All the protocols involving animals were reviewed and approved by our Institutional Animal Care Committee. We used C57BL/6 mice, weighing 20–25 g, that were raised at the Central Animal Laboratory of the School of Medicine of Ribeirão Preto, SP, Brazil.

B16-F10 melanoma cells were maintained in RPMI-1640 medium supplemented with 10% inactivated fetal calf serum, 2 mM L-glutamine all from (Invitrogen, Carlsbad, CA, USA) and 1% penicillin/streptomycin (100 U/ml; Gibco Life Technologies, Carlsbad, CA, USA) in a humidified atmosphere at 37°C and 5% CO₂.

Two shRNA target sequences were selected from different positions of mouse CXCR4 cDNA sequence (GenBank, accession no. BC031665) corresponding to nucleotides 85-103 (CXCR4-1 shRNA) and 409-427 (CXCR4-2 shRNA). Each shRNA contains a sense strand of 19 nucleotides followed by a short spacer (AAGTTCTCT), the antisense strand and a stop signal (TTTTT) for RNA polymerase III.

The selection of shRNA sequences was based on the shRNA Target Finder and Design Tool available at Dharmacon website. These target sequences were submitted to a BLAST search to ensure that only the CXCR4 gene was the target. The target sequence of the negative control group named control shRNA has no homology with that of human or mice. The targeting sequence and location of each shRNA in CXCR4 cDNA are shown in Table I.

Two micrograms of sense and antisense oligonucleotides were mixed and diluted in annealing buffer to a final concentration of 40 ng/µl. The mixture was then heated to 90°C for 3 min and then transferred to a water bath at 37°C and incubated for 15 min. The annealed oligonucleotides were diluted in nuclease-free water to a final concentration of 4 ng/µl. Each paired oligonucleotide was ligated overnight by enzyme T4 DNA ligase (3 U/µl) to the plasmid psiTRIKE (50 ng/µl). The DH5α E. coli strain was used for cloning. Transformation of plasmid DNA into competent E. coli was performed using the heat shock method. After a short incubation in ice, a mixture of chemically competent bacteria and plasmid DNA was placed at 42°C for 45 sec and then placed back in ice. LB media were added and the transformed cells were incubated at 37°C for 30 min with agitation. The E. coli was plated and transformed bacteria was selected based on resistance to ampicillin. Plasmid DNA was extracted of transformed E. coli with FlexiPrep kit (Amersham Biosciences, Little Chalfont, UK). The method employs a standard alkaline lysis procedure, including treatment with RNase and precipitation with isopropanol. Plasmid DNA was subsequently purified by Sephaglas PF resin (Amersham Biosciences) according to the manufacturer's instructions. Screening for the two inserts was performed by digestion of one microgram of plasmid DNA with PstI overnight for 37°C.

In vitro transfection of B16-F10 melanoma cells were plated on tissue culture flasks at a density of 7×10⁵ cells. After an overnight incubation and at a confluence ~70–80%, these cells were transfected with 30 µg of each CXCR4 shRNA and 30 µl of Lipofectamine 2000 (Invitrogen). The plasmid and Lipofectamine 2000 were diluted in serum-free medium left at room temperature for 5 min, mixed immediately and incubated for 20 min at room temperature at a v/w ratio of liposomes to shRNA of 1:1. The culture medium was removed and the shRNA-lipid complex (1.5 ml total volume) was added. The transfection efficiency (~75–80%) was evaluated by using the green fluorescent protein (GFP) expressing plasmid. Prior to the in vivo study, we examined whether the two plasmid-based CXCR4-specific shRNAs (CXCR4-1 shRNA and CXCR4-2 shRNA) were effective in reducing the CXCR4 expression in cultured B16-F10 cells. Thus, tumor cells were transfected with CXCR4-1, CXCR4-2 or control shRNA for 5 h and thereafter the cells were washed, suspended in medium and maintained in culture for 24 or 48 h. To determine the amount of mRNA CXCR4 and CXCR4 protein, lysates of the B16-F10 melanoma cells were used for RNA isolation and western blot analysis.

Total cellular RNA was extracted using TRIzol-LS Reagent (Invitrogen). The integrity of RNA was assessed using the Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA).

Reverse transcription-PCR was performed with 1.2 µg of the isolated total RNA and synthesized to cDNA in a 25 µl reaction system using reverse transcriptase (Promega, Madison, WI, USA). RT conditions were 5 min denaturation at 65°C, 60 min at 37°C and 5 min at 75°C in a thermocycler (Abgene, Epsom, UK). Reverse transcription was carried out with 0.5 µg of the oligodT primer, 1 unit of reverse transcriptase, 1 unit of RNase inhibitor (all from Invitrogen), 5 µl of 5X buffer and 4 µl MgCl₂. The β-actin mRNA was used as a loading control. PCR conditions for β-actin were 4 min denaturation at 94°C, 40 cycles of 1 min at 94°C, 1 min at 52°C and 2 min at 72°C and 10 min elongation at 72°C in a thermocycler (Abgene). PCR conditions for CXCR4 were 5 min denaturation at 94°C, 35 cycles of 1 min at 94°C, 1 min at 51°C and 1 min at 72°C, and 10 min elongation at 72°C in a thermocycler (Abgene). The primers sequences and GenBank Accession number are shown in Table II. PCR products of β-actin (364 bp) and CXCR4 (291 bp) were analysed by electrophoresis in a 1.5% agarose gel and visualized using UV fluorescence after staining with ethidium bromide. Quantification of CXCR4 bands was performed by using ImageQuant software, version 3.3 (Molecular Dynamics, Inc., Sunnyvale, CA, USA) and the results were expressed in terms of percentage.

