The irony of highly-effective bacterial therapy of a patient-derived orthotopic xenograft (PDOX) model of Ewing's sarcoma,which was blocked by Ewing himself 80 years ago

William B. Coley developed bacterial therapy of cancer more than 100 years ago and had clinical success. James Ewing, a very famous cancer pathologist for whom the Ewing sarcoma is named, was Coley's boss at Memorial Hospital in New York and terminated Coley's bacterial therapy of cancer. A tumor from a patient with soft-tissue Ewing's sarcoma, who failed doxorubicin (DOX) therapy, was previously implanted in nude mice to establish a patient-derived orthotopic xenograft (PDOX) model. In the present study, the Ewing's sarcoma PDOX was treated with tumor-targeting S. typhimurium A1-R expressing green fluorescent (GFP), alone and in combination with DOX. S. typhimurium A1-R-GFP was detected in the tumors after intratumor (i.t.) or intravenous (i.v.) injection. The combination of S. typhimurium A1-R and DOX significantly reduced tumor weight (37.8 ± 15.6 mg) compared to the untreated control (73.8 ± 10.1 mg, P < 0.01). S. typhimurium A1-R monotherapy-treated tumors tended to be smaller (50.9 ± 17.8 mg, P = 0.051). DOX monotherapy did not show efficacy (66.3 ± 26.4 mg, P = 0.82), as was the case with the patient. The PDOX model faithfully replicated the DOX resistance the Ewing's sarcoma had in the patient. S. typhimurium A1-R converted the Ewing's sarcoma from DOX resistant to sensitive. One can only wonder how bacterial therapy and immunotherapy of cancer would have developed over the past 80 years if Ewing did not stop Coley.     

High ALDH activity identifies chemotherapy-resistant Ewing's sarcoma stem cells that retain sensitivity to EWS-FLI1 inhibition

Background

Cancer stem cells are a chemotherapy-resistant population capable of self-renewal and of regenerating the bulk tumor, thereby causing relapse and patient death. Ewing''s sarcoma, the second most common form of bone tumor in adolescents and young adults, follows a clinical pattern consistent with the Cancer Stem Cell model – remission is easily achieved, even for patients with metastatic disease, but relapse remains frequent and is usually fatal.

Methodology/Principal Findings

We have isolated a subpopulation of Ewing''s sarcoma cells, from both human cell lines and human xenografts grown in immune deficient mice, which express high aldehyde dehydrogenase (ALDH^high) activity and are enriched for clonogenicity, sphere-formation, and tumor initiation. The ALDH^high cells are resistant to chemotherapy in vitro, but this can be overcome by the ATP binding cassette transport protein inhibitor, verapamil. Importantly, these cells are not resistant to YK-4-279, a small molecule inhibitor of EWS-FLI1 that is selectively toxic to Ewing''s sarcoma cells both in vitro and in vivo.

Conclusions/Significance

Ewing''s sarcoma contains an ALDH^high stem-like population of chemotherapy-resistant cells that retain sensitivity to EWS-FLI1 inhibition. Inhibiting the EWS-FLI1 oncoprotein may prove to be an effective means of improving patient outcomes by targeting Ewing''s sarcoma stem cells that survive standard chemotherapy.     

Eribulin regresses a doxorubicin‐resistant Ewing's sarcoma with a FUS‐ERG fusion and CDKN2A‐deletion in a patient‐derived orthotopic xenograft (PDOX) nude mouse model

