Hepatic SPARC Expression Is Associated with Inflammasome Activation during the Progression of Non-Alcoholic Fatty Liver Disease in Both Mice and Morbidly Obese Patients.

The severity of non-alcoholic fatty liver disease (NAFLD) ranges from simple steatosis to steatohepatitis, and it is not yet clearly understood which patients will progress to liver fibrosis or cirrhosis. SPARC (Secreted Protein Acidic and Rich in Cysteine) has been involved in NAFLD pathogenesis in mice and humans. The aim of this study was to investigate the role of SPARC in inflammasome activation, and to evaluate the relationship between the hepatic expression of inflammasome genes and the biochemical and histological characteristics of NAFLD in obese patients. In vitro studies were conducted in a macrophage cell line and primary hepatocyte cultures to assess the effect of SPARC on inflammasome. A NAFLD model was established in SPARC knockout (SPARC-/-) and SPARC+/+ mice to explore inflammasome activation. A hepatic RNAseq database from NAFLD patients was analyzed to identify genes associated with SPARC expression. The results were validated in a prospective cohort of 59 morbidly obese patients with NAFLD undergoing bariatric surgery. Our results reveal that SPARC alone or in combination with saturated fatty acids promoted IL-1ß expression in cell cultures. SPARC-/- mice had reduced hepatic inflammasome activation during the progression of NAFLD. NAFLD patients showed increased expression of SPARC, NLRP3, CASP1, and IL-1ß. Gene ontology analysis revealed that genes positively correlated with SPARC are linked to inflammasome-related pathways during the progression of the disease, enabling the differentiation of patients between steatosis and steatohepatitis. In conclusion, SPARC may play a role in hepatic inflammasome activation in NAFLD.

Chromatographic Scalable Method to Isolate Engineered Extracellular Vesicles Derived from Mesenchymal Stem Cells for the Treatment of Liver Fibrosis in Mice.

New therapeutic options for liver cirrhosis are needed. Mesenchymal stem cell (MSC)-derived extracellular vesicles (EVs) have emerged as a promising tool for delivering therapeutic factors in regenerative medicine. Our aim is to establish a new therapeutic tool that employs EVs derived from MSCs to deliver therapeutic factors for liver fibrosis. EVs were isolated from supernatants of adipose tissue MSCs, induced-pluripotent-stem-cell-derived MSCs, and umbilical cord perivascular cells (HUCPVC-EVs) by ion exchange chromatography (IEC). To produce engineered EVs, HUCPVCs were transduced with adenoviruses that code for insulin-like growth factor 1 (AdhIGF-I-HUCPVC-EVs) or green fluorescent protein. EVs were characterized by electron microscopy, flow cytometry, ELISA, and proteomic analysis. We evaluated EVs' antifibrotic effect in thioacetamide-induced liver fibrosis in mice and on hepatic stellate cells in vitro. We found that IEC-isolated HUCPVC-EVs have an analogous phenotype and antifibrotic activity to those isolated by ultracentrifugation. EVs derived from the three MSCs sources showed a similar phenotype and antifibrotic potential. EVs derived from AdhIGF-I-HUCPVC carried IGF-1 and showed a higher therapeutic effect in vitro and in vivo. Remarkably, proteomic analysis revealed that HUCPVC-EVs carry key proteins involved in their antifibrotic process. This scalable MSC-derived EV manufacturing strategy is a promising therapeutic tool for liver fibrosis.

Glycosylated 4-methylumbelliferone as a targeted therapy for hepatocellular carcinoma.

