From the identification of actionable molecular targets to the generation of faithful neuroblastoma patient-derived preclinical models.

BACKGROUND: Neuroblastoma (NB) represents the most frequent and aggressive form of extracranial solid tumor of infants. Although the overall survival of patients with NB has improved in the last years, more than 50% of high-risk patients still undergo a relapse. Thus, in the era of precision/personalized medicine, the need for high-risk NB patient-specific therapies is urgent. METHODS: Within the PeRsonalizEd Medicine (PREME) program, patient-derived NB tumors and bone marrow (BM)-infiltrating NB cells, derived from either iliac crests or tumor bone lesions, underwent to histological and to flow cytometry immunophenotyping, respectively. BM samples containing a NB cells infiltration from 1 to 50 percent, underwent to a subsequent NB cells enrichment using immune-magnetic manipulation. Then, NB samples were used for the identification of actionable targets and for the generation of 3D/tumor-spheres and Patient-Derived Xenografts (PDX) and Cell PDX (CPDX) preclinical models. RESULTS: Eighty-four percent of NB-patients showed potentially therapeutically targetable somatic alterations (including point mutations, copy number variations and mRNA over-expression). Sixty-six percent of samples showed alterations, graded as "very high priority", that are validated to be directly targetable by an approved drug or an investigational agent. A molecular targeted therapy was applied for four patients, while a genetic counseling was suggested to two patients having one pathogenic germline variant in known cancer predisposition genes. Out of eleven samples implanted in mice, five gave rise to (C)PDX, all preserved in a local PDX Bio-bank. Interestingly, comparing all molecular alterations and histological and immunophenotypic features among the original patient's tumors and PDX/CPDX up to second generation, a high grade of similarity was observed. Notably, also 3D models conserved immunophenotypic features and molecular alterations of the original tumors. CONCLUSIONS: PREME confirms the possibility of identifying targetable genomic alterations in NB, indeed, a molecular targeted therapy was applied to four NB patients. PREME paves the way to the creation of clinically relevant repositories of faithful patient-derived (C)PDX and 3D models, on which testing precision, NB standard-of-care and experimental medicines.

Antitumor activity of the investigational B7-H3 antibody-drug conjugate, vobramitamab duocarmazine, in preclinical models of neuroblastoma.

INTRODUCTION: B7-H3 is a potential target for pediatric cancers, including neuroblastoma (NB). Vobramitamab duocarmazine (also referred to as MGC018 and herein referred to as vobra duo) is an investigational duocarmycin-based antibody-drug conjugate (ADC) directed against the B7-H3 antigen. It is composed of an anti-B7-H3 humanized IgG1/kappa monoclonal antibody chemically conjugated through a cleavable valine-citrulline linker to a duocarmycin-hydroxybenzamide azaindole (vc-seco-DUBA). Vobra duo has shown preliminary clinical activity in B7-H3-expressing tumors. METHODS: B7-H3 expression was evaluated by flow-cytometry in a panel of human NB cell lines. Cytotoxicity was evaluated in monolayer and in multicellular tumor spheroid (MCTS) models by the water-soluble tetrazolium salt,MTS, proliferation assay and Cell Titer Glo 3D cell viability assay, respectively. Apoptotic cell death was investigated by annexin V staining. Orthotopic, pseudometastatic, and resected mouse NB models were developed to mimic disease conditions related to primary tumor growth, metastases, and circulating tumor cells with minimal residual disease, respectively. RESULTS: All human NB cell lines expressed cell surface B7-H3 in a unimodal fashion. Vobra duo was cytotoxic in a dose-dependent and time-dependent manner against all cell lines (IC50 range 5.1-53.9 ng/mL) and NB MCTS (IC50 range 17.8-364 ng/mL). Vobra duo was inactive against a murine NB cell line (NX-S2) that did not express human B7-H3; however, NX-S2 cells were killed in the presence of vobra duo when co-cultured with human B7-H3-expressing cells, demonstrating bystander activity. In orthotopic and pseudometastatic mouse models, weekly intravenous treatments with 1 mg/kg vobra duo for 3 weeks delayed tumor growth compared with animals treated with an irrelevant (anti-CD20) duocarmycin-ADC. Vobra duo treatment for 4 weeks further increased survival in both orthotopic and resected NB models. Vobra duo compared favorably to TOpotecan-TEMozolomide (TOTEM), the standard-of-care therapy for NB relapsed disease, with tumor relapse delayed or arrested by two or three repeated 4-week vobra duo treatments, respectively. Further increased survival was observed in mice treated with vobra duo in combination with TOTEM. Vobra duo treatment was not associated with body weight loss, hematological toxicity, or clinical chemistry abnormalities. CONCLUSION: Vobra duo exerts relevant antitumor activity in preclinical B7-H3-expressing NB models and represents a potential candidate for clinical translation.

