Mice, tumors, cell line, drugs testing
DBA/2 mice, aged 8 to 12 weeks, were obtained from a local breeding facility SRICIM, Vilnius, Lithuania. Mice were used and cared for in accordance with the Guide for the Care and Use of Laboratory Animals. All research protocols were approved by the Institutional Animal Care Committee. Animals had ad libitum access to pelleted feed, food supplements and water. All animals were determined specific pathogen free. Dox or dox-dgr were injected iv via retroorbital plexus as a single push at a dose of 15 mg/kg. Light inhalation anesthesia with isoflurane was used for iv injections and blood sampling procedures. The SL2 lymphoma occurred as a spontaneous tumor in a DBA/2 mouse of the Chester Beatty Research Institute, UK, and was acquired through prof. Den Otter, University Medical Center Utrecht, Netherlands. SL2 cells were maintained in RPMI-1640 medium (Thermo Fisher scientific, Waltham, USA) containing 10% heat-inactivated fetal bovine serum (Thermo Fisher scientific, Waltham, USA). All tumors in our TSDR study were induced by ip injection of 5x105 cells per mouse. The therapeutic efficacy of dox and dox-dgr was tested by injecting drug iv at specific time intervals (24 hr, 48 hr, 120 hr, 168 hr and 192 hr) following ip implantation of 5x104 cells on day 0.
Drugs and reagents
Doxorubicin hydrochloride (2mg/ml) (Ebewe, Unterach, Austria) was obtained from a hospital pharmacy. Daunorubicin hydrochloride (analytical grade), chloroform (HPLC grade). Acetonitrile, orthophosphoric acid and hydrochloric acid were purchased from Sigma Chemical Company (St Louis, MO, USA) and HPLC grade methanol was purchased from Carl Roth GmbH (Karlsruhe, Germany).
Two step tumor dormancy/recurrence (TSDR) model
The time course of dox-loaded viable SL2 cell ip implantation into DBA2 mice is shown in Fig 1. SL2 cells were cultured for 48 h to obtain 5x107 cells (viability > 95%). Then the suspension was washed and incubated for 30 min. within RPMI-1640 medium containing 10% of fetal bovine serum (FBS) and variable amounts of dox or dox-dgr (10 µg/ml, 1.0 µg/ml, 0.5 µg/ml, 0.1 µg/ml, 0.05 µg/ml). Following 30 min. of incubation, the media containing dox or dox-dgr was washed with phosphate buffered saline (PBS) and put on ice before the transplantation procedure. Then SL2 dox loaded cells were diluted in PBS and a total of 5x105 cells per mouse (inoculation volume 0.2 ml, viability > 95%) were injected ip. The viable cell number was determined by performing trypan blue stains on the cells. Control mice received the same number of cells exposed to RPMI-1640 medium containing no dox. Total time of the manipulation procedure from cell harvesting to inoculation into mice did not exceed 60 min. The number of surviving mice in each experimental group was checked daily until day 30, then checked twice a week until day 60. All mice that died had an evident ascitic tumor. Mice surviving more than 60 days were considered as cured.
Immunophenotyping
SL2 cells were centrifuged at 300 g for 5 min at 20 °C and washed twice with FACS buffer (Becton-Dickinson, San Jose, CA, USA). For flow cytometry (FCM) analyses, 1 x 106 cells per sample were incubated with FACS buffer containing 0.1 µg of anti-mouse FcgIII/II receptor (clone 2.4G2) for 20 min at 40C. Cells were then washed with FACS buffer and staining was performed using 0.4 µg APC anti-CD3 (clone 17A2), 0.5 µg of FITC anti-CD4 (clone GK1.5), 0.5 µg of PerCP anti-CD8a (clone 53-6.7), 0.02 µg of PE anti-CD44 (clone IM7), 2 µg of Pacific blue anti-CD45 (clone 30-F11). All antibodies were purchased from Thermo Fisher scientific (Waltham, USA). Cells were stained at 4 °C for 30 min and then washed with FACS buffer. At least 10,000 cells were acquired on a FACS-LSRII flow cytometer (Becton-Dickinson, San Jose, CA, USA) and analysis was performed using FlowJo 8.6.3 software (Tree Star, Ashland, OR, USA).
Flow cytometry analysis for dox and dox-dgr uptake
Cells loaded with dox or dox-dgr were washed for 30 min. twice with PBS and resuspended in 1.0 ml of PBS. Fluorescence histograms were then recorded with a FACS LSRII flow cytometer (Beckton Dickinson, San Jose, USA) in FL2 channel with 488 nm excitation. Mean channel fluorescence intensity (FL 2 height) was used as an uptake value. Non-viable cells and duplets were excluded by gating out their light scatter characteristics. A total of 20,000 events to generate each histogram were acquired.
