MIF/CXCR4 signaling axis contributes to survival, invasion, and drug resistance of metastatic neuroblastoma cells in the bone marrow microenvironment

Background: The bone marrow (BM) is the most common site of dissemination in patients with aggressive, metastatic neuroblastoma (NB). However, the molecular mechanisms underlying the aggressive behavior of NB cells in the BM niche are still greatly unknown. In the present study, we explored biological mechanisms that play a critical role in NB cell survival and progression in the BM and investigated potential therapeutic targets. Methods: Patient-derived bone marrow (BM) primary cultures were generated using fresh BM aspirates obtained from NB patients. NB cell lines were cultured in the presence of BM conditioned media containing cell-secreted factors, and under low oxygen levels (1% O2) to mimic specific features of the BM microenvironment of high-risk NB patients. The BM niche was explored using cytokine profiling assays, cell migration-invasion and viability assays, flow cytometry and analysis of RNA-sequencing data. Selective pharmacological inhibition of factors identified as potential mediators of NB progression within the BM niche was performed in vitro and in vivo. Results: We identified macrophage migration inhibitory factor (MIF) as a key inflammatory cytokine involved in BM infiltration. Cytokine profiling and RNA-sequencing data analysis revealed NB cells as the main source of MIF in the BM, suggesting a potential role of MIF in tumor invasion. Exposure of NB cells to BM-conditions increased NB cell-surface expression of the MIF receptor CXCR4, which was associated with increased cell viability, enhanced migration-invasion, and activation of PI3K/AKT and MAPK/ERK signaling pathways. Moreover, subcutaneous co-injection of NB and BM cells enhanced tumor engraftment in mice. MIF inhibition with 4-IPP impaired in vitro NB aggressiveness, and improved drug response while delayed NB growth, improving survival of the NB xenograft model. Conclusions: Our findings suggest that BM infiltration by NB cells may be mediated, in part, by MIF-CXCR4 signaling. We demonstrate the antitumor efficacy of MIF targeting in vitro and in vivo that could represent a novel therapeutic target for patients with disseminated high-risk NB

The PARP inhibitor olaparib enhances the sensitivity of Ewing sarcoma to trabectedin

Producción CientíficaRecent preclinical evidence has suggested that Ewing Sarcoma (ES) bearing EWSR1-ETS fusions could be particularly sensitive to PARP inhibitors (PARPinh) in combination with DNA damage repair (DDR) agents. Trabectedin is an antitumoral agent that modulates EWSR1-FLI1 transcriptional functions, causing DNA damage. Interestingly, PARP1 is also a transcriptional regulator of EWSR1-FLI1, and PARPinh disrupts the DDR machinery. Thus, given the impact and apparent specificity of both agents with regard to the DNA damage/DDR system and EWSR1-FLI1 activity in ES, we decided to explore the activity of combining PARPinh and Trabectedin in in vitro and in vivo experiments. The combination of Olaparib and Trabectedin was found to be highly synergistic, inhibiting cell proliferation, inducing apoptosis, and the accumulation of G2/M. The drug combination also enhanced γH2AX intranuclear accumulation as a result of DNA damage induction, DNA fragmentation and global DDR deregulation, while EWSR1-FLI1 target expression remained unaffected. The effect of the drug combination was corroborated in a mouse xenograft model of ES and, more importantly, in two ES patient-derived xenograft (PDX) models in which the tumors showed complete regression. In conclusion, the combination of the two agents leads to a biologically significant deregulation of the DDR machinery that elicits relevant antitumor activity in preclinical models and might represent a promising therapeutic tool that should be further explored for translation to the clinical setting.Ministerio de Economía y Competitividad (PI081828)Ministerio de Economía y Competitividad (RD06/0020/0059 )Ministerio de Economía y Competitividad (RD12/0036/0017)Ministerio de Economía y Competitividad (PT13/0010/0056

SN-38-loaded nanofiber matrices for local control of pediatric solid tumors after subtotal resection surgery

