Inhibition of experimental lung metastasis by systemic lentiviral delivery of kallistatin

Human rhabdomyosarcoma cells (TE671), 293T and mouse Lewis lung carcinoma (LL2) were cultured in Dulbecco's Modified Minimal Essential Medium (DMEM) supplemented with 10% cosmic calf serum (Hyclone, Logan, Utah, USA), 2 mM L-glutamine and 50 μg/ml gentamicin at 37°C in 5% CO₂. Human umbilical vein endothelial cells (HUVEC) were cultured in EGM medium (Cambrex, East Rutherford, NJ, USA). The pcDNA3.1-hKallistatin plasmid was linearized with HindIII and filled in with Klenow fragment to form blunt end, and then the human kallistatin fragment was released with EcoRI from the linearized plasmid. The human kallistatin expression vector pWPXL-Kallistatin, under the control of elongation factor 1-α (EF-1α) promoter, was constructed by cloning the 1.3-kb cDNA fragment of human kallistatin into the PmeI/EcoRI sites of pWPXL. To produce recombinant lentiviruses encoding kallistatin or green fluorescence protein (GFP), 293T cells were transfected with pWPXL-Kallistatin or pWPXL, together with psPAX2 packaging vector and pMD2.G envelope vector by calcium phosphate precipitation, and the conditioned medium (CM) containing viral particles was harvested 48 h after transfection [9]. The titers of virus were determined on TE671 cells by fluorescence-activated cell sorter (FACS) analysis for GFP expression (BD Biosciences, San Jose, CA, USA). Briefly, approximately 2 × 10⁵ TE671 cells were plated in six-well culture dishes and infected with serial 10-fold dilutions of viral vectors (10^-1 to 10^-5) in serum free medium. Cells were collected 72 h after infection, and the number of GFP-positive cells was used to quantify the titer (transduction unit, TU).

Meanwhile, the titer of viral particles was performed using the Quick Titer lentivirus titer kit (Cell Biolabs, San Diego, CA, USA), which measures lentivirus-associated HIV p24 by enzyme-linked immunosorbent assay (ELISA). One ng/ml of lentiviral p24 corresponds to 1 × 10⁵ TU/ml of the GFP-encoding lentiviral vector. The vector pWPXLLuc was derived from pWPXL where the GFP cDNA was replaced with firefly luciferase cDNA obtained from the pGL3-Basic vector (Promega, Madison, WI, USA). The EF1-α promoter was removed from pWPXLLuc at ClaI and SwaI sites and then replaced by the NF-κB responsive elements obtained from pGL2-IL8, resulting in pWPXL-NF-κBLuc [10]. The recombinant lentiviruses encoding luciferase were produced as previously described. For transduction of LL2 cells with the luciferase gene, cells were infected with recombinant lentiviruses carrying luciferase gene under the control of NF-κB responsive elements. As the lentiviral vectors did not contain selectable markers, luciferase-expressing stable LL2 clones were identified by monitoring luciferase expression in each isolated clone. LL2/NF-κBLuc clones with high level luciferase expression were used for further studies. Male C57BL/6 mice (6-8 weeks old) were obtained from the Laboratory Animal Center of the National Cheng Kung University. The experimental protocol adhered to the rules of the Animal Protection Act of Taiwan and was approved by the Laboratory Animal Care and Use Committee of the National Cheng Kung University. At indicated time points, mice were sacrificed, and the tissue lysates were assessed for luciferase activity with luciferase reporter gene assay system (Applied Biosystems, Foster City, CA, USA) using a luminometer (MiniLumat LB 9506; Berthold Technologies, Bad Wildbad, Germany).

