Female BALB/c nude mice aged 5–6 weeks were purchased from Charles River Laboratories, Japan. All animals were treated in accordance with the guidelines approved by the Committee on Animal Experiments at Tohoku University. All surgical processes were performed on mice under anesthesia with the subcutaneous injection of a combination of 0.3 mg/kg of medetomidine, 4.0 mg/kg of midazolam, and 5.0 mg/kg of butorphanol.
Cell culture and drugs
Hepa1-6 cells, derived from murine hepatoma, were purchased from RIKEN BRC, Japan. The cells were cultured in Dulbecco’s minimum essential medium (DMEM) containing 10% fetal bovine serum and incubated at 37 °C in a mixture of 5% CO2.
Lenvatinib was obtained from Chemscene (Monmouth Junction, NJ), and sorafenib was obtained from Toronto Research Chemicals (North York, Canada). Lenvatinib was dissolved in 0.5 w/v% methylcellulose for in vivo studies and in dimethyl sulfoxide (DMSO) for the in vitro studies. Sorafenib was dissolved in DMSO for both in vivo and in vitro studies.
Cell experiments in vitro
Hepa1-6 cells were seeded at a density of 5 × 104 cells in 35-mm well plates and were treated the next day with various concentrations of lenvatinib (1–30 μM; dissolved in 0.1% DMSO), sorafenib (0.3–30 μM; dissolved in 0.1% DMSO), or control medium (0.1% DMSO). After 24 h, 48 h, and 72 h, the cells were counted under a microscope (n = 3).
Preparation of the tumor-bearing mouse models
Hepa1-6 cells (1 × 107) suspended in DMEM were subcutaneously transplanted into the right flanks of mice. Approximately seven days after the injection, tumor-bearing mice with tumor sizes of 150–200 mm3 were randomly assigned to one of five groups: control group (orally treated with saline), sorafenib groups (30 mg/kg and 50 mg/kg, dissolved in 10% DMSO), or lenvatinib groups (3 mg/kg and 10 mg/kg, dissolved in methylcellulose). Saline, sorafenib, and lenvatinib were administered via oral gavage once daily (Fig. 1a). The tumor dimensions were measured using a caliper every alternate day and the tumor volume was calculated using the following formula:
Tumor volume =1/2 × (long axis) × (short axis) 2.
Day 1 was the first day of drug administration and the mice were treated with saline, sorafenib, or lenvatinib until day 4 or day 14. On day 4 or day 15, the mice were anesthetized and injected intravenously with AuNPs, followed by μX-ray CT imaging. After the CT imaging, the mice were euthanized, and tumors were harvested and fixed.
To administer lenvatinib and radiation combination therapy, tumor-bearing mice (tumor size 150–200 mm3) were randomly assigned to one of four groups: control group (with saline and without irradiation), irradiation group (with saline and with 6 Gy irradiation on day 4), lenvatinib group (with 3 mg/kg lenvatinib and without irradiation), and combination group (with 3 mg/kg lenvatinib and 6 Gy irradiation on day 4). On day 15, the mice were scanned using μX-ray CT and their tumors were harvested after euthanasia.
Fabrication of AuNPs as a contrast agent
The AuNPs were synthesized according to the method described in our previous study . Briefly, 99.4 mg of HAuCl4 (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan, 99%) was dissolved in 233 mL ultrapure water and heated to a boil. The HAuCl4 solution was vigorously stirred, and 28 mL of 39 mM sodium citrate was added to the solution under constant stirring. The sample was boiled for 30 min. After cooling the sample solution to 25°C, 99.4 mg of HS-PEG-COOH (thiol carboxylic polyethylene glycol (PEG), MW 5000, Nanocos Inc., New York, NY) was added to the sample and incubated at 25°C for 12 h, with constant stirring to modify PEG chains on the surface of AuNPs.
μX-ray CT imaging was performed using a μCT scanner (SkyScan1176, Bruker, Billerica MA). The CT system can minimize motion artifacts when scanning live mice administered inhalation anesthesia with isoflurane. CT images were acquired at 50 kVp and 490 µA. The CT image size was 18 × 18 µm and the slice thickness was 18 µm. An aluminum 0.5-mm compensating filter was used. To evaluate enhancement by the contrast agent, the quantification of CT signals was expressed in Hounsfield units (HUs). The CT data were analyzed based on the HUs in a region of interest.
μX-ray CT imaging with AuNPs
Tumor-bearing mice were anesthetized and injected intravenously with 200 μL of 1.0 M 15 nm AuNPs. CT scanning was then performed. The acquired CT data were reconstructed using NRecon software (Bruker) and displayed in 3D using CTvox software (Bruker). The 3D blood vessel images were displayed using maximum intensity projection. The tumor area was manually set to the regions of interest using CTAn software (Bruker) and tumor and vessel volumes were calculated. The tumor blood vessel volume was calculated by considering regions with CT values of ≥570–600 HU to indicate a blood vessel as tumor CT values scanned with plain CT were 80–100 HU and voxels over 570–600 HU comprised only 0.1% of all tumor voxels. In this protocol, the CT images were visualized and analyzed in vessels measuring over 50 μm.