BACKGROUND & AIMS: Reaching efficacious drug delivery to target cells/tissues represents a major obstacle in the current treatment of solid malignancies including hepatocellular carcinoma (HCC). In this study, we developed a pipeline to selective add complex-sugars to the aglycone 4-methylumbelliferone (4MU) to help their bioavailability and tumour cell intake. METHODS: The therapeutic efficacy of sugar-modified rutinosyl-4-methylumbelliferone (4MUR) and 4MU were compared in vitro and in an orthotopic HCC model established in fibrotic livers. The mechanistic bases of its selective target to liver tumour cells were evaluated by the interaction with asialoglycoprotein receptor (ASGPR), the mRNA expression of hyaluronan synthases (HAS2 or HAS3) and hyaluronan deposition. RESULTS: 4MUR showed a significant antiproliferative effect on liver tumoural cells as compared to non-tumoural cells in a dose-dependent manner. Further analysis showed that 4MUR is incorporated mostly into HCC cells by interaction with ASGPR, a receptor commonly overexpressed in HCC cells. 4MUR-treatment decreased the levels of HAS2 and HAS3 and the cytoplasmic deposition of hyaluronan. Moreover, 4MUR reduced CFSC-2G activation, hence reducing the fibrosis. In vivo efficacy showed that 4MUR treatment displayed a greater tumour growth inhibition and increased survival in comparison to 4MU. 4MUR administration was associated with a significant reduction of liver fibrosis without any signs of tissue damage. Further, 60% of 4MUR treated mice did not present macroscopically tumour mass post-treatment. CONCLUSION: Our results provide evidence that 4MUR may be used as an effective HCC therapy, without damaging non-tumoural cells or other organs, most probably due to the specific targeting.

Bioinformatic analysis of RHO family of GTPases identifies RAC1 pharmacological inhibition as a new therapeutic strategy for hepatocellular carcinoma.

OBJECTIVE: The RHO family of GTPases, particularly RAC1, has been linked with hepatocarcinogenesis, suggesting that their inhibition might be a rational therapeutic approach. We aimed to identify and target deregulated RHO family members in human hepatocellular carcinoma (HCC). DESIGN: We studied expression deregulation, clinical prognosis and transcription programmes relevant to HCC using public datasets. The therapeutic potential of RAC1 inhibitors in HCC was study in vitro and in vivo. RNA-Seq analysis and their correlation with the three different HCC datasets were used to characterise the underlying mechanism on RAC1 inhibition. The therapeutic effect of RAC1 inhibition on liver fibrosis was evaluated. RESULTS: Among the RHO family of GTPases we observed that RAC1 is upregulated, correlates with poor patient survival, and is strongly linked with a prooncogenic transcriptional programme. From a panel of novel RAC1 inhibitors studied, 1D-142 was able to induce apoptosis and cell cycle arrest in HCC cells, displaying a stronger effect in highly proliferative cells. Partial rescue of the RAC1-related oncogenic transcriptional programme was obtained on RAC1 inhibition by 1D-142 in HCC. Most importantly, the RAC1 inhibitor 1D-142 strongly reduce tumour growth and intrahepatic metastasis in HCC mice models. Additionally, 1D-142 decreases hepatic stellate cell activation and exerts an anti-fibrotic effect in vivo. CONCLUSIONS: The bioinformatics analysis of the HCC datasets, allows identifying RAC1 as a new therapeutic target for HCC. The targeted inhibition of RAC1 by 1D-142 resulted in a potent antitumoural effect in highly proliferative HCC established in fibrotic livers.

Hepatokines and adipokines in NASH-related hepatocellular carcinoma.

The incidence of hepatocellular carcinoma (HCC) is increasing in industrialised societies; this is likely secondary to the increasing burden of non-alcoholic fatty liver disease (NAFLD), its progressive form non-alcoholic steatohepatitis (NASH), and the metabolic syndrome. Cumulative studies suggest that NAFLD-related HCC may also develop in non-cirrhotic livers. However, prognosis and survival do not differ between NAFLD- or virus-associated HCC. Thus, research has increasingly focused on NAFLD-related risk factors to better understand the biology of hepatocarcinogenesis and to develop new diagnostic, preventive, and therapeutic strategies. One important aspect thereof is the role of hepatokines and adipokines in NAFLD/NASH-related HCC. In this review, we compile current data supporting the use of hepatokines and adipokines as potential markers of disease progression in NAFLD or as early markers of NAFLD-related HCC. While much work must be done to elucidate the mechanisms and interactions underlying alterations to hepatokines and adipokines, current data support the possible utility of these factors - in particular, angiopoietin-like proteins, fibroblast growth factors, and apelin - for detection or even as therapeutic targets in NAFLD-related HCC.