In addition to surgery, local tumor control in pediatric oncology requires new treatments as an alternative to radiotherapy. SN-38 is an anticancer drug with proved activity against several pediatric solid tumors including neuroblastoma, rhabdomyosarcoma and Ewing sarcoma. Taking advantage of the extremely low aqueous solubility of SN-38, we have developed a novel drug delivery system (DDS) consisting of matrices made of poly(lactic acid) electrospun polymer nanofibers loaded with SN-38 microcrystals for local release in difficult-to-treat pediatric solid tumors. To model the clinical scenario, we conducted extensive preclinical experiments to characterize the biodistribution of the released SN-38 using microdialysis sampling in vivo. We observed that the drug achieves high concentrations in the virtual space of the surgical bed and penetrates a maximum distance of 2 mm within the tumor bulk. Subsequently, we developed a model of subtotal tumor resection in clinically relevant pediatric patient-derived xenografts and used such models to provide evidence of the activity of the SN-38 DDS to inhibit tumor regrowth. We propose that this novel DDS could represent a potential future strategy to avoid harmful radiation therapy as a primary tumor control together with surgery.Fil: Monterrubio, Carles. Fundació Sant Joan de Déu; España. Esplugues de Llobregat; EspañaFil: Pascual Pasto, Guillem. Fundació Sant Joan de Déu; España. Esplugues de Llobregat; EspañaFil: Cano, Francisco. Universidad Politécnica de Catalunya; EspañaFil: Vila Ubach, Monica. Fundació Sant Joan de Déu; España. Esplugues de Llobregat; EspañaFil: Manzanares, Alejandro. Fundació Sant Joan de Déu; España. Esplugues de Llobregat; EspañaFil: Schaiquevich, Paula Susana. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Gobierno de la Ciudad de Buenos Aires. Hospital de Pediatría "Juan P. Garrahan"; ArgentinaFil: Tornero, Jose A.. Universidad Politécnica de Catalunya; EspañaFil: Sosnik, Alejandro Dario. Technion-Israel Institute of Technology; IsraelFil: Mora, Jaume. Fundació Sant Joan de Déu; España. Esplugues de Llobregat; EspañaFil: Montero Carcaboso, Angel. Fundació Sant Joan de Déu; España. Esplugues de Llobregat; Españ

Preclinical platform of retinoblastoma xenografts recapitulating human disease and molecular markers of dissemination

Translational research in retinoblastoma – a pediatric tumor that originates during the development of the retina – would be improved by the creation of new patient-derived models. Using tumor samples from enucleated eyes we established a new battery of preclinical models that grow in vitro in serum-free medium and in vivo in immunodeficient mice. To examine whether the new xenografts recapitulate human disease and disseminate from the retina to the central nervous system, we evaluated their histology and the presence of molecular markers of dissemination that are used in the clinical setting to detect extraocular metastases. We evaluated GD2 synthase and CRX as such markers and generated a Taqman real-time quantitative PCR method to measure CRX mRNA for rapid, sensitive and specific quantification of local and metastatic tumor burden. This approach was able to detect 1 human retinoblastoma cell in 100.000 mouse brain cells. Our research adds novel preclinical tools for the discovery of new retinoblastoma treatments for clinical translation.Fil: Pascual Pasto, Guillem. Fundacio Sant Joan de Deu; España. Hospital Sant Joan de Deu Barcelona; EspañaFil: Olaciregui, Nagore G.. Fundacio Sant Joan de Deu; España. Hospital Sant Joan de Deu Barcelona; EspañaFil: Vila Ubach, Monica. Fundacio Sant Joan de Deu; España. Hospital Sant Joan de Deu Barcelona; EspañaFil: Paco, Sonia. Fundacio Sant Joan de Deu; España. Hospital Sant Joan de Deu Barcelona; EspañaFil: Monterrubio, Carles. Fundacio Sant Joan de Deu; España. Hospital Sant Joan de Deu Barcelona; EspañaFil: Rodriguez, Eva. Fundacio Sant Joan de Deu; España. Hospital Sant Joan de Deu Barcelona; EspañaFil: Winter, Ursula Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Hospital Sant Joan de Deu Barcelona; España. Gobierno de la Ciudad de Buenos Aires. Hospital de Pediatría "Juan P. Garrahan"; ArgentinaFil: Batalla Vilacis, Mireia. Fundacio Sant Joan de Deu; EspañaFil: Catala, Jaume. Hospital Sant Joan de Deu Barcelona; EspañaFil: Salvador, Hector. Fundacio Sant Joan de Deu; España. Hospital Sant Joan de Deu Barcelona; EspañaFil: Parareda, Andreu. Fundacio Sant Joan de Deu; España. Hospital Sant Joan de Deu Barcelona; EspañaFil: Schaiquevich, Paula Susana. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Gobierno de la Ciudad de Buenos Aires. Hospital de Pediatría "Juan P. Garrahan"; ArgentinaFil: Suñol, Mariona. Hospital Sant Joan de Deu Barcelona; EspañaFil: Mora, Jaume. Fundacio Sant Joan de Deu; España. Hospital Sant Joan de Deu Barcelona; EspañaFil: Lavarino, Cinzia. Fundacio Sant Joan de Deu; España. Hospital Sant Joan de Deu Barcelona; EspañaFil: de Torres, Carmen. Fundacio Sant Joan de Deu; España. Hospital Sant Joan de Deu Barcelona; EspañaFil: Chantada, Guillermo Luis. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Fundacio Sant Joan de Deu; España. Hospital Sant Joan de Deu Barcelona; España. Gobierno de la Ciudad de Buenos Aires. Hospital de Pediatría "Juan P. Garrahan"; ArgentinaFil: Carcaboso, Angel M.. Fundacio Sant Joan de Deu; España. Hospital Sant Joan de Deu Barcelona; Españ