Augmented efficacy of nano-formulated docetaxel plus curcumin in orthotopic models of neuroblastoma.

Neuroblastoma is a biologically heterogeneous extracranial tumor, derived from the sympathetic nervous system, that affects most often the pediatric population. Therapeutic strategies relying on aggressive chemotherapy, surgery, radiotherapy, and immunotherapy have a negative outcome in advanced or recurrent disease. Here, spherical polymeric nanomedicines (SPN) are engineered to co-deliver a potent combination therapy, including the cytotoxic docetaxel (DTXL) and the natural wide-spectrum anti-inflammatory curcumin (CURC). Using an oil-in-water emulsion/solvent evaporation technique, four SPN configurations were engineered depending on the therapeutic payload and characterized for their physico-chemical and pharmacological properties. All SPN configurations presented a hydrodynamic diameter of ∼ 185 nm with a narrow size distribution. A biphasic release profile was observed for all the configurations, with almost 90 % of the total drug mass released within the first 24 h. SPN cytotoxic potential was assessed on a panel of human neuroblastoma cells, returning IC50 values in the order of 1 nM at 72 h and documenting a strong synergism between CURC and DTXL. Therapeutic efficacy was tested in a clinically relevant orthotopic model of neuroblastoma, following the injection of SH-SY5Y-Luc+ cells in the left adrenal gland of athymic mice. Although ∼ 2 % of the injected SPN per mass tissue reached the tumor, the overall survival of mice treated with CURC/DTXL-SPN was extended by 50 % and 25 % as compared to the untreated control and the monotherapies, respectively. In conclusion, these results demonstrate that the therapeutic potential of the DTXL/CURC combination can be fully exploited only by reformulating these two compounds into systemically injectable nanoparticles.

Combined mitoxantrone and anti-TGFß treatment with PD-1 blockade enhances antitumor immunity by remodelling the tumor immune landscape in neuroblastoma.

BACKGROUND: Poor infiltration of functioning T cells renders tumors unresponsive to checkpoint-blocking immunotherapies. Here, we identified a combinatorial in situ immunomodulation strategy based on the administration of selected immunogenic drugs and immunotherapy to sensitize poorly T-cell-infiltrated neuroblastoma (NB) to the host antitumor immune response. METHODS: 975A2 and 9464D NB cell lines derived from spontaneous tumors of TH-MYCN transgenic mice were employed to study drug combinations able of enhancing the antitumor immune response using in vivo and ex vivo approaches. Migration of immune cells towards drug-treated murine-derived organotypic tumor spheroids (MDOTS) were assessed by microfluidic devices. Activation status of immune cells co-cultured with drug-treated MDOTS was evaluated by flow cytometry analysis. The effect of drug treatment on the immune content of subcutaneous or orthotopic tumors was comprehensively analyzed by flow-cytometry, immunohistochemistry and multiplex immunofluorescence. The chemokine array assay was used to detect soluble factors released into the tumor microenvironment. Patient-derived organotypic tumor spheroids (PDOTS) were generated from human NB specimens. Migration and activation status of autologous immune cells to drug-treated PDOTS were performed. RESULTS: We found that treatment with low-doses of mitoxantrone (MTX) recalled immune cells and promoted CD8+ T and NK cell activation in MDOTS when combined with TGFß and PD-1 blockade. This combined immunotherapy strategy curbed NB growth resulting in the enrichment of a variety of both lymphoid and myeloid immune cells, especially intratumoral dendritic cells (DC) and IFNγ- and granzyme B-expressing CD8+ T cells and NK cells. A concomitant production of inflammatory chemokines involved in remodelling the tumor immune landscape was also detected. Interestingly, this treatment induced immune cell recruitment against PDOTS and activation of CD8+ T cells and NK cells. CONCLUSIONS: Combined treatment with low-dose of MTX and anti-TGFß treatment with PD-1 blockade improves antitumor immunity by remodelling the tumor immune landscape and overcoming the immunosuppressive microenvironment of aggressive NB.

