Optimal Therapeutic Strategy for PD-L1 Negative Metastatic Non-Small Cell Lung Cancer: A Decision-Making Guide Based on Clinicopathological and Molecular Features.

OPINION STATEMENT: Strategies using immune checkpoint inhibitors (ICI), which can enhance antitumor immune responses, have revolutionized the lung cancer therapeutic landscape. The ICI mechanism of action involves the blockade of regulatory cell surface molecules using antibodies against the Cytotoxic T-Lymphocyte Antigen 4 (CTLA-4) (ipilimumab, tremelimumab); the programmed death receptor-1 (PD-1; nivolumab, pembrolizumab); or the PD ligand-1 (PD-L1; atezolizumab, durvalumab). Notably, anti-PD-1 demonstrated long-term survival benefits, durable objective responses, and a manageable safety profile in patients with non-small cell lung cancer (NSCLC). The combination of anti-PD1 or anti-PD-L1 and platinum chemotherapy achieved better survival outcomes than chemotherapy alone, which was observed irrespective of PD-L1 expression on cancer cells. Although promising results have been reported from large clinical trials, especially for patients with high PD-L1 expression, the optimal treatment approach for patients with PD-L1-negative NSCLC has yet to be defined. We propose a guide for clinicians in the therapeutic decision-making process based on the latest data available about treatments, prognostic factors, predictive biomarkers, and real-world evidence in PD-L1-negative NSCLC patients.

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.

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.

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.

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.

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.

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.

4-methylumbelliferone-mediated polarization of M1 macrophages correlate with decreased hepatocellular carcinoma aggressiveness in mice.

Hepatocellular carcinoma (HCC) arises in the setting of advanced liver fibrosis, a dynamic and complex inflammatory disease. The tumor microenvironment (TME) is a mixture of cellular components including cancer cells, cancer stem cells (CSCs), tumor-associated macrophages (TAM), and dendritic cells (DCs), which might drive to tumor progression and resistance to therapies. In this work, we study the effects of 4-methylumbelliferone (4Mu) on TME and how this change could be exploited to promote a potent immune response against HCC. First, we observed that 4Mu therapy induced a switch of hepatic macrophages (Mϕ) towards an M1 type profile, and HCC cells (Hepa129 cells) exposed to conditioned medium (CM) derived from Mϕ treated with 4Mu showed reduced expression of several CSCs markers and aggressiveness. HCC cells incubated with CM derived from Mϕ treated with 4Mu grew in immunosuppressed mice while presented delayed tumor progression in immunocompetent mice. HCC cells treated with 4Mu were more susceptible to phagocytosis by DCs, and when DCs were pulsed with HCC cells previously treated with 4Mu displayed a potent antitumoral effect in therapeutic vaccination protocols. In conclusion, 4Mu has the ability to modulate TME into a less hostile milieu and to potentiate immunotherapeutic strategies against HCC.

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.

[Cancer immunotherapy. Importance of overcoming immune suppression]. / Inmunoterapia del cáncer. Importancia de controlar la inmunosupresión.

Increasing evidence indicates that the immune system is involved in the control of tumor progression. Effective antitumor immune response depends on the interaction between several components of the immune system, including antigen-presenting cells and different T cell subsets. However, tumor cells develop a number of mechanisms to escape recognition and elimination by the immune system. In this review we discuss these mechanisms and address possible therapeutic approaches to overcome the immune suppression generated by tumors.

Immunotherapy for Hepatocellular Carcinoma: Is Latin America Ready for Primetime?

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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.

SPARC gene deletion protects against toxic liver injury and is associated to an enhanced proliferative capacity and reduced oxidative stress response.

SPARC, also known as osteonectin and BM-40, is a matricellular protein with a number of biological functions. Hepatic SPARC expression is induced in response to thioacetamide, bile-duct ligation, and acute injuries such as concanavalin A and lipopolysacharide (LPS)/D-galactosamine. We have previously demonstrated that the therapeutic inhibition of SPARC or SPARC gene deletion protected mice against liver injury. We investigated the mechanisms involved in the protective effect of SPARC inhibition in mice. We performed a proteome analysis of livers from SPARC+/+ and SPARC-/- mice chronically treated with thioacetamide. Catalase activity, carbonylation levels, oxidative stress response, and mitochondrial function were studied. Genomic analysis revealed that SPARC-/- mice had an increased expression of cell proliferation genes. Proteins involved in detoxification of reactive oxygen species such as catalase, peroxirredoxine-1, and glutathione-S-transferase P1 and Mu1 were highly expressed as evidenced by proteome analysis; hepatic catalase activity was increased in SPARC-/- mice. Oxidative stress response and carbonylation levels were lower in livers from SPARC-/- mice. Hepatic mitochondria showed lower levels of nitrogen reactive species in the SPARC-/- concanavalin A-treated mice. Mitochondrial morphology was preserved, and its complex activity reduced in SPARC-/- mice. In conclusion, our data suggest that the protection associated with SPARC gene deletion may be partially due to a higher proliferative capacity of hepatocytes and an enhanced oxidative stress defense in SPARC-/- mice after liver injury.