Primary human brain tumor culture

Tumor tissue was harvested during resection surgery after patient consent in accordance with the IRB-12418 protocol, approved by the Institutional Review Boards of Tufts University and Connecticut Children’s Hospital. Following de-identification, each patient tumor sample was transferred in ice-cold DMEM-F12 supplemented with 1% antibiotic-antimycotic (Sigma–Aldrich). Resected tumor tissue was portioned for immediate culturing, and for cryopreserving for future cultures. For viably freezing, each tumor sample was chopped into ~2 mm2 pieces and frozen in equal volumes of growth media and fetal bovine serum (FBS) supplemented with 5% dimethyl sulfoxide (DMSO). All the frozen tumor samples were completely used during the course of this study.

Anaplastic ependymoma (Grade III) from a pediatric patient (2-year-old female patient) was mechanically dissociated by sequential pipetting using 10 mL, 5 mL, 2 mL, and 1 mL pipettes until a homogenous cell suspension was obtained. The cell suspension was centrifuged at 1200 r.p.m. for 5 min, followed by resuspension in either FBS free growth media, EGM 2MV-NBM [50% complete neurobasal (NBM) containing base neurobasal with 1% glutamax, 1% B27 supplement, 1% antibiotic-antimycotic; 50% endothelial growth media (EGM 2MV BulletKit minus FBS)] or FBS-containing growth media (DMEM F12 with 10% FBS and 1% antibiotic-antimycotic).

For the adult GBM sample (55-year-old male patient, primary IDH-wild-type, EGFR-positive), the tumor tissue was minced into 1 cm2 pieces and incubated in TrypLE Select enzyme (Thermo Fischer Scientific, 12563029) for 15 min at 37 °C, with regular pipetting every 5 min. Upon completion of incubation, twice the volume of GBM growth media was added to the tumor sample. GBM growth media consisted of vitamin-A deprived neurobasal, 1% B27 supplement, 1% antibiotic-antimycotic and the following human growth factors: 20 ng mL−1 epidermal growth factor (EGF, Peprotech AF10015100UG), 20 ng mL−1 basic fibroblast growth factor (bFGF, vWR 100-18B-100UG), 10 ng mL−1 heparin, 20 ng mL−1 platelet-derived growth factor-BB (PDGF-BB, Peprotech 100-14B). Next, the minced and digested GBM samples in media were further mechanically dissociated by pipetting using a 1 mL pipette with a broad tip. Since the adult tumor tissue was much more fibrous and stiffer than the pediatric samples, a balance had to be obtained between the pipetting repetitions and complete dissociation of the tumor tissue. Once a relatively homogeneous cell suspension was obtained, it was centrifuged at 1200 rpm for 5 min and aspirated to remove any TrypLE Select enzyme, followed by resuspension in GBM growth media.

Additional steps involved filtering of the adult GBM cell suspension using a 100 µm cell strainer to remove undigested tissue debris. The filtrate was plated on 2D laminin and Matrigel coated plates, as well as grown as spheroids in low adhesion six-well plates. Laminin (Sigma, 11243217001) and Matrigel (Corning, 354277) coatings were prepared by performing dilutions in PBS and DMEM, respectively. The incubation period for the coatings was either overnight at 4 °C or ~2 h at 37 °C. During the passaging of the 2D cultures, TrypLE Select enzyme was used to detach cells and frozen stocks were created from the early passages. For the initial media changes of the spheroid cultures, 40 um cell strainers were used to retain the formed spheroids and the filtrate with single dead cells was discarded. Eventually, to avoid losing spheroids that remained adhered to the cell strainer, media changes for spheroid cultures involved centrifuging the spheroids and resuspending them gently in half fresh media.

3D bioengineered brain tissue model with primary tumor cells

The assembly of the bioengineered cortical tissue was performed as previously described with further optimization64. Briefly, silk porous 3D scaffolds were coated with 0.1 mg mL−1 poly-D-lysine (Sigma–Aldrich) either overnight at 4 °C or for 2 h at 37 °C. The scaffolds were washed with PBS three times and incubated in media at 37 °C for at least 30 min to equilibrate the scaffolds for cell seeding. Subsequently, tumor cells from either the mechanically dissociated pediatric tumor, TrypLE Select digested adult GBM or single cells obtained from primary GBM expanded as spheroids (P2) were seeded in the 3D ring-shaped silk scaffolds. After overnight incubation of 100 µL tumor cell suspension per scaffold in a 96-well plate, the unattached cells were washed away with the growth media for the corresponding tumor type. Next, the tumor cell-seeded scaffolds were infused with 3 mg mL−1 rat tail collagen type I (Corning) or HA hydrogels.

