Chiara Brignole

Targeting Liposomal Chemotherapy via Both Tumor Cell–Specific and Tumor Vasculature–Specific Ligands Potentiates Therapeutic Efficacy

Neuroblastoma, the most common solid tumor of infancy derived from the sympathetic nervous system, continues to present a formidable clinical challenge. Sterically stabilized immunoliposomes (SIL) have been shown to enhance the selective localization of entrapped drugs to solid tumors, with improvements in therapeutic indices. We showed that SIL loaded with doxorubicin (DXR) and targeted to the disialoganglioside receptor GD(2) [aGD(2)-SIL(DXR)] led to a selective inhibition of the metastatic growth of experimental models of human neuroblastoma. By coupling NGR peptides that target the angiogenic endothelial cell marker aminopeptidase N to the surface of DXR-loaded liposomes [NGR-SL(DXR)], we obtained tumor regression, pronounced destruction of the tumor vasculature, and prolonged survival of orthotopic neuroblastoma xenografts. Here, we showed good liposome stability, long circulation times, and enhanced time-dependent tumor accumulation of both the carrier and the drug. Antivascular effects against animal models of lung and ovarian cancer were shown for formulations of NGR-SL(DXR). In the chick embryo chorioallantoic assay, NGR-SL(DXR) substantially reduced the angiogenic potential of various neuroblastoma xenografts, with synergistic inhibition observed for the combination of NGR-SL(DXR) with aGD(2)-SIL(DXR). A significant improvement in antitumor effects was seen in neuroblastoma-bearing animal models when treated with the combined formulations compared with control mice or mice treated with either tumor- or vascular-targeted liposomal formulations, administered separately. The combined treatment resulted in a dramatic inhibition of tumor endothelial cell density. Long-term survivors were obtained only in animals treated with the combined tumor- and vascular-targeted formulations, confirming the pivotal role of combination therapies in treating aggressive metastatic neuroblastoma.

Macrophages are alternatively activated in patients with endometriosis and required for growth and vascularization of lesions in a mouse model of disease.

The mechanisms that sustain endometrial tissues at ectopic sites in patients with endometriosis are poorly understood. Various leukocytes, including macrophages, infiltrate endometriotic lesions. In this study, we depleted mouse macrophages by means of either clodronate liposomes or monoclonal antibodies before the injection of syngeneic endometrial tissue. In the absence of macrophages, tissue fragments adhered and implanted into the peritoneal wall, but endometriotic lesions failed to organize and develop. When we depleted macrophages after the establishment of endometriotic lesions, blood vessels failed to reach the inner layers of the lesions, which stopped growing. Macrophages from patients with endometriosis and experimental mice, but not nonendometriotic patients who underwent surgery for uterine leiomyomas or control mice, expressed markers of alternative activation. These markers included high levels of scavenger receptors, CD163 and CD206, which are involved in both the scavenging of hemoglobin with iron transfer into macrophages and the silent clearance of inflammatory molecules. Macrophages in both inflammatory liquid and ectopic lesions were equally polarized, suggesting a critical role of environmental cues in the peritoneal cavity. Adoptively transferred, alternatively activated macrophages dramatically enhanced endometriotic lesion growth in mice. Inflammatory macrophages effectively protected mice from endometriosis. Therefore, endogenous macrophages involved in tissue remodeling appear as players in the natural history of endometriosis, required for effective vascularization and ectopic lesion growth.

Expression and methylation of CASP8 in neuroblastoma: identification of a promoter region.

Neuroblastoma (NB) is a tumor of infancy that presents a high rate of spontaneous regression, a phenomenon that likely reflects the activation of an apoptotic and/or differentiation program. An attractive hypothesis suggested that the caspase-8 gene (CASP8), which encodes a key enzyme at the top of the apoptotic cascade, is an anti-oncogene that can be inactivated by methylation or deletion in MYCN-amplified neuroblastoma1. However, subsequent reports have not fully confirmed this model of NB development2, 3. To clarify this question, we have studied CASP8 expression in primary tumors and NB cell lines and its relationship to CpG methylation at the putative 5 regulatory region of the gene.

