Cell culture and drug treatments
Raji Burkitt’s lymphoma cells (CCL-86; ATCC, Manassas, VA, USA) were propagated as per supplier recommendations, in complete growth medium consisting of RPMI-1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS, Sigma, St. Louis, MO, USA), 100 IU/mL penicillin, 100 μg/ml streptomycin, and 2mM L-Glutamine (Invitrogen, Carlsbad, CA, USA) at 37°C in a 5% CO2 incubator. Cell cultures were maintained at a density of no more than 350,000 cells/mL, and cultured for no more than 30 total passages. Prior to treatment, cells were washed free of complete growth medium and re-suspended in minimum essential medium (MEM) (that is, without FBS) to a density of approximately 100,000 cells/mL. Drug-treated cell cultures were split into aliquots for extraction and LC-MS/MS analysis, or for NIMS analysis as described below. For studies of endogenous metabolite responses to chemotherapy, Raji cells were treated with 50 μM rapamycin or 0.5% (v/v) dimethyl sulfoxide (DMSO) as a vehicle control for up to 90 minutes at 37°C in a 5% CO2 incubator. Four experimental replicate samples were prepared. For studies of xenobiotic metabolism, Raji cells were treated with 0.5mM FLT or 0.5% distilled water vehicle control at 37°C in a 5% CO2 incubator for 60 minutes. Two experimental replicate samples were prepared, and two technical replicate spots from each of these independent samples were deposited on the NIMS surface.
LC-MS/MS analysis and metabolite profiling of lymphoma cell extracts
Drug- or vehicle-treated Raji cells were centrifuged at 400 × g for 1 minute and the supernatant was removed. In order to remove confounding media components, cells were washed three times in 1 mL ice cold phosphate-buffered saline (PBS). After the third wash, cells were suspended in 100 μL of extraction solvent containing 10% chloroform, 40% methanol, and 50% nanopure water, and centrifuged at 15,000 × g for 5 minutes. Thus clarified, the supernatant was collected and stored at −80°C for subsequent LC-MS/MS analysis. All Raji cell LC-MS/MS analyses were performed on an Agilent 1200 series high-performance liquid chromatography (HPLC) system coupled to an Agilent 6538 Q-TOF mass spectrometer (Agilent Technologies, Santa Clara, CA, USA) operated in positive electrospray ionization mode. A 4-μL injection volume of rapamycin- or vehicle-treated Raji cell extracts was used. Comparative analysis of rapamycin- and vehicle-treated LC/MS data was performed with XCMS
 to identify rapamycin-sensitive metabolites, a subset of which were further analyzed by targeted LC-MS/MS using the same HPLC conditions and Q-TOF acquisition parameters. MS/MS spectra were compared to the METLIN metabolite database for metabolite identification
Single cell NIMS imaging and metabolite profiling
NIMS substrates were prepared as described
. In brief, p-type silicon wafers, 500 to 550 μm thick with 0.01 to 0.02 Ω cm resistivity (Silicon Quest International, Santa Clara, CA) were cut into 33 mm2 pieces. The wafers were soaked in Piranha solution (sulfuric acid and hydrogen peroxide (2:1)) overnight, washed thoroughly with nanopure water and then dried using nitrogen gas. Etching was carried out by clamping the wafer in a Teflon chamber. Gold foil was used for the anode and a platinum loop as the cathode; a 25% ethanolic hydrogen fluoride solution was then added to the chamber. A BIO-RAD PowerPack1000 (Hercules, CA, USA) was connected and run at a constant-current mode (300 mA) for 30 minutes. The etched wafers were washed in methanol and evaporated to dryness using nitrogen gas. Bis(heptadecafluoro-1,1,2,2-tetrahydrodecyl)tetramethyldisiloxane (Gelest, Morrisvilles, PA, USA) (100 μL) was applied to the surface of the chip and allowed to sit at room temperature for 1 h before using nitrogen gas to remove excess from the surface. The drug- or vehicle-treated Raji cells were washed and centrifuged as described above, and the supernatant was carefully removed. Cell pellets were immediately re-suspended to a density of 5 to 10 cells/μL in cold PBS by gentle pipetting. Thus diluted, cell suspensions were immediately applied to the NIMS surface in 10-μL aliquots and immediately transferred to a desiccated, room temperature vacuum. Sample spots were visibly dry within one minute. NIMS imaging data was acquired at 5-μm intervals using an AB/SCIEX 5800 mass spectrometer in negative-mode. NIMS images of isolated cells were generated using an FLT-MP/FLT intensity ratio, which was calculated for each pixel to generate a ratiometric image. Individual FLT-MP and FLT intensities were calculated as an integrated peak area within a 100-ppm window centered on the calibrated FLT-MP or FLT peak, using Matlab (Version 2010b, The Mathworks Inc., Natick, MA, USA). Single-cell mass spectra and 2-dimensional ratiometric images with four-fold nearest neighbor interpolation were generated with Matlab and 3-dimensional images were generated with Fiji (
