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Supplementary Figure 1 from Therapeutic Targeting of TLR9 Inhibits Cell Growth and Induces Apoptosis in Neuroblastoma

Objects touched by patients and healthcare workers in hospitals may harbor pathogens, including multi-drug resistant (MDR) staphylococci, enterococci (VRE), Escherichia coli, Acinetobacter, and Pseudomonas species. Medical devices contaminated by these pathogens may also act as a source of severe and difficult-to-treat human infections, thus becoming a critical public health concern requiring urgent resolutions. To this end, we recently reported the bactericidal effects of a cationic copolymer (CP1). Here, aiming at developing a bactericidal formulation possibly to be used either for surfaces disinfection or to treat skin infections, CP1 was formulated as a hydrogel (CP1_1.1-Hgel). Importantly, even if not cross-linked, CP1 formed the gel upon simple dispersion in water, without requiring gelling agents or other additives which could be skin-incompatible or interfere with CP1 bactericidal effects in possible future topical applications. CP1_1.1-Hgel was characterized by attenuated-total-reflectance Fourier transform infrared (ATR-FTIR) and UV-Vis spectroscopy, as well as optic and scanning electron microscopy (OM and SEM) to investigate its chemical structure and morphology. Its stability was assessed by monitoring its inversion properties over time at room temperature, while its mechanical characteristics were assessed by rheological experiments. Dose-dependent cytotoxicity studies performed on human fibroblasts for 24 h with gel samples obtained by diluting CP_1.1-Hgel at properly selected concentrations established that the 3D network formation did not significantly affect the cytotoxic profile of CP1. Also, microbiologic investigations carried out on two-fold serial dilutions of CP1-gel confirmed the minimum inhibitory concentrations (MICs) previously reported for the not formulated CP1.Selectivity indices values up to 12 were estimated by the values of LD50 and MICs determined here on gel samples.

Background The oncolytic viruses have shown promising results for the treatment of multiple myeloma. However, the use of human viruses is limited by the patients’ antiviral immune response. In this study, we investigated an alternative oncolytic strategy using non-human pathogen viruses as the bovine viral diarrhea virus (BVDV) that were able to interact with CD46. Methods We treated several human myeloma cell lines and non-myeloma cell lines with BVDV to evaluate the expression of CD46 and to study the effect on cell viability by flow cytometry. The possible synergistic effect of bortezomib in combination with BVDV was also tested. Moreover, we infected the bone marrow mononuclear cells obtained from myeloma patients and we checked the BVDV effect on different cell populations, defined by CD138, CD14, CD3, CD19, and CD56 expression evaluated by flow cytometry. Finally, the in vivo BVDV effect was tested in NOD-SCID mice injected subcutaneously with myeloma cell lines. Results Human myeloma cells were selectively sensitive to BVDV treatment with an increase of cell death and, consequently, of apoptotic markers. Consistently, bone marrow mononuclear cells isolated from myeloma patients treated with BVDV, showed a significant selective decrease of the percentage of viable CD138+ cells. Interestingly, bortezomib pre-treatment significantly increased the cytotoxic effect of BVDV in myeloma cell lines with a synergistic effect. Finally, the in vitro data were confirmed in an in vivo myeloma mouse model showing that BVDV treatment significantly reduced the tumoral burden compared to the vehicle. Conclusions Overall, our data indicate, for the first time, a direct oncolytic effect of the BVDV in human myeloma cells suggesting its possible use as novel alternative anti-myeloma virotherapy strategy.