SPARC inhibition accelerates NAFLD-associated hepatocellular carcinoma development by dysregulating hepatic lipid metabolism.

BACKGROUND AND AIMS: Non-alcoholic fatty liver (NAFLD) and its more serious form non-alcoholic steatohepatitis increase risk of hepatocellular carcinoma (HCC). Lipid metabolic alterations and its role in HCC development remain unclear. SPARC (Secreted Protein, Acidic and Rich in Cysteine) is involved in lipid metabolism, NAFLD and diabetes, but the effects on hepatic lipid metabolism and HCC development is unknown. The aim of this study was to evaluate the role of SPARC in HCC development in the context of NAFLD. METHODS: Primary hepatocyte cultures from knockout (SPARC-/- ) or wild-type (SPARC+/+ ) mice, and HepG2 cells were used to assess the effects of free fatty acids on lipid accumulation, expression of lipogenic genes and de novo triglyceride (TG) synthesis. A NAFLD-HCC model was stabilized on SPARC-/- or SPARC+/+ mice. Correlations among SPARC, lipid metabolism-related gene expression patterns and clinical prognosis were studied using HCC gene expression dataset. RESULTS: SPARC-/- mice increases hepatic lipid deposits over time. Hepatocytes from SPARC-/- mice or inhibition of SPARC by an antisense adenovirus in HepG2 cells resulted in increased TG deposit, expression of lipid-related genes and nuclear translocation of SREBP1c. Human HCC database analysis revealed that SPARC negatively correlated with genes involved in lipid metabolism, and with poor survival. In NAFLD-HCC murine model, the absence of SPARC accelerates HCC development. RNA-seq study revealed that pathways related to lipid metabolism, cellular detoxification and proliferation were upregulated in SPARC-/- tumour-bearing mice. CONCLUSIONS: The absence of SPARC is associated with an altered hepatic lipid metabolism, and an accelerated NAFLD-related HCC development.

Acceleration of TAA-Induced Liver Fibrosis by Stress Exposure Is Associated with Upregulation of Nerve Growth Factor and Glycopattern Deviations.

Liver fibrosis results from many chronic injuries and may often progress to cirrhosis and hepatocellular carcinoma (HCC). In fact, up to 90% of HCC arise in a cirrhotic liver. Conversely, stress is implicated in liver damage, worsening disease outcome. Hence, stress could play a role in disrupting liver homeostasis, a concept that has not been fully explored. Here, in a murine model of TAA-induced liver fibrosis we identified nerve growth factor (NGF) to be a crucial regulator of the stress-induced fibrogenesis signaling pathway as it activates its receptor p75 neurotrophin receptor (p75NTR), increasing liver damage. Additionally, blocking the NGF decreased liver fibrosis whereas treatment with recombinant NGF accelerated the fibrotic process to a similar extent than stress challenge. We further show that the fibrogenesis induced by stress is characterized by specific changes in the hepatoglycocode (increased ß1,6GlcNAc-branched complex N-glycans and decreased core 1 O-glycans expression) which are also observed in patients with advanced fibrosis compared to patients with a low level of fibrosis. Our study facilitates an understanding of stress-induced liver injury and identify NGF signaling pathway in early stages of the disease, which contributes to the established fibrogenesis.

Human umbilical cord perivascular cells-derived extracellular vesicles mediate the transfer of IGF-I to the liver and ameliorate hepatic fibrogenesis in mice.

