original article
© The American Society of Gene & Cell Therapy
Delivery of siRNA Using CXCR4-targeted Nanoparticles Modulates Tumor Microenvironment and Achieves a Potent Antitumor Response in Liver Cancer Jia-Yu Liu1, Tsaiyu Chiang1, Chun-Hung Liu1, Guann-Gen Chern1, Ts-Ting Lin1, Dong-Yu Gao1 and Yunching Chen1 Institute of Biomedical Engineering, National Tsing Hua University, Hsinchu, Taiwan, ROC
1
Antiangiogenic therapy has recently emerged as a highly promising therapeutic strategy for treating hepatocellular carcinoma (HCC). However, the only clinically approved systemic antiangiogenic agent for advanced HCC is sorafenib, which exerts considerable toxicity. Moreover, acquired resistance to antiangiogenic therapy often develops and restricts the therapeutic efficacy of this treatment. Hence, in this study, we develop a CXCR4targeted lipid-based nanoparticle (NP) formulation to specifically deliver vascular endothelial growth factor (VEGF) siRNA as an antiangiogenic substance into HCC. AMD3100, a CXCR4 antagonist, is added into NPs to serve as both a targeting moiety and a sensitizer to antiangiogenic therapy. We demonstrate that AMD-modified NPs (AMD-NPs) can efficiently deliver VEGF siRNAs into HCC and downregulate VEGF expression in vitro and in vivo. Despite the upregulation of the SDF1α/CXCR4 axis upon the induction of hypoxia after antiangiogenic therapy, CXCR4 inhibition by AMD-NPs in combination with either conventional sorafenib treatment or VEGF siRNA prevents the infiltration of tumor-associated macrophages. These dual treatments also induce synergistic antiangiogenic effects and suppress local and distant tumor growth in HCC. In conclusion, the tumor-targeted multifunctional AMD-NPs that co-deliver VEGF siRNA and AMD3100 provide an effective approach for overcoming tumor evasion of antiangiogenic therapy, leading to delayed tumor progression in HCC. Received 19 January 2015; accepted 28 July 2015; advance online publication 29 September 2015. doi:10.1038/mt.2015.147
INTRODUCTION
Hepatocellular carcinoma (HCC) is one of the most fatal cancers worldwide. HCC arises from damaged liver tissues with a preexisting inflammatory microenvironment, fibrosis, and subsequent
hypervascularization and thus has been identified as a highly vascular tumor.1–7 Therefore, antiangiogenic therapy has become a highly promising therapeutic strategy for treating HCC.8–10 The tyrosine kinase inhibitor sorafenib is the only approved systemic treatment for advanced HCC. Sorafenib suppresses angiogenesis and tumor progression by blocking VEGFR/PDGFR in the tumor vasculature and the RAF/MEK/ERK pathway in HCC cells.11–15 Although sorafenib shows moderate therapeutic outcomes in some HCC patients, antiangiogenic therapy remains challenging. There are multiple hurdles involved in efficient antiangiogenic cancer treatment, including the rapid development of resistance and considerable toxicity. Several mechanisms are involved in the rapid escape from antiangiogenic therapy, leading to transient therapeutic effects, as well as local and distant recurrences of HCC.16–18 We have recently found that sorafenib can trigger a tumor-promoting microenvironment in HCC by increasing hypoxia, inflammation, and fibrosis in the tumor tissues.1 The SDF1α/CXCR4 axis is upregulated after sorafenib treatment and plays a key role in enhancing cancer cell survival, recruiting tumor-associated bone marrow–derived cells, increasing desmoplasia and promoting metastatic phenotypes in HCC.10,19–21 In addition, angiogenic blockades can also be taken by the normal tissues, causing unwanted side effects and toxicity.22 Thus, there is an urgent need for the development of a new therapeutic strategy. Herein, we developed multifunctional lipid-based nanoparticles (NPs) to co-deliver a small interfering RNA (siRNA) against vascular endothelial growth factor (VEGF) and a small molecule CXCR4 antagonist. Compared to tyrosine kinase inhibitors such as sorafenib, siRNAs (short double-stranded RNAs) with 20–25 base pairs can silence the targeted genes more specifically and efficiently with lower unwanted toxicity. Thus, siRNAs can serve as a safe and effective cancer therapy. We showed that VEGF siRNA delivered by NPs downregulated VEGF expression and suppressed angiogenesis in HCC. Furthermore, the CXCR4 antagonist AMD3100 was not only
Correspondence: Yunching Chen, Institute of Biomedical Engineering, National Tsing Hua University, Hsinchu 30013, Taiwan, ROC. E-mail: [email protected]
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Figure 1 Illustration of the preparation of AMD-NPs containing siRNA and in vitro cytotoxicity of AMD-NPs in combination with sorafenib. (a) Sizes and zeta potentials of NPs in different stages of preparation. The TEM characterization of AMD-NPs is shown. The data are the mean values ± SEM (n = 3–4). (b,c) Exposure to recombinant SDF1α (50 ng/ml) increased the viability of JHH-7 and HCA-1 cells despite sorafenib (Sor) treatment (0.25 μmol/l). Cell viability was measured using the MTT assay. Inhibition of CXCR4 with free AMD3100 or AMD-NPs (0.5 μmol/l) prevented the pro-proliferative effects of SDF1α. The data are the mean values ± SEM (n = 4–6). *P < 0.05, **P < 0.01. MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; PDI, polydispersity index; TEM, transmission electronic microscope.
encapsulated in NPs but added to modify the surfaces of the NPs. To this end, AMD3100 carries dual effects, working as a ligand for the intracellular delivery of siRNA into malignant HCC as well as a CXCR4 blocker, achieving enhanced anticancer effects when combined with VEGF siRNA.1,23–25 In conclusion, our tumor-targeting multifunctional NP co-delivering VEGF siRNA and AMD3100 may serve as a novel strategy for overcoming local and distant tumor evasion of antiangiogenic therapy, leading to delayed tumor progression in HCC. Molecular Therapy vol. 23 no. 11 nov. 2015
RESULTS Preparation and characterization of siRNA-loaded, CXCR4-targeted lipid-based NPs
The preparation and proposed structure of the siRNA-loaded, CXCR4-targeted NP is shown in Figure 1a. First, the protamine/ nucleic acids (calf thymus DNA and therapeutic siRNA) complex was prepared by mixing the protamine and nucleic acids at a weight ratio of approximately 4:7. The average size of the protamine/DNA complex was 169.8 nm and the zeta potential was 1773
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Figure 2 Changes in tumor microenvironments after treatment with sorafenib in different combinations. (a) Immunostaining for vWF, CAIX, SDF1α, and F4/80 in HCA-1 tumor tissue samples from mice treated with different treatments. Representative confocal images stained with different marker proteins are shown. Bar = 50 μm. (b–e) Samples were imaged and quantified with a Zeiss LSM 780 confocal microscope (n = 5–10). The data are the mean values ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001. vWF, von Willebrand factor.
−28.6 mV. Condensation of the NPs occurred when AMD3100 was present. Adding AMD3100 to the protamine/nucleic acids complex reduced the particle size to 110.7 nm and the zeta potential became positive (27 mV). Subsequently, the surface of the protamine/nucleic acids/AMD3100 complex was coated with an anionic liposome composed of DOPC, DOPA, and cholesterol. The liposome-coated NPs carrying siRNA and AMD3100 had an average size of 113.3 nm and were negatively charged (−52.9 mV). To achieve tumor targeting, the surface of the anionic liposome-wrapped NPs was coated with the CXCR4 ligand AMD3100, resulting in NPs with less negative charges (−23.4 mV) compared to the AMD3100-free NPs. Finally, the AMD3100-coated NPs were PEGylated to prolong their systemic circulation in the blood. The transmission electronic microscope images revealed that the 1774
PEGylated AMD3100-coated NPs (AMD-NPs) were spherical. The average AMD-NP diameter was 144.7 ± 14 nm, with a polydispersity index of 0.299 ± 0.03. siRNA and AMD3100 encapsulations in the NPs were about 90 and 80%, respectively.
AMD-NP modulation of the tumor microenvironment and sensitization of HCC to sorafenib treatment in vitro and in vivo To determine if the SDF1α/CXCR4 axis mediates HCC resistance to sorafenib treatment, we first exposed both murine HCC (HCA-1) and human HCC (JHH-7) cell lines to recombinant SDF1α in the presence of sorafenib. We found that SDF1α increased the viability of both JHH-7 and HCA-1 cells despite sorafenib treatment (Figure 1b,c). Furthermore, the inhibition of CXCR4 by AMD3100 prevented the effects of SDF1α and sensitized HCC www.moleculartherapy.org vol. 23 no. 11 nov. 2015
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Figure 3 Sorafenib induces a minor tumor growth inhibition in HCC, and CXCR4 inhibition with AMD-NPs synergizes with sorafenib treatment and suppresses distant metastasis. (a) Tumor sizes in orthotopic tumor-bearing mice after the treatment of sorafenib in different combinations (n = 4–5). (b,c) The number of lung metastatic nodules was significantly reduced in mice treated with AMD-NPs either alone or in combination with sorafenib. Lung metastases of HCC tumor cells were quantified and imaged with an optical microscope (n = 6). The data are the mean values ± SEM. *P < 0.05; **P < 0.01.
to sorafenib treatment. We next examined the cytotoxicity of NPs containing AMD3100 (AMD-NPs). Consistent with free AMD3100, the rescue effect of SDF1α on the sorafenib-induced cytotoxicity could be reversed by AMD-NPs in both the HCA-1 and JHH-7 cell lines (Figure 1b,c). To establish a syngeneic orthotopic murine HCC model, we intrahepatically implanted HCA-1 cells into C3H mice. We first evaluated the effects of sorafenib either alone or in combination with free AMD3100 or AMD-NPs on the mean vessel density (MVD) and hypoxic induction in the HCA-1 transplanted HCC models. After HCC tumors were established, sorafenib was given at a dose of 50 mg/kg daily for 14 days either alone or in combination with free AMD3100 or AMD-NPs (Supplementary Figure S1). We further evaluated the changes in MVD and tissue hypoxia in implanted tumor tissues. Sorafenib treatment alone significantly reduced MVD and increased carbonic anhydrase IX expression, indicating hypoxia induction (Figure 2a–c). We next examined the changes in SDF1α expression as a consequence of treatment-induced hypoxia. We found a threefold increase in SDF1α expression in HCA-1 tumor tissues after sorafenib treatment (Figure 2a,d). The combination of sorafenib and AMD-NPs further decreased the MVD and increased the fraction of hypoxic tissue and SDF1α expression in orthotopic HCA-1 tumor grafts when compared to sorafenib treatment alone (Figure 2a–d). We next examined the effects of sorafenib either alone or in combination with free AMD3100 or AMD-NPs on inflammatory cell infiltration in HCC. We found that the number of F4/80+ macrophages increased by over 3-fold in HCA-1 transplanted HCC models after sorafenib treatment (Figure 2a,e). Finally, blocking the SDF1α/CXCR4 axis with AMD-NPs in combination with Molecular Therapy vol. 23 no. 11 nov. 2015
sorafenib decreased the number of tumor-infiltrating F4/80+ macrophages to levels comparable to those of control-treated HCA-1 tumors (Figure 2a,e). However, due to poor pharmacokinetics, the addition of free AMD to the sorafenib treatment did not show significant differences for all of the parameters compared to sorafenib treatment alone (Figure 2). We further evaluated the effects of sorafenib either alone or in combination with free AMD3100 or AMD-NPs on HCC progression in HCA-1 transplanted HCC models. As shown in Figure 3a, treatment with sorafenib alone only showed a moderate tumor growth inhibition effect. Due to the poor pharmacokinetics of free AMD3100, the systemic injection of free AMD3100 did not significantly sensitize HCC to sorafenib treatment in vivo. However, the systemic administration of AMD-NPs in combination with sorafenib led to the synergistic tumor growth inhibition of orthotopic HCA-1 tumors in immunocompetent C3H mice compared to treatment with AMD-NP or sorafenib alone (Figure 3a). In the HCA-1 orthotopic model, spontaneous lung metastases developed around day 14 after tumor implantation. Neither treatment with sorafenib alone nor sorafenib in combination with free AMD3100 showed a reduction in metastasis formation compared to control-treated mice (Figure 3b,c). In contrast, both AMDNPs alone and a combination of AMD-NPs and sorafenib significantly inhibited lung metastasis in the orthotopic HCA-1 model (Figure 3b,c). It indicated that the SDF1α/CXCR4 pathway might serve as a direct regulator for metastasis or indirectly mediate metastasis via stromal cells (Figure 2e, Supplementary Figure S2, and Supplementary Materials and Methods). AMD-NPs blocked CXCR4 and thus could efficiently reduce the metastatic burden. 1775
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Figure 4 Intracellular uptake and gene silencing effects in HCC cells treated with siRNAs delivered by AMD-NPs in vitro. (a) HCA-1 cells were treated with FAM-labeled siRNAs in different formulations for 4 h and observed by confocal microscopy. The arrowhead represents the fluorescence of FAM-labeled siRNA in the cytoplasm. Bar = 50 μm. The nuclei are blue (DAPI). (b) Fluorescent siRNA uptake was imaged and quantified with a Zeiss LSM 780 confocal microscope (n = 5–10). (c) Fluorescent siRNA uptake was quantified by flow cytometry (n = 3). (d) Competitive inhibition was performed with excess free AMD3100. AMD-NPs containing VEGF siRNAs (300 nmol/l) significantly decreased VEGF expression in HCA-1 cells. VEGF mRNA expression was detected and quantified in HCA-1 cells by using (e) real-time PCR and (f) western blotting. The data are the mean values ± SEM. *P < 0.05; ***P < 0.001. DAPI, 4′,6-diamidino-2-phenylindole; FAM, fluorescein amidite.
