Activity of the polyamine-vectorized anti-cancer drug F14512 against pediatric glioma and neuroblastoma cell lines.

The poor prognosis of children with high-grade glioma (HGG) and high-risk neuroblastoma, despite multidisciplinary therapeutic approaches, demands new treatments for these indications. F14512 is a topoisomerase II inhibitor containing a spermine moiety that facilitates selective uptake by tumor cells via the Polyamine Transport System (PTS) and increases topoisomerase II poisoning. Here, F14512 was evaluated in pediatric HGG and neuroblastoma cell lines. PTS activity and specificity were evaluated using a fluorescent spermine-coupled probe. The cytotoxicity of F14512, alone or in combination with ionizing radiation and chemotherapeutic agents, was investigated in vitro. The antitumor activity of F14512 was assessed in vivo using a liver-metastatic model of neuroblastoma. An active PTS was evidenced in all tested cell lines, providing a specific and rapid transfer of spermine-coupled compounds into cell nuclei. Competition experiments confirmed the essential role of PTS in the cell uptake and cytotoxicity of F14512. This cytotoxicity appeared greater in neuroblastoma cells compared with HGG cells but appeared independent of PTS activity levels. In vivo evaluation confirmed a marked and prolonged antitumoral effect in neuroblastoma cells. The combinations of F14512 with cisplatin and carboplatin were often found to be synergistic, and we demonstrated the significant radiosensitizing potential of F14512 in the MYCN-amplified Kelly cell line. Thus, F14512 appears more effective than etoposide in pediatric tumor cell lines, with greater efficacy in neuroblastoma cells compared with HGG cells. The synergistic effects observed with platinum compounds and the radiosensitizing effect could lead to a clinical development of the drug in pediatric oncology.

Use of an image restoration process to improve spatial resolution in bioluminescence imaging.

To improve spatial resolution in in vivo bioluminescence imaging, a photon scattering correction, image restoration method was tested. The chosen algorithm was tested on in vivo bioluminescent images acquired on three representative tumor models: subcutaneous, pulmonary, and disseminated peritoneal. Tumor size was chosen as a quantitative criterion, such that the tumor reference measurements (determined photographically or by computed tomography) were compared to those derived from bioluminescent images, before and after restoration. This technique allowed a significant reduction to be achieved in the relative error between reference measurements and dimensions derived from bioluminescent images. In addition, improved delineation of the tumor foci was achieved. The restoration method allows spatial resolution in bioluminescence imaging to be improved, with interesting perspectives in terms of staging and quantitation in experimental oncology.

Preclinical activity of F14512, designed to target tumors expressing an active polyamine transport system.

We have exploited the polyamine transport system (PTS) to deliver selectively a spermine-drug conjugate, F14512 to cancer cells. This study was aimed to define F14512 anticancer efficacy against tumor models and to investigate whether fluorophor-labeled polyamine probes could be used to identify tumors expressing a highly active PTS and that might be sensitive to F14512 treatments. Eighteen tumor models were used to assess F14512 antitumor activity. Cellular uptake of spermine-based fluorescent probes was measured by flow cytometry in cells sampled from tumor xenografts by needle biopsy. The accumulation of the fluorescent probe within B16 tumors in vivo was assessed using infrared fluorescence imaging. This study has provided evidence of a major antitumor activity for F14512. Significant responses were obtained in 67% of the tumor models evaluated, with a high level of activity recorded in 33% of the responsive models. Complete tumor regressions were observed after i.v., i.p. or oral administrations of F14512 and its antitumor activity was demonstrated over a range of 2-5 dose levels, providing evidence of its good tolerance. The level of cellular fluorescence emitted by the fluorescent probes was higher in cells sampled from tumors sensitive to F14512 treatments than from F14512-refractory tumors. We suggest that these probes could be used to identify tumors expressing a highly active PTS and guide the selection of patients that might be treated with F14512. These results emphasize the preclinical interest of this novel molecule and support its further clinical development.

99mTc-HYNIC-spermine for imaging polyamine transport system-positive tumours: preclinical evaluation.

