Etv4 regulates nociception by controlling peptidergic sensory neuron development and peripheral tissue innervation.

The perception of noxious environmental stimuli by nociceptive sensory neurons is an essential mechanism for the prevention of tissue damage. Etv4 is a transcriptional factor expressed in most nociceptors in dorsal root ganglia (DRG) during the embryonic development. However, its physiological role remains unclear. Here, we show that Etv4 ablation results in defects in the development of the peripheral peptidergic projections in vivo, and in deficits in axonal elongation and growth cone morphology in cultured sensory neurons in response to NGF. From a mechanistic point of view, our findings reveal that NGF regulates Etv4-dependent gene expression of molecules involved in extracellular matrix (ECM) remodeling. Etv4-null mice were less sensitive to noxious heat stimuli and chemical pain, and this behavioral phenotype correlates with a significant reduction in the expression of the pain-transducing ion channel TRPV1 in mutant mice. Together, our data demonstrate that Etv4 is required for the correct innervation and function of peptidergic sensory neurons, regulating a transcriptional program that involves molecules associated with axonal growth and pain transduction.

Schwann cell precursors from nerve innervation are a cellular origin of melanocytes in skin.

Current opinion holds that pigment cells, melanocytes, are derived from neural crest cells produced at the dorsal neural tube and that migrate under the epidermis to populate all parts of the skin. Here, we identify growing nerves projecting throughout the body as a stem/progenitor niche containing Schwann cell precursors (SCPs) from which large numbers of skin melanocytes originate. SCPs arise as a result of lack of neuronal specification by Hmx1 homeobox gene function in the neural crest ventral migratory pathway. Schwann cell and melanocyte development share signaling molecules with both the glial and melanocyte cell fates intimately linked to nerve contact and regulated in an opposing manner by Neuregulin and soluble signals including insulin-like growth factor and platelet-derived growth factor. These results reveal SCPs as a cellular origin of melanocytes, and have broad implications on the molecular mechanisms regulating skin pigmentation during development, in health and pigmentation disorders.

Cell-based experimental strategies for myelin repair in multiple sclerosis.

Multiple sclerosis (MS) is an autoimmune demyelinating disorder of the central nervous system (CNS), diagnosed at a mean age of 32 years. CNS glia are crucial players in the onset of MS, primarily involving astrocytes and microglia that can cause/allow massive oligodendroglial cells death, without immune cell infiltration. Current therapeutic approaches are aimed at modulating inflammatory reactions during relapsing episodes, but lack the ability to induce very significant repair mechanisms. In this review article, different experimental approaches based mainly on the application of different cell types as therapeutic strategies applied for the induction of myelin repair and/or the amelioration of the disease are discussed. Regarding this issue, different cell sources were applied in various experimental models of MS, with different results, both in significant improvements in remyelination and the reduction of neuroinflammation and glial activation, or in neuroprotection. All cell types tested have advantages and disadvantages, which makes it difficult to choose a better option for therapeutic application in MS. New strategies combining cell-based treatment with other applications would result in further improvements and would be good candidates for MS cell therapy and myelin repair.

Diverse cellular origins of adult blood vascular endothelial cells.

During embryonic stages, vascular endothelial cells (ECs) originate from the mesoderm, at specific extraembryonic and embryonic regions, through a process called vasculogenesis. In the adult, EC renewal/replacement mostly depend on local resident ECs or endothelial progenitor cells (EPCs). Nevertheless, contribution from circulating ECs/EPCs was also reported. In addition, cells lacking from EC/EPC markers with in vitro extended plasticity were shown to originate endothelial-like cells (ELCs). Most of these cells consist of mesenchymal stromal progenitors, which would eventually get mobilized from the bone marrow after injury. Based on that, current knowledge on different mouse and human bone marrow stromal cell (BM-SC) subpopulations, able to contribute with mesenchymal stromal/stem cells (MSCs), is herein reviewed. Such analyses underline an unexpected heterogeneity among sinusoidal LepR+ stromal/CAR cells. For instance, in a recent report a subgroup of LepR+ stromal/CAR progenitors, which express GLAST and is traced in Wnt1Cre;R26RTom mice, was found to contribute with ELCs in vivo. These GLAST â€‹+ â€‹Wnt1+ BM-SCs were shown to get mobilized to the peripheral blood and to contribute with liver regeneration. Other sources of ELCs, such as adipose, neural and dental pulp tissues, were also published. Finally, mechanisms likely involved in the enhanced cellular plasticity properties of bone marrow/adipose tissue stromal cells, able to originate ELCs, are assessed. In the future, strategies to analyze the in vivo expression profile of stromal cells, with MSC properties, in combination with screening of active genomic regions at the single cell-level, during early postnatal development and/or after injury, will likely help understanding properties of these ELC sources.

