Diego M. Avella

Nanoliposomal ceramide prevents in vivo growth of hepatocellular carcinoma

Background and objectives Hepatocellular carcinoma (HCC) affects an increasing number of people worldwide. The poor survival rate of patients with HCC is manifested by an aggressive and metastatic phenotype, as well as a poor response to common therapeutic strategies. The purpose of this study was to evaluate the efficacy of nanoliposomal C6-ceramide as an antineoplastic agent in an in vivo model of human HCC. Methods The growth-arresting and pro-apoptotic properties of nanoliposomal C6-ceramide were first evaluated in vitro in human SK-HEP-1 cells by assessing cellular viability, caspase 3/7 activity, annexin-V expression, DNA fragmentation, cell cycle distribution and AKT phosphorylation. SK-HEP-1 cells were then engrafted subcutaneously into athymic nude mice and nanoliposomal C6-ceramide was administered by tail vein injection. Tumour size was monitored over time, followed by excision of tumours to evaluate tumour vascularisation, proliferation, apoptosis and cellular signalling. Results Nanoliposomal C6-ceramide, but not ghost (no ceramide) nanoliposomes, induced apoptotic cell death of SK-HEP-1 cells in vitro, concomitant with an accumulation of cells in the G2 phase of the cell cycle and decreased phosphorylation of AKT. Systemic administration of nanoliposomal C6-ceramide to mice engrafted with SK-HEP-1 tumours reduced tumour vascularisation and proliferation, induced tumour cell apoptosis, decreased phosphorylation of AKT and ultimately blocked tumour growth. Conclusions These studies show that nanoliposomal ceramide is an efficacious antineoplastic agent for the treatment of in vitro and in vivo models of human HCC.

The opioid growth factor-opioid growth factor receptor axis regulates cell proliferation of human hepatocellular cancer

Hepatocellular carcinoma (HCC) is the third leading cause of cancer deaths worldwide, with a mortality rate approximating its incidence. Understanding the biology of these tumors, as well as treatment modalities, has been challenging. The opioid growth factor (OGF; [Met(5)]-enkephalin) and the OGF receptor (OGFr) form an endogenous growth-regulating pathway in homeostasis and neoplasia. In this investigation, we examined the relationship of the OGF-OGFr axis in HCC and define its presence, function, and mechanism. Using SK-HEP-1, Hep G2, and Hep 3B human HCC cell lines, we found that OGF and OGFr were present and functional. Exogenous OGF was observed to have a dose-dependent, reversible, and receptor-mediated inhibitory action on cell proliferation. Endogenous OGF was found to be constitutively produced and tonically active on cell replicative activities, with neutralization of this peptide accelerating cell proliferation. Silencing of OGFr using siRNA stimulated cell replication, even when exogenous OGF was added to the cultures, documenting its importance in mediating OGF activity. The mechanism of OGF-OGFr action on cell number was related to inhibition of DNA synthesis and not to apoptotic or necrotic pathways. Both OGF and OGFr were detected in surgical specimens of HCC, and no quantitative differences were recorded in peptide or receptor between pathological and normal specimens. These data are the first to report that the OGF-OGFr system is a native biological regulator of cell proliferation in HCC. The findings may provide important insight in designing treatment strategies for this deadly disease.

Regression of established hepatocellular carcinoma is induced by chemoimmunotherapy in an orthotopic murine model.

The high rate of mortality and frequent incidence of recurrence associated with hepatocellular carcinoma (HCC) reveal the need for new therapeutic approaches. In this study we evaluated the efficacy of a novel chemoimmunotherapeutic strategy to control HCC and investigated the underlying mechanism that increased the antitumor immune response. We developed a novel orthotopic mouse model of HCC through seeding of tumorigenic hepatocytes from SV40 T antigen (Tag) transgenic MTD2 mice into the livers of syngeneic C57BL/6 mice. These MTD2‐derived hepatocytes form Tag‐expressing HCC tumors specifically within the liver. This approach provides a platform to test therapeutic strategies and antigen‐specific immune‐directed therapy in an immunocompetent murine model. Using this model we tested the efficacy of a combination of oral sunitinib, a small molecule multitargeted receptor tyrosine kinase (RTK) inhibitor, and adoptive transfer of tumor antigen‐specific CD8+ T cells to eliminate HCC. Sunitinib treatment alone promoted a transient reduction in tumor size. Sunitinib treatment combined with adoptive transfer of tumor antigen‐specific CD8+ T cells led to elimination of established tumors without recurrence. In vitro studies revealed that HCC growth was inhibited through suppression of STAT3 signaling. In addition, sunitinib treatment of tumor‐bearing mice was associated with suppression of STAT3 and a block in T‐cell tolerance. Conclusion: These findings indicate that sunitinib inhibits HCC tumor growth directly through the STAT3 pathway and prevents tumor antigen‐specific CD8+ T‐cell tolerance, thus defining a synergistic chemoimmunotherapeutic approach for HCC. (HEPATOLOGY 2012;55:141–152)