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Figure 5 In vivo tumor uptake and gene silencing in orthotopic HCC models treated with siRNA delivered by AMD-NPs. (a) Tissue distribution of FAM-labeled siRNAs in different formulations. (n = 4–6). siRNA-loaded NPs, siRNA-loaded AMD-NPs, and free siRNAs were compared statistically. (b) Tumor uptake of FAM-labeled siRNAs in different formulations 4 hours after i.v. administration was observed by confocal microscopy. Bar = 50 μm. AMD-NPs containing VEGF siRNA significantly decreased the (c) mRNA and (d) protein expression of VEGF in the tumor tissues. (n = 3–6). The data are the mean values ± SEM. *P < 0.05; ***P < 0.001. FAM, fluorescein amidite.
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Figure 6 Changes in tumor microenvironments after treatment with VEGF siRNAs in AMD-NPs. (a) Immunostaining for vWF, CAIX, SDF1α, and F4/80 in HCA-1 tumor tissue samples from mice treated with different treatments. Representative confocal images stained with different marker proteins are shown. Bar = 50 μm. (b–e) Samples were imaged and quantified with a Zeiss LSM 780 confocal microscope (n = 5–10). The data are the mean values ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001. vWF, von Willebrand factor.
Delivery of VEGF siRNA using AMD-NPs showed significant gene silencing in HCC in vitro and in vivo Although our data showed that the combination of sorafenib and AMD-NPs synergistically suppressed HCC progression, the adverse effects associated with sorafenib resulted in an urgent demand to develop a new therapeutic strategy. In an attempt to block angiogenesis in HCC more effectively and specifically, VEGF siRNAs were loaded into AMD-NPs to replace conventional sorafenib treatment. As shown in Figure 4a–c and Supplementary Figures S3 and S4, the in vitro uptake of fluorescently labeled siRNAs was much greater in both JHH-7 Molecular Therapy vol. 23 no. 11 nov. 2015
and HCA-1 cells when delivered via AMD-NPs than in cells treated with nontargeted NPs. The uptake of siRNA-containing AMD-NPs was competitively suppressed by the addition of free AMD3100 in a dose-dependent manner (Figure 4d). Moreover, VEGF siRNAs delivered by AMD-NPs showed significant inhibition of VEGF expression. In contrast, free VEGF siRNAs or VEGF siRNAs formulated into nontargeted NPs did not affect the expression of VEGF in either JHH-7 or HCA-1 cells (Figure 4e,f and Supplementary Figure S4). Our results indicated that the AMD-NPs efficiently delivered VEGF siRNAs into CXCR4expressing HCC cells and achieved a significant gene silencing 1777
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Figure 7 Treatment with AMD-NPs containing VEGF siRNAs significantly inhibits primary tumor growth and incidence of lung metastasis formation. (a) Systemic injections of VEGF siRNAs encapsulated in AMD-NPs caused a significant tumor growth inhibition (n = 4–7). (b,c) The number of lung metastatic nodules was significantly reduced in mice treated with AMD-NPs containing control or VEGF siRNAs (n = 6–10). The data are the mean values ± SEM. *P < 0.05; **P < 0.01.
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Figure 8 Schematic illustration of mechanisms to deliver VEGF siRNAs into HCC cells, overcome stroma-mediated resistance to sorafenib, and achieve enhanced therapeutic effects in HCC using CXCR4-targeted lipid-based NPs. AMD3100 has dual effects. First, it works as a ligand for the intracellular delivery of VEGF siRNAs into malignant HCC tumors, leading to antiangiogenesis in HCC. Later on, the decreased MVD in tumors induces hypoxia, SDF1α expression and TAM infiltration, resulting in resistance to antiangiogenic therapy. However, the second function of AMD3100 is to block the SDF1α/CXCR4 axis to further inhibit the stromal cell recruitment, thus achieving an enhanced anticancer effect when combined with VEGF siRNAs.
effect. In addition, both the delivery property and the silencing activity were ligand (AMD3100) dependent. We next studied the tissue distribution of fluorescently labeled siRNA in orthotopic HCA-1 tumor models 4 hours after i.v. 1778
injections. As shown in Figure 5a,b, AMD-NPs showed higher siRNA delivery in the tumor tissue than free siRNAs and s iRNAs in nontargeted NPs. The strong cytosolic delivery and heterogeneous distribution of fluorescently labeled siRNAs in the www.moleculartherapy.org vol. 23 no. 11 nov. 2015
© The American Society of Gene & Cell Therapy
HCA-1 tumor tissues were observed after delivery by AMD-NPs (Figure 5b). However, the intracellular uptake of free siRNAs or siRNAs encapsulated in nontargeted NPs was hardly detected in the HCA-1 tumor tissues. In other organs (Figure 5a), the kidney showed higher uptake of free siRNAs than siRNAs formulated in nontargeted or targeted NPs. Only low concentrations of siRNAs encapsulated in AMD-NPs (less than 2% injected dose/g) were present in organs such as the heart, spleen, lung, and blood 4 hours after i.v. injections. We further demonstrated that systemic injections of VEGF siRNAs in AMD-NPs significantly silenced VEGF expression in HCA-1 tumors, whereas VEGF expression in tumor tissues remains unchanged after treatment with control siRNAs in nontargeted NPs or in AMD-NPs (Figure 5c,d). VEGF siRNAs in nontargeted NPs only caused minimal silencing effect on VEGF expression. These results revealed that CXCR4-targeted NPs could efficiently deliver siRNAs into HCC cells and silence the target gene in vitro and in vivo. AMD3100, a CXCR4 antagonist, serves as a targeting ligand for the delivery of siRNAs into HCC and a therapeutic agent for modulating the tumor microenvironment and sensitizing HCC to antiangiogenic therapy. We further evaluated the effects of VEGF siRNAs in AMDNPs on MVD, hypoxic induction, SDF1α expression, and tumor-associated macrophage (TAM) infiltration in HCA-1 transplanted HCC models. After the HCC tumor was established, AMD-NPs containing VEGF siRNAs were i.v. injected into tumor-bearing mice (Supplementary Figure S5). AMD-NPs containing VEGF siRNAs significantly reduced MVD, increased the hypoxic fraction and SDF1α expression compared to nontargeted NPs containing VEGF siRNAs or AMD-NPs containing control siRNAs (Figure 6a–d). Moreover, treatment with VEGF siRNAs in AMD-NPs showed better angiogenic effects compared to conventional sorafenib treatment. Despite the observance of increased hypoxic tissue fraction and SDF1α expression in the tumor tissues after treatment with VEGF siRNAs in AMD-NPs, the infiltration of F4/80+ macrophages into the tumor tissues remained the same as in the control treatment (Figure 6a,e). This indicated that the incorporation of AMD3100 into the siRNA- containing NPs blocked the recruitment of TAMs into the tumor microenvironment. Finally, tumor growth inhibition was observed after treatment with VEGF siRNA in AMD-NPs in the HCA-1 orthotopic model (Figure 7a). Control siRNAs in nontargeted NPs or AMD-NPs or VEGF siRNA in nontargeted NPs did not show significant HCC growth inhibition. In addition, AMD-NPs carrying either therapeutic VEGF siRNAs or nontherapeutic control siRNAs significantly suppressed metastasis formation in the lungs of HCA-1 orthotopic models (Figure 7b,c).
VEGF siRNA encapsulated in AMD-NPs is an effective and safe therapeutic agent for HCC In addition to therapeutic efficacy, evaluation of the toxicity is an essential part of drug development process. The hepatotoxicity makers—aspartate aminotransferase, alanine aminotransferase, and alkaline phosphatase—and the proinflammatory cytokines in the serum were measured in C3H mice 24 hours after Molecular Therapy vol. 23 no. 11 nov. 2015
Delivery of siRNA Using CXCR4-targeted Nanoparticles
administration of VEGF siRNA-loaded AMD-NPs. Aspartate aminotransferase, alanine aminotransferase, and alkaline phosphatase levels remained minimally changed after treatment of VEGF siRNA-loaded AMD-NPs when compared to the untreated mice (Supplementary Table S1). Besides, VEGF siRNA formulated in AMD-NPs did not induce proinflammatory cytokines such as IL-6 and IFN-γ significantly (Supplementary Table S1). The addition of AMD3100 to the siRNA delivery system showed a significant improvement in delivery and therapeutic efficacy with low toxicity. Our data indicate that VEGF siRNA delivered by CXCR4-targeted NPs may serve as a safe and effective therapeutic option for HCC.