Ursolic acid (UA), a pentacyclic triterpenoid acid found in many medicinal plants and aromas, is known for its antibacterial effects against multi-drug-resistant (MDR) Gram-positive bacteria, which seriously threaten human health. Unfortunately, UA water-insolubility, low bioavailability, and systemic toxicity limit the possibilities of its application in vivo. Consequently, the beneficial activities of UA observed in vitro lose their potential clinical relevance unless water-soluble, not cytotoxic UA formulations are developed. With a nano-technologic approach, we have recently prepared water-soluble UA-loaded dendrimer nanoparticles (UA-G4K NPs) non-cytotoxic on HeLa cells, with promising physicochemical properties for their clinical applications. In this work, with the aim of developing a new antibacterial agent based on UA, UA-G4K has been tested on different strains of the Enterococcus genus, including marine isolates, toward which UA-G4K has shown minimum inhibitory concentrations (MICs) very low (0.5–4.3 µM), regardless of their resistance to antibiotics. Time-kill experiments, in addition to confirming the previously reported bactericidal activity of UA against E. faecium, also established it for UA-G4K. Furthermore, cytotoxicity experiments on human keratinocytes revealed that nanomanipulation of UA significantly reduced the cytotoxicity of UA, providing UA-G4K NPs with very high LD50 (96.4 µM) and selectivity indices, which were in the range 22.4–192.8, depending on the enterococcal strain tested. Due to its physicochemical and biological properties, UA-G4K could be seriously evaluated as a novel oral-administrable therapeutic option for tackling difficult-to-treat enterococcal infections.

Background & Aim Viral infections remain a leading cause of morbidity and mortality after transplantation. Although prophylactic pharmacotherapy is effective in reducing the risk for some viral infections, therapeutic options for breakthrough infections are still unsatisfactory. Conventional pharmacologic agents against Adenovirus (AdV) and Epstein-Barr virus (EBV) have limited efficacy and relevant toxicity, whereas in the case of cytomegalovirus (CMV) are documented better response rates, but toxicity and reactivation after treatment stop are frequent. T-cell reconstitution is a key requirement for effective antiviral control following transplantation, and factors that influence the speed of T-cell recovery impact also the risk of viral infection in this period. The use of lymphocyte infusions derived from seropositive donors is an effective salvage therapy for viral infections in transplanted recipients prior to T-cell recovery, but the risk of severe graft-versus-host disease (GVHD) is a critical concern. Thus, we propose a novel immunotherapeutic approach based on gd T cells specific for AdV, CMV and EBV. Many studies highlighted the importance of gd T cells in antiviral immunity, uniquely combining conventional adaptive features with rapid, innate-like responses. These lymphocytes are effector cells that: i) recognize target cells in a MHC-independent manner, thus do not cause GvHD; ii) participate CMV and EBV responses, particularly when conventional adaptive immunity is insufficient to clear the viral infection; iii) may develop memory responses, and iv) cross-talk with other immune cells, including NK lymphocytes, alfa beta T cells and dendritic cells. These features render gd T cells extremely appealing for clinical purposes. We believe that an ATMP containing Vd1 and Vd2 gd T cells will be the ideal immunotherapeutic approach to prevent and treat viral infections after transplantation. Methods, Results & Conclusion To this end, we are developing a new method to obtain a cell product containing expanded Vd1 and Vd2 gd T cells using exclusively GMP-graded reagents, including zoledronic acid, cytokines, viral peptides (related to AdV, CMV and EBV) and ab depletion techniques. Such cell product has been tested for i) phenotype, ii) allo-reactivity, iii) cytotoxicity and iv) specificity. Our preliminary results strongly support the feasibility for development of new immunotherapeutic protocols using gd T cells, with the aim of preventing and treating viral infections after transplantation.

