Supported by NIH R21 grant (PI: C. Willey, CA185712-01), Brain Tumor SPORE award (PD: G.Y. Gillespie, P20CA 151129-03) and SBIR contract (PI: R. Singh, N43CO-2013-00026).

NOTE: All xenograft therapy evaluations were done using an orthotopic tumor model for glioblastoma on a protocol approved by the Institutional Animal Care and Use Committee.
1. Isolation of Patient-derived GBM Xenograft Cells
<ol>
	<li>Preparation of Reagents
	<ol>
		<li>Re-constitute collagenase-I in sterile water to a concentration of 5 mg/ml and sterile filter. Store in 1 ml aliquots at -20 &#176;C (final concentration is 50 &#181;g/ml in 100 ml enzyme solution).</li>
		<li>Dissolve 100 &#181;g Epidermal Growth Factor (EGF) in 2 ml sterile Phosphate-Buffered Saline (PBS). Store in 100 &#181;l (5 &#181;g/100 &#181;l) aliquots at -20 &#176;C for a final concentration of 10 ng/ml.</li>
		<li>Dissolve 100 &#181;g FGF-&#946; in 2 ml sterile PBS. Store in 100 &#181;l (5 &#181;g/100 &#181;l) aliquots at -20 &#176;C for a final concentration of 10 ng/ml.</li>
		<li>Add the following to one 500 ml bottle of Neurobasal Media (NBM) to prepare complete NBM: 10 ml B-27 supplement without vitamin A, 5 ml N2 supplement, 100 &#181;l EGF, 100 &#181;l Fibroblast Growth Factor (FGF)-Basic, 5 ml amphotericin B, 0.5 ml gentamycin, 5 ml L-glutamine.</li>
		<li>Prepare fresh enzyme solution by combining: 98.5 ml PBS, 1 ml collagenase-1, 0.5 ml 10x trypsin/EDTA and sterilize by filtration through 0.22 &#181;m pore filter.</li>
		<li>Prepare or obtain a small volume sterile Dulbecco&#39;s Modified Eagle Medium (DMEM)/F12 cell culture medium with 7% fetal bovine serum (FBS) and 1x PBS without calcium or magnesium.</li>
	</ol>
	</li>
	<li>Tumor Disaggregation 
	NOTE: Athymic nu/nu mouse previously injected with tumor cells in the right flank and allowed to from a palpable tumor mass is necessary for this procedure. In the examples shown here, we used patient-derived xenograft GBM cells, JX10 and JX1217.
	<ol>
		<li>Sterilize work area by spraying 2% chlorhexidine and set up work area with necessary instruments/supplies including disposable chuck, forceps, scalpel, glass Petri dish, enzyme solution, PBS, and chlorhexidine (See Figure 2A).</li>
		<li>Euthanize mouse/mice harboring a flank tumor. Aim for a 20%/min displacement rate of CO2 using 30% CO2. After the mouse shows respiratory arrest, maintain CO2 flow for about 1 min (typically 3 min total). Mice are removed from the chamber and cervical dislocation is performed as a secondary euthanasia confirmation. Spray the animal with 3% chlorhexidine to sanitize skin.</li>
		<li>While holding the mouse steady, make semi-circular skin incision ~ 1.5 cm from tumor mass (flank implanted tumor) using a sterile disposable scalpel with #11 blade beginning cranial to the tumor mass and incising toward the belly of the mouse and around to the caudal end.</li>
		<li>Reflect skin over tumor mass using fingers with gentle traction on caudal end of the incision and then push on skin surface to elevate tumor to have best access without touching the tumor (See Figure 2B).</li>
		<li>Use blunt dissection with forceps to gently free tumor mass from peritoneal wall and from skin (See Figure 2C).</li>
		<li>Transfer tumor mass with forceps to sterile glass Petri dish (DO NOT use plastic dishes to prevent breaking during mincing) and properly discard carcass and place these instruments aside.</li>
		<li>Use new set of sterile instruments (scalpel with #11 blade, semi-curved forceps) to debride tumor tissue of necrotic tissue and membranous host connective tissue covering tumor.</li>
		<li>Place 15 ml enzyme solution in a sterile vented-trypsinizing flask with stir bar and begin stirring slowly in the hood.</li>
		<li>In a sterile glass Petri dish, wash harvested tumors 3 - 5 times with sterile PBS to remove excess blood (may pour or use syringe to rinse them with the PBS). Gently tilt dish and pipet or aspirate the wash material. Mince finely using two #11 scalpel blades (See Figure 2D).</li>
		<li>Add 14 ml of enzyme solution to minced tumor and pipet gently up and down 2 - 3 times. Transfer mixture to vented-trypsinizing flask and stir slowly for 20 min. Prepare 5 50 ml conical tubes by adding 1.5 ml FBS to each in order to neutralize the trypsin in step 1.2.11.</li>
		<li>After 20 min, collect 14 ml of cell solution from trypsinizing flask and add to one tube with FBS. Add 14 ml fresh enzyme solution to flask and continue stirring.</li>
		<li>Centrifuge collected cells at 150 x g for 8 min at room temperature and discard the supernatant. Add 45 ml of complete NBM to the pellet, mix gently, and repeat centrifugation to rinse out serum. Re-suspend pellet in 5ml complete NBM and hold on ice.</li>
		<li>Repeat cell harvest steps for a total of 5 harvests, combining all harvested-cells in a single conical tube on ice.</li>
		<li>Place a 40 &#181;m cell strainer on the top of a 50 ml conical tube. Prep the cell strainer by passing 10 ml of DMEM/F12 + 7% FBS through and then washing with 10 ml PBS.</li>
		<li>Slowly add cell suspension to strainer, allowing it to drip through (See Figure 2M). Gently lift the tab of the cell strainer in between each new addition to break any vacuum and allow the suspended cells to pass freely (See Figure 2N).</li>
		<li>Centrifuge the filtered cell suspension as above. Re-suspend in 10 ml complete NBM and count to determine total number of viable cells using 0.04% trypan blue and a hemocytometer. Hold cells on ice until microtumor generation.</li>
	</ol>
	</li>
</ol>
2. Alternate Tissue Disaggregation Protocol
<ol>
	<li>Automated tissue dissociator and disaggregation system.
	<ol>
		<li>Place the disaggregation tube into the cellular dissociator, select the program &#34;Tumor _02_02&#34; and press start and run for 32 sec. Transfer tube to rotator, place in 37 &#176;C incubator for 40 min.</li>
		<li>Centrifuge suspended-cells at 150 x g for 8 min at room temperature and discard the supernatant. While centrifuging the cells, set up reagents for the processing of the cell suspension. Add 10 ml serum-free NBM and gently triturate suspension.</li>
		<li>After centrifugation, obtain a cell pellet containing ~ 10 - 30 x 106 viable cells (~ 0.3 ml). Re-suspended the pellet in 10 ml serum-free NBM (See Figure 2O). Determine the viable cells by exclusion assay (See Step 3.2.2).</li>
	</ol>
	</li>
</ol>
3. Microtumor Generation
<ol>
	<li>Preparation of Reagents
	<ol>
		<li>Prepare complete NBM as in 1.1.4 and prepare neutralized High Density human biogel (HuBiogel) (HDHG at 3 mg/ml) per internal protocol. Similar biogel matrices can be used as well.</li>
	</ol>
	</li>
	<li>Microtumor Production and Culture
	<ol>
		<li>Obtain freshly dissociated PDX cells as single cell suspension in complete NBM, on ice.</li>
		<li>Mix the cell suspension with an equal volume of trypan blue solution (0.4% in PBS) and analyze using hemacytometer to determine cell number and viability by trypan blue exclusion18. Remove volume necessary to generate 50,000 cells/microtumor where volume = (50,000 cells/tumor * # tumors)/(viable cell count / 1 ml) and place into a fresh conical tube.</li>
		<li>Concentrate cells by centrifugation at 150 x g, for 8 min, at room temperature. Discard the supernatant and resuspend the cell pellet with ice-cold HDHG solution in a final ratio of 1 part cells in FBS and 4 parts HDHG.</li>
		<li>Use an electronic multichannel pipette to dispense 10 &#181;l per pin cell-HDHG mixture onto a 96-pin steel plate (with hydrophobic coating) to generate microtumors (2 mm beads each containing 50,000 cells).</li>