The TE671 cells were infected with lentiviruses encoding GFP (LV-GFP) or LV-Kallistatin in different multiplicity of infection (MOI). The virus-containing medium from the infected cells was removed 8 h post-infection and fresh medium was added. The expression of kallistatin was verified by ELISA as described previously [11]. Forty-eight h after infection, the supernatant was collected for the migration and proliferation assays. Cell migration was assessed using a modified Boyden Chamber (Corning Costar, Cambridge, MA, USA). The membrane was coated with gelatin (100 ng/ml) for migration assay or collagen (100 ng/ml) for invasion assay. VEGF (10 ng/ml) or bFGF (15 ng/ml) was added at the lower compartment of the chamber. HUVEC (1 × 10⁴ cells/well) or LL2 cells (1 × 10⁴ cells/well) treated with CM were added to the upper compartment and incubated for 4 h. The filter was then fixed with 100% methanol for 8 minutes and stained by Giemsa stain solution for 1 h. Cells were counted randomly three images per well under a microscope. For the cell proliferation assay, cells were treated with serum-free medium for 24 h, and then incubated with 100 μl of CM from TE671 cells infected with lentiviruses in 10 MOI or mock infection in the presence of VEGF (10 ng/ml) or bFGF (15 ng/ml) for 48 h. Cell viability was measured with WST-1 assay (Dojindo Laboratories, Tokyo, Japan).

To determine the expression of kallistatin after LV-Kallistatin injection, mice were inoculated with LL2 cells (5 × 10⁵) via the tail vein at day 0. LV-Kallistatin, LV-GFP (10⁶ TU), or saline were injected into the mice via tail vein at day 15. The distribution of kallistatin in tissue was determined by ELISA at day 17. In the experimental lung metastatic model, mice were injected with LL2 cells (5 × 10⁵) via tail vein at day 0. Then the mice were treated with lentivirus (10⁸ TU) by intravenous injection at day 1. To detect the protein and cytokine expressions, the lungs were collected at day 12. Levels of TNF-α and VEGF in the lung were determined by ELISA (R & D, Minneapolis, MN, USA). The protein content in each sample was determined by bicinchoninic acid (BCA) protein assay (Pierce Biotechnology, Rockford, IL, USA). Moreover, lungs from the tumor-bearing mice treated with lentivirus (10⁸ TU) or saline were collected and weighted at day 25. To analyze infiltrating macrophages and microvessel density in the tumor sites, tumor-bearing mice were injected intravenously (i.v.) with LV-Kallistatin, LV-GFP (10⁸ TU), or saline at day 1. The whole tumors were excised and snap frozen at day 20. Tumor angiogenesis and infiltrating macrophages were assessed by immunostaining as previously described [12].

To test the feasibility of exploiting recombinant lentiviruses for gene delivery in cells, the expression of kallistatin was examined and characterized in TE671 cells infected with LV-Kallistatin or LV-GFP. TE671 cells transduced with lentiviral vectors were shown 1.3~2.1 times more efficiently transduction rate than 293T and HepG2 cells [13]. In this study, TE671 cells were used as the target cells to express the transgene. ELISA analysis was performed to examine the protein levels of kallistatin in the supernatant from TE671 cells. Kallistatin was detectable in LV-Kallistatin-treated cells, but not in LV-GFP- or saline-treated cells. To determine the gene expression in mice, we injected saline, LV-GFP, or LV-Kallistatin into tumor-bearing mice at day 15 and examined the expression of kallistatin at day 17. Low levels of signal in serum, lung, and tumor sites were detected in the saline-treated and LV-GFP-treated group. After LV-Kallistatin injection, the expression of human kallistatin was detected in all examined organs including tumor sites (Figure 1A). The CM from LV-Kallistatin-infected cells was tested for bioactive kallistatin. As shown in Figure 1, the CM from LV-Kallistatin-infected cells significantly reduced VEGF- (Figure 1B) and bFGF-(Figure 1C) induced the proliferation of endothelial cells. The inhibitive phenotype was in a dose-dependent manner. Together, these results showed that lentivirus-mediated gene transfer could produce biological active kallistatin.

