Soluble Flt-1 gene therapy for peritoneal metastases using HVJ-cationic liposomes

Abstract

Many studies have reported a close association between VEGF and tumor angiogenesis. The aim of the present study was to evaluate the effectiveness of gene therapy against cancer, including peritoneal metastasis, using a cDNA encoding a soluble type of Flt-1, one of the VEGF receptors. In a peritoneal metastasis model of MKN45 human gastric cancer cells, mice repetitively treated with intraperitoneal injections of HVJ-Fex, a type of HVJ-cationic liposome encapsulating a plasmid expressing soluble mFlt-1, exhibited smaller disseminated foci with fewer microvessels, thus resulting in a significantly longer survival period than the control mice. In another peritoneal metastasis model using HT1080S cells, a clone of HT1080 human fibrosarcoma cells stably transfected with hVEGF, treatments with HVJ-Fex also reduced the growth of disseminated foci without ascites formation. In conclusion, this study demonstrated that the peritoneal metastases of some cancers were largely dependent on VEGF, and that the repeated intraperitoneal transduction of a soluble flt-1 gene using HVJ-cationic liposomes suppressed peritoneal metastases, thereby contributing to a longer survival period.

Introduction

The growth of solid tumors greater than 1–2 mm3 is critically dependent on angiogenesis.1 Among the many angiogenic molecules produced by tumor cells, vascular endothelial growth factor (VEGF), a potent growth factor specific for vascular endothelial cells,2 is overexpressed in most tumors and appears to play a crucial role in tumor angiogenesis.345 Many studies have demonstrated a positive correlation between VEGF and tumor growth, as well as a close association between VEGF and tumor angiogenesis.67891011121314 The angiogenic actions of VEGF are mediated via two endothelium-specific receptor tyrosine kinases, Flt-1 and Flk-1/KDR.1516171819 The structures of these membrane-spanning receptors are highly homologous, characterized by the presence of seven immunoglobulin-like domains in the extracellular domain, a transmembrane domain and an intracellular tyrosine kinase domain.15 Flt-1 shows at least a 10-fold higher affinity for VEGF than Flk-1/KDR, even if present as the soluble form of the extracellular domain only,20 and functions as a trapping protein to regulate negatively the levels of VEGF around endothelial cells during embryogenesis.2122

Recently, several studies have reported that not only angiogenesis but also tumor growth was suppressed by the soluble form of the VEGF receptors. For example, Lin et al23 reported that a recombinant form of the soluble Flk-1 protein inhibited tumor growth and vascular density in a cutaneous tumor window chamber. Goldman et al24 reported that the ex vivo transfection of human tumor cells with a cDNA encoding a soluble Flt-1 receptor inhibited the tumor growth rate after either subcutaneous or intracranial injection, and also lung metastases after i.v. injection. Finally, Kong et al25 reported that liver metastases, lung metastases and primary subcutaneous tumors were inhibited by the intravenous, intratracheal and intratumoral administration of an adenovirus-mediated soluble flt-1 cDNA, respectively.

We have previously demonstrated that VEGF induced strong tumorigenicity in the peritoneal cavity as well as in various organs.14 However, no studies on the use of gene therapy targeting VEGF for peritoneal dissemination have been performed, although peritoneal metastases remains a critical disease for cancer patients.26 These lines of evidence led us to hypothesize that an inhibition of VEGF function might be a novel strategy against disseminated cancer in the peritoneal cavity. In the present study, we evaluated the effectiveness of gene therapy against peritoneal metastases, using repeated intraperitoneal transductions of a soluble flt-1 gene mediated by HVJ-cationic liposome.272829

Results

Treatments with soluble Flt-1 gene transduction for subcutaneous and peritoneal disseminated tumors

We prepared three types of HVJ-opDC liposome solutions, designated HVJ-Fex, HVJ-CD4 and HVJ-GFP, made from a plasmid expressing soluble mFlt-1, a plasmid expressing hCD4 as a control and a plasmid expressing GFP, respectively.

