclinical proof-of-concept for both the feasibility and the antitumor efficacy of these cellular immunotherapies that rely on stereotactic injections of allogeneic human lymphocytes within intrabrain tumor beds.

Here, we present a protocol for the preparation and the stereotaxic administration of allogeneic human lymphocytes in immunodeficient mice carrying orthotopic human primary brain tumors. This study provides a proof-of-concept for both the feasibility and the antitumor efficacy of intrabrain-delivered cellular immunotherapies.

Glioblastoma multiforme (GBM), the most frequent and aggressive primary brain cancer in adults, is generally associated with a poor prognosis, and scarce ...

Intranasal Delivery of Therapeutic Stem Cells to Glioblastoma in a Mouse Model

The intrinsic tropism towards brain malignancies renders stem cells as promising carriers of therapeutic agents against malignant tumors. The delivery of therapeutic stem cells via the intranasal route is a recently discovered alternative strategy, with strong potential for clinical translation, due to its non-invasive nature compared to intracranial implantation or delivery via systemic routes. The lack of blood brain barrier further strengthens the therapeutic potential of stem cells undergoing intranasal brain entry. This article summarizes the essential techniques utilized in our studies and outlines the basic principles of intranasal strategy for stem cell delivery using a mouse model of intracranial glioma xenografts. We demonstrate the optimized procedures that generate consistent and reproducible results with specific predetermined experimental parameters and offer guidelines for streamlined work flow that ensure efficient execution and reliable experimental outcome. The article is designed to serve as a baseline for further experimental customization based on hypothesis, stem cell types, or tumor specifics.

Stem cells are promising therapeutic carriers to treat brain tumors due to their intrinsic tumor tropism. Non-invasive intranasal stem cell delivery bypasses the blood brain barrier and demonstrates strong potential for clinical translation. This article summarizes the basic principles of intranasal stem cell delivery in a mouse model of glioma.

The intrinsic tropism towards brain malignancies renders stem cells as promising carriers of therapeutic agents against malignant tumors. The delivery of ...

Image-Guided Resection of Glioblastoma and Intracranial Implantation of Therapeutic Stem Cell-seeded Scaffolds

Glioblastoma (GBM), the most common and aggressive primary brain cancer, carries a life expectancy of 12-15 months. The short life expectancy is due in part to the inability of the current treatment, consisting of surgical resection followed by radiation and chemotherapy, to eliminate invasive tumor foci. Treatment of these foci may be improved with tumoricidal human mesenchymal stem cells (MSCs). MSCs exhibit potent tumor tropism and can be engineered to express therapeutic proteins that kill tumor cells. Advancements in preclinical models indicate that surgical resection induces premature MSC loss and reduces therapeutic efficacy. Efficacy of MSC treatment can be improved by seeding MSCs on a biodegradable poly(lactic acid) (PLA) scaffold. MSC delivery into the surgical resection cavity on a PLA scaffold restores cell retention, persistence, and tumor killing. To study the effects of MSC-seeded PLA implantation on GBM, an accurate preclinical model is needed. Here we provide a preclinical surgical protocol for image-guided tumor resection of GBM in immune-deficient mice followed by MSC-seeded scaffold implantation. MSCs are engineered with lentiviral constructs to constitutively express and secrete therapeutic TNF&#945;-related apoptosis-inducing ligand (TRAIL) as well as green fluorescent protein (GFP) to allow fluorescent tracking. Similarly, the U87 tumor cells are engineered to express mCherry and firefly luciferase, providing dual fluorescent/luminescent tracking. While currently used for investigating stem cell mediated delivery of therapeutics, this protocol could be modified to investigate the impact of surgical resection on other GBM interventions.

Tumor-seeking therapeutic mesenchymal stem cells (MSCs) show promise as a treatment for invasive glioblastoma. Optimal transplantation involves delivery of MSCs into the tumor resection cavity on scaffolds. Here, preclinical techniques to study MSC treatment of glioblastoma are provided including: image-guided tumor resection; implantation of MSC-seeded scaffolds; and postoperative therapy tracking.

Glioblastoma (GBM), the most common and aggressive primary brain cancer, carries a life expectancy of 12-15 months. The short life expectancy is due in ...

Isolating Tumor-Infiltrating Immune Cells from a Murine Brain

Source: Baker, G. J., et al., Lowenstein, P. R. <a href="https://app.jove.com/v/53676">Isolation and Flow Cytometric Analysis of Glioma-infiltrating Peripheral Blood Mononuclear Cells.</a> J. Vis. Exp. (2015)
The video demonstrates the isolation of tumor-infiltrating peripheral blood mononuclear cells from a mouse brain using mechanical dissociation and enzymatic digestion. This is followed by density gradient centrifugation to collect the immune cells for further analysis.

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
Prepare density centrifugation media ...

Isolation and Purification of Fungal β-Glucan as an Immunotherapy Strategy for Glioblastoma

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