Synthesis of FA-PEG4000-SH
FA (0.75 mol, 331.05 mg) was dissolved in 10 mL of dried DMSO, EDC (0.48 mmol, 92.02 mg) and NHS (0.48 mmol, 55.25 mg) were rapidly added to the system, and then the system reacted for 2 h at 37 °C in the dark. The above solution was slowly added dropwise into a solution of PEG4000 (0.2 mmol, 800 mg) in DMSO. The reaction continued for 24 h at room temperature. Meanwhile, EDC (0.8 mmol, 153.36 mg) and NHS (0.8 mmol, 92.08 mg) were added to a solution containing 3-mercaptopropionic acid (0.4 mmol, 42.46 mg) in 5 mL of DMSO, which was activated at 37 °C with stirring, and then the solution was added to the above solution. The system continued stirring for 48 h at room temperature. Finally, the reaction stopped and the solution was dialyzed with a dialysis membrane (3.5 kDa) against distilled water for 48 h to remove the solvent and unreacted materials. The free FA in dialysis solution was removed by centrifugation at 16,000 rpm to give a pure pale yellow solution. The pure pale yellow solution was lyophilized to give a pale yellow fluffy solid.
Preparation of water-solution targeting pseudopolyrotaxane (FA-PR-SH)
The pseudopolyrotaxane was formed by self-assembly of carboxylated cyclodextrin (Detailed preparation process was presented in support information) and FA-PEG-SH. The prepared α-CD-COOH (60.8 mg, 0.044 mmol) was dissolved in as little deionized water as possible, then FA-PEG-SH (20 mg, 0.0044 mmol) was added to the above solution, and then the system was sonicated for 10 min. Finally, the reaction continued for 36 h at room temperature in the dark to obtain FA-PR. The reaction solution was processed by dialysis, and the dialysate was lyophilized to collect the product.
Preparation of FA-PR modified AuNR hybrid nanomaterials (AuNR@FA-PR/PEG)
The AuNRs were prepared with the method reported in literature [20] and the detail preparation process was presented in supporting information. The formation of hybrid nanomaterials (AuNR@FA-PR/PEG) was through the Au-S bond of and FA-PR-SH, mPEG-SH and AuNR. Specifically, a solution of AuNR (1 mL, 1 mg mL−1) was added to FA-PR-SH (5 mL, 5 mg mL−1), the reaction system continued reacting with stirring for 24 h at room temperature. After that the reaction was stopped and the solution was centrifuged to get AuNR@FA-PR. The sulfhydrylation of mPEG is mainly carried out by the method reported in the literature [26]. The detailed preparation process was presented in supporting information. The prepared AuNR@FA-PR was dispersed in mPEG-SH (2 mL, 0.75 mg mL−1) solution, and the mixture reacted for 36 h at room temperature, finally the reaction was stopped and the solution was centrifuged to collect precipitation, the precipitation was washed twice with deionized water to obtain AuNR@FA-PR/PEG. AuNR@FA-PR/PEG was suspended in deionized water and stored in a refrigerator at 4 °C.
The morphology and structures of AuNRs and AuNR@FA-PR/PEG
The morphology of AuNR and AuNR@FA-PR/PEG was mainly characterized by transmission electron microscopy (TEM). In detail, the sample was dissolved in distilled water, and dispersed uniformly by ultrasound, and 6 µL of solution is taken out with a pipette. The solution was dropped on ordinary carbon films, and naturally dried at room temperature, finally, the morphology was observed by a transmission electron microscopy.
In order to further prove the successful modification of AuNR, the Fourier transform infrared spectroscopy (FT-IR) of AuNR and AuNR@FA-PR/PEG were detected. In detail, small amount of sample was mixed with dry potassium bromide, and fully ground with an agate mortar. Then the mixture was put in a clean compression mold (spread evenly in the compression mold), and pressed using a tablet press for 1–2 min under a pressure of 20 MPa into a transparent sheet, which can be used for measurement. The prepared materials were also measured by an ultraviolet spectrophotometer (UV-vis). The samples were dissolved in distilled water to prepare the same concentration of an aqueous solution. The distilled water was used as a blank control, and the absorbance at 200–1000 nm was measured.
