Marta Oteo

In Vivo Disruption of an Rb–E2F–Ezh2 Signaling Loop Causes Bladder Cancer

Bladder cancer is a highly prevalent human disease in which retinoblastoma (Rb) pathway inactivation and epigenetic alterations are common events. However, the connection between these two processes is still poorly understood. Here, we show that the in vivo inactivation of all Rb family genes in the mouse urothelium is sufficient to initiate bladder cancer development. The characterization of the mouse tumors revealed multiple molecular features of human bladder cancer, including the activation of E2F transcription factor and subsequent Ezh2 expression and the activation of several signaling pathways previously identified as highly relevant in urothelial tumors. These mice represent a genetically defined model for human high-grade superficial bladder cancer. Whole transcriptional characterizations of mouse and human bladder tumors revealed a significant overlap and confirmed the predominant role for Ezh2 in the downregulation of gene expression programs. Importantly, the increased tumor recurrence and progression in human patients with superficial bladder cancer is associated with increased E2F and Ezh2 expression and Ezh2-mediated gene expression repression. Collectively, our studies provide a genetically defined model for human high-grade superficial bladder cancer and demonstrate the existence of an Rb-E2F-Ezh2 axis in bladder whose disruption can promote tumor development.

Structural characterization of two CD1A allelic variants

CD1 molecules are specialized in presenting lipidic antigens to T lymphocytes. They are structurally and evolutionary related to MHC molecules and show very limited polymorphism. We have previously described and partially characterized a new human CD1A allele differing from the wild type CD1A by a substitution of Cysteine by Tryptophan at position 52 in the alpha1 domain of the CD1A molecule. The frequency of this allele varies from 10% in individuals of Caucasian origin to 56% in Chinese people. The aim of the present work was to structurally characterize this CD1A allele. To do this we have cloned and sequenced the full-length cDNA encoding the new CD1A allele. The cDNA sequence of this allele encodes a protein differing the wild type in two amino acids at positions 14 (Threonine versus Isoleucine) and 52 (Cysteine versus Tryptophan). The cDNAs encoding both wild type and mutant CD1A were cloned in the expression vector pSRalphaNeo and transfected into C1R and L721.221 cells. Cell surface expression of the protein products in transfected cell lines were analyzed by flow cytometry and immunoprecipitation using CD1a-specific monoclonal antibodies. Our results indicate that both allelic products are efficiently expressed on the cell surface.

Meningiomas: A Comparative Study of 68Ga-DOTATOC, 68Ga-DOTANOC and 68Ga-DOTATATE for Molecular Imaging in Mice

Purpose The goal of this study was to compare the tumor uptake kinetics and diagnostic value of three 68Ga-DOTA-labeled somatostatin analogues (68Ga-DOTATOC, 68Ga-DOTANOC, and 68Ga-DOTATATE) using PET/CT in a murine model with subcutaneous meningioma xenografts. Methods The experiment was performed with 16 male NUDE NU/NU mice bearing xenografts of a human meningioma cell line (CH-157MN). 68Ga-DOTATOC, 68Ga-DOTANOC, and 68Ga-DOTATATE were produced in a FASTLab automated platform. Imaging was performed on an Argus small-animal PET/CT scanner. The SUVmax of the liver and muscle, and the tumor-to-liver (T/L) and tumor-to-muscle (T/M) SUV ratios were computed. Kinetic analysis was performed using Logan graphical analysis for a two-tissue reversible compartmental model, and the volume of distribution (Vt) was determined. Results Hepatic SUVmax and Vt were significantly higher with 68Ga-DOTANOC than with 68Ga-DOTATOC and 68Ga-DOTATATE. No significant differences between tracers were found for SUVmax in tumor or muscle. No differences were found in the T/L SUV ratio between 68Ga-DOTATATE and 68Ga-DOTATOC, both of which had a higher fraction than 68Ga-DOTANOC. The T/M SUV ratio was significantly higher with 68Ga-DOTATATE than with 68Ga-DOTATOC and 68Ga-DOTANOC. The Vt for tumor was higher with 68Ga-DOTATATE than with 68Ga-DOTANOC and relatively similar to that of 68Ga-DOTATOC. Conclusions This study demonstrates, for the first time, the ability of the three radiolabeled somatostatin analogues tested to image a human meningioma cell line. Although Vt was relatively similar with 68Ga-DOTATATE and 68Ga-DOTATOC, uptake was higher with 68Ga-DOTATATE in the tumor than with 68Ga-DOTANOC and 68Ga-DOTATOC, suggesting a higher diagnostic value of 68Ga-DOTATATE for detecting meningiomas.

Targeting MT1-MMP as an ImmunoPET-Based Strategy for Imaging Gliomas.

