In vitro invasion assays

GL261 invasion was assessed in vitro using 12-well (Millipore PI8P01250) or 96-well (Corning 3374) tissue culture inserts1. Cancer cells were seeded at 1 × 106 cells/mL in 3D hydrogels comprising 1.5% rat tail collagen (Corning 354236), 0.2% thiolated hyaluronic acid (Glycosil®; ESI Bio GS220), and 0.1% PEGDA (ESI Bio GS3006). After 20 minutes of gelation, 15 μL of fresh medium was applied on top of the gels. Flow was initiated three hours later using serum-free medium, and cultures were maintained overnight. AMD3100 was used at 10 μM (Sigma A5602) to block the receptor CXCR4 or an excess of 100 nM CXCL12 (Peprotech 300–28 A) was added to prevent chemokine gradient formation. The membranes were then fixed in 4% paraformaldehyde and counterstained using DAPI (Thermo Fisher D1306). An EVOS FL fluorescence microscope was used to acquire 20X images of the porous membrane bottom at five random locations for each sample50. The number of invading cells was manually counted for each technical replicate for n ≥ 4 biological replicates.

Lentiviral transfection and in vivo tumor model

All animal procedures were approved by the Institutional Animal Care and Use Committees (IACUC) at the University of Virginia and Virginia Polytechnic Institute and State University. Lentivirus conferring expression of green fluorescent protein (GFP) under puromycin antibiotic selection was a generous gift from the laboratory of Dr. Kevin Janes. Murine GL261 were serially transfected with GFP lentivirus and purified by selection with 2 μg/mL puromycin (Thermo Fisher A1113803). For in vivo tumor studies, a burr hole was drilled into the skull of anesthetized C57BL/6 mice (5–8 weeks; Harlan Laboratories) at coordinates −2, +2, −2.2 (AP, ML, DV) from bregma. 100,000 GFP+ GL261 cells were inoculated in 5 μL at 1 μL/min, and the bur hole was sealed with bone wax. Ketoprofen was administered at 2 mg/kg for 48 hours to manage pain. One week later, the inoculation site was re-exposed, and a blunt-end 26 gauge needle was used to infuse 10 μL of 1 mg/mL biotinylated dextran amine at 1 μL/min. Ketoprofen was again administered at 2 mg/kg for 48 hours to manage pain.

Flow cytometry

Triplicate wells of 100,000 GL261 cells were cultured in serum-containing medium overnight, either on 2D tissue culture plastic or in 3D hydrogels, as described above. The following day, cells were cultured with 10 μM Brefeldin A in serum-free medium for 6 hours, harvested, pooled, and subjected to antibody labeling7. To assess expression in vivo, mice were inoculated with GFP + tumor cells as above, and 14 days post-implantation mice were treated with 0.25 mg Brefeldin A for 6 hours via intraperitoneal injection51. The brains were then dissociated for analysis. Briefly, the ipsilateral cortical hemisphere was isolated into HBSS and slightly trimmed to reduce the number of non-cancerous cells. The tissue was minced using a scalpel blade, incubated in 5 mL of ACK RBC lysis buffer for 3–5 minutes at room temperature, and centrifuged at 1100 rpm for 5 minutes. An approximately equal volume of 1.5 mg/mL Liberase DL (Sigma 5466202001) was then added to digest the tissue for 30 minutes on a rocker at 37 °C, pipetting up and down to ensure complete digestion.

The tissue slurry was then strained through a 40 µm cell strainer followed by 35 mL of HBSS. This solution was centrifuged at 1100 rpm for 5 minutes, and the isolated cells were resuspended and counted for flow cytometry. Primary-conjugated antibodies were used to stain for CXCR4 (eBiosciences 17-9991-80) and CXCL12 (R&D IC350C), along with appropriate isotype controls. Dead cells were stained using LIVE/DEAD® Fixable Green Dead cell stain kit (Thermo Fisher L23101). Stained samples were run on a Millipore Guava flow cytometer for a minimum of 50,000 events, and the data was analyzed using Incyte software. A flow chart of the gating strategy is shown in Supplemental Fig. 1. For data analysis, plots were gated based on data from single stained controls. In vivo samples were further gated on GFP+ cells to assess only GL261. All numbers are shown as percent of live, single cells.