PURPOSE: F14512 exploiting the polyamine transport system (PTS) for tumour cell delivery has been described as a potent antitumour agent. The optimal use of this compound will require a probe to identify tumour cells expressing a highly active PTS that might be more sensitive to the treatment. The aim of this study was to design and characterize a scintigraphic probe to evaluate its uptake in cancer cells expressing the PTS. METHODS: Three polyamines coupled to a hydrazinonicotinamide (HYNIC) moiety were synthesized and labelled with 99mTc. Their radiochemical purity was determined by HPLC. The plasma stability of the 99mTc-HYNIC-spermine probe and its capacity to accumulate into PTS-active cells were also evaluated. In vitro internalization was tested using murine melanoma B16/F10 cells and human lung carcinoma A549 cells. Biodistribution was determined in healthy mice and tumour uptake was studied in B16/F10 tumour-bearing mice. A HL-60-Luc human leukaemia model was used to confront single photon emission computed tomography (SPECT) images obtained with the 99mTc-labelled probe with those obtained by bioluminescence. RESULTS: The 99mTc-HYNIC-spermine probe was selected for its capacity to accumulate into PTS-active cells and its stability in plasma. In vitro studies demonstrated that the probe was internalized in the cells via the PTS. In vivo measurements indicated a tumour to muscle scintigraphic ratio of 7.9±2.8. The combined bioluminescence and scintigraphic analyses with the leukaemia model demonstrated that the spermine conjugate accumulates into the tumour cells. CONCLUSION: The 99mTc-HYNIC-spermine scintigraphic probe is potentially useful to characterize the PTS activity of tumours. Additional work is needed to determine if this novel conjugate may be useful to analyse the PTS status of patients with solid tumours.

Biological analysis of endocrine-disrupting compounds in Tunisian sewage treatment plants.

Endocrin-disrupting compounds (EDCs) are frequently found in wastewater treatment plants (WWTPs). So far, research has been mainly focused on the detection of estrogenic compounds and very little work has been carried out on other receptors activators. In this study, we used reporter cell lines, which allow detecting the activity of estrogen (ERalpha), androgen (AR), pregnane X (PXR), glucocorticoid (GR), progesterone (PR), mineralocorticoid (MR), and aryl hydrocarbon (AhR) receptors, to characterise the endocrine-disrupting profile of the aqueous, suspended particulate matter, and sludge fractions from three Tunisian WWTPs. The aqueous fraction exhibited estrogenic and androgenic activities. Suspended particulate matter and sludge extracts showed estrogenic, aryl hydrocarbon and pregnane X receptor activities. No GR, MR, or PR (ant) agonistic activity was detected in the samples, suggesting that environmental compounds present in sewage might have a limited spectrum of activity. By performing competition experiments with recombinant ERalpha, we demonstrated that the estrogenic activity detected in the aqueous fraction was due to EDCs with a strong affinity for ERalpha. Conversely, in the sludge fraction, it was linked to the presence of EDCs with weak affinity. Moreover, by using different incubation times, we determined that the EDCs present in suspended particulate matter and sludge, which can activate AhR, are metabolically labile compounds. Finally, we showed in this study that environmental compounds are mainly ER, AR, PXR, and AhR activators. Concerning AR and PXR ligands, we do not to know the nature of the molecules. Concerning ER and AhR compounds, competition experiments with recombinant receptor and analysis at different times of exposure of the AhR activation gave some indications of the compound's nature that need to be confirmed by chemical analysis.

Peroxisome proliferator-activated receptor gamma regulates E-cadherin expression and inhibits growth and invasion of prostate cancer.

Peroxisome proliferator-activated receptor gamma (PPARgamma) might not be permissive to ligand activation in prostate cancer cells. Association of PPARgamma with repressing factors or posttranslational modifications in PPARgamma protein could explain the lack of effect of PPARgamma ligands in a recent randomized clinical trial. Using cells and prostate cancer xenograft mouse models, we demonstrate in this study that a combination treatment using the PPARgamma agonist pioglitazone and the histone deacetylase inhibitor valproic acid is more efficient at inhibiting prostate tumor growth than each individual therapy. We show that the combination treatment impairs the bone-invasive potential of prostate cancer cells in mice. In addition, we demonstrate that expression of E-cadherin, a protein involved in the control of cell migration and invasion, is highly up-regulated in the presence of valproic acid and pioglitazone. We show that E-cadherin expression responds only to the combination treatment and not to single PPARgamma agonists, defining a new class of PPARgamma target genes. These results open up new therapeutic perspectives in the treatment of prostate cancer.