The administration of HVJ-GFP into the peritoneal cavity revealed that disseminated MKN45 foci, especially the marginal portions of the foci, were selectively transfected (Figure 1), while other nontumorous organs including liver, kidney, spleen, pancreas, omentum, mesentery, intestine, colon, testis and ovary exhibited no specific fluorescence.

Figure 1
figure1

In vivo transfection into peritoneal disseminated tumors by intraperitoneal injection of the HVJ-cationic liposome. Mice bearing established, peritoneal-disseminated MKN45 tumors received intraperitoneal injections of HVJ-GFP (a–e) or HVJ-CD4 (f) four times over 2 weeks. (a) and (b) Macroscopic findings of the intraperitoneal cavity. Original magnification ×4. (c) and (d) Fluorescent stereomicroscopic findings of the disseminated tumors indicated by the square in (a) and (b). Original magnification ×50. (e) and (f) The disseminated tumors of mice were fixed in 4% paraformaldehyde and sliced into 8 μm sections, and were examined under a fluorescent microscope to detect GFP expression. The pictures were taken under the same conditions. Arrows: margin of the tumor; original magnification ×400.

In the HT1080S i.p. tumor model, when the mice were killed on day 11, the disseminated tumors treated with HVJ-Fex were much smaller than those treated with HVJ-CD4; the former was 42 ± 33 mm3 (mean ± s.e., n = 5), whereas the latter was 471 ± 54 mm3 (mean ± s.e., n = 5) in volume (P = 0.009) (Figure 2). Moreover, ascites formation was never detected in the mice treated with HVJ-Fex, but was observed in four of the five mice treated with HVJ-CD4.

Figure 2
figure2

Tumor volume after treatment with HVJ-Fex in the intraperitoneal disseminated tumor model using HT1080S cells. The mice received intraperitoneal injections of HT1080S cells (3 × 106 cells) on day 0, and two intraperitoneal injections of HVJ-Fex or HVJ-CD4 on day 2 and day 6. The mice were killed on day 11. The tumor volume was represented by the sum of all tumor volumes calculated as 1/2 × length × width2 (length > width). HVJ-Fex: mice treated with HVJ-Fex; HVJ-CD4: mice treated with HVJ-CD4. Bars represent the s.e.

Repeated treatments with HVJ-Fex decreased the tumor growth of MKN45 cells not only in the s.c. but also in the i.p. tumor model. On day 33, the subcutaneous tumors treated with peritumoral injections of HVJ-Fex had grown to only 395 ± 77 mm3 (mean ± s.e., n = 4), whereas the tumor treated with those of HVJ-CD4 had developed to 1506 ± 540 mm3 (mean ± s.e., n = 4) in volume (P = 0.043) (Figure 3). In the i.p. tumor model, when the mice were killed on day 33, the total volumes of the disseminated foci treated with intraperitoneal injections of HVJ-Fex and HVJ-CD4 were 260 ± 129 mm3 (mean ± s.e., n = 10) and 1397 ± 505 mm3 (mean ± s.e., n = 10), respectively (P = 0.013). All disseminated tumor foci larger than 7 mm in diameter in the above two groups were then subjected to an assessment of their microvessel density. The microvessel densities in the disseminated tumors treated with HVJ-Fex and HVJ-CD4 were 17.6 ± 2.6 per field (mean ± s.e., n = 3) and 28.8 ± 1.3 per field (mean ± s.e., n = 5), respectively (P = 0.025). The survival periods of the 10 mice treated with HVJ-Fex for 4 weeks were statistically longer than those of the mice treated with HVJ-CD4 (P = 0.033, log rank analysis) (Figure 4).

Figure 3
figure3

Growth rate of tumors treated with HVJ-Fex in a subcutaneous tumor model using MKN45 cells. The mice received subcutaneous injections of MKN45 cells (1 × 107 cells) on day 0. From day 1, repeated peritumoral injections of HVJ-Fex or HVJ-CD4 were performed twice a week for 4 weeks. HVJ-Fex: mice treated with HVJ-Fex; HVJ-CD4: mice treated with HVJ-CD4. Bars represent the s.e. Arrows: treatment; *P < 0.05 versus the HVJ-CD4 group at the same time.