		<li>Place 3D tumor beads inside tissue culture incubator (37 &#176;C, 5% CO2, humidified) for 15 min to gelate the beads.</li>
		<li>After gelation, transfer the microtumors (10 &#181;l) to a custom suspension culture chamber (50 ml) or large volume culture dish (10 cm) containing complete NBM using the electronic multi-channel pipette and custom pin-device.</li>
		<li>After 1 - 2 days in tissue culture incubator, transfer microtumors to 96-well culture plates containing 50 &#181;l NBM/well using a wide-mouth dispensing pipette and perform various assay and analysis protocols.</li>
	</ol>
	</li>
	<li>Drug Treatment and Maintenance of microtumors
	<ol>
		<li>Select final concentrations for drug testing. 
		NOTE: If the drug has known efficacy in 2D culture, select 2x the IC50 as the middle dose concentration and select 3-fold serial dilutions above and below for a total of 5 dose levels. For example, if the IC50 is 9 &#181;M for the drug in 2D culture, select 2 &#181;M, 6 &#181;M, 18 &#181;M, 54 &#181;M, and 162 &#181;M as final concentration for drug testing.</li>
		<li>Prepare 2x drug dosing solution in complete NBM from dimethyl sulfoxide (DMSO) stock. Dilute dosing solution in 1% DMSO medium to prepare a 5 dose, 3-fold serial dilution.</li>
		<li>Add 50 &#181;l of dosing solution to the microtumor well containing 50 &#181;l in assay plates to achieve a final DMSO concentration of 0.5%. Repeat for each replicate (e.g., 4) at each drug dose determined in 3.3.1.</li>
		<li>Maintain cultures in 37 &#176;C, 5% CO2, humidified tissue culture incubator for 1 - 14 days. Feed cultures twice weekly by refreshing media and drug solution as above.</li>
	</ol>
	</li>
</ol>
4. Morphological and Phenotypic Analysis of Microtumors
<ol>
	<li>Determine cell morphology in microtumors using standard live-cell staining.
	<ol>
		<li>Prepare 1 mM stock of Calcein-AM in DMSO. Aliquot and store at -20 &#176;C.</li>
		<li>At desired culture intervals (1, 7, and 14 days), add Calcein-AM solution prepared in PBS (without Ca/Mg) to 96-well plate for 1 &#181;M final concentration.</li>
		<li>Incubate 20 min in a 37 &#176;C, 5% CO2, humidified incubator, and image using a fluorescence microscope at 2X, 4X, and 10X (excitation: 450 - 490 nm band pass, emission: 515 nm long pass, dichroic 500 nm).</li>
	</ol>
	</li>
	<li>Microtumor Growth/Proliferation Assay
	<ol>
		<li>Dissolve MTT powder in PBS (without Ca/Mg) to prepare 12 mM stock. Sterile filter, aliquot, and store at -20 &#176;C.</li>
		<li>At desired culture intervals (1, 7, and 14 days), add 20 &#181;l of MTT solution to 96-well plate per 100 &#181;l culture volume. Incubate 2 hr in tissue culture incubator.</li>
		<li>Prepare fresh lysis solution of 10% SDS in 0.01 M HCl and add equal amount to culture volume to plates. Incubate sealed plates overnight in 37 &#176;C incubator.</li>
		<li>Read absorbance at 570 nm using a multi-plate reader.</li>
	</ol>
	</li>
	<li>Microtumor Preparation for Kinomic Analysis
	<ol>
		<li>Prepare lysis buffer by pre-chilling to 4 &#176;C and then adding a 1:100 ratio each of 100x Protein Phosphatase Inhibitor (PPI) and 100x Protein Protease Inhibitor (PI). Mix well and keep on ice.</li>
		<li>Transfer 2 microtumors to each of three 1.5 ml microcentrifuge tube. Remove supernatant and add 40 &#181;l of lysis buffer containing PPI and PI to each tube and lyse for 30 min at 4 &#176;C.</li>
		<li>Pipette samples vigorously to break tumor beads. Centrifuge at 16,000 x g for 10 min at 4 &#176;C and store samples at -80 &#176;C until kinomic analysis.</li>
	</ol>
	</li>
</ol>
5. Kinomic Profiling of Microtumors
<ol>
	<li>Protein Tyrosine Kinase (PTK) profiling
	<ol>
		<li>Thaw reagents (10x Protein Kinase (PK) buffer and 2% bovine serum albumin (BSA)) to 4 &#176;C Load PK buffer (300 &#181;l 10x PK buffer stock in 2.7 ml dH2O) into syringe in position # 2 on the profiling platform.</li>
		<li>Open the kinase assay software program and load the PTK protocol file and press &#39;START&#39;, annotating samples within software program after scanning chips. Place chips onto profiling platform and load 25 &#181;l of 2% Bovine Serum Albumin (BSA) per array and then press LOAD to begin the software-controlled protocol blocking step.</li>
		<li>Dilute 6 &#181;M of 100 mM ATP stock with 54 &#181;l dH2O to make 10 mM adenosine triphosphate (ATP). When the software begins the 3rd, and final wash step (visible on screen) bring 15 &#181;g of lysate brought up to 28 &#181;l with dH2O, mix and add to array.</li>
		<li>Prepare PTK master mix (MM) (for up to 12 samples). Add 32.6 &#181;l dH2O to reconstitute dithiothreitol (DTT) and add 6 &#181;l DTT, and 60 &#181;l 10x PK solution, to PTK-MM1 tube (already contains 60 &#181;l 10x BSA).</li>
		<li>Wait until &#39;Load&#39; prompt in the kinase assay software appears then add 126 &#181;l of PTK-MM1, 60 &#181;l PTK additive, and 60 &#181;l 10 mM ATP to MM2 (contains previously aliquoted 4.5 &#181;l PY20 FITC antibody) and mix. Add 16 &#181;l of this PTK master mix to each sample lysate tube and pipette mix 5 times.</li>
		<li>Ensure cover glass is clean and then add 35 &#181;l lysate/master mix per array, close carousel lid, and press LOAD button.</li>
	</ol>
	</li>
	<li>Serine/Threonine Kinase (STK) Profiling:
	<ol>
		<li>Thaw reagents (10x PK buffer, 10x STK buffer, and 2% BSA) to 4 &#176;C. Load PK buffer (300 &#181;l in 2.7 ml dH20) into syringe position # 1, 1:10 and STK buffer (300 &#181;l in 2.7 ml dH20) into syringe position # 2.</li>
		<li>Open the kinase assay computer software program and load the STK protocol file and press START, annotating samples within the program after scanning chips. Place chips onto profiling platform and load 25 &#181;l of 2% BSA per array, and then press LOAD to begin the software-controlled protocol blocking step.</li>
		<li>Dilute 6 &#181;M of 100 mM ATP with 54 &#181;l dH2O. During final (3rd) wash step mix 2 &#181;g of lysate and bring up to 32.6 &#181;l with dH2O, mix and add to array.</li>
		<li>Make STK master mix: Add 70 &#181;l 10x PK to STK-MM1 tube (contains 7 &#181;l 100x BSA), and then add 42 &#181;l dH2O to STK-MM1 tube.</li>
		<li>Wait until &#39;Load&#39; prompt in the kinase assay software then: Add 18 &#181;l 10 mM ATP to STK-MM1, and add 9 &#181;l MM1 to each sample lysate. Mix well.</li>
		<li>Ensure cover glass is clean and add 35 &#181;l lysate/master mix per array, close carousel lid, and press LOAD.</li>
		<li>During Final (3rd) Wash Step add 1.05 &#181;l STK-FITC secondary antibody to DMAB tube (contains 3.03 &#181;l STK primary antibody mix), add 39.6 &#181;l AB Buffer to DMAB, add 356.0 &#181;l dH2O to DMAB, add 30 &#181;l to DMAB to each array, and press LOAD.</li>
	</ol>
	</li>
	<li>Analysis of Kinomic Data 19,20
	<ol>
		<li>Open analysis software and load the Image Analysis App. Select image folder containing barcoded and time-stamped whole array images for PTK or STK data, select article number matched array layout file (86312 for PTK and 87102 for STK), and load previously generated array annotation File.</li>
		<li>Verify correct gridding of all images, and run Exposure Time Scaling App. Statistically compare exposure/slope log2 transformed values, and validate with prewash kinetic curves19-21.</li>
		<li>Run upstream kinase prediction software to query altered kinomic profiles between conditions for upstream kinases22,23.</li>
	</ol>
	</li>
</ol>

