This work was supported by internal funds of the Department of Neurosurgery, Brown University to N.T.

The protocols for collection, isolation, and propagation of patient-derived human glioma cells were approved by the IRB committee of Rhode Island Hospital. All animals were maintained according to the NIH Guide for the Care and Use of Laboratory Animals. All animal use protocols were approved by the Institutional Animal Care and Use Committee of Rhode Island Hospital.
1. Media and buffer preparations
<ol>
	<li>Prepare 50 mL of Neurosphere Media: 1x Neuronal basal medium w/o vitamin A, 1x serum free supplement w/o vitamin A, 2 mM L-glutamine, 20 ng/mL epidermal growth factor (EGF), 20 ng/mL basic-fibroblast growth factor (FGF), 2 &#181;g/mL heparin, and 1x antibiotic-antimycotic (anti-anti).</li>
	<li>Prepare 50 mL of supplemented Neuronal Basal Medium: 1x Neuronal basal medium, 1x serum free supplement, 4 g/L D-glucose, 2 mM L-glutamine, and 50 ng/mL nerve growth factor (NGF).</li>
	<li>Prepare 500 mL of N2B2 Medium: 1:1 Dulbecco&#39;s Modified Eagle Medium-Ham&#39;s F12 Nutrient Mixture (DMEM-F12), 1x Insulin-Transferrin-Selenium (ITS-G), 66 mg/mL bovine serum albumin (BSA), 0.1 mg/mL transferrin, 0.01 mg/mL biotin, 6.29 mg/mL progesterone, 5 &#181;g/mL N-Acetyl-L-cystine (NAC), and 1 mM putriscine. Aliquot and freeze at -20 &#176;C.</li>
	<li>Prepare 500 mL of C-Medium: 1x Minimum Essential Medium (MEM), D-glucose (final 4 g/L), 10% fetal bovine serum (FBS), and 2 mM L-glutamine. Aliquot and freeze at -20 &#176;C. Add nerve growth factor (NGF) fresh before use (50 ng/mL).</li>
	<li>Prepare 200 mL of Papain Buffer: 1x Earle&#39;s Balanced Salt Solution (EBSS), 100 mM Mg2SO4, 30% Glucose, 0.25 M EGTA, and 1 M NaHCO3.</li>
	<li>Prepare 10 mL of DMEM-ITS-G Medium: 1x DMEM, 0.5% BSA, and 1x ITS-G.</li>
	<li>Prepare 200 mL of PB Buffer: 1x Dulbecco&#39;s phosphate-buffered saline (DPBS) without Ca2+ and Mg2+ and 0.5% BSA. Degas the buffer before use and keep on ice.</li>
</ol>
2. Isolation and culture of Glioma Stem Cell Neurospheres
<ol>
	<li>Isolation of Neurospheres
	<ol>
		<li>Collect IRB approved and patient consented fresh human glioblastoma (GBM) tissue from the operating room. Transfer GBM samples on ice in DPBS containing 2x anti-anti to a BL2 certified biological safety cabinet.</li>
		<li>Transfer one 1 cm3 GBM sample with minimal necrosis or red blood cell contamination to a 60 mm plate and cut into 1 mm3 fragments; remove any excess DPBS.</li>
		<li>Digest the tissue with 5 mL of collagenase/dispase (1 mg/mL) for 30 min at 37 &#176;C and gently swirl the dish every 5-10 min.</li>
		<li>Transfer digested fragments to a 50 mL tube and triturate by pipetting with a 10 mL pipette several times to dissociate the tissue.</li>
		<li>Add an equal volume of Neurosphere Media and triturate the tissue again; allowing the large fragments to settle.</li>
		<li>Remove media without tissue fragments and pass slowly through a 70 &#181;M cell strainer placed over a fresh 50 mL tube.</li>
		<li>Repeat steps 2.1.5-2.1.6 until all the tissue has been dissociated, changing the cell strainer as needed.</li>
		<li>Spin the cell suspension at 110 x g for 10 min.</li>
		<li>Remove the supernatant and resuspend the pellet in 10 mL of ACK lysing buffer to remove red blood cell contamination. Incubate for 10 min at room temperature.</li>
		<li>Spin the cell suspension at 110 x g for 5 min.</li>
		<li>Remove the supernatant and resuspend cells in 10 mL of Neurosphere Media. Take 10 &#181;L of the cell suspension and dilute with 10 &#181;L of trypan blue. Count 10 &#181;L of the cell suspension using an automated cell counter or hemocytometer and plate in 10 mL of Neurosphere Media at a density of 3 x 106 cells in 100 mm suspension culture plates.</li>
		<li>Add 2 mL of fresh Neurosphere Media to the culture 2-3 times a week. 
		NOTE: Neurospheres will form in suspension within 3-4 weeks. After subculturing for 2-4 times, confirm the stemness via the limiting dilution assay and tumor recapitulation via xenographic transplantation in immunocompromised mice as previously described 22.</li>
	</ol>
	</li>
	<li>Sub-culturing Neurospheres
	<ol>
		<li>When spheres form and reach a diameter between 200-500 &#181;m, transfer neurospheres to a 15 mL tube and spin at 110 x g for 5 min.</li>
		<li>Resuspend neurospheres in 1 mL of pre-warmed cell detachment solution.</li>
		<li>Transfer to a 1.5 mL tube and incubate at 37 &#176;C for 5 min.</li>
		<li>Set a P200 pipette to 150 &#181;L and triturate by pipetting up and down to dissociate neurospheres.</li>
		<li>Transfer dissociated cells to a 15 mL tube. Add 4 mL of Neurosphere Media and spin at 300 x g for 5 min.</li>
		<li>Remove the supernatant and resuspend cells in 5 mL of Neurosphere Media. Take 10 &#181;L of the cell suspension and dilute with 10 &#181;L of trypan blue. Count 10 &#181;L of the cell suspension using an automated cell counter or hemocytometer and plate at a density of 1 x 106 cells/60 mm dish in a final volume of 4 mL.</li>
		<li>Refresh media 2 times a week and subculture cells as needed. Freeze whole neurospheres when desired in Neuronal basal media w/o vitamin A supplemented with 10% DMSO. Neurospheres may be used for experiments after P4.</li>
	</ol>
	</li>
</ol>
3. Compartmented culture of Rat Dorsal Root Ganglia (DRGs), Oligodendrocytes (OPCs) and hGCs
<ol>
	<li>Preparation of compartmented culture dishes 
	NOTE: Perform the following steps the days before the planned harvest of the DRGs.
	<ol>
		<li>Assemble compartmented culture dishes.</li>
		<li>Dilute collagen stock solution to 500 &#181;g/mL in sterile distilled H2O; mix thoroughly.</li>
		<li>With a sterile transfer pipette, fill a 35 mm culture dish with 2 mL of collagen solution; remove the solution, leaving a thin film of collagen behind and place it into the next 35 mm dish. Repeat this process, adding more collagen solution as needed, until all dishes have been coated.</li>
		<li>Once all plates are coated, place the plates in a 245 mm x 245 mm culture tray and lay three 1 mm x 1 mm gauze pads in the center of the tray.</li>
		<li>To polymerize the collagen, wet gauze pads with 1 mL of concentrated ammonium hydroxide and cover the trays for 15 min.</li>
		<li>Remove gauze pads and allow the 35 mm dishes to dry in the laminar flow hood.</li>
		<li>While dishes are drying, load the barrel of the syringe grease applicator with high vacuum grease. Place the compartmented chambers in a large mouth media bottle filled with distilled water. Sterilize both by autoclaving and allow to cool.</li>
		<li>File off the point of an 18-G needled to make a blunt tip. Sterilize in 70% ethanol. Attach the needle to the grease syringe.</li>
		<li>Sterilize the pin rake by soaking in 70% ethanol; allow it to air-dry in the laminar flow hood.</li>
		<li>Remove the lid from a dry, collagen coated 35 mm dish. Hold the dish between the thumb and pointer finger. Hold the pin rake with the other hand. Apply a firm pressure to create even 200 &#181;M wide scratches across the center of the dish.</li>
		<li>Using a pasture pipette, place two drops of Supplemented Neuronal basal Medium in the center of the scratches.</li>
		<li>Repeat steps 3.1.10-3.1.11 until all dishes have been scratched.</li>
		<li>Dry the compartmented chambers in a laminar flow hood.</li>
		<li>With sterile hemostatic forceps, grasp one compartmented chamber by the center divider. Flip the hemostatic forceps so the bottom of the chamber is facing up.</li>
		<li>Apply silicone grease to the compartmented chamber, starting at the top. Ensure that grease is placed neatly and overlaps at all corners.</li>
		<li>Remove the lid from a 35 mm dish. Invert the dish and place the scratches over the chamber. Tap down on the bottom of the plate gently with a pair of forceps.</li>
		<li>Gently flip the plate over by using the hemostatic forceps. Release the forceps.</li>
		<li>Place a mound of grease at the base of the center compartment. Fill each chamber with Supplemented Neuronal Basal Medium (NB) and check for leaks. Seal leaks with silicone grease as needed.</li>
		<li>Continue assembling all culture dishes and store overnight at 37&#176;, 5% CO2.</li>
	</ol>
	</li>
	<li>Isolation of Rat DRG Neurons and Culture in the Compartmented Chamber
	<ol>
		<li>Sacrifice a timed pregnant E16 Sprague-Dawley rat by CO2 asphyxiation or by chemical overdose.</li>
		<li>Place the animal in the supine position on a clean surface; disinfect the abdomen with 70% ethanol.</li>
		<li>Grasp the skin of the lower abdomen with forceps and lift gently.</li>
		<li>Using scissors, make an &#34;I&#34; incision along the midline of the animal, taking care not to puncture the muscles of the abdominal wall.</li>
		<li>With a clean pair of forceps, grasp the muscle wall and make a transverse incision with a clean pair of scissors using caution not to puncture the uterus or intestines.</li>
		<li>With a pair of blunt forceps, grasp the uterus and gently lift straight up out of the peritoneal cavity.</li>