Figure 4
figure4

Repeated treatments with HVJ-Fex in an intraperitoneal disseminated tumor model using MKN45 cells. The mice received intraperitoneal injections of MKN45 cells (3 × 106 cells) on day 0. From day 1, repeated intraperitoneal injections of HVJ-Fex (a and d) or HVJ-CD4 (b and e) were performed twice a week for 4 weeks. The mice were killed on day 33. (a) and (b) Macroscopic findings of disseminated tumors on the mesentery. (c) Total volume of the disseminated tumors. (d) and (e) All disseminated tumors larger than 7 mm in diameter were subjected to immunohistochemical staining with an antibody against factor VIII-related antigen. (f) Microvessel density in the disseminated tumors was represented by the average number of immunopositive stained spots in five fields at 200-fold magnification. (g) Kaplan–Meier curves for mice treated for 4 weeks. Bars represent the s.e. Differences in survival were evaluated by the log rank test.

Discussion

VEGF and its receptors, Flt-1 and Flk-1/KDR, have been shown to play an important role in normal and pathological angiogenesis. From the viewpoint of tumor angiogenesis, VEGF is commonly overexpressed in most human tumors. Recently, several studies have reported that angiogenesis was suppressed by a soluble type of VEGF receptor. Either endogenous membrane-spanning VEGF receptor, Flt-1 or Flk-1/KDR, is activated by ligand-mediated dimerization, followed by phosphorylation of the intracellular tyrosine kinase domain. In the present study, we focused on the soluble Flt-1 receptor, because Flt-1 has a much higher affinity for VEGF even in its soluble form, but has lower kinase activity than Flk-1/KDR. Therefore, soluble Flt-1 receptors attenuate VEGF activity by trapping VEGF itself.

Although it is generally recognized that angiogenesis is essential for the growth of solid tumors, no direct evidence that angiogenesis plays a critical role in peritoneal metastasis has been provided. We have previously reported that VEGF facilitates peritoneal dissemination as well as tumorigenicity in various organs. The HT1080 cells transfected with the VEGF cDNA, HT1080S, showed an augmented potential for peritoneal metastasis with accompanying ascites. The present study demonstrated that intraperitoneal treatment with HVJ-Fex suppressed peritoneal dissemination and ascites formation in the HT1080S model. Furthermore, the peritoneal dissemination model using MKN45 cells also showed that intraperitoneal injections of HVJ-Fex inhibited tumor angiogenesis and tumor growth, resulting in a prolongation of the survival period even after the treatment was discontinued. These data demonstrate that VEGF is one of the key molecules responsible for peritoneal metastasis, and suggest that high concentrations of soluble Flt-1 receptors in the intraperitoneal closed cavity could attenuate the biological activity of VEGF. Since VEGF is also known to be a vascular permeability factor,30 two different functions of VEGF might contribute to peritoneal dissemination. First, VEGF may act as an angiogenic factor which augments tumor vascularity. Second, it may act as a permeability factor to promote the supply of nutrients to the tumor by diffusion, and thus occasionally causes ascites. This study suggested that both of these functions were suppressed by treatment with HVJ-Fex.