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<li>Hothi, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((High-throughput+chemical+screens+identify+disulfiram+as+an+inhibitor+of+human+glioblastoma+stem+cells%5BTitle%5D)+AND+%22Oncotarget%22%5BJournal%5D)">High-throughput chemical screens identify disulfiram as an inhibitor of human glioblastoma stem cells.</a> Oncotarget. 3 (10), 1124-1136 (2012).</li>
<li>Quartararo, C. E., Reznik, E., deCarvalho, A. C., Mikkelsen, T., Stockwell, B. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((High-Throughput+Screening+of+Patient-Derived+Cultures+Reveals+Potential+for+Precision+Medicine+in+Glioblastoma%5BTitle%5D)+AND+%22ACS+Med+Chem+Lett%22%5BJournal%5D)">High-Throughput Screening of Patient-Derived Cultures Reveals Potential for Precision Medicine in Glioblastoma.</a> ACS Med Chem Lett. 6 (8), 948-952 (2015).</li>
<li>Ma, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Towards+personalized+medicine+with+a+three-dimensional+micro-scale+perfusion-based+two-chamber+tissue+model+system%5BTitle%5D)+AND+%22Biomaterials%22%5BJournal%5D)">Towards personalized medicine with a three-dimensional micro-scale perfusion-based two-chamber tissue model system.</a> Biomaterials. 33 (17), 4353-4361 (2012).</li>
<li>Pedron, S., Becka, E., Harley, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Regulation+of+glioma+cell+phenotype+in+3D+matrices+by+hyaluronic+acid%5BTitle%5D)+AND+%22Biomaterials%22%5BJournal%5D)">Regulation of glioma cell phenotype in 3D matrices by hyaluronic acid.</a> Biomaterials. 34 (30), 7408-7417 (2013).</li>
<li>Rape, A., Ananthanarayanan, B., Kumar, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Engineering+strategies+to+mimic+the+glioblastoma+microenvironment%5BTitle%5D)+AND+%22Adv+Drug+Deliv+Rev%22%5BJournal%5D)">Engineering strategies to mimic the glioblastoma microenvironment.</a> Adv Drug Deliv Rev. 79-90, 172-183 (2014).</li>
<li>Willey, C. D., Gilbert, A. N., Anderson, J. C., Gillespie, G. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Patient-Derived+Xenografts+as+a+Model+System+for+Radiation+Research%5BTitle%5D)+AND+%22Semin+Radiat+Oncol%22%5BJournal%5D)">Patient-Derived Xenografts as a Model System for Radiation Research.</a> Semin Radiat Oncol. 25 (4), 273-280 (2015).</li>
</ol>

R.S. and R.S.S. are employees of Vivo Biosciences. J.C.A. is partially funded by the UAB Kinome Core.

