1. Volume 19, Issue 12, Pages 2201-2212 (December 2011)
Selective Therapeutic Targeting of the Anaplastic Lymphoma Kinase With Liposomal siRNA Induces Apoptosis and Inhibits Angiogenesis in Neuroblastoma 
Daniela Di Paolo, Chiara Ambrogio, Fabio Pastorino, Chiara Brignole, Cinzia Martinengo, Roberta Carosio, Monica Loi, Gabriella Pagnan, Laura Emionite, Michele Cilli, Domenico Ribatti, Theresa M Allen, Roberto Chiarle, Mirco Ponzoni, Patrizia Perri 
Molecular Therapy 
Volume 19, Issue 12, Pages (December 2011)
DOI: /mt
Copyright © 2011 The American Society of Gene & Cell Therapy Terms and Conditions

2. Figure 1
ALK-shRNA inhibits cell growth and induces cell death of human neuroblastoma (NB) cell lines. (a) SK-N-BE2 and SH-SY5Y cells were transduced with two different lentiviral constructs, inducible for specific shRNAs against ALK (A5) and a control sequence (M5). Native cells, and those treated with 1 µg/ml doxycycline (doxy) for 72 hours, were then lysed and immunoblotted with the indicated antibodies. (b) Inducible SK-N-BE2 and SH-SY5Y, described in a, were mixed in 1:1 ratio with native cells and then cultivated in the presence of 1 µg/ml doxy to induce enhanced green fluorescent protein (EGFP) expression and ALK downmodulation. Growth rate was evaluated by fluorescence-activated cell sorter (FACS) for detecting the percentages of EGFP positive cells over time. (c) Inducible SK-N-BE2 and SH-SY5Y cells were treated as described in a, then percentages of apoptotic cells were measured by tetramethyl rhodamine methyl ester (TMRM) staining. (***P < 0.001). (d) SK-N-BE2 and SH-SY5Y cells, inducible for A5 or M5, were treated with doxy and analyzed by western blot with the indicated antibodies. (e) The ratio between phosphorylated ERK1/2 and total ERK1/2 was calculated by densitometric analysis of blots from three independent experiments as in d. Histograms indicate the relative phosphorylation levels of ERK1/2 in SK-N-BE2 and SH-SY5Y cells. The statistical difference in ERK1/2 phosphorylation was calculated using the Student's t-test, and was significantly reduced in A5 transduced cells as compared to M5 controls (*P < 0.05). ALK, anaplastic lymphoma kinase; shRNA, short hairpin RNA.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2011 The American Society of Gene & Cell Therapy Terms and Conditions

3. Figure 2
ALK-shRNA inhibits neuroblastoma (NB) tumor growth and progression in vivo. Survival curves of SCID mice (n = 7 for each treatment) (a–c) orthotopically or (d,e) subcutaneously transplanted with NB cell lines transducing a Tet-On inducible ALK-shRNA in response to doxy treatment. (a) SK-N-BE2 TTA A5 cells were pretreated in vitro for 48 hours with doxy and then transplanted in mice kept under doxy treatment or transplanted in mice, and treated with doxy 7 days after orthotopic xenografts. Cells kept under ALK ablation grew far less efficiently than control cells (P = ). (b,c) Doxy administered 7 days postimplantation of SH-SY5Y (TTA A5) or IMR-5 (TTA A5) cells [sorted by a statistically significant inhibition of tumor growth with respect to untreated control mice: SH-SY5Y (TTA A5): P = ; IMR-5 (TTA A5): P = )]. (d,e) Doxy administered at time 0 or 10 days postimplantation of SK-N-BE2 TTA A5 (***P < 0.001; in panel e, P = ). ALK, anaplastic lymphoma kinase; shRNA, short hairpin RNA.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2011 The American Society of Gene & Cell Therapy Terms and Conditions

4. Figure 3
Small interfering RNA (siRNA)-mediated silencing of ALK and apoptosis induction in neuroblastoma (NB) cell lines. (a) Effects of ALK knockdown evaluated by real-time reverse transcriptase (RT)-qPCR 48 hours post-transfection in SH-SY5Y cells. A marked ALK knockdown was obtained by in vitro transfection with TL[ALK-siRNA], demonstrating the high specificity of siRNA sequence, not affecting other tested genes. Relative gene expression analysis is reported on y-axis as expression percentage with respect to the scrambled control. Values are the mean ± 95% confidence interval (CI) of N = 3 independent experiments performed in duplicate. (b) Western blot showing an extremely selective effects of ALK protein ablation obtained after transfection with TL[ALK-siRNA] in GD2-positive SH-SY5Y cells but not in GD2-negative cells (LB24Dagi). Both native cell lines (control) and cells treated with TL[scr-siRNA] were loaded as negative controls. (c,d) TL[ALK-siRNA] selectively promoted apoptosis in GD2-positive cells (d), but not in GD2-negative cells (e). Annexin V-positive cells after TL[ALK-siRNA] treatment are reported on y-axis as percentage with respect to the control and the TL[scr-siRNA]-treated cells. Values are the mean ± 95% confidence interval (CI) of N = 3 independent assays performed in quadruplicate. ***P < versus control. ALK, anaplastic lymphoma kinase.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2011 The American Society of Gene & Cell Therapy Terms and Conditions

