2.1 Cell lines and culture
HT-1080 (a fibrosarcoma cell line) was cultured in minimum essential medium (MEM) (Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS) (Biological Industries, Cromwell, CT, USA), 100 U/mL penicillin, and 100 mg/mL streptomycin (Invitrogen, Carlsbad, CA). Human pancreatic epithelioid carcinoma cells (PANC-1), human embryonic kidney cells 293T (HEK 293T), and HT-29 (a human
colorectal adenocarcinoma cell line with epithelial morphology) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Thermo Fisher Scientific, Waltham,
MA, USA) supplemented with 10% FBS, 100 U/mL penicillin, and 100 mg/mL streptomycin (Thermo Fisher Scientific, Waltham, MA, USA). PANC-1 was purchased from Food Industry Research and Development Institute (Taipei, Taiwan), HT-29, HEK293T were kindly provided by Professor Ming-Jium Shieh (National Taiwan University). All cells were maintained in a humidified atmosphere containing 5% CO2
at 37°C. HEK 293T, HT-1080, PANC-1, and HT-29 passaged with 0.5% trypsin (Thermo Fisher Scientific, Waltham, MA, USA) for 0.5, 2,4, and 10 min, and the ratios for passage were 1/4, 1/6, 1/2, and 1/6, respectively. The cell preservation was frozen at -80 °C in the concentration of 1x106 per 100μL Bambanker (Lymphotec Inc., Japan).
2.2 Vector construction and cell transfection and transduction
The lentiviral vector, pWPXL-MCS-PuroR, was a generous gift from Dr. Ming-Jium Shieh and the GFP control plasmid was from Dr. Yong-Chong Lin in National Taiwan University. Besides, pLAS5w.PtRFP-I2-Puro (Fig. 1B) was purchased from the RNAi consortium at Academia Sinica. OATP1B3 sequence was cloned from the Mammalian Gene Collection (MGC) full-length cDNA clone (Open BioSystems, Lafayette, CO, USA). Subsequently, OATP1B3 was amplified and cloned into the pWPXL-MCS-PuroR and pLAS5w.PtRFP-I2-Puro plasmid and the final construct were named pWPXL-OATP1B3-ires-Puro (Fig. 1C) and pLAS5w.OATP1B3-new-I2-Puro (Fig. 1D). The SLC10A1 sequence (NM_003049) and SLC10A2 sequence (NM_ 000452) were cloned from plasmids {RC210241L2 (Fig. 1F) and RC221202L2 (Fig. 1G);
Origene, Rockville, MD, United States}. Subsequently, the SLC10A1 and SLC10A12 sequences were amplified and cloned into the pWPXL-MCS-PuroR plasmid, and the final constructs were named pWPXL NTCP-ires-Puro (Fig. 1E) and pWPXL ASBT-ires-Puro (Fig. 1G), respectively. In total, 3.5 × 106 293T cells were seeded on a
10-cm2 dish one day before transfection. Before transfection, the medium was changed with fresh culture medium 1 h. Cotransfection was performed with target plasmids, psPAX2, and pMD2.G plasmids in a ratio of 4.5:3.6:0.9 (9 μg in total) by using PolyJet (SignaGen, Rockville, US) with vortexing or pipetting to mix homogenously and maintained in a still position for 15 min. Next, well-mixed transfection reagents were added to the cells and incubated at 37°C. At 16 h after transfection, the transfection medium was replaced with a culture medium. Viruses were collected at 30 h after transfection and were filtered through a 0.45-μm filter (Millipore-Sigma, Billerica, MA, USA). To infect cells with viral particles, 3 × 105 PANC-1 cells, 3 × 105 HT-1080 cells, and 2 × 105 HT-29 cells were seeded in the 6-well plates for one day and transduced at a multiplicity of infection (MOI) of 50, 5, and 5, respectively. The infection process was performed through centrifugation at 2000 rpm at room temperature for 30 min. Cells were selected using 2 to 4 µg/mL puromycin (Millipore-Sigma, Billerica, MA, USA) for 10 days and change the medium every 2 days after infection. Cells without transduction were served as blank control. Cells with pLAS5w.PtRFP-I2-Puro transduction were served as vector control.
