Cell culture and labeling
Lewis cells were purchased from the Institute of Biochemistry and Cell Biology of the Chinese Academy of Sciences (Shanghai, China). The cells were propagated in RPMI 1640 medium (Gibco BRL) supplemented with 10% fetal bovine serum (ScienCell, USA), 100 U/ml penicillin and 100 U/ml streptomycin at 37 °C in a 5% CO2 humidified atmosphere. Luc-lentivirus was purchased from Genechem Co., Ltd. The cells were seeded on culture plates, and a moderate amount of Luc-lentivirus was added to the plate according to the instructions. Clones stably expressing luciferase were selected with neomycin.
Athymic nu/nu mice (7 to 8 weeks of age, 21-25 g each) were purchased from Shanghai, China. The mice were maintained under controlled conditions (room temperature and 12-h light/dark cycle) and had access to standard food and water ad libitum. All experiments were conducted according to the guidelines of the National Institutes of Health. The experimental procedures were approved by the Institutional Animal Care and Use Committee of Nanjing Medical University.
Stereotactic intracranial injection of tumor cells
Mice were anesthetized with 4% chloral hydrate and placed in a stereotactic frame (RWD Life Science, Shenzhen, China). A middle incision was made, followed by a 0.5-mm burr hole that was 2 mm lateral and 2 mm posterior to the bregma. Then, 4 µl of cell suspension containing approximately 5 × 104 Lewis cells was slowly injected intracranially using a 10 µl microinjector syringe at 3 mm below the skull surface over a period of 3 min. The needle was then removed over a 3-min period, and the incision was closed. The mice were monitored until regaining consciousness and were returned to their cages.
In vivo bioluminescent imaging
Mice were i.p. injected with 150 mg/kg D-luciferin potassium salt (15 mg/ml in phosphate-buffered saline (PBS), Shanghai Sciencelight Biology Science & Technology Co., Ltd.) and anesthetized with isoflurane (2-3%). Ten minutes after injection, images were acquired using an IVIS 200 system. The bioluminescent signal was measured as photons per second per square centimeter in regions of interest using Living Image software.
Intravascular injection and detection of fluorescence tracers
A solution of 2% Evans blue (EB, 4 ml/kg, Sigma, USA) was injected into each animal via the femoral vein and circulated for 30 min. After 25 min of EB circulation, fluorescein sodium (F-Na, 300 mg/kg, Sigma, USA) was administered intravenously to circulate for 5 min. Anesthetized mice were perfused with saline to wash out excess fluorescence tracers, and then euthanized by cervical dislocation, after which the brains were quickly removed and embedded in optimum cutting temperature (OCT, Sakura, USA) cryostat-embedding compound. Next, the brains were frozen in liquid nitrogen and sliced into 40-μm thick sections at -19 °C. The penetration of fluorescence tracers through the BBB was further assessed by fluorescence microscopy (Leica, Germany).
Hematoxylin and eosin staining
To confirm the presence of intracranial xenografts, anesthetized mice euthanized by cervical dislocation and whole brains of mice were removed and fixed in a neutral-buffered 10% formaldehyde solution overnight. Then, the brains were embedded in paraffin, sectioned into 4 μm slices and stained with hematoxylin and eosin (H&E).
Transmission electron microscopy analysis
Mice were anesthetized by intraperitoneal injection of pentobarbital sodium (70 mg/kg) and perfused through the left ventricle with 100 ml saline followed by 100 ml 4% paraformaldehyde. The brains were removed, fixed in glutaraldehyde, dehydrated in acetone, embedded with epoxy resin, and sliced into ultrathin sections. Then, the sections were observed under a transmission electron microscope (JEM-1010, Japan).