5. Figure 4
ALK gene silencing by TL[ALK-siRNA] induces apoptosis and inhibits angiogenesis in vivo. Immunohistochemistry (IHC) and immunofluorescence (IF) staining performed on formalin-fixed and paraffin-embedded tumor sections of SH-SY5Y subcutaneous (s.c.) xenografts treated with TL[ALK-siRNA] and compared with untreated controls, or those treated with free ALK-siRNA and TL[scr-siRNA]. Black bar: 100 µm. White bar in b,c: 50 µm; in d,e: 100 µm. (a) IHC staining for ALK on neuroblastoma (NB) xenografts showing a complete ablation of ALK protein by TL[ALK-siRNA], and a partial downmodulation by free ALK-siRNA. (b) IF staining for Ki-67 highlights a dramatic decrease of cell proliferation in tumors treated with TL[ALK-siRNA]. (c) Terminal deoxynucleotidyl transferase-mediated end labeling (TUNEL) staining remarks a massive induction of apoptosis by TL[ALK-siRNA] treatment. (d,e) Endothelial (CD34) and perivascular [smooth muscle actin (SMA)] cell staining, respectively, after ALK silencing by TL[ALK-siRNA] in xenograft tumor reveals a strong antiangiogenic effect. On the right side, percentage of positive cells for each specific marker are reported on y-axis of graphs as the mean ± 95% confidence interval (CI) of nine randomly selected fields every three sections of different tumor areas. *P < 0.05, **P < 0.01, ***P < ALK, anaplastic lymphoma kinase.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2011 The American Society of Gene & Cell Therapy Terms and Conditions

6. Figure 5
Effect of ALK silencing by TL[ALK-siRNA] on neuroblastoma (NB) angiogenesis and metalloproteinases expression in vivo. Immunohistochemistry (IHC) performed on tumor sections as reported in Figure 4. Black bar: 100 µm. (a,b) IHC staining for matrix metalloproteinases (MMP)-2 and MMP-9 show highly significant expression inhibition of both matrix metalloproteinases in NB tumors derived from TL[ALK-siRNA]-treated mice versus control, TL[scr-siRNA]- and free ALK-siRNA-treated tumors. Similar inhibitory effects by TL[ALK-siRNA] treatment were observed for (c) VEGF and for (d) VEGF receptor-2 (VEGF-R2) expression. On the right side, percentage of positive cells for each specific marker are reported on y-axis of graphs as the mean ± 95% confidence interval (CI) of nine randomly selected fields every three sections of different tumor areas. *P < 0.05, **P < 0.01, ***P < ALK, anaplastic lymphoma kinase.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2011 The American Society of Gene & Cell Therapy Terms and Conditions

7. Figure 6
Tumor growth inhibition in vivo by TL[ALK-siRNA]. Survival curves of pseudometastatic neuroblastoma (NB)-bearing mice in response to treatment with liposomal small interfering RNA (siRNA) formulations and with free ALK-siRNA. (a) nu/nu (n = 7) and (b) SCID/bg (n = 8) mice were treated, two-times a week for five times, with L[siRNA] or TL[siRNA] formulations 24 hours after intravenous (i.v.) inoculation of HTLA-230 cells. (c) SCID/bg (n = 8) mice were treated, as above, after i.v. inoculation of LAN-5 cells. (d) SCID/bg (n = 7) mice were treated, as above, with Fab′-TL[siRNA] formulations after i.v. inoculation of HTLA-230 cells. (a) Treatment with TL[ALK-siRNA] showed a significant increased life span compared to control and to TL[scr-siRNA]-treated mice (P = and P = 0.004, respectively). (b) TL[ALK-siRNA] significantly increased life span as compared to mice treated with the other formulations, i.e., P = versus TL[scr-siRNA]-treated mice. (c) TL[ALK-siRNA] significantly increased life span as compared to mice treated with the other formulations, i.e., P = versus TL[scr-siRNA]-treated mice. (d) Fab′-TL[ALK-siRNA] significantly increased life span as compared to mice treated with the other formulations, i.e., P = versus Fab′-TL[scr-siRNA]-treated mice. ALK, anaplastic lymphoma kinase.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2011 The American Society of Gene & Cell Therapy Terms and Conditions

8. Figure 7
Immunostimulatory activity of the innate immune system by TL[ALK-siRNA]. The immunostimulatory activity was evaluated in terms of interferon-α (IFN-α) and tumor necrosis factor-α (TNF-α) production in human peripheral blood mononuclear cells (PBMC) supernatants, and of IFN-α, TNF-α, and interleukin 1-α (IL1-α) secretion in mouse serum. Both TL[ALK-siRNA] and naked ALK-siRNA at the indicated doses, which were the same used in the in vivo experiments, induced a very mild production of the cytokines analyzed in vitro (upper panel) and in vivo (lower panel) compared to stimulations induced by the TLR9-agonist CpG or the TLR4-agonist LPS. ALK, anaplastic lymphoma kinase; CpG, CpG-rich oligonucleotides; LPS, lipopolysaccharides.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2011 The American Society of Gene & Cell Therapy Terms and Conditions