Background A critical challenge in the management of Glioblastoma Multiforme (GBM) tumors is the accurate diagnosis and assessment of tumor progression in a noninvasive manner. We have identified Membrane-type 1 matrix metalloproteinase (MT1-MMP) as an attractive biomarker for GBM imaging since this protein is actively involved in tumor growth and progression, correlates with tumor grade and is closely associated with poor prognosis in GBM patients. Here, we report the development of an immunoPET tracer for effective detection of MT1-MMP in GBM models. Methods An anti-human MT1-MMP monoclonal antibody (mAb), LEM2/15, was conjugated to p-isothiocyanatobenzyl-desferrioxamine (DFO-NCS) for 89Zr labeling. Biodistribution and PET imaging studies were performed in xenograft mice bearing human GBM cells (U251) expressing MT1-MMP and non-expressing breast carcinoma cells (MCF-7) as negative control. Two orthotopic brain GBM models, patient-derived neurospheres (TS543) and U251 cells, with different degrees of blood-brain barrier (BBB) disruption were also used for PET imaging experiments. Results 89Zr labeling of DFO-LEM2/15 was achieved with high yield (>90%) and specific activity (78.5 MBq/mg). Biodistribution experiments indicated that 89Zr-DFO-LEM2/15 showed excellent potential as a radiotracer for detection of MT1-MMP positive GBM tumors. PET imaging also indicated a specific and prominent 89Zr-DFO-LEM2/15 uptake in MT1-MMP+ U251 GBM tumors compared to MT1-MMP- MCF-7 breast tumors. Results obtained in orthotopic brain GBM models revealed a high dependence of a disrupted BBB for tracer penetrance into tumors. 89Zr-DFO-LEM2/15 showed much higher accumulation in TS543 tumors with a highly disrupted BBB than in U251 orthotopic model in which the BBB permeability was only partially increased. Histological analysis confirmed the specificity of the immunoconjugate in all GBM models. Conclusion A new anti MT1-MMP-mAb tracer, 89Zr-DFO-LEM2/15, was synthesized efficiently. In vivo validation showed high-specific-contrast imaging of MT1-MMP positive GBM tumors and provided strong evidence for utility of MT1-MMP-targeted immunoPET as an alternate to nonspecific imaging of GBM.

Preparation of ⁶⁸Ga-labelled DOTA-peptides using a manual labelling approach for small-animal PET imaging.

(68)Ga-DOTA-peptides are a promising PET radiotracers used in the detection of different tumours types due to their ability for binding specifically receptors overexpressed in these. Furthermore, (68)Ga can be produced by a (68)Ge/(68)Ga generator on site which is a very good alternative to cyclotron-based PET isotopes. Here, we describe a manual labelling approach for the synthesis of (68)Ga-labelled DOTA-peptides based on concentration and purification of the commercial (68)Ga/(68)Ga generator eluate using an anion exchange-cartridge. (68)Ga-DOTA-TATE was used to image a pheochromocytoma xenograft mouse model by a microPET/CT scanner. The method described provides satisfactory results, allowing the subsequent (68)Ga use to label DOTA-peptides. The simplicity of the method along with its implementation reduced cost, makes it useful in preclinical PET studies.

Ablating all three retinoblastoma family members in mouse lung leads to neuroendocrine tumor formation

Lung cancer is a deadly disease with increasing cases diagnosed worldwide and still a very poor prognosis. While mutations in the retinoblastoma (RB1) tumor suppressor have been reported in lung cancer, mainly in small cell lung carcinoma, the tumor suppressive role of its relatives p107 and p130 is still a matter of debate. To begin to investigate the role of these two Rb family proteins in lung tumorigenesis, we have generated a conditional triple knockout mouse model (TKO) in which the three Rb family members can be inactivated in adult mice. We found that ablation of all three family members in the lung of mice induces tumorlets, benign neuroendocrine tumors that are remarkably similar to their human counterparts. Upon chemical carcinogenesis, DHPN and urethane accelerate tumor development; the TKO model displays increased sensitivity to DHPN, and urethane increases malignancy of tumors. All the tumors developing in TKO mice (spontaneous and chemically induced) have neuroendocrine features but do not progress to fully malignant tumors. Thus, loss of Rb and its family members confers partial tumor susceptibility in neuroendocrine lineages in the lungs of mice. Our data also imply the requirement of other oncogenic signaling pathways to achieve full transformation in neuroendocrine lung lesions mutant for the Rb family.