Demethylation by low-dose 5-aza-2'-deoxycytidine impairs 3D melanoma invasion partially through miR-199a-3p expression revealing the role of this miR in melanoma.

BACKGROUND: Efficient treatments against metastatic melanoma dissemination are still lacking. Here, we report that low-cytotoxic concentrations of 5-aza-2'-deoxycytidine, a DNA demethylating agent, prevent in vitro 3D invasiveness of metastatic melanoma cells and reduce lung metastasis formation in vivo. RESULTS: We unravelled that this beneficial effect is in part due to MIR-199A2 re-expression by promoter demethylation. Alone, this miR showed an anti-invasive and anti-metastatic effect. Throughout integration of micro-RNA target prediction databases with transcriptomic analysis after 5-aza-2'-deoxycytidine treatments, we found that miR-199a-3p downregulates set of genes significantly involved in invasion/migration processes. In addition, analysis of data from melanoma patients showed a stage- and tissue type-dependent modulation of MIR-199A2 expression by DNA methylation. CONCLUSIONS: Thus, our data suggest that epigenetic- and/or miR-based therapeutic strategies can be relevant to limit metastatic dissemination of melanoma.

Profiling of benzophenone derivatives using fish and human estrogen receptor-specific in vitro bioassays.

Benzophenone (BP) derivatives, BP1 (2,4-dihydroxybenzophenone), BP2 (2,2',4,4'-tetrahydroxybenzophenone), BP3 (2-hydroxy-4-methoxybenzophenone), and THB (2,4,4'-trihydroxybenzophenone) are UV-absorbing chemicals widely used in pharmaceutical, cosmetics, and industrial applications, such as topical sunscreens in lotions and hair sprays to protect skin and hair from UV irradiation. Studies on their endocrine disrupting properties have mostly focused on their interaction with human estrogen receptor alpha (hERalpha), and there has been no comprehensive analysis of their potency in a system allowing comparison between hERalpha and hERbeta activities. The objective of this study was to provide a comprehensive ER activation profile of BP derivatives using ER from human and fish origin in a battery of in vitro tests, i.e., competitive binding, reporter gene based assays, vitellogenin (Vtg) induction in isolated rainbow trout hepatocytes, and proliferation based assays. The ability to induce human androgen receptor (hAR)-mediated reporter gene expression was also examined. All BP derivatives tested except BP3 were full hERalpha and hERbeta agonists (BP2>THB>BP1) and displayed a stronger activation of hERbeta compared with hERalpha, the opposite effect to that of estradiol (E2). Unlike E2, BPs were more active in rainbow trout ERalpha (rtERalpha) than in hERalpha assay. All four BP derivatives showed anti-androgenic activity (THB>BP2>BP1>BP3). Overall, the observed anti-androgenic potencies of BP derivatives, together with their proposed greater effect on ERbeta versus ERalpha activation, support further investigation of their role as endocrine disrupters in humans and wildlife.

Micro-CT combined with bioluminescence imaging: a dynamic approach to detect early tumor-bone interaction in a tumor osteolysis murine model.