DISCUSSION
The Aim of this study is to develop gene delivery system for delivering antiangiogenic siRNAs into HCC and targeting inflammation tumor stroma in order to inhibit HCC progression and metastasis. Given overexpression of VEGF and hypoxia-induced SDF1α secretion in HCC, we developed CXCR4-targeted lipidbased NPs to co-deliver siRNA against VEGF and a small molecule CXCR4 antagonist. We showed that VEGF siRNA delivered by NPs silenced VEGF expression and inhibited angiogenesis in HCC. Furthermore, the CXCR4 antagonist AMD3100 not only served as a targeting moiety for the intracellular delivery of siRNA into malignant HCC but also suppressed recruitment of TAMs and synergistically inhibited angiogenesis when combined with VEGF siRNA. This combination therapy achieved in one multifunctional formulation led to effective tumor growth inhibition effects and significant metastasis reduction. siRNAs serve as potential therapeutic agents to treat cancer by specifically silencing the targeted oncogenes, but efficient siRNA delivery has remained challenging. Here, we show that CXCR4targeted NPs can encapsulate siRNA and deliver siRNA into HCC with high efficiency, silence the proangiogenic factor, and simultaneously block SDF1α/CXCR4 axis in HCC cells. The targeted siRNA delivery to tumors was mediated by the enhanced permeability and retention effect and CXCR4-dependent internalization on HCC cells. Although CXCR4 is expressed on several cell types such as lymphocytes, hematopoietic stem cells, endothelial cells, and epithelial cells,26 the expression level is higher in malignant HCC.23 Abundant CXCR4 expression in primary HCCs is associated with risk of distant metastasis and poor clinical outcome.23 Furthermore, treatment-induced hypoxia enhances CXCR4 expression through activation of HIF-1α in tumor cells and stromal cells in tumor microenvironment. Thus, the CXCR4-targeted NPs can target both malignant cancer cells and the surrounding stroma, providing a new strategy for efficient cancer therapy. Tumor Hypoxia and inflammation in the tumor microenvironment play important roles in development of drug resistance and promotion of cancer progression and metastasis. We have previously shown that antiangiogenic therapy resulted in hypoxic induction, elevated SDF1α expression, tumor inflammation and immunosuppression in HCC, leading to poor therapeutic outcome.1,27 Furthermore, inhibition of SDF1α/CXCR4 axis in combination with antiangiogenic treatment prevented the polarization of tumor-promoting microenvironment and resulted in enhanced therapeutic effect. In this study, we co-formulated 1779
Delivery of siRNA Using CXCR4-targeted Nanoparticles
VEGF siRNA together with AMD3100, a CXCR4 antagonist in a multifunctional NP formulation. We found that treatment of AMD-NPs containing VEGF siRNAs significantly reduced MVD and prevented the increase in TAM infiltration—despite persistently elevated hypoxia—in HCC. Interestingly, we only observed increased hypoxia and SDF1α expression when MVD was profoundly decreased. Treatment of AMD-NPs alone or VEGF siRNAs in nontargeted NPs caused moderate MVD reduction and thus did not increased hypoxia and SDF1α secretion in the tumor microenvironment. This phenomenon may be due to the paradoxical effects of antiangiogenic drugs. Dose and time dependence of antivascular effects or vascular normalization was observed in solid tumors treated with antiangiogenic agents.28,29 It has been reported that treatment of anti-VEGFR2 antibody (DC101) at low dose could alleviate tumor hypoxia and normalize tumor vasculature, whereas the increased tumor hypoxia was observed only when treated with antiangiogenic agents at high dose.30 To this end, although the optimal dose of antiangiogenic treatment remains debatable, it is an important consideration for future clinical applications. TAM plays a key role in regulating the tumor microenvironment toward proangiogenesis, immunosuppression, and metastasis.31,32 To this end, our study showed ablation of TAMs using AMD-NPs synergistically suppressed angiogenesis, metastasis, and tumor progression when combined with either sorafenib or VEGF siRNA treatment. Of note, the in vitro study only showed a minor effect of treatment with VEGF siRNA in AMD-NPs on HCC viability. Therefore, the suppression of HCC progression induced by VEGF siRNA in AMD-NPs is mainly mediated by the tumor stroma. This study highlights the potential of targeting tumor microenvironment as efficient cancer therapy as well as for the future development of nanotechnology-based gene therapy in HCC. In conclusion, AMD3100 incorporated into our multifunctional NPs carries dual effects, working as a ligand for the intracellular delivery of siRNA into malignant HCC as well as a CXCR4 blocker, synergistically suppressing HCC progression when combined with antiangiogenic therapy (Figure 8).
MATERIALS AND METHODS
Materials. Protamine sulfate salt (fraction X from salmon), calf thymus DNA, and AMD3100 octahydrochloride hydrate were purchased from Sigma-Aldrich (St Louis, MO). VEGF siRNA with sequence 5′-AUGUGAAUGCAGACCAAAGAA-3′ and control siRNA with sequence 5′-AATTCTCCGAACGTGTCACGT-3′ were purchased from Sigma-Aldrich. For cellular and tumor uptake studies, fluorescein amidite (FAM) was conjugated to the 5′ end of the sense sequence. 5′-FAM-labeled VEGF siRNA was purchased from MDBio (Taipei, Taiwan). DOPA, DOPC, cholesterol, and DSPE-PEG (2000) were purchased from Avanti Polar Lipids (Alabaster, AL). Sorafenib (Nexavar) was purchased from Bayer Schering Pharma (Berlin, Germany), and recombinant SDF1α was purchased from ProSpec TechnoGene (Rehovot, Israel). Cell culture. We used the murine HCC cell line HCA-1 (refs. 1,33) and human HCC cell line JHH-7, kindly provided by Dr Dan Duda, MGH Boston. HCA-1 and JHH-7 were maintained in Dulbecco’s modified Eagle’s medium/high glucose and Dulbecco’s modified Eagle medium/ nutrient mixture F-12 (Hyclone, Logan, UT), respectively. All media were supplemented with 10% fetal bovine serum and antibiotics of penicillin and streptomycin (Hyclone).
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Animals. Four to five-week-old male C3H/HeNCrNarl mice were purchased from National Laboratory Animal Center (Taipei, Taiwan). HCA-1 cells were orthotopically implanted in mice, as previously described.34 All animals received humane care in compliance with the “Guide for the Care and Use of Laboratory Animals” published by the National Academy of Sciences. Preparation of AMD-NPs. Liposomes and NPs were prepared according to a previously described method with modifications.35–37 Briefly, anionic liposomes composed of DOPA, DOPC, and cholesterol (1:2:1 molar ratio) were prepared by thin film hydration followed by sonication (Q125, Qsonica) at 50 W for 2 minutes on ice to reduce the particle size. To prepare the cores of NPs, a mixture of 18 μl AMD3100 (2 mg/ml) and 16 μl of protamine (2 mg/ml), 120 μl of deionized water, and 22 μl of a mixture of siRNA (12 μl, 3 mg/ml) and calf thymus DNA (10 μl, 2 mg/ml) were mixed and kept at room temperature (RT) for 5 minutes before 90 μl of anionic liposomes (20 mmol/l) were added. After mixing the anionic liposomes with the DNA-Protamine-AMD cores, the mixture solution stood at RT for 10 minutes. To attach the AMD3100 into the NPs, the solution was then mixed with AMD3100 in phosphate-buffered saline (PBS) and kept at RT for 5 minutes. To post-insert DSPE-PEG into AMD-LPD, the solution was further mixed with 20 μl of DSPE-PEG (10 mg/ml) and kept at 55 °C for 10 minutes to form AMD-NPs as final product. Characterization of AMD-NPs. The morphology of the NPs was observed
using the transmission electronic microscope in the Microscopy Center of Chang Gung University (Taipei, Taiwan). 0.5 μl of AMD-NPs solution was dropped onto carbon-stabilized Formvar-coated 200-mesh copper grids (Ted Pella, Redding, CA) and dried under ambient conditions. Transmission electronic microscope images were captured using a Hitachi H-7500 microscope (Hitachi High-Technologies, Tokyo, Japan) operated at 75 kV. The particle size and surface charge were measured using a Zetasizer (3000HS; Malvern Instruments, Worcestershire, UK) at RT. The parameters of viscosity and refraction index were set equal to those of water for all samples during testing.
Cell viability assays. Cell viability was assessed using 3-(4,5‐dimethylthiazol‐2-yl)‐2,5-diphenyltetrazolium bromide (MTT); Serva Electrophoresis, Heidelberg, Germany) assay. HCA-1 or JHH-7 cells (2,000 per well) were seeded in 96-well transparent plates (Costar, IL) and incubated for 12 hours. Later, the medium was replaced with serum-free medium (Hyclone), and the cells were exposed to different treatments. After 48–72 hours, cells were incubated with MTT (3-(4,5‐dimethylthiazol‐ 2-yl)‐2,5-diphenyltetrazolium bromide) for 3 hours, and cell viability was assessed by absorbance measurement using a Multiskan reader (at 570 nm; Thermo Scientific, Asheville, NC). The fraction of control was calculated by dividing the absorbance obtained from the cells with different treatments by the absorbance obtained from the control-treated cells. Immunofluorescence. Frozen sections (10 µm thick) were fixed in acetone at −20 °C for 10 minutes and washed with PBS. The sections were then blocked with 5% bovine serum albumin (in PBS) for 1 hour and then incubated overnight with primary antibodies against von Willebrand factor (Dako, Glostrup, Denmark), SDF1α (BioVision, Mountain View, CA), CAIX (Abcam, Cambridge, MA), or F4/80 (Affymetrix eBiosciencem, San Diego, CA) at 4 °C. After washed with PBS, the sections were further incubated with secondary antirabbit IgG-Alexa Fluor 488 antibodies (Life Technologies, Grand Island, NY) for 1 hour. Unbound secondary antibodies were washed by PBS, counterstained with 4′,6‐diamidino‐2‐phenylindole (DAPI); Vector Laboratories, Burlingame, CA) and imaged using a confocal microscope (LSM 780; Carl Zeiss, Heidelberg, Germany). Cellular uptake study and AMD3100 competitive cellular uptake assay.
HCA-1 and JHH-7 cells (1 × 105/1 ml/well) were seeded in 12-well plates (Corning, Corning, NY) overnight before the experiments were conducted. www.moleculartherapy.org vol. 23 no. 11 nov. 2015
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The cells were treated in serum-containing media at 37 °C for 4 hours with different formulations containing FAM-labeled siRNA. Cells were washed twice with PBS, fixed in 4% paraformaldehyde (in PBS) for 10 minutes, counterstained with DAPI (4′,6‐diamidino‐2‐phenylindole), and imaged using a Zeiss LSM 780 confocal microscope (Carl Zeiss). For evaluation and quantification of cellular uptake using flow cytometry, cells were washed twice with PBS and harvested with trypsin/ ethylenediaminetetraacetic acid and then resuspended in PBS. The fluorescent signals were measured using a BD FACSAria III sorter (BD Biosciences, San Jose, CA). The cell fluorescence intensity distributions were obtained by counting a minimum of 10,000 cells for each replica. For the AMD3100 competitive cellular uptake assay, HCA-1 cells were prepared as previously mentioned. Free AMD3100 was added into the media at the final concentration of 0, 100, and 200 μmol/l for 10 minutes, and cells were treated with AMD-NPs containing FAM-labeled siRNA for 4 hours. The cellular uptake of FAM-labeled siRNA was examined and calculated using a confocal microscope. Tissue distribution study. Tumor-bearing mice were i.v. injected with
FAM-labeled siRNA (1.5 mg/kg) in different formulations. After 4 hours, mice were sacrificed, and tissues were collected and homogenized in lysis buffer and incubated on ice for 30 minutes. The tissue lysate was transferred to a black 96-well plate (Corning). The fluorescence intensity of the sample was measured by a plate reader (Fluoroskan Ascent FL, Thermo Scientific) at excitation wavelength 485nm and emission wavelength 538 nm. The concentration of FAM-labeled siRNA in each sample was calculated from a standard curve.
Tumor uptake study. C3H mice with a tumor size of ~1 cm2 were i.v.
injected with FAM-labeled siRNA (1.5 mg/kg) in nontargeted NPs or AMD-NPs. Four hours after injections, mice were sacrificed, and tissues were collected and homogenized in lysis buffer and incubated on ice for 30 minutes. The tissue lysate was transferred to a black 96-well plate (Corning). The fluorescence intensity of the sample was measured by a plate reader (Fluoroskan Ascent FL, Thermo Scientific) at excitation wavelength 485nm and emission wavelength 538 nm. The concentration of FAM-labeled siRNA in each sample was calculated using a standard curve. To examine the intracellular uptake of FAM-labeled siRNA in tumor tissues, the tissues were collected and fixed in 4% paraformaldehyde (in PBS) overnight before cryo-embedding. Tumor tissues were sectioned (10 μm thick) and imaged using a Zeiss LSM 780 confocal microscope. Hematoxylin and eosin staining. The lung tissue was cut into small pieces
and fixed in 4% paraformaldehyde (in PBS) overnight before embedded in paraffin wax. The sections were then stained with hematoxylin and eosin and observed with a Nikon microscope (Eclipse E800, Tallahassee, FL).