Immunohistochemistry (IHC) methods
Mice were euthanized as mentioned above, the brains were removed for alcohol dehydration and paraffin embedding. Paraffin-embedded tissue slices were deparaffinized in xylene, rehydrated in graded ethanol, and rinsed with PBS. Then, the slices were placed in a repair box filled with EDTA antigen repair buffer (pH 8.0) and incubated with BSA. The sections were then incubated overnight at 4 °C with polyclonal antibodies against GFAP (Abcam, USA) and CD31 (Santa Cruz, USA). After washing, the slides were incubated with secondary antibody (Life, USA) for 1 h at room temperature. Finally, the slides were visualized by incubation with 3, 3′-diaminobenzidine (DAB) and counterstained with hematoxylin (37%).
Immunofluorescence (IF) methods
The procedure was similar to that used for IHC analysis. Briefly, paraffin-embedded tissue slices were deparaffinized in xylene, rehydrated in graded ethanol, rinsed with PBS, and incubated with primary and secondary antibodies. Then, the slices were incubated with Hoechst staining buffer for 30 min at room temperature and analyzed via fluorescence microscopy (Leica, Germany).
The chemotherapeutic drugs were administered 7 days after Lewis cells implantation in mice. All drugs were prepared immediately before use and were given at a dose volume of 10 ml/kg via tail vein. Paclitaxel was administered as an alcohol solution of Cremophor (Cremophor dose, 1.2 ml/kg), and gemcitabine and pemetrexed were given as a water solution. The drugs were administered at the dose level of 30 mg/kg to mice bearing tumors (n = 5 per group).
As mentioned above, the mice in each group were anesthetized and then euthanized by cervical dislocation. Blood, brain tumors, healthy brain tissues and subcutaneous tumors were rapidly obtained at 15 min, 30 min, 1 h, 2 h and 4 h after injection of the chemotherapeutic drugs for liquid chromatography-mass spectrometry analysis. Liquid chromatography with tandem mass spectrometry (LC-MS/MS) was performed using an Agilent 1290 series HPLC system (Agilent Technologies, Palo Alto, CA, USA) coupled to an Agilent 6460 triple-quadrupole mass spectrometer (Agilent Technologies, Palo Alto, CA, USA) equipped with electrospray ionization (ESI). Analytes were separated with matrices using a Waters Symmetry 300 C18 column (2.1*100 mm, 3.5 μm; Torrance, CA, USA) maintained at 20 °C. The mobile phase of the pemetrexed group samples was 1% formic acid and 0.5% methanol, and the flow rate was 0.3 ml/min. The mobile phases of the gemcitabine and paclitaxel group samples were 10 mM ammonium acetate in aqueous solution and acetonitrile, and the flow rates were 0.4 ml/min and 0.45 ml/min, respectively. A triple-quadrupole mass spectrometer was used to detect the analytes by ESI in the positive mode. MRM mode was used to detect the product ions. The MS parameters were as follows: pemetrexed group samples: Gas Temp: 350 °C, Gas Flow: 10 l/min, Nebulizer: 40 psi, Sheath Gas Heater: 350 °C, Sheath Gas Flow: 9 l/min, and Capillary: 4000; gemcitabine group samples: Gas Temp: 350 °C, Gas Flow: 8 l/min, Nebulizer: 40 psi, and Capillary: 4000; and paclitaxel group samples: Gas Temp: 350 °C, Gas Flow: 10 l/min, Nebulizer: 40 psi, Sheath Gas Heater: 350 °C, Sheath Gas Flow: 8 l/min, and Capillary: 3500. The column efﬂuent was monitored at the following precursor–product ion transitions: m/z 428.1 → 163.1 for pemetrexed, m/z 641.1→112.0 for gemcitabine and m/z 876.3 → 308.0 for paclitaxel, with a dwell time of 100 ms for each ion transition. The total run time was 6 min for pemetrexed, 8 min for gemcitabine and 5 min for paclitaxel.
All experiments were performed at least three times. Data are expressed as the mean ± SEM. All statistical analyses were performed using one-way analysis of variance (ANOVA) followed by a Dunnett post hoc test with Prism 6.00 software (GraphPad Software, San Diego, CA, USA) and SPSS version 20 (SPSS Inc., Chicago, IL, USA). P < 0.05 was considered to indicate significant differences.