http://fiji.sc/). Median normalized total-ion intensity images were generated with Biomap (
Mouse solid tumor xenograft models, drug treatments, and tissue preparation
All animal experimental procedures complied with the Guide for the Care and Use of Laboratory Animals (Institute for Laboratory Animal Research, 1996) and were approved by the Pfizer Worldwide Research and Development Institutional Animal Care and Use Committee. LC-MS/MS studies of FLT metabolism in tumor tissues were performed using a HCT116 colorectal carcinoma xenograft model. 2.5 million HCT116 cells (CCL247; ATCC) were implanted in the dorsal region of five female Nu/Nu mice (Charles River Breeding Laboratories, Boston, MA, USA) per treatment group. When tumors reached 100 to 120 mm3 in volume, typically eight days after implantation, animals received an intra-peritoneal injection of 30 mg/kg docetaxel or vehicle. At 22 or 46 h after docetaxel treatment, each animal received an intra-peritoneal injection of non-radioactive FLT (2μg/kg; approximately 0.2 nanomoles). Two hours later, the animals were euthanized, and tissues were excised then immediately frozen in liquid nitrogen until extraction and LC-MS/MS analysis (see below).
NIMS tissue imaging and immunofluorescence microscopy were performed on tissues excised from SCID-beige mice bearing MDA-MB-231Luc breast tumor xenografts. Two million MDA-MB-231Luc cells (Xenogen Corp., Alameda, CA, USA) were implanted in the dorsal region of female SCID-beige mice (Charles River Labs). When tumors were in the range of 200 to 600 mm3, two mice (per group) received an intra-peritoneal injection of 15 mg/kg docetaxel or vehicle 22 hours prior to intra-peritoneal administration of non-radioactive FLT (210 μg/kg; approximately 22 nanomoles).
LC-MS/MS quantification of FLT and FLT-MP from xenograft tumors and liver tissues
HCT116 xenograft tumors and liver samples were excised and homogenized in methanol at a 3:1 ratio (v/w) using a Mini BeadBeater (BioSPEC Bartlesville, OK, USA). Analytes were extracted from the sample matrix via protein precipitation, through the addition of 250 μL of methanol containing internal standards to 100 μL of reference standard samples, quality control samples, and tissue homogenates
. After 15 minutes of centrifugation at 3200 × g and 4°C, the supernatant was transferred to a 1-mL 96-well injection plate, dried under nitrogen at ambient temperature, and reconstituted with 50 μL of injection solvent composed of 0.5mM ethylenediaminetetraacetic acid, and 1 mM citric acid. Chromatographic separation was carried out on a reverse phase analytical column (Phenomenex AQ C18, 100 × 2 mm, 5 μm) coupled with Shimadzu LC-20AC pumps and a SIL-20AC auto-sampler. The mobile phase consisted of solvent A (0.1% formic acid) and solvent B (acetonitrile/0.1% formic acid) at a flow rate of 0.2 mL/minute. A linear gradient of 5 to 30% solvent B was applied during the first 3 minutes. Within 1 minute, solvent B was increased to 70% and held for an additional 0.5 minutes. At the end of the run, solvent B was decreased to 5% in 0.2 minutes, and held at 5% for 2.3 minutes for column re-equilibration. Analytes were then detected on a triple quadrupole mass spectrometer (API4000, Applied Biosystems, Foster City, CA, USA) by monitoring their specific precursor and respective product ions in positive electrospray ionization mode. The mass transitions for FLT, FLT-MP and their correspondent internal standard d3-FLT and d3-(FLT-MP) were 245.1 → 127.1, 325.3 → 81.2, 248 → 130.2, and 328.2 → 81.2 respectively. For HCT-116 xenograft tumor extracts, FLT-MP peak areas and FLT mass (ng/g tissue) were quantified using d3-FLT as an internal standard.