		<li>Using fresh, sterile scissors, clip the connective tissue at the base of each uterine horn.</li>
		<li>Place the entire uterus in a sterile 100 mm tissue culture dish.</li>
		<li>Carry the uterus to a laminar flow hood for dissection.</li>
		<li>Remove embryos from the uterus, one at a time, by clipping through the amniotic sac and gently teasing the embryo out and into a 60 mm dish containing 5 mL of L-15 with 1x pen-strep.</li>
		<li>With fine forceps, place 3-4 embryos into a 60 mm dish containing 5 mL of L-15 with 1x pen-strep.</li>
		<li>Working under a dissecting microscope and with one embryo at a time, euthanize by decapitation.</li>
		<li>Lie the embryo ventral side up and remove the limbs and the tail.</li>
		<li>With fine forceps, make a midline incision in the animal.</li>
		<li>Remove internal organs and tissue to expose the dorsal structures, particularly the spinal cord which should be visible upon completion.</li>
		<li>Place one blade of micro-dissecting scissors between the vertebral column and the spinal canal and carefully cut through the vertebral column to expose the spinal cord. Take care not to clip through the spinal cord.</li>
		<li>With fine forceps, lightly grasp the rostral end of the spinal cord and slowly lift out of the embryo. The DRGs will be attached to the spinal cord.</li>
		<li>Transfer spinal cords and attached DRGs to a 35 mm dish containing 2 ml of L-15 with 1x pen-strep. Place on the ice.</li>
		<li>Once all spinal cords have been isolated, use fine forceps to individually pluck DRGs from the spinal cord. Place DRGs into a fresh 35 mm dish.</li>
		<li>If nerve roots are present on DRGs, clip them away.</li>
		<li>Remove prepared 35 mm dishes from the 37 &#176;C, 5% CO2 incubator. Remove the media from the previous day. Place 80 &#181;L of Supplemented Neuronal Basal Media (NBF) containing 10 &#181;M 5-Fluoro-2&#39;-deoxyuridine (FUDR) in the center compartment and 250 &#181;L of media in each outer compartment.</li>
		<li>Place 2 ganglia in each center compartment. Return dishes to the 37 &#176;C, 5% CO2 incubator.</li>
		<li>The following day add 2.5 mL of NBF.</li>
		<li>Feed the cultures following the schedule in Table 1, altering the schedule based on the day chosen to begin the DRG prep.</li>
		<li>On day 21 (or when axons reach the end of the distal compartments), either seed cultures with a GBM neurosphere (Proceed to step 3.2.26) and live image or myelinate the cultures with oligodendrocytes cultures (Proceed to step 3.3) and then seed with a GBM neurosphere after myelination.</li>
		<li>Replace Supplemented NB Medium in each distal compartmented chamber that will be seeded with a GBM neurosphere with Supplemented NB Medium containing 10% FBS.</li>
		<li>With a P20 pipette set to 10 &#181;L, remove one GBM neurosphere from the culture dish. The GBM neurosphere should measure approximately 200 microns in size.</li>
		<li>Place the tip of the pipette in the distal chamber and expel the GBM neurosphere slowly so that it gently falls onto the axons in the portion of the distal chamber that is closest to the center chamber, over the axons near the center chamber. Be careful not to let the pipette tip disrupt the axons.</li>
		<li>Leave the culture in the biosafety cabinet for 1 h at room temperature to allow the GBM neurosphere to attach.</li>
		<li>Once the neurosphere has attached, very carefully replace the media in the distal compartment with Supplemented NB Medium.</li>
		<li>Live image cultures for 3-7 days, adding media as needed. In this case, a controlled CO2 live cell enclosure attached to the microscope (e.g., Zeiss Axiovert) was used to continuously monitor cell migration for 7 days using brightfield. Images were acquired every 10 min using the associated software. Repeat the same protocol using hGCs that have been stably transfected to express GFP and images were captured using a combination of brightfield and 488 nm laser. 
		NOTE: Neurospheres may be transduced with lentivirus or transfected with plasmids or siRNAs. Compartments may also be treated with desired small molecule inhibitors to study migration.</li>
	</ol>
	</li>
	<li>Myelination of DRG Axons with Oligodendrocytes
	<ol>
		<li>The day before oligodendrocyte isolation, replace the NB Medium in the DRGs with C-Medium.</li>
		<li>Before dissection, place 10 mL of Papain Buffer into a 60 mm dish in the incubator to equilibrate.</li>
		<li>In a laminar flow hood, fill one 100 mm dish and one 60 mm dish with ice cold HBSS. Place on ice.</li>
		<li>Sacrifice a P2 rat pup by decapitation. Remove the skin using a pair of scissors. After the skin is removed, cut the skull along the midline with a pair of fine scissors.</li>
		<li>Gently remove the skull with fine forceps. Using a spatula, gently scoop the brain from the bottom of the skull and transfer to an inverted tissue culture plate lid.</li>
		<li>Remove the cerebellum and divide the cerebrum into two cerebral hemispheres. Remove the olfactory bulbs, hippocampus, and basal ganglia below the cerebral cortex of each hemisphere. Place the cerebral cortex in the 100 mm dish containing HBSS. Repeat steps 3.3.4-3.3.6 for the remaining animals.</li>
		<li>Working with one cortex at a time, remove the meninges with fine Dumont forceps. Place all meninges-free cortices into fresh 60 mm dishes with HBSS.</li>
		<li>Dice the cortical tissue into 1 mm3 pieces. Place on ice.</li>
		<li>Place equilibrated Papain Buffer into a 15 mL tube. Add 200 units of papain and 2 mg L-cysteine. Filter sterilize and add 200 &#181;L of sterile DNase I.</li>
		<li>Remove HBSS from the diced brain tissue and replace with Papain Buffer from 3.3.2. Place dish in 37 &#176;C, 5% CO2 incubator for 80 min, gently shaking every 15 min.</li>
		<li>Transfer the digested tissue to a 50 mL tube and add 2 mL of C-medium.</li>
		<li>Triturate with a 5 mL serological pipette to dissociate the tissue; allow larger pieces of tissue to settle. Remove supernatant and place into a sterile 15 mL tube.</li>
		<li>Add 2 mL of C-Medium and repeat trituration with a 5 mL serological pipette once more. Switch to a 1 mL pipette tip and triturate until the tissue is completely dissociated. Transfer to the 15 mL tube.</li>
		<li>Spin the triturated tissue at 300 x g for 15 min. Carefully remove supernatant and resuspend pellet in 8 mL of DMEM-ITS-G Medium.</li>
		<li>Pre-wet a sterile 30 &#181;M cell strainer with 2 mL of PB Buffer. Place cell strainer over a 50 mL tube and filter the cell suspension 1 mL at a time. Rinse the filter with 5 mL of PB Buffer.</li>
		<li>Transfer the cell suspension to a 100 mm bacteriological plate; incubate for 15 min in a 37 &#176;C, 5% CO2 incubator to allow microglia to attach.</li>
		<li>Remove media and place into a 15 mL tube. Rinse the plate gently with 2 mL of DMEM-ITS-G medium and transfer to the 15 mL tube.</li>
		<li>Resuspend the cell suspension gently. Take 10 &#181;L of the cell suspension and dilute with 10 &#181;l of trypan blue. Count 10 &#181;l of the cell suspension using an automated cell counter or hemocytometer.</li>
		<li>Spin the cell suspension at 300 x g for 10 min.</li>
		<li>Aspirate supernatant completely and resuspend cells in 70 &#181;L of PB Buffer for every 1 x 107 cells. Mix well and incubate at 4 &#176;C for 10 min.</li>
		<li>Add 20 &#181;l of anti-A2B5 microbeads for every 1 x 107 cells. Mix well and incubate at 4 &#176;C.</li>
		<li>Add 1-2 mL of PB Buffer for every 1 x 107 cells and centrifuge at 300 x g for 10 min in a 4 &#176;C centrifuge.</li>
		<li>Aspirate supernatant completely. Resuspend up to a total of 108 cells in 500 &#181;L of PB Buffer. Keep on ice.</li>
		<li>Place a magnetic bead column in the magnetic field of a separator.</li>
		<li>Prepare the column by rinsing with 500 &#181;L of PB Buffer. Apply the cell suspension to the column. Discard flow-through in a waste container (this contains un-labeled cells).</li>
		<li>Wash the column with 500 &#181;L of PB Buffer 3 times. Discard flow-through.</li>
		<li>Remove the column from magnetic separator and place in a collection tube.</li>
		<li>Pipette 1 mL of PB Buffer onto the column. Flush the magnetically labeled cells by firmly pushing the plunger into the column.</li>
		<li>Add 4 mL of C-Medium to the cell fraction and spin at 300 x g for 10 min.</li>
		<li>Remove supernatant and resuspend in 5 mL of N2B2 Medium. Take 10 &#181;L of the cell suspension and dilute with 10 &#181;L of trypan blue. Count 10 &#181;L of the cell suspension using an automated cell counter or hemocytometer.</li>
		<li>Plate oligodendrocytes at a concentration of 150,000 cells per compartmented chamber containing a DRG with fully extended axons.</li>
		<li>The following day change the media in the compartmented chamber to N2B2 to allow for myelination. Replace media every 2-3 days. Myelination will be complete in 14 days.</li>
		<li>On day 14, seed culture with a GBM neurosphere as in 3.2.26-3.2.32. Maintain myelinated cultures in N2B2 Medium for the duration of any experiments. 
		NOTE: The protocol is presented as a flow chart schematic in Figure 1.</li>
	</ol>
	</li>
</ol>