The preparation of drug-loaded AuNR@FA-PR/PEG (AuNR@FA-PR/PEG/CDDP)
The surface of the AuNR@FA-PR/PEG is rich in carboxyl groups, thus CDDP can be loaded in the hybrid nanomaterials through chelation bonds. The detailed procedure is as follows: 25 mg of AuNR@FA-PR/PEG was resuspended in 5 mL of deionized water, and then 3 mg of CDDP was added into the system, the samples were uniformly mixed by ultrasound. The system was placed on a shaker for 48 h in the dark. After that, the reaction solution was centrifuged, and the precipitation was collected and washed twice with distilled water to remove unreacted CDDP. The product AuNR@FA-PR/PEG/CDDP was obtained and stored at 4 °C for use. Unreacted CDDP was measured by the o-phenylenediamine colorimetric method mentioned in reported reference [19], and the drug loading content was calculated to be about 7.4% by using Eq. (1).
$${\text{DLC }}(\% ) \, = \, \frac{{\text{Weight of drug in hybrid nanomaterials }}}{{\text{Weight of hybrid nanomaterials }}} \, \times \, 100\% \,$$
(1)
The release of CDDP loaded in AuNR@FA-PR/PEG/CDDP
AuNR@FA-PR/PEG/CDDP (74 μg/mL CDDP, 1 mL) was placed in a 3.5 KDa dialysis bag and immersed in 20 mL of PBS solution with various pH values (7.4, 6.5, 5) in 50 mL centrifuge tubes. The centrifuge tubes were placed in a shaker with room temperature at a speed of 150 r/min, and 1 mL of dialysate was taken at predetermined times (0.25, 1, 2, 4, 8, 12, 24, 48, 72, 96, 120, 168 h), and then 1 mL of new PBS solution with different pH value was added into the corresponding centrifuge tubes. Simultaneously, the photothermal effect dependent drug release of AuNR@FA-PR/PEG/CDDP was carried out. The sample was irradiated with an 808 nm laser (1.5 W cm−2) for 10 min in every time stage of drug release. Finally, the absorption value at 703 nm was measured by an ultraviolet spectrophotometer and the drug release was calculated by an accumulation method. The formula is as follows:
$$E_{r} = \frac{{V_{e} \sum\limits_{1}^{n - 1} {C_{i} } + V_{0} C_{n} }}{{m_{\text{drug}} }} \times 100\%$$
(2)
(Where, Er: Cumulative drug release (%); Ve: Sample volume (mL); V0: Total volume of the released medium (mL); Ci: Concentration of the drug in the i th sample (μg/mL); Cn: Concentration of the drug in the n th sample (μg/mL); m: Total amount of drug in the drug-loaded micelle (μg)).
Photothermal performance of AuNR@FA-PR/PEG/CDDP
In order to measure the photothermal conversion capability AuNR@FA-PR/PEG/CDDP, an in vitro photothermal conversion experiment was carried out, which was briefly described as follows: AuNR@FA-PR/PEG/CDDP was prepared into 50 µg mL−1 aqueous solution. 1 mL of the solution was taken and placed in a quartz cuvette. Then the solution was irradiated with an 808 nm laser (1.5 W cm−2) for 10 min, the change in temperature was recorded every 15 s during the irradiation, and the distilled water is used as a blank control group.
To study the relation between the concentration of AuNR@FA-PR/PEG/CDDP and the rate of heat generation, AuNR@FA-PR/PEG/CDDP solution with various concentration (0, 25, 50, 100, 200 µg mL−1) were irradiated with an 808 nm laser (1 W cm−2) for 10 min, and the change in temperature was recorded every 10 s during irradiating. Simultaneously, AuNR@FA-PR/PEG/CDDP (50 µg mL−1) was exposed to an 808 nm laser with various power densities (0.75, 1.5, 2.25, 3 W cm−2), and the temperature change at the scheduled irradiation time point is recorded.