We demonstrated that γδ T cells of patients given HLA-haploidentical HSCT after removal of αβ+ T cells and CD19+ B cells are endowed with the capacity of killing leukemia cells after ex vivo treatment with zoledronic acid (ZOL). Thus, we tested the hypothesis that infusion of ZOL in patients receiving this type of graft may enhance γδ T-cell cytotoxic activity against leukemia cells. ZOL was infused every 28 d in 43 patients; most were treated at least twice. γδ T cells before and after ZOL treatments were studied in 33 of these 43 patients, till at least 7 mo after HSCT by high-resolution mass spectrometry, flow-cytometry, and degranulation assay. An induction of Vδ2-cell differentiation, paralleled by increased cytotoxicity of both Vδ1 and Vδ2 cells against primary leukemia blasts was associated with ZOL treatment. Cytotoxic activity was further increased in Vδ2 cells, but not in Vδ1 lymphocytes in those patients given more than one treatment. Proteomic analysis of γδ T cells purified from patients showed upregulation of proteins involved in activation processes and immune response, paralleled by downregulation of proteins involved in proliferation. Moreover, a proteomic signature was identified for each ZOL treatment. Patients given three or more ZOL infusions had a better probability of survival in comparison to those given one or two treatments (86% vs. 54%, respectively, p = 0.008). Our data indicate that ZOL infusion in pediatric recipients of αβ T- and B-cell-depleted HLA-haploidentical HSCT promotes γδ T-cell differentiation and cytotoxicity and may influence the outcome of patients.

Purpose: The interleukin (IL)-27 cytokine subunit p28, also called IL-30, has been recognized as a novel immunoregulatory mediator endowed with its own functions. These are currently the subject of discussion in immunology, but completely unexplored in cancer biology. We set out to investigate the role of IL-30 in prostate carcinogenesis and its effects on human prostate cancer (hPCa) cells. Experimental Design: IL-30 expression, as visualized by immunohistochemistry and real-time reverse transcriptase PCR on prostate and draining lymph nodes from 125 patients with prostate cancer, was correlated with clinicopathologic data. IL-30 regulation of hPCa cell viability and expression of selected gene clusters was tested by flow cytometry and PCR array. Results: IL-30, absent in normal prostatic epithelia, was expressed by cancerous epithelia with Gleason ≥ 7% of 21.3% of prostate cancer stage I to III and 40.9% of prostate cancer stage IV. IL-30 expression by tumor infiltrating leukocytes (T-ILK) was higher in stage IV that in stage I to III prostate cancer (P = 0.0006) or in control tissue (P = 0.0011). IL-30 expression in prostate draining lymph nodes (LN)-ILK was higher in stage IV than in stage I to III prostate cancer (P = 0.0031) or in control nodes (P = 0.0023). The main IL-30 sources were identified as CD68+ macrophages, CD33+/CD11b+ myeloid cells, and CD14+ monocytes. In vitro, IL-30 stimulated proliferation of hPCa cells and also downregulated CCL16/LEC, TNFSF14/LIGHT, chemokine-like factor (CKLF), and particularly CKLF-like MARVEL transmembrane domain containing 3 (CMTM3) and greatly upregulated ChemR23/CMKLR. Conclusions: We provide the first evidence that IL-30 is implicated in prostate cancer progression because (i) its expression by prostate cancer or T- and LN-ILK correlates with advanced disease grade and stage; and (ii) IL-30 exerts protumor activity in hPCa cells. Clin Cancer Res; 20(3); 585–94. ©2013 AACR.

