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Systemic miRNA-7 delivery inhibits tumor angiogenesis and growth in murine xenograft glioblastoma.

Babae N, Bourajjaj M, Liu Y, Van Beijnum JR, Cerisoli F, Scaria PV, Verheul M, Van Berkel MP, Pieters EH, Van Haastert RJ, Yousefi A, Mastrobattista E, Storm G, Berezikov E, Cuppen E, Woodle M, Schaapveld RQ, Prevost GP, Griffioen AW, Van Noort PI, Schiffelers RM - Oncotarget (2014)

Bottom Line: Introduction of miR-7 in EC resulted in strongly reduced cell viability, tube formation, sprouting and migration.Application of miR-7 in the chick chorioallantoic membrane assay led to a profound reduction of vascularization, similar to anti-angiogenic drug sunitinib.Transcriptome analysis of miR-7 transfected EC in combination with in silico target prediction resulted in the identification of OGT as novel target gene of miR-7.

View Article: PubMed Central - PubMed

Affiliation: Utrecht Institute for Pharmaceutical Sciences, University Utrecht, Utrecht, the Netherlands. These authors contributed equally to this work.

ABSTRACT
Tumor-angiogenesis is the multi-factorial process of sprouting of endothelial cells (EC) into micro-vessels to provide tumor cells with nutrients and oxygen. To explore miRNAs as therapeutic angiogenesis-inhibitors, we performed a functional screen to identify miRNAs that are able to decrease EC viability. We identified miRNA-7 (miR-7) as a potent negative regulator of angiogenesis. Introduction of miR-7 in EC resulted in strongly reduced cell viability, tube formation, sprouting and migration. Application of miR-7 in the chick chorioallantoic membrane assay led to a profound reduction of vascularization, similar to anti-angiogenic drug sunitinib. Local administration of miR-7 in an in vivo murine neuroblastoma tumor model significantly inhibited angiogenesis and tumor growth. Finally, systemic administration of miR-7 using a novel integrin-targeted biodegradable polymeric nanoparticles that targets both EC and tumor cells, strongly reduced angiogenesis and tumor proliferation in mice with human glioblastoma xenografts. Transcriptome analysis of miR-7 transfected EC in combination with in silico target prediction resulted in the identification of OGT as novel target gene of miR-7. Our study provides a comprehensive validation of miR-7 as novel anti-angiogenic therapeutic miRNA that can be systemically delivered to both EC and tumor cells and offers promise for miR-7 as novel anti-tumor therapeutic.

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Inhibitory effect of miR-7 on tumor growth by systemic delivery(a) miR-7 treated animals show pale and less vascularized tumors. Macroscopic images of the tumors after the 7th dose administration. (b) miR-7 inhibits tumor growth after systemic delivery. Athymic Nude-Foxn1nu mice bearing U-87 MG tumors were injected intravenously with αvβ3/αvβ5 targeted miR-7 nanoparticles (3 mg/kg miRNA). Arrows in the graph indicate the days of treatment (8 treatments, every other day). miR-7 treated mice showed significant tumor growth inhibition compared to vehicle treated mice. Data are plotted as mean values ± SEM (n=10), *p<0.05. (c-d) miR-7 reduces angiogenesis after systemic delivery. Tumor sections were stained for CD31 (in brown) and quantified by counting CD31 positive staining area (pixels) in 6 random fields in each tumor. Data are plotted as mean values ± s.d. (n=5), *p<0.001. Magnification of Fig 5c in Supplementary Fig. S13. (e-f) miR-7 reduces cell-proliferation in U-87 MG tumors after systemic delivery. Anti-proliferative effect of systemically delivered miR-7 was determined by Ki-67 staining, indicated as brown spots. Quantification of Ki-67 was performed by counting of the stains in 6 random fields in each tumor section and proliferation was expressed as percentage of PBS treated mice. Data are plotted as mean values ± s.d. (n=5), *p<0.001. (g-h) miR-7 reduces OGT in U-87 MG tumors after systemic delivery. Tumor sections stained for OGT (brown nuclei and brownish cytoplasm) and quantified by counting of the stains in 6 random fields in each tumor section. Data are plotted as mean values ±s.d. (n=5), * p=0.0004.
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Figure 5: Inhibitory effect of miR-7 on tumor growth by systemic delivery(a) miR-7 treated animals show pale and less vascularized tumors. Macroscopic images of the tumors after the 7th dose administration. (b) miR-7 inhibits tumor growth after systemic delivery. Athymic Nude-Foxn1nu mice bearing U-87 MG tumors were injected intravenously with αvβ3/αvβ5 targeted miR-7 nanoparticles (3 mg/kg miRNA). Arrows in the graph indicate the days of treatment (8 treatments, every other day). miR-7 treated mice showed significant tumor growth inhibition compared to vehicle treated mice. Data are plotted as mean values ± SEM (n=10), *p<0.05. (c-d) miR-7 reduces angiogenesis after systemic delivery. Tumor sections were stained for CD31 (in brown) and quantified by counting CD31 positive staining area (pixels) in 6 random fields in each tumor. Data are plotted as mean values ± s.d. (n=5), *p<0.001. Magnification of Fig 5c in Supplementary Fig. S13. (e-f) miR-7 reduces cell-proliferation in U-87 MG tumors after systemic delivery. Anti-proliferative effect of systemically delivered miR-7 was determined by Ki-67 staining, indicated as brown spots. Quantification of Ki-67 was performed by counting of the stains in 6 random fields in each tumor section and proliferation was expressed as percentage of PBS treated mice. Data are plotted as mean values ± s.d. (n=5), *p<0.001. (g-h) miR-7 reduces OGT in U-87 MG tumors after systemic delivery. Tumor sections stained for OGT (brown nuclei and brownish cytoplasm) and quantified by counting of the stains in 6 random fields in each tumor section. Data are plotted as mean values ±s.d. (n=5), * p=0.0004.