Increased delivery of chemotherapy to the vitreous by inhibition of the blood-retinal barrier

Treatment of retinoblastoma -a pediatric cancer of the developing retina- might benefit from strategies to inhibit the blood-retinal barrier (BRB). The potent anticancer agent topotecan is a substrate of efflux transporters BCRP and P-gp, which are expressed at the BRB to restrict vitreous and retinal distribution of xenobiotics. In this work we have studied vitreous and retinal distribution, tumor accumulation and antitumor activity of topotecan, using pantoprazole as inhibitor of BCRP and P-gp. We used rabbit and mouse eyes as BRB models and patient-derived xenografts as retinoblastoma models. To validate the rabbit BRB model we stained BCRP and P-gp in the retinal vessels. Using intravitreous microdialysis we showed that the penetration of the rabbit vitreous by lactone topotecan increased significantly upon concomitant administration of pantoprazole (P = 0.0285). Pantoprazole also increased topotecan penetration of the mouse vitreous, measured as the vitreous-to-plasma topotecan concentration ratio at the steady state (P = 0.0246). Pantoprazole increased topotecan antitumor efficacy and intracellular penetration in retinoblastoma in vitro, but did not enhance intratumor drug distribution and survival in mice bearing the intraocular human tumor HSJD-RBT-2. Anatomical differences with the clinical setting likely limited our in vivo study, since xenografts were poorly vascularized masses that loaded most of the vitreous compartment. We conclude that pharmacological modulation of the BRB is feasible, enhances anticancer drug distribution into the vitreous and might have clinical implications in retinoblastoma.Fil: Pascual-Pasto, Guillem. Hospital Sant Joan de Deu Barcelona; España. Institut de Recerca Sant Joan de Deu; EspañaFil: Olaciregui, Nagore G.. Hospital Sant Joan de Deu Barcelona; España. Institut de Recerca Sant Joan de Deu; EspañaFil: Opezzo, Javier A. W.. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Farmacología; ArgentinaFil: Castillo Ecija, Helena. Hospital Sant Joan de Deu Barcelona; España. Institut de Recerca Sant Joan de Deu; EspañaFil: Cuadrado Vilanova, Maria. Hospital Sant Joan de Deu Barcelona; España. Institut de Recerca Sant Joan de Deu; EspañaFil: Paco, Sonia. Hospital Sant Joan de Deu Barcelona; España. Institut de Recerca Sant Joan de Deu; EspañaFil: Rivero, Ezequiel Mariano. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Vila Ubach, Monica. Hospital Sant Joan de Deu Barcelona; España. Institut de Recerca Sant Joan de Deu; EspañaFil: Restrepo Perdomo, Camilo A.. Hospital Sant Joan de Deu Barcelona; EspañaFil: Torrebadell, Montserrat. Hospital Sant Joan de Deu Barcelona; España. Institut de Recerca Sant Joan de Deu; EspañaFil: Suñol, Mariona. Hospital Sant Joan de Deu Barcelona; EspañaFil: Schaiquevich, Paula Susana. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Gobierno de la Ciudad de Buenos Aires. Hospital de Pediatría "Juan P. Garrahan"; ArgentinaFil: Mora, Jaume. Hospital Sant Joan de Deu Barcelona; España. Institut de Recerca Sant Joan de Deu; EspañaFil: Bramuglia, Guillermo Federico. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Farmacología; ArgentinaFil: Chantada, Guillermo Luis. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Gobierno de la Ciudad de Buenos Aires. Hospital de Pediatría "Juan P. Garrahan"; Argentina. Hospital Sant Joan de Deu Barcelona; España. Institut de Recerca Sant Joan de Deu; EspañaFil: Carcaboso, Angel M.. Hospital Sant Joan de Deu Barcelona; España. Institut de Recerca Sant Joan de Deu; Españ

Targeted drug distribution in tumor extracellular fluid of GD2-expressing neuroblastoma patient-derived xenografts using SN-38-loaded nanoparticles conjugated to the monoclonal antibody 3F8