Ewing's sarcoma is a recalcitrant tumor greatly in need of more effective therapy. The aim of this study was to determine the efficacy of eribulin on a doxorubicin (DOX)‐resistant Ewing's sarcoma patient derived orthotopic xenograft (PDOX) model. The Ewing's sarcoma PDOX model was previously established in the right chest wall of nude mice from tumor resected form the patient's right chest wall. In the previous study, the Ewing's sarcoma PDOX was resistant to doxorubicin (DOX) and sensitive to palbociclib and linsitinib. In the present study, the PDOX models were randomized into three groups when the tumor volume reached 60 mm³: G1, untreated control (n = 6); G2, DOX treated (n = 6), intraperitoneal (i.p.) injection, weekly, for 2 weeks); G3, Eribulin treated (n = 6, intravenous (i.v.) injection, weekly for 2 weeks). All mice were sacrificed on day 15. Changes in body weight and tumor volume were assessed two times per week. Tumor weight was measured after sacrifice. DOX did not suppress tumor growth compared to the control group (P = 0.589), consistent with the previous results in the patient and PDOX. Eribulin regressed tumor size significantly compared to G1 and G2 (P = 0.006, P = 0.017) respectively. No significant difference was observed in body weight among any group. Our results demonstrate that eribulin is a promising novel therapeutic agent for Ewing's sarcoma.     

Targeted morphoproteomic profiling of Ewing's sarcoma treated with insulin-like growth factor 1 receptor (IGF1R) inhibitors: response/resistance signatures

Background

Insulin-like growth factor 1 receptor (IGF1R) targeted therapies have resulted in responses in a small number of patients with advanced metastatic Ewing''s sarcoma. We performed morphoproteomic profiling to better understand response/resistance mechanisms of Ewing''s sarcoma to IGF1R inhibitor-based therapy.

Methodology/Principal Findings

This pilot study assessed two patients with advanced Ewing''s sarcoma treated with IGF1R antibody alone followed by combined IGF1R inhibitor plus mammalian target of rapamycin (mTOR) inhibitor treatment once resistance to single-agent IGF1R inhibitor developed. Immunohistochemical probes were applied to detect p-mTOR (Ser2448), p-Akt (Ser473), p-ERK1/2 (Thr202/Tyr204), nestin, and p-STAT3 (Tyr 705) in the original and recurrent tumor. The initial remarkable radiographic responses to IGF1R-antibody therapy was followed by resistance and then response to combined IGF1R plus mTOR inhibitor therapy in both patients, and then resistance to the combination regimen in one patient. In patient 1, upregulation of p-Akt and p-mTOR in the tumor that relapsed after initial response to IGF1R antibody might explain the resistance that developed, and the subsequent response to combined IGF1R plus mTOR inhibitor therapy. In patient 2, upregulation of mTOR was seen in the primary tumor, perhaps explaining the initial response to the IGF1R and mTOR inhibitor combination, while the resistant tumor that emerged showed activation of the ERK pathway as well.

Conclusion/Significance

Morphoproteomic analysis revealed that the mTOR pathway was activated in these two patients with advanced Ewing''s sarcoma who showed response to combined IGF1R and mTOR inhibition, and the ERK pathway in the patient in whom resistance to this combination emerged. Our pilot results suggests that morphoproteomic assessment of signaling pathway activation in Ewing''s sarcoma merits further investigation as a guide to understanding response and resistance signatures.     

Sarcome d’Ewing agressif apparaissant comme une lésion froide sur la scintigraphie osseuse

Ewing's sarcoma classically presents as a hot spot on bone scan as a result of increased vascularity of the tumor and new bone formation.PurposeWe report and analyze an uncommon pattern of a “cold” lesion in Ewing's sarcoma on bone scan and its physiopathologic significance.Case reportA 15-year-old boy complaining of thigh pain. CT scan evocated Ewing's sarcoma or osteitis. MRI evocated chronic osteitis. Scintigraphy showed a fairly intense and heterogeneous uptake on the femoral lesion and no abnormal uptake elsewhere. Biopsy showed none pathologic pattern. Three months later, a second biopsy concluded to Ewing's sarcoma. Bone scan showed a larger lesion with peripheral intense uptake centered by enlarged “cold” area in the left femoral diaphysis and no evident bone metastasis. The patient underwent chemotherapy and surgery. Three months later, bone scan showed extensive skeletal metastasis.ConclusionEwing's sarcoma appears usually as an intense lesion on bone scan. Nevertheless, decreased radiopharmaceutical uptake or “cold” lesion may be seen in aggressive Ewing's sarcoma with lytic tumor, growth of which is very rapid and bony reaction is minimal.     

