The parental PC-9 cells (EGFR exon19 E746–A750 deletion) were provided by Dr. Lori Hazlehurst’s laboratory, and came transduced to display Tomato Red and Firefly luciferase (Luc2 = tdT), allowing for fluorescence quantification and bioluminescence tracking. The pcDNA3.1(+)/Luc2 = tdT was a gift from Christopher Contag (addgene plasmid # 32904). Cells were grown in RPMI supplemented with 10% fetal bovine serum, 1% penicillin-streptomycin, and 10 μL/mL of G418 to ensure selection of transduced cells. Cells were kept at 37 °C and 5% CO2. All cells used for in vivo and in vitro experiments were between passages 1–10.
Animals and brain tumor model development
Female athymic nu/nu mice (~ 25 g) were purchased from Charles River Laboratories (Wilmington, MA). All animals were aged approximately 6–8 weeks on time of model initiation. Mice were anesthetized using 2% isoflurane. After placement into a stereotactic device (Stoelting), approximately 150,000 of PC-9 cells in 100 μL of PBS were injected into the left cardiac ventricle. Bioluminescence was used to verify presence of PC-9 cells in the brain. Upon termination, animals were euthanized and brains were extracted to begin ex-vivo creation of the PC-9 brain seeking line (PC-9-Br). The protocol developed by Yoneda et al.  was similarly followed to establish the PC-9-Br line. Tumor-bearing brains were extracted, partially homogenized, and digested in a collegenase solution in DMEM. The preparation was then extruded through a 19G needle and strained with a 70 μm cell strainer. The preparation was then centrifuged multiple times, following addition of DMEM and FBS, PBS, and 25% BSA in PBS, respectively. The pellet was collected and cultured in media containing G418 to select for transfected cells. After cells had sufficiently proliferated, they were washed with PBS and re-plated for at least 24 h prior to re-injection in mice. This process was repeated until the extracted population predominantly formed intracranial lesions, which was 6 times for the PC-9 line, named as PC-9-Br.
Longitudinal bioluminescence and survival model
To demonstrate the high morbidity and progression associated with LCBM, we monitored the survival and bioluminescence (BLI) signal after injection of 150,000 PC-9-Br and PC-9 parental cells. Animals were given an intraperitoneal 150 mg/kg injection of d-luciferin potassium salt and anesthetized with 2% isoflurane. Based on the results from unpublished preliminary work, after 10 min of circulation, animals were transferred to the IVIS Spectra CT (PerkinElmer) and BLI was captured at auto-exposure and one-minute time spans on Stage D with medium binning, fitting within the optimal imaging time for the PC-9-Br line. For quantification, a region of interest (ROI) was drawn based on cranial circumference. BLI based on ROI is reported as radiance (photons/sec/cm2/steridian). These mice were monitored regularly for survival until all the mice in PC-9 parental expired. The time and number of deaths in PC-9-Br and PC-9 parental groups were recorded regularly. The experiment was performed under the strict compliance of IACUC of West Virginia University. Data was plotted on a Kaplan Meier curve, which was used to analyze the survival pattern of mice in PC-9 parental and PC-9-Br groups. Mice were euthanized via exanguination under deep ketamine/xylazine (100 mg/kg and 8 mg/kg, respectively) anesthesia.
Chemotherapy preparation and administration
On day 21, mice were randomized into treatment or vehicle groups and began treatment. Cisplatin (5 mg/kg, weekly) and either etoposide (100 mg/kg, days 2 through 5 after cisplatin administration) or pemetrexed (100 mg/kg, days 3 through 5 after cisplatin administration) were selected to represent the most common nonspecific platinum doublet therapy given to lung cancer patients. Cisplatin and pemetrexed were dissolved in saline, and etoposide was dissolved in 5% DMSO, 5% Tween 80, and 90% saline prior to intravenous dosing. All chemotherapy was purchased from SelleckChem. BLI was taken twice weekly to measure chemotherapy response and tumor burden, performed at least an hour prior to drug administration to avoid interactions.
Brain extraction, tissue processing, and quantification
Upon reaching survival endpoints, mice were anesthetized and given tail vein injections of 150 μg of OG dissolved in PBS, along with 10 μCi of 14C-AIB. Following a 10-min circulation, the descending aorta and inferior vena cava were clamped off. A solution of 6 mg of ICG bound to 0.27% bovine serum albumin (270 mg in 10 mL) was perfused through the left ventricle at 5 mL/min to provide a washout. Brains were then rapidly removed and flash-frozen in isopentane (− 80 °C) and stored at − 80 °C prior to tissue slicing and visualization.