Extracellular vesicles (EVs) can mediate mesenchymal stromal cells (MSCs) paracrine effects. We aimed to evaluate the therapeutic potential of human umbilical cord perivascular cells (HUCPVCs) engineered to produce Insulin Growth Factor like-I (IGF-I) in experimental liver fibrosis and the role of EVs in this effect. HUCPVCs were engineered to produce human IGF-I (AdhIGF-I) or green fluorescence protein (AdGFP) using adenoviruses, and EVs were isolated from their conditioned medium (CM). In vitro effects of CM and EVs on hepatic stellate cells and hepatic macrophages were studied. Cells or EVs-based treatments were evaluated in thioacetamide-induced liver fibrosis in mice. The application of AdhIGF-I-HUCPVCs resulted in a further amelioration of liver fibrosis when compared to AdGFP-HUCPVCs and saline. Similarly, treatment with AdhIGF-I-HUCPVCs-derived EVs resulted in a reduction of collagen deposition and gene expression of the fibrogenic related molecules TGF-ß1, α-SMA, and COL1A2. In vitro incubation of hepatic stellate cells with EVs-AdhIGF-I-HUCPVCs significantly reduced activation of fibrogenic cells. In addition, EVs-AdhIGF-I-HUCPVCs trigger hepatic macrophages to switch their phenotype towards anti-inflammatory phagocytes, which might be involved in the antifibrotic effect. Consistently, high levels of IGF-I were observed within EVs-AdhIGF-I-HUCPVCs but not in controls EVs. Our results showed that hIGF-I carrying EVs could mediate the paracrine mechanism by which AdhIGF-I-HUCPVCs reduce liver fibrosis.

Argentinian clinical practice guideline for surveillance, diagnosis, staging and treatment of hepatocellular carcinoma.

The A.A.E.E.H has developed this guideline for the best care of patients with hepatocellular carcinoma (HCC) from Argentina. It was done from May 2018 to March 2020. Specific clinical research questions were systematically searched. The quality of evidence and level of recommendations were organized according to GRADE. HCC surveillance is strongly recommended with abdominal ultrasound (US) every six months in the population at risk for HCC (cirrhosis, hepatitis B or hepatitis C); it is suggested to add alpha-feto protein (AFP) levels in case of inexeperienced sonographers. Imaging diagnosis in patients at risk for HCC has high specificity and tumor biopsy is not mandatory. The Barcelona Clinic Liver Cancer algorithm is strongly recommended for HCC staging and treatment-decision processes. Liver resection is strongly recommended for patients without portal hypertension and preserved liver function. Composite models are suggested for liver transplant selection criteria. Therapies for HCC with robust clinical evidence include transarterial chemoembolization (TACE) and first to second line systemic treatment options (sorafenib, lenvatinib, regorafenib, cabozantinib and ramucirumab). Immunotherapy with nivolumab and pembrolizumab has failed to show statistical benefit but the novel combination of atezolizumab plus bevacizumab has recently shown survival benefit over sorafenib in frontline.

A comprehensive study of epigenetic alterations in hepatocellular carcinoma identifies potential therapeutic targets.

BACKGROUND & AIMS: A causal link has recently been established between epigenetic alterations and hepatocarcinogenesis, indicating that epigenetic inhibition may have therapeutic potential. We aimed to identify and target epigenetic modifiers that show molecular alterations in hepatocellular carcinoma (HCC). METHODS: We studied the molecular-clinical correlations of epigenetic modifiers including bromodomains, histone acetyltransferases, lysine methyltransferases and lysine demethylases in HCC using The Cancer Genome Atlas (TCGA) data of 365 patients with HCC. The therapeutic potential of epigenetic inhibitors was evaluated in vitro and in vivo. RNA sequencing analysis and its correlation with expression and clinical data in the TCGA dataset were used to identify expression programs normalized by Jumonji lysine demethylase (JmjC) inhibitors. RESULTS: Genetic alterations, aberrant expression, and correlation between tumor expression and poor patient prognosis of epigenetic enzymes are common events in HCC. Epigenetic inhibitors that target bromodomain (JQ-1), lysine methyltransferases (BIX-1294 and LLY-507) and JmjC lysine demethylases (JIB-04, GSK-J4 and SD-70) reduce HCC aggressiveness. The pan-JmjC inhibitor JIB-04 had a potent antitumor effect in tumor bearing mice. HCC cells treated with JmjC inhibitors showed overlapping changes in expression programs related with inhibition of cell proliferation and induction of cell death. JmjC inhibition reverses an aggressive HCC gene expression program that is also altered in patients with HCC. Several genes downregulated by JmjC inhibitors are highly expressed in tumor vs. non-tumor parenchyma, and their high expression correlates with a poor prognosis. We identified and validated a 4-gene expression prognostic signature consisting of CENPA, KIF20A, PLK1, and NCAPG. CONCLUSIONS: The epigenetic alterations identified in HCC can be used to predict prognosis and to define a subgroup of high-risk patients that would potentially benefit from JmjC inhibitor therapy. LAY SUMMARY: In this study, we found that mutations and changes in expression of epigenetic modifiers are common events in human hepatocellular carcinoma, leading to an aggressive gene expression program and poor clinical prognosis. The transcriptional program can be reversed by pharmacological inhibition of Jumonji enzymes. This inhibition blocks hepatocellular carcinoma progression, providing a novel potential therapeutic strategy.

