Hypoxia as a potential inducer of immune tolerance, tumor plasticity and a driver of tumor mutational burden: Impact on cancer immunotherapy.

In cancer patients, immune cells are often functionally compromised due to the immunosuppressive features of the tumor microenvironment (TME) which contribute to the failures in cancer therapies. Clinical and experimental evidence indicates that developing tumors adapt to the immunological environment and create a local microenvironment that impairs immune function by inducing immune tolerance and invasion. In this context, microenvironmental hypoxia, which is an established hallmark of solid tumors, significantly contributes to tumor aggressiveness and therapy resistance through the induction of tumor plasticity/heterogeneity and, more importantly, through the differentiation and expansion of immune-suppressive stromal cells. We and others have provided evidence indicating that hypoxia also drives genomic instability in cancer cells and interferes with DNA damage response and repair suggesting that hypoxia could be a potential driver of tumor mutational burden. Here, we reviewed the current knowledge on how hypoxic stress in the TME impacts tumor angiogenesis, heterogeneity, plasticity, and immune resistance, with a special interest in tumor immunogenicity and hypoxia targeting. An integrated understanding of the complexity of the effect of hypoxia on the immune and microenvironmental components could lead to the identification of better adapted and more effective combinational strategies in cancer immunotherapy. Clearly, the discovery and validation of therapeutic targets derived from the hypoxic tumor microenvironment is of major importance and the identification of critical hypoxia-associated pathways could generate targets that are undeniably attractive for combined cancer immunotherapy approaches.

Hypoxia, a Targetable Culprit to Counter Pancreatic Cancer Resistance to Therapy.

Pancreatic ductal adenocarcinoma (PDAC) is the most common type of pancreatic cancer, and it is a disease of dismal prognosis. While immunotherapy has revolutionized the treatment of various solid tumors, it has achieved little success in PDAC. Hypoxia within the stroma-rich tumor microenvironment is associated with resistance to therapies and promotes angiogenesis, giving rise to a chaotic and leaky vasculature that is inefficient at shuttling oxygen and nutrients. Hypoxia and its downstream effectors have been implicated in immune resistance and could be contributing to the lack of response to immunotherapy experienced by patients with PDAC. Paradoxically, increasing evidence has shown hypoxia to augment genomic instability and mutagenesis in cancer, suggesting that hypoxic tumor cells could have increased production of neoantigens that can potentially enable their clearance by cytotoxic immune cells. Strategies aimed at relieving this condition have been on the rise, and one such approach opts for normalizing the tumor vasculature to reverse hypoxia and its downstream support of tumor pathogenesis. An important consideration for the successful implementation of such strategies in the clinic is that not all PDACs are equally hypoxic, therefore hypoxia-detection approaches should be integrated to enable optimal patient selection for achieving improved patient outcomes.

Phase Ib dose-escalation study of the hypoxia-modifier Myo-inositol trispyrophosphate in patients with hepatopancreatobiliary tumors.

Hypoxia is prominent in solid tumors and a recognized driver of malignancy. Thus far, targeting tumor hypoxia has remained unsuccessful. Myo-inositol trispyrophosphate (ITPP) is a re-oxygenating compound without apparent toxicity. In preclinical models, ITPP potentiates the efficacy of subsequent chemotherapy through vascular normalization. Here, we report the results of an unrandomized, open-labeled, 3 + 3 dose-escalation phase Ib study (NCT02528526) including 28 patients with advanced primary hepatopancreatobiliary malignancies and liver metastases of colorectal cancer receiving nine 8h-infusions of ITPP over three weeks across eight dose levels (1'866-14'500 mg/m2/dose), followed by standard chemotherapy. Primary objectives are assessment of the safety and tolerability and establishment of the maximum tolerated dose, while secondary objectives include assessment of pharmacokinetics, antitumor activity via radiological evaluation and assessment of circulatory tumor-specific and angiogenic markers. The maximum tolerated dose is 12,390 mg/m2, and ITPP treatment results in 32 treatment-related toxicities (mostly hypercalcemia) that require little or no intervention. 52% of patients have morphological disease stabilization under ITPP monotherapy. Following subsequent chemotherapy, 10% show partial responses while 60% have stable disease. Decreases in angiogenic markers are noted in ∼60% of patients after ITPP and tend to correlate with responses and survival after chemotherapy.

Parastomal gallbladder herniation: A case report and review of the literature.