In order to further investigate the photothermal stability of AuNR@FA-PR/PEG/CDDP, the solution of AuNR@FA-PR/PEG/CDDP (100 µg mL−1) was irradiated with an 800 nm laser (1 W cm−2) for four cycles of laser on/off irradiation experiment.
s, and every cycle contained a 10 min NIR-light exposure period followed by a period of cooling to room temperature. All of the temperature changes were recorded with a thermocouple device. The photothermal conversion efficiency (η) was calculated as follows:
$$\eta = \frac{{hS(T_{\max ,M} - T_{S} )}}{{I(1 - 10^{ - A808} )}}$$
(3)
$$t = - \tau_{S} {\kern 1pt} {\text{ln(}}\theta {)}$$
(4)
$$\tau_{S} = \frac{mC}{{hS}}$$
(5)
$$\theta = \frac{{T - T_{S} }}{{T_{{\max ,{\text{M}}}} - T_{S} }}$$
(6)
where m and C are the mass and heat capacity of water, respectively. h and S represent the heat transfer coefficient and the surface area of the container, respectively. I represents the power density of an 808 nm laser. A808 is the absorbance of AuNR@FA-PR/PEG/CDDP at 808 nm. Tmax,M and TS are the maximum temperature during irradiation and room temperature, respectively. τs is the time constant. The photothermal conversion efficiency was calculated as η = 26.35%.
Cell uptake experiment
Firstly, AuNR@FA-PR/PEG/CDDP was labeled with fluorescein isothiocyanate (FICT), and the preparation process was as follows: 1 mL solution of FITC dissolved anhydrous DMSO (1.0 mg mL−1) was added into 10 mL solution of AuNR@FA-PR/PEG/CDDP, and the system was stirred for 24 h at room temperature under dark condition. After that, the reaction was stopped and the mixture was centrifuged to collect the precipitation, then the precipitation was washed three times with distilled water to remove the unreacted FITC, finally, the FITC-labeled AuNR@FA-PR/PEG/CDDP was redispersed in distilled water for cellular uptake use.
HepG2 cells maintained in DMEM supplemented with 10% fetal bovine serum (FBS), streptomycin (100 U/mL), penicillin (100 U/mL) culture medium were inoculated into a 6-well plate, and placed in a 5% CO2 incubator at 37 °C for 24 h. Then, the medium was discarded, and 2 mL of medium solution containing 240 μg of FITC-labeled AuNR@FA-PR/PEG/CDDP was added into the plates and further incubated for 4 h. The medium was aspirated and the cells were washed three times with PBS to remove the free FITC-labeled AuNR@FA-PR/PEG/CDDP. 4% paraformaldehyde was added into the plates and fixed for 10 min, and then the cells were washed three times with PBS and stained with nuclear dye DAPI for 15 min in the dark. Finally, the cells were observed with a confocal laser scanning microscopy (CLSM).
Cytotoxicity and photothermal cytotoxicity of AuNR@FA-PR/PEG
The cytotoxicity and photothermal cytotoxicity of AuNR and AuNR@FA-PR/PEG were investigated by 1-(4,5-dimethylthiazol-2-yl)-3,5-diphenyl-formazan (MTT) assay. Human liver cancer cells (HepG2) were selected as a cell experimental model. DMEM containing 10% fetal bovine serum was used as a medium, and HepG2 cells were placed in a 96-well plate at 7000 cells per well, 90 µL medium was added into every well and cultured for 24 h, and then 10 µL various concentrations (10, 30, 60, 90, 120 µg mL−1) of AuNR, AuNR+Laser, AuNR@FA-PR/PEG and AuNR@FA-PR/PEG+Laser were added into plates, each concentration was designed four replicate wells. The cells in AuNR and AuNR@FA-PR/PEG treatment groups continued culturing for 48 h. After that, MTT in PBS solution (50 µL, 1 mg mL−1) (tetramethylazozolium salt) was added into per well, and the cells continued incubating for another 4 h. Then the culture medium was discarded, and DMSO (100 µL/well) was added to each well at 37 °C for 10 min, and the plate was gently shaken on a shaker for 5 min to completely dissolve the crystalline material. The absorbance at 570 nm was measured with a microplate reader.
The cells in AuNR + Laser and AuNR@FA-PR/PEG + Laser treatment groups incubated 24 h, then irradiated with an 808 nm laser (1 W cm−2) for 3 min, and continued to cultivate for 24 h. MTT (50 µL, 1 mg mL−1) (tetramethylazozolium salt) in PBS solution was added into each well. The culture medium was discarded, and 100 µL of DMSO was added to each well to dissolve the crystalline material at 37° C for 10 min. The plate was gently shaken on a shaker for 5 min to completely dissolve the crystalline material, and the absorbance at 570 nm was measured with a microplate reader.