Background & Aim Leukemia is the most common cancer in childhood, accounting for almost 1 out of 3 cases. Of these malignancies, most are acute lymphoblastic leukemia (ALL), with the remaining cases largely acute myeloid leukemia (AML). Despite remarkable advances in treatments, 50%-70% of all children with ALL and AML who relapse after allogeneic hematopoietic stem cell transplantation (HSCT), succumb to their disease. Thus, there is an urgent clinical need of novel therapies for improving prognosis. γδ T cells are a minor subset (1-5%) of CD3+ combining conventional adaptive features with rapid, innate-like responses that place them in the initiation phase of immune cascade. In addition, they recognize tumor cells in a major histocompatibility complex (MHC) independent manner, thereby bypassing the occurrence of Graft versus Host Disease (GvHD). gd T cells is mainly constituted of Vg9Vd2 and Vd1 subpopulations which has demonstrated a potent Graft versus Leukemia (GvL) effect. The Vg9Vd2 subset, which represents the main circulating proportion of gd T cells, recognizes phospoantigens presented by leukemic blasts. The Vd1 subset, conversely, residing within the epithelial tissues, especially at sites of cytomegalovirus (CMV) replication, exerts a protective anti-infections post-transplant role. Methods, Results & Conclusion Here we purpose to develop a novel strategy for ex vivo expanding both Vg9Vd2 and Vd1 gdT-cells in a clinical-scale fashion. The ultimate goal is to post-HSCT administer multiple infusions of expanded gd T cells in order to reduce the risk of leukemia recurrence. We have previously demonstrated that the in vitro exposure of gd T cells to zoledronic acid (ZOL) combined with IL-2, allows robust expansion and activation of the Vd2 subset alone. Herein, we developed a novel protocol for the expansion of both Vd1 and Vd2 by exploiting the mitogenic effect of Concanavalin-A (Con-A), together with ZOL and a cocktail of different cytokines (Table 1). Preliminary results obtained from 5 different healthy donors show that 1) we succeeded in expanding gd T cells up to 50 folds, 2) the use of Con-A is essential for the Vd1 subset expansion, 3) gd T cells ex vivo expanded acquire an effector memory phenotype (Fig 1) and are cytotoxic in vitro against pediatric acute leukemia cell lines. Therefore, these encouraging results lay the foundation for advancing the translational use of gd T cells.

// Emma Di Carlo 1,2 , Carlo Sorrentino 1,2 , Alessia Zorzoli 3 , Serena Di Meo 1,2 , Maria Grazia Tupone 1,2 , Emanuela Ognio 4 , Gabriella Mincione 2,5 and Irma Airoldi 3 1 Department of Medicine and Sciences of Aging, Section of Anatomic Pathology and Molecular Medicine, “G. d’Annunzio” University, Chieti, Italy 2 Ce.S.I. Aging Research Center, “G. d’Annunzio” University Foundation, Chieti, Italy 3 Laboratory of Oncology, Istituto Giannina Gaslini, Genova, Italy 4 Animal Facility, IRCCS AOU San Martino-IST Istituto Nazionale per la Ricerca sul Cancro, Genova, Italy 5 Department of Experimental and Clinical Sciences, “G. d’Annunzio” University, Chieti, Italy Correspondence: Emma Di Carlo, email: // Keywords : prostate cancer, interleukin-27, cytokines, immunotherapy, anti-tumor activity, tumor microenvironment Received : September 19, 2013 Accepted : December 28, 2013 Published : December 28, 2013 Abstract Prostate cancer (PCa) is of increasing significance worldwide as a consequence of the population ageing. Fragile elderly patients may particularly benefit from noninvasive and well tolerable immunotherapeutic approaches. Preclinical studies have revealed that the immune-regulatory cytokine IL-27 may exert anti-tumor activities in a variety of tumor types without discernable toxicity. We, thus, investigated whether IL-27 may function as anti-tumor agent in human (h) PCa and analyzed the rationale for its clinical application. In vitro, IL-27 treatment significantly inhibited proliferation and reduced the angiogenic potential of hPCa cells by down-regulating the pro-angiogenesis-related genes fms-related tyrosine kinase (FLT)1, prostaglandin G/H synthase 1/cyclooxygenase-1 (PTGS1/COX-1) and fibroblast growth factor receptor (FGFR)3. In addition, IL-27 up-regulated the anti-angiogenesis-related genes such as CXCL10 and TIMP metallopeptidase inhibitor 3 (TIMP3). In vivo, IL-27 reduced proliferation and vascularization in association with ischemic necrosis of tumors developed after PC3 or DU145 cell injection in athymic nude mice. In patients’ prostate tissues, IL-27R was expressed by normal epithelia and low grade PCa and lost by high tumor grade and stages. Nevertheless, IL-27R was expressed by CD11c + , CD4 + and CD8 + leukocytes infiltrating the tumor and draining lymph nodes. These data lead to the conclusion that i) IL-27’s anti-PCa potential may be fully exploited in patients with well-differentiated, localized IL-27R positive PCa, since in this case it may act on both cancerous epithelia and the tumor microenvironment; ii) PCa patients bearing high grade and stage tumor that lack IL-27R may benefit, however, from IL-27’s immune-stimulatory properties.