For the generation of ECM-collagen type I hydrogels, porcine brain-ECM from different developmental stages was obtained via a previously developed decellularization process51. The lyophilized ECM was solubilized with 1 mg mL−1 pepsin from porcine gastric mucosa (Sigma–Aldrich) in 0.1 N hydrochloric acid (Sigma–Aldrich). The solubilization time for fetal and adult ECM at room temperature was ~16 and 24 h, respectively. Once solubilized, the ECM was mixed with 10x DMEM (final 1x in gel) and neutralized using 1 M NaOH (Sigma–Aldrich). The neutralized ECM solution was mixed with 3 mg mL−1 rat tail collagen type I (Corning) for a final ECM concentration of 1000 μg mL−1 and the gelation process started using NaOH. The ECM-collagen solution was kept on ice until complete gelation was required and was used within 2 h of preparation. For HA hydrogels, 5.5% tyramine-substituted HA (Lifecore) was reconstituted sterile at 10 mg mL−1 in ultrapure water overnight at 4 °C on a shaker. To obtain HA gels of ~ 1kPa bulk modulus, final optimized concentrations of HA (4 mg mL^−1), horseradish peroxidase (1 U mL−1), hydrogen peroxide (0.005% v/v) and pH adjusted 10x DMEM (1x in gel) were mixed together on ice. The remaining volume was adjusted by addition of ultrapure water. In the case of ECM-HA hydrogels, 10x DMEM (with a final concentration of 1x in gel) was added to solubilized ECM, which was further pH adjusted and then mixed with the rest of the components. HA hydrogels were prepared in small volumes (~1 mL) due to their rapid gelation time and added to scaffolds immediately. After introduction within the cell-seeded scaffolds, the gelation was completed within 30 min at 37 °C, following which more media was added to each well with the constructs. The next day, each of the ECM-containing tumor cell-seeded 3D constructs was moved to a larger well of a 24-well plate with sufficient media.

We tested the response of the 3D tumor cultures to the chemotherapy drugs that were administered to the patients, cisplatin (Sigma–Aldrich, 232120) and TMZ (Selleckchem, S1237) for anaplastic ependymoma and GBM, respectively. Anaplastic ependymoma samples were treated at 4 mo with multiple doses (2–3) of cisplatin over a period of 2 weeks; while GBM samples were treated at ~7mo in culture with a single dose of 200 µM TMZ for 72 h. We started with TMZ concentrations based on published in vitro concentrations, which were tested mostly in 2D glioblastoma cell lines65. We performed in vitro screening to check for TMZ concentration in 3D cultures that reduced viability, such that we could image the changes by metabolic imaging. Control samples were exposed to DMSO in media for the same time frame as drug treated samples.

Co-culture with differentiated healthy hiNSCs

3D bioengineered constructs with healthy differentiated induced neural stem cells (hiNSCs) were generated using a similar methodology as described for tumor cultures and previously detailed for hiNSCs in the silk-scaffold system66, with further optimization. The differentiating hiNSC constructs were transfected with a viral vector, AAVdj-hSyn-eYFP (Charu Ramakrishnan, Karl Deisseroth, Stanford University), such that the differentiated synapsin (Syn)-expressing neurons would express eYFP (Yellow fluorescent protein). GBM cells expanded as spheres from the patient-tumor were placed in close proximity to the 3D hiNSC constructs 7 mo post-differentiation. Before starting the co-cultures, the GBM cells were separately incubated with 5-aminolevulinic acid (5-ALA, Sigma–Aldrich A3785) for 48 h, for visualization following 5-ALA preferential accumulation. The invasion of healthy hiNSC cultures by the GBM cells and vice versa was observed 48 h post co-culture, by simultaneous live imaging of the differentiated hiNSCs by eYFP and GBM by 5-ALA.

Viability assay

Cell proliferation was performed via WST-1 assay (Sigma–Aldrich) according to the instructions provided by the manufacturer, to assess tumor cell proliferation. Briefly, the samples were incubated for 1 h with WST-1 reagent diluted 1:10 (v:v) in culture medium, followed by a reading of the medium absorbance with plate reader (Molecular Devices) at 450 and 600 nm. Fresh medium was used as a baseline control and its average absorbance was subtracted from the value of the samples. A total of N = 3−6 samples/condition were used in the assay depending on the tumor type.