Critical steps within the protocol predominantly relate to microtumor generation, as well as drug dosing and maintenance. Because the microtumor beads are fragile and easily torn, extreme care is needed in both developmental stages of an assay and maintenance. If an error occurs during either of these processes, experimental interpretation can be compromised, causing extension or unnecessary repetition of the experiments or even exclusion of data.
Modifications and troubleshooting, especially of the microtumor development process, included the design and production of a custom hydrophobic tool for the use of making the microtumor beads. This tool allows for faster and more accurate production of microtumors. Additionally, small modifications to the maintenance of the microtumors helped to speed up the process of changing medium and dosing the cells. Several of these modifications included, but were not limited to, using a multichannel electronic pipette to remove and replace medium from the 96-well plates and pre-mixing fresh drug dosing solutions and arranging the 2x solutions in a corresponding 2 ml 96-well plate.
Major optimizations for culture conditions in 3D modeling includes determining appropriate dosages for drug compounds, as there is a poor relation between 2D modeling and 3D modeling. Therefore, serial dilutions to establish a dose titration curve is often necessary for moving a 2D model system to the 3D microtumor model.
Potential issues to consider with microtumor generation from xenoline tumor cells harvested from a mouse include bacterial or fungal contamination, inconsistent availability of primary xenolines, as unique growth properties in mice can produce differences in harvest time and variance in cell yields from separate dissociations. With each cell line, the number of cells received, and similarly, how many microtumors can be produced will vary. As such, there is a need to prioritize which assays are required and which ones are &#34;optional&#34; should the microtumor yield be insufficient. Potential limitations with the kinomic profiling of microtumors include the difficulty in correcting for inert proteinaceous material loading, as samples are loaded in a manner to correct for BCA determined protein levels, that may not distinguish between biogel based proteins (ECM proteins) and those of the cellular kinase component that is intended to be measured. Measuring of gross microtumor mass via Calcein-AM, or using a housekeeping protein as a correction factor may mitigate this, although issues with housekeeping protein levels in tumors being altered may confound this. For short time-course signaling experiments, assuming equally sized (kinase containing live cell number) and paired treated and untreated samples, this is less of an issue.
With this research, the goal is to provide a more cost-effective and disease-representative preclinical model for accurate drug therapy testing in comparison to orthotopic patient-derived xenografts, the current gold standard model for GBM testing. In recent years, several groups have attempted to model the in vivo GBM environment for drug testing purposes. Some groups have simply tried to grow GBM tumor cells in non-differentiating conditions to preserve GBM stem cells or at least brain tumor initiating cells (BTIC)24,25. These tumorsphere or neurosphere cultures can be used for drug testing and are likely superior to traditional models. However, since they still lack the tumor-stroma interplay that is preserved in our microtumor model, the tumorsphere approach is still limited. Other groups have attempted to apply engineering principles to improve GBM models26-28. These approaches have included flow chambers, variable ECM proteins and tumor cell/normal cell mixtures. While promising, almost all of these reports have used immortalized cells lines with the caveats mentioned previously. As such, we believe that the PDX-based microtumor model described here is more clinically relevant.
In the future, the microtumor model could be used as either a true tumor avatar or a &#39;proband&#39; system. With the proband system, the PDX developed from a particular patient does not directly inform the clinician regarding that specific patient&#39;s therapy, as with the tumor avatar system. Instead, a patient&#39;s tumor is &#34;matched&#34; to a pre-existing, well-characterized (both molecularly and phenotypically) PDX29. Indeed, this model could serve as a &#39;go-to&#39; avatar or reside in a proband library as a comparative profile. With traditional avatars, growing a patient&#39;s tumor cells in mice in parallel to the patient&#39;s treatment is not only time consuming, as it may take several months for the primary passage, but also costly, as testing several drug therapies in mice generate an overwhelming price tag. To combat this, the primary patient tumor can be implanted directly into the biogel matrix. With the microtumors, results could be generated quickly and at a lower cost.
As a proband model, the microtumor kinome data profiles and drug response could be added into a proband &#39;library,&#39; along with profiles and data from previous 3D in vitro models, existing PDXs, animal studies, other patients, etc. In theory, the patient&#39;s tumor cells could then be &#39;omically&#39; (genomically, kinomically, transcriptomically, etc.) matched to a similar existing microtumor profile to determine accurate treatment for the best possible outcome.
Summary: Here we identify that these microtumor models can be used to measure both phenotypic growth and can be queried at both the basal kinase activity level and in response to drug treatment that may be useful as a translational tool for further preclinical research. Developing accurate, and molecularly valid testable models for human tumors is imperative for effective drug development.

The most common primary intracranial malignant brain tumors are grade III astrocytomas and grade IV glioblastoma multiforme (glioblastoma or GBM). These tumors offer poor prognoses with median one-year survival between 12 - 15 months with current therapies for GBM in the US1-3. Multimodality therapies include surgery, radiation, and chemotherapy including temozolomide (TMZ) and kinase-targeted agents. Kinase signaling is frequently dysregulated in GBM, including subsets of tumors with amplification or activating mutations in the Epidermal Growth Factor Receptor (EGFR), increases in Platelet Derived Growth Factor Receptor (PDGFR) signaling, increased Phosphatidyl-Inositol-3 Kinase (PI3K) and tumor supporting angiogenic signaling through Vascular Endothelial Growth Factor Receptor (VEGFR) as well as other kinase driven pathways4-6. Current in vitro and in vivo models frequently lose these representative alterations7. Additionally, genetic profiling has not offered the anticipated benefits that may reflect the fact that genetic and epigenetic changes do not always predict changes at the level of protein activity, where most kinase targeting agents act directly, and where therapies with other mechanisms of action may act indirectly.
The traditional immortalized cell line that can be passaged ad infinitum has long been the standard for drug testing due to their ease of maintenance and reproducibility. However, this model suffers from a high nutrient (and artificial) growth environment that selects for fast growing cells that differ greatly from the original tumor. As such, there has been considerable interest in developing more realistic model systems that reflect a more complex tumor biological system as is present in the patient. Tumor xenografts developed directly from a primary tumor grown in mice (&#34;xenoline,&#34; patient-derived xenograft or PDX) provide a more reflective model system, particularly in the setting of cancer therapeutics, as they are felt to more reliably predict clinical success.8 Despite the more reflective biology, these models are expensive and are difficult to establish and maintain. Moreover, they are not amenable to high-throughput studies. The need to better develop biologic models that more accurately reflect molecular alterations in the primary tumors, and to profile and test these models using direct measures of kinase activity, not surrogate genetic markers, is clear.
It is well recognized that unlike two-dimensional (2D) monolayer cultures, 3D or multicellular assay models can provide more physiologically relevant endpoints9-11. Common 3D culture approaches involve matrix-coated microcarriers and cell spheroid formation. Tumor spheroids can be generated via cellular aggregation using spinner flask, pHEMA plate and hanging drop techniques. Limitations for these approaches include: inability for some cells to form stable spheroids, variability in growth and challenges with mixed cell types. Alternatively, many synthetic (hydrogel, polymer) and animal-derived Engelbreth-Holm-Swarm (EHS) matrix from mouse sarcomas, bovine collagen) matrices have been developed for 3D culture studies12-14. Mouse EHS matrix is extensively used but known to promote cell growth and differentiation in vitro and in vivo15.
In order to replicate 3D tumor biology, a human biomatrix system was developed by Dr. Raj Singh et al.16. The natural, growth factor-free human biogel allows 3D culture scaffolds (beads, discs), which support long-term cultivation of multiple cell types. A series of 3D human biogel culture designs are established for studying tumor growth, adhesion, angiogenesis and invasion properties. Advantages and properties of human biogel as compared to common mouse EHS gels are summarized in Table 1 and Table 2.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Source:</td>
			<td>Human Amnions (Pooled tissue) 
			Pathogen-free, IRB-exempt/approved</td>
		</tr>
		<tr>
			<td>ECM nature:</td>
			<td>Non-denatured Biogel (GLP-production)</td>
		</tr>
		<tr>
			<td>Key 
			Components:</td>
			<td>Col-I (38%), Laminin (22%), Col-IV (20%), Col-III (7%), Entactin &#38; HSPG (&#60; 3%)</td>
		</tr>
		<tr>
			<td>GF-free:</td>
			<td>Undetectable EGF, FGF, TGF, VEGF, PDGF (Non-angiogenic, Non-toxic)</td>
		</tr>
	</tbody>
</table>
Table 1: Properties of Human Biogel as Compared to Common EHS Gels. 
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Human Biogel</td>
			<td>EHS gels</td>
		</tr>
		<tr>
			<td>Natural human matrix</td>
			<td>Reconstituted mouse matrix</td>
		</tr>
		<tr>
			<td>Controlled cell growth &#38; differentiation</td>
			<td>Can promote cell growth &#38; differentiation</td>
		</tr>
		<tr>
			<td>Physiologic gene expression</td>
			<td>Variable gene expression</td>
		</tr>
		<tr>
			<td>3D tissue-like culture model</td>
			<td>Plate-based culture model</td>
		</tr>
	</tbody>
</table>
Table 2: Advantages of Human Biogel as Compared to Common EHS Gels.