Interleukin (IL)-23 and IL-27 are pro-inflammatory cytokines that share functional and structural similarities and may exert anti-tumor activities against solid and hematological malignancies. Here, we asked whether IL-23 and IL-27, alone or in combination, may act directly against human follicular lymphoma (FL) and diffuse large B-cell lymphoma (DLBCL) cells. In this study, we demonstrated for the first time that human primary FL and DLBCL cells expressed complete and functional IL-23 and IL-27 receptors (R) and that IL-23 and IL-27 exerted anti-tumor activities in vitro and in vivo through different and complementary mechanisms. In vivo studies using severe combined immunodeficiency /non-obese diabetic mice-injected subcutaneously with human SU-DHL-4 cell line revealed that IL-23 inhibited directly tumor-cell proliferation, whereas IL-27 impaired the angiogenic program of lymphoma cells resulting in strong reduction of cell growth. In addition, combined treatment of IL-23 and IL-27 amplified the anti-tumor effects in vivo as compared with administration of each cytokine alone. These anti-tumor mechanisms were confirmed by in vitro experiments performed with primary lymphoma cells and cell lines. Our results strongly encourage the development of future clinical trials to evaluate the toxicity and efficacy of the IL-23 and IL-27 in lymphoma patients.

B-acute lymphoblastic leukemia (B-ALL) represents the most common pediatric hematological tumor that derives from the aberrant proliferation of early B lymphocytes in the bone marrow. Although most of the B-ALL children take advantage from current therapeutic protocols, some patients relapse and need alternative therapies. With this background, we investigated whether interleukin (IL)-27, an immunomodulatory cytokine with antitumor properties, may function as an antitumor agent against pediatric B-ALL cells. Here we show for the first time that pediatric B-ALL cells functional IL-27R and that IL-27 dampens directly tumor growth in vivo and in vitro through mechanisms elucidated in this study. The novelty of these results deals with the first demonstration that (1) B-ALL cells from pediatric patients injected intravenously (i.v.) into NOD/SCID/Il2rg(-/-) (NSG) mice gave rise to leukemic spreading that was severely hampered by IL-27; (2) IL-27-treated mice, compared with controls, showed significant reduction of putative B-ALL-initiating cells and blasts in the peripheral blood (PB), bone marrow (BM) and spleen; and that (3) IL-27 reduced in vitro B-ALL cell proliferation and angiogenesis, induced apoptosis and downregulated miR-155. Our results strongly encourage the development of future clinical trials to evaluate the toxicity and efficacy of IL-27 in childhood B-ALL patients.

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. Cancer Res; 70(23); 9816–26. ©2010 AACR.

We prospectively assessed functional and phenotypic characteristics of γδ T lymphocytes up to 7 months after HLA-haploidentical hematopoietic stem cell transplantation (haplo-HSCT) depleted of αβ+ T cells and CD19+ B cells in 27 children with either malignant or nonmalignant disorders. We demonstrate that (1) γδ T cells are the predominant T-cell population in patients during the first weeks after transplantation, being mainly, albeit not only, derived from cells infused with the graft and expanding in vivo; (2) central-memory cells predominated very early posttransplantation for both Vδ1 and Vδ2 subsets; (3) Vδ1 cells are specifically expanded in patients experiencing cytomegalovirus reactivation and are more cytotoxic compared with those of children who did not experience reactivation; (4) these subsets display a cytotoxic phenotype and degranulate when challenged with primary acute myeloid and lymphoid leukemia blasts; and (5) Vδ2 cells are expanded in vitro after exposure to zoledronic acid (ZOL) and efficiently lyse primary lymphoid and myeloid blasts. This is the first detailed characterization of γδ T cells emerging in peripheral blood of children after CD19+ B-cell and αβ+ T-cell–depleted haplo-HSCT. Our results can be instrumental to the development of clinical trials using ZOL for improving γδ T-cell killing capacity against leukemia cells. This trial was registered at www.clinicaltrials.gov as #{"type":"clinical-trial","attrs":{"text":"NCT01810120","term_id":"NCT01810120"}}NCT01810120.