Gene silencing study. For evaluation of gene silencing by quantitative
reverse transcription polymerase chain reaction (qRT-PCR), 48 hours after treatment with different formulations, cells were washed twice with PBS, and mRNA was isolated by using RNeasy Mini kit (Qiagen, Valencia, CA) according to manufacturer’s instructions. In order to avoid genomic DNA contamination, RNA samples were subjected to RNase-free DNase (Qiagen) treatment at RT for 15 minutes. After isolation, 1-μg RNA samples were used for cDNA synthesis. cDNA synthesis was carried out using High-Capacity cDNA Reverse Transciption kit (Applied Biosystems, Foster City, CA) in a Piko Thermal Cycler, 24-well (Thermo Scientific) according to the manufacturer’s instruction. Quantitative PCR was performed using SYBR Green PCR Master Mix (Applied Biosystems) with the quantitative PCR primer pairs. The primer sequences were as follows: sense, 5′-CTGTGCAGGCTGCTGTAACG-3′ and antisense, 5′-GTTCCCGAAACCCTGAGGAG-3′ for VEGF-A; sense, 5′- TGAGAGGGAAATCGTGCGTG-3′ and antisense, 5′- TTGCTGA TCCACATCTGCTGG-3′ for β-actin; sense, 5′- CTGCCACCCAGAAG ACTGTG -3′ and antisense, 5′- GGTCCTCAGTGTAGCCCAAG -3′ for
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glyceraldehyde 3-phosphate dehydrogenase. The relative expression levels were normalized to β-actin or glyceraldehyde 3-phosphate dehydrogenase to obtain their relative gene expression. Primers employed in these studies were based on previous studies.38–40 For evaluation of protein expression by immunoblotting, cells were lysed in radioimmunoprecipitation assay buffer with ethylenediaminetetraacetic acid and ethylene glycol tetraacetic acid (BioBasic, Toronto, Ontario, Canada) for 20 minutes on ice, and the supernatant was collected after centrifugation at 12,700 rpm for 20 minutes. Cell lysates were separated on a 10% acrylamide gel and transferred to a polyvinylidene difluoride membrane. The membrane was blocked with 5% nonfat milk in PBS for 1 hour and then incubated overnight with primary antibodies against VEGF (Abcam, Cambridge, MA) or β-actin (Sigma-Aldrich) at 4 °C. After the membrane was washed with PBST (0.1% Tween 20 in PBS), it was further incubated with the horseradish peroxidase–conjugated secondary antibodies (Cell Signaling Technology, Beverly, MA) for 1 hour. The membrane was washed and developed by enhanced chemiluminescence using the ECL Select western blotting detection reagent (GE Healthcare, Little Chalfont, Buckinghamshire, UK), followed by autoradiography. For the in vivo studies, HCA-1 tumor-bearing mice were i.v. injected with siRNA in different formulations (1.44 mg siRNA/kg/dose, 1 dose per day). The day after the third injection, the mice were sacrificed, and tumor samples were collected. The total protein (40 μg) isolated from the tumors was loaded onto a polyacrylamide gel, electrophoresed, and blotted as described above. Statistical analysis. Comparisons between treatment groups were per-
formed using Mann–Whitney’s U-test. P value of less than 0.05 was considered to denote statistical significance.
SUPPLEMENTARY MATERIAL Figure S1. Treatment schedule of sorafenib combined with AMDNPs in the orthotopic HCC tumor model. Figure S2. AMD-NPs reduce HCA-1 cell migration. Figure S3. Uptake of FAM-labeled siRNAs in different formulations was examined by flow cytometry. Figure S4. Intracellular uptake and gene silencing effects in JHH-7 cells treated with siRNAs delivered by AMD-NPs in vitro. Figure S5. Treatment schedule of VEGF siRNAs in different formulations in the orthotopic HCC tumor model. Table S1. Toxicity profile of VEGF siRNA in CXCR4-targeted nanoparticles. Materials and Methods
ACKNOWLEDGMENTS This work was supported by Ministry of Science and Technology through grant no: NSC 102-2320-B-007-011-MY2 and MOST 103-2221-E-007032-MY2 and by Chang Gung Memorial Hospital-National Tsing Hua University Joint Research Grant 104N2744E1 to Y.C.
References
1. Chen, Y, Huang, Y, Reiberger, T, Duyverman, AM, Huang, P, Samuel, R et al. (2014). Differential effects of sorafenib on liver versus tumor fibrosis mediated by stromalderived factor 1 alpha/C-X-C receptor type 4 axis and myeloid differentiation antigenpositive myeloid cell infiltration in mice. Hepatology 59: 1435–1447. 2. Hernandez–Gea, V, Toffanin, S, Friedman, SL, and Llovet, JM (2013). Role of the microenvironment in the pathogenesis and treatment of hepatocellular carcinoma. Gastroenterology 144: 512–527. 3. Hui, CK, Leung, N, Shek, TW, Yao, H, Lee, WK, Lai, JY et al.; Hong Kong Liver Fibrosis Study Group. (2007). Sustained disease remission after spontaneous HBeAg seroconversion is associated with reduction in fibrosis progression in chronic hepatitis B Chinese patients. Hepatology 46: 690–698. 4. Luedde, T and Schwabe, RF (2011). NF-κB in the liver–linking injury, fibrosis and hepatocellular carcinoma. Nat Rev Gastroenterol Hepatol 8: 108–118. 5. Murakata, A, Tanaka, S, Mogushi, K, Yasen, M, Noguchi, N, Irie, T et al. (2011). Gene expression signature of the gross morphology in hepatocellular carcinoma. Ann Surg 253: 94–100. 6. Sato, K, Tanaka, S, Mitsunori, Y, Mogushi, K, Yasen, M, Aihara, A et al. (2013). Contrast-enhanced intraoperative ultrasonography for vascular imaging of
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7. 8. 9. 10. 11.
12.
13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24.
hepatocellular carcinoma: clinical and biological significance. Hepatology 57: 1436–1447. Semela, D and Dufour, JF (2004). Angiogenesis and hepatocellular carcinoma. J Hepatol 41: 864–880. Kerbel, RS (2006). Antiangiogenic therapy: a universal chemosensitization strategy for cancer? Science 312: 1171–1175. Meng, H, Xing, G, Sun, B, Zhao, F, Lei, H, Li, W et al. (2010). Potent angiogenesis inhibition by the particulate form of fullerene derivatives. ACS Nano 4: 2773–2783. Zhu, AX, Duda, DG, Sahani, DV and Jain, RK (2011). HCC and angiogenesis: possible targets and future directions. Nat Rev Clin Oncol 8: 292–301. Adnane, L, Trail, PA, Taylor, I and Wilhelm, SM (2006). Sorafenib (BAY 43-9006, Nexavar), a dual-action inhibitor that targets RAF/MEK/ERK pathway in tumor cells and tyrosine kinases VEGFR/PDGFR in tumor vasculature. Methods Enzymol 407: 597–612. Liu, L, Cao, Y, Chen, C, Zhang, X, McNabola, A, Wilkie, D et al. (2006). Sorafenib blocks the RAF/MEK/ERK pathway, inhibits tumor angiogenesis, and induces tumor cell apoptosis in hepatocellular carcinoma model PLC/PRF/5. Cancer Res 66: 11851–11858. Llovet, JM, Ricci, S, Mazzaferro, V, Hilgard, P, Gane, E, Blanc, JF et al.; SHARP Investigators Study Group. (2008). Sorafenib in advanced hepatocellular carcinoma. N Engl J Med 359: 378–390. Méndez-Sánchez, N, Vásquez-Fernández, F, Zamora-Valdés, D and Uribe, M (2008). Sorafenib, a systemic therapy for hepatocellular carcinoma. Ann Hepatol 7: 46–51. Xie, B, Wang, DH and Spechler, SJ (2012). Sorafenib for treatment of hepatocellular carcinoma: a systematic review. Dig Dis Sci 57: 1122–1129. Carmeliet, P and Jain, RK (2011). Molecular mechanisms and clinical applications of angiogenesis. Nature 473: 298–307. Jain, RK (2013). Normalizing tumor microenvironment to treat cancer: bench to bedside to biomarkers. J Clin Oncol 31: 2205–2218. Semenza, GL (2012). Hypoxia-inducible factors in physiology and medicine. Cell 148: 399–408. Gao, F, Liang, B, Reddy, ST, Farias-Eisner, R and Su, X (2014). Role of inflammationassociated microenvironment in tumorigenesis and metastasis. Curr Cancer Drug Targets 14: 30–45. Kryczek, I, Wei, S, Keller, E, Liu, R and Zou, W (2007). Stroma-derived factor (SDF-1/ CXCL12) and human tumor pathogenesis. Am J Physiol Cell Physiol 292: C987–C995. Gentilini, A, Rombouts, K, Galastri, S, Caligiuri, A, Mingarelli, E, Mello, T et al. (2012). Role of the stromal-derived factor-1 (SDF-1)-CXCR4 axis in the interaction between hepatic stellate cells and cholangiocarcinoma. J Hepatol 57: 813–820. Kamba, T and McDonald, DM (2007). Mechanisms of adverse effects of anti-VEGF therapy for cancer. Br J Cancer 96: 1788–1795. Xiang, ZL, Zeng, ZC, Tang, ZY, Fan, J, Zhuang, PY, Liang, Y et al. (2009). Chemokine receptor CXCR4 expression in hepatocellular carcinoma patients increases the risk of bone metastases and poor survival. BMC Cancer 9: 176. Yu, M, Gang, EJ, Parameswaran, R, Stoddart, S, Fei, F, Schmidhuber, S et al. (2011). AMD3100 sensitizes acute lymphoblastic leukemia cells to chemotherapy in vivo. Blood Cancer J 1: e14.
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25. Domanska, UM, Timmer-Bosscha, H, Nagengast, WB, Oude Munnink, TH, Kruizinga, RC, Ananias, HJ et al. (2012). CXCR4 inhibition with AMD3100 sensitizes prostate cancer to docetaxel chemotherapy. Neoplasia 14: 709–718. 26. Teicher, BA and Fricker, SP (2010). CXCL12 (SDF-1)/CXCR4 pathway in cancer. Clin Cancer Res 16: 2927–2931. 27. Chen, Y, Ramjiawan, RR, Reiberger, T, Ng, MR, Hato, T, Huang, Y et al. (2015). CXCR4 inhibition in tumor microenvironment facilitates anti-programmed death receptor-1 immunotherapy in sorafenib-treated hepatocellular carcinoma in mice. Hepatology 61: 1591–1602. 28. Jain, RK (2005). Normalization of tumor vasculature: an emerging concept in antiangiogenic therapy. Science 307: 58–62. 29. Huang, Y, Yuan, J, Righi, E, Kamoun, WS, Ancukiewicz, M, Nezivar, J et al. (2012). Vascular normalizing doses of antiangiogenic treatment reprogram the immunosuppressive tumor microenvironment and enhance immunotherapy. Proc Natl Acad Sci USA 109: 17561–17566. 30. Huang, Y, Stylianopoulos, T, Duda, DG, Fukumura, D and Jain, RK (2013). Benefits of vascular normalization are dose and time dependent–letter. Cancer Res 73: 7144–7146. 31. Capece, D, Fischietti, M, Verzella, D, Gaggiano, A, Cicciarelli, G, Tessitore, A et al. (2013). The inflammatory microenvironment in hepatocellular carcinoma: a pivotal role for tumor-associated macrophages. Biomed Res Int 2013: 187204. 32. Zhou, D, Huang, C, Kong, L and Li, J (2014). Novel therapeutic target of hepatocellular carcinoma by manipulation of macrophage colony-stimulating factor/ tumor-associated macrophages axis in tumor microenvironment. Hepatol Res 44: E318–E319. 33. Tofilon, PJ, Basic, I and Milas, L (1985). Prediction of in vivo tumor response to chemotherapeutic agents by the in vitro sister chromatid exchange assay. Cancer Res 45: 2025–2030. 34. Kim, W, Seong, J, Oh, HJ, Koom, WS, Choi, KJ and Yun, CO (2011). A novel combination treatment of armed oncolytic adenovirus expressing IL-12 and GM-CSF with radiotherapy in murine hepatocarcinoma. J Radiat Res 52: 646–654. 35. Chen, Y, Wu, JJ and Huang, L (2010). Nanoparticles targeted with NGR motif deliver c-myc siRNA and doxorubicin for anticancer therapy. Mol Ther 18: 828–834. 36. Cui, Z, Han, SJ, Vangasseri, DP and Huang, L (2005). Immunostimulation mechanism of LPD nanoparticle as a vaccine carrier. Mol Pharm 2: 22–28. 37. Li, SD, Chen, YC, Hackett, MJ and Huang, L (2008). Tumor-targeted delivery of siRNA by self-assembled nanoparticles. Mol Ther 16: 163–169. 38. Clifford, RL, Deacon, K and Knox, AJ (2008). Novel regulation of vascular endothelial growth factor-A (VEGF-A) by transforming growth factor (beta)1: requirement for Smads, (beta)-CATENIN, AND GSK3(beta). J Biol Chem 283: 35337–35353. 39. Kim, IS, Song, YM, Cho, TH, Park, YD, Lee, KB, Noh, I et al. (2008). In vitro response of primary human bone marrow stromal cells to recombinant human bone morphogenic protein-2 in the early and late stages of osteoblast differentiation. Dev Growth Differ 50: 553–564. 40. Albrektsen, T, Sørensen, BB, Hjortø, GM, Fleckner, J, Rao, LV and Petersen, LC (2007). Transcriptional program induced by factor VIIa-tissue factor, PAR1 and PAR2 in MDAMB-231 cells. J Thromb Haemost 5: 1588–1597.
