Limits...
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.

Show MeSH

Related in: MedlinePlus

Inhibitory effect of miR-7 on tumor growth by local delivery(a) miR-7 inhibits tumor growth after local delivery. AJ mice bearing tumors with Neuro2A cells were treated locally with 10 μg miR-7 or 10 μg miR-Scr or PBS by intratumoral injection and electroporation. Arrows below the graph indicate treatment schedule (6 treatments, every other day). Data are plotted as mean values ± SEM (n=7), p<0.05. (b) Increased presence of miR-7 in miR-7 treated animals. The day after the last injection tumors were removed and total RNA was isolated. Delivery of miR-7 by electroporation into the tumor tissue was determined by stem loop RT-PCR. Values were normalized to U6 expression in the tumors. Data are plotted as mean values ± s.d. (n=3), *p<0.001. (c-d) miR-7 reduces angiogenesis in vivo after local delivery. Tumor sections were stained for CD31 (in brown) and microvessel density (MVD) was quantified by counting blood vessels in 6 random high magnification fields in each sample. Data are plotted as mean values ± s.d. (n=4), *p< 0.01. (e-f) miR-7 does not affect proliferation in Neuro2A tumors. The effect of miR-7 on tumor cell proliferation was determined by Ki-67 staining (in brown). Quantification of the images was performed in the same way as described in (c-d), and expressed as percentage of the PBS treated group. Data are plotted as mean values ± s.d. (n=4). Magnification of Fig 4c and 4e in Supplementary Fig. S12.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4196156&req=5

Figure 4: Inhibitory effect of miR-7 on tumor growth by local delivery(a) miR-7 inhibits tumor growth after local delivery. AJ mice bearing tumors with Neuro2A cells were treated locally with 10 μg miR-7 or 10 μg miR-Scr or PBS by intratumoral injection and electroporation. Arrows below the graph indicate treatment schedule (6 treatments, every other day). Data are plotted as mean values ± SEM (n=7), p<0.05. (b) Increased presence of miR-7 in miR-7 treated animals. The day after the last injection tumors were removed and total RNA was isolated. Delivery of miR-7 by electroporation into the tumor tissue was determined by stem loop RT-PCR. Values were normalized to U6 expression in the tumors. Data are plotted as mean values ± s.d. (n=3), *p<0.001. (c-d) miR-7 reduces angiogenesis in vivo after local delivery. Tumor sections were stained for CD31 (in brown) and microvessel density (MVD) was quantified by counting blood vessels in 6 random high magnification fields in each sample. Data are plotted as mean values ± s.d. (n=4), *p< 0.01. (e-f) miR-7 does not affect proliferation in Neuro2A tumors. The effect of miR-7 on tumor cell proliferation was determined by Ki-67 staining (in brown). Quantification of the images was performed in the same way as described in (c-d), and expressed as percentage of the PBS treated group. Data are plotted as mean values ± s.d. (n=4). Magnification of Fig 4c and 4e in Supplementary Fig. S12.

Mentions: Based on the potent anti-angiogenic activity of miR-7 observed both in vitro and in vivo in the CAM assay, the anti-angiogenic potency and inhibitory effect on tumor growth was investigated in a subcutaneous neuroblastoma (N2A) mouse tumor model using intratumoral injections and electroporation. The miR-7 mimic (10 μg) treated mice exhibited a 43% reduction in tumor growth compared to both the PBS and miR-Scr negative control treated mice (Fig. 4a). Stem-loop RT-PCR was used to determine the relative tumor amounts of miR-7 in the different treatment groups. Tumors of miR-7 treated animals showed significantly higher miR-7 levels compared to the control groups (Fig. 4b). The biochemical process underlying tumor growth inhibition by miR-7 mimics was investigated using immunohistochemical (IHC) detection of CD31, an endothelial cell marker for microvessel density (Fig. 4c). MiR-7 mimic treated tumors displayed a reduced microvessel density, indicative of anti-angiogenic activity of the treatment (Fig. 4d). However, no differences in expression of Ki-67, a marker for proliferation, were detected among the treatment groups (Fig. 4e and f). These data suggest that inhibition of angiogenesis is the prime mechanism for the N2A tumor growth suppression upon intratumoral delivery of miR-7. The lack of efficacy on tumor cell proliferation in vivo is corroborated by the observation that miR-7 did not inhibit cell viability of N2A cells in vitro (Supplementary Fig. S5).