Nicotine Neurotoxicity Involves Low Wnt1 Signaling in Spinal Locomotor Networks of the Postnatal Rodent Spinal Cord.

The postnatal rodent spinal cord in-vitro is a useful model to investigate early pathophysiological changes after injury. While low dose nicotine (1 µM) induces neuroprotection, how higher doses affect spinal networks is unknown. Using spinal preparations of postnatal wild-type Wistar rat and Wnt1Cre2:Rosa26Tom double-transgenic mouse, we studied the effect of nicotine (0.5-10 µM) on locomotor networks in-vitro. Nicotine 10 µM induced motoneuron depolarization, suppressed monosynaptic reflexes, and decreased fictive locomotion in rat spinal cord. Delayed fall in neuronal numbers (including motoneurons) of central and ventral regions emerged without loss of dorsal neurons. Conversely, nicotine (0.5-1 µM) preserved neurons throughout the spinal cord and strongly activated the Wnt1 signaling pathway. High-dose nicotine enhanced expression of S100 and GFAP in astrocytes indicating a stress response. Excitotoxicity induced by kainate was contrasted by nicotine (10 µM) in the dorsal area and persisted in central and ventral regions with no change in basal Wnt signaling. When combining nicotine with kainate, the activation of Wnt1 was reduced compared to kainate/sham. The present results suggest that high dose nicotine was neurotoxic to central and ventral spinal neurons as the neuroprotective role of Wnt signaling became attenuated. This also corroborates the risk of cigarette smoking for the foetus/newborn since tobacco contains nicotine.

Contribution of neural crest and GLAST+ Wnt1+ bone marrow pericytes with liver fibrogenesis and/or regeneration.

BACKGROUND AND AIMS: Liver fibrosis results from cycles of liver damage and scar formation. We herein aimed at analysing neural crest cells and/or bone marrow stromal cells contribution to the liver. METHODS: Two liver fibrosis and one hepatectomy model were applied on double-transgenic loxP-Cre mouse lines. RESULTS: Increased numbers of glia with more complex processes were found in fibrotic livers. During embryonic development, only few cells were traced in the liver and bone marrow, in a minor fraction of mice of different neural crest reporter strains analysed: therefore, a neural crest origin of such cells is doubtful. In the fibrotic liver, a significantly higher incidence of endothelial cells and hepatocyte-like cells expressing the reporter gene Tomato were found in Wnt1-Cre-Tom and GLAST-CreERT2-Tom mice. Consistently, during early fibrogenesis stromal Wnt1-traced cells, with progenitor (CFU-F) properties, get likely mobilized to peripheral blood. Circulating adult Wnt1-traced cells are stromal cells and lack from the expression of other bone marrow and endothelial progenitor cells markers. Furthermore, in a 70% hepatectomy model GLAST+ Wnt1-traced pericytes were found to be mobilized from the bone marrow and the incidence of GLAST-traced hepatocyte-like cells was increased. Finally, GLAST-traced hepatocyte like-cells were found to maintain the expression of stromal markers. CONCLUSIONS: Our data suggest a gliosis process during liver fibrogenesis. While neural crest cells probably do not contribute with other liver cell types than glia, GLAST+ Wnt1-traced bone marrow pericytes are likely a source of endothelial and hepatocyte-like cells after liver injury and do not contribute to scarring.

Schwann cell precursors in health and disease.