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P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinctive+properties+of+human+adult+brain-derived+myelin+progenitor+cells.">Distinctive properties of human adult brain-derived myelin progenitor cells.</a> American Journal of Pathology. 165 (6), 2167-2175 (2004).</li><li>Zepecki, J. P., Snyder, K. M., Moreno, M. M., Fajardo, E., Fiser, A., Ness, J., Sarkar, A., Toms, S. A., Tapinos, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+human+glioma+cell+migration,+tumor+growth,+and+stemness+gene+expression+using+a+Lck+targeted+inhibitor.">Regulation of human glioma cell migration, tumor growth, and stemness gene expression using a Lck targeted inhibitor.</a> Oncogene. 38, 1734-1750 (2018).</li><li>Chen, H. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Boyden+chamber+assay.">Boyden chamber assay.</a> Methods in Molecular Bioliogy. 294, 15-22 (2005).</li><li>Merz, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organotypic+slice+cultures+of+human+glioblastoma+reveal+different+susceptibilities+to+treatments.">Organotypic slice cultures of human glioblastoma reveal different susceptibilities to treatments.</a> Neuro-Oncology. 15 (6), 670-681 (2013).</li><li>Singh, S. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+human+brain+tumour+initiating+cells.">Identification of human brain tumour initiating cells.</a> Nature. 432 (7015), 396-401 (2004).</li><li>Aubert, M., Badoual, M., Christov, C., Grammaticos, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+model+for+glioma+cell+migration+on+collagen+and+astrocytes.">A model for glioma cell migration on collagen and astrocytes.</a> Journal of the Royal Society Interface. 5 (18), 75-83 (2008).</li><li>Jia, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+three-dimensional+collagen+scaffolds+on+the+expression+profiles+and+biological+functions+of+glioma+cells.">Effects of three-dimensional collagen scaffolds on the expression profiles and biological functions of glioma cells.</a> International Journal of Oncology. 52 (6), 1787-1800 (2018).</li><li>Kaphle, P., Li, Y., Yao, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mechanical+and+pharmacological+regulation+of+glioblastoma+cell+migration+in+3D+matrices.">The mechanical and pharmacological regulation of glioblastoma cell migration in 3D matrices.</a> Journal of Cellular Physiology. 234 (4), 3948-3960 (2019).</li><li>Gritsenko, P., Leenders, W., Friedl, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recapitulating+in+vivo-like+plasticity+of+glioma+cell+invasion+along+blood+vessels+and+in+astrocyte-rich+stroma.">Recapitulating in vivo-like plasticity of glioma cell invasion along blood vessels and in astrocyte-rich stroma.</a> Histochemistry and Cell Biology. 148 (4), 395-406 (2017).</li><li>Gritsenko, P. G., Friedl, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adaptive+adhesion+systems+mediate+glioma+cell+invasion+in+complex+environments.">Adaptive adhesion systems mediate glioma cell invasion in complex environments.</a> Journal of Cell Science. 131 (15), (2018).</li><li>Kaur, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cadherin-11,+a+marker+of+the+mesenchymal+phenotype,+regulates+glioblastoma+cell+migration+and+survival+in+vivo.">Cadherin-11, a marker of the mesenchymal phenotype, regulates glioblastoma cell migration and survival in vivo.</a> Molecular Cancer Research. 10 (3), 293-304 (2012).</li><li>Rao, S. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mimicking+white+matter+tract+topography+using+core-shell+electrospun+nanofibers+to+examine+migration+of+malignant+brain+tumors.">Mimicking white matter tract topography using core-shell electrospun nanofibers to examine migration of malignant brain tumors.</a> Biomaterials. 34 (21), 5181-5190 (2013).</li><li>Huang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+hydrogel+is+suitable+for+targeted+investigation+of+amoeboid+migration+of+glioma+cells.">Three-dimensional hydrogel is suitable for targeted investigation of amoeboid migration of glioma cells.</a> Molecular Medicine Reports. 17 (1), 250-256 (2018).</li></ol>