INTRODUCTION: Parastomal hernia is a common complication of patients living with an enterostomy. However, a parastomal hernia involving the gallbladder is a rare condition with only eight cases documented in the literature. PRESENTATION OF CASE: We report the case of a 69-year old female who underwent an open right hemicolectomy with creation of a colostomy and terminal ileostomy. She presented with parastomal swelling and pain 16 months later. A computed tomography scan revealed a parastomal herniation of the gallbladder. We elected to proceed with a cholecystectomy and hernia repair, the patient was asymptomatic at her last follow-up. DISCUSSION: A systematic search of the literature found eight previously published cases. This condition primarily affects elderly females. Five patients were treated surgically and three conservatively, all with a favorable outcome. In frail patients without complicating factors, a conservative treatment approach with surveillance may be safe. We chose a surgical approach due to the symptomatic nature of the presentation and the gallstone containing hernia. This is the first case of a parastomal gallbladder herniation containing a large gallstone. CONCLUSION: This report should help broadening the physician's differential diagnosis in dealing with patients with symptomatic parastomal hernias and provide an example for diagnosis and management of this complication.

Exercise Improves Outcomes of Surgery on Fatty Liver in Mice: A Novel Effect Mediated by the AMPK Pathway.

OBJECTIVE: To investigate whether exercise improves outcomes of surgery on fatty liver, and whether pharmacological approaches can substitute exercising programs. SUMMARY OF BACKGROUND DATA: Steatosis is the hepatic manifestation of the metabolic syndrome, and decreases the liver's ability to handle inflammatory stress or to regenerate after tissue loss. Exercise activates adenosine monophosphate-activated kinase (AMPK) and mitigates steatosis; however, its impact on ischemia-reperfusion injury and regeneration is unknown. METHODS: We used a mouse model of simple, diet-induced steatosis and assessed the impact of exercise on metabolic parameters, ischemia-reperfusion injury and regeneration after hepatectomy. The same parameters were evaluated after treatment of mice with the AMPK activator 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR). Mice on a control diet served as age-matched controls. RESULTS: A 4-week-exercising program reversed steatosis, lowered insulin levels, and improved glucose tolerance. Exercise markedly enhanced the ischemic tolerance and the regenerative capacity of fatty liver. Replacing exercise with AICAR was sufficient to replicate the above benefits. Both exercise and AICAR improved survival after extended hepatectomy in mice challenged with a Western diet, indicating protection from resection-induced liver failure. CONCLUSIONS: Exercise efficiently counteracts the metabolic, ischemic, and regenerative deficits of fatty liver. AICAR acts as an exercise mimetic in settings of fatty liver disease, an important finding given the compliance issues associated with exercise. Exercising, or its substitution through AICAR, may provide a feasible strategy to negate the hepatic consequences of energy-rich diet, and has the potential to extend the application of liver surgery if confirmed in humans.

Perioperative omega-3 fatty acids fail to confer protection in liver surgery: Results of a multicentric, double-blind, randomized controlled trial.

BACKGROUND & AIMS: In a variety of animal models, omega-3 polyunsaturated fatty acids (Ω3-FAs) conferred strong protective effects, alleviating hepatic ischemia/reperfusion injury and steatosis, as well as enhancing regeneration after major tissue loss. Given these benefits along with its safety profile, we hypothesized that perioperative administration of Ω3-FAs in patients undergoing liver surgery may ameliorate the postoperative course. The aim of this study was to investigate the perioperative use of Ω3-FAs to reduce postoperative complications after liver surgery. METHODS: Between July 2013 and July 2018, we carried out a multicentric, double-blind, randomized, placebo-controlled trial designed to test whether 2 single intravenous infusions of Omegaven® (Ω3-FAs) vs. placebo may decrease morbidity. The primary endpoints were postoperative complications by severity (Clavien-Dindo classification) integrated within the comprehensive complication index (CCI). RESULTS: A total of 261 patients (132 in the Omegaven and 129 in the placebo groups) from 3 centers were included in the trial. Most cases (87%, n = 227) underwent open liver surgery and 56% (n = 105) were major resections (≥3 segments). In an intention-to-treat analysis including the dropout cases, the mortality rate was 4% and 2% in the Omegaven and placebo groups (odds ratio0.40;95% CI 0.04-2.51; p = 0.447), respectively. Any complications and major complications (Clavien-Dindo ≥ 3b) occurred in 46% vs. 43% (p = 0.709) and 12% vs. 10% (p = 0.69) in the Omegaven and placebo groups, respectively. The mean CCI was 17 (±23) vs.14 (±20) (p = 0.417). An analysis excluding the dropouts provided similar results. CONCLUSIONS: The routine perioperative use of 2 single doses of intravenous Ω3-FAs (100 ml Omegaven) cannot be recommended in patients undergoing liver surgery (Grade A recommendation). LAY SUMMARY: Despite strong evidence of omega-3 fatty acids having liver-directed, anti-inflammatory and pro-regenerative action in various rodent models, 2 single omega-3 fatty acid infusions given to patients before and during liver surgery failed to reduce complications. Because single omega-3 fatty acid infusions failed to confer liver protection in this trial, they cannot currently be recommended. TRIAL REGISTRATION: ClinicalTrial.gov: ID: NCT01884948; Institution Ethical Board Approval: KEK-ZH-Nr. 2010-0038; Swissmedic Notification: 2012DR3215.