Lactate dehydrogenase assay

Lactate dehydrogenase (LDH) released into media by ruptured tumor cells was used as a measure of cell death at different time points during the 3D culture without having to sacrifice the samples. LDH assay was performed according to the manufacturer instructions (Sigma–Aldrich). Briefly, culture medium was mixed with the assay reagents in a 1:2 ratio. Following 30 min incubation at room temperature, the reaction was stopped by addition of 1 N HCl. The absorbance was measured at 490 nm and 690 nm. Once again, fresh medium without any construct was used as a baseline control and its average was subtracted from the sample values. A total of N = 3−6 samples/condition were used in the assay depending on the tumor type.

CSPG release ELISA

CSPGs released by the tumor cells in media were measured using an ELISA based assay67. Media samples from the 3D tumor constructs were incubated overnight at 4 °C in a 96-well immuno plate (Thermo Fischer Scientific). Alongside the sample media incubation, chicken extracellular CSPGs (Millipore, CC117) were used over a range of serial dilutions for the generation of standard curves. Following washes with PBS-Tween, monoclonal anti-chondroitin sulfate antibody produced in mouse/clone CS-56, ascites fluid (Sigma–Aldrich, C8035) was added for overnight incubation at 4 °C. After the next round of washes, HRP conjugated goat anti-mouse secondary antibody (Abcam) was incubated at room temperature for 2 h. TMB (3,3′,5,5′-tetramethylbenzidine) 1-C Substrate (Fischer Scientific) was introduced following the last round of washes with PBS-Tween. Finally, after the color developed for 10 min at room temperature, the reaction was stopped with 1 N HCl. The absorbance readings were measured at 450 nm wavelength and the fresh media readings were subtracted from the sample readings. The standard curve was utilized for calculating the quantities of CSPGs released in the different conditions and reported in pg mL−1.

Immunostaining

The samples were fixed at different time points with 4% paraformaldehyde (PFA) solution in PBS (Santa Cruz Biotechnology). Fixation time was 10 min for 2D samples and 20–30 min for the 3D constructs. The cells were stained with glial fibrillary acidic protein (GFAP)primary antibody (Sigma G3893) at a 1:500 dilution and incubations were performed at 4 °C overnight, while the secondary antibody (Invitrogen A-11001, 1:250 dilution) incubations were carried out at room temperature for 2 h. The stained samples were imaged using the Leica SP8 confocal microscope with ×10 or ×25 objectives.

Live mitochondrial imaging

2D ependymoma cells or 3D ependymoma constructs were incubated in media containing diluted TMRE (30 nM) (Thermo Fischer Scientific) and Mitotracker (300 nM) (Thermo Fischer Scientific) at 37 °C for 10 min and 1 h, respectively. Following incubation, the samples were imaged using the Keyence BZ-X700 or the Leica SP8 confocal equipped with an incubator setup maintained at 37 °C and with 5% carbon dioxide inflow. The images were taken with ×60 objective using the same exposure time, and light power across all sample conditions when using the Keyence BZ-X700 for the 2D samples.

5-ALA imaging

During long-term culture (4 mo) of GBM cells, tumor cell invasion in different matrices was examined via migration into the ECM filled central window of the cell-seeded ring-shaped scaffold via 5-ALA (Sigma–Aldrich A3785) accumulation within tumor cells. 5-ALA was excited at 405 nm and emission was collected within 620–720 nm, corresponding to the protoporphyrin IX which accumulates in glioma cells due to low ferrochelatase activity. The 3D samples were imaged using the Leica SP8 confocal microscope.

RNA sequencing and qRT-PCR

Samples were flash frozen in liquid nitrogen and stored in −80 oC in individual RNAase free eppendorf tubes until RNA extraction was performed. All samples were placed on dry ice during extraction, sequentially disrupted using a liquid nitrogen chilled bio-pulverizer. Between each sample, the pulverizer was wiped with 70% ethanol to remove remnants of the previous sample, and between each sample set (different conditions), all the tools were cleaned with RNAzap. Lysis buffer was immediately added to the powdered frozen sample and placed on ice. Once all the samples were in lysis buffer on ice, a 22-gauge needle and syringe were used for sample homogenization one by one using a fresh needle and syringe every time. All the samples were spun down to remove undigested material (mainly silk scaffold) and the supernatant was transferred to clean RNAase free eppendorfs. Following this, the SurePrep All Prep kit (Fischer Scientific) protocol was followed until RNA was eluted from the columns. Preliminary RNA concentrations were measured using nanodrop (Nanodrop 2000, Thermo Fischer Scientific) and sent to Jackson labs for Genomic Medicine in Connecticut, MA for sequencing. Quality control was performed on the RNA samples post cleanup and DNAase treatment for improved RIN values. Next, a KAPA mRNA stranded library was generated for RNA sequencing (RNA-seq) on the Illumina HiSeq 4000.