The authors have nothing to disclose.

Migration studies for hGCs may be performed by using Boyden chamber systems or scratch assays. However, while these experiments fail to give any information regarding the interactions of tumor cells with other surrounding tissues, the present system can recapitulate GC interactions with myelinated and non-myelinated fibers. Furthermore, to study tumor formation and end-point migration, organotypic slice cultures of the rodent brain or in vivo implantation of glioma cells into the rodent brain or flank have previously bee...

Glioblastoma is one of the most aggressive and lethal tumors of the human brain. The current standard of care includes surgical resection of the tumor followed by radiation1 plus concomitant and adjuvant administration of temozolomide2. Even with this multi-therapeutic approach, tumor recurrence is inevitable3. This is partly due to the extensive migratory nature of the tumor cells, which invade the brain parenchyma creating multiple finger-like projections within the brain4 that make complete resection unlikely.
In recent years, it has become evident that the aggressiveness of glioblastoma is due, in part, to the presence of a population of cancer stem cells within the tumor mass5,6, which exhibit high migratory potential7,8, resistance to chemotherapy and radiation9,10 and the ability to form secondary tumors11. GSCs are capable of recapitulating original polyclonal tumors when xenografted to nude mice5.
Despite the wealth of knowledge regarding the genetic background of glioblastomas, studies on glioma cell (GC) migration are currently hindered by a lack of efficient in vitro or in vivo migration models. Notably, while glioma cell-axonal interactions modulated by cellular and environmental factors are a core component of glioma invasion, to our knowledge there is currently no experimental system with the ability to model these interactions12,13,14. To address this deficiency, we developed an ex vivo culture system of primary hGCs co-cultured with purified DRG axon-oligodendrocytes that results in elevated expression of differentiated tumor markers as well as extensive migration and interaction of hGCs with myelinated and non-myelinated fibers. This ex vivo platform, due to its compartmentalized layout, is suitable for testing the effects of novel therapeutics on hGC migration patterns.,