Brains were mounted and 20 μm slices were created with the Leica CM3050S cryotome (Leica Microsystems, Wetzlar, Germany), which were transferred to charged microscope slides. Each slide contains 3 slices for a total of approximately 120 slices per brain. Brain slice fluorescence was acquired using a stereomicroscope (Olympus MVX10; Olympus, Center Valley, PA) equipped with a 0.5 NA 2X objective and a monochromatic cooled CCD scientific camera (Retiga 4000R, QIMaging, Surrey, BC, Canada). Tomato Red fluorescence was imaged using a DsRed sputter filter (excitation/band λ 545/25 nm, emission/band λ 605/70 nm and dichromatic mirror at λ 565 nm) (Chroma Technologies, Bellows Falls, VT), OG using an ET-GFP sputter filter (excitation/band λ 470/40 nm, emission/band λ 525/50 nm and dichromatic mirror at λ 495 nm) (Chroma Technologies, Bellows Falls, VT), and ICG using a Cy7 sputter filter (excitation/band λ 710/75 nm, emission/band λ 810/90 nm and dichromatic mirror at λ 760 nm) (Chroma Technologies, Bellows Falls, VT). Fluorescence was captured and analyzed using CellSens (Olympus) software. OG intensity increases were determined by sum intensity per unit of metastatic lesion area relative to non-tumor brain regions.
Fluorescence imaging slides and 14C-AIB slides were placed in quantitative autoradiography (QAR) cassettes (FujiFilm Life Sciences, Stanford, CT) along with 14C autoradiographic standards (American Radiochemicals, St. Louis, MO). A phosphor screen (FujiFilm Life Sciences, 20 × 40 super-resolution) was placed with the slides and standards and allowed to develop for 21 days. QAR phosphor screens were developed in a high-resolution phosphor-imager (GE Typhoon FLA 7000, Uppsala, Sweden) and converted to digital images, which were then calibrated to 14C standards and analyzed using MCID Analysis software (InterFocus Imaging LTD, Linton, Cambridge, England). Metastases permeability fold-changes were calculated based on 14C-AIB signal intensity within confirmed metastases locations (determined using cresyl violet and Tomato Red fluorescence intensity overlays) relative to non-tumor brain 14C-AIB signal intensity.
Tissue sections were processed as described above. After allowing tissues to become adherent to charged slides overnight, slides were briefly dipped in PBS. Staining was performed using 0.1% cresyl violet acetate (Sigma-Aldrich, St. Louis, MO) (2 min) followed by briefly rinsing in tap water. Sections were cleared in 70% ethanol (15 s), 95% ethanol (30 s), 100% ethanol (30 s), respectively. Images were obtained with a 2× objective on the Olympus MVX microscope.
Cell viability assay
Cell viability was evaluated by the MTT assay as described previously [14, 15]. PC-9 parental and PC-9-Br were treated by gefitinib at different concentrations for 48 and/or 72 h. Experiments were repeated independently three times.
Western blot analyses, PCR, and T790M mutation analyses
Protein expressions in PC-9 parental and PC-9-Br were analyzed by Western blot as previously described [14, 15]. α-tubulin was used as an internal control.
Genomic DNAs from PC-9 parental and PC-9-Br were isolated using a DNeasy Blood & Tissue Kit (Qiagen, Valencia, CA, USA). EGFR exon 20 were amplified by PCR according to the method established previously . The PCR products were purified by QIAquick PCR Purification Kit (Qiagen, Hilden, Germany) and sequenced as described in our previous study . For MET, METFR (endogenous control for MET), HER2, and EFTUD2 (endogenous control for HER2), 75 ng of genomic DNA was amplified using SYBR Green Supermix (BioRad). Experiment was performed in triplicate for each group. The PCR primer sequences were reported in the previous studies [14,15,16].
Total RNA was isolated from PC-9 parental and PC-9-Br using the RNeasy Plus Mini Kit (Qiagen) following the manufacturer protocol. One-step RT-PCR Kit with SYBR green was used for amplification of total mRNA (75 ng) following the manufacturer’s protocol (BioRad, Hercules, CA, USA) and our previous studies [14, 15]. Experiment was performed in triplicate for each group. The PCR primer sequences were reported in the previous studies [14,15,16].
All statistics were performed on GraphPad Prism software. XY plots were analyzed by linear regression. Median and interquartile ranges are used for permeability changes and size of metastases. A D’Agostino and Pearson omnibus test was performed and determined a non-Gaussian distribution of data. Statistical analysis of permeability and size was performed using the non-parametric Kruskal-Wallis test followed by Dunn’s multiple comparison test. On survival endpoints, mice were sacrificed and date of death recorded. Kaplan-Meier curves were generated and compared using log-rank statistics. Prism was used for calculation of the 50% inhibitory concentrations (IC50s). Student’s t test and one-way ANOVA followed by a Fisher’s LSD test were applied to determine the difference in the results of cell viabilities and qRT-PCR. Significance for all tests was defined as p < 0.05.