MiR-146b-5p regulates IL-23 receptor complex expression in chronic lymphocytic leukemia cells.

Chronic lymphocytic leukemia (CLL) cells express the interleukin-23 receptor (IL-23R) chain, but the expression of the complementary IL-12Rß1 chain requires cell stimulation via surface CD40 molecules (and not via the B-cell receptor [BCR]). This stimulation induces the expression of a heterodimeric functional IL-23R complex and the secretion of IL-23, initiating an autocrine loop that drives leukemic cell expansion. Based on the observation in 224 untreated Binet stage A patients that the cases with the lowest miR-146b-5p concentrations had the shortest time to first treatment (TTFT), we hypothesized that miR-146b-5p could negatively regulate IL-12Rß1 side chain expression and clonal expansion. Indeed, miR-146b-5p significantly bound to the 3'-UTR region of the IL-12Rß1 mRNA in an in vitro luciferase assay. Downregulation of miR-146b-5p with specific miRNA inhibitors in vitro led to the upregulation of the IL-12Rß1 side chain and expression of a functional IL-23R complex similar to that observed after stimulation of the CLL cell through the surface CD40 molecules. Expression of miR-146b-5p with miRNA mimics in vitro inhibited the expression of the IL-23R complex after stimulation with CD40L. Administration of a miR-146b-5p mimic to NSG mice, successfully engrafted with CLL cells, caused tumor shrinkage, with a reduction of leukemic nodules and of IL-12Rß1-positive CLL cells in the spleen. Our findings indicate that IL-12Rß1 expression, a crucial checkpoint for the functioning of the IL-23 and IL-23R complex loop, is under the control of miR-146b-5p, which may represent a potential target for therapy since it contributes to the CLL pathogenesis. This trial is registered at www.clinicaltrials.gov as NCT00917540.

Ischemia-reperfusion damage is attenuated by GQ-11, a peroxisome proliferator-activated receptor (PPAR)-α/γ agonist, after aorta clamping in rats.

INTRODUCTION: Ischemia-Reperfusion (I/R) damage is one of the major challenges in cardiothoracic surgeries and in a pathological manner, is identified by exacerbated damage signals resulted from blood supply restriction and subsequent flow restoration and re­oxygenation. I/R damage includes cellular dysfunction and death, impairing tissue and organ function. Inflammation and oxidative stress are known to underlie either ischemia or reperfusion, leaded by HIF, TNF-α, NF-κB, IL-6 and ROS formation. However, the available approaches to prevent I/R damage has been unsuccessful so far. As agonists of peroxisome-proliferation activation receptor (PPAR) are described as transcription factors related to anti-inflammatory factors, we proposed to observe the effects of novel dual agonist, GQ-11, in I/R-related damage. METHODS: Male, Wistar rats, 60 days age and 305 g body weight average were treated with vehicle, pioglitazone or GQ-11 (20 mg/kg) for 7 consecutive days and were submitted to aorta clamping for 30 min followed by 3 h of reperfusion. 18F-fluorodeoxyglucose (18F-FDG), an analog of glucose associated with inflammation when accumulated, was observed in liver and bowel by positron emission tomography (PET). RESULTS: GQ-11 decreased 18F-FDG uptake in liver and bowel when compared to vehicle and pioglitazone. The treatment also modulated inflammatory markers IL-10, TGF-ß, IL-6, IL1-ß, TNFα, and CCL-2, besides antioxidant enzymes such as catalase, GPx and SOD. CONCLUSION: Inflammation and oxidative stress showed to be important processes to be regulated in I/R in order to prevent exacerbated responses that leads to cell/tissue dysfunction and death. PPAR agonists - including GQ-11 - might be promising agents in a strategy to avoid tissue dysfunction and death after cardiothoracic surgeries.