SPARC is required for the maintenance of glucose homeostasis and insulin secretion in mice.

Obesity, metabolic syndrome, and type 2 diabetes, three strongly interrelated diseases, are associated to increased morbidity and mortality worldwide. The pathogenesis of obesity-associated disorders is still under study. Secreted protein acidic and rich in cysteine (SPARC) is a matricellular glycoprotein expressed in many cell types including adipocytes, parenchymal, and non-parenchymal hepatic cells and pancreatic cells. Studies have demonstrated that SPARC inhibits adipogenesis and promotes insulin resistance; in addition, circulating SPARC levels were positively correlated with body mass index in obese individuals. Therefore, SPARC is being proposed as a key factor in the pathogenesis of obesity-associated disorders. The aim of this study is to elucidate the role of SPARC in glucose homeostasis. We show here that SPARC null (SPARC-/-) mice displayed an abnormal insulin-regulated glucose metabolism. SPARC-/- mice presented an increased adipose tissue deposition and an impaired glucose homeostasis as animals aged. In addition, the absence of SPARC worsens high-fat diet-induced diabetes in mice. Interestingly, although SPARC-/- mice on high-fat diet were sensitive to insulin they showed an impaired insulin secretion capacity. Of note, the expression of glucose transporter 2 in islets of SPARC-/- mice was dramatically reduced. The present study provides the first evidence that deleted SPARC expression causes diabetes in mice. Thus, SPARC deficient mice constitute a valuable model for studies concerning obesity and its related metabolic complications, including diabetes.

4Mu Decreases CD47 Expression on Hepatic Cancer Stem Cells and Primes a Potent Antitumor T Cell Response Induced by Interleukin-12.

The tumor microenvironment (TME) represents a complex interplay between different cellular components, including tumor cells and cancer stem cells (CSCs), with the associated stroma; such interaction promotes tumor immune escape and sustains tumor growth. Several experimental approaches for cancer therapy are focused on TME remodeling, resulting in increased antitumor effects. We previously demonstrated that the hyaluronan synthesis inhibitor 4-methylumbelliferone (4Mu) decreases liver fibrosis and induces antitumor activity in hepatocellular carcinoma (HCC). In this work, 4Mu, in combination with an adenovirus encoding interleukin-12 genes (AdIL-12), elicited a potent antitumor effect and significantly prolonged animal survival (p < 0.05) in an orthotopic HCC model established in fibrotic livers. In assessing the presence of CSCs, we found reduced mRNA levels of CD133+, CD90+, EpCAM+, CD44+, and CD13+ CSC markers within HCC tumors (p < 0.01). Additionally, 4Mu downregulated the expression of the CSC marker CD47+ on HCC cells, promoted phagocytosis by antigen-presenting cells, and, combined with Ad-IL12, elicited a potent cytotoxic-specific T cell response. Finally, animal survival was increased when CD133low HCC cells, generated upon 4Mu treatment, were injected in a metastatic HCC model. In conclusion, the combined strategy ameliorates HCC aggressiveness by targeting CSCs and as a result of the induction of anticancer immunity.

Immunostimulatory monoclonal antibodies for hepatocellular carcinoma therapy. Trends and perspectives.