2.3 Cell viability
The good indicator for cell viability is 3-[4,5-dimethylthiazol-2-yl]-2,5- diphenyltetrazolium bromide (MTT) which turns to purple formazan in living cells
through mitochondrial reductase58. The PANC-1control cell and OATP1B3-expressing PANC-1 cell number was 1 × 103 cells to seed in 96-well plates for 3 days and treated 0.5 mg/mL MTT for 1 h. Subsequently, the medium was removed from 96-well plates carefully and replaced 100μL dimethyl sulfoxide (DMSO) to dissolve MTT. The plate was detected by SpectraMax® M5 (Molecular Devices, Sunnyvale, CA, USA), the filter is 590nm after 1 min shaking at 200 rpm.
2.4 Reactive oxygen species reaction
For the adaptation of environmental stress, reactive oxygen species (ROS) are produced through mitochondrial oxidative metabolism or cellular response59. Oxidative stress occurs when excess ROS or oxidants imbalance the homeostasis of antioxidant response59. After 1 × 106 cells were seeded in 6-well plates for one day, cells were treated with 10μM H2DCFDA (Millipore-Sigma, Billerica, MA, USA) in 1 mL of DMEM for 30 min at 37 °C. Then, the cells were washed three times with phosphate buffered saline (PBS) and collected. Cells which treated with 10uM H2O2 for 2 h before H2DCFDA treatment regards as a positive control. The fluorescent intensities were detected and quantified by SpectraMax® M5 (Molecular Devices, Sunnyvale, CA, USA)
2.5 Mitochondria membrane potential
Mitochondria membrane potential is an indicator of the apoptosis60. The apoptosis
signaling pathway will be triggered while cells under a toxic or unfavorable environment. Mitochondria membrane potential will decrease during apoptosis. Thus, it could be served as an apoptosis indicator61. On day 1, 6-well plates were cultured 4 × 105 cells. Cells which treated with 1μg/mL doxorubicin from day 2 to day 5 were
regarded as a negative control. Subsequently, cells were trypsinized and treated with 3,3’-dihexyloxacarbocyanine (DiOC6(3), Millipore-Sigma, Billerica, MA, USA) at
37°C for 30 min in a cell culture incubator on day 6. The cells were washed and suspended in PBS for the measurement of fluorescent intensities (FITC as the filter) by FACSCalibur (BD Biosciences, San Jose, CA, USA).
2.6 Differentiation Capacities
Hardikar et al have introduced the PANC-1 cells into hormone-expressing islet-like cells62. Generally, 1 × 106 cells were sprayed in 6-well plates using DMEM/F12 (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with insulin, transferrin, selenium solution (ITS-G) (Thermo Fisher Scientific, Waltham, MA, USA), 100 U/mL penicillin, and 100 mg/mL streptomycin (Thermo Fisher Scientific, Waltham, MA, USA) at day 0. The medium was replaced every 2 or 3 days. Cells were harvested at day 4 and day 7 for the analysis of differentiation.
2.7 Quantitative-PCR
In total, 2 to 20 x 105 cells were used for the RNA extraction by Direct-zol RNA Kits
(ZYMO RESEARCH, CA, USA). One or 2 μg RNA was reversely transcribed to cDNA by ReverTra Ace® qPCR RT Kit (TOYOBO, Japan). After reverse transcription, cDNA was diluted 1/5 fold with ddH2O. TaqMan master mix (Thermo Fisher Scientific, Waltham, MA, USA) was mixed with 2 μL diluted cDNA and specific primers to final volume 20 μL. Q-PCR was performed by Applied Biosystems 7900HT Fast (Thermo Fisher Scientific, Waltham, MA, USA). The analysis of relative expression differences was through the 2-∆∆CT method and samples were normalized to beta-actin and relative to the vector control 63. The primers are shown in supporting information Table S1.