B16-F10 adherent cells were detached using EDTA with RPMI without fetal bovine serum and centrifuged at 4,000 rpm for 15 min. The cell pellet was resuspended in 300 µl PBS plus the proteases inhibitors 0.1% aprotinin, 0.1% leupepsin and 1% Triton. The sample was incubated under agitation on ice for 20 min and after centrifuged at 12,000 rpm for 15 min at 4°C and the protein concentration determined by Cadman method. Total cellular protein (30 µg) was separated by electrophoresis through a 10% SDS-PAGE resolving gel with an SDS-PAGE stacking gel. After electrophoresis, proteins were transferred onto a Hybond-C supported nitrocellulose membrane (Amersham Biosciences) by electroblotting for 4 h at 45 V, 25°C, in transfer buffer (3.94 g Tris-HCl, 18.80 g glycine, 240 ml methanol, 10% SDS). The membrane was then blocked with 10% dried milk in TBS (20 mM Tris, 500 mM NaCl) at room temperature overnight, after washed twice followed by incubation at room temperature with 1:250 off rabbit anti-CXCR4 polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) in TBS-Tween-20 buffer for 90 min. The membrane was washed in TBS 1X Tween-20 for 30 min and secondary anti-rabbit antibodies labeled with horseradish peroxidase (Amersham Biosciences) was added and the membrane incubated at room temperature for 60 min under agitation. Membranes were washed twice in TBS-T for 20 min and in TBS for 5 min. Antibody labeled protein bands were visualized with ECL detection reagents (Amersham Biosciences) applied following the manufacturer's protocol. To use β-actin as a loading control, a second gel was loaded with identical volume of the experimental sample followed by blotting with the anti-β-actin antibody and the detection was performed as described for CXCR4. Quantification of bands was performed by using Image Quant software, version 3.3 (Molecular Dynamics Inc.) and the results were expressed in terms of percentage.

Based on the in vitro findings, CXCR4-1 shRNA was selected for in vivo experiments. B16-F10 melanoma cells were transfected with CXCR4-1 shRNA or control shRNA for 5 h and thereafter the cells were washed, suspended in medium and maintained in culture for 24 h to inject into mice. After this treatment, tumor cells were detached with EDTA, washed twice in PBS and finally resuspended in RPMI. The viability of cells was assessed by trypan blue staining and was >95%. Tumor cells transfected were then inoculated subcutaneously (2×10⁵ cells/animal) into the right flank of mice (n=10 per group), animals were euthanized 14 days after treatment according to Kumar et al (18) and tumors were excised and weighted on a microbalance Sartorius Supermicro (model S4; Sartorius, Goettingen, Germany).

Each animal received 2×10⁵ tumor cells subcutaneously into the right flank (n=10 per group). The tumors were monitored, and treatment began when the average tumor diameter reached 5–7 mm, typically 7 days after of tumor cell inoculation. This tumor size allows the intratumoral injection to be performed with safety. Thus, after 7 days of tumor inoculation mice received a single intratumoral injection of 2 µg of CXCR4-1 shRNA complexed with 2 µl of Lipofectamine 2000 dissolved in 50 µl of RPMI. Some mice received an intratumoral injection of 2 µg control shRNA-expressing plasmid complexed with 2 µl of Lipofectamine 2000 as a negative control. The mice were sacrificed 7 days after the injection and the tumors weighed.

One-way analysis of variance (ANOVA) was used to analyze the significance between groups. All data represent mean ± SD. P<0.05 was considered to indicate a statistically significant difference.

Several studies have demonstrated the involvement of chemokines and their receptors in tumor growth and metastasis. In order to investigate the role of CXCR4 chemokine receptor in a model of murine melanoma, we first examined whether B16-F10 melanoma cells express this chemokine receptor. Our results indicate that these tumor cells constitutively express CXCR4 as shown in Fig. 1.

To reduce the expression of CXCR4 mRNA in B16-F10 melanoma cells, two CXCR4 shRNAs were designed. Each CXCR4 shRNA was annealed and ligated into the psiSTRIKE vector controlled by Pol III U6 promoter. The tumor cells were transfected for 24 and 48 h with the plasmid-based CXCR4-1 shRNA, CXCR4-2 shRNA or control shRNA expressing plasmids with Lipofectamine 2000. After transfection, CXCR4 mRNA degradation was monitored by RT-PCR. Fig. 2A shows that only the plasmid-based CXCR4-1 shRNA significantly reduced the level of CXCR4 mRNA (85%, P<0.001) after 48 h of transfection and this effect remains for up to 4 days (data not shown).

The level of CXCR4 protein in B16-F10 melanoma cells transfected with CXCR4-1 shRNA, CXCR4-2 shRNA or control shRNA was evaluated by western blot analysis. The downregulation of CXCR4 protein expression was also significantly (70%, P<0.001) observed after 48 h of transfection with CXCR4-1 shRNA as shown in Fig. 2B.

The B16-F10 melanoma cells transfected with CXCR4-1 shRNA or control shRNA were injected subcutaneously into C57BL/6 mice. The animals were sacrificed 14 days after the inoculation of B16-F10 cells and tumors were excised and weighted. Fig. 3 shows that tumor growth was significantly inhibited (66%, P<0.001) when B16-F10 melanoma cells were transfected with CXCR4-1 shRNA when compared to control shRNA.

To investigate the effect of the intratumoral injection of the CXCR4-1 shRNA-expressing plasmids on melanoma growth, mice were inoculated subcutaneously with B16-F10 melanoma cells. Fig. 4 shows a significant reduction of tumor weight (70%, P<0.001) in mice that had received a single intratumoral injection of the CXCR4-1 shRNA compared to animals injected with the plasmid-based control shRNA.