The raw sequencing data were evaluated at Tufts Genomic Core where the fastq reads were quality checked, preprocessed and aligned to the reference human genome, Hg38 using HISAT2. The aligned data were mapped to possible transcripts using Cufflinks. The final assembled transcriptome or the output FPKM (Reads Per Kilobase of transcript per Million mapped reads) values were imported in Qlucore Omics Explorer for visualization and plotting.

For qRT-PCR, RT2 First Strand Kit with an incorporated gDNA removal step with buffer GE (Qiagen) was utilized for cDNA synthesis from the eluted RNA. cDNA samples were mixed with RT2 SYBR Green Fluor qPCR Mastermix and added to the Qiagen RT2 PCR Array (including a housekeeping gene, genomic DNA control and RT control). PCR was run on BioRad CFX96.

Microarrays for MMP/TIMP and cytokine release

Multiplex Quantibody matrix metalloproteinases/tissue inhibitors of matrix metalloproteinases (MMP/TIMP) and cytokine arrays (RayBiotech) were used to semi-quantitatively or qualitatively compare the proteases/protease inhibitors and cytokines released by the tumor cells cultured in the 3D bioengineered brain tumor model with different ECM environments. Small volumes of control media samples/cell culture supernatants (50 µL or 2 mL) from the 3D constructs were incubated in the capture antibody spotted glass slides or the membranes, along with the standards provided that corresponded to known concentrations of the targets for the Quantibody arrays. This overnight incubation was followed by another overnight step at 4 °C, involving the biotinylated detection antibody cocktail. Next, streptavidin-conjugated fluorophore or HRP-streptavidin was added for 1 h at room temperature. Finally, the slide was disassembled from the removable gasket, dried, and scanned using a fluorescence microarray laser scanner (Ray Biotech). For the membrane array, chemiluminescence detection agent was added right before imaging the membrane.

The human MMP array Q1 (Ray Biotech) allowed for simultaneous detection of six MMPs and three TIMPs: MMP-1,2,3,9,10,13, TIMP-1,2,4. Additionally, the human cytokine array C5 (Ray Biotech) was used to determine the release of 80 different growth factors and inflammatory cytokines (Supplementary Table 1). Protein expression profiles of the pediatric and adult brain tumors across the varying ECM conditions were quantified using the Q-analyzer software (Ray Biotech) to obtain the final values in pg mL−1 for the Quantibody arrays. For the membrane array, chemiluminescence measurements were quantified using ImageJ and fold change over the control media was reported in heat maps.

Metabolic imaging of 3D bioengineered brain tumor model

Tissue samples were imaged with a multiphoton confocal microscope (TCS SP8, Leica) equipped with a Ti-Sapphire laser and time-correlated single-photon counting electronics. During imaging, each sample was placed in a well of a glass bottomed (No. 1.5 coverslip) 24-well plate. The imaging chamber was maintained at 37 °C and humidified along with a continuous supply of 5% CO 2 . Endogenous two-photon excited fluorescence (TPEF) images were acquired at 1024 by 1024 pixels with FOV 232.50 µm by 232.50 µm, and 2x zoom using a 25× (0.95 NA) water objective following excitations at 755 nm and 860 nm for capturing NADH and FAD signals, 460 ± 25 nm (NADH) and 525 ± 25 nm (FAD), respectively.

During endogenous imaging, multiple single planes (n > 3) at varying depths were acquired within each 3D construct. Power measurements of the laser at the two excitation wavelengths were taken regularly and used to normalize the signals during analysis. Incident laser power was ~20 mW at the acquisition focal plane with a pixel dwell time of 600 ns. CARS imaging targeting the C–H molecular stretch was also performed on a subset of samples to validate lipid content of the droplets observed within the GBM cultures. CARS imaging was performed using a commercial Leica TCS SP8 CARS microscope (Wetzlar, Germany), equipped with a picoEmerald picosecond laser and optical parametric oscillator (Stokes beam = 1064 nm, 7 ps; pump beam = 770–850 nm, 5–6 ps). Images (295 × 295 microns, 1024 × 1024 pixels, scanning speed of 400 Hz) were acquired with the pump wavelength set at 816 nm in order to optimally excite the C–H vibrational lipid stretch at 2856 wavenumbers. The CARS signal was detected at 650 ± 105 nm. A HC PL IRAPO 40 × /1.10 water immersion objective was used and the average power of each beam at the sample was held below 50 mW to avoid photodamage of the sample68.