2.8 Western blotting
Cells were lysed in buffer containing protease
inhibitor cocktail (Roche, Mannheim, Germany). Protein concentrations were measured using the Pierce™ Coomassie Protein Assay (Bradford, Thermo Fisher Scientific, Waltham, MA, USA). Equal amounts of proteins (40 µg/lane) were loaded in 10%
sodium dodecyl sulfate (SDS)-polyacrylamide gel for 20 min at 80 V and 60~80 min (depending on the size of protein) at 110 V, and electrotransferred onto phosphatidylcholine membranes (Sartorius, G¨ottingen, Germany) for 2 h at 200 mA.
The membranes were blocked with 5% nonfat powdered milk in Tris-buffered saline-Tween 20 (TBST). Subsequently, the blots incubated with the primary antibodies against OATP8 (1:1000, Santa Cruz, CA, US), RFP (1:1000, MA5-15257, Thermo
Fisher Scientific, Waltham, MA, USA), β-actin (1:5000, MA5-15739, Thermo Fisher
Scientific, Waltham, MA, USA), NTCP (1:1000; Thermo Fisher Scientific), α-tubulin (1:5000; Merck Millipore, Burlington, MA, USA), and GAPDH (1:5000; Cell Signaling Technology, Danvers, MA, USA) separately at 4°C overnight. Afterward, membranes incubated with 1:5000 HRP-conjugated rabbit/mouse anti-IgG for 1 h at room temperature. Detection of protein bands was run by enhanced chemiluminescence (Millipore-Sigma, Billerica, MA, USA) using the BioSpectrum® 810 Imaging System (UVP, CA, US).
2.9 Immunofluorescence
Cells (2 × 104) were seeded in the 8-well chamber slide for one day and fixed with 4%
formaldehyde and penetrated with 1X phosphate-buffered saline with Tween-20 (PBST) for 1 h. After washed with PBST, cells were blocked with 5% bovine serum albumin (BSA) (FocusBio, Dunedin, New Zealand) at room temperature for 1 h. Afterward, the samples were incubated with 1:100 rabbit polyclonal anti-OATP8 antibodies (Santa Cruz, CA, US), 1:50 anti-insulin antibodies (Novus Biological, US), 1:50 anti-glucagon antibodies (Thermo Fisher Scientific, Waltham, MA, USA), 1:1000 anti-RFP antibodies (Thermo Fisher Scientific, Waltham, MA, USA), 1:100 anti-NTCP antibodies (Thermo Fisher Scientific, Waltham, MA, USA), or/and rhodamine-phalloidin (staining actin;
Invitrogen, Carlsbad, CA, USA) at 4°C overnight. After a short wash in PBST, the
slides were incubated with 1:100~1:200 488-conjugated goat anti-rabbit/mouse IgG antibodies (Thermo Fisher Scientific, Waltham, MA, USA) or/and 1:100~1:200 546-conjugated goat anti-rabbit/mouse IgG antibodies (Thermo Fisher Scientific, Waltham, MA, USA) at room temperature for 1 h. The slides were mounted in SlowFade® Gold Antifade Reagent with 4'-6-diamidino-2-phenylindole (DAPI) (Thermo Fisher Scientific, Waltham, MA, USA) and were visualized using a ZOE Fluorescent Cell Imager (Bio-Rad, Hercules, CA, USA). Negative control was only treated with 5% BSA.