HVJ-liposomes can deliver exogenous DNA into cells by means of HVJ-mediated membrane fusion. Among the many approaches for gene transfer in vivo using viral or nonviral vectors, the HVJ-liposome method is thought to be the most suitable for antiangiogenic gene therapy because HVJ-liposomes can maintain gene expression for long periods by repeated injection, without apparent toxicity, inflammation or significant immunogenicity in vivo.31323334 Although the transfection efficiency of HVJ-liposomes is relatively lower than that of viral vectors such as adenoviral vectors, repeated transductions with HVJ-liposomes make it feasible to express continuously an indicated gene, and thus may be more advantageous for antiangiogenic therapy against cancer. Boehm et al35 reported that cyclical therapy with the antiangiogenic proteins, angiostatin and endostatin, resulted in permanent tumor arrest at a microscope dormant size with blocked angiogenesis, even after the therapy was discontinued. Antiangiogenic gene therapy does not always require a direct, selective transduction of the gene into the cancer cells, but rather around the tumor to create an antiangiogenic environment.36 Fortunately, the HVJ-opDC liposomes used in this study selectively transduced the gene into the peritoneal disseminated tumors themselves, as demonstrated in the experiment using HVJ-GFP. The exact mechanism remains unknown, but we speculate that the transduction specificity of the HVJ-opDC liposomes depends mainly on the electric charge of the liposomes and the mitotic activity of the cells.37383940 The plasma membrane of cancer cells has a negative charge in comparison with normal cells, whereas the HVJ-opDC liposomes generated from cationic liposomes have a net neutral charge.29 Therefore, HVJ-opDC liposomes might be attracted more strongly to cancer cells than to normal cells. In addition, since DNA translocates into the nucleus easily during the M phase of the cell cycle, in which the nuclear membrane disappears, proliferating cancer cells are more effectively transfected than normal cells. These characteristics of the HVJ-opDC liposomes and the cancer cells themselves favor the treatment of peritoneal disseminated tumors, and hence HVJ-opDC liposomes preferentially fuse to the exposed cancer cells in the closed peritoneal cavity, resulting in selective transduction into the tumors.

In conclusion, this study was the first to demonstrate that the peritoneal metastases from some cancers are largely dependent on VEGF, and that the repeated intraperitoneal transduction of the soluble flt-1 gene using HVJ-cationic liposomes can suppress peritoneal metastases, thereby contributing to a longer survival period.

Materials and methods

Cell culture

The human fibrosarcoma cell line HT1080 and the human gastric cancer cell line MKN45 were cultured in MEM (Nissui, Tokyo, Japan) containing 10% heat-inactivated fetal bovine serum (BioWhittaker, Walkersville, MD, USA), 100 units/ml penicillin and 100 μg/ml streptomycin in a humidified atmosphere of 5% CO2. As previously reported, we established a stably transfected clone of HT1080 with hVEGF121 cDNA, named HT1080S. This clone possesses stronger tumorigenicity in the peritoneal cavity than the parental HT1080, with massive ascites formation, as well as in the subcutaneous space with vigorous angiogenesis.14

Plasmids

The eukaryotic expression vector for the soluble mouse Flt-1 receptor was kindly donated by Dr M Hirashima (Dept of Molecular Genetics, Graduate School of Medicine, Kyoto University, Japan).41 This plasmid vector, named pFex-IgG, was constructed with pCDM8 containing the cDNA encoding the seven extracellular immunoglobulin-like domains of mouse flt-1 followed by the human IgG1 Fc gene.42 The result was a chimeric protein consisting of soluble mFlt-1 fused to the Fc portion of hIgG1. A control expression vector, named pCD4-IgG, was constructed to express a chimeric protein consisting of human CD4 fused to hIgG1 Fc. A reporter system using the green fluorescent protein (GFP) expressed by the plasmid vector pEGFP-N1 (Clontech, Palo Alto, CA, USA) was used for the evaluation of the transfection efficiency in vivo.

Preparation of the HVJ-opDC liposome

The cationic liposome conjugated with HVJ (hemagglutinating virus of Japan, Sendai virus), HVJ-opDC liposome, was prepared as previously described.29 Briefly, HVJ was collected and purified by two centrifugations at 27 000 g for 30 min, and suspended in balanced salt solution (BSS; 137 mM NaCl, 5.4 mM KCl, 10 mM Tris-HCl pH 7.6). The RNA genome of HVJ was inactivated by ultraviolet irradiation (198 mJ/cm2) just before use. Lipid solution in chloroform at a concentration of 30 mM was prepared by mixing egg yolk phosphatidylcholine (ePC) (Sigma, St Louis, MO, USA), egg yolk sphingomyelin (eSph) (Sigma), dioleoylphosphatidylethanolamine (DOPE) (Sigma), cholesterol (Chol) (Sigma) and DC-cholesterol (DC-Chol) (synthesized as previously reported43) (ePC:eSph:DOPE:Chol:DC-Chol = 5:5:5:12:3 in molar ratio). A dried thin film of the lipid was then generated by evaporating 500 μl of the lipid solution in a glass tube. The dried lipid film was hydrated in 200 μl of BSS containing 200 μg plasmid DNA, and then liposomes encapsulating the DNA were prepared by the vortexing-filtration method. After the liposome suspension was shaken with the inactivated HVJ suspension (30 000 hemagglutinating units), the HVJ-opDC liposome complexes were separated from the free HVJ by sucrose density gradient centrifugation. The HVJ-opDC liposomes were collected and diluted with BSS to the appropriate concentration for each experiment.

