Mice, cells and parasites
Female C57BL/6 mice (aged 6-8 weeks) were purchased from Beijing Vital River Experimental Animals Co., Ltd. The murine Hepa1-6 hepatoma cell line was obtained from the First Affiliated Hospital of Sun Yat-Sen University and cultured in Dulbecco’s modified Eagle’s medium (DMEM) (GIBCO) supplemented with 10% fetal bovine serum (FBS), 1% penicillin and streptomycin. The murine H22 hepatoma cell line was purchased from the China Center for Type Culture Collection and maintained in RPMI 1640 medium supplemented with 10% FBS, 1% penicillin and streptomycin. The murine RAW264.7 macrophage cell line was kindly provided by Dr. Chiwei Huang and cultured in DMEM supplemented with 10% FBS, 1% penicillin and streptomycin. Plasmodium yoelii 17 XNL (P.y), a Plasmodium parasite strain that is not lethal to C57BL/6 mice, was kindly provided by BEI Resources (formerly the Malaria Research and Reference Reagent Resource Center, MR4) and was recovered in 7-week-old female C57BL/6 mice. The antibodies (Table S1) and gene specific primers (Table S2) in this paper can be found in supplementary material.
Design of animal experiments
To evaluate the effects of Plasmodium infection on tumors in mice, mice were subcutaneously inoculated with 2×106 Hepa1-6 or H22 cells in 100 μl of serum-free RPMI 1640 and simultaneously intraperitoneally (i.p.) inoculated with 5×105 parasitized erythrocytes or uninfected RBCs as a control in 200 μl of saline. The palpable spherical tumor mass emerged 4-6 days after tumor cell inoculation. The tumor size was measured every 3 or 4 days using a caliper and calculated using the formula 0.52 × a × b2 (a: long diameter of the tumor and b: short diameter of the tumor). Blood samples were collected from the tail vein of the mice every 2 days for 30 days, and parasitemia was determined by analyzing a thin Giemsa-stained blood film. The mice were observed until death or until their tumor size reached 2000 mm3. In some experiments, the mice were sacrificed on day 8 or 17 after parasite inoculation for biopsy. The tumor tissue was used for further analysis. For assaying the blockage of parasite infection, Plasmodium parasites were killed with a dose of 10 mg of chloroquine per kilogram of mouse body weight on day 8 after infection, and the mice were then euthanized on day 17 for further analysis.
To establish the orthotopic tumor model, 50 μl of 1×106 Hepa1-6 cells were injected into the left lobe of the liver of anesthetized mice. After recovery, the mice were i.p. inoculated with 5×105 parasitized erythrocytes or uninfected RBCs. On day 17 after parasite inoculation, the mice were sacrificed for biopsy. The liver and spleen were removed and photographed, and the liver sections were stained with H&E.
Alginate-encapsulated tumor cell assay
An in vivo alginate-encapsulated tumor cell assay was performed as previously described with slight modifications . Briefly, 1.5% (W/V) sodium alginate solution was prepared in sterile saline. Hepal-6 or H22 hepatoma cells were harvested once 80% confluence was reached in cell culture. After centrifugation, the cells were resuspended in the sodium alginate solution to a concentration of 2×107/ml. The alginate solution was extruded through a point-cut 200-µl tip to produce droplets (20 µl per droplet). The addition of droplets into a swirling solution of 250 mmol/l calcium chloride at 37℃ resulted in the formation of alginate beads containing tumor cells in the solution. After incubation in the CaCl2 bath for an additional 30 min, the beads were washed twice with saline, centrifuged and resuspended in 10% RPMI1640 at 37℃.
Four alginate beads (1×105 Hepal-6 or H22 cells per bead) were subcutaneously implanted into an incision made on the dorsal side of the mice (aged 6-8 weeks) under anesthetic conditions. The mice were randomly divided into two groups (n = 10 per group). One group was i.p. inoculated with 5×105 parasitized erythrocytes, and the other group, which served as the control, was i.p. inoculated with the same number of uninfected RBCs. After 14 days, the mice were i.v. injected with 0.1 ml of 1% FITC-dextran (Sigma-Aldrich) solution (100 mg/kg) through the lateral tail vein. After 20 min, the beads were rapidly removed and photographed. After dissection of the capsular implant, the beads were transferred to tubes containing 2 ml of saline. The tubes were mixed by vortexing for 30 s and centrifuged (5 min, 1000 × g). After dilution (1:1), the fluorescence of the supernatant was measured. The uptake of FITC-dextran was assessed using a standard FITC-dextran curve. After removal of the implants from the mice, the following procedures were performed in the dark.
