Insights into tissue microstructure using a double diffusion encoding sequence on a clinical scanner: Validation and application to experimental tumor models.

PURPOSE: To present a double diffusion encoding MRI sequence on a clinical scanner to analyze micro-structure and micro-vasculature of tumors. METHODS: The sequence was tested on phantoms, asparaguses, and 2 tumors allografts in a rodent. Results were analyzed using an adapted VERDICT model to estimate microstructural parameters. The technical feasibility of the sequence on a 3T clinical system was assessed on a water phantom. The accuracy of cell size estimation was assessed on asparaguses by comparison with light microscopy. Cell size estimations were also validated when limiting relative angles of diffusion encodings to 0 and 180°. Sensitivities to restricted diffusion and incoherent flow from the vasculature were investigated in experimental tumor models. Values of microstructural parameters in viable and decaying tumor tissue were compared with those obtained from histological analysis. RESULTS: Measurements on the water phantom revealed no significant sequence artifacts and accurate apparent diffusion coefficient values within a 4% relative error. In asparaguses, quartiles and medians of pore size distributions typically deviated less than 6% from light microscopy regardless of whether the full or reduced set of relative angles was used. Signal analyses in tumors showed mixed effects of both blood flow and diffusion restriction. Microstructural parameter estimations in tumors were consistent with histology and allowed clear and histology-proven distinctions between decaying and viable tumor tissue. CONCLUSIONS: Double diffusion encoding with clinical gradients and scan times allows characterization of restricted diffusion and micro-circulation flow in tumors. Our estimated microstructural parameters are promising for further investigations in assessing microstructural evolutions in tumors.

Double Diffusion Encoding for Probing Radiation-Induced Microstructural Changes in a Tumor Model: A Proof-of-Concept Study With Comparison to the Apparent Diffusion Coefficient and Histology.

BACKGROUND: Microstructure analyses are gaining interest in cancer MRI as an alternative to the conventional apparent diffusion coefficient (ADC), of which the determinants remain unclear. PURPOSE: To assess the sensitivity of parameters calculated from a double diffusion encoding (DDE) sequence to changes in a tumor's microstructure early after radiotherapy and to compare them with ADC and histology. STUDY TYPE: Cohort study on experimental tumors. ANIMAL MODEL: Sixteen WAG/Rij rats grafted with one rhabdomyosarcoma fragment in each thigh. Thirty-one were imaged at days 1 and 4, of which 17 tumors received a 20 Gy radiation dose after the first imagery. FIELD STRENGTH/SEQUENCE: 3T. Diffusion-weighted imaging, DDE with flow compensated, and noncompensated measurements. ASSESSMENTS: 1) To compare, after irradiation, DDE-derived parameters (intracellular fraction, cell size, and cell density) to their histological counterparts (fraction of stained area, minimal Feret diameter, and nuclei count, respectively). 2) To compare percentage changes in DDE-derived parameters and ADC. 3) To evaluate the evolution of DDE-derived parameters describing perfusion. STATISTICAL TESTS: Wilcoxon rank sum test. RESULTS: 1) Intracellular fraction, cell size, and cell density were respectively lower (-24%, P < 0.001), higher (+7.5%, P < 0.001) and lower (-38%, P < 0.001) in treated tumors as compared to controls. Fraction of stained area, minimal Feret diameter, and nuclei count were respectively lower (-20%, P < 0.001), higher (+28%, P < 0.001), and lower (-34%, P < 0.001) in treated tumors. 2) The magnitude of ADC's percentage change due to irradiation (16.4%) was superior to the one of cell size (8.4%, P < 0.01) but inferior to those of intracellular fraction (35.5%, P < 0.001) and cell density (42%, P < 0.001). 3) After treatment, the magnitude of the vascular fraction's decrease was higher than the increase of flow velocity (33.3%, vs. 13.3%, P < 0.001). DATA CONCLUSION: The DDE sequence allows quantitatively monitoring the effects of radiotherapy on a tumor's microstructure, whereas ADC only reveals global changes. EVIDENCE LEVEL: 2. TECHNICAL EFFICACY: Stage 4. J. Magn. Reson. Imaging 2020;52:941-951.

Participation of liver progenitor cells in liver regeneration: lack of evidence in the AAF/PH rat model.