MiR‐125b inhibits cell biological progression of Ewing's sarcoma by suppressing the PI3K/Akt signalling pathway

Objectives: Increasing evidence has suggested the close relationship between microRNAs (miRNAs) dysregulation and the carcinogenesis of Ewing's sarcoma (ES), among of which miR‐125b has been reported to be decreased in ES tissues recently. Strikingly, ectopic expression of miR‐125b could suppress cell proliferation of ES cell line A673, suggesting the tumor suppressor role of miR‐125b in ES. However, the other accurate mechanistic functions and relative molecule mechanisms are largely unknown. Materials and Methods: Herein, we completed a series of experiments to investigate the role of miR‐125b in Ewing's sarcoma. We restored the expression of miR‐125b in ES cell line A673 through transfection with miR‐125b mimics. To further understand the role of miR‐125b in ES, we detected the effects of miR‐125b on the cell proliferation, migration and invasion, cell cycle as well as cell apoptosis. Results: We found that restored expression of miR‐125b in ES cell line A673 inhibited cell proliferation, migration and invasion, arrested cell cycle progression, and induced cell apoptosis. Moreover, bioinformatic prediction suggested the oncogene, phosphoinositide‐3‐kinase catalytic subunit delta (PIK3CD), was a target gene of miR‐125b in ES cells. Further quantitative RT‐PCR and western blot assays identified over‐expression of miR‐125b suppressed the expression of PIK3CD mRNA and protein. PIK3CD participates in regulating the PI3K signaling pathway, which has been reported to play an important role in the development of ES. Suppression of PIK3CD down‐regulated the expression of phospho‐AKT and phospho‐mTOR proteins and inhibited the biologic progression of A673 cells. Conclusions: Collectively, these data suggest that miR‐125b functions as a tumor suppressor by targeting the PI3K/Akt/mTOR signaling pathway, and may provide potential therapy strategy for ES patients by targeting miRNA expression.     

Construction of a prognostic signature in Ewing's sarcoma: Based on metabolism-related genes

ObjectiveBy combining the expression profiles of metabolism-related genes (MRGS) with clinical information, the expression quantities of MRGS and the influence on development and prognosis were systematically analyzed, so as to provide a theoretical basis for the clinical study on the prognosis of Ewing's sarcoma.MethodsMRGs expression profiles of 64 patients with Ewing's sarcoma were obtained from GEO dataset. Univariate Cox regression analysis was used to identify metabolization-related differentially expressed genes (DEGs) related with prognosis in Ewing's sarcoma patients. Then, multivariate Cox analysis was used to calculate novel prognostic markers based on metabolism-related DEGs. Besides, We validate the model using ICGC datasets. Finally, the new prognostic index was verified on the basis of the prognostic models.ResultsMultivariate Cox regression analysis identified 74 metabolization-related DEGs, 25 of which were associated with Ewing's sarcoma patients' overall survival. Subsequently, we used 25 DEGs to construct metabolism-related prognostic signature for patients with Ewing's sarcoma. Based on the 18 DEGs regression coefficient, we propose the formula of each patient's risk score, and then divided the patients into high-risk group and low-risk group. The results indicated that the survival rate and survival time were higher in the low-risk group and lower in the high-risk group. Multivariate Cox analysis showed that risk score index was an independent prognostic factor for Ewing's sarcoma.ConclusionThe experimental results suggest that the 18 metabolism-related DEGs marker may be effective in predicting the prognosis of Ewing's sarcoma to some extent, helping to individualize treatment of patients at different risks.     