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Delivery of siRNA Using CXCR4-targeted Nanoparticles Modulates Tumor Microenvironment and Achieves a Potent Antitumor Response in Liver Cancer Jia-Yu Liu1, Tsaiyu Chiang1, Chun-Hung Liu1, Guann-Gen Chern1, Ts-Ting Lin1, Dong-Yu Gao1 and Yunching Chen1 Institute of Biomedical Engineering, National Tsing Hua University, Hsinchu, Taiwan, ROC
1
Antiangiogenic therapy has recently emerged as a highly promising therapeutic strategy for treating hepatocellular carcinoma (HCC). However, the only clinically approved systemic antiangiogenic agent for advanced HCC is sorafenib, which exerts considerable toxicity. Moreover, acquired resistance to antiangiogenic therapy often develops and restricts the therapeutic efficacy of this treatment. Hence, in this study, we develop a CXCR4targeted lipid-based nanoparticle (NP) formulation to specifically deliver vascular endothelial growth factor (VEGF) siRNA as an antiangiogenic substance into HCC. AMD3100, a CXCR4 antagonist, is added into NPs to serve as both a targeting moiety and a sensitizer to antiangiogenic therapy. We demonstrate that AMD-modified NPs (AMD-NPs) can efficiently deliver VEGF siRNAs into HCC and downregulate VEGF expression in vitro and in vivo. Despite the upregulation of the SDF1α/CXCR4 axis upon the induction of hypoxia after antiangiogenic therapy, CXCR4 inhibition by AMD-NPs in combination with either conventional sorafenib treatment or VEGF siRNA prevents the infiltration of tumor-associated macrophages. These dual treatments also induce synergistic antiangiogenic effects and suppress local and distant tumor growth in HCC. In conclusion, the tumor-targeted multifunctional AMD-NPs that co-deliver VEGF siRNA and AMD3100 provide an effective approach for overcoming tumor evasion of antiangiogenic therapy, leading to delayed tumor progression in HCC. Received 19 January 2015; accepted 28 July 2015; advance online publication 29 September 2015. doi:10.1038/mt.2015.147
INTRODUCTION
Hepatocellular carcinoma (HCC) is one of the most fatal cancers worldwide. HCC arises from damaged liver tissues with a preexisting inflammatory microenvironment, fibrosis, and subsequent
hypervascularization and thus has been identified as a highly vascular tumor.1–7 Therefore, antiangiogenic therapy has become a highly promising therapeutic strategy for treating HCC.8–10 The tyrosine kinase inhibitor sorafenib is the only approved systemic treatment for advanced HCC. Sorafenib suppresses angiogenesis and tumor progression by blocking VEGFR/PDGFR in the tumor vasculature and the RAF/MEK/ERK pathway in HCC cells.11–15 Although sorafenib shows moderate therapeutic outcomes in some HCC patients, antiangiogenic therapy remains challenging. There are multiple hurdles involved in efficient antiangiogenic cancer treatment, including the rapid development of resistance and considerable toxicity. Several mechanisms are involved in the rapid escape from antiangiogenic therapy, leading to transient therapeutic effects, as well as local and distant recurrences of HCC.16–18 We have recently found that sorafenib can trigger a tumor-promoting microenvironment in HCC by increasing hypoxia, inflammation, and fibrosis in the tumor tissues.1 The SDF1α/CXCR4 axis is upregulated after sorafenib treatment and plays a key role in enhancing cancer cell survival, recruiting tumor-associated bone marrow–derived cells, increasing desmoplasia and promoting metastatic phenotypes in HCC.10,19–21 In addition, angiogenic blockades can also be taken by the normal tissues, causing unwanted side effects and toxicity.22 Thus, there is an urgent need for the development of a new therapeutic strategy. Herein, we developed multifunctional lipid-based nanoparticles (NPs) to co-deliver a small interfering RNA (siRNA) against vascular endothelial growth factor (VEGF) and a small molecule CXCR4 antagonist. Compared to tyrosine kinase inhibitors such as sorafenib, siRNAs (short double-stranded RNAs) with 20–25 base pairs can silence the targeted genes more specifically and efficiently with lower unwanted toxicity. Thus, siRNAs can serve as a safe and effective cancer therapy. We showed that VEGF siRNA delivered by NPs downregulated VEGF expression and suppressed angiogenesis in HCC. Furthermore, the CXCR4 antagonist AMD3100 was not only
Correspondence: Yunching Chen, Institute of Biomedical Engineering, National Tsing Hua University, Hsinchu 30013, Taiwan, ROC. E-mail: [email protected]
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a VEGF siRNA or scrambled siRNA
Calf thymus DNA
Protamine
AMD 3100
Liposome
DSPE-PEG 2000
100 nm
AMD-core
Core
AMD-LPD
LPD
Post-inserted PEG
Size (nm) 169.8 ± 9.8
110.7 ± 7.6
113.3 ± 5.9
138.5 ± 2.4
144.7 ± 14
Zeta (mV) –28.6 ± 1.1
27 ± 0.3
–52.9 ± 1.5
–23.4 ± 0.4
–21.8 ± 0.2
0.441 ± 0.01
0.311 ± 0.03
0.117 ± 0.01
PDI
0.142 ± 0.01
b
JHH-7
0.299 ± 0.03 HCA-1
SDF1α
*
SDF1α
125
*
Viability (% of control)
*
100
100 75 50 25
50 25
SDF1α
100
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120
40 20
Viability (% of control)
**
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100
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Viability (% of control)
SDF1α
120
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HCA-1
JHH-7
80
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Viability (% of control)
125
Figure 1 Illustration of the preparation of AMD-NPs containing siRNA and in vitro cytotoxicity of AMD-NPs in combination with sorafenib. (a) Sizes and zeta potentials of NPs in different stages of preparation. The TEM characterization of AMD-NPs is shown. The data are the mean values ± SEM (n = 3–4). (b,c) Exposure to recombinant SDF1α (50 ng/ml) increased the viability of JHH-7 and HCA-1 cells despite sorafenib (Sor) treatment (0.25 μmol/l). Cell viability was measured using the MTT assay. Inhibition of CXCR4 with free AMD3100 or AMD-NPs (0.5 μmol/l) prevented the pro-proliferative effects of SDF1α. The data are the mean values ± SEM (n = 4–6). *P < 0.05, **P < 0.01. MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; PDI, polydispersity index; TEM, transmission electronic microscope.
encapsulated in NPs but added to modify the surfaces of the NPs. To this end, AMD3100 carries dual effects, working as a ligand for the intracellular delivery of siRNA into malignant HCC as well as a CXCR4 blocker, achieving enhanced anticancer effects when combined with VEGF siRNA.1,23–25 In conclusion, our tumor-targeting multifunctional NP co-delivering VEGF siRNA and AMD3100 may serve as a novel strategy for overcoming local and distant tumor evasion of antiangiogenic therapy, leading to delayed tumor progression in HCC. Molecular Therapy vol. 23 no. 11 nov. 2015
RESULTS Preparation and characterization of siRNA-loaded, CXCR4-targeted lipid-based NPs
The preparation and proposed structure of the siRNA-loaded, CXCR4-targeted NP is shown in Figure 1a. First, the protamine/ nucleic acids (calf thymus DNA and therapeutic siRNA) complex was prepared by mixing the protamine and nucleic acids at a weight ratio of approximately 4:7. The average size of the protamine/DNA complex was 169.8 nm and the zeta potential was 1773
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−28.6 mV. Condensation of the NPs occurred when AMD3100 was present. Adding AMD3100 to the protamine/nucleic acids complex reduced the particle size to 110.7 nm and the zeta potential became positive (27 mV). Subsequently, the surface of the protamine/nucleic acids/AMD3100 complex was coated with an anionic liposome composed of DOPC, DOPA, and cholesterol. The liposome-coated NPs carrying siRNA and AMD3100 had an average size of 113.3 nm and were negatively charged (−52.9 mV). To achieve tumor targeting, the surface of the anionic liposome-wrapped NPs was coated with the CXCR4 ligand AMD3100, resulting in NPs with less negative charges (−23.4 mV) compared to the AMD3100-free NPs. Finally, the AMD3100-coated NPs were PEGylated to prolong their systemic circulation in the blood. The transmission electronic microscope images revealed that the 1774
PEGylated AMD3100-coated NPs (AMD-NPs) were spherical. The average AMD-NP diameter was 144.7 ± 14 nm, with a polydispersity index of 0.299 ± 0.03. siRNA and AMD3100 encapsulations in the NPs were about 90 and 80%, respectively.
AMD-NP modulation of the tumor microenvironment and sensitization of HCC to sorafenib treatment in vitro and in vivo To determine if the SDF1α/CXCR4 axis mediates HCC resistance to sorafenib treatment, we first exposed both murine HCC (HCA-1) and human HCC (JHH-7) cell lines to recombinant SDF1α in the presence of sorafenib. We found that SDF1α increased the viability of both JHH-7 and HCA-1 cells despite sorafenib treatment (Figure 1b,c). Furthermore, the inhibition of CXCR4 by AMD3100 prevented the effects of SDF1α and sensitized HCC www.moleculartherapy.org vol. 23 no. 11 nov. 2015
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Figure 3 Sorafenib induces a minor tumor growth inhibition in HCC, and CXCR4 inhibition with AMD-NPs synergizes with sorafenib treatment and suppresses distant metastasis. (a) Tumor sizes in orthotopic tumor-bearing mice after the treatment of sorafenib in different combinations (n = 4–5). (b,c) The number of lung metastatic nodules was significantly reduced in mice treated with AMD-NPs either alone or in combination with sorafenib. Lung metastases of HCC tumor cells were quantified and imaged with an optical microscope (n = 6). The data are the mean values ± SEM. *P < 0.05; **P < 0.01.