We prepared three types of HVJ-opDC liposome solutions, designated HVJ-Fex, HVJ-CD4 and HVJ-GFP, made from pFex-IgG, pCD4-IgG and pEGFP-N1, respectively.

In vivo treatment of tumors with HVJ-opDC liposomes

All in vivo experiments were performed in accordance with the Guideline for Animal Experiments of Kyoto University.

We used the HVJ-GFP to determine the sites which were transfected with the HVJ-opDC liposomes in the peritoneal disseminated (i.p.) tumor model. Four-week-old male BALB/cAnCrj-nu/nu mice (Charles River, Yokohama, Japan) bearing peritoneal disseminated MKN45 tumors were prepared by an intraperitoneal inoculation of 3 × 106 cells in 0.5 ml PBS. One week later, when each disseminated focus was still less than 5 mm in diameter, the mice received intraperitoneal injections twice a week of 0.5 ml of HVJ-GFP solution generated from 30 μg of plasmid DNA. After this treatment for 2 weeks, the mice were killed and those tissues expressing GFP were detected in the peritoneal cavity using a fluorescent stereomicroscope (Olympus, Tokyo, Japan) at low magnification. The disseminated tumors were then fixed in 4% paraformaldehyde and sliced into 8 μm sections, and were also examined with a fluorescent microscope at high magnification. Mice bearing peritoneal disseminations of MKN45 which received injections of HVJ-CD4 were also examined as controls.

To evaluate the effects of treatment with HVJ-Fex, we used HT1080S cells in the intraperitoneal disseminated (i.p.) model, and MKN45 cells in the subcutaneous (s.c.) and i.p. tumor models. At day 0, the mice received a subcutaneous and an intraperitoneal inoculation at doses of 1 × 107 cells in 0.2 ml PBS and 3 × 106 cells in 0.5 ml PBS, respectively. On day 1, repeated injections of HVJ-Fex or HVJ-CD4 as the control were started. The s.c. and i.p. tumor model mice received peritumoral and intraperitoneal injections of 0.5 ml of the solution generated from 15 μg and 30 μg plasmid DNA, respectively. The peritumoral injection means injection into subcutaneous space around the tumor. The mice with the HT1080S tumors received treatments on days 2 and 6 and were killeed on day 11. The mice with the MKN45 tumors received treatments twice per week for 4 weeks and were killed on day 33. The tumor volume per mouse was determined by the sum of all tumor volumes calculated as 1/2 × length × width2 (length > width).

The microvessel density in tumor was assessed by immunohistochemical staining with the antibody against factor VIII-related antigen (Dako, Carpinteria, CA, USA). The average number of immunopositive stained spots from five fields at 200-fold magnification was used to represent the microvessel density of the tumor.

Statistical analysis

The vessel count and tumor growth rate were evaluated by a Mann–Whitney U test. The statistical significance of differences in the survival times among groups was determined using the log rank test and a Kaplan–Meier survival analysis.

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Acknowledgements

We thank Dr G Breier from the Max Planck Institute for Physiological and Clinical Research for providing us with the plasmid containing the full length murine flt-1 cDNA.

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Correspondence to A Mori.

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Mori, A., Arii, S., Furutani, M. et al. Soluble Flt-1 gene therapy for peritoneal metastases using HVJ-cationic liposomes. Gene Ther 7, 1027–1033 (2000). https://doi.org/10.1038/sj.gt.3301202

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Keywords

  • soluble Flt-1
  • HVJ-liposome
  • peritoneal metastasis
  • gene therapy

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