Prostate cancer (CaP) cells possess high affinity for bone marrow and predilection to induce bone metastasis. Although the end result of metastasis is predominantly osteoblastic, most patients present mixed lesions with osteolytic component which could initiate and precede bone formation. A precise characterization of tumor-induced bone resorption is thus necessary for early evaluation of therapeutic efficiency. Herein, we investigate the advantage of combining micro-computed tomography (microCT) and in vivo bioluminescence imaging (BLI) to determine the kinetics of the intraosseous CaP growth and bone lesions appearance in an experimental murine model. To mimic established osteolytic bone metastasis, the left tibiae of SCID mice were injected with the human CaP cell line PC-3 expressing luciferase (PC-3 Luc). Noninvasive monitoring of tumor progression was followed weekly by BLI during 4 weeks and bone morphometric parameters were quantified by microCT. Data were compared with conventional radiological and histological analyses. While BLI monitoring in vivo revealed an exponential growth of PC-3 Luc after 2 weeks, a decrease of bone density and bone mineral content was evidenced by microCT as early as 7 days post-injection, reaching significant values at day 21 (30% and 25% loss, respectively), compared with mock-injected controls. Enhanced osteoclast TRAP activity was observed during the first two weeks, highlighting an active interaction between low proliferative PC-3 cells and osteoclasts at the early stage of tumor establishment in bone. Tumor growth detected by BLI was tightly correlated to the osteolysis assessed by microCT (p<0.05). Our results show that the combination of microCT and BLI applied to this tumor osteolysis murine model allows early measurement of intraosseous tumor growth and bone destruction, as well as correlation between both processes kinetics. This model will help to assess new therapeutic approaches targeting intraosseous tumor growth or tumor/osteoclast crosstalk.

Estrogens and antiestrogens activate hPXR.

The pregnane X receptor (PXR, NR1I2) and the estrogen receptors (ERalpha, NR3A1 and ERbeta, NR3A2) bind a large number of compounds, including environmental pollutants and drugs, which exhibit remarkably diverse structural features. This prompted us to investigate if ER ligands could be PXR activators. We focused our attention on known estrogens from various chemical classes: physiological and synthetic estrogens and antiestrogens, plant and fungus estrogens, and other man-made chemicals belonging to phthalate plasticizers, surfactant-derived alkylphenols and cosmetics. Altogether, nearly 50 compounds were thus analyzed for their ability to activate human PXR in stably transfected cells, HGPXR cells, derived from HeLa cells and expressing luciferase under the control of a chimeric hPXR. Some of the newly identified hPXR activators were also checked for their ability to induce cytochrome P450 3A4 and 2B6 expressions in a primary culture of human hepatocytes. A significant proportion (54%) of compounds with estrogenic activity or able to bind ER were found to be hPXR activators: in particular, antiestrogens, mycoestrogens and phthalates. An even greater proportion is observed if estrogenic pesticides are included. Altogether, these results raise the question of the meaning and consequences of compounds with double PXR/ER activation ability.

[Endocrine xenoestrogenics disrupters: molecular mechanisms and detection methods]. / Les perturbateurs endocriniens xénooestrogéniques: mécanismes moléculaires et méthodes de détection.

The attention paid to endocriniens modulators for purpose micropolluants (endocrine disrupters) has been increasingly studied these last years particularly on animals. The results of this study raised big concerns from Doctors and Biologists on the eventual risks human health can face. Indeed, endocrine systems of the body play an essential and pervasive role in both the short- and long-term regulation of metabolic processes. Nutritional, behavioural, and reproductive processes are intricately regulated by endocrine systems, as are growth (including bone growth/remodelling), gut, cardiovascular, and kidney function and responses to all forms of stress. Disorders of any of the endocrine system, involving both over- and under-active hormone secretion, result inevitably in disease, the effects of which may extend to many different organs and functions and are often debilitating or life-threatening. Viewed from this general perspective, the threat posed from environmental chemicals with endocrine activity (either agonist or antagonistic) is potentially serious. However, the fact that humans and wildlife are exposed to such chemicals does not necessarily mean that clinically manifest disturbance of the relevant endocrine system will result, because much depends on the level and duration of exposure and on the timing of exposure. Indeed, a large numbers of environmental estrogens are suspected of altering human health as well as the marine ecosystem balance. The objective of this review is to study the different molecular mechanisms of these xenoestrogenes micropolluants, in order to emphasize their potential risk and to present some of the different experimental methods for their detection.

Actinic keratosis modelling in mice: A translational study.