Following the last session of endogenous imaging, some representative samples were co-stained with CellEvent Caspase-3/7 green (Thermo Fischer Scientific), NucBlue Dead (Thermo Fischer Scientific), and TMRE (30 nM) (Thermo Fischer Scientific) reagents to confirm the presence of dead cells and for morphological comparison post-TMZ treatment. Similarly, a few samples were co-stained with a NucBlue Live (Thermo Fischer Scientific), BODIPY 493/503 (2 µg mL−1 in PBS) (Thermo Fischer Scientific) and TMRE (30 nM) to test for the presence of lipids within the droplets observed during imaging.

Field based redox ratio calculation (FAD/FAD + NADH), an intensity based-metric, was determined using MATLAB processing routines as described previously41,69. In brief, all acquired images were normalized for power and detector gain prior to processing. For a given field, the 755 nm excitation, 460 nm emission (NADH channel) and 860 nm excitation, 525 nm emission (FAD channel) were then spatially co-registered by determining the shift maximizing correlation between the two channels. A mean normalized redox ratio value was computed for each field as the normalized fluorescence intensity contributions from FAD over the sum of the intensity contributions from NADH and FAD and reported as the mean redox value per field of view. Additionally, a lifetime based filter was utilized to remove silk contributions. Briefly, silk autofluorescence has a longer fluorescent lifetime during excitation at 755 nm than that of NADH found in cellular regions. Silk scaffold areas were removed by masking out regions that possessed lifetime values beyond a cutoff threshold value when this lifetime was transformed into phasor space (transforming the exponential decay of fluorescence lifetime with sines and cosines), so that there was minimal loss of cellular signal23,41,70. The overall image-based redox ratios were measured after drug treatment with TMZ or control DMSO, and across different ECM conditions. The signals captured at the excitation/emission pair 755/460 ± 25 nm could be the contributions of both NADH and NADPH, as these are indistinguishable from each other. However, we have shown previously NADPH to be present at negligible levels as opposed to NADH in multiple cell types and settings, and thus we assess the optical redox ratio as based on the concentrations of NADH and FAD.

We further proceeded with the determination of redox ratio distribution components using a subset of representative images (n = 3–11). Pixel based redox ratio histograms were first generated by applying a spatial Gaussian low-pass filter to each redox map image and then creating 50 bins between redox ratio values 0 and 1. Images from different groups were compiled and used in the gmdistribution.fit function in MATLAB to fit the aggregate histograms with Gaussian components. The basic components for the model were determined as an average of means and standard deviations of 10 iterations. We then fit these basis components to each histogram and normalized relative weights to sum to 100%. To quantify changes in redox histograms between groups, image-wise ratios of the various Gaussian component weights were used (i.e., W1/ (W2 + W3 + W4), W2/ (W1 + W3 + W4), etc.). Component 1 (centered at the lowest redox ratio) is typically associated with glycolysis, while higher components have been paired to enhanced oxidative phosphorylation and oxidative stress conditions.

Statistics

The graphs were prepared, and statistical analysis performed using GraphPad Prism 7 software (GraphPad, CA, USA). All data are expressed as mean ± s.d arising from sample size of n ≥ 3. The analysis methods utilized included one-way ANOVA, followed by Tukey’s post-hoc test to determine the statistically significant differences for multiple comparisons with one independent variable (ECM, media) and unpaired two-tailed t-test for comparison of two groups unless stated otherwise in the figure captions. A 95% confidence interval or 0.05 was used as the threshold for significance (*p < 0.05) throughout all the statistical tests. Stars associated with p-values have no bearing on the results and conclusion, and are simply reported as obtained from Graphpad prism for the ease of showing individual p-values. Appropriate regression curve was used to fit a trendline on the standard curve generated for ELISAs, such that R2 < 0.97. During the generation of heat maps with hierarchical clustering and principal component analysis (PCA) plots from RNA-seq data, false discovery rate q < 0.2 or p-values < 0.005 were used to identify discriminating variables between comparison groups. Additionally, z-score method, which centers the data to zero mean and unit variance, was used to scale the data presented in heat maps corresponding to RNA-seq data22.