Cell surface Nucleolin represents a novel cellular target for neuroblastoma therapy.

BACKGROUND: Neuroblastoma (NB) represents the most frequent and aggressive form of extracranial solid tumor of infants. Nucleolin (NCL) is a protein overexpressed and partially localized on the cell surface of tumor cells of adult cancers. Little is known about NCL and pediatric tumors and nothing is reported about cell surface NCL and NB. METHODS: NB cell lines, Schwannian stroma-poor NB tumors and bone marrow (BM)-infiltrating NB cells were evaluated for the expression of cell surface NCL by Flow Cytometry, Imaging Flow Cytometry and Immunohistochemistry analyses. The cytotoxic activity of doxorubicin (DXR)-loaded nanocarriers decorated with the NCL-recognizing F3 peptide (T-DXR) was evaluated in terms of inhibition of NB cell proliferation and induction of cell death in vitro, whereas metastatic and orthotopic animal models of NB were used to examine their in vivo anti-tumor potential. RESULTS: NB cell lines, NB tumor cells (including patient-derived and Patient-Derived Xenografts-PDX) and 70% of BM-infiltrating NB cells show cell surface NCL expression. NCL staining was evident on both tumor and endothelial tumor cells in NB xenografts. F3 peptide-targeted nanoparticles, co-localizing with cell surface NCL, strongly associates with NB cells showing selective tumor cell internalization. T-DXR result significantly more effective, in terms of inhibition of cell proliferation and reduction of cell viability in vitro, and in terms of delay of tumor growth in all NB animal model tested, when compared to both control mice and those treated with the untargeted formulation. CONCLUSIONS: Our findings demonstrate that NCL could represent an innovative therapeutic cellular target for NB.

Enhanced therapeutic index of liposomal doxorubicin Myocet locally delivered by fibrin gels in immunodeficient mice bearing human neuroblastoma.

Caelyx and Myocet are clinically used liposomal forms of doxorubicin (Dox). To explore ways to improve their therapeutic index, we have studied their activity in vitro and in vivo when locally delivered by fibrin gels (FBGs). In vivo local toxic and anti-tumour activities of loaded FBGs were assessed in two immunodeficient mouse orthotopic human neuroblastoma (NB) models after application in the visceral space above the adrenal gland, either still tumour-bearing or after tumour removal. In parallel, in vitro assays were used to mimic the in vivo overlaying of FBGs on the tumour surface. FBGs were prepared with different concentrations of fibrinogen (FG) and clotted in the presence of Ca2+ and thrombin. The in vitro assays showed that FBGs loaded with Myocet possess a cytotoxic activity against NB cell lines generally greater than those loaded with free Dox or Caelyx. In vivo FBGs loaded with Myocet showed lower general and local toxicities as compared to gels loaded with Caelyx or free Dox, and also to free Dox administered i.v. (all treatments with Dox at 2.5 mg/Kg). The anti-tumour activity, evaluated in the two mouse orthotopic NB models of adjuvant and neo-adjuvant therapy, resulted in a better performance of FBGs loaded with Myocet compared to the other local (FBGs loaded with Caelyx or free Dox) or systemic (free Dox) treatments (administered at 2.5 and 5 mg/Kg Dox). Specifically, the application of FBGs at 40 mg/mL in the adjuvant model caused 92 % tumour volume reduction, while by the neo-adjuvant application of FBGs at 22 mg/mL a re-growing tumour volume reduction of 89 % was obtained. Taken together, our in vitro and in vivo results indicate a significantly higher activity for the FBGs loaded with Myocet. In particular, the lower toicity coupled with the higher anti-tumour activity on both the local treatment modalities strongly suggest a better therapeutic index when Myocet is administered through FBGs. Therefore, FBGs loaded with Myocet may be considered as a possible new tool for the loco-regional treatment of NB or even other tumour histotypes treatable by loco-regional chemotherapy.

