The following reagents were used in the study: Rat anti-mouse F4/80 mAb (AbD; Serotec, Kidlington, UK), a rat anti-MΦ mAb [RM0029-11H3] (a rat monoclonal antibody against isolated mouse MΦ; Abcam, Cambridge, MA, USA), rat anti-mouse MHC class II (I-Ab,d,q/I-Ed,k) mAb (BioLegend, San Diego, CA, USA), and rat anti-mouse-IL-10/TGF-β and rabbit-anti-mouse-CD31/VEGF/MMP-9 mAb (Santa Cruz Biotechnology, Santa Cruz, CA, USA). The IL-10 and TGF-β enzyme-linked immunosorbent assay (ELISA) kits were purchased from Bender Medsystems (GmbH, Vienna, Austria). All fluorescently-labeled mAbs and isotype control mAbs were obtained from eBioscience (San Diego, CA, USA). Clodronate (dichloromethylene diphosphonate, Cl2MDP) was a generous gift from Roche Diagnostics (GmbH, Mannheim, Germany). All other reagents were obtained from Sigma. (St Louis, MO, USA) unless otherwise indicated.
Female C57BL/6 and BALB/c mice (6-8 weeks old) were obtained from Beijing Vital River Experimental Animals, Co. Ltd. All animal experiments were approved by the Laboratory Animal Committee of Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Science.
To investigate the effects of Cl2MDP-liposomes on tumor growth, naïve mice were first injected i.v. with 200 μl of C12MDP-liposome suspension 2 days prior to tumor inoculation. Peripheral blood (50 μl) was collected retro-orbitally 0, 24, and 48 h after treatment and immediately transferred to ethylenediaminetetraacetic acid (EDTA) -containing tubes (BD Bioscience). Whole blood cells were stained with APC-conjugated anti-CD11b and FITC-conjugated anti-F4/80 mAbs for 30 min in the dark on ice. The cells were then washed and the red blood cells lysed with FACS lysis buffer (BD Bioscience). The remaining white blood cells were washed again and resuspended in PBS containing 1% fetal calf serum (FCS). Flow cytometry data were acquired using a BD FACSAria and analyzed using FLOWJO software version 7.6.0 (Tree Star, Inc., Ashland, OR, USA). After tumor inoculation, mice were treated with Cl2MDP-liposomes via both i.v. (200 μl) and i.t. (50 μl) routes on Days 3, 8, 13, and 18. PBS-liposomes and saline were used as the controls. Tumor size was measured every 4 days using calipers and calculated using the formula: 0.52 × a × b2 (a: long diameter of the tumor; b: short diameter of the tumor). After 20 days, tumor-bearing mice were sacrificed for further analysis.
For the orthotopic tumor model, 200 μl of Cl2MDP-liposomes were injected i.v. on Day 2 before surgery, and at 3, 8, and 13 days after surgery. After 15 days, mice were sacrificed and the tumors were removed and weighed.
Preparation of Cl2MDP-liposomes
Cl2MDP-liposomes were prepared as described previously [21, 22]. Briefly, a mixture of 8 mg of cholesterol and 86 mg of phosphatidylcholine was prepared in chloroform in a round-bottom flask. The thin film on the interior of the flask after low-vacuum evaporation at 37°C was dissolved in 10 ml of 0.7 M Cl2MDP solution and incubated for 2 h at room temperature under Argon gas protection, followed by 3 min of sonication and another 2 h of incubation at room temperature. The liposomes were washed five times by centrifugation to remove any free drugs. The final pellet was resuspended in 4 ml of sterile PBS. The control liposomes were prepared using PBS. Cl2MDP-liposomes and PBS-liposomes were stored at 4°C for 2 weeks.
Cell culture and tumor models
The murine HCC cell line, Hepa1-6, and the murine MΦ cell line, RAW264.7, were cultured in DMEM supplemented with 10% FCS and 1% penicillin-streptomycin at 37°C. For the s.c. tumor model, 100 μl of serum-free RPMI 1640 containing 2 × 106 Hepa1-6 cells was injected s.c. into the right anterior axillary fossa of naïve mice. A palpable spherical mass emerged after 4 to 6 days.