2.10 Immunohistochemistry (IHC)
The entire tumor tissues were collected from mice and sectioned in the same direction as the view shown in the MRI. Samples were fixed with 10% formalin and incubated with series dehydration buffers and wax to be prepared as paraffin-embedded sections (5 µm thick). Slide sections were deparaffinized in Sub-X Xylene Substitute (Leica, USA) and then were incubated in Trilogy (CELL MARQUE, Rocklin, CA, USA) to perform rehydration and antigen retrieval at 121°C for 10 min. Endogenous peroxidase was blocked by 0.3% hydrogen peroxide for 10 min. After washed with PBST, the tissue slides were blocked with 5% BSA at room temperature for 1 h. Subsequently, the slides were incubated with 1:100 rabbit polyclonal anti-OATP8 antibodies (Santa Cruz Biotechnology, Dallas, TX, USA) or 1:1000 anti-NTCP (Thermo Fisher Scientific,
Waltham, MA, USA) at 4°C overnight. After washed with PBST, the slides were treated with the EnVision kit (Agilent Technologies Inc., Santa Clara, CA, USA) and were counterstained with hematoxylin. All cover slides were examined using the ECLIPSE TE2000-U microscope (Nikon, Melville, NY, USA). Negative control was only treated with 5% BSA.
2.11 Apoptosis analysis
Tissue slide sections were deparaffinized in Sub-X Xylene Substitute (Leica, USA) and
were rehydrated in a graded alcohol series ending with water. The slides were stained using a DeadEnd™ Colorimetric Apoptosis Detection System (Promega, USA). The
operation of photo acquisition was described in the IHC section.
2.12 Animal experiments
Female BALB/cAnN.Cg-Foxnlnu/CrlNarl nude mice and severe combined immunodeficient (SCID) mice (aged 6–8 weeks) were purchased from the National Laboratory Animal Center and raised at the animal center of Taipei Tzu Chi Hospital.
All experimental procedures were approved by the Institutional Animal Use and Care Committee of Taipei Tzu Chi Hospital, Buddhist Tzu Chi Medical Foundation (102-IACUC-024, 106-IACUC-004). The mice caring were followed to the recommendations of the Guide for the Care and Use of Laboratory Animals (NIH).
2.13 Xenograft
Six to eight-week-old SCID mice were subcutaneously inoculated 1 ×106 OATP1B3-expressing PANC-1 cells in 100 μL PBS near the hind limb at the right side of the mice and another injection of 1 ×106 PANC-1 cells in 100 μL PBS on the other side. Six to eight-week-old nude mice were performed the same inoculations, except for the cell line was HT-1080. In total, 2.5 × 106 HT-29 control cells and NTCP-expressing cells were injected subcutaneously to the nude mice on the left and right sides, respectively. The cell number used to inoculate control cells and OATP1B3-expressing HT-29 cells was 1.0 × 106. The tumor sizes were measured with a digital caliper before performing fluorescence imaging by using the following formula: [(longest length) × (shortest)2]/2. If a subcutaneous nodule could be visualized (normally it was easier to observe once tumors exceeded 13.5 mm3), the size of the xenograft was recorded and the mice were sent for MRI analysis and fluorescence imaging by using the following formula: [(longest length) × (shortest)2]/2.
2.14 Magnetic resonance imaging (MRI) in vitro
For the confirmation of Primovist intake, 5 × 105 cells (except for PANC-1 which was 1
× 106) were seeded in 6-well plates overnight and were treated with 1.25 mM Primovist (Bayer Pharma AG, Berlin, Germany) for 4 h. Cells were washed three times with PBS before trypsinization. After one more time washed with PBS, cells were centrifuged at
1200 rpm for 5 min in 0.2-mL tubes at 4 °C, the cells were analyzed using a clinical 1.5T MRI system (Siemens Magnetom Aera, Erlangen Germany) or 3T MR system (Signa Excite, GE Healthcare, US).
For the evaluation of the change of MR signal intensity in the treated PANC-1 cells over time, 5 × 105 cells were seeded in 6-well plates overnight and further incubated with
For comparison of MRI contrasts, 1 × 106 cells were seeded in 6-well plates for one day
and were incubated with 1.25 μM Primovist (Bayer Pharma AG, Berlin, Germany) or 1.25 μM gadodiamide (GE Healthcare, Waukesha, Wisconsin, USA) for 4 h. Some
groups were also co-incubated with 100 μM rifampicin, a competitive inhibitor of OATP1B3 and NTCP. Cells were washed 3 times with PBS before trypsinization. And then, cells were washed once more with PBS. After cells were centrifuged at 1200 rpm for 5 min in 0.2-mL tubes at 4°C, the cells were imaged with a clinical 3T-MR system.
