Validation of an engineered cell model for in vitro and in vivo HIF-1α evaluation by different imaging modalities.

PURPOSE: The aim of this study was to characterize a cell-based model for the molecular study of hypoxia-inducible factor (HIF)-1α activity, in the context of hypoxia, by means of different imaging techniques. PROCEDURES: Engineered U251-HRE glioma cells were used to analyze the molecular mechanisms underlying HIF-1α activity in vitro in relation to luciferase expression. The same cells were orthotopically implanted in mice to evaluate tumor progression and hypoxia induction by bioluminescence imaging, fluorescence imaging, positron emission tomography (PET), and magnetic resonance imaging (MRI). RESULTS: In vitro analyses highlighted the relationship between HIF-1α and luciferase activity in hypoxic conditions and after pharmacological treatments in U251-HRE cells. Through in vivo studies, it was possible to assess hypoxia establishment in relation to tumor growth by optical imaging, PET and MRI. CONCLUSIONS: The findings of this study indicate that the U251-HRE orthotopic murine model can be used to reliably evaluate processes modulating HIF-1α activity, using both molecular and preclinical non-invasive imaging techniques.

Anti-tumour efficacy on glioma models of PHA-848125, a multi-kinase inhibitor able to cross the blood-brain barrier.

BACKGROUND AND PURPOSE: Malignant gliomas, the most common primary brain tumours, are highly invasive and neurologically destructive neoplasms with a very bad prognosis due to the difficulty in removing the mass completely by surgery and the limited activity of current therapeutic agents. PHA-848125 is a multi-kinase inhibitor with broad anti-tumour activity in pre-clinical studies and good tolerability in phase 1 studies, which could affect two main pathways involved in glioma pathogenesis, the G1-S phase progression control pathway through the inhibition of cyclin-dependent kinases and the signalling pathways mediated by tyrosine kinase growth factor receptors, such as tropomyosin receptors. For this reason, we tested PHA-848125 in glioma models. EXPERIMENTAL APPROACH: PHA-848125 was tested on a panel of glioma cell lines in vitro to evaluate inhibition of proliferation and mechanism of action. In vivo efficacy was evaluated on two glioma models both as single agent and in combination with standard therapy. KEY RESULTS: When tested on a subset of representative glioma cell lines, PHA-848125 blocked cell proliferation, DNA synthesis and inhibited both cell cycle and signal transduction markers. Relevantly, PHA-848125 was also able to induce cell death through autophagy in all cell lines. Good anti-tumour efficacy was observed by oral route in different glioma models both with s.c. and intracranial implantation. Indeed, we demonstrate that the drug is able to cross the blood-brain barrier. Moreover, the combination of PHA-848125 with temozolomide resulted in a synergistic effect, and a clear therapeutic gain was also observed with a triple treatment adding PHA-848125 to radiotherapy and temozolomide. CONCLUSIONS AND IMPLICATIONS: All the pre-clinical data obtained so far suggest that PHA-848125 may become a useful agent in chemotherapy regimens for glioma patients and support its evaluation in phase 2 trials for this indication.

Immunohistopathological and neuroimaging characterization of murine orthotopic xenograft models of glioblastoma multiforme recapitulating the most salient features of human disease.

Tumorigenesis in human glioblastoma multiforme (GBM) is driven by several genetic abnormalities with disruption of important molecular pathways, such as p53/MDM2/p14ARF and EGFR/PTEN/Akt/mTOR. The malignant progression of human GBM is also primarily associated with a peculiar multistep pathophysiological process characterized by intratumoral ischemic necrosis (i.e. pseudopalisading necrosis) and activation of the hypoxia-inducible factor (HIF)-1alpha pathway with consequent peritumoral microvascular proliferation and infiltrative behaviour. Predictable preclinical animal models of GBM should recapitulate the main pathobiological hallmarks of the human disease. In this study we describe two murine orthotopic xenograft models using U87MG and U251 human cell lines. Ten Balb/c nude male mice were orthotopically implanted with either U87MG (5 mice) or U251 (5 mice) cell lines. Intracranial tumor growth was monitored through Magnetic Resonance Imaging (MRI). Immunohistopathological examination of the whole cranium was performed 30 days after implantation. U251 orthotopic xenografts recapitulated the salient pathobiological features described for human GBM, including invasive behaviour, wide areas of pseudopalisading necrosis, florid peripheral angiogenesis, GFAP and vimentin expression, nonfunctional p53 expression, striking active-caspase-3 and HIF-1alpha expression along pseudopalisades. U87MG orthotopic xenografts proved to be very dissimilar from human GBM, showing expansile growth, occasional necrotic foci without pseudopalisades, intratumoral lacunar pattern of angiogenesis, lack of GFAP expression, functional p53 expression and inconsistent HIF-1alpha expression. Expression of pAkt was upregulated in both models. The results obtained suggest that the U251 orthotopic model may be proposed as a predictive and reliable tool in preclinical studies since it recapitulates the most salient pathobiological features reported for human GBM.

