TSPO PET Imaging as a Potent Non-Invasive Biomarker for Diffuse Intrinsic Pontine Glioma in a Patient-Derived Orthotopic Rat Model

Diffuse intrinsic pontine gliomas (DIPG), the first cause of cerebral pediatric cancer death, will greatly benefit from specific and non-invasive biomarkers for patient follow-up and monitoring of drug efficacy. Since biopsies are challenging for brain tumors, molecular imaging may be a technique of choice to target and follow tumor evolution. So far, MR remains the imaging technique of reference for DIPG, although it often fails to define the extent of tumors, an essential parameter for therapeutic efficacy assessment. Thanks to its high sensitivity, positron emission tomography (PET) offers a unique way to target specific biomarkers in vivo. We demonstrated in a patient-derived orthotopic xenograft (PDOX) model in the rat that the translocator protein of 18 kDa (TSPO) may be a promising biomarker for monitoring DIPG tumors. We studied the distribution of 18F-DPA-714, a TSPO radioligand, in rats inoculated with HSJD-DIPG-007 cells. The primary DIPG human cell line HSJD-DIPG-007 highly represents this pediatric tumor, displaying the most prevalent DIPG mutations, H3F3A (K27M) and ACVR1 (R206H). Kinetic modeling and parametric imaging using the brain 18F-DPA-714 PET data enabled specific delineation of the DIPG tumor area, which is crucial for radiotherapy dose management

S100 protein, an early and prognostic serum marker for brain damage following cardiac surgery

S100 protein is a marker for brain damage easily analysed in blood. The serum level immediatly after termination of extracorporeal circulation (ECC) is increased and associated to age and the duration of perfusion. A prolonged residual concentration indicates cellular brain damage. The objective of this study was to ascertain if the prognosis of individual patients, with stroke following ECC, could be predicted by the S100 levels. 224 patients with an uneventful outcome constituted a reference population. Blood samples for the analysis of S100 were collected at termination of ECC (TO) and at 5, 15 and 48 hours thereafter. Eight patients with stroke following ECC were analysed. In 4, S100 levels continued to increase after ECC. The volume of brain infarctions were large and in 3 of the patients outcome was fatal. In 4 other patients with small volume brain injury S100 dropped initially, but after 5 hours the elimination slowed down, indicating a small but persistant release. In 3 of these patients recovery was complete or with minor residuals. The fourth patient never regained consciousness and died. The levels from serial S100 measurements early after cardiac surgery were related to the size of brain injury and a prognostic value is therefore suggested

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

Panobinostat decreases proliferation and viability and induces apoptosis independent of H3.3-K27M status <i>in vitro</i>.

<p>Three independent murine brainstem glioma H3.3-K27M and H3.3-WT cell lines (driven by PDGF-B, Cre-induced p53 loss, and either H3.3-K27M or H3.3-WT, respectively) were treated with varying doses of panobinostat for 48 h. (<b>A</b>) Proliferation assessment using a BrdU assay. (<b>B</b>) Cell viability assessment using a CellTiter-Glo assay. (<b>C</b>) Apoptosis assessment using a Caspase-Glo 3/7 assay. Results from the different drug concentrations were normalized to the control (0 nM Panobinostat, 0.1% DMSO). Each experiment was performed in triplicate and independently repeated three times for each tumor cell line. Error bars represent mean with SEM. Statistical significance to compare H3.3-K27M and H3.3-WT cells at each drug concentration were determined using an unpaired two-tailed t-test (* p = 0.035).</p

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

Panobinostat shows efficacy against brainstem glioma cells <i>in vitro</i>.

<p><b>(A-C)</b> Mouse brainstem glioma cells driven by PDGF-B, H3.3-K27M, and Cre-induced p53 loss from three separate tumors were cultured as neurospheres and treated with varying doses of panobinostat for 48 h. (A) Proliferation assessment using a BrdU assay (**** p < 0.0001). (B) Cell viability assessment using a Celltiter-Glo assay (* p = 0.0252, **** p < 0.0001). (C) Apoptosis assessment using a Caspase-Glo 3/7 assay (* p = 0.0149, *** p = 0.0005, **** p < 0.0001). Values from each drug concentration were normalized to the control (0 nM panobinostat, 0.1% DMSO). IC50 values for each tumor cell line are shown in the legends. Each experiment was performed in triplicate and independently repeated three times for each tumor cell line. For all panels, error bars represent mean with SEM. Statistical significance to compare drug concentration groups to controls were determined using an unpaired two-tailed t-test.</p

No overall survival benefit with panobinostat treatments <i>in vivo</i>.

<p><b>(A)</b> Mice with tumors driven by PDGF-B and p53 loss harboring H3.3-K27M mutations (generated as described in Materials and Methods) were treated with 20 mg/kg panobinostat (n = 11) or vehicle (25% DMSO, 0.25x PBS, 5% glucose, n = 8) administered via intraperitoneal (i.p.) injection once per day twice a week beginning 21 days post-virus injection and continuing until mice reached humane endpoints. The mice were monitored daily and sacrificed upon moribund condition (lethargy, enlarged head circumference, ataxia, and/or > 25% weight loss) (p = 0.756, log rank test). <b>(B-D)</b> Effect of panobinostat treatment on the survival of mice-bearing H3.3-K27M HSJD-DIPG-007 orthotopic xenografts. NOD-SCID mice (7 weeks old) were orthotopically injected with HSJD-DIPG-007 cells (passage 39) into the brainstem via stereotactic coordinates. (<b>B</b>) Representative H&E (top panel) and IHC staining for Ki67 (middle panel) and Vimentin (bottom panel) of control mice (treated with vehicle and sacrificed immediately after the last dose, as described in the Materials and Methods). 400x magnification, scale bar = 50 μm. (<b>C-D</b>) Starting on day 28 post-implantation, mice were treated with panobinostat prepared in a vehicle containing 5% dextrose (<b>C</b>) or 2.5% DMSO, 5% PEG400 and 5% Tween80 in 0.9% saline (<b>D</b>) via intraperitoneal (i.p.) injection at 10 mg/kg, three times a week for four weeks (<i>p</i>>0.05, log-rank test). Shaded areas under the curves in <b>A, C-D</b> indicate treatment duration.</p

Short-term <i>in vivo</i> treatments with panobinostat reduces tumor cell proliferation and increases H3 acetylation without evidence of apoptosis.

<p>Neonatal Ntv-a;p53-fl/fl mice were injected with RCAS-PDGF-B, -H3.3-K27M, and–Cre viruses to induce tumor formation. Upon the first appearance of brain tumor symptoms (3–5 weeks post-injection), mice were treated with five doses of 20 mg/kg panobinostat (Drug) or vehicle (Veh, 25% DMSO, 0.25x PBS, 5% glucose, n = 6 in each group) once daily via i.p. injections and then sacrificed 1 hour after the final treatment. Shown is immunohistochemistry of brain tumor tissue for cell proliferation (phospho histone H3, pH3) <b>(A)</b>, cell apoptosis (cleaved caspase-3, cc3) (<b>B</b>), and H3 acetylation (AcH3) <b>(C</b>), 400x, scale bar = 50 μm, with quantification of total positive nuclear staining as a percentage of total nuclear area below in each panel. Statistical significance was determined using unpaired two-tailed t-test to compare groups. (ns: nonsignificant).</p

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