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.

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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.

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.

IntroductionSecreted Protein, Acidic and Rich in Cysteine (SPARC) is a matricellular protein involved in many biological processes and found over-expressed in cirrhotic livers. By mean of a genetic approach we herein provide evidence from different in vivo liver disease models suggesting a profibrogenic role for SPARC.MethodsTwo in vivo models of liver fibrosis, based on TAA administration and bile duct ligation, were developed on SPARC wild-type (SPARC+/+) and knock-out (SPARC−/−) mice. Hepatic SPARC expression was analyzed by qPCR. Fibrosis was assessed by Sirius Red staining, and the maturation state of collagen fibers was analyzed using polarized light. Necroinflammatory activity was evaluated by applying the Knodell score and liver inflammatory infiltration was characterized by immunohistochemistry. Hepatic stellate cell activation was assessed by α-SMA immunohistochemistry. In addition, pro-fibrogenic genes and inflammatory cytokines were measured by qPCR and/or ELISA. Liver gene expression profile was analyzed in SPARC−/− and SPARC+/+ mice using Affymetrix Mouse Gene ST 1.0 array.ResultsSPARC expression was found induced in fibrotic livers of mouse and human. SPARC−/− mice showed a reduction in the degree of inflammation, mainly CD4+ cells, and fibrosis. Consistently, collagen deposits and mRNA expression levels were decreased in SPARC−/− mice when compared to SPARC+/+ mice; in addition, MMP-2 expression was increased in SPARC−/− mice. A reduction in the number of activated myofibroblasts was observed. Moreover, TGF-β1 expression levels were down-regulated in the liver as well as in the serum of TAA-treated knock-out animals. Ingenuity Pathway Analysis (IPA) analysis suggested several gene networks which might involve protective mechanisms of SPARC deficiency against liver fibrogenesis and a better established machinery to repair DNA and detoxify from external chemical stimuli.ConclusionsOverall our data suggest that SPARC plays a significant role in liver fibrogenesis. Interventions to inhibit SPARC expression are suggested as promising approaches for liver fibrosis treatment.

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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 CD133 low 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.

Purpose: Interleukin-12 (IL-12) is an immunostimulatory cytokine with potent antitumor effects in several animal models. However, serious toxicity has been associated with its systemic application in humans. Gene transfer has emerged as a tool to specifically express therapeutic genes into the tumor/peritumoral milieu, thus avoiding systemic toxicity. The aim of this study was to analyze whether subtherapeutic doses of an adenovirus encoding IL-12 (AdIL-12) might synergize with low immunopotentiating doses of cyclophosphamide in the treatment of colorectal carcinoma. Experimental Design: The antitumor effect of combining a single low dose of cyclophosphamide with an intratumoral injection of AdIL-12 was evaluated in an in vivo murine colorectal carcinoma model. The immune responses achieved with different treatments were monitored, comparing the effect of combining both therapies with individual treatments. Results: The combined therapy induced a complete tumor regression in &gt;50% of mice in a synergistic fashion, and it significantly prolonged their survival. This strategy was superior to each single treatment in reducing both peripheral and splenic CD4+CD25+Foxp3+ regulatory T cells, increasing the number of activated dendritic cells, and inducing IFN-γ-secreting CD4-positive T lymphocytes. Importantly, the combined treatment generated a powerful tumor-specific CTL response. Consistently, a significant reduction in IL-10 levels was found. Our data suggest that the combination of nontoxic doses of cyclophosphamide with AdIL-12 allows the generation of good antitumoral responses, thus avoiding undesired side effects of both agents. Conclusions: Our data strongly support the use of a combination of cyclophosphamide and AdIL-12 as a novel therapeutic strategy against colorectal carcinoma. (Clin Cancer Res 2009;15(23):7256–65)