Inhibition of tyrosine kinase receptors by SU6668 promotes abnormal stromal development at the periphery of carcinomas.

Dynamic contrast-enhanced (albumin-Gd-DTPA) magnetic resonance imaging, performed during 2 weeks of daily administration of an inhibitor of tyrosine kinase receptors (SU6668) in an HT-29 colon carcinoma model, revealed the onset of a hyper-enhancing rim, not observed in untreated tumours. To account for tissue heterogeneity in the quantitative analysis, we segmented tumours into three subunits automatically identified by cluster analysis of the enhancement curves using a k-means algorithm. Transendothelial permeability (Kps) and fractional plasma volume (fPV) were calculated in each subunit. An avascular and necrotic region, an intermediate zone and a well-vascularised periphery were reliably identified. During untreated tumour growth, the identified sub-regions did not substantially change their enhancement pattern. Treatment with SU6668 induced major changes at tumour periphery where a significant increase of Kps and fPV was observed with respect to control tumours. Histology revealed a sub-capsular layer composed of hyper-dense viable tumour cells in the periphery of untreated tumours. The rim of viable neoplastic cells was reduced in treated tumours, and replaced by loose connective tissue characterised by numerous vessels, which explains the observed hyper-enhancement. The present data show a peripheral abnormal development of cancer-associated stroma, indicative of an adaptive response to anti-angiogenic treatment.

A new oxygenator change-out system and procedure.

Official reports relate that, in the US, one patient/month dies as a result of the emergency oxygenator change-out procedure, and the permanent injury of some patients is the result of current oxygenator change-out procedures or oxygenator failures, both in extracorporeal circulation (ECC) and extracorporeal membrane oxygenation (ECMO). The aim of this article is to evaluate a new system and procedure, dedicated to oxygenator change-out, represented by two three-way stopcocks inserted in the ECC line in use. A dedicated back-up oxygenator and circuit can be easily primed and connected to the dedicated connector on the stopcocks, then blood flow is diverted to the new oxygenator without interruption of the ECC. Tests performed showed that oxygenator change-out can be completed by perfusionists in 62.13 +/- 11.12 sec. Results obtained show that the new system and procedure allows fast, safe and reproducible oxygenator change-out without interruption of the ECC.

A closed system for the clinical banking of umbilical cord blood.

Umbilical cord blood (UCB) is a source of hematopoietic progenitor cells and is used as an alternative to the bone marrow or peripheral blood for treatment of several onco-hematological diseases. Because of the limited number of CD34+ hematopoietic stem cells present in UCB units and of the elevated costs of cryopreservation, it is of paramount importance to select the UCB units that are clinically useful before storage and optimize banking efficiency by designing reliable procedures to process and freeze the selected units. Among the different parameters characterizing UCB, nucleated cell (NC) and CD34+ cell content provides useful criteria to select UCB units since clinical data documented that the infused cell load (both NC and CD34+ cells) plays an important role in the successful outcome of transplants. By evaluating volume, CD34+ cell content, NC total amount, and NC density of 117 UCB units, we found a significant association between CD34+ cell content and NC density and total amount, indicating these parameters as useful to decide UCB clinical utility. Furthermore, we set up a fast procedure to process UCB units for storage. A system for NC separation and volume reduction of UCB samples in a dedicated, germ-free, closed circuit was developed, where plasma and red blood cells (RBC) depletion was obtained by sedimentation in the presence of a 3.5% Polygeline solution. By this separation system, both RBC depletion and high NC and CD34+ cell recoveries were achieved in 60 min, and the yield was comparable to the one obtained by other separation methods. Since Polygeline has been clinically used as a plasma expander and no toxic effects on patients were reported, the protocol can be applied in the large-scale banking of UCB.

Evaluation of ex vivo expansion and engraftment in NOD-SCID mice of umbilical cord blood CD34+ cells using the DIDECO "Pluricell System".