to sorafenib treatment. We next examined the cytotoxicity of NPs containing AMD3100 (AMD-NPs). Consistent with free AMD3100, the rescue effect of SDF1α on the sorafenib-induced cytotoxicity could be reversed by AMD-NPs in both the HCA-1 and JHH-7 cell lines (Figure 1b,c). To establish a syngeneic orthotopic murine HCC model, we intrahepatically implanted HCA-1 cells into C3H mice. We first evaluated the effects of sorafenib either alone or in combination with free AMD3100 or AMD-NPs on the mean vessel density (MVD) and hypoxic induction in the HCA-1 transplanted HCC models. After HCC tumors were established, sorafenib was given at a dose of 50 mg/kg daily for 14 days either alone or in combination with free AMD3100 or AMD-NPs (Supplementary Figure S1). We further evaluated the changes in MVD and tissue hypoxia in implanted tumor tissues. Sorafenib treatment alone significantly reduced MVD and increased carbonic anhydrase IX expression, indicating hypoxia induction (Figure 2a–c). We next examined the changes in SDF1α expression as a consequence of treatment-induced hypoxia. We found a threefold increase in SDF1α expression in HCA-1 tumor tissues after sorafenib treatment (Figure 2a,d). The combination of sorafenib and AMD-NPs further decreased the MVD and increased the fraction of hypoxic tissue and SDF1α expression in orthotopic HCA-1 tumor grafts when compared to sorafenib treatment alone (Figure 2a–d). We next examined the effects of sorafenib either alone or in combination with free AMD3100 or AMD-NPs on inflammatory cell infiltration in HCC. We found that the number of F4/80+ macrophages increased by over 3-fold in HCA-1 transplanted HCC models after sorafenib treatment (Figure 2a,e). Finally, blocking the SDF1α/CXCR4 axis with AMD-NPs in combination with Molecular Therapy vol. 23 no. 11 nov. 2015
sorafenib decreased the number of tumor-infiltrating F4/80+ macrophages to levels comparable to those of control-treated HCA-1 tumors (Figure 2a,e). However, due to poor pharmacokinetics, the addition of free AMD to the sorafenib treatment did not show significant differences for all of the parameters compared to sorafenib treatment alone (Figure 2). We further evaluated the effects of sorafenib either alone or in combination with free AMD3100 or AMD-NPs on HCC progression in HCA-1 transplanted HCC models. As shown in Figure 3a, treatment with sorafenib alone only showed a moderate tumor growth inhibition effect. Due to the poor pharmacokinetics of free AMD3100, the systemic injection of free AMD3100 did not significantly sensitize HCC to sorafenib treatment in vivo. However, the systemic administration of AMD-NPs in combination with sorafenib led to the synergistic tumor growth inhibition of orthotopic HCA-1 tumors in immunocompetent C3H mice compared to treatment with AMD-NP or sorafenib alone (Figure 3a). In the HCA-1 orthotopic model, spontaneous lung metastases developed around day 14 after tumor implantation. Neither treatment with sorafenib alone nor sorafenib in combination with free AMD3100 showed a reduction in metastasis formation compared to control-treated mice (Figure 3b,c). In contrast, both AMDNPs alone and a combination of AMD-NPs and sorafenib significantly inhibited lung metastasis in the orthotopic HCA-1 model (Figure 3b,c). It indicated that the SDF1α/CXCR4 pathway might serve as a direct regulator for metastasis or indirectly mediate metastasis via stromal cells (Figure 2e, Supplementary Figure S2, and Supplementary Materials and Methods). AMD-NPs blocked CXCR4 and thus could efficiently reduce the metastatic burden. 1775
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Figure 4 Intracellular uptake and gene silencing effects in HCC cells treated with siRNAs delivered by AMD-NPs in vitro. (a) HCA-1 cells were treated with FAM-labeled siRNAs in different formulations for 4 h and observed by confocal microscopy. The arrowhead represents the fluorescence of FAM-labeled siRNA in the cytoplasm. Bar = 50 μm. The nuclei are blue (DAPI). (b) Fluorescent siRNA uptake was imaged and quantified with a Zeiss LSM 780 confocal microscope (n = 5–10). (c) Fluorescent siRNA uptake was quantified by flow cytometry (n = 3). (d) Competitive inhibition was performed with excess free AMD3100. AMD-NPs containing VEGF siRNAs (300 nmol/l) significantly decreased VEGF expression in HCA-1 cells. VEGF mRNA expression was detected and quantified in HCA-1 cells by using (e) real-time PCR and (f) western blotting. The data are the mean values ± SEM. *P < 0.05; ***P < 0.001. DAPI, 4′,6-diamidino-2-phenylindole; FAM, fluorescein amidite.
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Delivery of VEGF siRNA using AMD-NPs showed significant gene silencing in HCC in vitro and in vivo Although our data showed that the combination of sorafenib and AMD-NPs synergistically suppressed HCC progression, the adverse effects associated with sorafenib resulted in an urgent demand to develop a new therapeutic strategy. In an attempt to block angiogenesis in HCC more effectively and specifically, VEGF siRNAs were loaded into AMD-NPs to replace conventional sorafenib treatment. As shown in Figure 4a–c and Supplementary Figures S3 and S4, the in vitro uptake of fluorescently labeled siRNAs was much greater in both JHH-7 Molecular Therapy vol. 23 no. 11 nov. 2015
and HCA-1 cells when delivered via AMD-NPs than in cells treated with nontargeted NPs. The uptake of siRNA-containing AMD-NPs was competitively suppressed by the addition of free AMD3100 in a dose-dependent manner (Figure 4d). Moreover, VEGF siRNAs delivered by AMD-NPs showed significant inhibition of VEGF expression. In contrast, free VEGF siRNAs or VEGF siRNAs formulated into nontargeted NPs did not affect the expression of VEGF in either JHH-7 or HCA-1 cells (Figure 4e,f and Supplementary Figure S4). Our results indicated that the AMD-NPs efficiently delivered VEGF siRNAs into CXCR4expressing HCC cells and achieved a significant gene silencing 1777
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Figure 7 Treatment with AMD-NPs containing VEGF siRNAs significantly inhibits primary tumor growth and incidence of lung metastasis formation. (a) Systemic injections of VEGF siRNAs encapsulated in AMD-NPs caused a significant tumor growth inhibition (n = 4–7). (b,c) The number of lung metastatic nodules was significantly reduced in mice treated with AMD-NPs containing control or VEGF siRNAs (n = 6–10). The data are the mean values ± SEM. *P < 0.05; **P < 0.01.
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Figure 8 Schematic illustration of mechanisms to deliver VEGF siRNAs into HCC cells, overcome stroma-mediated resistance to sorafenib, and achieve enhanced therapeutic effects in HCC using CXCR4-targeted lipid-based NPs. AMD3100 has dual effects. First, it works as a ligand for the intracellular delivery of VEGF siRNAs into malignant HCC tumors, leading to antiangiogenesis in HCC. Later on, the decreased MVD in tumors induces hypoxia, SDF1α expression and TAM infiltration, resulting in resistance to antiangiogenic therapy. However, the second function of AMD3100 is to block the SDF1α/CXCR4 axis to further inhibit the stromal cell recruitment, thus achieving an enhanced anticancer effect when combined with VEGF siRNAs.
effect. In addition, both the delivery property and the silencing activity were ligand (AMD3100) dependent. We next studied the tissue distribution of fluorescently labeled siRNA in orthotopic HCA-1 tumor models 4 hours after i.v. 1778
injections. As shown in Figure 5a,b, AMD-NPs showed higher siRNA delivery in the tumor tissue than free siRNAs and s iRNAs in nontargeted NPs. The strong cytosolic delivery and heterogeneous distribution of fluorescently labeled siRNAs in the www.moleculartherapy.org vol. 23 no. 11 nov. 2015
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HCA-1 tumor tissues were observed after delivery by AMD-NPs (Figure 5b). However, the intracellular uptake of free siRNAs or siRNAs encapsulated in nontargeted NPs was hardly detected in the HCA-1 tumor tissues. In other organs (Figure 5a), the kidney showed higher uptake of free siRNAs than siRNAs formulated in nontargeted or targeted NPs. Only low concentrations of siRNAs encapsulated in AMD-NPs (less than 2% injected dose/g) were present in organs such as the heart, spleen, lung, and blood 4 hours after i.v. injections. We further demonstrated that systemic injections of VEGF siRNAs in AMD-NPs significantly silenced VEGF expression in HCA-1 tumors, whereas VEGF expression in tumor tissues remains unchanged after treatment with control siRNAs in nontargeted NPs or in AMD-NPs (Figure 5c,d). VEGF siRNAs in nontargeted NPs only caused minimal silencing effect on VEGF expression. These results revealed that CXCR4-targeted NPs could efficiently deliver siRNAs into HCC cells and silence the target gene in vitro and in vivo. AMD3100, a CXCR4 antagonist, serves as a targeting ligand for the delivery of siRNAs into HCC and a therapeutic agent for modulating the tumor microenvironment and sensitizing HCC to antiangiogenic therapy. We further evaluated the effects of VEGF siRNAs in AMDNPs on MVD, hypoxic induction, SDF1α expression, and tumor-associated macrophage (TAM) infiltration in HCA-1 transplanted HCC models. After the HCC tumor was established, AMD-NPs containing VEGF siRNAs were i.v. injected into tumor-bearing mice (Supplementary Figure S5). AMD-NPs containing VEGF siRNAs significantly reduced MVD, increased the hypoxic fraction and SDF1α expression compared to nontargeted NPs containing VEGF siRNAs or AMD-NPs containing control siRNAs (Figure 6a–d). Moreover, treatment with VEGF siRNAs in AMD-NPs showed better angiogenic effects compared to conventional sorafenib treatment. Despite the observance of increased hypoxic tissue fraction and SDF1α expression in the tumor tissues after treatment with VEGF siRNAs in AMD-NPs, the infiltration of F4/80+ macrophages into the tumor tissues remained the same as in the control treatment (Figure 6a,e). This indicated that the incorporation of AMD3100 into the siRNA- containing NPs blocked the recruitment of TAMs into the tumor microenvironment. Finally, tumor growth inhibition was observed after treatment with VEGF siRNA in AMD-NPs in the HCA-1 orthotopic model (Figure 7a). Control siRNAs in nontargeted NPs or AMD-NPs or VEGF siRNA in nontargeted NPs did not show significant HCC growth inhibition. In addition, AMD-NPs carrying either therapeutic VEGF siRNAs or nontherapeutic control siRNAs significantly suppressed metastasis formation in the lungs of HCA-1 orthotopic models (Figure 7b,c).
VEGF siRNA encapsulated in AMD-NPs is an effective and safe therapeutic agent for HCC In addition to therapeutic efficacy, evaluation of the toxicity is an essential part of drug development process. The hepatotoxicity makers—aspartate aminotransferase, alanine aminotransferase, and alkaline phosphatase—and the proinflammatory cytokines in the serum were measured in C3H mice 24 hours after Molecular Therapy vol. 23 no. 11 nov. 2015
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administration of VEGF siRNA-loaded AMD-NPs. Aspartate aminotransferase, alanine aminotransferase, and alkaline phosphatase levels remained minimally changed after treatment of VEGF siRNA-loaded AMD-NPs when compared to the untreated mice (Supplementary Table S1). Besides, VEGF siRNA formulated in AMD-NPs did not induce proinflammatory cytokines such as IL-6 and IFN-γ significantly (Supplementary Table S1). The addition of AMD3100 to the siRNA delivery system showed a significant improvement in delivery and therapeutic efficacy with low toxicity. Our data indicate that VEGF siRNA delivered by CXCR4-targeted NPs may serve as a safe and effective therapeutic option for HCC.
DISCUSSION
The Aim of this study is to develop gene delivery system for delivering antiangiogenic siRNAs into HCC and targeting inflammation tumor stroma in order to inhibit HCC progression and metastasis. Given overexpression of VEGF and hypoxia-induced SDF1α secretion in HCC, we developed CXCR4-targeted lipidbased NPs to co-deliver siRNA against VEGF and a small molecule CXCR4 antagonist. We showed that VEGF siRNA delivered by NPs silenced VEGF expression and inhibited angiogenesis in HCC. Furthermore, the CXCR4 antagonist AMD3100 not only served as a targeting moiety for the intracellular delivery of siRNA into malignant HCC but also suppressed recruitment of TAMs and synergistically inhibited angiogenesis when combined with VEGF siRNA. This combination therapy achieved in one multifunctional formulation led to effective tumor growth inhibition effects and significant metastasis reduction. siRNAs serve as potential therapeutic agents to treat cancer by specifically silencing the targeted oncogenes, but efficient siRNA delivery has remained challenging. Here, we show that CXCR4targeted NPs can encapsulate siRNA and deliver siRNA into HCC with high efficiency, silence the proangiogenic factor, and simultaneously block SDF1α/CXCR4 axis in HCC cells. The targeted siRNA delivery to tumors was mediated by the enhanced permeability and retention effect and CXCR4-dependent internalization on HCC cells. Although CXCR4 is expressed on several cell types such as lymphocytes, hematopoietic stem cells, endothelial cells, and epithelial cells,26 the expression level is higher in malignant HCC.23 Abundant CXCR4 expression in primary HCCs is associated with risk of distant metastasis and poor clinical outcome.23 Furthermore, treatment-induced hypoxia enhances CXCR4 expression through activation of HIF-1α in tumor cells and stromal cells in tumor microenvironment. Thus, the CXCR4-targeted NPs can target both malignant cancer cells and the surrounding stroma, providing a new strategy for efficient cancer therapy. Tumor Hypoxia and inflammation in the tumor microenvironment play important roles in development of drug resistance and promotion of cancer progression and metastasis. We have previously shown that antiangiogenic therapy resulted in hypoxic induction, elevated SDF1α expression, tumor inflammation and immunosuppression in HCC, leading to poor therapeutic outcome.1,27 Furthermore, inhibition of SDF1α/CXCR4 axis in combination with antiangiogenic treatment prevented the polarization of tumor-promoting microenvironment and resulted in enhanced therapeutic effect. In this study, we co-formulated 1779
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VEGF siRNA together with AMD3100, a CXCR4 antagonist in a multifunctional NP formulation. We found that treatment of AMD-NPs containing VEGF siRNAs significantly reduced MVD and prevented the increase in TAM infiltration—despite persistently elevated hypoxia—in HCC. Interestingly, we only observed increased hypoxia and SDF1α expression when MVD was profoundly decreased. Treatment of AMD-NPs alone or VEGF siRNAs in nontargeted NPs caused moderate MVD reduction and thus did not increased hypoxia and SDF1α secretion in the tumor microenvironment. This phenomenon may be due to the paradoxical effects of antiangiogenic drugs. Dose and time dependence of antivascular effects or vascular normalization was observed in solid tumors treated with antiangiogenic agents.28,29 It has been reported that treatment of anti-VEGFR2 antibody (DC101) at low dose could alleviate tumor hypoxia and normalize tumor vasculature, whereas the increased tumor hypoxia was observed only when treated with antiangiogenic agents at high dose.30 To this end, although the optimal dose of antiangiogenic treatment remains debatable, it is an important consideration for future clinical applications. TAM plays a key role in regulating the tumor microenvironment toward proangiogenesis, immunosuppression, and metastasis.31,32 To this end, our study showed ablation of TAMs using AMD-NPs synergistically suppressed angiogenesis, metastasis, and tumor progression when combined with either sorafenib or VEGF siRNA treatment. Of note, the in vitro study only showed a minor effect of treatment with VEGF siRNA in AMD-NPs on HCC viability. Therefore, the suppression of HCC progression induced by VEGF siRNA in AMD-NPs is mainly mediated by the tumor stroma. This study highlights the potential of targeting tumor microenvironment as efficient cancer therapy as well as for the future development of nanotechnology-based gene therapy in HCC. In conclusion, AMD3100 incorporated into our multifunctional NPs carries dual effects, working as a ligand for the intracellular delivery of siRNA into malignant HCC as well as a CXCR4 blocker, synergistically suppressing HCC progression when combined with antiangiogenic therapy (Figure 8).