Novel Benefits of Remote Ischemic Preconditioning Through VEGF-dependent Protection From Resection-induced Liver Failure in the Mouse.

OBJECTIVE: To investigate the impact of remote ischemic preconditioning (RIPC) on liver regeneration after major hepatectomy. SUMMARY BACKGROUND DATA: RIPC is a strategy applied at remote sites to mitigate ischemic injury. Unlike other preconditioning approaches, RIPC spares target organs as it acts via systemic VEGF elevations. In the liver, however, VEGF is an important driver of regeneration following resection. Therefore, RIPC may have pro-regenerative effects. METHODS: RIPC was applied to C57BL/6 mice through intermittent clamping of the femoral vessels prior to standard 68%-hepatectomy or extended 86%-hepatectomy, with the latter causing liver failure and impaired survival. Liver regeneration was assessed through weight gain, proliferative markers (Ki67, pH3, mitoses), cell cycle-associated molecules, and survival. The role of the VEGF-ID1-WNT2 signaling axis was assessed through WIF1 (a WNT antagonist) and recombinant WNT2 injected prior to hepatectomy. RESULTS: RIPC did not affect regeneration after 68%-hepatectomy, but improved liver weight gain and hepatocyte mitoses after 86%-hepatectomy. Importantly, RIPC raised survival from 40% to 80% after 86%-hepatectomy, indicating the promotion of functional recovery. Mechanistically, the RIPC-induced elevations in VEGF were accompanied by increases in the endothelial transcription factor Id1, its target WNT2, and its hepatocellular effector ß-catenin. WIF1 injection prior to 86%-hepatectomy abrogated the RIPC benefits, while recombinant WNT2 had pro-regenerative effects akin to RIPC. CONCLUSION: RIPC improves the regenerative capacity of marginal liver remnants in a VEGF-dependent way. If confirmed in patients, RIPC may become the preconditioning strategy of choice in the setting of extended liver resections.

Impact of associating liver partition and portal vein ligation for staged hepatectomy (ALPPS) on growth of colorectal liver metastases.

BACKGROUND: Associating liver partition and portal vein ligation for staged hepatectomy induces an unprecedented liver hypertrophy and enables resection of otherwise unresectable liver tumors. The effect of associating liver partition and portal vein ligation for staged hepatectomy on tumor proliferation, however, remains a concern. This study investigated the impact of associating liver partition and portal vein ligation for staged hepatectomy on growth of colorectal metastases in mice and in humans. METHODS: The effect of associating liver partition and portal vein ligation for staged hepatectomy and 90% portal vein ligation on colorectal liver and lung metastases was investigated in mice. In vivo tumor progression was assessed by magnetic resonance imaging, histology, and survival experiments. The effects of associating liver partition and portal vein ligation for staged hepatectomy, portal vein ligation, and control sera on cultures of several colorectal cancer cell lines (MC38 and CT26) were tested in vitro. Additionally, the international associating liver partition and portal vein ligation for staged hepatectomy registry enabled us to identify patients with remaining tumor in the future liver remnant after associating liver partition and portal vein ligation for staged hepatectomy stage 1. RESULTS: Two and 3 weeks after associating liver partition and portal vein ligation for staged hepatectomy stage 1, portal vein ligation, or sham surgery, liver magnetic resonance images showed similar numbers (P=.14/0.82), sizes (P=.45/0.98), and growth kinetics (P=.58/0.68) of intrahepatic tumor. Tumor growth was not different between the associating liver partition and portal vein ligation for staged hepatectomy and portal vein ligation groups after completion of stage 2. Median survival after tumor cell injection was similar after sham surgery (36 days; 95% confidence interval; 27-57 days), completion of associating liver partition and portal vein ligation for staged hepatectomy (42 days; 95% confidence interval; 35-49 days), and portal vein ligation (39 days; 95% confidence interval; 34-43 days, P=.237). Progression of pulmonary metastases and in vitro cell proliferation were comparable among groups. Observations in humans failed to identify any accelerated tumor growth in the future liver remnant within the regenerative phase after associating liver partition and portal vein ligation for staged hepatectomy stage 1. CONCLUSION: The accelerated regeneration process associated with associating liver partition and portal vein ligation for staged hepatectomy does not appear to enhance growth of colorectal metastases.