When hepatocyte proliferation is impaired, liver progenitor cells (LPC) are activated to participate in liver regeneration. We used the 2-acetaminofluorene/partial hepatectomy (AAF/PH) model to evaluate the contribution of LPC to liver cell replacement and function restoration. Fischer rats subjected to AAF/PH (or PH alone) were investigated 7, 10 and 14 days post-hepatectomy. Liver mass recovery (LMR) was estimated, and the liver mass to body weight ratio calculated. We used serum albumin and bilirubin levels, and liver albumin mRNA levels to assess the liver function. LPC expansion was analyzed by cytokeratin 19 (CK19), glutathione S-transferase protein (GSTp) immunohistochemistry and by CK19, CD133, transforming growth factor-ß1 and hepatocyte growth factor mRNA expression in livers. Cell proliferation was evaluated by Ki67 and BrdU immunostaining. Compared with PH alone where LMR was ∼100% 14 days post-PH, LMR was defective in AAF/PH rats (64.1±15.5%, P=0.0004). LPC expansion was scarce in PH livers (0.5±0.4% of CK19(+) area), but significant in AAF/PH livers (8.5±7.2% of CK19(+)), and inversely correlated to LMR (r(2)=0.63, P<0.0001). A quarter of AAF/PH animals presented liver failure (low serum albumin and high serum bilirubin) 14 days post-PH. Compared with animals with preserved function, this was associated with a lower LMR (50±6.8 vs 74.6±9.4%, P=0.0005), a decreased liver to body weight ratio (2±0.3 vs 3.5±0.6%, P=0.001), and a larger LPC expansion such as proliferating Ki67(+) LPC covered 17.4±4.2% of the liver parenchyma vs 3.1±1.5%, (P<0.0001). Amongst those, rare LPC with an intermediate hepatocyte-like phenotype were seen. Also, less than 2% of hepatocytes were engaged into the cell cycle (Ki67(+)), while more numerous (∼25% of hepatocytes) in the livers with preserved function. These observations suggest that, in this model, the efficient recovery of the liver function was ensured rather by the proliferation of mature hepatocytes than by the LPC expansion and differentiation into hepatocytes.

Kupffer cells influence parenchymal invasion and phenotypic orientation, but not the proliferation, of liver progenitor cells in a murine model of liver injury.

Activation of myofibroblasts (MF) and extracellular matrix (ECM) deposition predispose the expansion and differentiation of liver progenitor cells (LPC) during chronic liver injury. Because Kupffer cells (KC) are active modulators of tissue response and fibrosis, we analyzed their role in a model of LPC proliferation. A choline-deficient diet, supplemented by ethionine (CDE) was administrated to C57Bl/6J mice that were depleted of KC by repeated injections of clodronate (CLO) and compared to PBS-injected mice. On CDE, massive KC activation was observed in the PBS group, but this was blunted in CLO-treated mice. The depletion of KC did not influence LPC proliferation but reduced their invasive behavior. Instead of being found far into the parenchyma, as was found in the PBS group (mean distance from portal vein: 209 µm), LPC of CLO mice remained closer to the portal area (138 µm), forming aggregates and phenotypically resembling cells of biliary lineage. Notably, removal of KC was also associated with a significant decrease in amount of MF and ECM and in the expression of profibrotic factors. Thus, besides ECM and MF, KC are also a significant component of the microenvironmental changes preceding LPC expansion. Depletion of KC may limit the LPC parenchymal invasion through a deficiency in chemoattracting factors, reduced activation of MF, and/or a paucity of the ECM framework necessary for cell motility.

Early (72-hour) detection of radiotherapy-induced changes in an experimental tumor model using diffusion-weighted imaging, diffusion tensor imaging, and Q-space imaging parameters: a comparative study.

PURPOSE: To assess and compare the potential of various diffusion-related magnetic resonance imaging (MRI) parameters to detect early radiotherapy (RT)-induced changes in tumors. MATERIALS AND METHODS: Nineteen tumors in a rat model were imaged on a clinical 3T system before and 72 hours after a single RT session. Diffusion imaging was performed using an echo planar sequence containing 16 b-factors and six gradient directions. This allowed us to perform a tensor analysis of mono- and biexponential decays and a q-space analysis. Parametric maps (both trace and fractional anisotropy) were reconstructed for: 2-point apparent diffusion coefficient (ADC), 16-point ADC, biexponential amplitudes and ADCs, and height, width, and kurtosis of the probability density function (PDF). A texture analysis yielded quantities such as average and contrast. The sensitivity of diffusion-related parameters was quantified in terms of the mean relative difference (when comparing pre- and post-RT status). RESULTS: Traces and anisotropies display differences in response to RT. Average traces are most sensitive for ADCs and kurtosis. Average anisotropies are all very sensitive except the slow biexponential component. The best contrast (traces) was found for the ADCs and the width of the PDF. CONCLUSION: ADC performed well, but high b-values analysis added extra sensitive parameters for monitoring early RT-induced changes.