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>100 mm Suspension Culture Dish</td>
			<td>Corning</td>
			<td>430591</td>
			<td></td>
		</tr>
		<tr>
			<td>2.5S NGF</td>
			<td>ENVIGO</td>
			<td>B.5025</td>
			<td></td>
		</tr>
		<tr>
			<td>60 mm Suspension Culture Dish</td>
			<td>Corning</td>
			<td>430589</td>
			<td></td>
		</tr>
		<tr>
			<td>ACK Lysing Buffer</td>
			<td>Thermo Fisher</td>
			<td>A1049201</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium Hydroxide Solution</td>
			<td>Fisher Scientific</td>
			<td>A669-500</td>
			<td>Concentrated</td>
		</tr>
		<tr>
			<td>Animal-Free Recombinant Human EGF</td>
			<td>Peprotech</td>
			<td>AF-100-15</td>
			<td></td>
		</tr>
		<tr>
			<td>Animal-Free Recombinant Human FGF-basic (154 a.a.)</td>
			<td>Peprotech</td>
			<td>AF-100-18B</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-A2B5 MicroBeads, human, mouse, rat</td>
			<td>Miltenyi Biotec</td>
			<td>130-093-392</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibiotic-Antimycotic (100X)</td>
			<td>Thermo Fisher</td>
			<td>15240062</td>
			<td></td>
		</tr>
		<tr>
			<td>AutoMACS Rinsing Solution (PBS, pH 7.2)</td>
			<td>Miltenyi Biotec</td>
			<td>130-091-222</td>
			<td></td>
		</tr>
		<tr>
			<td>B27 Supplement</td>
			<td>Thermo Fisher</td>
			<td>17504044</td>
			<td></td>
		</tr>
		<tr>
			<td>B27 Supplement, minus vitamin A</td>
			<td>Thermo Fisher</td>
			<td>12587001</td>
			<td></td>
		</tr>
		<tr>
			<td>Bacteriological Plate</td>
			<td>BD Falcon</td>
			<td>351029</td>
			<td></td>
		</tr>
		<tr>
			<td>Biotin</td>
			<td>Sigma</td>
			<td>B4639</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Sigma</td>
			<td>A9418</td>
			<td></td>
		</tr>
		<tr>
			<td>Campenot Chamber</td>
			<td>Tyler Research</td>
			<td>CAMP-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Culture Dish</td>
			<td>Corning</td>
			<td>430165</td>
			<td>35mm X 10mm</td>
		</tr>
		<tr>
			<td>Cell Strainer</td>
			<td>BD Falcon</td>
			<td>352350</td>
			<td>70 uM, Nylon</td>
		</tr>
		<tr>
			<td>Cell Strainer</td>
			<td>BD Falcon</td>
			<td>352340</td>
			<td>30 uM, Nylon</td>
		</tr>
		<tr>
			<td>Collagenase/Dispase</td>
			<td>Roche</td>
			<td>11097113001</td>
			<td></td>
		</tr>
		<tr>
			<td>Cultrex Rat Collagen I</td>
			<td>Trevigen</td>
			<td>3440-100-01</td>
			<td></td>
		</tr>
		<tr>
			<td>D-Glucose</td>
			<td>Sigma</td>
			<td>G5146</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Thermo Fisher</td>
			<td>10313021</td>
			<td></td>
		</tr>
		<tr>
			<td>DNase I</td>
			<td>Sigma</td>
			<td>D7291</td>
			<td></td>
		</tr>
		<tr>
			<td>Dow Corning High-Vacuum Grease</td>
			<td>Fisher Scientific</td>
			<td>14-635-5D</td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont #5 Forceps</td>
			<td>Roboz</td>
			<td>RS-5045</td>
			<td></td>
		</tr>
		<tr>
			<td>E16 Timed Pregnant Sprague Dawley Rat</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>EBSS</td>
			<td>Sigma</td>
			<td>E7510</td>
			<td></td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>Sigma</td>
			<td>E3889</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Hyclone</td>
			<td>SH30070.02</td>
			<td></td>
		</tr>
		<tr>
			<td>FUDR</td>
			<td>Sigma</td>
			<td>F0503</td>
			<td></td>
		</tr>
		<tr>
			<td>GlutaMAX Supplement</td>
			<td>Thermo Fisher</td>
			<td>35050061</td>
			<td></td>
		</tr>
		<tr>
			<td>Ham&#39;s F-12 Nutrient Mix</td>
			<td>Thermo Fisher</td>
			<td>11765054</td>
			<td></td>
		</tr>
		<tr>
			<td>HBSS</td>
			<td>Thermo Fisher</td>
			<td>14175095</td>
			<td></td>
		</tr>
		<tr>
			<td>Hemostatic Forceps</td>
			<td>Roboz</td>
			<td>RS-7035</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin Sodium Salt, 0.2% in PBS</td>
			<td>Stem Cell Technologies</td>
			<td>07980</td>
			<td></td>
		</tr>
		<tr>
			<td>Hypodermic Needle, 18G</td>
			<td>BD</td>
			<td>511097</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin-Transferrin-Selenium G</td>
			<td>Thermo Fisher</td>
			<td>41400045</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Cysteine</td>
			<td>Sigma</td>
			<td>C7477</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>Thermo Fisher</td>
			<td>25030081</td>
			<td></td>
		</tr>
		<tr>
			<td>Leibovitz&#39;s L-15 Medium</td>
			<td>Thermo Fisher</td>
			<td>11415064</td>
			<td></td>
		</tr>
		<tr>
			<td>MACS BSA Stock Solution</td>
			<td>Miltenyi Biotec</td>
			<td>130-091-376</td>
			<td></td>
		</tr>
		<tr>
			<td>MACS MultiStand</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-303</td>
			<td></td>
		</tr>
		<tr>
			<td>MEM</td>
			<td>Thermo Fisher</td>
			<td>1190081</td>
			<td></td>
		</tr>
		<tr>
			<td>Mg2SO4</td>
			<td>Sigma</td>
			<td>M2643</td>
			<td></td>
		</tr>
		<tr>
			<td>MiniMACS Separator</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-102</td>
			<td></td>
		</tr>
		<tr>
			<td>MS Columns plus tubes</td>
			<td>Miltenyi Biotec</td>
			<td>130-041-301</td>
			<td></td>
		</tr>
		<tr>
			<td>NAC</td>
			<td>Sigma</td>
			<td>A8199</td>
			<td></td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>Sigma</td>
			<td>S5761</td>
			<td></td>
		</tr>
		<tr>
			<td>Neurobasal Medium</td>
			<td>Thermo Fisher</td>
			<td>21103049</td>
			<td></td>
		</tr>
		<tr>
			<td>Neurobasal-A Medium</td>
			<td>Thermo Fisher</td>
			<td>10888022</td>
			<td></td>
		</tr>
		<tr>
			<td>Ordinary forceps</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>P2 Sprague Dawley Rat Pups</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Papain</td>
			<td>Worthington</td>
			<td>LS003126</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Thermo Fisher</td>
			<td>15140148</td>
			<td></td>
		</tr>
		<tr>
			<td>Pin Rake</td>
			<td>Tyler Research</td>
			<td>CAMP-PR</td>
			<td></td>
		</tr>
		<tr>
			<td>Progesterone</td>
			<td>Sigma</td>
			<td>P8783</td>
			<td></td>
		</tr>
		<tr>
			<td>StemPro Accutase Cell Dissociation Reagent</td>
			<td>Thermo Fisher</td>
			<td>A1110501</td>
			<td></td>
		</tr>
		<tr>
			<td>Syrine Grease Applicator</td>
			<td>Tyler Research</td>
			<td>CAMP-GLSS</td>
			<td></td>
		</tr>
		<tr>
			<td>Transferrin</td>
			<td>Sigma</td>
			<td>T2036</td>
			<td></td>
		</tr>
		<tr>
			<td>Uridine</td>
			<td>Sigma</td>
			<td>U3003</td>
			<td></td>
		</tr>
	</tbody>
</table>