Construction of a prognostic signature in Ewing's sarcoma: Based on metabolism-related genes

ObjectiveBy combining the expression profiles of metabolism-related genes (MRGS) with clinical information, the expression quantities of MRGS and the influence on development and prognosis were systematically analyzed, so as to provide a theoretical basis for the clinical study on the prognosis of Ewing's sarcoma.MethodsMRGs expression profiles of 64 patients with Ewing's sarcoma were obtained from GEO dataset. Univariate Cox regression analysis was used to identify metabolization-related differentially expressed genes (DEGs) related with prognosis in Ewing's sarcoma patients. Then, multivariate Cox analysis was used to calculate novel prognostic markers based on metabolism-related DEGs. Besides, We validate the model using ICGC datasets. Finally, the new prognostic index was verified on the basis of the prognostic models.ResultsMultivariate Cox regression analysis identified 74 metabolization-related DEGs, 25 of which were associated with Ewing's sarcoma patients' overall survival. Subsequently, we used 25 DEGs to construct metabolism-related prognostic signature for patients with Ewing's sarcoma. Based on the 18 DEGs regression coefficient, we propose the formula of each patient's risk score, and then divided the patients into high-risk group and low-risk group. The results indicated that the survival rate and survival time were higher in the low-risk group and lower in the high-risk group. Multivariate Cox analysis showed that risk score index was an independent prognostic factor for Ewing's sarcoma.ConclusionThe experimental results suggest that the 18 metabolism-related DEGs marker may be effective in predicting the prognosis of Ewing's sarcoma to some extent, helping to individualize treatment of patients at different risks.     

The Ewing's sarcoma fusion protein,EWS‐FLI,binds Runx2 and blocks osteoblast differentiation

Ewing's sarcomas are highly aggressive round cell tumors of bone and soft tissues that afflict children and young adults. The majority of these tumors harbor the t(11;22) translocation and express the fusion protein EWS‐FLI. Modern molecular profiling experiments indicate that Ewing's tumors originate from mesenchymal precursors in young individuals. EWS‐FLI alters the morphology of mesenchymal cells and prevents lineage specification; however, the molecular mechanisms for differentiation arrest are unclear. We recently showed that EWS‐FLI binds Runx2, a master regulator of osteoblast differentiation. In this report, we demonstrate that FLI sequences within EWS‐FLI are responsible for interactions with Runx2. EWS‐FLI blocks the expression of osteoblastic genes in a multipotent progenitor cell line that requires Runx2 to integrate bone morphogenic protein (Bmp)2 signaling while increasing proliferation and altering cell morphology. These results demonstrate that EWS‐FLI blocks the ability of Runx2 to induce osteoblast specification of a mesenchymal progenitor cell. Disrupting interactions between Runx2 and EWS‐FLI1 may promote differentiation of the tumor cell. J. Cell. Biochem. 111: 933–943, 2010. © 2010 Wiley‐Liss, Inc.     

Hyaluronic acid complexed to biodegradable poly L‐arginine for targeted delivery of siRNAs

Background

Small interfering RNA (siRNA) has been recognized as a new therapeutic drug to treat various diseases by inhibition of oncogene or viral gene expression. Because hyaluronic acid (HA) has been described as a biocompatible biomaterial, we tested the nanoparticles formed by electrostatic complexation of negatively‐charged HA and cationic poly L ‐arginine (PLR) for siRNA delivery systems.

Methods

Different electrostatic complexes of HA and PLR (HPs) were formulated: HP101 with 50% (w/w) HA and HP110 with 9% (w/w) HA.

Results

Gel retardation assays showed that HP101 and HP110 could form complexes with siRNAs. The diameters of these complexes were less than 200 nm. Cellular delivery efficiency of siRNAs by HPs depended on cell surface CD44 density. The HP‐mediated delivery of siRNAs was highest in WM266.4 cells followed by B16F10 cells and COS‐7 cells, in parallel with CD44 surface densities of these cell lines. TC₅₀ values (i.e. the HP concentrations at which 50% of cells were viable after treatment) were used as indicators of cytotoxicity. HP101 showed TC₅₀ values that were 2‐fold and 23‐fold higher than those of HP110 and PLR, respectively. After delivery into cells, siRNA exerted target‐specific RNA interference effects on mRNA and protein levels. Three days after treatment of red fluorescent protein (RFP)‐expressing B16F10 cells with RFP‐specific siRNA complexed to HP101, cellular fluorescence signals were reduced. Intratumoral administration of RFP‐specific siRNA via HP101 delivery significantly reduced the expression of RFP in tumor tissues.