The cell survival rate is calculated as follows:
$${\text{Cell viability\% = }}\frac{{{\text{OD}}_{{{\text{sample}}}} \, - {\text{OD}}_{{{\text{blank}}}} }}{{{\text{OD}}_{{{\text{control}}}} - {\text{OD}}_{{{\text{blank}}}} }} \, \times {\text{ 100\% }}$$
(7)
Cytotoxicity and photothermal cytotoxicity of AuNR@FA-PR/PEG/CDDP
HepG2 cells in logarithmic growth phase were seeded at a density of 7000/well in 96-well plates with 90 μL culture medium per well. After incubating for 24 h, various concentrations (1, 2, 5, 8, 10 μg/mL, CDDP) of CDDP, CDDP+Laser, AuNR@FA-PR/PEG/CDDP and AuNR@FA-PR/PEG/CDDP+Laser were added into the plate, and each concentration was designed four replicate wells. The cells in CDDP and AuNR@FA-PR/PEG/CDDP treatment rows continued cultivating for 48 h. After that, 50 µL of 1 mg mL−1 MTT in PBS solution was added into the plate, and the cells continued incubating for 4 h, then the medium was removed. 100 µL of DMSO was added into per well, and the plate was maintained at 37 °C for 10 min, then gently shaken to make intracellular crystalline formamidine sufficiently dissolve. Finally, the absorption value was measured at a wavelength of 570 nm using a microplate reader.
The cells in CDDP+Laser and AuNR@FA-PR/PEG/CDDP+Laser treatment rows continued to culture 24 h, and irradiated with an 808 nm laser (1 W cm−2) for 3 min per well, then cultivated for another 24 h. 50 µL of MTT solution was added into per well, and the cells continued to culture for 4 h. The medium was removed, and 100 µL of DMSO was added into per well. The pate was maintained at 37 °C for 10 min, and the crystal formamidine was sufficiently dissolved by gently shaking. The absorption was measured at a wavelength of 570 nm using a microplate reader.
To investigate the cytotoxicity of photothermal and chemotherapy to normal cells, human normal liver cells HL-7702 were selected as cell experimental model. DMEM containing 10% fetal bovine serum was used as a medium, and HL-7702 cells were placed in a 96-well plate at 7000 cells per well, 90 µL medium was added into every well and cultured for 24 h, and then 10 µL various concentrations (1, 2, 5, 8, 10 µg mL−1) of CDDP, CDDP+Laser, AuNR@FA-PR/PEG/CDDP, AuNR@FA-PR/PEG/CDDP+Laser were added into plates, each concentration was designed four replicate wells. The cells in CDDP and AuNR@FA-PR/PEG/CDDP treatment groups continued culturing for 48 h. The cells in CDDP + Laser and AuNR@FA-PR/PEG/CDDP+Laser treatment groups were incubated 24 h, then irradiated with an 808 nm laser (1 W cm−2) for 3 min, and continued to cultivate for another 24 h. After that, MTT in PBS solution (50 µL, 1 mg mL−1) (tetramethylazozolium salt) was added into per well, and the cells continued incubating for another 4 h. Then the culture medium was discarded, and DMSO (100 µL/well) was added to each well, and the plate was gently shaken on a shaker for 5 min to completely dissolve the crystalline material. The absorbance at 570 nm was measured with a microplate reader. The survival rate was calculated, and the average value was measured three times. The survival rate was also calculated by Eq. (7).
Labeling AuNR@FA-PR/PEG/CDDP with NIR-797 isothiocyanate and in vivo real-time imaging
Firstly, AuNR@FA-PR/PEG/CDDP was labeled with NIR-797 Isothiocyanate (NIR797), and the preparation process was as follows: 1 mL solution of NIR-797 isothiocyanate dissolved anhydrous DMSO (1.0 mg mL−1) was added into 10 mL solution of AuNR@FA-PR/PEG/CDDP, and the system was stirred for 24 h at room temperature under dark conditions. After that, the reaction was stopped and the mixture was centrifuged to collect the precipitation, then the precipitation was washed three times with distilled water to remove the unreacted NIR797, finally, the NIR797-labeled AuNR@FA-PR/PEG/CDDP was redispersed in distilled water for real-time imaging.