A new method of graft manipulation based on physical removal of αβ+ T cells and CD19+ B cells, leaving mature NK cells and γδ T cells in the graft, has been recently developed for HLA-haploidentical HSCT. We demonstrated that γδ T cells collected from transplanted patients are endowed with capacity of killing leukemia cells after ex vivo treatment with zoledronic acid (ZOL). Thus, we hypothesized that infusion of ZOL in patients receiving this type of graft, may boost γδ T cell cytotoxic activity against leukemia cells. Thirty-three patients were treated with ZOL every 28 days at least twice. γδ T cells before and after ZOL treatments were studied till at least 7 months after HSCT by high-resolution mass spectrometry, flow-cytometry, and degranulation assay. Proteomic analysis of γd T cells purified from patients showed that, starting from the first infusion, ZOL caused up-regulation of proteins involved in activation processes and immune response, paralleled by down-regulation of proteins involved in proliferation. These findings are consistent with an induction of Vδ2 cell differentiation, paralleled by increased cytotoxicity of both Vδ1 and Vδ2 cells against primary leukemia blasts. Furthermore, a proteomic signature was identified for each individual ZOL treatment. Patients given 3 or more ZOL infusions had a better probability of survival in comparison to those given 1 or 2 treatments. In conclusion,ZOL influences Vδ2 cell activity, determines a specific proteomic signature and enhances anti-leukemia activity, this potentially resulting into an increased anti-tumor effect. Disclosures No relevant conflicts of interest to declare.

Purpose: Acute myeloid leukemia (AML) accounts for more than half of fatal cases in all pediatric leukemia patients; this observation highlights the need of more effective therapies. Thus, we investigated whether interleukin (IL)-27, an immunomodulatory cytokine, functions as an antitumor agent against pediatric AML cells. Experimental Design: Expression of WSX-1 and gp130 on AML cells from 16 pediatric patients was studied by flow cytometry. Modulation of leukemia cell proliferation or apoptosis upon IL-27 treatment in vitro was tested by bromodeoxyuridine/propidium iodide (PI) and Ki67, or Annexin V/PI staining and flow cytometric analysis. The angiogenic potential of AML cells treated or not with IL-27 was studied by chorioallantoic membrane assay and PCR array. In vivo studies were carried out using nonobese diabetic/severe combined immunodeficient (NOD/SCID)/Il2rg−/− mice injected intravenously with five pediatric AML cell samples. Leukemic cells engrafted in PBS and IL-27–treated animals were studied by immunohistochemical/morphologic analysis and by PCR array for expression angiogenic/dissemination-related genes. Results: We provided the first demonstration that (i) AML cells injected into NOD/SCID/Il2rg−/− mice gave rise to leukemia dissemination that was severely hampered by IL-27, (ii) compared with controls, leukemia cells harvested from IL-27–treated mice showed significant reduction of their angiogenic and spreading related genes, and (iii) similarly to what was observed in vivo, IL-27 reduced in vitro AML cell proliferation and modulated the expression of different genes involved in the angiogenic/spreading process. Conclusion: These results provide an experimental rationale for the development of future clinical trials aimed at evaluating the toxicity and efficacy of IL-27. Clin Cancer Res; 18(6); 1630–40. ©2012 AACR.