Single-cell suspensions were prepared from tumor tissue as described previously with some modifications . Briefly, tumor-bearing mice were sacrificed 17 days after parasite inoculation. The solid tumors were dissected, chopped into small pieces using scissors and incubated with a mixture of enzymes dissolved in RPMI 1640 (400 U/ml collagenase type IV, 0.05 mg/ml collagenase type I, 0.025 mg/ml hyaluronidase, all from Sigma-Aldrich; 0.01 mg/ml DNase I and 0.2 unit/ml soybean trypsin inhibitor, both from Boehringer Mannheim) for 30 min at 37℃. The cells were harvested by centrifugation. The RBCs were lysed using ammonium chloride lysing buffer. The suspension was filtered through a 70-μm BD Falcon cell strainer (BD Biosciences) to generate a single-cell suspension. The single-cell suspension was centrifuged, washed twice with phosphate-buffered saline (PBS) containing 1% FBS and resuspended in PBS containing 1% FBS and 2 mM EDTA. The cell suspension was layered onto Ficoll-PaqueTM PLUS (GE Healthcare) and centrifuged at 450 g for 30 min without breakage. Mononuclear cells were obtained by removing the middle white layer, washed twice with phosphate-buffered saline (PBS) containing 1% FBS and resuspended in PBS containing 1% FBS. F4/80+ cells were isolated from the cell suspension by flow cytometry (BD FACSAria). Briefly, a total of 1×107 cells were incubated with 10 μg of FITC- or APC-conjugated anti-F4/80 monoclonal antibody (mAb) for 30 min on ice and then washed with cold buffer to remove any unbound antibody. The F4/80+ cell populations were sorted by flow cytometry (BD FACSAria). The purity of the cell populations was between 85% and 90%.
Induction of TAMs in vitro
Murine nonhypoxic TAMs were induced in vitro by incubation with soluble factors released from the murine hepatoma cell line Hepa1-6 as described previously with some modifications .
Tumor cell-free supernatant (TSN) was obtained by culturing 4×106 Hepa1-6 cells in 24-well flat-bottom tissue culture plates for 72 h and stored in aliquots at −80°C. For in vitro induction of the TAM phenotype, 5×105 RAW 264.7 cells were seeded in a six-well plate, and TSN (diluted 1:1 with fresh RPMI 1640 medium supplemented with 1% FBS) was added. After 48 h, the supernatant was removed, and the cells were washed twice with PBS. Total RNA was isolated using the TRIzol reagent according to the manufacturer’s instructions (Invitrogen). The expression levels of alternatively activated genes (ym1, mgl1/2, and arginase 1) and classically activated genes (il-12 and inos) were analyzed by real-time qRT-PCR.
Hepa1-6 cell-implanted mice were sacrificed on day 17 after Plasmodium infection. TAM isolation was performed as described in a previous section. The TAMs were sorted by FACS using FITC-conjugated anti-F4/80 mAb. Gene expression was compared using Roche NimbleGen mouse gene chips (CapitalBio, China). Genes that showed an expression fold-change ≥ 2 with a q-value ≤ 0.05 in triplicate arrays were considered to be significantly induced in response to treatment. These differentially expressed genes were annotated using the Molecule Annotation System (CapitalBio, China).
The preparation and immunostaining of histological specimens were performed as described previously .
Paraffin-embedded tissue sections were deparaffinized in xylene and rehydrated through a graded ethanol series. High-pressure antigen retrieval was performed in 10 mM EDTA buffer (pH 8.0) before the sections were incubated with a primary antibody against F4/80, a rat mAb against mouse macrophages (clone No. RM0029-11H3), an anti-CD31 antibody and an anti-MMP-9 antibody. Bright field images were captured and analyzed.
The number of TAMs, density of CD31-positive microvessels and expression of MMP-9 in TAMs were assessed under 400× magnification (0.17-mm2 field) in eight random fields, and the results are expressed as the mean numbers per field ± SDs (eight fields per tumor, four tumors per group).
RNA isolation and real-time RT-PCR
Total RNA was isolated using TRIzol (Invitrogen) according to the manufacturer’s instructions. Real-time RT-PCR for the ym1, arg-1, mgl1, mgl2, fizz1, inos, mmp-2, mmp-9, leyve1, vegf, igf1 and b-actin genes was performed with an MJ chromo 4 real-time PCR machine (Bio-Rad) using the OneStep SYBR® PrimeScriptTM RT-PCR Kit (Takara) according to the manufacturer’s instructions. The b-actin gene was used as a reference gene.