Multiparametric magnetic resonance imaging to differentiate high-grade gliomas and brain metastases.

PURPOSE: To assess the performance of parameters used in conventional magnetic resonance imaging (MRI), perfusion-weighted MR imaging (PWI) and visual texture analysis, alone and in combination, to differentiate a single brain metastasis (MET) from glioblastoma multiforme (GBM). PATIENTS AND METHODS: In a retrospective study of 50 patients (41 GBM and 14 MET) who underwent T2/FLAIR/T1(post-contrast) imaging and PWI, morphological (circularity, surface area), perfusion (rCBV in the ring-like tumor area, rCBV in the peritumoral area, percentage of signal intensity recovery at the end of first pass) and texture parameters in the peritumoral area were estimated. Statistical differences and performances were assessed using Wilcoxon's test and receiver operating characteristic curves, respectively. Multiparametric classification of tumors was performed using k-means clustering. RESULTS: Significant statistical differences in circularity, surface area, rCBVs, percentage of signal intensity recovery and texture parameters (energy, entropy, homogeneity, correlation, inverse differential moment, sum average) were observed between MET and GBM (P<0.05). Moderate-to-good classification performances were found with these parameters. Clustering based on rCBV and texture parameters (contrast, sum average) differentiated MET from GBM with a sensitivity of 92% and a specificity of 71%. CONCLUSION: Combining perfusion and visual texture parameters within a statistical classifier significantly improved the differentiation of a single brain MET and GBM.

Relation between liver progenitor cell expansion and extracellular matrix deposition in a CDE-induced murine model of chronic liver injury.

UNLABELLED: In chronic liver injury, liver progenitor cells (LPCs) proliferate in the periportal area, migrate inside the lobule, and undergo further differentiation. This process is associated with extracellular matrix (ECM) remodeling. We analyzed LPC expansion and matrix accumulation in a choline-deficient, ethionine-supplemented (CDE) model of LPC proliferation. After day 3, CDE induced collagen deposits in the periportal area. Expansion of LPCs as assessed by increased number of cytokeratin 19 (CK19)-positive cells was first observed at day 7, while ECM accumulated 10 times more than in controls. Thereafter, LPCs and ECM increased in parallel. Furthermore, ECM not only accumulates prior to the increase in number of LPCs, but is also found in front of LPCs along the porto-venous gradient of lobular invasion. Double immunostaining revealed that LPCs are embedded in ECM at all times. Moreover, LPCs infiltrating the liver parenchyma are chaperoned by alpha-smooth muscle actin (alpha-SMA)-positive cells. Gene expression analyses confirmed these observations. The expression of CK19, alpha-fetoprotein, E-cadherin, and CD49f messenger RNA (mRNA), largely overexpressed by LPCs, significantly increased between day 7 and day 10. By contrast, at day 3 there was a rapid burst in the expression of components of the ECM, collagen I and laminin, as well as in alpha-SMA and connective tissue growth factor expression. CONCLUSION: Our data demonstrate that, in a CDE model, ECM deposition and activation of matrix-producing cells occurred as an initial phase, prior to LPC expansion, and in front of LPCs along the porto-venous gradient of lobular invasion. Those observations may reveal a fundamental role for the established hepatic microenvironment or niche during the process of activation and differentiation of liver progenitor cells.

MRI of iron-oxide labelled transplanted hepatocytes in mice: effect of treatment with cyclophosphamide.