The orthotopic model reproduces aspects of the tumour microenvironment and emulates a number of important biological features of cancer progression, angiogenesis, metastasis and resistance. Due to its parallels with human cancer, the model can be used to evaluate therapeutic responses to various therapies. This review outlines the importance of using the orthotopic implantation of tumour cells in mice models for evaluating the effectiveness of antivascular therapies.

RNA interference molecules have some advantages as cancer therapeutics, including a proved efficacy on both wild-type (WT) and mutated transcripts and an extremely high sequence-specificity. The most significant hurdle to be overcome if exogenous small interfering RNAs (siRNA) is to be used therapeutically is the specific, effective, nontoxic delivery of siRNA to its intracellular site of action. At present, human applications are confined almost exclusively to targets within the liver, where the delivery systems naturally accumulate, and extra-hepatic targets remain a challenge. Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase that has recently been shown to contribute to the cell growth and progression of human neuroblastoma (NB). We investigated its potential as a therapeutic target in NB by generating anti-GD₂-targeted nanoparticles that carry ALK-directed siRNA, which are specifically and efficiently delivered to GD₂-expressing NB cells. Relative to free ALK-siRNA, anti-GD₂-targeted liposomal formulations of ALK-siRNA had low plasma clearance, increased siRNA stability, and improved binding, uptake, silencing and induction of cell death, and specificity for NB cells. In NB xenografts, intravenous (i.v.) injection of the targeted ALK-siRNA liposomes showed gene-specific antitumor activity with no side effects. ALK-selective siRNA entrapped in anti-GD₂-targeted nanoparticles is a promising new modality for NB treatment.

Novel anti-vasculature strategies that are emerging for the treatment of cancer and for the inhibition of angiogenesis may be a promising new tool for the adjuvant therapy of malignant tumours. Over the last fifteen years, several reports have been published concerning the relationship between tumour progression and angiogenesis in experimental models of neuroblastoma in vitro and in vivo. Moreover, a high vascular index in neuroblastoma correlates with poor prognosis, suggesting dependence of aggressive tumour growth on active angiogenesis. Here, we present an overview of the most recent advances in anti-vasculature therapy of neuroblastoma, and describe some preclinical results as well as future perspectives.

Neuroblastoma (NB) is the most common extracranial solid tumor in childhood and the most frequently diagnosed neoplasm during infancy. Despite of aggressive treatment strategies, the 5-year survival rate for metastatic disease is still less than 60% and, consequently, novel therapeutic approaches are needed. For increasing the therapeutic index of anticancer drugs, while reducing side effects, one of the most promising strategies in modern chemotherapy is based on the development of innovative drug delivery systems, such as liposomes. "Anticancer drug"-loaded liposomes have demonstrated enhanced ability to target to the affected area, as well as increased antitumor efficacy compared to conventional drugs. Liposomes tend to extravasate preferentially and to accumulate into tumor interstitial fluids, due to the defective structure of the new angiogenic vessels within the tumor masses. This inherent tumor selectivity can be increased further by coupling tumor-specific antibodies or other targeting moieties to the surface of the lipid envelope. Here, we describe the methodology used in these studies, as well as the antitumor results obtained by the use of several "anticancer drugs," encapsulated into antibody- and peptide-targeted liposomal formulations, against NB.