Conclusions

HP101 may function as a biocompatible polymeric carrier of siRNAs and have possible application to localized siRNA delivery in vivo. Copyright © 2009 John Wiley & Sons, Ltd.     

Polyethylenimine/small interfering RNA‐mediated knockdown of vascular endothelial growth factor in vivo exerts anti‐tumor effects synergistically with Bevacizumab

Background

RNA interference is a powerful method for the knockdown of pathologically relevant genes. The in vivo delivery of siRNAs, preferably through systemic, nonviral administration, poses the major challenge in the therapeutic application of RNAi. Small interfering RNA (siRNA) complexation with polyethylenimines (PEI) may represent a promising strategy for siRNA‐based therapies and, recently, the novel branched PEI F25‐LMW has been introduced in vitro. Vascular endothelial growth factor (VEGF) is frequently overexpressed in tumors and promotes tumor growth, angiogenesis and metastasis and thus represents an attractive target gene in tumor therapy.

Methods

In subcutaneous tumor xenograft mouse models, we established the therapeutic efficacy and safety of PEI F25‐LMW/siRNA‐mediated knockdown of VEGF. In biodistribution and siRNA quantification studies, we optimized administration strategies and, employing chemically modified siRNAs, compared the anti‐tumorigenic efficacies of: (i) PEI/siRNA‐mediated VEGF targeting; (ii) treatment with the monoclonal anti‐VEGF antibody Bevacizumab (Avastin®); and (iii) a combination of both.

Results

Efficient siRNA delivery is observed upon systemic administration, with the biodistribution being dependent on the mode of injection. Toxicity studies reveal no hepatotoxicity, proinflammatory cytokine induction or other side‐effects of PEI F25‐LMW/siRNA complexes or polyethylenimine, and tumor analyses show efficient VEGF knockdown upon siRNA delivery, leading to reduced tumor cell proliferation and angiogenesis. The determination of anti‐tumor effects reveals that, in pancreas carcinoma xenografts, single treatment with PEI/siRNA complexes or Bevacizumab is already highly efficacious, whereas, in prostate carcinoma, synergistic effects of both treatments are observed.

Conclusions

PEI F25‐LMW/siRNA complexes, which can be stored frozen as opposed to many other carriers, represent an efficient, safe and promising avenue in anti‐tumor therapy, and PEI/siRNA‐mediated, therapeutic VEGF knockdown exerts anti‐tumor effects. Copyright © 2010 John Wiley & Sons, Ltd.     

Expression of PAX3 in Ewing's Sarcoma Family of Tumors

The Ewing's sarcoma family of tumors (ESFT) is the second most common pediatric malignancy originating in the bone and is characterized by the t(11; 22) translocation. PAX3, a member of the paired box family of genes, is expressed during embryonal development of neural crest cells and is involved in the t(2; 13) translocation found in alveolar rhabdomyosarcoma. Since ESFTs are believed to be derived from neural crest tissue, we screened a series of Ewing's sarcoma and peripheral neuroectodermal tumor cell lines and tumor specimens for expression of PAX3. We found expression of PAX3 in most, but not all, of the specimens analyzed, including cell lines and patient material.     

Ewing's sarcoma: Analysis of single nucleotide polymorphism in the EWS gene

We aimed to investigate single nucleotide polymorphisms (SNPs) in the EWS gene breaking region in order to analyze Ewing's sarcoma susceptibility. The SNPs were investigated in a healthy subject population and in Ewing's sarcoma patients from Southern Brazil. Genotyping was performed by TaqMan® assay for allelic discrimination using Real-Time PCR. The analysis of incidence of SNPs or different SNP-arrangements revealed a higher presence of homozygote TT-rs4820804 in Ewing's sarcoma patients (p = 0.02; Chi Square Test). About 300 bp from the rs4820804 SNP lies a palindromic hexamer (5′-GCTAGC-3′) and three nucleotides (GTC), which were previously identified to be in close vicinity of the breakpoint junction in both EWS and FLI1 genes. This DNA segment surrounding the rs4820804 SNP is likely to indicate a breakpoint region. If the T-rs4820804 allele predisposes a DNA fragment to breakage, homozygotes (TT-rs4820804) would have double the chance of having a chromosome break, increasing the chances for a translocation to occur. In conclusion, the TT-rs4820804 EWS genotype can be associated with Ewing's sarcoma and the SNP rs4820804 can be a candidate marker to understand Ewing's sarcoma susceptibility.     