An orthotopic hepatoma model was created by transplantation of a small tumor mass into the left liver lobe of naïve mice. Briefly, 2 × 106 Hepa1-6 cells was injected s.c. into the right anterior axillary fossa of naïve mice. The mice were sacrificed when the hepatoma grew progressively and the tumor diameter exceeded 10 mm. The tumors were removed and chopped into small pieces (about 1 mm3) using a scalpel and forceps. The small masses were surgically implanted under anesthesia (a small tumor mass/mouse) into one site in the left liver lobe of mice treated with Cl2MDP-liposomes, PBS-liposomes, or saline.
Preparation of histological specimens (from tumors on Day 20 after treatment with Cl2MDP-liposomes or from tumors on Day 10 after adoptive transfer of sorted TAM populations) and immunostaining were carried out as described previously . Antigen retrieval was performed under high pressure in 10 mM EDTA buffer (pH 8.0) before incubation with primary antibodies to F4/80, CD68, or rat monoclonal anti-MΦ [RM0029-11H3] or IL-10, TGF-β, VEGF, MMP-9, CD31 and MHC class II. Brightfield images were then captured and analyzed.
The number of infiltrating MΦ and the CD31-positive microvessel density were counted in 10 random fields at × 400 magnification (a 0.17 mm2 field), and the results were expressed as the mean number per field ± SD (10 fields per tumor, 5 tumors per group).
Isolation of TAMs and splenocytes
Single cells within the tumor tissue were isolated as described previously with some modifications . Briefly, solid tumors were harvested from tumor-bearing mice on Days 7, 14, 21, and 28 after tumor inoculation or harvested under sterile conditions from tumor-bearing mice untreated or treated with Cl2MDP-liposomes. The tumors were chopped into small pieces using scissors and forceps before incubation 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, 0.01 mg/ml DNase I and 0.2 units/ml soybean trypsin inhibitor) for 30 min at 37°C. Cells were recovered by centrifugation and resuspended in PBS, containing 1% FBS. The debris and red blood cells were removed using the Ficoll density gradient method. F4/80+MHC IIhi and F4/80+MHC IIlow cells were analyzed and sorted by flow cytometry. Briefly, cells were resuspended in PBS supplemented with 1% FCS, and 1 × 107 cells were incubated with 10 μg of FITC-conjugated F4/80 mAb and 0.2 μg of PE-conjugated MHC class II mAb for 30 min on ice. The cells were then washed with PBS supplemented with 1% FCS to remove unbound Abs. The F4/80+MHC IIhi and F4/80+MHC IIlow cell populations were analyzed and sorted using a FACSAria flow cytometer. All data were analyzed using FLOWJO software version 7.6.0. The purity of the cell populations exceeded 90%.
Single cells were also prepared as described above on Day 10 after tumor inoculation. Briefly, 1 × 107 sorted cells were incubated with 2 μg of APC-conjugated F4/80 mAb and 0.2 μg of PE-conjugated MHC class II mAb for 30 min on ice. The cells were then washed with PBS supplemented with 1% FCS. After fixation, permeation, and washing, the cells were stained with 10 μg of FITC-conjugated IL-10 mAb. The unbound antibodies were removed by washing. The expression of IL-10 by F4/80+MHC IIhi and F4/80+MHC IIlow cells was then analyzed.
Naïve BALB/c mice (6-8 weeks old) were sacrificed and the spleens were harvested using syringes and forceps and single cell suspensions obtained. Red blood cells were removed with ACK lysis buffer (0.15 M NH4Cl, 1 mM KHCO3, 0.1 mM EDTA, pH 7.3), and the remaining splenocytes were washed twice by centrifugation. The splenocytes were counted, diluted to the appropriate concentration in RPMI 1640 supplemented with 10% FCS and 1% penicillin-streptomycin for further investigation.