PURPOSE: To assess if 1.5T MRI can be used to study the transport to the liver, the intrahepatic distribution and engraftment of iron-oxide labelled hepatocytes in cyclophosphamide-treated and untreated mice. MATERIALS AND METHODS: Hepatocytes were isolated from C57bl/6 mice and were labelled with 1.63 microm iron-oxide particles. Seventeen mice were pretreated with cyclophosphamide to disrupt the sinusoidal endothelium and 15 were left untreated. Seven days after splenic injection of the labelled hepatocytes, T2*-weighted gradient-echo images at 1.5T were acquired. The hepatic transport, distribution and engraftment of the labelled hepatocytes were assessed with signal intensity (SI) and T2* measurements, electron paramagnetic resonance (EPR), texture analysis and histology. RESULTS: Lower hepatic SI (P = 0.005), lower T2* (P = 0.033) and larger number of particles at histology (P = 0.006) suggested increased transport to the liver of labelled hepatocytes in cyclophosphamide-treated mice versus untreated mice. At histology, most particles were located in Kupffer cells. Particles distribution was heterogeneous. No difference between both groups was observed at texture analysis. CONCLUSION: MRI is useful to assess the transport to the liver and intrahepatic distribution of transplanted labelled hepatocytes. The preferential location of iron-oxide particles within Kupffer cells after seven days limits the value of MRI for assessing hepatocyte engraftment.

Prevention of steatohepatitis by pioglitazone: implication of adiponectin-dependent inhibition of SREBP-1c and inflammation.

BACKGROUND/AIMS: Peroxisome proliferator-activated receptor gamma (PPARgamma) agonist drugs, like pioglitazone (PGZ), are proposed as treatments for steatohepatitis. Their mechanisms of action remain ill-clarified. METHODS: To test the hypothesis that PGZ improves steatohepatitis through adiponectin-dependent stimulation of AMPK and/or PPARalpha, mice lacking adiponectin (Adipo(-/-)) or the AMPKalpha1 catalytic subunit (AMPKalpha1(-/-)) or wild-type (Wt) mice were fed the methionine and choline deficient (MCD) diet, supplemented or not with PGZ. RESULTS: In Wt mice, PGZ increased circulating levels of adiponectin and reduced the severity of MCD-induced steatohepatitis but there was no evidence of activation of AMPK or PPARalpha and their downstream targets. By contrast, PGZ completely repressed nuclear translocation of SREBP-1c, a key transcription factor for de novo lipogenesis. This effect was lacking in Adipo(-/-) mice in which PGZ failed to prevent steatohepatitis. Surprisingly, AMPKalpha1(-/-) mice were resistant to MCD-induced steatohepatitis, a status also associated with repression of SREBP-1c. CONCLUSIONS: The preventive effect of PGZ on MCD-induced steatohepatitis depends on adiponectin upregulation but apparently does not involve AMPK or PPARalpha activation. The inhibition of SREBP-1c and dependent repression of lipogenesis are likely to participate in this effect. The mechanisms by which PGZ and adiponectin control SREBP-1c and inflammation remain to be elucidated.

Early detection of steatohepatitis in fatty rat liver by using MR elastography.

PURPOSE: To assess the potential value of magnetic resonance (MR) elastographic imaging to help detect nonalcoholic steatohepatitis in the fatty rat liver. MATERIALS AND METHODS: This study was approved by the regional ethics committee. Fifty-four rats were imaged after being fed either a standard diet, a choline-deficient diet for up to 8 weeks to induce steatohepatitis, or a 2-week orotic acid diet to induce steatosis; or were imaged 48 hours after carbon tetrachloride injection to model acute liver injury. MR elastography was performed at 7.0 T to assess viscoelastic liver parameters. Steatosis and fibrosis were quantified with morphometric and biochemical analysis. Myofibroblast activation was assessed with morphometric analysis of alpha-smooth muscle actin. Expression of transforming growth factor beta1 and procollagens 1 and 3 as markers of fibrogenesis was evaluated with real-time reverse transcription polymerase chain reaction. Inflammation was scored at histologic analysis. RESULTS: In rats with steatohepatitis, mean elasticity (2.24 kPa +/- 0.19 [standard deviation] vs 1.82 kPa +/- 0.22) and mean viscosity (0.86 kPa +/- 0.10 vs 0.59 kPa +/- 0.12) increased significantly (P < .005) after the 2-week orotic acid diet, while steatosis, inflammation, myofibroblast activation, and increase of other fibrogenesis markers were observed. Fibrosis appeared only after 5 weeks. In rats with steatosis, viscosity increased (0.77 kPa +/- 0.11, P < .005), elasticity remained constant. In rats with acute liver injury, elasticity (2.96 kPa +/- 0.63) and viscosity (0.85 kPa +/- 0.22) increased (P < .005), while fibrogenesis and inflammation were observed without substantial fibrosis or steatosis. At multivariate analysis in all rats, liver elasticity correlated only with myofibroblast activation (P < .001, r > 0.6). CONCLUSION: The results suggest that in nonalcoholic fatty rat liver, MR elastography may be useful in the early detection of steatohepatitis by showing increased elasticity and appearing before fibrosis development, which was linked to myofibroblast activation. SUPPLEMENTAL MATERIAL: http://radiology.rsna.org/lookup/suppl/doi:10.1148/radiol.2523081817/-/DC1.