<table><tbody><tr><td>Collagenase-I&amp;nbsp;</td><td>Sigma-Aldrich</td><td>CO130</td><td></td></tr><tr><td>Trypsin EDTA (10x)</td><td>Invitrogen</td><td>15400-054&amp;nbsp;</td><td></td></tr><tr><td>Neurobasal-A</td><td>Life Technologies</td><td>10888-022</td><td></td></tr><tr><td>N-2 Supplement</td><td>Life Technologies</td><td>17502-048</td><td>1x final concentration</td></tr><tr><td>B-27 Supplement w/o Vitamin A</td><td>Life Technologies</td><td>12587-010</td><td>1x final concentration</td></tr><tr><td>Recombinant Human FGF-basic</td><td>Life Technologies</td><td>PHG0266</td><td>10 ng/ml final concentration</td></tr><tr><td>Recombinant Human EGF</td><td>Life Technologies</td><td>PGH0315</td><td>10 ng/ml final concentration</td></tr><tr><td>L-Glutamine</td><td>Corning Cellgro Mediatech</td><td>25-005-CI</td><td>2 mM final concentration</td></tr><tr><td>Fungizone</td><td>Omega Scientific</td><td>FG-70</td><td>2.5 &amp;micro;g/ml final concentration</td></tr><tr><td>Penicillin Streptomycin</td><td>Omega Scientific</td><td>PS-20</td><td>100 U/ml Penicillin G, 100 &amp;micro;g/ml Streptomycin final concentration</td></tr><tr><td>Gentamicin</td><td>Life Technologies</td><td>15750-060</td><td>50 ng/ml final concentration</td></tr><tr><td>MTT</td><td>Life Technologies</td><td>M6494</td><td>prepared to 5 mg/ml in PBS and sterile filtered, 1 mg/ml in well</td></tr><tr><td>SDS</td><td>Fisher</td><td>BP166</td><td>for MTT lysis buffer, prepared to 10% in 0.01&amp;nbsp;M HCl, 5% in well</td></tr><tr><td>HCl</td><td>Fisher</td><td>A144SI-212</td><td>for MTT lysis buffer, prepared to 0.01&amp;nbsp;M with SDS, 5 mM in well</td></tr><tr><td>Calcein AM</td><td>Life Technologies</td><td>C1430</td><td>1 mM in DMSO stock, 2&amp;nbsp;&amp;micro;M in PBS staining solution, 1&amp;nbsp;&amp;micro;M in well</td></tr><tr><td>Halt&amp;rsquo;s Protein Phosphatase Inhibitor cocktail&amp;nbsp;</td><td>Pierce ThermoScientific</td><td>78420</td><td>1:100 ratio in MPER&amp;nbsp;</td></tr><tr><td>Halt&#039;s Protein Protease Inhibitor&amp;nbsp;</td><td>Pierce ThermoScientific</td><td>87786</td><td>1:100 ratio in MPER</td></tr><tr><td>Mammalian Protein Extraction Reagent (MPER)</td><td>Pierce ThermoScientific</td><td>PI78501</td><td></td></tr><tr><td>Trypan Blue</td><td>Pierce ThermoScientific</td><td>15250-061</td><td></td></tr><tr><td>DMSO</td><td>Fisher</td><td>BP231</td><td>for dissolution of calcein AM &amp;amp; compounds</td></tr><tr><td>Phosphate-Buffered Saline without Ca/Mg</td><td>Lonza</td><td>17-517Q</td><td>diluted to 1x with MiliQ ultrapure water and sterile filtered (for cell culture)</td></tr><tr><td>Dulbecco&#039;s Phosphate-Buffered Saline with Ca/Mg</td><td>Corning Cellgro Mediatech</td><td>20-030-CV</td><td>diluted to 1x with MiliQ ultrapure water (for pre-fixation wash)</td></tr><tr><td>10% Neutral Buffered Formalin</td><td>Protocol</td><td>032-060</td><td></td></tr><tr><td>Trypan Blue</td><td>Pierce ThermoScientific</td><td>15250-061</td><td></td></tr><tr><td>High Density Hubiogel</td><td>Vivo Biosciences</td><td>HDHG-5</td><td></td></tr><tr><td>Halt&#039;s Protein Phosphatase Inhibitor</td><td>Pierce</td><td>78420</td><td></td></tr><tr><td>Halt&#039;s Protein Protease Inhibitor</td><td>Pierce</td><td>87786</td><td></td></tr><tr><td>Mammalian Protein Extraction Reagent (MPER)</td><td>Thermo Scientific</td><td>78501</td><td></td></tr><tr><td>Protein Tyrosine Kinase (PTK) Array Profiling chip</td><td>PamGene</td><td>86312</td><td></td></tr><tr><td>PTK kinase buffer</td><td>PamGene</td><td>36000</td><td>300 &amp;micro;l 10x PK buffer stock in 2.7&amp;nbsp;ml dH&lt;sub&gt;2&lt;/sub&gt;O, catalog number for PTK reagent kit</td></tr><tr><td>ATP</td><td>PamGene</td><td>36000</td><td>catalog number for PTK reagent kit</td></tr><tr><td>PY20- FITC-conjugated antibody</td><td>PamGene</td><td>36000</td><td>catalog number for PTK reagent kit</td></tr><tr><td>PTK Additive</td><td>PamGene</td><td>32114</td><td></td></tr><tr><td>PTK-MM1 tube (10x BSA)</td><td>PamGene</td><td>36000</td><td>catalog number for PTK reagent kit</td></tr><tr><td>Serine/Threonine Kinase (STK) Array Profiling chip</td><td>PamGene</td><td>87102</td><td></td></tr><tr><td>STK kinase buffer</td><td>PamGene</td><td>32205</td><td>catalog number for STK reagent kit</td></tr><tr><td>STK Primary Antibody Mix (DMAB tube)</td><td>PamGene</td><td>32205</td><td>catalog number for STK reagent kit</td></tr><tr><td>FITC-conjugated Secondary Antibody</td><td>PamGene</td><td>32203</td><td></td></tr><tr><td>STK-MM1 tube (100x BSA)</td><td>PamGene</td><td>32205</td><td>catalog number for STK reagent kit</td></tr><tr><td>STK Antibody Buffer</td><td>PamGene</td><td>32205</td><td>catalog number for STK reagent kit</td></tr><tr><td>Equipment</td><td></td><td></td><td></td></tr><tr><td>#11 Blades, sterile</td><td>Fisher</td><td>3120030</td><td></td></tr><tr><td>#3 scalpel handles, sterile</td><td>Fisher</td><td>08-913-5</td><td></td></tr><tr><td>100 mm glass Petri dishes</td><td>Fisher</td><td>08-748D</td><td></td></tr><tr><td>Semicurved forceps</td><td>Fisher</td><td>12-460-318</td><td></td></tr><tr><td>Trypsinizing flask</td><td>Fisher</td><td>10-042-12B</td><td></td></tr><tr><td>Magnetic stirrer</td><td>Fisher</td><td>14-490-200</td><td></td></tr><tr><td>3/4&quot; stir bar</td><td>Fisher</td><td>14-512-125</td><td></td></tr><tr><td>B-D cell strainer&amp;nbsp;</td><td>Fisher</td><td>#352340</td><td></td></tr><tr><td>B-D 50 ml Centrifuge tube</td><td>Fisher</td><td>#352098</td><td></td></tr><tr><td>PamStation 12</td><td>PamGene</td><td></td><td></td></tr><tr><td>BioNavigator 6.0 kinomic analysis software&amp;nbsp;</td><td>PamGene</td><td></td><td></td></tr><tr><td>Evolve Kinase Assay Software</td><td>PamGene</td><td></td><td></td></tr><tr><td>UpKin App software (upstream kinase prediction)</td><td>PamGene</td><td></td><td></td></tr><tr><td>gentleMACS Dissociator</td><td>Miltenyi Biotec</td><td>130-093-235</td><td></td></tr><tr><td>Rotary Cell Culture System (RCCS)</td><td>Synthecon</td><td>RCCS-D</td><td>with 10 ml disposable HARV</td></tr></tbody></table>