Schwann cell precursors (SCPs) are frequently regarded as neural crest-derived cells (NCDCs) found in contact with axons during nerve formation. Nevertheless, cells with SCPs properties can be found up to the adulthood. They are well characterized with regard to both gene expression profile and cellular behavior -for instance, proliferation, migratory capabilities and survival requirements-. They differ in origin regarding their anatomic location: even though most of them are derived from migratory NCCs, there is also contribution of the boundary cap neural crest cells (bNCCs) to the skin and other tissues. Many functions are known for SCPs in normal development, including nerve fasciculation and target innervation, arterial branching patterning and differentiation, and other morphogenetic processes. In addition, SCPs are now known to be a source of many neural (glia, endoneural fibroblasts, melanocytes, visceral neurons, and chromaffin cells) and non-neural-like (mesenchymal stromal cells, able e.g., to generate dentine-producing odontoblasts) cell types. Until now no reports of endoderm-like derivatives were reported so far. Interestingly, in the Schwann cell lineage only early SCPs are likely able to differentiate into melanocytes and bone marrow mesenchymal stromal cells. We have also herein discussed the literature regarding their role in repair as well as in disease mechanisms, such as in diverse cancers. Moreover, many caveats in our knowledge of SCPs biology are highlighted all through this article. Future research should expand more into the relevance of SCPs in pathologies and in other regenerative mechanisms which might bring new unexpected clinically-relevant knowledge.

[Mesenchymal stem cells and regenerative medicine in liver cirrhosis]. / Células madre mesenquimales y medicina regenerativa en la cirrosis hepática.

Mesenchymal stem cells (MSCs) are multipotent cells with self-renewal capacity which are present in diverse tissues. Recently, significant progresses have been made in the field of MSCs because of its therapeutic potential in regenerative medicine. MSCs selectively migrate toward sites of damage and remodeling, and have the ability to evade the immune system and to promote tissue repair through the production of a number of growth factors and cytokines. Many pre-clinical and clinical studies have been carried out to study its therapeutic effect in liver cirrhosis with promising results. In addition, experimental studies showed that this therapeutic effect can be improved by engineering MSCs to produce therapeutic genes. In this work, the role of MSCs in regenerative medicine and its clinical and pre-clinical applications are reviewed, with an emphasis on its potential as vehicles for therapeutic genes.

Positional differences of axon growth rates between sensory neurons encoded by Runx3.

The formation of functional connectivity in the nervous system is governed by axon guidance that instructs nerve growth and branching during development, implying a similarity between neuronal subtypes in terms of nerve extension. We demonstrate the molecular mechanism of another layer of complexity in vertebrates by defining a transcriptional program underlying growth differences between positionally different neurons. The rate of axon extension of the early subset of embryonic dorsal root ganglion sensory neurons is encoded in neurons at different axial levels. This code is determined by a segmental pattern of axial levels of Runx family transcription factor Runx3. Runx3 in turn determines transcription levels of genes encoding cytoskeletal proteins involved in axon extension, including Rock1 and Rock2 which have ongoing activities determining axon growth in early sensory neurons and blocking Rock activity reverses axon extension deficits of Runx3(-/-) neurons. Thus, Runx3 acts to regulate positional differences in axon extension properties apparently without affecting nerve guidance and branching, a principle that could be relevant to other parts of the nervous system.

Tumor Microenvironment Remodeling by 4-Methylumbelliferone Boosts the Antitumor Effect of Combined Immunotherapy in Murine Colorectal Carcinoma.

We have previously demonstrated that a low dose of cyclophosphamide (Cy) combined with gene therapy of interleukin-12 (AdIL-12) has a synergistic, although limited, antitumoral effect in mice with colorectal carcinoma. The main mechanism involved in the efficacy of Cy+AdIL-12 was the induction of a specific immune response mediated by cytotoxic T lymphocytes. Our current aims were to evaluate the effects of 4-methylumbelliferone (4Mu), a selective inhibitor of hyaluronan (HA) synthesis, on tumor microenvironment (TME) and to investigate how 4Mu affects the therapeutic efficacy of Cy+AdIL-12. The results showed that 4Mu significantly reduced the amount of tumoral HA leading to a significant decrease in tumor interstitial pressure (TIP). As a consequence, tumor perfusion was improved allowing an increased adenoviral transgene expression. In addition, treatment with 4Mu boosted the number of cytotoxic T lymphocytes that reach the tumor after adoptive transfer resulting in a potent inhibition of tumor growth. Importantly, we observed complete tumor regression in 75% of mice when 4Mu was administrated in combination with Cy+AdIL-12. The triple combination 4Mu+Cy+AdIL-12 also induced a shift toward antiangiogenic factors production in tumor milieu. Our results showed that TME remodeling is an interesting strategy to increase the efficacy of anticancer immunotherapies based on gene and/or cell therapy.