Development of hepatic fibrosis occurs normally in AMPK-deficient mice.

Inhibition or blockade of HSCs (hepatic stellate cells), the main matrix-producing cells involved in the wound-healing response, represents an attractive strategy for the treatment of liver fibrosis. In vitro studies have shown that activation of AMPK (AMP-activated protein kinase), a key player in the regulation of cellular energy homoeostasis, inhibits proliferation of myofibroblasts derived from HSCs. If AMPK is a true regulator of fibrogenesis then defective AMPK activity would enhance fibrogenesis and hepatic fibrosis. To test this, in the present work, in vitro studies were performed on mouse primary HSCs treated or not with the AMPK activator AICAR (5-amino-4-imidazolecarboxamide ribonucleotide) or isolated from mice lacking the AMPKalpha1 catalytic subunit (AMPKalpha1-/-) or their littermates (AMPKalpha1+/+). Liver fibrosis was induced in vivo in AMPKalpha1-/- and AMPKalpha1+/+ mice by repeated injections of CCl(4) (carbon tetrachloride). During culture activation of HSCs, AMPK protein and activity significantly increased and regulatory AMPKgamma3 mRNA was specifically up-regulated. Stimulation of AMPK activity by AICAR inhibited HSC proliferation, as expected, as well as collagen alpha1(I) expression. Importantly, AMPKalpha1 deletion inhibited proliferation of HSCs, but not fibrogenesis, in vivo. Moreover, AMPKalpha1 deletion was not associated with enhanced CCl(4)-induced fibrosis in vivo. In conclusion, our present findings demonstrate that HSC transdifferentiation is associated with increased AMPK activity that could relate to the stabilization of AMPK complex by the gamma3 subunits. Activation of AMPK in HSCs inhibits in vitro fibrogenesis. By contrast, low AMPK activity does not prevent HSC activation in vitro nor in in vivo fibrosis.

Alginate moulding: an empirical method for magnetic resonance imaging/positron emission tomography co-registration in a tumor rat model.

BACKGROUND AND PURPOSE: In the experimental field of animal models, co-registration between positron emission tomography (PET) and magnetic resonance imaging (MRI) data still relies on non-automated post-processing using sophisticated algorithms and software developments. We assessed the value of an empirical method using alginate moulding for PET-MR co-registration in a tumor rat model. METHODS: Male WAG/RijHsd rats bearing grafted syngenic rhabdomyosarcoma were examined under general anesthesia by MRI using a clinical whole-body 3-T system equipped with a sensitivity-encoding four-channel wrist coil and by a small animal PET system using labelled [(18)F]-fluorocholine as tracer. An alginate mould including a system of external fiducials was manufactured for each animal, allowing strict immobilization and similar positioning for both modalities. Fourteen rats (27 tumors) had only one MR/PET imaging session. Five rats (9 tumors) had a similar MR/PET session before and 3 days after external radiation therapy (13 Gy in one fraction) using the same mould. Co-registration was performed using the Pmod release 2.75 software (PMOD Technologies, Ltd., Adliswil, Switzerland) with mutual information algorithm. RESULTS: The manufacture of the alginate moulds was easy and innocuous. Imaging sessions were well tolerated. PET-MR co-registration based on mutual information was perfect at visual examination, which was confirmed by the superimposition of external fiducials on fused images. Reuse of the same mould for the post-therapeutic session was feasible 3 days after the pre-therapeutic one in spite of tumor growth. CONCLUSION: The empirical method using alginate moulding with external fiducials for PET-MR co-registration in a rodent tumor model was feasible and accurate.

Effect of curcumin on the cell surface markers CD44 and CD24 in breast cancer.