The Dideco "Pluricell System" is a commercially available closed device composed of an expansion chamber and a kit of certified reagents that allow haematopoietic stem cell expansion. We have expanded seven umbilical cord blood (UCB) samples following the manufacturer's instructions; two groups of irradiated NOD-SCID mice have been transplanted with expanded and nonexpanded cells from the same UCB, and bone marrow was analysed for the presence of human cells. Average UCB volume was 61.6+/-8.8 ml; mean nucleated cell content was 1090.5+/-189.9 x 10(6). Percentage and number of CD34+ cells were 0.37+/-0.13% and 3.9+/-1.2 x 10(6). After separation, CD34+ cell purity was 82+/-11%. Mean number of inoculated cells was 760 000; mean NC and CD34+ fold expansion at 12 days was 230.4+/-91.5 and 21.0+/-11.9. Both groups of mice showed successful engraftment: the percentage of human cells was higher in the group receiving expanded cells (3.4+/-2.01%) compared to the group receiving nonexpanded cells (1.5+/-0.66%) (P<0.00018, Mann-Whitney test). The cell population obtained after 12 days expansion consisted mainly of myeloid and megakaryocytic progenitors. The CD34+ antigen reached the maximum expression level at day 12 (7.5+/-2.0%). Analysis of lineage-markers for human myelomonocytic, megakaryocytic, B, T, CD34 and erythroid cells, gave evidence that all the lineages were represented in the marrow of transplanted mice.

Qualitative multiplex RT-PCR for simultaneous detection of hepatitis C virus and human immunodeficiency virus in plasma samples.

This report describes the development of a one-tube multiplex reverse transcriptase (RT)-PCR assay for the simultaneous detection of hepatitis C virus (HCV) and human immunodeficiency virus (HIV) in plasma samples. The assay was evaluated with two panels of HCV- and HIV-1-positive samples, as well as negative plasma specimens. Extraction and amplification of HCV and HIV-1 RNA from plasma samples were performed in a single reaction, and amplified genomes were detected with specific probes. Serial dilutions of the HCV and HIV-1 first World Health Organization International Standards were used to evaluate the sensitivity of the method. Two RNA controls were constructed to determine inter-assay variations and the sensitivity of the amplification step. The assay had good specificity and detected all the genotypes and subtypes tested. The analytical sensitivity of the entire assay was 100 IU/mL for HCV and 200 IU/mL for HIV-1, while the amplification step detected ten copies/reaction for HCV and 20 copies/reaction for HIV-1. The multiplex assay allowed the simultaneous extraction, amplification and detection of two virus genomes, thereby providing an important practical advantage and an efficient approach for analysing individual and pooled plasma donations.

Corticotropin-releasing factor, urocortin and endothelin-1 stimulate activin A release from cultured human placental cells.

Human placenta produces activin A, a glycoprotein belonging to the transforming growth factor beta superfamily, which modulates several placental immune and endocrine functions. However, substances involved in controlling placental activin A production are not yet completely elucidated. The aim of the present study was to investigate the effects of placental products, corticotropin-releasing factor (CRF), urocortin, prostaglandin E(2) (PGE(2)) and endothelin-1 (ET-1) on activin A release from cultured human placental cells. Placental tissue was collected at term from normal pregnancies and a trophoblast-enriched cell preparation was cultured for 48 h. The test substances were applied (concentration from 10(-9)-10(-7)M) and the medium was harvested after 3 h incubation; vehicle-treated cells (controls) were present in each experiment. Activin A concentrations in culture medium were measured by using a specific two-site enzyme immunoassay. The addition of CRF resulted in a dose-related increase of activin A concentrations (P < 0.01). The stimulatory effect of CRF was significantly reversed by alpha-helical CRF(9-41), the CRF receptor antagonist. Urocortin showed a stimulating effect on activin A release from placental cells (P < 0.05) but not dose-related; the effect of urocortin was reversed by an equimolar dose of CRF antagonist, astressin. ET-1 significantly increased activin A concentrations in the culture medium only at the highest concentration, 10(-7)M (P < 0.05). No difference in activin A release was observed after incubating the cells with PGE(2). The evidence that CRF, urocortin and ET-1 stimulate activin A secretion from cultured placental cells suggests that these vasoactive factors may affect the changes of placental activin A secretion in pre-eclamptic woman.

A novel protocol that allows short-term stem cell expansion of both committed and pluripotent hematopoietic progenitor cells suitable for clinical use.