Recombinant lentiviral vectors can transduce genes with great efficiency. Furthermore, systemic administration of lentiviruses resulted in the major transgene production in livers (Figure 1A) [14]. The results point out that host tissues can be transduced with lentiviral vectors and contribute to transgene production. We used the CM system in vitro to mimic the microenvironment in vivo. Figure 2A demonstrated that proliferation of LL2 cells was dramatically decreased upon addition of the CM from LV-Kallistatin-infected cells compared with that from LV-GFP-infected counterparts. Meanwhile, kallistatin in the CM dramatically reduced VEGF- or bFGF- induced LL2 cell migration and invasion (Figures 2B and 2C). In addition, the proliferation of LL2 cells was inhibited by LV-Kallistatin infection (Additional file 1). Taken together, these observations suggested that kallistatin inhibited the proliferation, migration, and invasion of tumor cells.

Since inhibition of metastatic tumor is still a major challenge for tumor treatment, we next investigated whether LV-Kallistatin could inhibit pulmonary tumor nodules. The in vivo antitumor effects of LV-Kallistatin were evaluated in terms of tumor growth and survival in mice bearing metastatic pulmonary nodules. We injected mice with LL2 cells via tail vein at day 0, and then treated them i.v. with LV-Kallistatin, LV-GFP or saline at day 1 after tumor inoculation. Tumor-bearing mice were sacrificed at day 25 to determine the wet lung weight for quantifying the tumor burden in the lungs. As shown in Figures 3A and 3B, the tumor nodules and total lung weight were dramatically reduced in LV-Kallistatin-treated mice. The lung weight of mice treated with LV-Kallistatin was 35% less than that treated with LV-GFP or saline. Meanwhile, the treatment of LV-Kallistatin prolonged the survival of mice with pulmonary metastasis (Figure 3C). Results of this study showed that systemic delivery of LV-Kallistatin delayed tumor growth and prolonged mice survival.

Microvessel density within the tumors was analyzed by immunohistochemistry at day 20. Tumors from LV-Kallistatin-treated mice revealed much less vessels than those from LV-GFP- or saline-treated counterparts, whereas no such difference was found between LV-GFP- and saline-treated groups (Figures 4A and 4B). Microvessel density in tumors receiving LV-Kallistatin reduced to 40% as compared with that in LV-GFP-injected group (19 ± 8 vessels/mm², n = 10 versus 10 ± 4 vessels/mm², n = 11) and in saline-injected group (19 ± 5 vessels/mm², n = 11 versus 10 ± 4 vessels/mm², n = 11). These results demonstrated that kallistatin gene delivery markedly suppressed the angiogenic response in the tumor sites.

Tumor progression depends, in part, on the density of tumor-associated macrophages (TAMs). There is a correlation between TAM abundance and poor prognosis [15]. TAMs are highlighted as not only a major participator but also an important regulator of inflammation. These macrophages actually promote the proliferation and metastasis of tumor cells by secreting a wide range of growth and pro-angiogenic factors, as well as pro-inflammatory cytokines [16]. In present study, the distribution of macrophages in the tumor sites was assayed by immunostaining. The numbers of infiltrating macrophages in mice treated with LV-Kallistatin were significantly decreased in comparison with those in mice treated with LV-GFP or saline (Figures 5A and 5B). As macrophages are major cytokine-secreting cells, we next examined the levels of TNF-α and VEGF in tumor microenvironment after different treatments. Tumors from LV-Kallistatin group contained lower concentrations of TNF-α and VEGF than those from control groups (Figures 5C and 5D). The presence of macrophages in solid tumors suggests that tumors grow at the sites of chronic inflammation. NF-κB signaling might play an important role in inflammation and tumor progression. To assess the activation of NF-κB signal transduction pathway, we performed a reporter assay using lentivirus-transduced LL2 cells containing the luciferase gene under the control of NF-κB responsive elements in vivo. LL2/NF-κBLuc cells were injected into the tail vein of mice, and the luciferase activity from lungs was determined. Figure 5E showed that luciferase gene expression was high, indicative of higher NF-κB activity, in the lungs of mice treated with saline or LV-GFP. Kallistatin inhibited the NF-κB transcriptional activity in tumor. These results suggest a potential role for kallistatin in the anti-inflammation and anti-angiogenesis, which may contribute to the inhibition of tumor growth.