Mouse tissue samples were immediately frozen in Tissue-Tek® O.C.T. compound (SAKURA, Tokyo, Japan) or fixed in 10% formalin and prepared in paraffin. OCT compound-embedded frozen samples were cut into 15-μm thick sections while paraffin-embedded samples were cut into 3-μm thick sections. Immunofluorescence analysis was performed to evaluate the area densities of the tumor microvessels, vascular normalization, and tumor hypoxia. The frozen tissue sections were fixed in 5% paraformaldehyde for 15 min and the paraffin-embedded tissue sections were deparaffinized in xylene and hydrated with a graded alcohol series and distilled water. These tissue slides were blocked using 5% goat or donkey serum for 1 h. The sections were then incubated with the following primary antibodies: anti-CD31 (1:200, Angio-Protemie, Boston, MA), anti-α-smooth muscle actin (SMA) (1:100, Abcam, Cambridge, UK), or anti-pimonidazole (1:50, Hypoxyprobe Inc., Burlington, MA) antibody overnight at 4 °C. The tissues were subsequently washed with phosphate-buffered saline (PBS) and incubated for 1 h at 25°C with a second antibody (diluted 1:400) conjugated to AF488, AF568, or Cy5 (Abcam). The cell nuclei were counterstained with 4', 6-diamidino-2-phenylindole (DAPI). The slides were observed under BZ-X800 fluorescence (KEYENCE, Osaka, Japan) or laser confocal LSM 780 (Zeiss, Oberkochen, Germany) microscopes.
Tumor vessel perfusion was quantified on tumor cryosections following intravenous injection with 50 μg of AF649-labeled Lycopersicon esculentum lectin (Vector Laboratories, Burlingame, CA) into tumor-bearing mice. The area densities of the tumor microvessels, percentages of vessels covered with pericytes (α-SMA and CD-31 colocalization area/CD-31 localization area), and percentages of perfused vessels (lectin positive area/CD-31 positive area) were captured in five fields at 20× magnification. Tumor hypoxia was determined using pimonidazole. Pimonidazole (60 mg/kg) was injected intraperitoneally and the tumor was harvested after 60 min. The tissue sections were analyzed by immunofluorescence with anti-pimonidazole antibody (1:100). All immunofluorescence images were analyzed using ImageJ software.
Hematoxylin and Eosin (H&E) staining was performed to evaluate tumor necrosis and organ dysfunction. The H&E-stained slides covered the entire tissue section at 20× magnification. For quantification of the necrotic area, all images were stitched using a BZ-X800 microscope to obtain an image of the entire tissue section. The percentage of hematoxylin-stained tissue was defined as the viable area. The necrotic areas were calculated using ImageJ software.
To evaluate numbers of microvessels and Ki-67-positive cells, paraffin-embedded tissues were deparaffinized and antigen retrieval was performed in 10 mM sodium citrate buffer for 5 min in a 121 ℃ autoclave. After antigen retrieval, 5% goat serum was used for blocking at 25 °C for 1 h and the sections were incubated with primary antibodies (CD31, 1:50; Ki-67, 1:100) (Abcam) overnight at 4 °C. The sections were then stained with 3,3'- diaminobenzidine tetrahydrochloride (DAB) and counterstained with hematoxylin.
MVD was determined as previously described . Briefly, two researchers independently calculated the MVD. Microvessels were defined as vascular regions surrounded by CD31-positive endothelial cells or cell clusters immunostained with anti-CD31 antibody and DAB, which were clearly separated from the surrounding tumor and stromal cells. The sections were screened at a lower magnification (10×) to identify five vascularized areas. Within the selected areas, microvessels were counted under high magnification (40×). MVD was the average number of microvessels in the five fields.
Ki-67 was quantified using a method similar to that described above for the MVD analysis. The sectioned samples were screened at lower magnifications to identify the representative areas. The numbers of DAB-positive nuclei were counted and compared to the total numbers of nuclei under high magnification. Five fields were calculated for each tumor and the percentage of Ki-67-positive cells was the average number from five fields.
IFP measurement with the wick-in-needle technique
IFP of the tumor was measured in the tumor-bearing mice using the wick-in-needle technique after lenvatinib, sorafenib, or control treatment [32, 33]. Briefly, a 23-gauge needle filled with multifilamentous nylon thread was connected to a transducer and amplifier (ADInstruments, Dunedin, New Zealand). The tumor-bearing mice were anesthetized and fixed in a position. The needle was then inserted into the HCC tumor 2 mm from the surface and left in place until the measured pressure had stabilized. The IFP was measured in at least two different tumor regions.
X-ray irradiation was performed using a device to administer a dose of 6 Gy (80 kV, 1.25 mA, MX-80Labo, mediXtec Japan (Chiba, Japan).
JMP software (SAS institute, Cary NC) was used to perform general statistical analyses. All measurements are expressed as means ± the standard error of the mean (SEM). Student’s t-tests and one-way analysis of variance test, followed by Tukey multiple comparison test were used for comparisons between two groups and multiple groups. P＜0.05 was considered statistically significant.