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 local delivery(a) miR-7 inhibits tumor growth after local delivery. AJ mice bearing tumors with Neuro2A cells were treated locally with 10 μg miR-7 or 10 μg miR-Scr or PBS by intratumoral injection and electroporation. Arrows below the graph indicate treatment schedule (6 treatments, every other day). Data are plotted as mean values ± SEM (n=7), p<0.05. (b) Increased presence of miR-7 in miR-7 treated animals. The day after the last injection tumors were removed and total RNA was isolated. Delivery of miR-7 by electroporation into the tumor tissue was determined by stem loop RT-PCR. Values were normalized to U6 expression in the tumors. Data are plotted as mean values ± s.d. (n=3), *p<0.001. (c-d) miR-7 reduces angiogenesis in vivo after local delivery. Tumor sections were stained for CD31 (in brown) and microvessel density (MVD) was quantified by counting blood vessels in 6 random high magnification fields in each sample. Data are plotted as mean values ± s.d. (n=4), *p< 0.01. (e-f) miR-7 does not affect proliferation in Neuro2A tumors. The effect of miR-7 on tumor cell proliferation was determined by Ki-67 staining (in brown). Quantification of the images was performed in the same way as described in (c-d), and expressed as percentage of the PBS treated group. Data are plotted as mean values ± s.d. (n=4). Magnification of Fig 4c and 4e in Supplementary Fig. S12.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4196156&req=5

Figure 4: Inhibitory effect of miR-7 on tumor growth by local delivery(a) miR-7 inhibits tumor growth after local delivery. AJ mice bearing tumors with Neuro2A cells were treated locally with 10 μg miR-7 or 10 μg miR-Scr or PBS by intratumoral injection and electroporation. Arrows below the graph indicate treatment schedule (6 treatments, every other day). Data are plotted as mean values ± SEM (n=7), p<0.05. (b) Increased presence of miR-7 in miR-7 treated animals. The day after the last injection tumors were removed and total RNA was isolated. Delivery of miR-7 by electroporation into the tumor tissue was determined by stem loop RT-PCR. Values were normalized to U6 expression in the tumors. Data are plotted as mean values ± s.d. (n=3), *p<0.001. (c-d) miR-7 reduces angiogenesis in vivo after local delivery. Tumor sections were stained for CD31 (in brown) and microvessel density (MVD) was quantified by counting blood vessels in 6 random high magnification fields in each sample. Data are plotted as mean values ± s.d. (n=4), *p< 0.01. (e-f) miR-7 does not affect proliferation in Neuro2A tumors. The effect of miR-7 on tumor cell proliferation was determined by Ki-67 staining (in brown). Quantification of the images was performed in the same way as described in (c-d), and expressed as percentage of the PBS treated group. Data are plotted as mean values ± s.d. (n=4). Magnification of Fig 4c and 4e in Supplementary Fig. S12.
Mentions: Based on the potent anti-angiogenic activity of miR-7 observed both in vitro and in vivo in the CAM assay, the anti-angiogenic potency and inhibitory effect on tumor growth was investigated in a subcutaneous neuroblastoma (N2A) mouse tumor model using intratumoral injections and electroporation. The miR-7 mimic (10 μg) treated mice exhibited a 43% reduction in tumor growth compared to both the PBS and miR-Scr negative control treated mice (Fig. 4a). Stem-loop RT-PCR was used to determine the relative tumor amounts of miR-7 in the different treatment groups. Tumors of miR-7 treated animals showed significantly higher miR-7 levels compared to the control groups (Fig. 4b). The biochemical process underlying tumor growth inhibition by miR-7 mimics was investigated using immunohistochemical (IHC) detection of CD31, an endothelial cell marker for microvessel density (Fig. 4c). MiR-7 mimic treated tumors displayed a reduced microvessel density, indicative of anti-angiogenic activity of the treatment (Fig. 4d). However, no differences in expression of Ki-67, a marker for proliferation, were detected among the treatment groups (Fig. 4e and f). These data suggest that inhibition of angiogenesis is the prime mechanism for the N2A tumor growth suppression upon intratumoral delivery of miR-7. The lack of efficacy on tumor cell proliferation in vivo is corroborated by the observation that miR-7 did not inhibit cell viability of N2A cells in vitro (Supplementary Fig. S5).

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