Real-time quantitative reverse transcription PCR (qRT-PCR) and reverse transcription PCR (RT-PCR)
Total RNA from the sorted TAM populations was isolated using Trizol according to the manufacturer's instructions. qRT-PCR to examine the expression of ym1, mgl1/2, fizz1, arg-1, msf, il-10, tgf-β, mmp-9, vegf, ptge2, il-1β, il-6, il-12, and inos was performed using a One Step SYBR® PrimeScript™ RT-PCR kit according to the manufacturer's instructions (Takara, Dalian, China). β2-mG was used as a the reference gene.
Two-step RT-PCR was performed to examine the expression of mmp-9 and vegf in the sorted TAM populations. cDNA was synthesized from 2 ug of total RNA using Moloney murine leukemia virus reverse transcriptase (Takara) after DNase treatment. The PCR conditions were as follows: 94°C for 5 min followed by 30 cycles at 94°C for 30 s, 60°C for 30 s, and 72°C for 40 s, with a final extension at 72°C for 7 min. The β2-mG gene was used as a reference. The PCR products were analyzed by gel electrophoresis.
Western blot analysis
Total proteins were extracted from the sorted TAM populations and electrophoresed on 10% SDS polyacrylamide gels. The proteins were then transferred to polyvinylidene fluoride membranes (Millipore, MA, USA). Non-specific binding was blocked with 5% (wt/vol) nonfat milk in PBS-Tween-20 (0.1% vol/vol). The membranes were then incubated with primary antibodies against MMP-9 and VEGF at 4°C overnight and with the appropriate secondary antibodies at room temperature for 1 h. Immobilon Western HRP Chemiluminescent Substrate (Cell Signaling Technology, MA, USA) was used to detect the specific signals, and the band intensity was then visualized.
Invasion and migration assays
Tumor cell invasion assays were performed using a Transwell apparatus (Corning, NY, USA) with an 8- μm-pore polycarbonate filter membrane. The upper sides of the membranes were pre-coated with Matrigel matrix (BD, NJ, USA) according to the manufacturer's instructions. Sorted TAMs (2 × 105) were pre-seeded in the lower chamber. The upper chamber was seeded with 1 × 104 Hepa1-6 cells and inserted into the lower chamber. After incubation for 24 h, the cells on the interior of the upper chamber were removed by swabbing with cotton swabs. The polycarbonate membranes were fixed in methanol for 20 min and stained with 0.1% crystal violet for 15 min. The number of cells migrating to the underside of the membranes was counted in five randomly selected fields under a microscope. For the migration assay, the same procedure was used as described above but without addition of Matrigel to the top of the upper chamber membrane.
T cell suppression assays
For the T cell suppression assays, 1 × 105 to 2.5 × 104 sorted TAMs were co-cultured with 2 × 105 naive splenocytes (1:2-1:8) and 1 μg/ml anti-CD3. To neutralize IL-10 and TGF-β, 500 ng/ml IL-10 and/or TGF-β mAb were added to the wells containing MHC class IIlow TAMs, followed by BrdU staining 24 h later. T cell proliferation was assessed using a commercial ELISA kit (Roche Diagnostics, GmbH, Mannheim, Germany), according to the manufacturer's instructions.
The co-culture supernatants were collected and stored at -80°C until used. IL-10 and TGF-β levels were measured using commercially available ELISA kits (Bender Medsystems) according to the manufacturer's instructions.
Adoptive transfer of distinct TAM subsets
For the adoptive transfer assays, 2 × 106 sorted F4/80+MHC IIhi and F4/80+MHC IIlow cells were injected, into the tumors of mice 2 days after inoculation of Hepa1-6 cells. PBS was used as the control. Tumor size was measured every 4 days. The mice were sacrificed 10 days after adoptive transfer and the tumors dissected out for further analysis.
Statistical significance was determined using the Student's t test and GraphPad Prism software. Data were expressed as the mean ± SD. A p value < 0.05 was considered significant.