Manganese-enhanced MRI (MEMRI) in breast and prostate cancers: Preliminary results exploring the potential role of calcium receptors.

PROCEDURES: To preliminary assess the relationship between Manganese Enhanced Magnetic Resonance Imaging (MEMRI) and the expression of calcium receptors in human prostate and breast cancer animal models. METHODS: NOD/SCID mice were inoculated with MDA-MB-231 breast cancer cells and prostate PC3 cancer cells to develop orthotopic or pseudometastatic cancer animal models. Mice were studied on a clinical 3T scanner by using a prototype birdcage coil before and after intravenous injection of MnCl2. Assessment of receptor's status was carried out after the MR images acquisition by immunohistochemistry on excised tumours. RESULTS: Manganese contrast enhancement in breast or prostate cancer animal models well correlated with CaSR expression (p<0.01), whereas TRPV6 expression levels appeared not relevant to the Mn uptake. CONCLUSION: Our preliminary results suggest that MEMRI appears an efficient tool to characterize human breast and prostate cancer animal models in the presence of different expression level of calcium receptors.

Fasting-mimicking diet and hormone therapy induce breast cancer regression.

Approximately 75% of all breast cancers express the oestrogen and/or progesterone receptors. Endocrine therapy is usually effective in these hormone-receptor-positive tumours, but primary and acquired resistance limits its long-term benefit1,2. Here we show that in mouse models of hormone-receptor-positive breast cancer, periodic fasting or a fasting-mimicking diet3-5 enhances the activity of the endocrine therapeutics tamoxifen and fulvestrant by lowering circulating IGF1, insulin and leptin and by inhibiting AKT-mTOR signalling via upregulation of EGR1 and PTEN. When fulvestrant is combined with palbociclib (a cyclin-dependent kinase 4/6 inhibitor), adding periodic cycles of a fasting-mimicking diet promotes long-lasting tumour regression and reverts acquired resistance to drug treatment. Moreover, both fasting and a fasting-mimicking diet prevent tamoxifen-induced endometrial hyperplasia. In patients with hormone-receptor-positive breast cancer receiving oestrogen therapy, cycles of a fasting-mimicking diet cause metabolic changes analogous to those observed in mice, including reduced levels of insulin, leptin and IGF1, with the last two remaining low for extended periods. In mice, these long-lasting effects are associated with long-term anti-cancer activity. These results support further clinical studies of a fasting-mimicking diet as an adjuvant to oestrogen therapy in hormone-receptor-positive breast cancer.

Combined Replenishment of miR-34a and let-7b by Targeted Nanoparticles Inhibits Tumor Growth in Neuroblastoma Preclinical Models.

Neuroblastoma (NB) tumor substantially contributes to childhood cancer mortality. The design of novel drugs targeted to specific molecular alterations becomes mandatory, especially for high-risk patients burdened by chemoresistant relapse. The dysregulated expression of MYCN, ALK, and LIN28B and the diminished levels of miR-34a and let-7b are oncogenic in NB. Due to the ability of miRNA-mimics to recover the tumor suppression functions of miRNAs underexpressed into cancer cells, safe and efficient nanocarriers selectively targeted to NB cells and tested in clinically relevant mouse models are developed. The technology exploits the nucleic acids negative charges to build coated-cationic liposomes, then functionalized with antibodies against GD2 receptor. The replenishment of miR-34a and let-7b by NB-targeted nanoparticles, individually and more powerfully in combination, significantly reduces cell division, proliferation, neoangiogenesis, tumor growth and burden, and induces apoptosis in orthotopic xenografts and improves mice survival in pseudometastatic models. These functional effects highlight a cooperative down-modulation of MYCN and its down-stream targets, ALK and LIN28B, exerted by miR-34a and let-7b that reactivate regulatory networks leading to a favorable therapeutic response. These findings demonstrate a promising therapeutic efficacy of miR-34a and let-7b combined replacement and support its clinical application as adjuvant therapy for high-risk NB patients.