MATERIALS AND METHODS
Materials. Protamine sulfate salt (fraction X from salmon), calf thymus DNA, and AMD3100 octahydrochloride hydrate were purchased from Sigma-Aldrich (St Louis, MO). VEGF siRNA with sequence 5′-AUGUGAAUGCAGACCAAAGAA-3′ and control siRNA with sequence 5′-AATTCTCCGAACGTGTCACGT-3′ were purchased from Sigma-Aldrich. For cellular and tumor uptake studies, fluorescein amidite (FAM) was conjugated to the 5′ end of the sense sequence. 5′-FAM-labeled VEGF siRNA was purchased from MDBio (Taipei, Taiwan). DOPA, DOPC, cholesterol, and DSPE-PEG (2000) were purchased from Avanti Polar Lipids (Alabaster, AL). Sorafenib (Nexavar) was purchased from Bayer Schering Pharma (Berlin, Germany), and recombinant SDF1α was purchased from ProSpec TechnoGene (Rehovot, Israel). Cell culture. We used the murine HCC cell line HCA-1 (refs. 1,33) and human HCC cell line JHH-7, kindly provided by Dr Dan Duda, MGH Boston. HCA-1 and JHH-7 were maintained in Dulbecco’s modified Eagle’s medium/high glucose and Dulbecco’s modified Eagle medium/ nutrient mixture F-12 (Hyclone, Logan, UT), respectively. All media were supplemented with 10% fetal bovine serum and antibiotics of penicillin and streptomycin (Hyclone).
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Animals. Four to five-week-old male C3H/HeNCrNarl mice were purchased from National Laboratory Animal Center (Taipei, Taiwan). HCA-1 cells were orthotopically implanted in mice, as previously described.34 All animals received humane care in compliance with the “Guide for the Care and Use of Laboratory Animals” published by the National Academy of Sciences. Preparation of AMD-NPs. Liposomes and NPs were prepared according to a previously described method with modifications.35–37 Briefly, anionic liposomes composed of DOPA, DOPC, and cholesterol (1:2:1 molar ratio) were prepared by thin film hydration followed by sonication (Q125, Qsonica) at 50 W for 2 minutes on ice to reduce the particle size. To prepare the cores of NPs, a mixture of 18 μl AMD3100 (2 mg/ml) and 16 μl of protamine (2 mg/ml), 120 μl of deionized water, and 22 μl of a mixture of siRNA (12 μl, 3 mg/ml) and calf thymus DNA (10 μl, 2 mg/ml) were mixed and kept at room temperature (RT) for 5 minutes before 90 μl of anionic liposomes (20 mmol/l) were added. After mixing the anionic liposomes with the DNA-Protamine-AMD cores, the mixture solution stood at RT for 10 minutes. To attach the AMD3100 into the NPs, the solution was then mixed with AMD3100 in phosphate-buffered saline (PBS) and kept at RT for 5 minutes. To post-insert DSPE-PEG into AMD-LPD, the solution was further mixed with 20 μl of DSPE-PEG (10 mg/ml) and kept at 55 °C for 10 minutes to form AMD-NPs as final product. Characterization of AMD-NPs. The morphology of the NPs was observed
using the transmission electronic microscope in the Microscopy Center of Chang Gung University (Taipei, Taiwan). 0.5 μl of AMD-NPs solution was dropped onto carbon-stabilized Formvar-coated 200-mesh copper grids (Ted Pella, Redding, CA) and dried under ambient conditions. Transmission electronic microscope images were captured using a Hitachi H-7500 microscope (Hitachi High-Technologies, Tokyo, Japan) operated at 75 kV. The particle size and surface charge were measured using a Zetasizer (3000HS; Malvern Instruments, Worcestershire, UK) at RT. The parameters of viscosity and refraction index were set equal to those of water for all samples during testing.
Cell viability assays. Cell viability was assessed using 3-(4,5‐dimethylthiazol‐2-yl)‐2,5-diphenyltetrazolium bromide (MTT); Serva Electrophoresis, Heidelberg, Germany) assay. HCA-1 or JHH-7 cells (2,000 per well) were seeded in 96-well transparent plates (Costar, IL) and incubated for 12 hours. Later, the medium was replaced with serum-free medium (Hyclone), and the cells were exposed to different treatments. After 48–72 hours, cells were incubated with MTT (3-(4,5‐dimethylthiazol‐ 2-yl)‐2,5-diphenyltetrazolium bromide) for 3 hours, and cell viability was assessed by absorbance measurement using a Multiskan reader (at 570 nm; Thermo Scientific, Asheville, NC). The fraction of control was calculated by dividing the absorbance obtained from the cells with different treatments by the absorbance obtained from the control-treated cells. Immunofluorescence. Frozen sections (10 µm thick) were fixed in acetone at −20 °C for 10 minutes and washed with PBS. The sections were then blocked with 5% bovine serum albumin (in PBS) for 1 hour and then incubated overnight with primary antibodies against von Willebrand factor (Dako, Glostrup, Denmark), SDF1α (BioVision, Mountain View, CA), CAIX (Abcam, Cambridge, MA), or F4/80 (Affymetrix eBiosciencem, San Diego, CA) at 4 °C. After washed with PBS, the sections were further incubated with secondary antirabbit IgG-Alexa Fluor 488 antibodies (Life Technologies, Grand Island, NY) for 1 hour. Unbound secondary antibodies were washed by PBS, counterstained with 4′,6‐diamidino‐2‐phenylindole (DAPI); Vector Laboratories, Burlingame, CA) and imaged using a confocal microscope (LSM 780; Carl Zeiss, Heidelberg, Germany). Cellular uptake study and AMD3100 competitive cellular uptake assay.
HCA-1 and JHH-7 cells (1 × 105/1 ml/well) were seeded in 12-well plates (Corning, Corning, NY) overnight before the experiments were conducted. www.moleculartherapy.org vol. 23 no. 11 nov. 2015
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The cells were treated in serum-containing media at 37 °C for 4 hours with different formulations containing FAM-labeled siRNA. Cells were washed twice with PBS, fixed in 4% paraformaldehyde (in PBS) for 10 minutes, counterstained with DAPI (4′,6‐diamidino‐2‐phenylindole), and imaged using a Zeiss LSM 780 confocal microscope (Carl Zeiss). For evaluation and quantification of cellular uptake using flow cytometry, cells were washed twice with PBS and harvested with trypsin/ ethylenediaminetetraacetic acid and then resuspended in PBS. The fluorescent signals were measured using a BD FACSAria III sorter (BD Biosciences, San Jose, CA). The cell fluorescence intensity distributions were obtained by counting a minimum of 10,000 cells for each replica. For the AMD3100 competitive cellular uptake assay, HCA-1 cells were prepared as previously mentioned. Free AMD3100 was added into the media at the final concentration of 0, 100, and 200 μmol/l for 10 minutes, and cells were treated with AMD-NPs containing FAM-labeled siRNA for 4 hours. The cellular uptake of FAM-labeled siRNA was examined and calculated using a confocal microscope. Tissue distribution study. Tumor-bearing mice were i.v. injected with
FAM-labeled siRNA (1.5 mg/kg) in different formulations. After 4 hours, mice were sacrificed, and tissues were collected and homogenized in lysis buffer and incubated on ice for 30 minutes. The tissue lysate was transferred to a black 96-well plate (Corning). The fluorescence intensity of the sample was measured by a plate reader (Fluoroskan Ascent FL, Thermo Scientific) at excitation wavelength 485nm and emission wavelength 538 nm. The concentration of FAM-labeled siRNA in each sample was calculated from a standard curve.
Tumor uptake study. C3H mice with a tumor size of ~1 cm2 were i.v.
injected with FAM-labeled siRNA (1.5 mg/kg) in nontargeted NPs or AMD-NPs. Four hours after injections, mice were sacrificed, and tissues were collected and homogenized in lysis buffer and incubated on ice for 30 minutes. The tissue lysate was transferred to a black 96-well plate (Corning). The fluorescence intensity of the sample was measured by a plate reader (Fluoroskan Ascent FL, Thermo Scientific) at excitation wavelength 485nm and emission wavelength 538 nm. The concentration of FAM-labeled siRNA in each sample was calculated using a standard curve. To examine the intracellular uptake of FAM-labeled siRNA in tumor tissues, the tissues were collected and fixed in 4% paraformaldehyde (in PBS) overnight before cryo-embedding. Tumor tissues were sectioned (10 μm thick) and imaged using a Zeiss LSM 780 confocal microscope. Hematoxylin and eosin staining. The lung tissue was cut into small pieces
and fixed in 4% paraformaldehyde (in PBS) overnight before embedded in paraffin wax. The sections were then stained with hematoxylin and eosin and observed with a Nikon microscope (Eclipse E800, Tallahassee, FL).
Gene silencing study. For evaluation of gene silencing by quantitative
reverse transcription polymerase chain reaction (qRT-PCR), 48 hours after treatment with different formulations, cells were washed twice with PBS, and mRNA was isolated by using RNeasy Mini kit (Qiagen, Valencia, CA) according to manufacturer’s instructions. In order to avoid genomic DNA contamination, RNA samples were subjected to RNase-free DNase (Qiagen) treatment at RT for 15 minutes. After isolation, 1-μg RNA samples were used for cDNA synthesis. cDNA synthesis was carried out using High-Capacity cDNA Reverse Transciption kit (Applied Biosystems, Foster City, CA) in a Piko Thermal Cycler, 24-well (Thermo Scientific) according to the manufacturer’s instruction. Quantitative PCR was performed using SYBR Green PCR Master Mix (Applied Biosystems) with the quantitative PCR primer pairs. The primer sequences were as follows: sense, 5′-CTGTGCAGGCTGCTGTAACG-3′ and antisense, 5′-GTTCCCGAAACCCTGAGGAG-3′ for VEGF-A; sense, 5′- TGAGAGGGAAATCGTGCGTG-3′ and antisense, 5′- TTGCTGA TCCACATCTGCTGG-3′ for β-actin; sense, 5′- CTGCCACCCAGAAG ACTGTG -3′ and antisense, 5′- GGTCCTCAGTGTAGCCCAAG -3′ for
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Delivery of siRNA Using CXCR4-targeted Nanoparticles
glyceraldehyde 3-phosphate dehydrogenase. The relative expression levels were normalized to β-actin or glyceraldehyde 3-phosphate dehydrogenase to obtain their relative gene expression. Primers employed in these studies were based on previous studies.38–40 For evaluation of protein expression by immunoblotting, cells were lysed in radioimmunoprecipitation assay buffer with ethylenediaminetetraacetic acid and ethylene glycol tetraacetic acid (BioBasic, Toronto, Ontario, Canada) for 20 minutes on ice, and the supernatant was collected after centrifugation at 12,700 rpm for 20 minutes. Cell lysates were separated on a 10% acrylamide gel and transferred to a polyvinylidene difluoride membrane. The membrane was blocked with 5% nonfat milk in PBS for 1 hour and then incubated overnight with primary antibodies against VEGF (Abcam, Cambridge, MA) or β-actin (Sigma-Aldrich) at 4 °C. After the membrane was washed with PBST (0.1% Tween 20 in PBS), it was further incubated with the horseradish peroxidase–conjugated secondary antibodies (Cell Signaling Technology, Beverly, MA) for 1 hour. The membrane was washed and developed by enhanced chemiluminescence using the ECL Select western blotting detection reagent (GE Healthcare, Little Chalfont, Buckinghamshire, UK), followed by autoradiography. For the in vivo studies, HCA-1 tumor-bearing mice were i.v. injected with siRNA in different formulations (1.44 mg siRNA/kg/dose, 1 dose per day). The day after the third injection, the mice were sacrificed, and tumor samples were collected. The total protein (40 μg) isolated from the tumors was loaded onto a polyacrylamide gel, electrophoresed, and blotted as described above. Statistical analysis. Comparisons between treatment groups were per-
formed using Mann–Whitney’s U-test. P value of less than 0.05 was considered to denote statistical significance.