Combined pancreaticoduodenectomy and extended right hemicolectomy: outcomes and indications

BACKGROUND Pancreaticoduodenectomy (PD) combined with an en bloc extended right hemicolectomy is required to achieve complete oncological resection of various malignancies. Information regarding the indications and outcomes of this procedure is limited. STUDY DESIGN Patients requiring PD combined with extended right hemicolectomy for primary tumours from 2002 to 2008 were identified. RESULTS PD combined with an en bloc extended right hemicolectomy was required in 14 patients, constituting 8% of pancreaticoduodenal resections. Pancreatic adenocarcinoma (8), retroperitoneal sarcoma (2) and colon cancer (2) were the main primary tumours resected. The indication for an extended right hemicolectomy was extensive tumour involvement of the transverse mesentery in seven patients. Clear tumour margins were achieved in 11 individuals. The median operating time was 10 h with intra-operative transfusions required in three patients. One or more complications were noted in eight, with delayed gastric emptying and pancreatic fistula the most common. The median length of hospital stay was 8 days. The overall 2-year survival in this series was 37%, with a median survival of 20 months in pancreatic cancer patients. CONCLUSIONS This series suggests that PD combined with an en bloc extended right hemicolectomy is feasible and can achieve complete tumour clearance with acceptable morbidity.

Reduction of surgical site infections by use of pulsatile lavage irrigation after prolonged intra-abdominal surgical procedures

BACKGROUND Surgical site infections cause significant postoperative morbidity and may be reduced by pressurized irrigation of high-risk laparotomy wounds before closure. This was a retrospective review (June 2007 to May 2008) from a surgical unit at a tertiary care center. METHODS Patients undergoing laparotomy extending beyond 4 hours, when a standard wound management strategy was instituted by either simple irrigation or pressurized pulsatile lavage (<15 psi) with saline before closure, were included. The outcome measures were the surgical site infections and factors contributing to them. RESULTS The median surgical time for the patients was 8 hours, with 34 wounds managed by simple irrigation and 42 wounds managed by pulse irrigation. Both groups had similar characteristics. Overall there were 15 (20%) surgical site infections. Significantly fewer infections occurred in the pulse irrigation group (10% vs 32%; P = .019). The use of a pulse irrigation device was the only factor associated with a reduction in wound infections (P = .019). CONCLUSIONS Surgical site infections appear to be reduced with pulsatile lavage irrigation of wounds before skin closure in patients undergoing prolonged intra-abdominal surgeries.

Human acellular dermal matrix: an innovative tool for diaphragmatic reconstruction in patients with large intra-abdominal tumors

BACKGROUND Patients presenting with large intra-abdominal tumors often require multivisceral organ resection including partial or complete hemidiaphragm excision to achieve complete gross and microscopic tumor resection. METHODS The ideal surgical approach to the repair of major diaphragmatic defects, particularly in a contaminated surgical field, is not well defined, but the use of prosthetic mesh in these situations is contraindicated. RESULTS We report our experience using human acellular dermal matrix for the repair of large diaphragmatic defects in these patients. CONCLUSIONS Human acellular dermal matrix is a safe and effective option in the surgical reconstruction of diaphragmatic defects in patients with large intra-abdominal tumors.

Successful chemoimmunotherapy against hepatocellular cancer in a novel murine model.

BACKGROUND & AIMS We have established a clinically relevant animal model of hepatocellular cancer (HCC) in immune competent mice to elucidate the complex dialog between host immunity and tumors during HCC initiation and progression. Mechanistic findings have been leveraged to develop a clinically feasible anti-tumor chemoimmunotherapeutic strategy. METHODS Intraperitoneal injection of carbon tetrachloride and intrasplenic inoculation of oncogenic hepatocytes were combined to induce progressive HCCs in fibrotic livers of immunocompetent mice. Immunization and adoptive cell transfer (ACT) were used to dissect the tumor antigen-specific immune response. The ability of the tyrosine kinase inhibitor sunitinib to enhance immunotherapy in the setting of HCC was evaluated. RESULTS This new mouse model mimics human HCC and reflects its typical features. Tumor-antigen-specific CD8+ T cells maintained a naïve phenotype and remained responsive during early-stage tumor progression. Late tumor progression produced circulating tumor cells, tumor migration into draining lymph nodes, and profound exhaustion of tumor-antigen-specific CD8+ T cells associated with accumulation of programmed cell death protein 1 (PD-1)hi CD8+ T cells and regulatory T cells (Tregs). Sunitinib-mediated tumoricidal effect and Treg suppression synergized with antibody-mediated blockade of PD-1 to powerfully suppress tumor growth and activate anti-tumor immunity. CONCLUSION Treg accumulation and upregulation of PD-1 provide two independent mechanisms to induce profound immune tolerance in HCC. Chemoimmunotherapy using Food and Drug Administration-approved sunitinib with anti-PD-1 antibodies achieved significant tumor control, supporting translation of this approach for the treatment of HCC patients. LAY SUMMARY In the current study, we have established a clinically relevant mouse model which mimics human liver cancer. Using this unique model, we studied the response of the immune system to this aggressive cancer. Findings from this trial have led to the development of an innovative and clinically feasible chemoimmunotherapeutic strategy.