Hepatocellular carcinoma (HCC) is the second cause of cancer-related death in the world and is the main cause of death in cirrhotic patients. Unfortunately, the incidence of HCC has grown significantly in the last decade. Curative treatments such as surgery, liver transplantation or percutaneous ablation can only be applied in less than 30% of cases. The multikinase inhibitor sorafenib is the first line therapy for advanced HCC. Regorafenib is the standard of care for second-line patients. However, novel and more specific potent therapeutic approaches for advanced HCC are still needed. The liver constitutes a unique immunological microenvironment, although anti-tumor immunity seems to be feasible with the use of checkpoint inhibitors such as nivolumab. Efficacy may be further increased by combining checkpoint inhibitors or by applying loco-regional treatments. The success of immune checkpoint blockade has renewed interest in immunotherapy in HCC.

[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.

Chronic hyperprolactinemia evoked by disruption of lactotrope dopamine D2 receptors impacts on liver and adipocyte genes related to glucose and insulin balance.

We studied the impact of high prolactin titers on liver and adipocyte gene expression related to glucose and insulin homeostasis in correlation with obesity onset. To that end we used mutant female mice that selectively lack dopamine type 2 receptors (D2Rs) from pituitary lactotropes (lacDrd2KO), which have chronic high prolactin levels associated with increased body weight, marked increments in fat depots, adipocyte size, and serum lipids, and a metabolic phenotype that intensifies with age. LacDrd2KO mice of two developmental ages, 5 and 10 mo, were used. In the first time point, obesity and increased body weight are marginal, although mice are hyperprolactinemic, whereas at 10 mo there is marked adiposity with a 136% increase in gonadal fat and a 36% increase in liver weight due to lipid accumulation. LacDrd2KO mice had glucose intolerance, hyperinsulinemia, and impaired insulin response to glucose already in the early stages of obesity, but changes in liver and adipose tissue transcription factors were time and tissue dependent. In chronic hyperprolactinemic mice liver Prlr were upregulated, there was liver steatosis, altered expression of the lipogenic transcription factor Chrebp, and blunted response of Srebp-1c to refeeding at 5 mo of age, whereas no effect was observed in the glycogenesis pathway. On the other hand, in adipose tissue a marked decrease in lipogenic transcription factor expression was observed when morbid obesity was already settled. These adaptive changes underscore the role of prolactin signaling in different tissues to promote energy storage.

Cell death mechanisms in human chronic liver diseases: a far cry from clinical applicability.

The liver is constantly exposed to a host of injurious stimuli. This results in hepatocellular death mainly by apoptosis and necrosis, but also due to autophagy, necroptosis, pyroptosis and in some cases by an intricately balanced combination thereof. Overwhelming and continuous cell death in the liver leads to inflammation, fibrosis, cirrhosis, and eventually hepatocellular carcinoma. Although data from various disease models may suggest a specific (predominant) cell death mode for different aetiologies, the clinical reality is not as clear cut. Reliable and non-invasive cell death markers are not available in general practice and assessment of cell death mode to absolute certainty from liver biopsies does not seem feasible, yet. Various aetiologies probably induce different predominant cell death modes within the liver, although the death modes involved may change during disease progression. Moreover, current methods applicable in patients are limited to surrogate markers for apoptosis (M30), and possibly for pyroptosis (IL-1 family) and necro(pto)sis (HMGB1). Although markers for some death modes are not available at all (autophagy), others may not be specific for a cell death mode or might not always definitely indicate dying cells. Physicians need to take care in asserting the presence of cell death. Still the serum-derived markers are valuable tools to assess severity of chronic liver diseases. This review gives a short overview of known hepatocellular cell death modes in various aetiologies of chronic liver disease. Also the limitations of current knowledge in human settings and utilization of surrogate markers for disease assessment are summarized.

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.

[Dendritic cell-based therapeutic cancer vaccines]. / Vacunas terapéuticas antitumorales basadas en células dendríticas.