BACKGROUND: Actinic keratoses (AK) are pre-malignant cutaneous lesions caused by prolonged exposure to ultraviolet radiation. As AKs lesions are generally accepted to be the initial lesions in a disease continuum that progresses to squamous cell carcinoma (SCC), AK lesions have to be treated. They are also the second most common reason for visits to the dermatologist. Several treatments are available but their efficacy still needs to be improved. The UV-B-induced KA lesion mouse model is used in preclinical studies to assess the efficacy of novel molecules, even though it is often more representative of advanced AK or SCC. OBJECTIVES: Here we report on a translational study, comparing the various stages of AK development in humans and in the UV-B irradiated mouse model, as well as the optimization of photograph acquisition of AK lesions on mouse skin. METHODS: Human and mouse skin lesions were analysed by histology and immunohistochemistry. Mouse lesions were also assessed using a digital dermatoscope. RESULTS: An histological and phenotypic analysis, including p53, Ki67 and CD3 expression detection, performed on human and mouse AK lesions, shows that overall AK modelling in mice is relevant in the clinical situation. Some differences are observed, such as disorganization of keratinocytes of the basal layer and a number of atypical nuclei which are more numerous in human AK, whereas much more pronounced acanthosis is observed in skin lesion in mice. Thanks to this translational study, we are able to select appropriate experimental conditions for establishing either early or advanced stage AK or an SCC model. Furthermore, we optimized photograph acquisition of AK lesions on mouse skin by using a digital dermatoscope which is also used in clinics and allows reproducible photograph acquisition for further reliable assessment of mouse lesions. Use of this camera is illustrated through a pharmacological study assessing the activity of CARAC®. CONCLUSION: These data demonstrate that this mouse model of UV-B-induced skin lesions is predictive for the identification of novel therapeutic treatments for both early and advanced stages of the disease.

Evaluation of ligand selectivity using reporter cell lines stably expressing estrogen receptor alpha or beta.

Estrogens control transcriptional responses through binding to two different nuclear receptors, estrogen receptor alpha (ERalpha) and beta (ERbeta). Since these two ER subtypes are thought to mediate different biological effects, there is intense interest in designing subtype-selective ER ligands. In this study, we evaluated the ERalpha and ERbeta selectivity of 19 known estrogens and antiestrogens using reporter cell lines previously developed in our laboratory. The HELN-ERalpha and HELN-ERbeta cells stably express full-length ERalpha and ERbeta, respectively, and are derived from HELN cells (HeLa cells stably transfected with an ERE-driven luciferase plasmid). We report that 16alpha-LE2, PPT and 3beta,5alpha-GSD have a high ERalpha-selective agonist potency while 8beta-VE2, DPN, genistein and biochanin A show ERbeta selectivity with 8beta-VE2 being the most potent and selective ERbeta agonist. We also tested ER antagonists and we showed that raloxifene and RU486 are ERalpha and ERbeta-selective antiestrogens, respectively. In all cases, selectivity is due to differences in binding affinities as indicated by whole-cell ligand-binding assays. Very interestingly, we demonstrate that a combination of genistein and raloxifene produces a full-ERbeta specific response. Together these results demonstrate the usefulness of our stably transfected cell lines to characterize ER ligands and indicate that treatments combining agonist/antagonist ligands produce full-ERbeta selectivity.

Identification of new human pregnane X receptor ligands among pesticides using a stable reporter cell system.