Enhanced Antitumor Efficacy of Clinical-Grade Vasculature-Targeted Liposomal Doxorubicin

Purpose:In vivo evaluation of good manufacturing practice-grade targeted liposomal doxorubicin (TVT-DOX), bound to a CD13 isoform expressed on the vasculature of solid tumors, in human tumor xenografts of neuroblastoma, ovarian cancer, and lung cancer. Experimental Design: Mice were implanted with lung, ovarian, or neuroblastoma tumor cells via the pulmonary, peritoneal, or orthotopic (adrenal gland) routes, respectively, and treated, at different days post inoculation, with multiple doses of doxorubicin, administered either free or encapsulated in untargeted liposomes (Caelyx) or in TVT-DOX. The effect of TVT-DOX treatment on tumor cell proliferation, viability, apoptosis, and angiogenesis was studied by immunohistochemical analyses of neoplastic tissues and using the chick embryo chorioallantoic membrane assay. Results: Compared with the three control groups (no doxorubicin, free doxorubicin, or Caelyx), statistically significant improvements in survival was seen in all three animal models following treatment with 5 mg/kg (maximum tolerated dose) of TVT-DOX, with long-term survivors occurring in the neuroblastoma group; increased survival was also seen at a dose of 1.7 mg/kg in mice bearing neuroblastoma or ovarian cancer. Minimal residual disease after surgical removal of neuroblastoma primary mass, and the enhanced response to TVT-DOX, was visualized and quantified by bioluminescence imaging and with magnetic resonance imaging. When treated with TVT-DOX, compared with Caelyx, all three tumor models, as assayed by immunohistochemistry and chorioallantoic membrane, showed statistically significant reductions in cell proliferation, blood vessel density, and microvessel area, showing increased cell apoptosis. Conclusion: TVT-DOX should be evaluated as a novel angiostatic strategy for adjuvant therapy of solid tumors.

Combined targeting of perivascular and endothelial tumor cells enhances anti-tumor efficacy of liposomal chemotherapy in neuroblastoma.

The therapeutic index of anti-cancer drugs is increased when encapsulating them in tumor-targeted liposomes. Liposome-entrapped doxorubicin (DXR), targeting the tumor vasculature marker, aminopeptidase N (APN), displayed enhanced anti-tumor effects and prolonged survival in human neuroblastoma (NB)-bearing mice. Here we exploited a peptide ligand of aminopeptidase A (APA), discovered by phage display technology for delivery of liposomal DXR to perivascular tumor cells. Immunohistochemistry, performed in NB-bearing mice, showed APA expression in the vascular wall of NB primary and metastatic lesions. APA-targeted peptides displayed specific binding to APA-transfected cells in vitro, and also accumulation in the tumor of NB-bearing mice. Consequently, novel, APA-targeted, DXR-liposomes were developed and in vivo proof-of-principle was established, alone and in combination with APN-targeted DXR-loaded liposomes, in NB-bearing mice. Mice receiving APA-targeted liposomal DXR exhibited an increased life span in comparison to control mice, but to a lesser extent relative to that in mice treated with APN-targeted formulation, moreover the greatest increase in TUNEL-positive tumor cells was observed in animals treated with APN-targeted formulations. Mice treated with a combination of APA- and APN-targeted, liposomal DXR had a significant increase in life span compared to each treatment administered separately. There was a significant increase in the level of apoptosis in the tumors of mice on the combination therapy, and a pronounced destruction of the tumor vasculature with nearly total ablation of endothelial cells and pericytes. The availability of novel ligands binding to additional tumor vasculature-associated antigens will allow the design of sophisticated combinations of ligand-targeted liposomal anti-cancer drugs.