A knockin mouse model for human ATP4aR703C mutation identified in familial gastric neuroendocrine tumors recapitulates the premalignant condition of the human disease and suggests new therapeutic strategies

ABSTRACT By whole exome sequencing, we recently identified a missense mutation (p.R703C) in the human ATP4a gene, which encodes the proton pump responsible for gastric acidification. This mutation causes an aggressive familial type I gastric neuroendocrine tumor in homozygous individuals. Affected individuals show an early onset of the disease, characterized by gastric hypoacidity, hypergastrinemia, iron-deficiency anemia, gastric intestinal metaplasia and, in one case, an associated gastric adenocarcinoma. Total gastrectomy was performed as the definitive treatment in all affected individuals. We now describe the generation and characterization of a knockin mouse model for the ATP4aR703C mutation to better understand the tumorigenesis process. Homozygous mice recapitulated most of the phenotypical alterations that were observed in human individuals, strongly suggesting that this mutation is the primary alteration responsible for disease development. Homozygous mice developed premalignant condition with severe hyperplasia, dysplasia and glandular metaplasia in the stomach. Interestingly, gastric acidification in homozygous mice, induced by treatment with 3% HCl acid in the drinking water, prevented (if treated from birth) or partially reverted (if treated during adulthood) the development of glandular metaplasia and dysplasia in the stomach and partially rescued the abnormal biochemical parameters. We therefore suggest that, in this model, achlorhydria contributes to tumorigenesis to a greater extent than hypergastrinemia. Furthermore, our mouse model represents a unique and novel tool for studying the pathologies associated with disturbances in gastric acid secretion. Summary: Gastric pathologies in an ATP4a knockin mouse model of a mutation responsible for the development of gastric neuroendocrine tumors in humans are prevented and reverted by adding HCl to drinking water.

MT1-MMP as a PET Imaging Biomarker for Pancreas Cancer Management

Pancreatic ductal adenocarcinoma (PDAC) continues to be one of the deadliest cancers for which optimal diagnostic tools are still greatly needed. Identification of PDAC-specific molecular markers would be extremely useful to improve disease diagnosis and follow-up. MT1-MMP has long been involved in pancreatic cancer, especially in tumour invasion and metastasis. In this study, we aim to ascertain the suitability of MT1-MMP as a biomarker for positron emission tomography (PET) imaging. Two probes were assessed and compared for this purpose, an MT1-MMP-specific binding peptide (MT1-AF7p) and a specific antibody (LEM2/15), labelled, respectively, with 68Ga and with 89Zr. PET imaging with both probes was conducted in patient-derived xenograft (PDX), subcutaneous and orthotopic, PDAC mouse models, and in a cancer cell line (CAPAN-2)-derived xenograft (CDX) model. Both radiolabelled tracers were successful in identifying, by means of PET imaging techniques, tumour tissues expressing MT1-MMP although they did so at different uptake levels. The 89Zr-DFO-LEM2/15 probe showed greater specific activity compared to the 68Ga-labelled peptide. The mean value of tumour uptake for the 89Zr-DFO-LEM2/15 probe (5.67 ± 1.11%ID/g, n=28) was 25-30 times higher than that of the 68Ga-DOTA-AF7p ones. Tumour/blood ratios (1.13 ± 0.51 and 1.44 ± 0.43 at 5 and 7 days of 89Zr-DFO-LEM2/15 after injection) were higher than those estimated for 68Ga-DOTA-AF7p probes (of approximately tumour/blood ratio = 0.5 at 90 min after injection). Our findings strongly point out that (i) the in vivo detection of MT1-MMP by PET imaging is a promising strategy for PDAC diagnosis and (ii) labelled LEM2/15 antibody is a better candidate than MT1-AF7p for PDAC detection.

Characterization of a PET prototype based on monolithic detectors

We have characterized a PET prototype based on monolithic scintillator detectors. The prototype has a modular design with 8 identical detector cassettes, each housing 4 monolithic blocks. The cassettes are mounted on a gantry in 2 groups of 4 cassettes facing each other, totaling 32 detectors. Each detector block is composed of a trapezoidal LYSO:Ce scintillator glued to a pair of Hamamatsu S8550-02 APD matrices, which are read out by dedicated VATA 241 ASICs from Gamma Medica Ideas. The ASIC collects and amplifies the charge pulses from the 64 photosensors in each detector block, sums them into 8 rows and 8 column outputs and generates a trigger signal whenever the charge in a channel is above a user-defined threshold. Positioning algorithms based on supervised training neural networks are used for the detector blocks. A rotating platform with precise movement control is aligned with the system axis allowing acquisition of coincidence data along different projection angles. Using this system, Na22 point sources and a NEMA NU4-2008 image quality phantom filled with F18-FDG have been imaged and the images obtained have been characterized and compared with those from a Sedecal Argus PET/CT small-animal scanner, used as reference. Results show that the data acquisition system performs according to specifications and that PET imaging based on a large number of monolithic detector blocks is an alternative to designs based on segmented detectors.