Inhibition of the growth of human hepatoma cell line bothin vitro andin vivo by transducing CKI genep21 WAF-1 with GE7 targeting gene delivery system

The EGF receptor-mediated targeting gene delivery system GE7 was used to transduce exogenous genepCEP-p21 ^WAF-1 into human hepatocellular carcinoma cell bothin vitro andin vivo. Afterin vitro transduction of the exogenous gene, the growth of the cell lines SMMC-7721 and BEL-7402 was significantly inhibited compared with the control. On day 8 the inhibition rates of the above cell lines reached 56.0% and 66.7%, respectively. Thein vivo experiment showed that the growth of human hepatoma transplanted in nude mice injected with GE7 gene delivery system subcutaneously once a week for 3 weeks was remarkably inhibited compared with that of untransfected control. The average tumor weight of the experiment group was (0.083 ±0.043) g, while that of the control group was (0.281 ±0.173) g. The difference is significant (P<0.05). It was indicated that GE7 gene delivery system could efficiently transduce exogenous genepCEP-p21 ^WAF-1 into hepatoma cell with high EGF receptor expression, and inhibit the cell growth with high efficacy bothin vivo andin vitro.     

Delivery of small interfering RNA with a synthetic collagen poly(Pro‐Hyp‐Gly) for gene silencing in vitro and in vivo

Silencing gene expression by small interfering RNAs (siRNAs) has become a powerful tool for the genetic analysis of many animals. However, the rapid degradation of siRNA and the limited duration of its action in vivo have called for an efficient delivery technology. Here, we describe that siRNA complexed with a synthetic collagen poly(Pro‐Hyp‐Gly) (SYCOL) is resistant to nucleases and is efficiently transferred into cells in vitro and in vivo, thereby allowing long‐term gene silencing in vivo. We found that the SYCOL‐mediated local application of siRNA targeting myostatin, coding a negative regulator of skeletal muscle growth, in mouse skeletal muscles, caused a marked increase in the muscle mass within a few weeks after application. Furthermore, in vivo administration of an anti‐luciferase siRNA/SYCOL complex partially reduced luciferase expression in xenografted tumors in vivo. These results indicate a SYCOL‐based non‐viral delivery method could be a reliable simple approach to knockdown gene expression by RNAi in vivo as well as in vitro.     

In‐vivo Tumor detection using diffusion reflection measurements of targeted gold nanorods – a quantitative study

The ability to quantitatively and non‐invasively detect nanoparticles has important implications on their development as an in‐vivo cancer diagnostic tool. The Diffusion Reflection (DR) method is a simple, non‐invasive imaging technique which has been proven useful for the investigation of tissue's optical parameters. In this study, Monte Carlo (MC) simulations, tissue‐like phantom experiments and in‐vivo measurements of the reflected light intensity from tumor bearing mice are presented. Following intravenous injection of antibody conjugated poly (ethylene glycol)‐coated (PEGylated) gold nanorods (GNR) to tumor‐bearing mice, accumulation of GNR in the tumor was clearly detected by the DR profile of the tumor. The ability of DR measurements to quantitate in‐vivo the concentration of the GNR in the tumor was demonstrated and validated with Flame Atomic Absorption spectroscopy results. With GNR as absorbing contrast agents, DR has important potential applications in the image guided therapy of superficial tumors such as head and neck cancer, breast cancer and melanoma. (© 2012 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Molecular photoacoustic tomography of breast cancer using receptor targeted magnetic iron oxide nanoparticles as contrast agents