HLA-haploidentical hematopoietic stem cell transplantation (HSCT) using CD34+ selected cells is a widely used procedure, which, however, is complicated by delayed immune reconstitution. We recently developed a new method of graft manipulation based on the physical removal of αβ+ T cells and CD19+ B cells, which permits to leave mature natural killer (NK) cells and γδ+ T cells in the graft. These cells can exert a graft-versus-leukemia (GvL) effect and reduce the risk of infection. In particular, unconventional γδ T cells play a critical role in both innate and adaptive immunity and exert HLA-unrestricted cytotoxicity against both solid and hematological tumors, thus potentially acting as beneficial effector cells in transplanted patients. Moreover, such grafts may limit the risk of graft-versus-host disease and prevent EBV-related lymphoproliferative disease. We performed phenotypic and functional studies on γδ T cells collected from 20 pediatric patients (pts, 13 males, 7 females, median age 10 years, range 6 months to 16 years) that received this type of allograft. Eighteen pts had acute leukemia and 2 non-malignant disorders. Ex vivo assays of peripheral blood γδ T cell phenotype and function were performed weekly until Hospital discharge and monthly until 6 months after HSCT. Phenotype of γδ T cells was analysed by flow cytometry. Analyses were performed on mononuclear cells labelled with mAb panels (CD3, CD45, pan-γδ, anti-Vδ1, -Vδ2, -Vγ9, CD45RO, CD45RA, CD27, CD16, CD56) allowing the identification of the main γδ+ T cell subsets, including Vδ1+ and Vδ2+ cells, naïve, central memory (CM), effector memory (EM) and terminally differentiated (TD) γδ T cells. Functional studies were performed using γδ T cells shortly after collection from pts, as well as after in vitro expansion with zoledronic acid and IL-2 for 10 days. Cytotoxic activity of γδ T cells was tested against primary leukemia cells, through CD107a degranulation assay and/or standard 51Cr-release assay. In the first 4 weeks after HSCT, T cells were consistently of the γδ subset (>90% of CD45+CD3+ cells); by contrast, αβ+ T cells gradually increased over time. In approximately half of the pts, the percentage of αβ T cells exceeded that of γδ T cells already starting from 30 days after HSCT. γδ T cells consisted of Vδ2+Vγ9+ and Vδ1+Vγ9+/- cells, and marginally of the Vδ1-Vδ2-Vγ9- population. Detailed phenotypic characterization of Vδ1+ and Vδ2+ γδ T cells revealed that, at day +20 after HSCT, 44% of Vδ1+ cells were CM (identified as CD45RO+CD27+ cells), 26% naïve (CD45RO-CD27+), 21.4% TD (CD45RO-CD27-) and 6.1% EM (CD45RO+CD27-). Similarly, 55.4% of Vδ2+ γδ T lymphocytes were CM, 9.8% naïve, 11.4% TD and 23.1% EM. The proportion of the different Vδ2+ γδ T cell subset did not change significantly over time, especially when comparing that present at day +20 after HSCT (time point, TP1) with that measured 30 days after the attainment of a 1:1 ratio of αβ-to- γδ T cells (TP2) (Figure 1, left panel). By contrast, by comparing TP1 and TP2, we found that Vδ1+ CM γδ T cells decreased and EM cells increased over time, while naïve or TD Vδ1+ γδ T cells did not change (Figure 1, right panel). In transplanted pts experiencing cytomegalovirus (CMV) reactivation, γδ T cells mostly consisted of Vδ1+ cells (mean 59.8% of γδ T cells), among which 49% were TD, 22.7% EM, 18.9% CM and 10.1% naïve. Noteworthy, in transplanted pts who did not have CMV reactivation, the main γδ T cells showed a Vδ2+ phenotype. Functional studies revealed that pt-derived γδ T cells consistently expanded in vitro after exposure to zoledronic acid and IL-2, the resulting Vγ9Vδ2 population expressing mainly an EM phenotype. These Vγ9Vδ2 cells exerted cytotoxic activities against primary allogeneic leukemia cells, especially when leukemia cells were pre-treated with zoledronic acid (Figure 2). More importantly, both Vδ1+ and Vδ2+ γδ T cells obtained from transplanted pts showed cytotoxic activity against primary leukemia cells, as assessed by CD107a degranulation assay. In conclusion, we provide the first phenotypic and functional characterization of γδ T cells, analyzed over time in children transplanted with grafts depleted of αβ+ T cells and of B lymphocytes. Our results support the concept that γδ T cells are important effector cells, which can be expanded and activated after exposure to bisphosphonates and IL-2 with the aim of improving their killing capacity against leukemia cells. Disclosures: No relevant conflicts of interest to declare.