real-time monitoring of human glioma cell migration on dorsal root ganglion axon-oligodendrocyte co-cultures

Glioblastoma is one of the most aggressive human cancers due to extensive cellular heterogeneity and the migration properties of hGCs. In order to better understand the molecular mechanisms underlying glioma cell migration, an ability to study the interaction between hGCs and axons within the tumor microenvironment is essential. In order to model this cellular interaction, we developed a mixed culture system consisting of hGCs and dorsal root ganglia (DRG) axon-oligodendrocyte co-cultures. DRG cultures were selected because they can be isolated efficiently and can form the long, extensive projections which are ideal for migration studies of this nature. Purified rat oligodendrocytes were then added on purified rat DRG axons and induced to myelinate. After confirming the formation of compact myelin, hGCs were finally added to the co-culture and their interactions with DRG axons and oligodendrocytes was monitored in real-time using time-lapse microscopy. Under these conditions, hGCs form tumor-like aggregate structures that express GFAP and Ki67, migrate along both myelinated and non-myelinated axonal tracks and interact with these axons through the formation of pseudopodia. Our ex vivo co-culture system can be used to identify novel cellular and molecular mechanisms of hGC migration and could potentially be used for in vitro drug efficacy testing.

In order to study the interaction of hGCs with axons, we generated purified DRG axons as previously described15,16,17,18. These purified DRG axons were then seeded with hGCs, which formed GFAP+/Ki67+ tumor-like structures integrated within the axonal network, while individual hGCs migrated either in association or between the axons (Figure 2). To determine how hGCs interact wit...

Watch this Scientific Journal Video about Real-Time Monitoring of Human Glioma Cell Migration on Dorsal Root Ganglion Axon-Oligodendrocyte Co-Cultures at JoVE.com

Real-Time Monitoring of Human Glioma Cell Migration on Dorsal Root Ganglion Axon-Oligodendrocyte Co-Cultures

Here we present an ex-vivo mixed monolayer culture system for the study of human glioma cell (hGC) migration in real-time. This model provides the ability to observe interactions between hGCs and both myelinated and non-myelinated axons within a compartmentalized chamber.

Glioblastoma is one of the most aggressive human cancers due to extensive cellular heterogeneity and the migration properties of hGCs. In order to better ...

real-time-monitoring-human-glioma-cell-migration-on-dorsal-root

Research

JoVE Journal

Cancer Research

6.1K Views.  Brown University, Rhode Island Hospital. Here we present an ex-vivo mixed monolayer culture system for the study of human glioma cell (hGC) migration in real-time. This model provides the ability to observe interactions between hGCs and both myelinated and non-myelinated axons within a compartmentalized chamber.

Video: Real-Time Monitoring of Human Glioma Cell Migration on Dorsal Root Ganglion Axon-Oligodendrocyte Co-Cultures

A Protocol for Rapid Post-mortem Cell Culture of Diffuse Intrinsic Pontine Glioma (DIPG)

Diffuse Intrinsic Pontine Glioma (DIPG) is a childhood brainstem tumor that carries a universally fatal prognosis. Because surgical resection is not a viable treatment strategy and biopsy is not routinely performed, the availability of patient samples for research is limited. Consequently, efforts to study this disease have been challenged by a paucity of faithful disease models. To address this need, we describe here a protocol for the rapid processing of post-mortem autopsy tissue samples in order to generate durable patient-derived cell culture models that can be used in in vitro assays or in vivo orthotopic xenograft experiments. These models can be used to screen for potential drug targets and to study fundamental pathobiological processes within DIPG. This protocol can further be extended to analyze and isolate tumor and microenvironmental cells using Fluorescence-activated Cell Sorting (FACS), which enables subsequent analysis of gene expression, protein expression, or epigenetic modifications of DNA at the bulk cell or single cell level. Finally, this protocol can also be adapted to generate patient-derived cultures for other central nervous system tumors.

A Protocol for Rapid Post-mortem Cell Culture of Diffuse Intrinsic Pontine Glioma DIPG

This protocol describes a method for the rapid processing of post-mortem diffuse intrinsic pontine glioma samples for the establishment of patient-derived cell culture models or direct characterization of tumor and microenvironmental cells.