In this report, we present a breast imaging technique combining high‐resolution near‐infrared (NIR) light induced photoacoustic tomography (PAT) with NIR dye‐labeled amino‐terminal fragments of urokinase plasminogen activator receptor (uPAR) targeted magnetic iron oxide nanoparticles (NIR830‐ATF‐IONP) for breast cancer imaging using an orthotopic mouse mammary tumor model. We show that accumulation of the targeted nanoparticles in the tumor led to photoacoustic contrast enhancement due to the high absorption of iron oxide nanoparticles (IONP). NIR fluorescence images were used to validate specific delivery of NIR830‐ATF‐IONP to mouse mammary tumors. We found that systemic delivery of the targeted IONP produced 4‐ and 10‐fold enhancement in photoacoustic signals in the tumor, compared to the tumor of the mice that received non‐targeted IONP or control mice. The use of targeted nanoparticles allowed imaging of tumors located as deep as 3.1 cm beneath the normal tissues. Our study indicates the potential of the combination of photoacoustic tomography and receptor‐targeted NIR830‐ATF‐IONP as a clinical tool that can provide improved specificity and sensitivity for breast cancer detection. (© 2014 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Cancer siRNA therapy by tumor selective delivery with ligand-targeted sterically stabilized nanoparticle

Potent sequence selective gene inhibition by siRNA ‘targeted’ therapeutics promises the ultimate level of specificity, but siRNA therapeutics is hindered by poor intracellular uptake, limited blood stability and non-specific immune stimulation. To address these problems, ligand-targeted, sterically stabilized nanoparticles have been adapted for siRNA. Self-assembling nanoparticles with siRNA were constructed with polyethyleneimine (PEI) that is PEGylated with an Arg-Gly-Asp (RGD) peptide ligand attached at the distal end of the polyethylene glycol (PEG), as a means to target tumor neovasculature expressing integrins and used to deliver siRNA inhibiting vascular endothelial growth factor receptor-2 (VEGF R2) expression and thereby tumor angiogenesis. Cell delivery and activity of PEGylated PEI was found to be siRNA sequence specific and depend on the presence of peptide ligand and could be competed by free peptide. Intravenous administration into tumor-bearing mice gave selective tumor uptake, siRNA sequence-specific inhibition of protein expression within the tumor and inhibition of both tumor angiogenesis and growth rate. The results suggest achievement of two levels of targeting: tumor tissue selective delivery via the nanoparticle ligand and gene pathway selectivity via the siRNA oligonucleotide. This opens the door for better targeted therapeutics with both tissue and gene selectivity, also to improve targeted therapies with less than ideal therapeutic targets.     

An RGD-Modified MRI-Visible Polymeric Vector for Targeted siRNA Delivery to Hepatocellular Carcinoma in Nude Mice

RNA interference (RNAi) has significant therapeutic promise for the genetic treatment of hepatocellular carcinoma (HCC). Targeted vectors are able to deliver small interfering RNA (siRNA) into HCC cells with high transfection efficiency and stability. The tripeptide arginine glycine aspartic acid (RGD)-modified non-viral vector, polyethylene glycol-grafted polyethylenimine functionalized with superparamagnetic iron oxide nanoparticles (RGD-PEG-g-PEI-SPION), was constructed as a magnetic resonance imaging (MRI)-visible nanocarrier for the delivery of Survivin siRNA targeting the human HCC cell line Bel-7402. The biophysical characterization of the RGD-PEG-g-PEI-SPION was performed. The RGD-modified complexes exhibited a higher transfection efficiency in transferring Survivin siRNA into Bel-7402 cells compared with a non-targeted delivery system, which resulted in more significant gene suppression at both the Survivin mRNA and protein expression levels. Then, the level of caspase-3 activation was significantly elevated, and a remarkable level of tumor cell apoptosis was induced. As a result, the tumor growth in the nude mice Bel-7402 hepatoma model was significantly inhibited. The targeting ability of the RGD-PEG-g-PEI-SPION was successfully imaged by MRI scans performed in vitro and in vivo. Our results strongly indicated that the RGD-PEG-g-PEI-SPION can potentially be used as a targeted non-viral vector for altering gene expression in the treatment of hepatocellular carcinoma and for detecting the tumor in vivo as an effective MRI probe.