In recent years immunotherapy has revolutionized the treatment of patients with advanced cancer. The increased knowledge in the tumor immune-biology has allowed developing rational treatments by manipulation of the immune system with significant clinical impact. This rapid development has significantly changed the prognosis of many tumors without treatment options up to date. Other strategies have explored the use of therapeutic vaccines based on dendritic cells (DC) by inducing antitumor immunity. DC are cells of hematopoietic origin, constitutively expressing molecules capable to present antigens, that are functionally the most potent inducers of the activation and proliferation of antigen specific T lymphocytes. The CD8+ T cells proliferate and acquire cytotoxic capacity after recognizing their specific antigen presented on the surface of DC, although only some types of DC can present antigens internalized from outside the cell to precursors of cytotoxic T lymphocytes (this function is called cross-presentation) requiring translocation mechanisms of complex antigens. The induction of an effective adaptive immune response is considered a good option given its specificity, and prolonged duration of response. The DC, thanks to its particular ability of antigen presentation and lymphocyte stimulation, are able to reverse the poor antitumor immune response experienced by patients with cancer. The DC can be obtained from various sources, using different protocols to generate differentiation and maturation, and are administered by various routes such as subcutaneous, intravenous or intranodal. The wide variety of protocols resulted in heterogeneous clinical responses.

4-methylumbelliferone inhibits hepatocellular carcinoma growth by decreasing IL-6 production and angiogenesis.

Cirrhosis is characterized by an excessive accumulation of extracellular matrix components including hyaluronic acid (HA) and is widely considered a preneoplastic condition for hepatocellular carcinoma (HCC). 4-Methylumbelliferone (4MU) is an inhibitor of HA synthesis and has anticancer activity in an orthotopic HCC model with underlying fibrosis. Our aim was to explore the effects of HA inhibition by 4MU orally administered on tumor microenvironment. Hepa129 tumor cells were inoculated orthotopically in C3H/HeJ male mice with fibrosis induced by thioacetamide. Mice were orally treated with 4MU. The effects of 4MU on angiogenesis were evaluated by immunostaining of CD31 and quantification of proangiogenic factors (vascular endothelial growth factor, VEGF, interleukin-6, IL-6 and C-X-C motif chemokine 12, CXCL12). IL-6 was also quantified in Hepa129 cells in vitro after treatment with 4MU. Migration of endothelial cells and tube formation were also analyzed. As a result, 4MU treatment decreases tumor growth and increased animal survival. Systemic levels of VEGF were significantly inhibited in 4MU-treated mice. Expression of CD31 was reduced after 4MU therapy in liver parenchyma in comparison with control group. In addition, mRNA expression and protein levels of IL-6 and VEGF were inhibited both in tumor tissue and in nontumoral liver parenchyma. Interestingly, IL-6 production was dramatically reduced in Kupffer cells isolated from 4MU-treated mice, and in Hepa129 cells in vitro. Besides, 4MU was able to inhibit endothelial cell migration and tube formation. In conclusion, 4MU has antitumor activity in vivo and its mechanisms of action involve an inhibition of angiogenesis and IL-6 production. 4MU is an orally available molecule with potential for HCC treatment.

The low-abundance transcriptome reveals novel biomarkers, specific intracellular pathways and targetable genes associated with advanced gastric cancer.

Studies on the low-abundance transcriptome are of paramount importance for identifying the intimate mechanisms of tumor progression that can lead to novel therapies. The aim of the present study was to identify novel markers and targetable genes and pathways in advanced human gastric cancer through analyses of the low-abundance transcriptome. The procedure involved an initial subtractive hybridization step, followed by global gene expression analysis using microarrays. We observed profound differences, both at the single gene and gene ontology levels, between the low-abundance transcriptome and the whole transcriptome. Analysis of the low-abundance transcriptome led to the identification and validation by tissue microarrays of novel biomarkers, such as LAMA3 and TTN; moreover, we identified cancer type-specific intracellular pathways and targetable genes, such as IRS2, IL17, IFNγ, VEGF-C, WISP1, FZD5 and CTBP1 that were not detectable by whole transcriptome analyses. We also demonstrated that knocking down the expression of CTBP1 sensitized gastric cancer cells to mainstay chemotherapeutic drugs. We conclude that the analysis of the low-abundance transcriptome provides useful insights into the molecular basis and treatment of cancer.