All the protocols for the animal tests have been reviewed and approved by the Animal Management and Ethics Committee of Henan University (Nos: HUSOM-2019-010) and performed in accordance with the guidelines provided by the National Institute of Animal Care. The murine H22 tumor-bearing Kunming mice models were established by inoculating subcutaneously 5 × 105 cells into the right armpit region of female mice. The mouse was kept with free access to food and water. When the tumor volume reached about 120 mm3, 0.2 mL of NIR797-labeled AuNR@FA-PR/PEG/CDDP were injected into tumor-bearing mice through a tail vein. The real-time distribution of NIR797-labeled AuNR@FA-PR/PEG/CDDP in tumor-bearing mice was imaged using an IVIS Lumina XRMS Series (USA, PerkinElmer) in vivo imaging system. The mice were anesthetized at 168 h after the tail vein injection. Finally, the main organics (heart, liver, spleen, lung, kidney, stomach, intestines and brain) and tumor were collected for isolated organ imaging to investigate the accumulation of AuNR@FA-PR/PEG/CDDP in different organs and tumors.
The accumulation of AuNR@FA-PR/PEG/CDDP in Tumor at different times
Real-time Imaging of NIR-797-labled AuNR@FA-PR/PEG/CDDP in tumor was carried out. Eighteen H22 tumor-bearing mice were randomly divided into six groups with 3 mice in each group. 0.2 mL NIR-797-labled AuNR@FA-PR/PEG/CDDP was intravenously injected into each mouse at a dose of 5 mg/kg (CDDP). Then six groups of mice were sacrificed at predetermined time points (6 h, 12 h, 24 h, 48 h, 72 h, 96 h), respectively, the tumors were harvested and weighed. Finally, the tumors were imaged using an IVIS Lumina XRMS Series.
Simultaneously, the collected tumors were digested in a mixed acid solution (perchloric acid: concentrated nitric acid, 1:3). Then the solution was heated at 100 ℃ for 24 h, the white crystal appears in the bottom and was dissolved with 5% dilute hydrochloric acid, and then the solution was made up to 2 ml. The ICP-AEs test was used to determine the Au element content in each tumor. Finally, the accumulation of AuNR@FA-PR/PEG/CDDP in tumor at different time points was calculated. The data were normalized to the tissue weight and expressed as percentage of dose/g at each test point.
Hemolysis assay
The eyeball blood was obtained from healthy Kunming mice, and then centrifuged to collect red blood cells. In detail, red blood cells were separated by centrifugation at 2000 rpm for 3 min, washed with 0.9% NaCl solution until the supernatant was colorless, and then separately mixed with 1 mL of water (negative control), saline (positive control), and AuNR@FA-PR/PEG/CDDP solution (12.5, 25, 50, 100, 200 µg mL−1). After incubating at 37 °C for 3 h, the supernatants were collected by centrifugation and the absorbance at 540 nm was recorded using a UV-vis spectrophotometer. The percentage of hemolysis was calculated by the following equation:
$${\text{Hemolysis (\%) = }}\frac{{{\text{Abs}}_{{{\text{sample}}}} \, - {\text{Abs}}_{{{\text{negative control}}}} }}{{{\text{Abs}}_{{{\text{positive control}}}} - {\text{Abs}}_{{{\text{negative control}}}} }} \, \times {\text{ 100\% }}$$
(8)
where Abssample, Absnegative, and Abspositive are the absorbance of samples, the negative control, and the positive control, respectively.
In vitro photothermal imaging
AuNR@FA-PR/PEG/CDDP was dissolved in distill water to prepare a solution with 50 µg mL−1 of concentration, and the solution was subjected to laser irradiation with different laser powers (0.75, 1.5, 2.25, 3 W cm−2) for 10 min. At the same time, AuNR@FA-PR/PEG/CDDP was also prepared various concentration water solution and subjected to an 808 nm laser irradiation with 1 W cm−2 of power for 10 min. The temperature change of AuNR@FA-PR/PEG/CDDP solution was recorded with an infrared thermal imaging camera (Thermo Shot F30, Nippon Avionics Co., Ltd, Japan), and the temperatures at tumor sites were measured.