The pan-caspase inhibitor Emricasan (IDN-6556) decreases liver injury and fibrosis in a murine model of non-alcoholic steatohepatitis.

BACKGROUND & AIMS: Hepatocyte apoptosis, the hallmark of non-alcoholic steatohepatitis (NASH) contributes to liver injury and fibrosis. Although, both the intrinsic and extrinsic apoptotic pathways are involved in the pathogenesis of NASH, the final common step of apoptosis is executed by a family of cysteine-proteases termed caspases. Thus, our aim was to ascertain if administration of Emricasan, a pan-caspase inhibitor, ameliorates liver injury and fibrosis in a murine model of NASH. METHODS: C57/BL6J-mice were fed regular chow or high fat diet (HFD) for 20 weeks. All mice were treated with vehicle or Emricasan. RESULTS: Mice fed a HFD diet demonstrate a five-fold increase in hepatocyte apoptosis by the TUNEL assay and a 1.5-fold and 1.3-fold increase in caspase-3 and-8 activities respectively; this increase in apoptosis was substantially attenuated in mice fed a HFD treated with Emricasan (HFD-Em). Likewise, liver injury and inflammation were reduced in mice fed HFD-Em as compare to HFD by measuring serum aspartate aminotransferase and alanine aminotransferase levels, NAS histological score and IL 1-ß, TNF-α, monocyte chemoattractant protein (MCP-1) and C-X-C chemokine ligand-2 (CXCL2) quantitative reverse-transcription polymerase chain reaction (qPCR). These differences could not be attributed to differences in hepatic steatosis as liver triglycerides content were similar in both HFD groups. Hepatic fibrosis was reduced by Emricasan in HFD animals by decreasing αSMA (a marker for hepatic stellate cell activation), fibrosis score, Sirius red staining, hydroxyproline liver content and profibrogenic cytokines by qPCR. CONCLUSION: In conclusion, these data demonstrate that in a murine model of NASH, liver injury and fibrosis are suppressed by inhibiting hepatocytes apoptosis and suggests that Emricasan may be an attractive antifibrotic therapy in NASH.

The therapeutic potential of bone marrow-derived mesenchymal stromal cells on hepatocellular carcinoma.

Mesenchymal stromal cells (MSCs) are more often obtained from adult and extraembryonic tissues, with the latter sources being likely better from a therapeutic perspective. MSCs show tropism towards inflamed or tumourigenic sites. Mechanisms involved in MSC recruitment into tumours are comprehensively analysed, including chemoattractant signalling axes, endothelial adhesion and transmigration. In addition, signals derived from hepatocellular carcinoma (HCC) tumour microenvironment and their influence in MSC tropism and tumour recruitment are dissected, as well as the present controversy regarding their influence on tumour growth and/or metastasis. Finally, evidences available on the use of MSCs and other selected progenitor/stem cells as vehicles of antitumourigenic genes are discussed. A better knowledge of the mechanisms involved in progenitor/stem cell recruitment to HCC tumours is proposed in order to enhance their tumour targeting which may result in improvements in cell-based gene therapy strategies.

Antitumor effects of hyaluronic acid inhibitor 4-methylumbelliferone in an orthotopic hepatocellular carcinoma model in mice.