The conditions in 1.5T MRI system: Two-dimensional T1-weighted fast spin-echo pulse sequences were repetition time/echo time (TR/TE) =700/20 ms; the scanning slice
thickness was 1.0 mm with a 0.5-mm gap; the field of view (FOV) was 14 cm × 6 cm, and the matrix size was 256 × 112. The conditions in 3T MR system: Two-dimensional T1-weighted fast spin-echo pulse sequences were TR/TE = 550/13 ms; slice thickness was 1.0 mm with a 0.5-mm gap; field of view was 14 cm× 10 cm; matrix size was 288 × 192; the number of excitations (NEX) was 2, and total scan time was 4 min and 5 s.
All images were analyzed using the open-source image processing program (ImageJ) or RadiAnt DICOM Viewer (64-bit) (Medixant, Poznan, Poland). The signal for quantification was circled within the edge of the signal and normalized the circled area to acquire the MR intensity under the unit area for comparison.
2.15 Magnetic resonance imaging (MRI) in vivo
After subcutaneous implantation of cells, the MRI of the nude and SCID mice were
acquired, with the injection of Primovist (0.25 mM, 200 μL) or gadodiamide (0.5 mM, 100 μL) intravenously. Before contrast enhancements administration, mice were taken
an MR imaging as prescan control. Images were acquired using a 7T-MRI (Biospec 70/30; Bruker, Billerica, MA, USA) provided by 7T Animal MRI Core Lab of the Molecular Imaging Center at National Taiwan University. Mice were imaged 1 h after intravenous injection of contrast enhancements. The parameters for acquiring MR imaging were: TR/TE = 841.9464/8.6404 ms; matrix size = 256 × 256, slice thickness = 5 mm, FOV = 5 cm × 5 cm, NEX = 10, total scan time = 3 min and 20 s. The images
were analyzed using an open-source image processing program (ImageJ) or RadiAnt DICOM Viewer (64-bit) (Medixant, Poznan, Poland). For homogeneous signals in the tumor, the signal for quantification was circled within the edge of the signal and normalized the circled area to acquire the MR intensity under the unit area for comparison. For heterogeneous signals in the tumor, the signal was acquired in each unit area using the crossline (Fig. 13A and 13C).
2.16 Inductively coupled plasma mass spectrometry (ICP-MS) to detect Gd
Cells were lysed in 1% SDS after in vitro MRI. The supernatants were gathered after centrifugation at 12000 rpm for 5 min. Afterward, nitrohydrochloric acid was added to the supernatants. After the dilution in 2% nitric acid (1/100X), the samples were filtered with a 0.45 μm filter. Eight standards containing GdCl3 concentrations of 0, 1, 3, 10, 30, 100, 300, and 1000 ppb were for calibration. The Gd concentration of the sample was acquired by 7700 e ICP-MS (Agilent Technologies Inc., Santa Clara, CA, USA)
2.17 Experiments on the cellular uptake of ICG
To test the intracellular uptake ability, 5 × 105 or 1 × 106 cells were seeded in 6-well plates for 1 day and treated with 50 or 300 µg/mL ICG (Millipore-Sigma, Missouri, USA) for 1 h. Cells were trypsinized before washed with PBS 3 times. Afterward, cells were washed once more with PBS. The ICG signal was detected by using a flow cytometer FACSCalibur instrument (BD, CA, USA) with the APC-Cy7 channel (785
nm) filter. In the same procedure mention in the above, ICG was examined by confocal microscopy using a TCS SP5 laser scanning microscope (Leica, Wetzlar, Germany) with a Cy5 filter. Besides, 2*104 cells or 2 × 105 cells were seeded in 96-well black plates for 1 day before adding 0.4, 2, 10, 50, and 200 µg/mL ICG for 1 h and 4 h. After cells were washed 3 times with 1X PBS, the ICG signal was acquired using Spark 10M (Tecan Trading AG, Switzerland) and an in vivo spectrum imaging system (Xenogen, Perkin Elmer, MA, USA). All images were acquired using the same parameters (emission/excitation channel: ICG/ICG; exposure time: 1 min; binning: medium; lens aperture [f/stop]: 2; the field of view: 12 cm). The imaging data were presented in the units of radiant efficiency (p/s/cm2/sr)/(µW/cm2). The quantification of ICG intensity was using the average efficiency in the region of interest (ROI).