Fibrin gels entrapment of a doxorubicin-containing targeted polycyclodextrin: Evaluation of in vivo antitumor activity in orthotopic models of human neuroblastoma.

In vivo local antitumor activity of fibrin gels (FBGs) loaded with the poly-cyclodextrin oCD-NH2/Dox, compared to free Dox, was evaluated in two mouse orthotopic neuroblastoma (NB) models, after positioning of the releasing devices in the visceral space. FBGs were prepared at the fibrinogen (FG) concentrations of 22 and 40 mg/ml clotted in the presence of 0.81 mM/mg FG Ca2+ and 1.32 U/mg FG thrombin. Our results indicate that FBGs loaded with oCD-NH2/Dox and applied as neoadjuvant loco-regional treatment, show an antitumor activity significantly greater than that displayed by the same FBGs loaded with identical dose of Dox or after free Dox administered intra venous (iv). In particular, FBGs prepared at 40 mg/ml showed a slightly lower antitumor activity, although after their positioning we observed a significant initial reduction of tumor burden lasting for several days after gel implantation. FBGs at 22 mg/ml loaded with oCD-NH2/Dox and applied after tumor removal (adjuvant treatment model) showed a significantly better antitumor activity than the iv administration of free Dox, with 90% tumor regrowth reduction compared to untreated controls. In all cases the weight loss post-treatment was limited after gel application, although in the adjuvant treatment the loss of body weight lasted longer than in the other treatment modality. In accordance with our recent published data on the low local toxic effects of FBGs, the present findings also underline an increase of the therapeutic index of Dox when locally administered through FBGs loaded with the oCD-NH2/Dox complex.

Fibrin Gels Entrapment of a Poly-Cyclodextrin Nanocarrier as a Doxorubicin Delivery System in an Orthotopic Model of Neuroblastoma: Evaluation of In Vitro Activity and In Vivo Toxicity.

PURPOSE: Fibrin gels (FBGs) are potential delivery vehicles for many drugs, and can be easily prepared from purified components. We previously demonstrated their applicability for the release of different doxorubicin (Dox) nanoparticles used clinically or in an experimental stage, such as its inclusion complex with the amino ß-cyclodextrin polymer (oCD-NH2/Dox). Here we extend these studies by in vitro and in vivo evaluations. METHODS: An in vitro cytotoxicity model consisting of an overlay of a neuroblastoma (NB) cell-containing agar layer above a drug-loaded FBG layer was used. Local toxicity in vivo (histology and blood analysis) was studied in a mouse orthotopic NB model (SHSY5YLuc+ cells implanted into the left adrenal gland). RESULTS: In vitro data show that FBGs loaded with oCD-NH2/Dox have a slightly lower cytotoxicity against NB cell lines than those loaded with Dox. Fibrinogen (FG), and Ca2+ concentrations may modify this activity. In vivo data support a lower general and local toxicity for FBGs loaded with oCD-NH2/Dox than those loaded with Dox. CONCLUSION: Our results suggest a possible increase of the therapeutic index of Dox when locally administered through FBGs loaded with oCD-NH2/Dox, opening the possibility of using these releasing systems for the treatment of neuroblastoma.

LncRNA EPR controls epithelial proliferation by coordinating Cdkn1a transcription and mRNA decay response to TGF-ß.