Liver cirrhosis is characterized by an excessive accumulation of extracellular matrix components, including hyaluronan (HA). In addition, cirrhosis is considered a pre-neoplastic disease for hepatocellular carcinoma (HCC). Altered HA biosynthesis is associated with cancer progression but its role in HCC is unknown. 4-Methylumbelliferone (4-MU), an orally available agent, is an HA synthesis inhibitor with anticancer properties. In this work, we used an orthotopic Hepa129 HCC model established in fibrotic livers induced by thioacetamide. We evaluated 4-MU effects on HCC cells and hepatic stellate cells (HSCs) in vitro by proliferation, apoptosis and cytotoxicity assays; tumor growth and fibrogenesis were also analyzed in vivo. Our results showed that treatment of HCC cells with 4-MU significantly reduced tumor cell proliferation and induced apoptosis, while primary cultured hepatocytes remained unaffected. 4-MU therapy reduced hepatic and systemic levels of HA. Tumors systemically treated with 4-MU showed the extensive areas of necrosis, inflammatory infiltrate and 2-3-fold reduced number of tumor satellites. No signs of toxicity were observed after 4-MU therapy. Animals treated with 4-MU developed a reduced fibrosis degree compared with controls (F1-2 vs F2-3, respectively). Importantly, 4-MU induced the apoptosis of HSCs in vitro and decreased the amount of activated HSCs in vivo. In conclusion, our results suggest a role for 4-MU as an anticancer agent for HCC associated with advanced fibrosis.

SPARC downregulation attenuates the profibrogenic response of hepatic stellate cells induced by TGF-ß1 and PDGF.

Liver fibrosis is an active process that involves changes in cell-cell and cell-extracellular matrix (ECM) interaction. Secreted protein, acidic and rich in cysteine (SPARC) is an ECM protein with many biological functions that is overexpressed in cirrhotic livers and upregulated in activated hepatic stellate cells (aHSCs). We have recently shown that SPARC downregulation ameliorates liver fibrosis in vivo. To uncover the cellular mechanisms involved, we have specifically knocked down SPARC in two aHSC lines [the CFSC-2G (rat) and the LX-2 (human)] and in primary cultured rat aHSCs. Transient downregulation of SPARC in hepatic stellate cells (HSCs) did not affect their proliferation and had only minor effects on apoptosis. However, SPARC knockdown increased HSC adhesion to fibronectin and significantly decreased their migration toward PDFG-BB and TGF-ß(1). Interestingly, TGF-ß(1) secretion by HSCs was reduced following SPARC small interfering RNA (siRNA) treatment, and preincubation with TGF-ß(1) restored the migratory capacity of SPARC siRNA-treated cells through mechanisms partially independent from TGF-ß(1)-mediated induction of SPARC expression; thus SPARC knockdown seems to exert its effects on HSCs partially through modulation of TGF-ß(1) expression levels. Importantly, collagen-I mRNA expression was reduced in SPARC siRNA-transfected HSCs. Consistent with previous results, SPARC knockdown in aHSCs was associated with altered F-actin expression patterns and deregulation of key ECM and cell adhesion molecules, i.e., downregulation of N-cadherin and upregulation of E-cadherin. Our data together suggest that the upregulation of SPARC previously reported for aHSCs partially mediates profibrogenic activities of TGF-ß(1) and PDGF-BB and identify SPARC as a potential therapeutic target for liver fibrosis.

Low molecular weight hyaluronan preconditioning of tumor-pulsed dendritic cells increases their migratory ability and induces immunity against murine colorectal carcinoma.

We have recently shown that systemic administration of low molecular weight hyaluronan (LMW HA) significantly reduces colorectal carcinoma (CRC) growth in vivo. The elicited response is partially mediated by activated dendritic cells (DC). To potentiate the ability of DC loaded with whole tumor lysate (DC/TL) to induce immunity against CRC in mice, we aimed to study the effects of preconditioning DC with LMW HA for therapeutic vaccination. LMW HA improved maturation of ex vivo generated DC, increased IL-12, decreased IL-10 production, and enhanced a MLR activity in vitro. Although TNF-α showed a similar capacity to mature DC, preconditioning of DC/TL with LMW HA increased their ability to migrate in vitro toward CCL19 and CCL-21 in a CD44- and a TLR4-independent manner; this effect was superior to Poly(I:C), LPS, or TNF-α and partially associated with an increase in the expression of CCR7. Importantly, LMW HA dramatically enhanced the in vivo DC recruitment to tumor-regional lymph nodes. When these LMW HA-treated CRC tumor lysate-pulsed DC (DC/TL/LMW HA) were administered to tumor-bearing mice, a potent antitumor response was observed when compared to DC pulsed with tumor lysate alone and matured with TNF-α. Then, we showed that splenocytes isolated from animals treated with DC/TL/LMW HA presented a higher proliferative capacity, increased IFN-γ production, and secreted lower levels of the immunosuppressive IL-10. Besides, increased specific CTL response was observed in DC/TL/LMW HA-treated animals and induced long-term protection against tumor recurrence. Our data show that LMW HA is superior to other agents at inducing DC migration; therefore, LMW HA could be considered a new adjuvant candidate in the preparation of DC-based anticancer vaccines with potent immunostimulatory properties.