Diffuse Intrinsic Pontine Glioma (DIPG) is a childhood brainstem tumor that carries a universally fatal prognosis. Because surgical resection is not a ...

Preparation and Metabolic Assay of 3-dimensional Spheroid Co-cultures of Pancreatic Cancer Cells and Fibroblasts

Many cancer types, including pancreatic cancer, have a dense fibrotic stroma that plays an important role in tumor progression and invasion. Activated cancer associated fibroblasts are a key component of the tumor stroma that interact with cancer cells and support their growth and survival. Models that recapitulate the interaction of cancer cells and activated fibroblasts are important tools for studying the stromal biology and for development of antitumor agents. Here, a method is described for the rapid generation of robust 3-dimensional (3D) spheroid co-culture of pancreatic cancer cells and activated pancreatic fibroblasts that can be used for subsequent biological studies. Additionally, described is the use of 3D spheroids in carrying out functional metabolic assays to probe cellular bioenergetics pathways using an extracellular flux analyzer paired with a spheroid microplate. Pancreatic cancer cells (Patu8902) and activated pancreatic fibroblast cells (PS1) were co-cultured and magnetized using a biocompatible nanoparticle assembly. Magnetized cells were rapidly bioprinted using magnetic drives in a 96 well format, in growth media to generate spheroids with a diameter ranging between 400-600 &#181;m within 5-7 days of culture. Functional metabolic assays using Patu8902-PS1 spheroids were then carried out using the extracellular flux technology to probe cellular energetic pathways. The method herein is simple, allows consistent generation of cancer cell-fibroblast spheroid co-cultures and can be potentially adapted to other cancer cell types upon optimization of the current described methodology.

Here, a method is described for the preparation of 3-dimensional (3D) spheroid co-culture of pancreatic cancer cells and fibroblasts, followed by measurement of metabolic functions using an extracellular flux analyzer.

Many cancer types, including pancreatic cancer, have a dense fibrotic stroma that plays an important role in tumor progression and invasion. Activated ...

Using the Dot Assay to Analyze Migration of Cell Sheets

Although complex organisms appear static, their tissues are under a continuous turnover. As cells age, die, and are replaced by new cells, cells move within tissues in a tightly orchestrated manner. During tumor development, this equilibrium is disturbed, and tumor cells leave the epithelium of origin to invade the local microenvironment, to travel to distant sites, and to ultimately form metastatic tumors at distant sites. The dot assay is a simple, two-dimensional unconstrained migration assay, to assess the net movement of cell sheets into a cell-free area, and to analyze parameters of cell migration using time-lapse imaging. Here, the dot assay is demonstrated using a human invasive, lung colony forming breast cancer cell line, MCF10CA1a, to analyze the cells' migratory response to epidermal growth factor (EGF), which is known to increase malignant potential of breast cancer cells and to alter the migratory phenotype of cells.

Cell migration is essential for development, tissue maintenance and repair, and tumorigenesis, and is regulated by growth factors, chemokines, and cytokines. This protocol describes the dot assay, a two-dimensional, unconstrained migration assay to assess the migratory phenotype of attached, cohesive cell sheets in response to microenvironmental cues.

Although complex organisms appear static, their tissues are under a continuous turnover. As cells age, die, and are replaced by new cells, cells move ...

Serum and Plasma Copy Number Detection Using Real-time PCR

Serum and plasma cell free DNA (cfDNA) has been shown as an informative, non-invasive source of biomarkers for cancer diagnosis, prognosis, monitoring, and prediction of treatment resistance. Starting from the hypothesis that androgen receptor (AR) gene copy number (CN) gain is a frequent event in metastatic castration resistance prostate cancer (mCRPC), we propose to analyze this event in cfDNA as a potential predictive biomarker.
We evaluated AR CN in cfDNA using 2 different real-time PCR assays and 2 reference genes (RNaseP and AGO1). DNA amount of 60 ng was used for each assay combination. AR CN gain was confirmed using Digital PCR as a more accurate method. CN variation analysis has already been demonstrated to be informative for the prediction of treatment resistance in the setting of mCRPC, but it could be useful also for other purposes in different patient settings. CN analysis on cfDNA has several advantages: it is non-invasive, rapid and easy to perform, and it starts from a small volume of serum or plasma material.

This manuscript describes the copy number variation analysis performed in serum or plasma DNA using real-time PCR approach. This method is suitable for the prediction of drug resistance in castration resistant prostate cancer patients, but it could be informative also for other diseases.

Serum and plasma cell free DNA (cfDNA) has been shown as an informative, non-invasive source of biomarkers for cancer diagnosis, prognosis, monitoring, ...

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 ...

Time-Lapse 2D Imaging of Phagocytic Activity in M1 Macrophage-4T1 Mouse Mammary Carcinoma Cells in Co-cultures

Tumor-associated macrophages (TAMs) have been identified as an important component for tumor growth, invasion, metastasis, and resistance to cancer therapies. However, tumor-associated macrophages can be harmful to the tumor depending on the tumor microenvironment and can reversibly alter their phenotypic characteristics by either antagonizing the cytotoxic activity of immune cells or enhancing anti-tumor response. The molecular actions of macrophages and their interactions with tumor cells (e.g., phagocytosis) have not been extensively studied. Therefore, the interaction between immune cells (M1/M2-subtype TAM) and cancer cells in the tumor microenvironment is now a focus of cancer immunotherapy research. In the present study, a live cell coculture model of induced M1 macrophages and mouse mammary 4T1 carcinoma cells was developed to assess the phagocytic activity of macrophages using a time-lapse video feature using phase-contrast, fluorescent, and differential interference contrast (DIC) microscopy. The present method can observe and document multipoint live-cell imaging of phagocytosis. Phagocytosis of 4T1 cells by M1 macrophages can be observed using fluorescent microscopy before staining 4T1 cells with carboxyfluorescein succinimidyl ester (CFSE). The current publication describes how to coculture macrophages and tumor cells in a single imaging dish, polarize M1 macrophages, and record multipoint events of macrophages engulfing 4T1 cells during 13 h of coculture.

Macrophage phagocytic activity against cancer cells, specifically 4T1 mouse mammary carcinoma cells, was imaged in this study. The live cell coculture model was established and observed using a combination of fluorescent and differential interference contrast microscopy. This assessment was imaged using imaging software to develop multipoint time-lapse video.