Long noncoding RNAs (lncRNAs) are emerging as regulators of fundamental biological processes. Here we report on the characterization of an intergenic lncRNA expressed in epithelial tissues which we termed EPR (Epithelial cell Program Regulator). EPR is rapidly downregulated by TGF-ß and its sustained expression largely reshapes the transcriptome, favors the acquisition of epithelial traits, and reduces cell proliferation in cultured mammary gland cells as well as in an animal model of orthotopic transplantation. EPR generates a small peptide that localizes at epithelial cell junctions but the RNA molecule per se accounts for the vast majority of EPR-induced gene expression changes. Mechanistically, EPR interacts with chromatin and regulates Cdkn1a gene expression by affecting both its transcription and mRNA decay through its association with SMAD3 and the mRNA decay-promoting factor KHSRP, respectively. We propose that EPR enables epithelial cells to control proliferation by modulating waves of gene expression in response to TGF-ß.

Enhancement of Tumor Homing by Chemotherapy-Loaded Nanoparticles.

Targeted delivery of anticancer drugs with nanocarriers can reduce side effects and ameliorate therapeutic efficacy. However, poorly perfused and dysfunctional tumor vessels limit the transport of the payload into solid tumors. The use of tumor-penetrating nanocarriers might enhance tumor uptake and antitumor effects. A peptide containing a tissue-penetrating (TP) consensus motif, capable of recognizing neuropilin-1, is here fused to a neuroblastoma-targeting peptide (pep) previously developed. Neuroblastoma cell lines and cells derived from both xenografts and high-risk neuroblastoma patients show overexpression of neuropilin-1. In vitro studies reveal that TP-pep binds cell lines and cells derived from neuroblastoma patients more efficiently than pep. TP-pep, after coupling to doxorubicin-containing stealth liposomes (TP-pep-SL[doxorubicin]), enhances their uptake by cells and cytotoxic effects in vitro, while increasing tumor-binding capability and homing in vivo. TP-pep-SL[doxorubicin] treatment enhances the Evans Blue dye accumulation in tumors but not in nontumor tissues, pointing to selective increase of vascular permeability in tumor tissues. Compared to pep-SL[doxorubicin], TP-pep-SL[doxorubicin] shows an increased antineuroblastoma activity in three neuroblastoma animal models mimicking the growth of neuroblastoma in humans. The enhancement of drug penetration in tumors by TP-pep-targeted nanoparticles may represent an innovative strategy for neuroblastoma.

Characterization of drug release from fibrin gels loaded with different pharmaceutical and experimental doxorubicin formulations.

BACKGROUND: Local delivery of anticancer drugs represents a desirable type of treatment. Nevertheless, characteristics such as availability, biocompatibility, ease of operation, and efficacy sometimes represent difficult to overcome hurdles. Fibrin gels (FBGs) may be attractive biomaterials for local treatment when loaded with different chemotherapeutics or with polymer-anticancer-drug conjugates and nanoparticles. These components, linked together, might represent candidates to counteract local recurrences or reduce the volume of inoperable tumors. In the present study we analyzed the features of in vitro release of different formulations of doxorubicin (DOXO) from FBGs, and in vivo FBGs degradation. METHODS: In vitro DOXO release from FBGs was studied as a function of thrombin and Ca2+ ion concentrations. DOXO was loaded in FBGs either alone or pre-incorporated in nanoparticles characterized by different physical features. The FBGs in vivo degradation was analyzed after sc or ip positioning. RESULTS: Our results suggest that each of the factors involved in the FBGs preparation may have different effects on drug release. In particular, the fibrinogen (FG) concentration and, above all, the DOXO formulation were found to have the greatest impact. Not surprisingly, we have also found a longer permanence in vivo of FBGs prepared at the highest thrombin, Ca2+ ion, and FG concentrations. CONCLUSIONS: The aim of this work was to study the effect of several conditions for preparing drug delivery systems based on FBGs loaded with different clinical or experimental formulations of DOXO. Our data identify some of these modalities that will be tested in vivo to evaluate their antitumor activity.

CHL1 gene acts as a tumor suppressor in human neuroblastoma.