Mentions: Clinical application of miRNA-based therapeutics is dependent on systemic administration and intracellular delivery of the miRNA (mimic) to the target site. Studies have shown an ability of cRGD-targeted nanoparticles to provide neovasculature targeting and VEGF pathway siRNA inhibition of angiogenesis in murine models [24-26]. Therefore, this approach was selected for systemic delivery of miR-7. Hereto, a novel biodegradable neovasculature targeted nanoparticles formulation was developed and used to investigate the anti-tumor activity of miR-7 following intravenous administration in human glioblastoma U-87 MG bearing mice. U-87 MG tumor bearing mice were treated with miR-7 mimic using a cyclic Arginine-Glycine-Aspartic acid (cRGD) peptide coupled biodegradable polyamide nanoparticles, targeting integrins αvβ3 and αvβ5. These integrins are present on tumor EC neovasculature, as well as on certain tumor cells [27], such as observed with the U-87 MG tumor cell line (Supplementary Fig. S6). The results demonstrate that targeted systemic delivery of miR-7 inhibited tumor angiogenesis and growth. Tumors of the miR-7 treatment group were pale and less vascularized compared to those in control groups (Fig. 5a) and systemic delivery of miR-7 mimic inhibited tumor growth by 42% after two weeks of treatment (Fig. 5b). The anti-angiogenic property of miR-7 was also observed microscopically with a statistically significant reduction of immunohistochemical staining of CD31 in tumor tissue from the miR-7 treated mice (Fig. 5c and d). Moreover, tumor tissue from the miR-7 treated mice contained considerable amounts of necrotic lesions, which is an indication of hypoxia from reduced angiogenesis. Systemic delivery of miR-7 mimic not only reduced tumor angiogenesis but also reduced tumor proliferation as demonstrated by the statistically significant reduction in Ki-67 staining of miR-7 treated tumor tissue (Fig. 5e and f). This was confirmed by in vitro studies that demonstrated reduced proliferation of U-87 MG cells upon transfection with miR-7 mimic (Supplementary Fig. S7). To confirm effective delivery of miR-7 into the tumor tissue we performed IHC staining of OGT, one of the target genes of miR-7 (Fig. 5g). miR-7 treated mice showed a statistically significant reduction in OGT levels (Fig. 5h). Downregulation of OGT by miR-7 is not EC specific, but was also confirmed in U-87 MG cells in vitro (Supplementary Fig. S8). The observed inhibition of tumor growth can thus be ascribed to a combination of an anti-angiogenic effect of miR-7 delivered to tumor-associated EC and an anti-proliferative effect of miR-7 delivered to tumor cells. Similar to EC, downregulation of OGT in U-87 MG cells by RNAi interference did not inhibit cell viability (Supplementary Fig. S9), indicating that also in tumor cells OGT is a good marker for miR-7 delivery but not for miR-7 efficacy.