Neuroblastoma is a pediatric solid tumor with high expression of the tumor associated antigen disialoganglioside GD2. Despite initial response to induction therapy, nearly 50% of high-risk neuroblastomas recur because of chemoresistance. Here we encapsulated the topoisomerase-I inhibitor SN-38 in polymeric nanoparticles (NPs) surface-decorated with the anti-GD2 mouse mAb 3F8 at a mean density of seven antibody molecules per NP. The accumulation of drug-loaded NPs targeted with 3F8 versus with control antibody was monitored by microdialysis in patient-derived GD2-expressing neuroblastoma xenografts. We showed that the extent of tumor penetration by SN-38 was significantly higher in mice receiving the targeted nano-drug delivery system when compared to non-targeted system or free drug. This selective penetration of the tumor extracellular fluid translated into a strong anti-tumor effect prolonging survival of mice bearing GD2-high neuroblastomas in vivo.Fil: Monterrubio, Carles. Institut de Recerca Sant Joan de Deu; España. Hospital Sant Joan de Deu Barcelona; EspañaFil: Paco, Sonia. Institut de Recerca Sant Joan de Deu; España. Hospital Sant Joan de Deu Barcelona; EspañaFil: Olaciregui, Nagore G.. Institut de Recerca Sant Joan de Deu; España. Hospital Sant Joan de Deu Barcelona; EspañaFil: Pascual Pasto, Guillem. Institut de Recerca Sant Joan de Deu; España. Hospital Sant Joan de Deu Barcelona; EspañaFil: Vila Ubach, Monica. Institut de Recerca Sant Joan de Deu; España. Hospital Sant Joan de Deu Barcelona; EspañaFil: Cuadrado Vilanova, Maria. Institut de Recerca Sant Joan de Deu; España. Hospital Sant Joan de Deu Barcelona; EspañaFil: Ferrandiz, M. Mar. Institut de Recerca Sant Joan de Deu; España. Hospital Sant Joan de Deu Barcelona; EspañaFil: Castillo Ecija, Helena. Institut de Recerca Sant Joan de Deu; España. Hospital Sant Joan de Deu Barcelona; EspañaFil: Glisoni, Romina Julieta. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Nanobiotecnología. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Nanobiotecnología; ArgentinaFil: Kuplennik, Nataliya. Technion-Israel Institute of Technology; IsraelFil: Jungbluth, Achim. Memorial Sloan-Kettering Cancer Center; Estados UnidosFil: de Torres, Carmen. Institut de Recerca Sant Joan de Deu; España. Hospital Sant Joan de Deu Barcelona; EspañaFil: Lavarino, Cinzia. Institut de Recerca Sant Joan de Deu; España. Hospital Sant Joan de Deu Barcelona; EspañaFil: Cheung, N. K. V.. Memorial Sloan-Kettering Cancer Center; Estados UnidosFil: Mora, Jaume. Institut de Recerca Sant Joan de Deu; España. Hospital Sant Joan de Deu Barcelona; EspañaFil: Sosnik, Alejandro Dario. Technion-Israel Institute of Technology; IsraelFil: Montero Carcaboso, Angel. Institut de Recerca Sant Joan de Deu; España. Hospital Sant Joan de Deu Barcelona; Españ

Therapeutic targeting of the RB1 pathway in retinoblastoma with the oncolytic adenovirus VCN-01