generation of microtumors using 3d human biogel culture system and patient-derived glioblastoma cells for kinomic profiling and drug response testing

The use of patient-derived xenografts for modeling cancers has provided important insight into cancer biology and drug responsiveness. However, they are time consuming, expensive, and labor intensive. To overcome these obstacles, many research groups have turned to spheroid cultures of cancer cells. While useful, tumor spheroids or aggregates do not replicate cell-matrix interactions as found in vivo. As such, three-dimensional (3D) culture approaches utilizing an extracellular matrix scaffold provide a more realistic model system for investigation. Starting from subcutaneous or intracranial xenografts, tumor tissue is dissociated into a single cell suspension akin to cancer stem cell neurospheres. These cells are then embedded into a human-derived extracellular matrix, 3D human biogel, to generate a large number of microtumors. Interestingly, microtumors can be cultured for about a month with high viability and can be used for drug response testing using standard cytotoxicity assays such as 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and live cell imaging using Calcein-AM. Moreover, they can be analyzed via immunohistochemistry or harvested for molecular profiling, such as array-based high-throughput kinomic profiling, which is detailed here as well. 3D microtumors, thus, represent a versatile high-throughput model system that can more closely replicate in vivo tumor biology than traditional approaches.

We have shown that 3D biogel culture system supports long-term growth and function of multiple cell types. In this collaborative project, patient-derived GBM xenolines (PDX) are used for producing hundreds of microtumors. Dissociated cells (3 x 105) or neurospheres (40 - 50) were embedded in biogel beads (2 mm) and after quick gelation they are cultured in a NB-media filled custom bioreactor. Cellular viability (Calcein-AM), growth profile (MTT), and kinomic activity array-based analysis were determined. PDX microtumors maintained multicellular organization and high viability (&#62; 80%) for &#62; 3 weeks. We believe this in vivo-like biology is due to maintenance of 3D cell-matrix architecture (not possible with 2D or spheroid culture). It is also observed that 3D tumors are difficult to produce with fragile gelatinous protein scaffold, possibly due to lack of collagen I14. A working scheme for microtumor production using PDX cells and 3D biogel system is summarized in Figure 1 and the dissociation process is depicted in Figure 2.
Through live cell imaging via Calcein-AM, we determined cell growth and viability as well as effects of the drugs on the GBM cells derived from the PDX tumor JX10, as indicated by a decrease in fluorescence, signaling cell death. Tumor growth was measured at day 0, 7 and 14 for microtumors displaying continuous growth as shown in Figure 3A. This microtumor, JX10, is known to be resistant to TMZ. When exposed to 1 - 10 &#181;M TMZ, minimal growth suppression was noted (Figure 3B). However, testing PDX tumor JX12, which is known to be sensitive to TMZ, demonstrated sensitivity by MTT assay at 14 days that was confirmed by Calcein-AM imaging (Figure 4).
In order to explore potential mechanisms of drug resistance, we measured kinase signaling in the TMZ resistant microtumors (JX10). Kinomic profiles for 144 tyrosine and 144 serine/threonine phosphopeptide targets were captured for DMSO and 10 &#181;M TMZ treated microtumors. Kinomic phosphorylation intensity for all peptide targets, per cycle, and per multiple exposure times was captured (Data not shown). A representative heatmap displaying peptides that had significantly altered intensities with 10 &#181;M TMZ treatment (p &#60; 0.05, unpaired students T-test) is displayed in Figure 5A. Kinomic profiles that were altered in TMZ treated JX10 microtumors were analyzed using the upstream kinase prediction software that identified SYK, LCK, and CTK kinases as increased in TMZ treated samples relative to DMSO (Figure 5B). Serine/threonine kinome upstream kinase analysis was also performed (Figure 5C).
<img alt="Figure 1" src="/files/ftp_upload/54026/54026fig1.jpg" /> 
Figure 1: Working Scheme for Microtumor Production. GBM-PDX tumors are dissociated from the murine host, and single cells are embedded in biogel matrix to form microtumors. Analyses are then performed on the microtumors. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54026/54026fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/54026/54026fig2.jpg" /> 
Figure 2: Dissociation of PDX Tumor Cells. Tumors are removed from host mice and minced prior to mixing with enzyme solution (A-G). Dissociation is shown after 40 min (H). Tumor cell dissociator is shown (I). Trituration in media and filtration (J-N) is shown with an end result of 0.3 ml pellet (O) containing 29.5 x 106 viable cells (P) that form spheroids in NBM in 24 hr (Q). Scale bar is 20 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54026/54026fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/54026/54026fig3.jpg" /> 
Figure 3: Growth of Microtumors. JX10 microtumor growth measured by optical density (O.D.) of MTT and representative images over 14 days, both basally (A), and in response to 1-10 &#181;M TMZ treatment (B). Images are shown at 4X magnification (scale bar = 500 &#181;m). Standard deviation indicated. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54026/54026fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/54026/54026fig4.jpg" /> 
Figure 4: Growth of Microtumors. JX12 microtumor growth measured by O.D. of MTT and representative images over 14 days, both basally (Day 1), and in response to 0, 1, or 10 &#181;M TMZ treatment (scale bar = 500 &#181;m). Standard deviation indicated. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54026/54026fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" src="/files/ftp_upload/54026/54026fig5.jpg" /> 
Figure 5: Kinomic Profiles of TMZ Treated Microtumors. Heatmap of exposure-integrated values (log2 transformed slopes (x 100) of 10, 20, 50, 100, and 200 ms median signal minus background exposure values) are displayed for significantly TMZ-altered altered peptides (p &#60; 0.05). Red indicates increased, and blue indicates decreased relative to DMSO mean per peptide. TMZ altered profiles were compared using the upstream kinase prediction software and altered kinases. Normalized Kinase Statistic (NKS) score &#62; 1.0 and specificity score &#62; 1.3 (red) are considered highly altered. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54026/54026fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Generation of Microtumors Using 3D Human Biogel Culture System and Patient-derived Glioblastoma Cells for Kinomic Profiling and Drug Response Testing at JoVE.