Human breast cell lines are often characterized based on the expression of the cell surface markers CD44 and CD24. CD44 is a type I transmembrane glycoprotein that regulates cell adhesion and cell-cell, as well as cell-extracellular matrix interactions. CD24 is expressed in benign and malignant solid tumors and is also involved in cell adhesion and metastasis. The aim of the present study was to investigate the effects of curcumin on the surface expression of CD44 and CD24 in breast epithelial cell lines. An established breast cancer model derived from the MCF-10F cell line was used. The results revealed that curcumin decreased CD44 and CD24 gene and protein expression levels in MCF-10F (normal), Alpha5 (premalignant) and Tumor2 (malignant) cell lines compared with the levels in their counterpart control cells. Flow cytometry revealed that the CD44+/CD24+ cell subpopulation was greater than the CD44+/CD24- subpopulation in these three cell lines. Curcumin increased CD44+/CD24+ to a greater extent and decreased CD44+/CD24- subpopulations in the normal MCF-10F and the pre-tumorigenic Alpha5 cells, but had no significant effect on Tumor2 cells compared with the corresponding control cells. Conversely, curcumin increased CD44 and decreased CD24 gene expression in MCF-7 breast cancer cells, and decreased CD44 gene expression in MDA-MB-231 cell line, while CD24 was not present in these cells. Curcumin did not alter the CD44+/CD24+ or CD44+/CD24- subpopulations in the MCF-7 cell line. However, it increased CD44+/CD24+ and decreased CD44+/CD24- subpopulations in MDA-MB-231 cells. In breast cancer specimens from patients, normal tissues were negative for CD44 and CD24 expression, while benign lesions were positive for both markers, and malignant tissues were found to be negative for CD44 and positive for CD24 in most cases. In conclusion, these results indicated that curcumin may be used to improve the proportion of CD44+/CD24+ cells and decrease the proportion of CD44+/CD24- cells. Therefore, it may be suggested that curcumin decreased cancerous types of breast cells.

Inhibition of early preneoplastic events in the rat liver by the somatostatin analog lanreotide.

Hepatocellular carcinoma (HCC) is the third most common cause of cancer-related death, and its incidence is increasing worldwide. Due to the known risk factors (mainly hepatitis B and C viruses), we believe there is a rationale for a chemopreventive approach to treat HCC. Here, based on described in vitro data, we evaluated the preventive effects of lanreotide, a somatostatin analog, on the induction of early carcinogenic events. We monitored preneoplastic foci induced by a two-stage initiation/promotion model of hepatocarcinogenesis in male Wistar rats, using diethylnitrosamine and 2-acetylaminofluorene. Lanreotide was given starting the day after the first diethylnitrosamine injection. By quantitative morphometry, we showed that lanreotide significantly decreases the size of induced preneoplastic foci. Analysis of proliferation and apoptosis assessed by immunohistochemistry, showed decreased proliferation and increased cell death in rats treated with lanreotide. As these events were associated with a significant decreased expression of the cell cycle regulator cyclin D1 and an increased expression of the cyclin-dependent kinase inhibitor p27(kip1) compared to the non-treated group, it is tempting to speculate that these factors are involved in the favorable effect of lanreotide. In conclusion, lanreotide significantly decreases early carcinogenic transformation in a two-step rat model. As lanreotide has a low toxicity profile, we believe it would be interesting to evaluate its effect in chemoprevention of HCC.

Peroxisome proliferated-activated receptor gamma ligand, Pioglitazone, does not prevent hepatic fibrosis in mice.

During hepatic fibrogenesis, quiescent hepatic stellate cells (HSCs) undergo phenotypic transformation into activated matrix-producing cells. This process is recapitulated in primary HSCs cultured on plastic. Based on studies in rats, peroxisome proliferator-activated receptor gamma (PPARgamma) has been suggested to play a key role in the control of HSC activation. Indeed, in rats, PPARgamma expression is depleted in activated HSCs. PPARgamma ligands inhibit HSC activation and prevent hepatic fibrosis in vivo. Here we evaluated the impact of PPARgamma agonists on hepatic fibrogenesis in mice both in vitro and in vivo. Primary HSCs from Balb/C mice were cultured with PPARgamma ligands Pioglitazone (PGZ) or 15-deoxy-Delta12,14 prostaglandin J2 (15d-PGJ2). PPARgamma mRNA expression was stable during culture-activation of HSCs. However, PPARgamma protein was only found in quiescent HSCs but not in fully activated cells. Exposure of HSCs to PPARgamma agonists maintained the expression of PPARgamma, and transactivated this transcription factor as demonstrated by gelshift assay and by induction of CD36, a PPARgamma-regulated gene. However, PPARgamma ligands did not alter the induction of Collagen-I mRNA or alpha-smooth muscle actin (alpha-SMA) in cultured HSCs. To test the effect of PPARgamma agonist PGZ in vivo, hepatic fibrosis was evaluated in Balb/C or C57BL6/J mice treated with CCl4 (three times a week for 4 weeks; or corn oil for controls), and fed a normal or a PGZ-supplemented diet (0.01% wt/wt). PGZ treatment was associated with increased serum adiponectin concentrations but did not decrease the severity of hepatic fibrosis induced by CCl4. Our data demonstrate that, although having anti-fibrotic properties in rats, PPARgamma agonists do not prevent activation of HSCs in vitro, nor hepatic fibrogenesis in vivo in mice.