Retinoblastoma is a pediatric solid tumor of the retina activated upon homozygous inactivation of the tumor suppressor RB1. VCN-01 is an oncolytic adenovirus designed to replicate selectively in tumor cells with high abundance of free E2F-1, a consequence of a dysfunctional RB1 pathway. Thus, we reasoned that VCN-01 could provide targeted therapeutic activity against even chemoresistant retinoblastoma. In vitro, VCN-01 effectively killed patient-derived retinoblastoma models. In mice, intravitreous administration of VCN-01 in retinoblastoma xenografts induced tumor necrosis, improved ocular survival compared with standard-of-care chemotherapy, and prevented micrometastatic dissemination into the brain. In juvenile immunocompetent rabbits, VCN-01 did not replicate in retinas, induced minor local side effects, and only leaked slightly and for a short time into the blood. Initial phase 1 data in patients showed the feasibility of the administration of intravitreous VCN-01 and resulted in antitumor activity in retinoblastoma vitreous seeds and evidence of viral replication markers in tumor cells. The treatment caused local vitreous inflammation but no systemic complications. Thus, oncolytic adenoviruses targeting RB1 might provide a tumor-selective and chemotherapy-independent treatment option for retinoblastoma.Fil: Pascual-Pasto, Guillem. Hospital Sant Joan de Déu; EspañaFil: Bazan-Peregrino, Miriam. No especifíca;Fil: Olaciregui, Nagore G.. Hospital Sant Joan de Déu; EspañaFil: Restrepo Perdomo, Camilo A.. Hospital Sant Joan de Déu; EspañaFil: Mato Berciano, Ana. No especifíca;Fil: Ottaviani, Daniela. Centre National de la Recherche Scientifique; FranciaFil: Weber, Klaus. No especifíca;Fil: Correa, Genoveva. Hospital Sant Joan de Déu; EspañaFil: Paco, Sonia. Hospital Sant Joan de Déu; EspañaFil: Vila Ubach, Monica. Hospital Sant Joan de Déu; EspañaFil: Cuadrado Vilanova, Maria. Hospital Sant Joan de Déu; EspañaFil: Castillo Ecija, Helena. Hospital Sant Joan de Déu; EspañaFil: Botteri, Gaia. Hospital Sant Joan de Déu; EspañaFil: Garcia Gerique, Laura. Hospital Sant Joan de Déu; EspañaFil: Moreno Gilabert, Helena. Hospital Sant Joan de Déu; EspañaFil: Gimenez Alejandre, Marta. No especifíca;Fil: Alonso Lopez, Patricia. No especifíca;Fil: Farrera Sal, Marti. No especifíca;Fil: Torres Manjon, Silvia. Instituto de Investigación Biomédica de Bellvitge; EspañaFil: Ramos Lozano, Dolores. Instituto de Investigación Biomédica de Bellvitge; EspañaFil: Moreno, Rafael. Instituto de Investigación Biomédica de Bellvitge; EspañaFil: Aerts, Isabelle. Centre National de la Recherche Scientifique; FranciaFil: Doz, François. Universite Paris Descartes; Francia. Centre National de la Recherche Scientifique; FranciaFil: Cassoux, Nathalie. Centre National de la Recherche Scientifique; Francia. Universite Paris Descartes; FranciaFil: Chapeaublanc, Elodie. Centre National de la Recherche Scientifique; FranciaFil: Torrebadell, Montserrat. Hospital Sant Joan de Déu; EspañaFil: Roldan, Monica. Hospital Sant Joan de Déu; EspañaFil: König, Andrés. No especifíca;Fil: Suñol, Mariona. Hospital Sant Joan de Déu; EspañaFil: Claverol, Joana. Hospital Sant Joan de Déu; EspañaFil: Lavarino, Cinzia. Hospital Sant Joan de Déu; EspañaFil: De Torres, Carmen. Hospital Sant Joan de Déu; EspañaFil: Fu, Ligia. Hospital Escuela Universitario; HondurasFil: Radvanyi, François. Centre National de la Recherche Scientifique; FranciaFil: Munier, Francis L.. Hopital Ophtalmique Jules Gonin; SuizaFil: Catalá-Mora, Jaume. Hospital Sant Joan de Déu; EspañaFil: Mora, Jaume. Hospital Sant Joan de Déu; EspañaFil: Alemany, Ramón. Instituto de Investigación Biomédica de Bellvitge; EspañaFil: Cascalló, Manel. No especifíca;Fil: Chantada, Guillermo Luis. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Gobierno de la Ciudad de Buenos Aires. Hospital de Pediatría "Juan P. Garrahan"; ArgentinaFil: Montero Carcaboso, Angel. Hospital Sant Joan de Déu; Españ

Influence of mechanical ventilation and sepsis on redox balance in diaphragm, myocardium, limb muscles, and lungs

Mechanical ventilation (MV), using high tidal volumes (VT), causes lung (ventilator-induced lung injury [VILI]) and distant organ injury. Additionally, sepsis is characterized by increased oxidative stress. We tested whether MV is associated with enhanced oxidative stress in sepsis, the commonest underlying condition in clinical acute lung injury. Protein carbonylation and nitration, antioxidants, and inflammation (immunoblotting) were evaluated in diaphragm, gastrocnemius, soleus, myocardium, and lungs of nonseptic and septic (cecal ligation and puncture 24 hours before MV) rats undergoing MV (n = 7 per group) for 150 minutes using 3 different strategies (low VT [VT = 9 mL/kg], moderate VT [VT = 15 mL/kg], and high VT [VT = 25 mL/kg]) and in nonventilated control animals.5.030 JCR (2014) Q1, 2/30 Medical laboratory technology, 17/153 Medicine, general & internal, 17/123 Medicine, research & experimentalUE