For the detection limit of cells in IVIS, 2 × 106 cells were sprayed into 6-well plates one day before the addition of 50 µg/mL of ICG for 1 h. After cells were washed with 1×
PBS three times to remove the excess ICG and were performed series dilution into a 96-well black plate. The ICG signal was acquired using an in vivo imaging system (IVIS Spectrum; Xenogen, Perkin Elmer, MA, USA). All images were acquired using the same parameters mentioned above.
For the evaluation of inhibitors of transporters, 4 × 104 cells were sprayed into 96-well plates one day before the treatments. Cells were pretreated with 1.25 mM Primovist, 2–
10 μM cyclosporin A (Millipore-Sigma, Billerica, MA, USA), emodin (Millipore-Sigma, Billerica, MA, USA), erythrosin B (Millipore-Sigma, Billerica, MA, USA), and 100 μM rifampicin (Millipore-Sigma, Billerica, MA, USA) for 30 min before adding 5 µM ICG for 1.5 h. Subsequently, cells were washed three times with 1× PBS and were detected using Spark 10M (Tecan Trading AG, Switzerland) and the IVIS. The ICG signal intensity was further normalized using data from an MTT assay.
2.18 Fluorescence and bioluminescence imaging in vivo and ex vivo
After the nude and SCID mice were subcutaneously administrated tumor cells for 3
For the drug screening platform in vivo, nude mice were administrated with 1 mg/kg of ICG through intravenous injection 4 h after the oral gavage of 10 and 50 mg/kg of cyclosporin A (solvents: olive oil or cream). Afterward, the ICG signal in the liver was acquired using the IVIS at 15 min, 1 h, and 4 h after ICG injection. For the drug
screening platform ex vivo, mice were euthanized to measure the ICG signal intensity in organs under the IVIS after cyclosporin A injection for 1 h and followed 10 mg/kg of ICG administration for 1 h.
All images were acquired using the same parameters and presented as mentioned in the section of “Experiments on the cellular uptake of ICG”.
2.19 Luciferase assay
The lentiviral vectors AS2w.FLuc.Ppuro (Fig. 1H; the RNAi consortium at Academia Sinica) were used for the viral production. The viral production was mentioned before in the same condition using HT-1080. HT-1080 were transduced FLuc first and OATP1B3 for one week later to obtain FLuc and OATP1B3 double-positive HT-1080.
There were 5 × 104 cells seeded in 96-well black plates for 1 day before the treatment of 94 µM d-Luciferin (Gold Biotechnology, St. Louis, MO). The OATP1B3 inhibitor, rifampicin, was pretreated for 30 mins before the addition of d-Luciferin. The plate was detected using TECAN SPARK 10M (Männedorf, Switzerland) after adding d-Luciferin at once. The RNA levels of FLuc and OATP1B3 were confirmed by reverse transcription-PCR (RT-PCR).
2.20 Statistical analyses
The data were presented as means ± standard errors (SEM) and had at least three biological replicates. Statistical analysis was performed using Student's t-test, Duncan's
new multiple range test, and Newman–Keuls and Dunnett’s multiple comparison tests were conducted to determine differences via GraphPad Prism 5 (* p < 0.05; # p < 0.01;
& p < 0.001).