In vivo temperature measurement and photothermal imaging
According to the in vivo fluorescence imaging of mice injected with NIR-797-labled AuNR@FA-PR/PEG/CDDP, the nanocomposite began to enter and accumulate in the tumor area at 6 h after the injection of AuNR@FA-PR/PEG/CDDP. Therefore, the thermal imaging of the tumor regions in H22 tumor-bearing mice was carried out at 6 h after injection of various samples through the tail vein to verify the accumulation and photothermal capabilities of AuNR@FA-PR/PEG/CDDP. The H22 tumor-bearing mice were divided into three groups when the tumor sizes reached about 120 mm3. Three groups mice were treated with saline, AuNR@FA-PR/PEG (Au: 55 mg of Au/kg) and AuNR@FA-PR/PEG/CDDP solution (Au: 55 mg of Au/kg), through the tail vein, respectively. At 24 h after injection, the tumors were irradiated with an 808 nm laser (1.0 W cm−2) for 10 min. The thermal imaging was captured every 2 min by using an infrared thermal imaging camera (Thermo Shot F30, Nippon Avionics Co., Ltd, Japan), and the temperatures at tumor sites were measured.
In vivo antitumor efficacy
The tumor models were established by implanting 5 × 105 murine hepatoma cell line H22 into the right armpit of Kunming mice (6−8 weeks). The antitumor activity of AuNR@FA-PR/PEG/CDDP was investigated using the established models. The “day 1” was determined when the tumor volume reached an average size of 90–100 mm3. On day 1, tumor-bearing mice were randomly divided into six groups. Saline and AuNR@FA-PR/PEG treatment groups were used as control groups. Two groups of mice were treated with saline and free CDDP (3 mg/kg), two groups of mice were injected with AuNR@FA-PR/PEG (50 mg/kg, 7.5 mg AuNR eq.) through the tail vein, the last two groups of mice were injected with AuNR@FA-PR/PEG/CDDP (50 mg/kg, 7.5 mg AuNR eq., 1.5 mg/kg cisplatin eq.) via the tail vein. The tail vein treatment was performed on the day 1, day 4 and day 7. At 24 h after tail vein injection, One of AuNR@FA-PR/PEG treatment group and one of AuNR@FA-PR/PEG/CDDP treatment group were irradiated using an 808 nm laser (1.0 W cm−2) for 5 min. Tumor sizes were measured in two dimensions every other day using a vernier caliper up to 14 days. Simultaneously, the weights of tumor-bearing mice were monitored. The tumor volume was calculated according to the following formula: V = d2 × D/2 (where d was the tumor width at the shortest dimension, and D was the longest dimension). On the 15th day, tumor-bearing mice in all treatment groups were sacrificed and the tumors were harvested. The antitumor activity and biocompatibility were evaluated via tumor growth and terminal tumor weight. The tumor growth inhibition (TGI) was calculated by the following formula:
$${\text{TGI = }}\frac{{\overline{{\text{V}}} {\text{ of saline control group - }}\overline{{\text{V}}} {\text{ of tested group}}}}{{\overline{{\text{V}}} {\text{ of saline control group}}}} \, \times {\text{ 100\% }}$$
(9)
Determination of cell apoptosis and proliferation and histology studies
Six group tumor-bearing mice were injected saline, CDDP, AuNR@FA-PR/PEG (two groups) and AuNR@FA-PR/PEG/CDDP (two groups) via tail vein, respectively. One AuNR@FA-PR/PEG treatment group and one AuNR@FA-PR/PEG/CDDP treatment group were irradiated with an 808 nm laser for 5 min at 24 h post tail vein injection. After 7 days, all tumor-bearing mice were sacrificed, and the main organs and tumors were harvested, fixed with paraformaldehyde, embedded with paraffin and cut into 4 μm thick sections for hematoxylin and eosin (H&E) staining and examined by optical microscopy.
The apoptotic cells in tumor tissue sections were detected using the Terminal deoxynucleotidyl transferase (TdT)-mediated d-UTP Nick End Labeling (TUNEL) assay (Apoptosis TUNEL assay IHC kit, AbD Serotec, MorphoSys, Oxford, UK, cat.no. APO002) according to the manufacturer’s protocol. The detection of proliferating cells was performed using an antibody against PCNA and the proliferating cells were visualized by incubation with 3,3'-diaminobenzidine tetrahydrochloride (Adamas-beta) for 2 min. After being rinsed with distilled water, the sections were counter-stained with hematoxylin. For quantification of PCNA and TUNEL expression, the number of positive cells was counted in 3 random high power fields (400 × magnification) and divided by the total number of cells for each tumor.