Systemic miRNA-7 delivery inhibits tumor angiogenesis and growth in murine xenograft glioblastoma.

Babae N, Bourajjaj M, Liu Y, Van Beijnum JR, Cerisoli F, Scaria PV, Verheul M, Van Berkel MP, Pieters EH, Van Haastert RJ, Yousefi A, Mastrobattista E, Storm G, Berezikov E, Cuppen E, Woodle M, Schaapveld RQ, Prevost GP, Griffioen AW, Van Noort PI, Schiffelers RM - Oncotarget (2014)

Inhibitory effect of miR-7 on tumor growth by systemic delivery(a) miR-7 treated animals show pale and less vascularized tumors. Macroscopic images of the tumors after the 7th dose administration. (b) miR-7 inhibits tumor growth after systemic delivery. Athymic Nude-Foxn1nu mice bearing U-87 MG tumors were injected intravenously with αvβ3/αvβ5 targeted miR-7 nanoparticles (3 mg/kg miRNA). Arrows in the graph indicate the days of treatment (8 treatments, every other day). miR-7 treated mice showed significant tumor growth inhibition compared to vehicle treated mice. Data are plotted as mean values ± SEM (n=10), *p<0.05. (c-d) miR-7 reduces angiogenesis after systemic delivery. Tumor sections were stained for CD31 (in brown) and quantified by counting CD31 positive staining area (pixels) in 6 random fields in each tumor. Data are plotted as mean values ± s.d. (n=5), *p<0.001. Magnification of Fig 5c in Supplementary Fig. S13. (e-f) miR-7 reduces cell-proliferation in U-87 MG tumors after systemic delivery. Anti-proliferative effect of systemically delivered miR-7 was determined by Ki-67 staining, indicated as brown spots. Quantification of Ki-67 was performed by counting of the stains in 6 random fields in each tumor section and proliferation was expressed as percentage of PBS treated mice. Data are plotted as mean values ± s.d. (n=5), *p<0.001. (g-h) miR-7 reduces OGT in U-87 MG tumors after systemic delivery. Tumor sections stained for OGT (brown nuclei and brownish cytoplasm) and quantified by counting of the stains in 6 random fields in each tumor section. Data are plotted as mean values ±s.d. (n=5), * p=0.0004.
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Related In: Results  -  Collection