The Allosteric Hemoglobin Effector ITPP Inhibits Metastatic Colon Cancer in Mice.

OBJECTIVE: To test the effects of enhanced intracellular oxygen contents on the metastatic potential of colon cancer. BACKGROUND: Colorectal cancer is the commonest gastrointestinal carcinoma. Distant metastases occur in half of patients and are responsible for most cancer-related deaths. Tumor hypoxia is central to the pathogenesis of metastases. Myo-Inositoltrispyrophosphate (ITPP), a nontoxic, antihypoxic compound, has recently shown significant benefits in experimental cancer, particularly when combined with standard chemotherapy. Whether ITPP protects from distant metastases in primary colon cancer is unknown. METHODS: ITPP alone or combined with FOLFOX was tested in a mouse model with cecal implantation of green fluorescent protein-labeled syngeneic colorectal cancer cells. Tumor development was monitored through longitudinal magnetic resonance imaging-based morphometric analysis and survival. Established serum markers of tumor spread were measured serially and circulating tumor cells were detected via fluorescence measurements. RESULTS: ITPP significantly reduced the occurrence of metastases as well as other indicators of tumor aggressiveness. Less circulating tumor cells along with reduction in malignant serum markers (osteopontin, Cxcl12) were noted. The ITPP benefits also affected the primary cancer site. Importantly, animals treated with ITPP had a significant survival benefit compared with respective controls, while a combination of FOLFOX with ITPP conferred the maximum benefits, including dramatic improvements in survival (mean 86 vs 188 d). CONCLUSIONS: Restoring oxygen in metastatic colon cancer through ITPP inhibits tumor spread and markedly improves animal survival; an effect that is enhanced through the application of subsequent chemotherapy. These promising novel findings call for a clinical trial on ITPP in patients with colorectal cancer, which is under way.

PTEN Down-Regulation Promotes ß-Oxidation to Fuel Hypertrophic Liver Growth After Hepatectomy in Mice.

In regenerating liver, hepatocytes accumulate lipids before the major wave of parenchymal growth. This transient, regeneration-associated steatosis (TRAS) is required for liver recovery, but its purpose is unclear. The tumor suppressor phosphatase and tensin homolog (PTEN) is a key inhibitor of the protein kinase B/mammalian target of rapamycin axis that regulates growth and metabolic adaptations after hepatectomy. In quiescent liver, PTEN causes pathological steatosis when lost, whereas its role in regenerating liver remains unknown. Here, we show that PTEN down-regulation promotes liver growth in a TRAS-dependent way. In wild-type mice, PTEN reduction occurred after TRAS formation, persisted during its disappearance, and correlated with up-regulated ß-oxidation at the expense of lipogenesis. Pharmacological modulation revealed an association of PTEN with TRAS turnover and hypertrophic liver growth. In liver-specific Pten-/- mice shortly after induction of knockout, hypertrophic regeneration was accelerated and led to hepatomegaly. The resulting surplus liver mass was functional, as demonstrated by raised survival in a lethal model of resection-induced liver failure. Indirect calorimetry revealed lipid oxidation as the primary energy source early after hepatectomy. The shift from glucose to lipid usage was pronounced in Pten-/- mice and correlated with the disappearance of TRAS. Partial inhibition of ß-oxidation led to persisting TRAS in Pten-/- mice and abrogated hypertrophic liver growth. PTEN down-regulation may promote ß-oxidation through ß-catenin, whereas hypertrophy was dependent on mammalian target of rapamycin complex 1. CONCLUSION: PTEN down-regulation after hepatectomy promotes the burning of TRAS-derived lipids to fuel hypertrophic liver regeneration. Therefore, the anabolic function of PTEN deficiency in resting liver is transformed into catabolic activities upon tissue loss. These findings portray PTEN as a node coordinating liver growth with its energy demands and emphasize the need of lipids for regeneration. (Hepatology 2017;66:908-921).