NIMS xenograft tumor activity imaging
Two hours after FLT administration, MDA-MB-231Luc tumor-bearing animals were sacrificed for tumor harvest, and tumors were frozen in optimum cutting temperature medium (OCT; Sakura Finetek, Torrance, CA, USA) for cryo-sectioning. Frozen OCT-embedded tumors were sliced into 4-μm sections with a cryostat (Leica Microsystems, Buffalo Grove, IL, USA), placed on the NIMS substrate, and immediately dried in a desiccated, room-temperature vacuum chamber. Adjacent tumor sections were collected and placed on glass tissue fixation slides for immunofluorescence microscopy and H&E staining. NIMS imaging data was acquired at 50-μm spatial resolution with an AB/SCIEX 5800 mass spectrometer (Applied Biosystems) in negative-mode across the entire tumor area. Tumor sections from docetaxel-treated and control mice were placed on a single NIMS substrate. A second NIMS chip was used to image tumor sections from the second set of docetaxel-treated and control mice. From the NIMS imaging data, an FLT-MP/FLT ratiometric image was calculated in an identical manner as was done for FLT treated single-cell analysis. In addition, median normalized total-ion intensity, FLT extracted-ion intensity, and FLT-MP extracted-ion intensity images with 4-fold nearest neighbor interpolation were generated in Matlab.
Optical imaging of xenograft tumors
Double indirect immunofluorescent staining was performed on OCT-embedded, cryostat sections of MDA-MB-231Luc tumor samples. Sections were air-dried, then baked at 37°C for 5 minutes. Sections were fixed in 10% neutral buffered formalin for 10 minutes. Following fixation, sections were washed in 10X Bonds Wash Solution (Leica Microsystems, AR9590) for 5 minutes then blocked in 10% Normal Donkey Serum (#017-000-121, Jackson Immunoresearch, West Grove, PA, USA) for 30 minutes at room temperature. The primary antibodies; anti-Luciferase (#G745A, Promega, Madison, WI, USA, dilution 1:200) and anti-TK1 (ab76495, Abcam, Cambridge, MA, USA, dilution 1:100), were applied to the tissue sections and incubated overnight at 4°C. Sections were washed in Bonds Wash Solution followed by the application of the fluorescent conjugate secondary antibodies applied for 1 h: Alexa Fluor 594 donkey anti rabbit (#A21207, Invitrogen, Carlsbad, CA, USA) and Alexa Fluor 488 donkey anti goat (Invitrogen, #A11055). DAPI (4′6- Diamidino-2-phenylindole dihydrochloride, Sigma #32670-5MG-F) was applied for 5 minutes at a dilution of 1:5000 from a working concentration of 5mg/mL as a nuclear counter stain. Slides were overlaid with DAKO Fluorescent mounting medium (S3023, Dako, Carpinteria, CA, USA) for imaging. All immunofluorescent images were generated using the Vectra™, a multispectral microscope slide analysis system (Perkin-Elmer, Waltham, MA, USA), equipped with epi-illumination, a Nuance™ camera, and appropriate filters. Filter cubes used included: (1) UV excitation; 350 nm+/− 25 nm 420 nm long pass emission; (2) blue excitation; 470 nm +/− 20 nm 515 nm long pass emission; (3) green excitation; 535 nm +/− 20 nm 610 nm long pass emission.