Magnetic resonance imaging

Animals were anesthetized and placed in a 7 T Clinscan small animal MRI (Bruker/Siemens, Ettlingen, Germany) equipped with a 30-mm head coil. A T2-weighted image was taken through the head with the following parameters: repetition time (TR) = 5500 ms, echo time (TE) = 65 ms, field of view (FOV) = 20 mm × 20 mm with a 192 × 192 matrix, slice thickness = 0.5 mm, number of slices = 30, two averages per phase-encode step requiring a total acquisition time of about 5 min per mouse. For T1-weighted MRI, a 33-Gauge, blunt-end catheter was placed into the same coordinates for tumor implantation, and 10 μL of 25 mg/mL Glowing Galbumin (BioPAL Inc.) was infused at a rate of 1 μL/min. Following an initial image, T1 images were acquired approximately 30 minutes, 1 hour, and 24 hours post-infusion according to the following parameters: TR = 500 ms, TE = 11 ms, FOV = 20 mm × 20 mm with a 192 × 192 matrix, slice thickness = 0.7 mm, number of slices = 22, two averages per phase-encode step requiring a total acquisition time of about 3 min per mouse. Contrast-enhanced T1-weighted images at time t = 0 were subtracted from images at t = 30 minutes to generate a difference heat map and visualize changes in contrast intensity over time.

Tissue harvest and immunohistochemistry

Two days after convection enhanced delivery, tumor-bearing mice were administered Euthasol solution and intracardially perfused with phosphate buffered saline (PBS). Brain tissue was quickly harvested and bisected coronally at the center of the injection site. The brains were fixed overnight in 4% paraformaldehyde, cryopreserved in 30% sucrose, and sectioned at 12 µm using a Leica 1950 cryostat. Tissue sections were blocked in 3% serum and 0.03% Triton X-100 in PBS for 1 hour, then were incubated overnight at 4 °C with rabbit anti-pCXCR4 (Abcam ab74012) diluted in blocker buffer. The samples were washed three times with PBS and incubated for 1 hour at room temperature with goat anti-rabbit 660 diluted in blocking buffer. After washing again, the nuclei were counterstained using DAPI (Thermo Fisher).

In vivo invasion quantification

Fluorescently labeled sections were imaged using an EVOS FL microscope. Five images were randomly taken around the tumor periphery for each of three sections 120 μm apart for each animal. The tumor border was identified based on GFP+ GL261 and nuclear staining, and a blinded investigator counted the number of GFP+ tumor cells beyond the border for each image. Data are presented as the number of invading cells per mm2 of tissue.

Patient sample collection and immunohistochemistry

All procedures involving human participants were performed in accordance with the ethical standards of the Institutional Review Board of the University of Virginia and the 1964 Helsinki declaration and its later amendments or comparable ethical standards. The tissue samples were initially banked with informed patient consent. The samples in this study were accessed through the University of Virginia Biorepository and Tissue Research Facility selected by a neuropathologist (Dr. James W. Mandell, UVA) based on a definitive diagnosis of GBM (astrocytoma, WHO grade IV) who had completed tumor resections at the University of Virginia between 2010 and 2013. Samples were de-identified and processed to identify ones containing primarily tumor bulk. Descriptive statistics and survival information for the patient cohort are presented in Table 1.

For quantitative analysis, we started with ten samples taken prior to therapy and seven taken after standard of care therapy. The eight micron sections were deparaffinized in xylene followed by four graded washed with an increasing ratio of ethanol:water to achieve rehydration. The samples were then subjected to boiling in citrate buffer for 30 minutes for antigen retrieval. The samples were blocked in 3% donkey serum and 0.03% Triton X-100 for 1 hr at room temperature, then primary antibodies against CXCR4 (Sigma GW21075) and CXCL12 (Abcam ab18919) were added overnight at 4 °C. The following day, the samples were washed three times in 1X PBS, treated with donkey anti-rabbit 488 and donkey anti-chicken 647 for 1 hr at room temperature, then washed again. Nuclei were counterstained using DAPI (Thermo D3571), and the samples were mounted in Fluoromount-G (SouthernBiotech) prior to coverslipping. Tissue samples were scanned at 10X using an EVOS-FL Auto2. Five regions were randomly selected from each sample. Thresholding was performed for each image in ImageJ prior to measuring the integrated densities, used to obtain an average integrated density for each sample. Grubb’s test was then used to identify statistical outliers. For both CXCR4 and CXCL12 analyses, one pre-therapy sample was identified as an outlier and was therefore excluded from each.

Statistics

Analysis of Variance (ANOVA) was performed for comparisons of more than two groups, using a significance level of 0.05. If significance was identified within the dataset, t-tests were performed to determine significance between individual groups. Ratio paired t-tests were used to analyze all in vitro data; unpaired t-tests were used to compare in vitro data to in vivo flow cytometry data; and unpaired student’s t-tests were used to compare experimental groups for in vivo invasion and patient sample quantification. All graphed and reported descriptive statistics in the text are presented as mean ± standard error of the mean, unless otherwise stated. Inferential statistics are reported as “(degrees of freedom) = value, n per group, p value” so that effect size can be determined from our reported data.