Pregnane X receptor (PXR, NR1I2) is activated by various chemically unrelated compounds, including environmental pollutants and drugs. We proceeded here to in vitro screening of 28 pesticides with a new reporter system that detects human pregnane X receptor (hPXR) activators. The cell line was obtained by a two-step stable transfection of cervical cancer HeLa cells. The first transfected cell line, HG5LN, contained an integrated luciferase reporter gene under the control of a GAL4 yeast transcription factor-binding site. The second cell line HGPXR was derived from HG5LN and stably expressed hPXR ligand-binding domain fused to GAL4 DNA-binding domain (DBD). The HG5LN cells were used as a control to detect nonspecific activities. Pesticides from various chemical classes were demonstrated, for the first time, to be hPXR activators: (1) herbicides: pretilachlor, metolachlor, and alachlor chloracetanilides, oxadiazon oxiconazole, and isoproturon urea; (2) fungicides: bupirimate and fenarimol pyrimidines, propiconazole, fenbuconazole, prochloraz conazoles, and imazalil triazole; and (3) insecticides: toxaphene organochlorine, permethrin pyrethroid, fipronil pyrazole, and diflubenzuron urea. Pretilachlor, metolachlor, bupirimate, and oxadiazon had an affinity for hPXR equal to or greater than the positive control rifampicin. Some of the newly identified hPXR activators were also checked for their ability to induce cytochrome P450 3A4 expression in a primary culture of human hepatocytes. HGPXR, with HG5LN as a reference, was grafted onto nude mice to assess compound bioavailability through in vivo quantification of hPXR activation. Altogether, our data indicate that HGPXR cells are an efficient tool for identifying hPXR ligands and establishing pesticides as hPXR activators.

Discovery bioanalysis and in vivo pharmacology as an integrated process: a case study in oncology drug discovery.

BACKGROUND: A bioanalytical team dedicated to in vivo pharmacology was set up to accelerate the selection and characterization of compounds to be evaluated in animal models in oncology. RESULTS: A DBS-based serial microsampling procedure was optimized from sample collection to extraction to obtain a generic procedure. UHPLC-high-resolution mass spectrometer configuration allowed for fast quantitative and qualitative analysis. Using an optimized lead compound, we show how bioanalysis supported in vivo pharmacology by generating blood and tumor exposure, drug monitoring and PK/PD data. CONCLUSION: This process provided unique opportunities for the characterization of drug properties, selection and assessment of compounds in animal models and to support and expedite proof-of-concept studies in oncology.

Enhancement of Fluorescent Probe Penetration into Tumors In Vivo Using Unseeded Inertial Cavitation.

Ultrasound-induced cavitation has found many applications in the field of cancer therapy. One of its beneficial effects is the enhancement of drug intake by tumor cells. Our group has developed a device that can create and control unseeded cavitation in tissue using ultrasound. We conducted experiments on tumor-bearing mice using our device to assess the impact of sonication on the penetration of fluorescent probes into tumor cells. We studied the influence of pressure level, timing of sonication and sonication duration on treatment efficiency. Our results indicate that fluorescent probes penetrate better into tumors exposed to ultrasound. The best results revealed an increase in penetration of 61% and were obtained when sonicating the tumor in presence of the probes with a peak negative pressure at focus of 19 MPa. At this pressure level, the treatment generated only minor skin damage. Treatments could be significantly accelerated as equivalent enhanced penetration of probes was achieved when multiplying the initial raster scan speed by a factor of four.

Binding of estrogenic compounds to recombinant estrogen receptor-alpha: application to environmental analysis.

Estrogenic activity in environmental samples could be mediated through a wide variety of compounds and by various mechanisms. High-affinity compounds for estrogen receptors (ERs), such as natural or synthetic estrogens, as well as low-affinity compounds such as alkylphenols, phthalates, and polychlorinated biphenyls are present in water and sediment samples. Furthermore, compounds such as polycyclic aromatic hydrocarbons, which do not bind ERs, modulate estrogen activity by means of the aryl hydrocarbon receptor (AhR). In order to characterize compounds that mediate estrogenic activity in river water and sediment samples, we developed a tool based on the ER-alphaligand-binding domain, which permitted us to estimate contaminating estrogenic compound affinities. We designed a simple transactivation assay in which compounds of high affinity were captured by limited amounts of recombinant ER-alpha and whose capture led to a selective inhibition of transactivation. This approach allowed us to bring to light that water samples contain estrogenic compounds that display a high affinity for ERs but are present at low concentrations. In sediment samples, on the contrary, we showed that estrogenic compounds possess a low affinity and are present at high concentration. Finally, we used immobilized recombinant ER-alpha to separate ligands for ER and AhR that are present in river sediments. Immobilized ER-alpha, which does not retain dioxin-like compounds, enabled us to isolate and concentrate ER ligands to facilitate their further analysis.