Tumor-associated macrophages (TAMs) have been identified as an important component for tumor growth, invasion, metastasis, and resistance to cancer ...

Impedance-based Real-time Measurement of Cancer Cell Migration and Invasion

Cancer arises due to uncontrolled proliferation of cells initiated by genetic instability, mutations, and environmental and other stress factors. These acquired abnormalities in complex, multilayered molecular signaling networks induce aberrant cell proliferation and survival, extracellular matrix degradation, and metastasis to distant organs. Approximately 90% of cancer-related deaths are estimated to be caused by the direct or indirect effects of metastatic dissemination. Therefore, it is important to establish a highly reliable, comprehensive system to characterize cancer cell behaviors upon genetic and environmental manipulations. Such a system can give a clear understanding of the molecular regulation of cancer metastasis and the opportunity for successful development of stratified, precise therapeutic strategies. Hence, accurate determination of cancer cell behaviors such as migration and invasion with gain or loss of function of gene(s) allows assessment of the aggressive nature of cancer cells. The real-time measurement system based on cell impedance enables researchers to continually acquire data during a whole experiment and instantly compare and quantify the results under various experimental conditions. Unlike conventional methods, this method does not require fixation, staining, and sample processing to analyze cells that migrate or invade. This method paper emphasizes detailed procedures for real-time determination of migration and invasion of glioblastoma cancer cells.

Cancer is a lethal disease due to its ability to metastasize to different organs. Determining the ability of cancer cells to migrate and invade under various treatment conditions is crucial to assessing therapeutic strategies. This protocol presents a method to assess the real-time metastatic abilities of a glioblastoma cancer cell line.&#160;

Cancer arises due to uncontrolled proliferation of cells initiated by genetic instability, mutations, and environmental and other stress factors. These ...

Measuring Real-time Drug Response in Organotypic Tumor Tissue Slices

Tumor tissues are composed of cancerous cells, infiltrated immune cells, endothelial cells, fibroblasts, and extracellular matrix. This complex milieu constitutes the tumor microenvironment (TME) and can modulate response to therapy in vivo or drug response ex vivo. Conventional cancer drug discovery screens are carried out on cells cultured in a monolayer, a system critically lacking the influence of TME. Thus, experimental systems that integrate sensitive and high-throughput assays with physiological TME will strengthen the preclinical drug discovery process. Here, we introduce ex vivo tumor tissue slice culture as a platform for medium-high-throughput drug screening. Organotypic tissue slice culture constitutes precisely-cut, thin tumor sections that are maintained with the support of a porous membrane in a liquid-air interface. In this protocol, we describe the preparation and maintenance of tissue slices prepared from mouse tumors and tumors from patient-derived xenograft (PDX) models. To assess changes in tissue viability in response to drug treatment, we leveraged a biocompatible luminescence-based viability assay that enables real-time, rapid, and sensitive measurement of viable cells in the tissue. Using this platform, we evaluated dose-dependent responses of tissue slices to the multi-kinase inhibitor, staurosporine, and cytotoxic agent, doxorubicin. Further, we demonstrate the application of tissue slices for ex vivo pharmacology by screening 17 clinical and preclinical drugs on tissue slices prepared from a single PDX tumor. Our physiologically-relevant, highly-sensitive, and robust ex vivo screening platform will greatly strengthen preclinical oncology drug discovery and treatment decision making.&#160;

We introduce a protocol for measuring real-time drug response in organotypic tumor tissue slices. The experimental strategy outlined here provides a platform to carry out medium-high throughput drug screens on tissue slices derived from clinical or mouse tumors in ex vivo conditions.

Tumor tissues are composed of cancerous cells, infiltrated immune cells, endothelial cells, fibroblasts, and extracellular matrix. This complex milieu ...

A Rapid Screening Workflow to Identify Potential Combination Therapy for GBM using Patient-Derived Glioma Stem Cells

The glioma stem cells (GSCs) are a small fraction of cancer cells which play essential roles in tumor initiation, angiogenesis, and drug resistance in glioblastoma (GBM), the most prevalent and devastating primary brain tumor. The presence of GSCs makes the GBM very refractory to most of individual targeted agents, so high-throughput screening methods are required to identify potential effective combination therapeutics. The protocol describes a simple workflow to enable rapid screening for potential combination therapy with synergistic interaction. The general steps of this workflow consist of establishing luciferase-tagged GSCs, preparing matrigel coated plates, combination drug screening, analyzing, and validating the results.

The glioma stem cells (GSCs) are a small fraction of cancer cells which play essential roles in tumor initiation, angiogenesis, and drug resistance in ...

Live-3D-Cell Immunocytochemistry Assays of Pediatric Diffuse Midline Glioma

Cell migration and invasion are specific hallmarks of Diffuse Midline Glioma (DMG) H3K27M-mutant tumors. We have already modeled these features using three-dimensional (3D) cell-based invasion and migration assays. In this study, we have optimized these 3D assays for live-cell immunocytochemistry. An Antibody Labeling Reagent was used to detect in real-time the expression of the adhesion molecule CD44, on the plasma membrane of migrating and invading cells of a DMG H3K27M primary patient-derived cell line. CD44 is associated with cancer stem cell phenotype and tumor cell migration and invasion and is involved in the direct interactions with the central nervous system (CNS) extracellular matrix. Neurospheres (NS) from the DMG H3K27M cell line were embedded into the basal membrane matrix (BMM) or placed onto a thin coating layer of BMM, in the presence of an anti-CD44 antibody in conjunction with the antibody labeling reagent (ALR). The live-3D-cell immunocytochemistry image analysis was performed on a live-cell analysis instrument to quantitatively measure the overall CD44 expression, specifically on the migrating and invading cells. The method also allows visualizing in real-time the intermittent expression of CD44 on the plasma membrane of migrating and invading cells. Moreover, the assay also provided new insights into the potential role of CD44 in the mesenchymal to amoeboid transition in DMG H3K27M cells.

This study presents a protocol of live-3D-cell immunocytochemistry applied to a pediatric diffuse midline glioma cell line, useful to study in real-time the expression of proteins on the plasma membrane during dynamic processes like 3D cell invasion and migration.

Cell migration and invasion are specific hallmarks of Diffuse Midline Glioma (DMG) H3K27M-mutant tumors. We have already modeled these features using ...