Confocal microscopy and spectral analysis
Following exposure to dox and dox-dgr, SL2 cells suspended in PBS were made adherent to glass coverslips placed at the bottom of a 12-well culture plate (TPP AG. Trasadingen, Switzerland) via 30 min. of gravity sedimentation as described by Tsang et al. (12). Adhered cells were fixed in 4% paraformaldehyde and then analyzed. The localization of dox and dox-dgr in SL2 cells were analyzed under a confocal microscope (Nikon Eclipse TE2000-S, C1 plus, Nikon, Tokyo, Japan) by scanning with the argon ion laser (488 nm) using oil immersion 60× NA 1.4 objective (Plan Apo VC, Nikon, Japan). Brightfield microscopy was performed to visualize morphology of the cells. The fluorescence of dox and dox-dgr was detected using a 515/30 bandpass filter (Semrock Inc., USA). In addition, spectrally-resolved intracellular fluorescence of dox and dox-dgr was registered using a microscope 32-channel spectral imaging unit. The fluorescence spectra of dox and dox-dgr in single cells and subcellular regions was analyzed. In the region of interest (ROI) in the stacked confocal images, cell fluorescence was measured. The images were further processed using EZ-C1 v3.91 (Nikon, Japan) and ImageJ v1.53a software (NIH, USA).
Optical characterization
Steady-state absorption and fluorescence spectra of doxorubicin in PBS were measured with a single-beam spectrophotometer Varian Cary Win UV (Varian Inc., Australia) and spectrofluorometer (FLS920, Edinburgh Instruments, Livingston, UK). Polystyrene cuvettes with an optical path length of 1 cm were used for all measurements.
Blood sampling, plasma preparation, complete blood counts
Blood samples (125 µL) were taken from the retroorbital plexus before and after the dox administration at several time points 5 min, 15 min, 30 min, 60 min, 6 hr, 12 hr, 48 hr and 72 hr in tubes containing EDTA as an anticoagulant. Dox-dgr measurements were not performed due to imprecision of the procedures (presence of byproducts with HPLC retention times close to dox and overlapping with internal standard). Blood sampling and dox injection were performed on two contralateral retroorbital plexus, while isoflurane anesthesia was applied. Each blood sample was gently inverted several times to ensure complete mixing with the anticoagulant. After centrifugation at 5000 x g for 10 min, plasma samples were separated and stored at – 20 °C until analysis. Complete blood counts (CBC) were analyzed using ABX Micros ESV 60 within an hour of sampling.
Pharmacokinetics
The microvolume method (13) was applied in this study for plasma Dox analysis. 50 µL of internal standard solution (800 ng/mL Daunorubicin hydrochloride) was added to 60 µL of a plasma sample and vortex-mixed for 30 seconds. The extraction of the drug was performed by adding 900 µL of a chloroform/methanol mixture (4:1, v/v). After vortex mixing for 10 min and centrifugation (10 min, 10,000 x g), the organic phase was collected, transferred to a clean tube, then evaporated to dryness under a stream of nitrogen. Dry residue from the plasma was dissolved in 60 µL of mobile phase, and after centrifugation for 5 min (10,000 x g), injected into the chromatographic column. The area under the concentration curve (AUC) were calculated by trapezoid rule.
Chromatographic conditions
High performance liquid chromatography (HPLC) analysis was performed on a Perkin Elmer system that consisted of a Flexar binary LC pump, a Kit – Flexar 3 CHNL VAC degasser, a Flexar LC autosampler and Flexar fluorescence detector (Xenon lamp). The chromatography data was acquired by Chromera software from Perkin Elmer. The chromatography separation was performed on a Brownlee Bio C18 column (4.6 × 150 mm, 5 μm particle size, Perkin Elmer, Shelton, USA). The optimum mobile phase consisting of acetonitrile and water (32:68, v/v), was pH adjusted to 2.6 with 85% orthophosphoric acid. Samples were delivered via isocratic flow at a rate of 0.25 mL/min. The column temperature was maintained at 37 °C and excitation and emission wavelengths were set at 475 and 555 nm respectively. The injection volume was 50 µL.
Statistical analysis
The statistical tests were performed using STATISTICA 12.0 (TIBCO Software Inc Palo Alto, California, USA).
All the results are presented as means and standard error (mean ± S.E.). Significance was considered at values of p < 0.05.