Angiogenesis in Neuroblastoma

Abstract: Angiogenesis is a biological process by which new capillaries are formed from preexisting vessels. It occurs in physiological and pathological conditions, such as tumors, where a specific turning point is the transition from the avascular to the vascular phase. Tumor angiogenesis depends mainly on the release by neoplastic cells of growth factors specific for endothelial cells able to stimulate the growth of the hosts blood vessels. In neuroblastoma, the most common extracranial solid tumor of infancy and childhood, angiogenesis also appears to play an important role in determining tumor phenotype. The nature of the angiogenic balance in neuroblastoma is complex, and a spectrum of angiogenesis stimulators, such as vascular endothelial growth factor (VEGF) and fibroblast growth factor‐2 (FGF‐2), and inhibitors, such as tissue inhibitors of matrix metalloproteinases (MMPs), have been detected in neuroblastoma tumors. Moreover, an increased production of MMP‐2 and ‐9 has been also observed in advanced stages of tumor, favoring degradation of extracellular matrix and enhancing tumor dissemination. High tumor vascularity is correlated with widely disseminated disease, MYCN amplification, unfavorable histology, and poor outcome. In contrast, low tumor vascularity is associated with prognostically favorable features, such as a localized disease and favorable histology. It is becoming increasingly evident that agents that interfere with blood vessel formation also block tumor progression. Preclinical studies suggest that antiangiogenic strategies may be effective in the treatment of neuroblastoma. A major goal is the determination of whether inhibition of angiogenesis is a realistic way of inhibiting tumor cell dissemination and formation of metastasis in neuroblastoma.

Combined Therapeutic Effects of Vinblastine and Rapamycin on Human Neuroblastoma Growth, Apoptosis, and Angiogenesis

Purpose: Vinblastine and rapamycin displayed synergistic inhibition of human neuroblastoma-related angiogenesis. Here, we studied the antitumor activity of vinblastine and rapamycin against human neuroblastoma. Experimental Design: Cell proliferation, cell cycle progression, and apoptosis were evaluated by measuring 3H-thymidine incorporation, bromodeoxyuridine uptake, and phosphatidylserine exposure, respectively. The in vivo sensitivity of neuroblastoma cells to vinblastine and rapamycin was determined in orthotopic neuroblastoma-engrafted mice. Angiogenesis was assessed by the chick embryo chorioallantoic membrane assay. Results: Each compound alone was able to induce a dose-dependent significant inhibition of cell proliferation, with a dramatically enhanced antiproliferative effect for the drugs used in combination. A marked G2-M cell cycle arrest with a nearly complete depletion of S phase was associated. The combined treatment triggered an increased apoptosis compared with either drug tested alone. A significant inhibition of tumor growth and microvessel area was obtained in neuroblastoma-bearing mice when treated with vinblastine or rapamycin alone, and a more dramatic effect with the combined treatment, compared with control mice. The therapeutic effectiveness, expressed as increased life span, was statistically improved by the combined therapy, compared with mice treated with either drug tested separately. Histologic evaluation of primary tumors showed that the combined treatment inhibited proliferation and angiogenesis and induced apoptosis. Combined treatment of neuroblastoma cells and neuroblastoma-bearing mice with vinblastine and rapamycin induced the down-modulation of both vascular endothelial growth factor production and vascular endothelial growth factor receptor 2 expression. In the chorioallantoic membrane assay, angiogenesis induced by human neuroblastoma biopsy specimens was significantly inhibited by vinblastine and rapamycin. Conclusions: These results may be relevant to design new therapeutic strategies against neuroblastoma.

Targeted delivery system for antisense oligonucleotides: a novel experimental strategy for neuroblastoma treatment