Hepatocellular carcinoma cells and their fibrotic microenvironment modulate bone marrow-derived mesenchymal stromal cell migration in vitro and in vivo.

Hepatocellular carcinoma (HCC) is the fifth most common cancer and the third cause of cancer-related death. Fibrogenesis is an active process characterized by the production of several proinflammatory cytokines, chemokines and growth factors. It involves the activation of hepatic stellate cells (HSCs) which accumulate at the site of injury and are the main source of the extracellular matrix deposits. There are no curative treatments for advanced HCC, thus, new therapies are urgently needed. Mesenchymal stromal cells (MSCs) have the ability to migrate to sites of injury or to remodeling tissues after in vivo administration; however, in several cancer models they demonstrated limited efficacy to eradicate experimental tumors partially due to poor engraftment. Thus, the aim of this work was to analyze the capacity of human MSCs (hMSCs) to migrate and anchor to HCC tumors. We observed that HCC and HSCs, but not nontumoral stroma, produce factors that induce hMSC migration in vitro. Conditioned media (CM) generated from established HCC cell lines were found to induce higher levels of hMSC migration than CM derived from fresh patient tumor samples. In addition, after exposure to CM from HCC cells or HSCs, hMSCs demonstrated adhesion and invasion capability to endothelial cells, type IV collagen and fibrinogen. Consistently, these cells were found to increase metalloproteinase-2 activity. In vivo studies with subcutaneous and orthotopic HCC models indicated that intravenously infused hMSCs migrated to lungs, spleen and liver. Seven days post-hMSC infusion cells were located also in the tumor in both models, but the signal intensity was significantly higher in orthotopic than in subcutaneous model. Interestingly, when orthotopic HCC tumors where established in noncirrhotic or cirrhotic livers, the amount of hMSCs localized in the liver was higher in comparison with healthy animals. A very low signal was found in lungs and spleens, indicating that liver tumors are able to recruit them at high efficiency. Taken together our results indicate that HCC and HSC cells produce factors that efficiently induce hMSC migration toward tumor microenvironment in vitro and in vivo and make MSCs candidates for cell-based therapeutic strategies to hepatocellular carcinoma associated with fibrosis.

GABAergic Mechanisms Can Redress the Tilted Balance between Excitation and Inhibition in Damaged Spinal Networks.

Correct operation of neuronal networks depends on the interplay between synaptic excitation and inhibition processes leading to a dynamic state termed balanced network. In the spinal cord, balanced network activity is fundamental for the expression of locomotor patterns necessary for rhythmic activation of limb extensor and flexor muscles. After spinal cord lesion, paralysis ensues often followed by spasticity. These conditions imply that, below the damaged site, the state of balanced networks has been disrupted and that restoration might be attempted by modulating the excitability of sublesional spinal neurons. Because of the widespread expression of inhibitory GABAergic neurons in the spinal cord, their role in the early and late phases of spinal cord injury deserves full attention. Thus, an early surge in extracellular GABA might be involved in the onset of spinal shock while a relative deficit of GABAergic mechanisms may be a contributor to spasticity. We discuss the role of GABA A receptors at synaptic and extrasynaptic level to modulate network excitability and to offer a pharmacological target for symptom control. In particular, it is proposed that activation of GABA A receptors with synthetic GABA agonists may downregulate motoneuron hyperexcitability (due to enhanced persistent ionic currents) and, therefore, diminish spasticity. This approach might constitute a complementary strategy to regulate network excitability after injury so that reconstruction of damaged spinal networks with new materials or cell transplants might proceed more successfully.