To obtain long-term engraftment and hematopoiesis in myeloablated patients, the cell population used for hematopoietic reconstitution should include a sufficient number of early pluripotent hematopoietic stem cells (HSCs), along with committed cells from the various lineages. For this purpose, the small subset of CD34+ cells purified from different sources must be expanded ex vivo. Since cytokines may induce both proliferation and differentiation, expansion would provide a cell population comprising committed as well as uncommitted cells. Optimization of HSC expansion methods could be obtained by a combination of cytokines able to sustain renewal of pluripotent cells yet endowed with poor differentiation potential. We used variations of the combinations of cytokines described by Brugger et al. [W. Brugger, S. Heimfels, R. J. Berenson, R. Mertelsmann, and L. Kanz (1995) N. Engl. J. Med. 333, 283-287] and Piacibello et al. [W. Piacibello, F. Sanavio, L. Garetto, A. Severino, D. Bergandi, J. Ferrario, F. Fagioli, M. Berger, and M. Aglietta (1997) Blood 89, 2644-2653] to expand UCB CD34+ cells and monitored proliferation rate and phenotype after 14 days of culture. Several hematopoietic lineage-associated surface antigens were evaluated. Our data show that flt3L and thrombopoietin in combination with IL-3, while sustaining a high CD34+ proliferation rate, provide a relatively low enrichment in very early uncommitted CD34+/CD38- cells. Conversely, in the absence of IL-3, they are less effective in inducing proliferation yet significantly increase the number of CD34+/CD38- cells. A combination of the above protocols, applied simultaneously to aliquots of the same sample, would allow expansion of both committed and pluripotent HSC. This strategy may represent a significant improvement for clinical applications.

Efficiency of doxorubicin handling by isolated hepatocytes is a valuable indicator for restored cell function.

Pig liver is a possible source of hepatocytes for extracorporeal bio-artificial liver devices. In order to evaluate recovered hepatocyte function following enzymatic isolation, we developed a cytochemical method that is based on the capacity of hepatocytes to sequester the anthracycline antitumour drug doxorubicin within intracellular acidic compartments. Doxorubicin is a naturally fluorescent molecule. Thus, the process of drug concentration within hepatocytes can be visualized in living conditions by fluorescence microscopy. Porcine hepatocytes harvested from heart-beating donors were grown either as isolated cell suspensions or as tissue monolayers. Immediately after isolation and at fixed culture times, cells were incubated with 0.1 mM doxorubicin in Hanks' balanced salt solution for 10 min at 37 degrees C in 5% CO2-humidified atmosphere and observed by fluorescence microscopy. Parallel electron microscopy was performed to compare fluorescence data with general cell morphology. To monitor lysosomal acidification capacity, the fluorescent pH-sensitive vital dye LysoSensor-Blue was used. Doxorubicin fluorescence showed different patterns of nuclear and cytoplasmic staining, according to the time allowed for cell recovery and the culture method. In particular, cytoplasmic fluorescence changed from a diffuse staining, that could be observed after cell isolation and in hepatocyte suspensions, to a punctate perinuclear and pericanalicular fluorescence detectable in fully recovered hepatocyte monolayers. This study indicates that the 'doxorubicin-fluorescence test' may be considered a simple and rapid procedure for assessing hepatocyte functional condition. It may provide valuable and 'real time' guidelines for judging the correct way these cells are to be collected, preserved and utilized for clinical purposes.

Increased blood volume and CD34(+)CD38(-) progenitor cell recovery using a novel umbilical cord blood collection system.

A major problem with the use of umbilical cord/placental blood (UCB) is the limited blood volume that can be collected from a single donor. In this study, we evaluated a novel system for the collection of UCB and analyzed the kinetics of output of hematopoietic stem cells in the collected blood. Sequential UCB fractions were collected from 48 placentas by gravity following common procedures. When UCB flow was ended, collection was continued using the device. Nucleated cell (NC) density in each fraction was evaluated and the expression of CD34, CD38 and other hematopoietic markers was assessed by flow cytometry. The total collected volume was 60.9 +/- 26.2 ml (mean +/- SD, range 17-141.5). The device yield (volume collected using the device/total volume) was 26.5 +/- 15.1%. No significant difference was observed in NC count in sequential fractions. A significant increase in CD34(+) cell content in sequential fractions and a 2.07 +/- 1.18-fold increase in the percentage of CD34(+) cells in the last versus first fraction were observed. Furthermore, within the CD34(+) population, the percentage of CD38(-) pluripotent stem cells in the first fraction was 3.24 +/- 1.39, while in the last fraction it raised to 34.43 +/- 22.62. Thus, at the end of a collection performed following current procedures, further blood rich in the most primitive progenitor cells can be recovered. Therefore, the optimization and standardization of collection procedures are required to obtain maximal recovery from each placenta and increase the percentage of UCB units suitable for clinical use.