Optical imaging of disseminated leukemia models in mice with near-infrared probe conjugated to a monoclonal antibody.

BACKGROUND: The assessment of anticancer agents to treat leukemia needs to have animal models closer to the human pathology such as implantation in immunodeficient mice of leukemic cells from patient samples. A sensitive and early detection of tumor cells in these orthotopic models is a prerequisite for monitoring engraftment of leukemic cells and their dissemination in mice. Therefore, we developed a fluorescent antibody based strategy to detect leukemic foci in mice bearing patient-derived leukemic cells using fluorescence reflectance imaging (FRI) to determine when to start treatments with novel antitumor agents. METHODS: Two mAbs against the CD44 human myeloid marker or the CD45 human leukocyte marker were labeled with Alexa Fluor 750 and administered to leukemia-bearing mice after having verified the immunoreactivity in vitro. Bioluminescent leukemic cells (HL60-Luc) were used to compare the colocalization of the fluorescent mAb with these cells. The impact of the labeled antibodies on disease progression was further determined. Finally, the fluorescent hCD45 mAb was tested in mice engrafted with human leukemic cells. RESULTS: The probe labeling did not modify the immunoreactivity of the mAbs. There was a satisfactory correlation between bioluminescence imaging (BLI) and FRI and low doses of mAb were sufficient to detect leukemic foci. However, anti-hCD44 mAb had a strong impact on the tumor proliferation contrary to anti-hCD45 mAb. The use of anti-hCD45 mAb allowed the detection of leukemic patient cells engrafted onto NOD/SCID mice. CONCLUSIONS: A mAb labeled with a near infrared fluorochrome is useful to detect leukemic foci in disseminated models provided that its potential impact on tumor proliferation has been thoroughly documented.

Potential role of estrogen receptor beta as a tumor suppressor of epithelial ovarian cancer.

Ovarian cancer is the gynecological cancer exhibiting the highest morbidity and improvement of treatments is still required. Previous studies have shown that Estrogen-receptor beta (ERß) levels decreased along with ovarian carcinogenesis. Here, we present evidence that reintroduction of ERß in BG-1 epithelial ovarian cancer cells, which express ERα, leads in vitro to a decrease of basal and estradiol-promoted cell proliferation. ERß reduced the frequency of cells in S phase and increased the one of cells in G2/M phase. At the molecular level, we found that ERß downregulated total retinoblastoma (Rb), phosphorylated Rb and phospho-AKT cellular content as well as cyclins D1 and A2. In addition, ERß had a direct effect on ERα, by strongly inhibiting its expression and activity, which could explain part of the anti-proliferative action of ERß. By developing a novel preclinical model of ovarian cancer based on a luminescent orthotopic xenograft in athymic Nude mice, we further revealed that ERß expression reduces tumor growth and the presence of tumor cells in sites of metastasis, hence resulting in improved survival of mice. Altogether, these findings unveil a potential tumor-suppressor role of ERß in ovarian carcinogenesis, which could be of potential clinical relevance for the selection of the most appropriate treatment for patients.

Quantitation in bioluminescence imaging by correction of tissue absorption for experimental oncology.

PURPOSE: We have developed a method of quantitation for correcting tissue absorption in in vivo bioluminescence imaging (BLI). PROCEDURES: Variations of luciferin emission spectrum were determined and were related to photon absorption to determine a correction curve. This was validated by combining BLI with tomoscintigraphy and tomodensitometry, which were applied to a lymphoma model. RESULTS: Tissue absorption affects luciferin emission spectrum, mainly for wavelengths less than 620 nm. So, we have selected two filters bordering 620 nm to quantify spectral modifications. A significant correlation was obtained between the spectral analysis and the percentage of transmitted light through tissues (R(2) = 0.97). On a disseminated tumour model, we have shown that such a methodology is of great interest to compare bioluminescent signals and to get more accurate quantitative data about tumour proliferation. CONCLUSIONS: Spectral analysis allows improved quantitation of BLI and could be of value to perform pharmacological studies and to follow tumour progression in models with tumours evolving in different locations.