Overexpression of SPARC obliterates the in vivo tumorigenicity of human hepatocellular carcinoma cells.

Hepatocellular carcinoma (HCC) is the sixth most common cancer and the third leading cause of cancer-related death worldwide. Current treatments are extremely disappointing. SPARC (Secreted protein, acidic and rich in cysteine) is a matricellular glycoprotein with differential expression in several tumors, including HCC, which significance remains unclear. We infected HCC cells (HepG2, Hep3B and Huh7) with an adenovirus expressing SPARC (AdsSPARC) to examine the role of SPARC expression on HCC cells and its effect on tumor aggressiveness. The in vitro HCC cells substrate-dependent proliferation and cell cycle profile were unaffected; however, SPARC overexpression reduced HCC proliferation when cells were grown in spheroids. A mild induction of cellular apoptosis was observed upon SPARC overexpression. SPARC overexpression resulted in spheroid growth inhibition in vitro while no effects were found when recombinant SPARC was exogenously applied. Moreover, the clonogenic and migratory capabilities were largely decreased in SPARC-overexpressing HCC cells, altogether suggesting a less aggressive HCC cell phenotype. Consistently, AdsSPARC-transduced cells showed increased E-cadherin expression and a concomitant decrease in N-cadherin expression. Furthermore, SPARC overexpression was found to reduce HCC cell viability in response to 5-FU-based chemotherapy in vitro, partially through induction of apoptosis. In vivo experiments revealed that SPARC overexpression in HCC cells inhibited their tumorigenic capacity and increased animal survival through a mechanism that partially involves host macrophages. Our data suggest that SPARC overexpression in HCC cells results in a reduced tumorigenicity partially through the induction of mesenchymal-to-epithelial transition (MET). These evidences point to SPARC as a novel target for HCC treatment.

Immunotherapy for liver tumors: present status and future prospects.

Increasing evidence suggests that immune responses are involved in the control of cancer and that the immune system can be manipulated in different ways to recognize and attack tumors. Progress in immune-based strategies has opened new therapeutic avenues using a number of techniques destined to eliminate malignant cells. In the present review, we overview current knowledge on the importance, successes and difficulties of immunotherapy in liver tumors, including preclinical data available in animal models and information from clinical trials carried out during the lasts years. This review shows that new options for the treatment of advanced liver tumors are urgently needed and that there is a ground for future advances in the field.

Differential expression and dynamic changes of murine NEDD9 in progenitor cells of diverse tissues.

NEDD9 is a scaffolding protein in the integrin signaling pathway that is involved in cell adhesion dynamics. Little is known of the cellular localization of NEDD9 expression during embryonic development. In the present study, we have analyzed NEDD9 mRNA expression in the mouse and identified new relevant expression sites. In addition, we have characterized NEDD9 protein expression pattern for the first time in mammals. At E9.5-E10.5, high levels of Nedd9 and the neurogenic transcription factor neurogenin-2 (Ngn2) were found to largely overlap in two discrete domains of the trunk neural tube along its dorso-ventral axis, with Nedd9 extending to more ventral regions of the ventricular zone and Ngn2 differentially expressed in neuronally committed progenitors of the intermediate zone. At encephalic and trunk levels of the neural tube, NEDD9 was present in Sox2(+) progenitor cell populations mostly generating Ngn2(+) and/or Nurr1(+) cells. A sharp down-regulation of NEDD9 expression was found in cells upon lineage commitment, as observed in Nurr1(+) and Ngn2(+) mesencephalic dopaminergic and brainstem neuronal progenitors. In other tissues/organs, i.e. prospective heart, retina, olfactory epithelium, gonads, cartilage, gut and pituitary gland, NEDD9 was found to be co-expressed with Sox2, RXR alpha and/or Nurr1-like proteins, suggesting that NEDD9 expression is confined to early progenitors involved in diverse organogenesis and that it may depend on the repertoire and levels of retinoic acid co-receptors expressed by those cells.