Generation of Microtumors Using 3D Human Biogel Culture System and Patient-derived Glioblastoma Cells for Kinomic Profiling and Drug Response Testing

Patient-derived xenografts of glioblastoma multiforme can be miniaturized into living microtumors using 3D human biogel culture system. This in vivo-like 3D tumor assay is suitable for drug response testing and molecular profiling, including kinomic analysis.

The use of patient-derived xenografts for modeling cancers has provided important insight into cancer biology and drug responsiveness. However, they are ...

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9.0K Views.  University of Alabama at Birmingham. Patient-derived xenografts of glioblastoma multiforme can be miniaturized into living microtumors using 3D human biogel culture system. This in vivo-like 3D tumor assay is suitable for drug response testing and molecular profiling, including kinomic analysis. 

Video: Generation of Microtumors Using 3D Human Biogel Culture System and Patient-derived Glioblastoma Cells for Kinomic Profiling and Drug Response Testing

Generation of High-Throughput Three-Dimensional Tumor Spheroids for Drug Screening

Cancer cells have routinely been cultured in two dimensions (2D) on a plastic surface. This technique, however, lacks the true environment a tumor mass is exposed to in vivo. Solid tumors grow not as a sheet attached to plastic, but instead as a collection of clonal cells in a three-dimensional (3D) space interacting with their neighbors, and with distinct spatial properties such as the disruption of normal cellular polarity. These interactions cause 3D-cultured cells to acquire morphological and cellular characteristics which are more relevant to in vivo tumors. Additionally, a tumor mass is in direct contact with other cell types such as stromal and immune cells, as well as the extracellular matrix from all other cell types. The matrix deposited is comprised of macromolecules such as collagen and fibronectin.
In an attempt to increase the translation of research findings in oncology from bench to bedside, many groups have started to investigate the use of 3D model systems in their drug development strategies. These systems are thought to be more physiologically relevant because they attempt to recapitulate the complex and heterogeneous environment of a tumor. These systems, however, can be quite complex, and, although amenable to growth in 96-well formats, and some now even in 384, they offer few choices for large-scale growth and screening. This observed gap has led to the development of the methods described here in detail to culture tumor spheroids in a high-throughput capacity in 1536-well plates. These methods represent a compromise to the highly complex matrix-based systems, which are difficult to screen, and conventional 2D assays. A variety of cancer cell lines harboring different genetic mutations are successfully screened, examining compound efficacy by using a curated library of compounds targeting the Mitogen-Activated Protein Kinase or MAPK pathway. The spheroid culture responses are then compared to the response of cells grown in 2D, and differential activities are reported. These methods provide a unique protocol for testing compound activity in a high-throughput 3D setting.

Across a wide variety of disease indications, more physiologically relevant models are being developed and implemented into drug discovery programs. The new model system described here demonstrates how three-dimensional tumor spheroids can be cultured and screened in a high-throughput 1536-well plate-based system to search for new oncology drugs.

Cancer cells have routinely been cultured in two dimensions (2D) on a plastic surface. This technique, however, lacks the true environment a tumor mass is ...

Cancer Research

Enhanced Viability for Ex vivo 3D Hydrogel Cultures of Patient-Derived Xenografts in a Perfused Microfluidic Platform

Patient-derived xenografts (PDX), generated when resected patient tumor tissue is engrafted directly into immunocompromised mice, remain biologically stable, thereby preserving molecular, genetic, and histological features, as well as heterogeneity of the original tumor. However, using these models to perform a multitude of experiments, including drug screening, is prohibitive both in terms of cost and time. Three-dimensional (3D) culture systems are widely viewed as platforms in which cancer cells retain their biological integrity through biochemical interactions, morphology, and architecture. Our team has extensive experience culturing PDX cells in vitro using 3D matrices composed of hyaluronic acid (HA). In order to separate mouse fibroblast stromal cells associated with PDXs, we use rotation culture, where stromal cells adhere to the surface of tissue culture-treated plates while dissociated PDX tumor cells float and self-associate into multicellular clusters. Also floating in the supernatant are single, often dead cells, which present a challenge in collecting viable PDX clusters for downstream encapsulation into hydrogels for 3D cell culture. In order to separate these single cells from live cell clusters, we have employed density step gradient centrifugation. The protocol described here allows for the depletion of non-viable single cells from the healthy population of cell clusters that will be used for further in vitro experimentation. In our studies, we incorporate the 3D cultures in microfluidic plates which allow for media perfusion during culture. After assessing the resultant cultures using a fluorescent image-based viability assay of purified versus non-purified cells, our results show that this additional separation step substantially reduced the number of non-viable cells from our cultures.

This protocol demonstrates methods to enable extended in vitro culture of patient-derived xenografts (PDX). One step enhances overall viability of multicellular cluster cultures in 3D hydrogels, through straightforward removal of non-viable single cells. A secondary step demonstrates best practices for PDX culture in a perfused microfluidic platform.

Patient-derived xenografts (PDX), generated when resected patient tumor tissue is engrafted directly into immunocompromised mice, remain biologically ...

A 3D Spheroid Model for Glioblastoma

Two-dimensional (2D) cell cultures do not mimic in vivo tumor growth satisfactorily. Therefore, three-dimensional (3D) culture spheroid models were developed. These models may be particularly important in the field of neuro-oncology. Indeed, brain tumors have the tendency to invade the healthy brain environment. We describe herein an ideal 3D glioblastoma spheroid-based assay that we developed to study tumor invasion. We provide all technical details and analytical tools to successfully perform this assay.

Here, we describe an easy-to-use invasion assay for glioblastoma. This assay is suitable for glioblastoma stem-like cells. A Fiji macro for easy quantification of invasion, migration, and proliferation is also described.

Two-dimensional (2D) cell cultures do not mimic in vivo tumor growth satisfactorily. Therefore, three-dimensional (3D) culture spheroid models were ...

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 ...

Maintaining Human Glioblastoma Cellular Diversity Ex vivo using Three-Dimensional Organoid Culture

Glioblastoma (GBM) is the most commonly occurring primary malignant brain cancer with an extremely poor prognosis. Intra-tumoral cellular and molecular diversity, as well as complex interactions between tumor microenvironments, can make finding effective treatments a challenge. Traditional adherent or sphere culture methods can mask such complexities, whereas three-dimensional organoid culture can recapitulate regional microenvironmental gradients. Organoids are a method of three-dimensional GBM culture that better mimics patient tumor architecture, contains phenotypically diverse cell populations, and can be used for medium-throughput experiments. Although three-dimensional organoid culture is more laborious and time-consuming compared to traditional culture, it offers unique benefits and can serve to bridge the gap between current in vitro and in vivo systems. Organoids have established themselves as invaluable tools in the arsenal of cancer biologists to better understand tumor behavior and mechanisms of resistance, and their applications only continue to grow. Here, details are provided about methods for generating and maintaining GBM organoids. Instructions of how to perform organoid sample embedding and sectioning using both frozen and paraffin-embedding techniques, as well as recommendations for immunohistochemistry and immunofluorescence protocols on organoid sections, and measurement of total organoid cell viability, are all also described.