Neuroblastoma is an aggressive, relapse-prone childhood tumor of the sympathetic nervous system that accounts for 15% of pediatric cancer deaths. A distal portion of human chromosome 3p is often deleted in neuroblastoma, this region may contain one or more putative tumor suppressor genes. A 2.54 Mb region at 3p26.3 encompassing the smallest region of deletion pinpointed CHL1 gene, the locus for neuronal cell adhesion molecule close homolog of L1. We found that low CHL1 expression predicted poor outcome in neuroblastoma patients. Here we have used two inducible cell models to analyze the impact of CHL1 on neuroblastoma biology. Over-expression of CHL1 induced neurite-like outgrowth and markers of neuronal differentiation in neuroblastoma cells, halted tumor progression, inhibited anchorage-independent colony formation, and suppressed the growth of human tumor xenografts. Conversely, knock-down of CHL1 induced neurite retraction and activation of Rho GTPases, enhanced cell proliferation and migration, triggered colony formation and anchorage-independent growth, accelerated growth in orthotopic xenografts mouse model. Our findings demonstrate unambiguously that CHL1 acts as a regulator of proliferation and differentiation of neuroblastoma cells through inhibition of the MAPKs and Akt pathways. CHL1 is a novel candidate tumor suppressor in neuroblastoma, and its associated pathways may represent a promising target for future therapeutic interventions.

A High-Content Screening of Anticancer Compounds Suggests the Multiple Tyrosine Kinase Inhibitor Ponatinib for Repurposing in Neuroblastoma Therapy.

Novel druggable targets have been discovered in neuroblastoma (NB), paving the way for more effective treatments. However, children with high-risk NB still show high mortality rates prompting for a search of novel therapeutic options. Here, we aimed at repurposing FDA-approved drugs for NB treatment by performing a high-content screening of a 349 anticancer compounds library. In the primary screening, we employed three NB cell lines, grown as three-dimensional (3D) multicellular spheroids, which were treated with 10 µmol/L of the library compounds for 72 hours. The viability of 3D spheroids was evaluated using a high-content imaging approach, resulting in a primary hit list of 193 compounds. We selected 60 FDA-approved molecules and prioritized drugs with multi-target activity, discarding those already in use for NB treatment or enrolled in NB clinical trials. Hence, 20 drugs were further tested for their efficacy in inhibiting NB cell viability, both in two-dimensional and 3D models. Dose-response curves were then supplemented with the data on side effects, therapeutic index, and molecular targets, suggesting two multiple tyrosine kinase inhibitors, ponatinib and axitinib, as promising candidates for repositioning in NB. Indeed, both drugs showed induction of cell-cycle block and apoptosis, as well as inhibition of colony formation. However, only ponatinib consistently affected migration and inhibited invasion of NB cells. Finally, ponatinib also proved effective inhibition of tumor growth in orthotopic NB mice, providing the rationale for its repurposing in NB therapy. Mol Cancer Ther; 17(7); 1405-15. ©2018 AACR.

Microenvironmental regulation of the IL-23R/IL-23 axis overrides chronic lymphocytic leukemia indolence.

Although the progression of chronic lymphocytic leukemia (CLL) requires the cooperation of the microenvironment, the exact cellular and molecular mechanisms involved are still unclear. We investigated the interleukin (IL)-23 receptor (IL-23R)/IL-23 axis and found that circulating cells from early-stage CLL patients with shorter time-to-treatment, but not of those with a more benign course, expressed a defective form of the IL-23R complex lacking the IL-12Rß1 chain. However, cells from both patient groups expressed the complete IL-23R complex in tissue infiltrates and could be induced to express the IL-12Rß1 chain when cocultured with activated T cells or CD40L+ cells. CLL cells activated in vitro in this context produced IL-23, a finding that, together with the presence of IL-23 in CLL lymphoid tissues, suggests the existence of an autocrine/paracrine loop inducing CLL cell proliferation. Interference with the IL-23R/IL-23 axis using an anti-IL-23p19 antibody proved effective in controlling disease onset and expansion in xenografted mice, suggesting potential therapeutic strategies.