Nanoliposome C6-Ceramide Increases the Anti-tumor Immune Response and Slows Growth of Liver Tumors in Mice

BACKGROUND & AIMS Ceramide, a sphingolipid metabolite, affects T-cell signaling, induces apoptosis of cancer cells, and slows tumor growth in mice. However, it has not been used as a chemotherapeutic agent because of its cell impermeability and precipitation in aqueous solution. We developed a nanoliposome-loaded C6-ceremide (LipC6) to overcome this limitation and investigated its effects in mice with liver tumors. METHODS Immune competent C57BL/6 mice received intraperitoneal injections of carbon tetrachlo-ride and intra-splenic injections of oncogenic hepatocytes. As a result, tumors resembling human hepatocellular carcinomas developed in a fibrotic liver setting. After tumors formed, mice were given an injection of LipC6 or vehicle via tail vein every other day for 2 weeks. This was followed by administration, also via tail vein, of tumor antigen-specific (TAS) CD8+ T cells isolated from the spleens of line 416 mice, and subsequent immunization by intraperitoneal injection of tumor antigen-expressing B6/WT-19 cells. Tumor growth was monitored with magnetic resonance imaging. Tumor apoptosis, proliferation, and AKT expression were analyzed using immunohistochemistry and immunoblots. Cytokine production, phenotype, and function of TAS CD8+ T cells and tumor-associated macrophages (TAMs) were studied with flow cytometry, real-time polymerase chain reaction (PCR), and ELISA. Reactive oxygen species (ROS) in TAMs and bone marrow-derived macrophages, induced by colony stimulating factor 2 (GMCSF or CSF2) or colony stimulating factor 1 (MCSF or CSF1), were detected using a luminescent assay. RESULTS Injection of LipC6 slowed tumor growth by reducing tumor cell proliferation and phosphorylation of AKT, and increasing tumor cell apoptosis, compared with vehicle. Tumors grew more slowly in mice given the combination of LipC6 injection and TAS CD8+ T cells followed by immunization compared with mice given vehicle, LipC6, the T cells, or immunization alone. LipC6 injection also reduced numbers of TAMs and their production of ROS. LipC6 induced TAMs to differentiate into an M1 phenotype, which reduced immune suppression and increased activity of CD8+ T cells. These results were validated by experiments with bone marrow-derived macrophages induced by GMCSF or MCSF. CONCLUSIONS In mice with liver tumors, injection of LipC6 reduces the number of TAMs and the ability of TAMs to suppress the anti-tumor immune response. LipC6 also increases the anti-tumor effects of TAS CD8+ T cells. LipC6 might therefore increase the efficacy of immune therapy in patients with hepatocellular carcinoma.

Laparoscopic repair of post-esophagectomy diaphragmatic hernias using human acellular dermal matrix

Diaphragmatic hernias occur in up to 2% of patients after esophagectomy with gastric pull-up, and the surgical repair in the setting of a previous esophagectomy is a challenge with high complication rates, in particular with regards to the gastric conduit and its critical vascular supply. We describe two cases and the technique of minimally invasive, laparoscopic repair of diaphragmatic defects with organ herniation after esophagectomy using absorbable human acellular dermal matrix.

Sunitinib represses regulatory T cells to overcome immunotolerance in a murine model of hepatocellular cancer

ABSTRACT Successful development of immunotherapeutic strategies for hepatocellular cancer (HCC) has been impeded by limited understanding of tumor-induced profound tolerance and lack of a clinically faithful HCC model. Recently, we developed a novel model that recapitulates typical features of human HCC. Using this clinically relevant model, we demonstrate that tumor growth impairs host immunity and causes a profound exhaustion of tumor antigen-specific (TAS) CD8+ T cells. Increase in frequency and suppressive function of regulatory T cells (Tregs) is critically involved in this tumor-induced immune dysfunction. We further demonstrate that sunitinib suppresses Tregs and prevents tumor-induced immune tolerance, allowing TAS immunization to activate endogenous CD8+ T cells. As a result, this combinational strategy delays tumor growth. Importantly, the additional integration of exogenous naïve TAS CD8+ T cells by adoptive cell transfer (ACT) leads to the elimination of the established tumors without recurrence and promotes long-term survival of the treated mice. Mechanistically, sunitinib treatment primes the antitumor immune response by significantly decreasing Treg frequency, reducing TGF-β and IL-10 production by Tregs, and also protecting TAS CD8+ T cells from tumor-induced deletion in the setting of HCC. Taken together, sunitinib quantitatively and qualitatively modifies Tregs to overcome tumor-induced immune deficiency, suggesting the potential of sunitinib as a therapeutic immune activator for HCC control.