Ras protein expression as a marker for breast cancer.

Breast cancer, the most common neoplasm in women of all ages, is the leading cause of cancer-related mortality in women worldwide. Markers to help to predict the risk of progression and ultimately provide non-surgical treatment options would be of great benefit. At present, there are no available molecular markers to predict the risk of carcinoma in situ progression to invasive cancer; therefore, all women diagnosed with this type of malignancy must undergo surgery. Breast cancer is a heterogeneous complex disease, and different patients respond differently to different treatments. In breast cancer, analysis using immunohistochemical markers remains an essential component of routine pathological examinations, and plays an import role in the management of the disease by providing diagnostic and prognostic strategies. The aim of the present study was to identify a marker that can be used as a prognostic tool for breast cancer. For this purpose, we firstly used an established breast cancer model. MCF-10F, a spontaneously immortalized breast epithelial cell line was transformed by exposure to estrogen and radiation. MCF-10F cells were exposed to low doses of high linear energy transfer (LET) α particles (150 keV/µm) of radiation, and subsequently cultured in the presence of 17ß-estradiol. Three cell lines were used: i) MCF-10F cells as a control; ii) Alpha5 cells, a malignant and tumorigenic cell line; and iii) Tumor2 cells derived from Alpha5 cells injected into nude mice. Secondly, we also used normal, benign and malignant breast specimens obtained from biopsies. The results revealed that the MCF-10F cells were negative for c-Ha-Ras protein expression; however, the Alpha5 and Tumor2 cell lines were positive for c-Ha-Ras protein expression. The malignant breast samples were also strongly positive for c-Ha-Ras expression. The findings of our study indicate that c-Ha-Ras protein expression may be used as a marker to predict the progression of breast cancer; this marker may also ultimately provide non-surgical treatment options for patients who are at a lower risk.

CPI-0004Na, a new doxorubicin prodrug, reduces growth of 3LL-H61 carcinoma lung metastases in C57BI/6 mice.

The ETAP concept (Extracellularly Tumor-Activated Prodrug) is a new approach developed to overcome the lack of selectivity and the side effects responsible for the limited efficacy of chemotherapeutic agents. CPI-0004Na, a doxorubicin (Dox) prototype prodrug of this type, is less toxic than free Dox and showed increased efficacy against subcutaneous human tumor xenografts. The aim of this study was to assess the efficacy of the prodrug vs Dox (given ip) at their maximal tolerated dose (MTD) for this administration schedule (129.3 micromol/kg and 12.93 micromol/kg, respectively) against experimentally induced 3LL-H61 carcinoma lung metastases in mice. Our results indicate that, Dox has no effect on the number of lung metastases while CPI-0004Na induces a 38.3% reduction on average. When considering the effect on the proportion of the lungs' surface covered by metastases, Dox induces a 39% reduction while the prodrug CPI-0004Na is about two fold more active with a 71% decrease.

Vimentin and Notch as biomarkers for breast cancer progression.