Maintaining Human Glioblastoma Cellular Diversity Ex vivo using Three-Dimensional Organoid Culture

Here, we describe a method of generating glioblastoma (GBM) organoids from primary patient specimens or patient-derived cell cultures and maintaining them to maturity. These GBM organoids contain phenotypically diverse cell populations and recreate tumor microenvironments ex vivo.

Glioblastoma (GBM) is the most commonly occurring primary malignant brain cancer with an extremely poor prognosis. Intra-tumoral cellular and molecular ...

Generation of 3D Tumor Spheroids for Drug Evaluation Studies

In recent decades, in addition to monolayer-cultured cells, three-dimensional tumor spheroids have been developed as a potentially powerful tool for the evaluation of anticancer drugs. However, the conventional culture methods lack the ability to manipulate the tumor spheroids in a homogeneous manner at the three-dimensional level. To address this limitation, in this paper, we present a convenient and effective method of constructing average-sized tumor spheroids. Additionally, we describe a method of image-based analysis using artificial intelligence-based analysis software that can scan the whole plate and obtain data on three-dimensional spheroids. Several parameters were studied. By using a standard method of tumor spheroid construction and a high-throughput imaging and analysis system, the effectiveness and accuracy of drug tests performed on three-dimensional spheroids can be dramatically increased.

This article demonstrates a standardized method for constructing three-dimensional tumor spheroids. A strategy for spheroid observation and image-based deep-learning analysis using an automated imaging system is also described.

In recent decades, in addition to monolayer-cultured cells, three-dimensional tumor spheroids have been developed as a potentially powerful tool for the ...

Spheroid Drug Sensitivity Screening in Glioma Stem Cell Lines

In vitro drug sensitivity screens are important tools in the discovery of anti-cancer drug combination therapies. Typically, these in vitro drug screens are performed on cells grown in a monolayer. However, these two-dimensional (2D) models are considered less accurate compared to three-dimensional (3D) spheroid cell models; this is especially true for glioma stem cell lines. Cells grown in spheres activate different signaling pathways and are considered more representative of in vivo models than monolayer cell lines. This protocol describes a method for in vitro drug screening of spheroid lines; mouse and human glioma stem cell lines are used as an example. This protocol describes a 3D spheroid drug sensitivity and synergy assay that can be used to determine if a drug or drug combination induces cell death and if two drugs synergize. Glioma stem cell lines are&#160;modified to express RFP. Cells are plated in low attachment round well bottom 96 plates, and spheres are allowed to form overnight. Drugs are added, and the growth is monitored by measuring the RFP signal over time using the Incucyte live imaging system,&#160;a fluorescence microscope embedded in the tissue culture incubator. Half maximal inhibitory concentration (IC50), median lethal dose (LD50), and synergy score are subsequently calculated to evaluate sensitivities to drugs alone or in combination. The three-dimensional nature of this assay&#160;provides a more accurate reflection of tumor growth, behavior, and drug sensitivities in vivo, thus forming the basis for further preclinical investigation.

In vitro drug sensitivity screens are important tools for discovering anti-cancer drug combinations. Cells grown in spheres activate different signaling pathways and are considered more representative of in vivo models than monolayer cell lines. This protocol describes a method for in vitro drug screening for spheroid lines.

In vitro drug sensitivity screens are important tools in the discovery of anti-cancer drug combination therapies. Typically, these in vitro drug screens ...

A Human Glioblastoma Organotypic Slice Culture Model for Study of Tumor Cell Migration and Patient-specific Effects of Anti-Invasive Drugs

Glioblastoma (GBM) continues to carry an extremely poor clinical prognosis despite surgical, chemotherapeutic, and radiation therapy. Progressive tumor invasion into surrounding brain parenchyma represents an enduring therapeutic challenge. To develop anti-migration therapies for GBM, model systems that provide a physiologically relevant background for controlled experimentation are essential. Here, we present a protocol for generating slice cultures from human GBM tissue obtained during surgical resection. These cultures allow for ex vivo experimentation without passaging through animal xenografts or single cell cultures. Further, we describe the use of time-lapse laser scanning confocal microscopy in conjunction with cell tracking to quantitatively study the migratory behavior of tumor cells and associated response to therapeutics. Slices are reproducibly generated within 90 min of surgical tissue acquisition. Retrovirally-mediated fluorescent cell labeling, confocal imaging, and tumor cell migration analyses are subsequently completed within two weeks of culture. We have successfully used these slice cultures to uncover genetic factors associated with increased migratory behavior in human GBM. Further, we have validated the model&#39;s ability to detect patient-specific variation in response to anti-migration therapies. Moving forward, human GBM slice cultures are an attractive platform for rapid ex vivo assessment of tumor sensitivity to therapeutic agents, in order to advance personalized neuro-oncologic therapy.

Current ex vivo models of glioblastoma (GBM) are not optimized for physiologically relevant study of human tumor invasion. Here, we present a protocol for generation and maintenance of organotypic slice cultures from fresh human GBM tissue. A description of time-lapse microscopy and quantitative cell migration analysis techniques is provided.

Glioblastoma (GBM) continues to carry an extremely poor clinical prognosis despite surgical, chemotherapeutic, and radiation therapy. Progressive tumor ...

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

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

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

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

Bioengineering

Generation of Microtumors: An In Vitro Culture Technique to Culture 3D Microtumors to Analyze Response to Drug Treatment

Source: Gilbert, A. N. et al. <a href="https://www-jove-com.remotexs.ntu.edu.sg/video/54026" target="_blank">Generation of Microtumors Using 3D Human Biogel Culture System and Patient-derived Glioblastoma Cells for Kinomic Profiling and Drug Response Testing.</a> J. Vis. Exp. (2016)
In this video, we generate microtumors from patient-derived tumor cells using 3D human biogel. These microtumors closely resemble the in vivo tumor microenvironment and serve as potential platforms for drug testing.

Source: Gilbert, A. N. et al. Generation of Microtumors Using 3D Human Biogel Culture System and Patient-derived Glioblastoma Cells for Kinomic Profiling ...

Generation of Microtumors Using 3D Human Biogel Culture System and Patient-derived Glioblastoma Cells for Kinomic Profiling and Drug Response Testing

Summary

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Generation of High-Throughput Three-Dimensional Tumor Spheroids for Drug Screening

Enhanced Viability for Ex vivo 3D Hydrogel Cultures of Patient-Derived Xenografts in a Perfused Microfluidic Platform

A 3D Spheroid Model for Glioblastoma

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

Maintaining Human Glioblastoma Cellular Diversity Ex vivo using Three-Dimensional Organoid Culture

Generation of 3D Tumor Spheroids for Drug Evaluation Studies

Spheroid Drug Sensitivity Screening in Glioma Stem Cell Lines

A Human Glioblastoma Organotypic Slice Culture Model for Study of Tumor Cell Migration and Patient-specific Effects of Anti-Invasive Drugs

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

Generation of Microtumors: An In Vitro Culture Technique to Culture 3D Microtumors to Analyze Response to Drug Treatment