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Figure 5: Inhibitory effect of miR-7 on tumor growth by systemic delivery(a) miR-7 treated animals show pale and less vascularized tumors. Macroscopic images of the tumors after the 7th dose administration. (b) miR-7 inhibits tumor growth after systemic delivery. Athymic Nude-Foxn1nu mice bearing U-87 MG tumors were injected intravenously with αvβ3/αvβ5 targeted miR-7 nanoparticles (3 mg/kg miRNA). Arrows in the graph indicate the days of treatment (8 treatments, every other day). miR-7 treated mice showed significant tumor growth inhibition compared to vehicle treated mice. Data are plotted as mean values ± SEM (n=10), *p<0.05. (c-d) miR-7 reduces angiogenesis after systemic delivery. Tumor sections were stained for CD31 (in brown) and quantified by counting CD31 positive staining area (pixels) in 6 random fields in each tumor. Data are plotted as mean values ± s.d. (n=5), *p<0.001. Magnification of Fig 5c in Supplementary Fig. S13. (e-f) miR-7 reduces cell-proliferation in U-87 MG tumors after systemic delivery. Anti-proliferative effect of systemically delivered miR-7 was determined by Ki-67 staining, indicated as brown spots. Quantification of Ki-67 was performed by counting of the stains in 6 random fields in each tumor section and proliferation was expressed as percentage of PBS treated mice. Data are plotted as mean values ± s.d. (n=5), *p<0.001. (g-h) miR-7 reduces OGT in U-87 MG tumors after systemic delivery. Tumor sections stained for OGT (brown nuclei and brownish cytoplasm) and quantified by counting of the stains in 6 random fields in each tumor section. Data are plotted as mean values ±s.d. (n=5), * p=0.0004.
Mentions: Clinical application of miRNA-based therapeutics is dependent on systemic administration and intracellular delivery of the miRNA (mimic) to the target site. Studies have shown an ability of cRGD-targeted nanoparticles to provide neovasculature targeting and VEGF pathway siRNA inhibition of angiogenesis in murine models [24-26]. Therefore, this approach was selected for systemic delivery of miR-7. Hereto, a novel biodegradable neovasculature targeted nanoparticles formulation was developed and used to investigate the anti-tumor activity of miR-7 following intravenous administration in human glioblastoma U-87 MG bearing mice. U-87 MG tumor bearing mice were treated with miR-7 mimic using a cyclic Arginine-Glycine-Aspartic acid (cRGD) peptide coupled biodegradable polyamide nanoparticles, targeting integrins αvβ3 and αvβ5. These integrins are present on tumor EC neovasculature, as well as on certain tumor cells [27], such as observed with the U-87 MG tumor cell line (Supplementary Fig. S6). The results demonstrate that targeted systemic delivery of miR-7 inhibited tumor angiogenesis and growth. Tumors of the miR-7 treatment group were pale and less vascularized compared to those in control groups (Fig. 5a) and systemic delivery of miR-7 mimic inhibited tumor growth by 42% after two weeks of treatment (Fig. 5b). The anti-angiogenic property of miR-7 was also observed microscopically with a statistically significant reduction of immunohistochemical staining of CD31 in tumor tissue from the miR-7 treated mice (Fig. 5c and d). Moreover, tumor tissue from the miR-7 treated mice contained considerable amounts of necrotic lesions, which is an indication of hypoxia from reduced angiogenesis. Systemic delivery of miR-7 mimic not only reduced tumor angiogenesis but also reduced tumor proliferation as demonstrated by the statistically significant reduction in Ki-67 staining of miR-7 treated tumor tissue (Fig. 5e and f). This was confirmed by in vitro studies that demonstrated reduced proliferation of U-87 MG cells upon transfection with miR-7 mimic (Supplementary Fig. S7). To confirm effective delivery of miR-7 into the tumor tissue we performed IHC staining of OGT, one of the target genes of miR-7 (Fig. 5g). miR-7 treated mice showed a statistically significant reduction in OGT levels (Fig. 5h). Downregulation of OGT by miR-7 is not EC specific, but was also confirmed in U-87 MG cells in vitro (Supplementary Fig. S8). The observed inhibition of tumor growth can thus be ascribed to a combination of an anti-angiogenic effect of miR-7 delivered to tumor-associated EC and an anti-proliferative effect of miR-7 delivered to tumor cells. Similar to EC, downregulation of OGT in U-87 MG cells by RNAi interference did not inhibit cell viability (Supplementary Fig. S9), indicating that also in tumor cells OGT is a good marker for miR-7 delivery but not for miR-7 efficacy.

Bottom Line: Introduction of miR-7 in EC resulted in strongly reduced cell viability, tube formation, sprouting and migration.Application of miR-7 in the chick chorioallantoic membrane assay led to a profound reduction of vascularization, similar to anti-angiogenic drug sunitinib.Transcriptome analysis of miR-7 transfected EC in combination with in silico target prediction resulted in the identification of OGT as novel target gene of miR-7.

View Article: PubMed Central - PubMed

Affiliation: Utrecht Institute for Pharmaceutical Sciences, University Utrecht, Utrecht, the Netherlands. These authors contributed equally to this work.

ABSTRACT
Tumor-angiogenesis is the multi-factorial process of sprouting of endothelial cells (EC) into micro-vessels to provide tumor cells with nutrients and oxygen. To explore miRNAs as therapeutic angiogenesis-inhibitors, we performed a functional screen to identify miRNAs that are able to decrease EC viability. We identified miRNA-7 (miR-7) as a potent negative regulator of angiogenesis. Introduction of miR-7 in EC resulted in strongly reduced cell viability, tube formation, sprouting and migration. Application of miR-7 in the chick chorioallantoic membrane assay led to a profound reduction of vascularization, similar to anti-angiogenic drug sunitinib. Local administration of miR-7 in an in vivo murine neuroblastoma tumor model significantly inhibited angiogenesis and tumor growth. Finally, systemic administration of miR-7 using a novel integrin-targeted biodegradable polymeric nanoparticles that targets both EC and tumor cells, strongly reduced angiogenesis and tumor proliferation in mice with human glioblastoma xenografts. Transcriptome analysis of miR-7 transfected EC in combination with in silico target prediction resulted in the identification of OGT as novel target gene of miR-7. Our study provides a comprehensive validation of miR-7 as novel anti-angiogenic therapeutic miRNA that can be systemically delivered to both EC and tumor cells and offers promise for miR-7 as novel anti-tumor therapeutic.

Show MeSH
Related in: MedlinePlus