Western blot analysis
The preparation of the samples from tissue or cells, gel electrophoresis, and transfer to polyvinylidene fluoride membranes (Millipore) were performed as described previously . The membranes were incubated with primary antibodies against MMP-2/9, VEGF, IGF-1, AKT, phospho-AKT, p44/42 MAPK (Erk1/2), and phospho-p44/42 MAPK (Erk1/2) overnight at 4°C and with secondary antibodies for 1 h at room temperature. Immobilon Western HRP Chemiluminescent Substrate (Cell Signaling Technology) was used to detect the specific signals. The band intensity was visualized.
Preparation of HZ
Natural HZ was obtained as described previously with some modifications . Spleen and liver tissues of P.y-infected C57BL/6 mice were chopped into small pieces using scissors and lysed with 1% saponin for 2 h, and the resulting suspension was centrifuged (14,000 g, 10 min, 10℃). The precipitate was washed three times with PBS by centrifugation. The precipitate was resuspended in 2% SDS and shaken for 1 h. The suspension was sonicated (3 s/time, 60 times) and centrifuged (25000 g, 30 min at 10℃). The precipitate was the crude extract of HZ, and this crude extract was further processed through the following steps. The precipitate was washed eight times with 2% SDS by centrifugation (25000 g, 30 min, 10℃), resuspended in 10 ml of 10 mM Tris-HCl (pH 8.0) containing 0.5% SDS, supplemented with proteinase K (2 mg/ml), DNase I (30 u/ml) and RNase A (10 mg/ml) and shaken overnight at 37℃. The suspension was centrifuged (25,000 g, 30 min, 10℃) and washed five times with 2% SDS by centrifugation (25000 g, 30 min, 10℃). The precipitate was resuspended with 6 M urea and shaken for 30 min at room temperature. The suspension was centrifuged (25000 g, 30 min, 10℃) with 2% SDS, and the precipitate was washed five times with 2% SDS by centrifugation (25000 g, 30 min, 10℃). After centrifugation, endotoxin contained by the extract was removed by addition of an endotoxin-binding reagent (Thermo Fisher Scientific). The extract was then washed with ultrapure H2O by centrifugation until complete removal of SDS was achieved. The pigment was lyophilized, dried, resuspended in endotoxin-free PBS at a final concentration of 10 mg/ml and maintained at -20℃. The endotoxin level of the isolated HZ was assessed using a Limulus amebocyte lysate kit (Sigma-Aldrich). The endotoxin level was less than 0.015 EU.
Assessment of IGF-1 derived from HZ-loaded TAMs
To assess the effect of HZ on the secretion of IGF-1, induced TAMs were treated with HZ (100 μg/ml) or left untreated for 48 h. The IGF-1 mRNA and protein levels were evaluated by real-time PCR and immunoblot analysis, respectively. In some experiments, the cells were treated with different concentrations of HZ (10 to 200 μg/ml) for different durations (3 to 48 h).
Preparation of dichloromethylene diphosphonate (Cl2MDP) liposomes and macrophage “in vivo knockout” assay
These experiments were performed by following previously described procedures . A mixture of 8 mg of cholesterol and 86 mg of phosphatidylcholine was prepared in chloroform. The thin film was dissolved in 10 ml of 0.7 M Cl2MDP solution, incubated for 2 h at room temperature under the protection of argon, sonicated for 3 min and incubated for 2 h at room temperature. Free drugs were removed by centrifugation, and the final pellet was resuspended in PBS.
Naïve mice (aged 6-8 weeks) were i.p. injected with 300 μl of C12MDP-liposomes and inoculated with tumor cells 2 days later. After tumor inoculation, the mice were treated with Cl2MDP-liposomes via both the intraperitoneal (300 μl) and intratumoral (50 μl) routes on days 4, 8, 13, and 18. PBS-liposomes and saline were used as controls. The tumor size was measured every 4 days and calculated. After 16 days, tumor-bearing mice were sacrificed for further analysis.
The statistical significance of the differences between values was determined by Student’s t test using GraphPad Prism 6.01 software. The correlation between two factors was evaluated by Pearson’s correlation analysis. Kaplan-Meier survival curves were plotted. The data are expressed as the means ± SDs, and P values < 0.05 were considered significant.