SUPPLEMENTARY MATERIAL Figure S1. Treatment schedule of sorafenib combined with AMDNPs in the orthotopic HCC tumor model. Figure S2. AMD-NPs reduce HCA-1 cell migration. Figure S3. Uptake of FAM-labeled siRNAs in different formulations was examined by flow cytometry. Figure S4. Intracellular uptake and gene silencing effects in JHH-7 cells treated with siRNAs delivered by AMD-NPs in vitro. Figure S5. Treatment schedule of VEGF siRNAs in different formulations in the orthotopic HCC tumor model. Table S1. Toxicity profile of VEGF siRNA in CXCR4-targeted nanoparticles. Materials and Methods
ACKNOWLEDGMENTS This work was supported by Ministry of Science and Technology through grant no: NSC 102-2320-B-007-011-MY2 and MOST 103-2221-E-007032-MY2 and by Chang Gung Memorial Hospital-National Tsing Hua University Joint Research Grant 104N2744E1 to Y.C.
References
1. Chen, Y, Huang, Y, Reiberger, T, Duyverman, AM, Huang, P, Samuel, R et al. (2014). Differential effects of sorafenib on liver versus tumor fibrosis mediated by stromalderived factor 1 alpha/C-X-C receptor type 4 axis and myeloid differentiation antigenpositive myeloid cell infiltration in mice. Hepatology 59: 1435–1447. 2. Hernandez–Gea, V, Toffanin, S, Friedman, SL, and Llovet, JM (2013). Role of the microenvironment in the pathogenesis and treatment of hepatocellular carcinoma. Gastroenterology 144: 512–527. 3. Hui, CK, Leung, N, Shek, TW, Yao, H, Lee, WK, Lai, JY et al.; Hong Kong Liver Fibrosis Study Group. (2007). Sustained disease remission after spontaneous HBeAg seroconversion is associated with reduction in fibrosis progression in chronic hepatitis B Chinese patients. Hepatology 46: 690–698. 4. Luedde, T and Schwabe, RF (2011). NF-κB in the liver–linking injury, fibrosis and hepatocellular carcinoma. Nat Rev Gastroenterol Hepatol 8: 108–118. 5. Murakata, A, Tanaka, S, Mogushi, K, Yasen, M, Noguchi, N, Irie, T et al. (2011). Gene expression signature of the gross morphology in hepatocellular carcinoma. Ann Surg 253: 94–100. 6. Sato, K, Tanaka, S, Mitsunori, Y, Mogushi, K, Yasen, M, Aihara, A et al. (2013). Contrast-enhanced intraoperative ultrasonography for vascular imaging of
1781
Delivery of siRNA Using CXCR4-targeted Nanoparticles
7. 8. 9. 10. 11.
12.
13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24.
hepatocellular carcinoma: clinical and biological significance. Hepatology 57: 1436–1447. Semela, D and Dufour, JF (2004). Angiogenesis and hepatocellular carcinoma. J Hepatol 41: 864–880. Kerbel, RS (2006). Antiangiogenic therapy: a universal chemosensitization strategy for cancer? Science 312: 1171–1175. Meng, H, Xing, G, Sun, B, Zhao, F, Lei, H, Li, W et al. (2010). Potent angiogenesis inhibition by the particulate form of fullerene derivatives. ACS Nano 4: 2773–2783. Zhu, AX, Duda, DG, Sahani, DV and Jain, RK (2011). HCC and angiogenesis: possible targets and future directions. Nat Rev Clin Oncol 8: 292–301. Adnane, L, Trail, PA, Taylor, I and Wilhelm, SM (2006). Sorafenib (BAY 43-9006, Nexavar), a dual-action inhibitor that targets RAF/MEK/ERK pathway in tumor cells and tyrosine kinases VEGFR/PDGFR in tumor vasculature. Methods Enzymol 407: 597–612. Liu, L, Cao, Y, Chen, C, Zhang, X, McNabola, A, Wilkie, D et al. (2006). Sorafenib blocks the RAF/MEK/ERK pathway, inhibits tumor angiogenesis, and induces tumor cell apoptosis in hepatocellular carcinoma model PLC/PRF/5. Cancer Res 66: 11851–11858. Llovet, JM, Ricci, S, Mazzaferro, V, Hilgard, P, Gane, E, Blanc, JF et al.; SHARP Investigators Study Group. (2008). Sorafenib in advanced hepatocellular carcinoma. N Engl J Med 359: 378–390. Méndez-Sánchez, N, Vásquez-Fernández, F, Zamora-Valdés, D and Uribe, M (2008). Sorafenib, a systemic therapy for hepatocellular carcinoma. Ann Hepatol 7: 46–51. Xie, B, Wang, DH and Spechler, SJ (2012). Sorafenib for treatment of hepatocellular carcinoma: a systematic review. Dig Dis Sci 57: 1122–1129. Carmeliet, P and Jain, RK (2011). Molecular mechanisms and clinical applications of angiogenesis. Nature 473: 298–307. Jain, RK (2013). Normalizing tumor microenvironment to treat cancer: bench to bedside to biomarkers. J Clin Oncol 31: 2205–2218. Semenza, GL (2012). Hypoxia-inducible factors in physiology and medicine. Cell 148: 399–408. Gao, F, Liang, B, Reddy, ST, Farias-Eisner, R and Su, X (2014). Role of inflammationassociated microenvironment in tumorigenesis and metastasis. Curr Cancer Drug Targets 14: 30–45. Kryczek, I, Wei, S, Keller, E, Liu, R and Zou, W (2007). Stroma-derived factor (SDF-1/ CXCL12) and human tumor pathogenesis. Am J Physiol Cell Physiol 292: C987–C995. Gentilini, A, Rombouts, K, Galastri, S, Caligiuri, A, Mingarelli, E, Mello, T et al. (2012). Role of the stromal-derived factor-1 (SDF-1)-CXCR4 axis in the interaction between hepatic stellate cells and cholangiocarcinoma. J Hepatol 57: 813–820. Kamba, T and McDonald, DM (2007). Mechanisms of adverse effects of anti-VEGF therapy for cancer. Br J Cancer 96: 1788–1795. Xiang, ZL, Zeng, ZC, Tang, ZY, Fan, J, Zhuang, PY, Liang, Y et al. (2009). Chemokine receptor CXCR4 expression in hepatocellular carcinoma patients increases the risk of bone metastases and poor survival. BMC Cancer 9: 176. Yu, M, Gang, EJ, Parameswaran, R, Stoddart, S, Fei, F, Schmidhuber, S et al. (2011). AMD3100 sensitizes acute lymphoblastic leukemia cells to chemotherapy in vivo. Blood Cancer J 1: e14.
1782
© The American Society of Gene & Cell Therapy
25. Domanska, UM, Timmer-Bosscha, H, Nagengast, WB, Oude Munnink, TH, Kruizinga, RC, Ananias, HJ et al. (2012). CXCR4 inhibition with AMD3100 sensitizes prostate cancer to docetaxel chemotherapy. Neoplasia 14: 709–718. 26. Teicher, BA and Fricker, SP (2010). CXCL12 (SDF-1)/CXCR4 pathway in cancer. Clin Cancer Res 16: 2927–2931. 27. Chen, Y, Ramjiawan, RR, Reiberger, T, Ng, MR, Hato, T, Huang, Y et al. (2015). CXCR4 inhibition in tumor microenvironment facilitates anti-programmed death receptor-1 immunotherapy in sorafenib-treated hepatocellular carcinoma in mice. Hepatology 61: 1591–1602. 28. Jain, RK (2005). Normalization of tumor vasculature: an emerging concept in antiangiogenic therapy. Science 307: 58–62. 29. Huang, Y, Yuan, J, Righi, E, Kamoun, WS, Ancukiewicz, M, Nezivar, J et al. (2012). Vascular normalizing doses of antiangiogenic treatment reprogram the immunosuppressive tumor microenvironment and enhance immunotherapy. Proc Natl Acad Sci USA 109: 17561–17566. 30. Huang, Y, Stylianopoulos, T, Duda, DG, Fukumura, D and Jain, RK (2013). Benefits of vascular normalization are dose and time dependent–letter. Cancer Res 73: 7144–7146. 31. Capece, D, Fischietti, M, Verzella, D, Gaggiano, A, Cicciarelli, G, Tessitore, A et al. (2013). The inflammatory microenvironment in hepatocellular carcinoma: a pivotal role for tumor-associated macrophages. Biomed Res Int 2013: 187204. 32. Zhou, D, Huang, C, Kong, L and Li, J (2014). Novel therapeutic target of hepatocellular carcinoma by manipulation of macrophage colony-stimulating factor/ tumor-associated macrophages axis in tumor microenvironment. Hepatol Res 44: E318–E319. 33. Tofilon, PJ, Basic, I and Milas, L (1985). Prediction of in vivo tumor response to chemotherapeutic agents by the in vitro sister chromatid exchange assay. Cancer Res 45: 2025–2030. 34. Kim, W, Seong, J, Oh, HJ, Koom, WS, Choi, KJ and Yun, CO (2011). A novel combination treatment of armed oncolytic adenovirus expressing IL-12 and GM-CSF with radiotherapy in murine hepatocarcinoma. J Radiat Res 52: 646–654. 35. Chen, Y, Wu, JJ and Huang, L (2010). Nanoparticles targeted with NGR motif deliver c-myc siRNA and doxorubicin for anticancer therapy. Mol Ther 18: 828–834. 36. Cui, Z, Han, SJ, Vangasseri, DP and Huang, L (2005). Immunostimulation mechanism of LPD nanoparticle as a vaccine carrier. Mol Pharm 2: 22–28. 37. Li, SD, Chen, YC, Hackett, MJ and Huang, L (2008). Tumor-targeted delivery of siRNA by self-assembled nanoparticles. Mol Ther 16: 163–169. 38. Clifford, RL, Deacon, K and Knox, AJ (2008). Novel regulation of vascular endothelial growth factor-A (VEGF-A) by transforming growth factor (beta)1: requirement for Smads, (beta)-CATENIN, AND GSK3(beta). J Biol Chem 283: 35337–35353. 39. Kim, IS, Song, YM, Cho, TH, Park, YD, Lee, KB, Noh, I et al. (2008). In vitro response of primary human bone marrow stromal cells to recombinant human bone morphogenic protein-2 in the early and late stages of osteoblast differentiation. Dev Growth Differ 50: 553–564. 40. Albrektsen, T, Sørensen, BB, Hjortø, GM, Fleckner, J, Rao, LV and Petersen, LC (2007). Transcriptional program induced by factor VIIa-tissue factor, PAR1 and PAR2 in MDAMB-231 cells. J Thromb Haemost 5: 1588–1597.
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