Breast cancer, the most common spontaneous malignancy diagnosed in women, is a classical model of hormone dependency as it is associated with prolonged exposure to female hormones. Different cytoplasmic proteins are important in the transformation of a normal cell to an invasive tumor cell, and these include vimentin and Notch. To investigate the importance of these two genes and proteins in breast carcinogenesis, we used an in vitro breast cancer model system, in which an immortalized human breast epithelial cell line, MCF-10F, was malignantly transformed by exposure to low doses of high linear energy transfer α particle (150 keV/µm) radiation and subsequent growth in the presence or absence of 17ß-estradiol. This model consisted of human breast epithelial cells in different stages of transformation: i) a parental cell line (MCF-10F), ii) an Estrogen cell line (MCF-l0F continuously grown with estradiol at 10-8), iii) a malignant and non-tumorigenic cell line (Alpha3), iv) a malignant and tumorigenic cell line (Alpha5) and v) a Tumor2 cell line derived from a xenograft of the Alpha5 cell line injected into nude mice. Vimentin and Notch showed greater expression in the Alpha5 and Tumor2 cell lines compared with that in the non-tumorigenic cell lines, MCF-10F, Estrogen and Alpha3. In the present study, positive staining for vimentin was found in 21% of cases. Vimentin and Notch protein expression was negative in noninvasive ductal carcinoma biopsies from breast cancer patients. However, positive cell expression was observed in invasive ductal carcinoma biopsies. These biomarkers can be considered important indicators of breast cancer progression and can be added to the diagnostic panel when overall survival is a primary end-point.

Enhanced choline metabolism in a rodent rhabdomyosarcoma model: correlation between RT-PCR and translational 3 T H-MRS.

PURPOSE: To investigate which transmembrane choline transporters and intracellular choline kinases play a prominent role at gene expression level in the rise of the total choline (tCho) peak at proton MR spectra in a rodent rhabdomyosarcoma model. MATERIALS AND METHODS: Twenty-two rats bearing grafted bilateral syngenic rhabdomyosarcoma were examined on a clinical 3 T MR system. Total choline concentration was measured from proton MR spectra using cubic centimeter volumes of interest (VOIs) located contiguously along the greater axis of the tumour. After euthanasia, cubic centimetre tissue specimens corresponding to Proton magnetic resonance spectroscopy (H-MRS) VOIs were frozen in liquid nitrogen. Out of 89 H-MRS voxels, only 39 with a corresponding tissue specimen suitable for biochemical processing were included in the analysis. RNA was extracted from all the 39 samples and reverse-transcribed into cDNA. Choline kinase α and ß gene expression as well as genes of the transmembrane transporters OCT1, OCT2, OCT3, CTL1, CTL3, CTL4 and CHT1 were studied using reverse transcriptase polymerase chain reaction. The expression level of each gene (ΔCt), was normalized referred to that of the RPL19 gene. The Spearman rank correlation coefficient was used to analyse variables. RESULTS: There was no overexpression of genes coding for kinases; however, significant correlation was observed between kinase α sub-type and the tCho peak (P=.002; r=0.51). OCT1 was the most overexpressed transporter gene. Less overexpressed CTL1 gene was significantly correlated with the tCho peak (P=.02; r=0.38). CONCLUSION: Choline transporters seem to play a predominant role in the increase in total choline concentration at the gene expression level in our model.

Early monitoring of external radiation therapy by [18F]-fluoromethylcholine positron emission tomography and 3-T proton magnetic resonance spectroscopy: an experimental study in a rodent rhabdomyosarcoma model.

PURPOSE: To assess early radiation therapy (RT)-induced variations in total choline (tCho) concentration measured by proton magnetic resonance spectroscopy (H-MRS) and in (18)F-labelled fluoromethylcholine (FCH) uptake measured by PET in a rodent tumour model. METHODS: Nine rats bearing syngenic rhabdomyosarcoma grafts in both thighs were irradiated (13 Gy, one fraction). H-MRS data and FCH-PET were acquired in the same imaging session prior to and 3, 9 and 16 days after external RT. Total choline concentration was expressed in arbitrary units as the area under the curve of the 3.2-ppm peak on H-MR spectra. FCH uptake was expressed as maximum standardized uptake value (SUV(max)) and as the % of injected dose per gram (%ID/g) after precise tumour delineation on hybrid PET-MR images. Pre- and post-RT data were compared using the Student's paired t test, and results were expressed as mean+/-S.D. RESULTS: Seventeen tumours were available for analysis. A mean drop in choline concentration of 45% was observed 3 days after irradiation (P<.001), whereas a concomitant mean increase in SUV(max) of 41% was observed (P=.006). Choline concentration reincreased on later time points. CONCLUSIONS: Opposite trend between increased FCH uptake and decreased tCho peak was observed at 3 days. Later (9 and 16 days), uptake remained stable and tCho peak reincreased.