Neuroblastoma (NB) is the most common neuroectoderma derived solid tumour of paediatric age. Since conventional treatments are often inefficient, novel therapeutic interventions are required. Among these, the use of antisense oligonucleotides (asODNs) as therapeutic antineoplastic agents has been recently investigated. Oligonucleotide in vivo applicability is impaired from their high sensitivity to cellular nuclease degradation. Encapsulating them within liposomes could nevertheless increase their stability. C-myb gene expression has been reported in several solid tumours of different embryonic origin, including NB, where it is linked to cell proliferation and/or differentiation. We performed a new technique to encapsulate c-myb antisense oligonucleotides within lipid particles. Liposomes resulting from this technique were called coated cationic liposomes (CCLs), since they were made up of a central core of a cationic phospholipid bound to myb-asODNs, and an outer shell of neutral lipids. A monoclonal antibody (mAb) specific for the neuroectoderma antigen disialoganglioside GD(2), has been covalently coupled to their external surface. The resulting anti-GD(2)-targeted CCLs showed high loading efficiency for the asODNs, small particle size and good stability. In vitro, they were able to deliver myb-asODNs selectively to GD(2)-positive NB cell lines more efficiently than non-targeted liposomes or free asODNs. Consequently, targeted formulations showed greater inhibition of cell proliferation than non-targeted formulations or free asODNs. Furthermore, we demonstrated that the inhibition of cell proliferation was dependent on the down-modulation of c-myb protein expression. Pharmacokinetic studies showed that these targeted liposomal formulations were long circulating in blood. Biodistribution studies presented differences between the free and the encapsulated myb-as ODN profiles, as well. While free myb-as ODNs are widely distributed (mainly liver, kidney and spleen) even after 30 min post-injection, myb-as ODN entrapped into CCL or anti-GD(2)-CCL presents only an accumulation in the spleen after 24 h. Future studies will be performed to evaluate the antitumour efficacy of the above formulations in animal models.

Therapeutic Targeting of TLR9 Inhibits Cell Growth and Induces Apoptosis in Neuroblastoma

The Toll-like receptor 9 (TLR9) evolved to cope with pathogens, but it is expressed in a variety of tumors for reasons that are unclear. In this study, we report that neuroblastoma (NB) cells express functional TLR9. Liposome-complexed CpG oligonucleotides inhibited the proliferation of TLR9-expressing NB cells and induced caspase-dependent apoptotic cell death. Inhibitory oligonucleotides (iODNs) abrogated these effects. RNA interference reduced TLR9 expression but not to the level where functional responses to CpG were abolished. Compared with free CpG, liposomal formulations of NB-targeted CpG (TL-CpG) significantly prolonged the survival of mice bearing NB tumor xenografts. While CpG alone lacked antitumor efficacy in NOD/SCID/IL2rg(-/-) mice, TL-CpG retained significant efficacy related to direct effects on tumor cells. TLR9 expression in primary human NB specimens was found to correlate inversely with disease stage. Our findings establish functional expression of TLR9 in NB and suggest that TLR9 may represent a novel theranostic target in this disease.

Development of Fab′ fragments of anti-GD2 immunoliposomes entrapping doxorubicin for experimental therapy of human neuroblastoma

Neuroblastoma (NB) is the most common extra-cranial solid tumor in children. Since intensive therapeutic intervention does not prolong the overall disease-free survival rate for this tumor, novel therapeutic strategies are required. NB tumor, but not normal tissues, over-express the disialoganglioside (GD(2)) at the cell surface. In this study we developed a novel immunoliposomal formulation by covalently coupled Fab fragments of the monoclonal antibody anti-GD(2) to Stealth liposomes (Fab-SIL). In vitro experiments showed specific, competitive binding to, and uptake by various NB cell lines. Moreover, doxorubicin-loaded immunoliposomes (Fab-SIL[DXR]) presented increased selectivity and efficacy in inhibiting NB cell proliferation compared to free drug and non-targeted liposomes (SL[DXR]). The in vivo cytotoxic effectiveness of different liposomal formulations encapsulating DXR was tested against an experimental metastatic model of human NB in nude mice. Long term survivors were obtained in mice treated with Fab-SIL[DXR], but not in untreated animals or those treated with free anti-GD(2) Fab fragments, Fab-SIL (no drug), free-DXR or SL[DXR] (P<0.0001). Fab-SIL[DXR] prevented the establishment and the metastatic growth of the tumor cells in all organs examined. In conclusion, Fab-SIL[DXR] formulations should receive clinical evaluation as adjuvant therapy of neuroblastoma.