Omega-3 Fatty Acids Protect Fatty and Lean Mouse Livers After Major Hepatectomy.

OBJECTIVE: The aim of this study was to assess the effect of Ω3 fatty acids (Ω3FA) on fatty and lean liver in hepatic surgery. BACKGROUND: The global spread of energy-dense diets has led to an endemic rise in fatty liver disease and obesity. Besides metabolic pathologies, steatosis enhances hepatic sensitivity to ischemia reperfusion (I/R) and impedes liver regeneration (LR). Steatosis limits the application of liver surgery, still the main curative option for liver cancer. Ω3FA are known to reverse steatosis, but how these lipids affect key factors defining surgical outcomes-that is, I/R, LR, and liver malignancy-is less clear. METHODS: We established a standardized mouse model of high fat diet (HFD)-induced steatosis followed by Ω3FA treatment and the subsequent assessment of Ω3FA effects on I/R, LR, and liver malignancy (n = 5/group), the latter through a syngeneic metastasis approach. Fatty liver outcomes were compared with lean liver to assess steatosis-independent effects. Nonparametric statistics were applied. RESULTS: Ω3FA reversed HFD-induced steatosis and markedly protected against I/R, improved LR, and prolonged survival of tumor-laden mice. Remarkably, these beneficial effects were also observed in lean liver, albeit at a smaller scale. Notably, mice with metastases in fatty versus lean livers were associated with improved survival. CONCLUSIONS: Ω3FA revealed multiple beneficial effects in fatty and lean livers in mice. The improvements in I/R injury, regenerative capacity, and oncological outcomes await confirmatory studies in humans.

Development of OXY111A, a novel hypoxia-modifier as a potential antitumor agent in patients with hepato-pancreato-biliary neoplasms - Protocol of a first Ib/IIa clinical trial.

BACKGROUND: Solid tumors, such as hepato-pancreato-biliary cancer, develop tumor hypoxia with tumor growth. Despite advances in surgery, a majority of these patients are in an unresectable condition. At this stage standard cytotoxic chemotherapy regimens are applied with limited success. Novel biological treatment options based on an antiangiogenic mechanism of action neglect other hypoxia mediated mechanisms (e.g. epithelial-mesenchymal transition, Warburg effect, and immunological response) leading to an increased invasiveness with a poor outcome. The novel antihypoxic molecule myo-inositoltrispyrophosphate (ITPP, OXY111A) acts as an allosteric effector of hemoglobin and promotes normoxia in hypoxic tumors. In preclinical studies, tumor growth was reduced and survival prolonged. Additionally, a beneficial side effect profile was observed. METHODS: In this first Ib/IIa clinical trial we will assess safety and tolerability of OXY111A as well as a proof of concept regarding efficacy in patients with non-resectable primary and secondary tumors of the liver, pancreas, and biliary tract. The study design is exploratory, prospective, open-labelled and mono-centric. The study is divided in a dose escalation part with a maximum of 48 subjects and an extension part, in which 21 subjects will be included. DISCUSSION: The novel antihypoxic compound OXY111A has been tested in several cancer animal models showing beneficial effects for both survival and low side effect profiles. This first in patient application of OXY111A will reveal potential beneficial outcomes if anti-hypoxic therapy is added to standard cytotoxic treatment in patients with primary and secondary hepatopancreatobiliary tumors. TRIAL REGISTRATION: Institution Ethical Board Approval ID: KEK-ZH-Nr. 2014-0374; Swiss regulatory authority Swissmedic (2015DR1009); ClinicalTrials.gov Identifier: NCT02528526 , prospectively registered on November 11th, 2014.

Remote Ischemic Preconditioning: A Novel Strategy in Rescuing Older Livers From Ischemia-reperfusion Injury in a Rodent Model.

OBJECTIVES: The aim of this study was to determine whether remote ischemic preconditioning (RIPC) protects aged liver against ischemia reperfusion (IR). SUMMARY OF BACKGROUND DATA: The demands for liver surgery in an aging population are growing. Clamping of vessels to prevent blood loss is integral to liver surgery, but the resulting IR injury (IRI) augments postoperative complications. More so, sensitivity to hepatic IRI increases with age; however, no strategies have been developed that specifically protect old liver. RIPC, a novel protective approach, was performed distant to the surgical site. Whether RIPC may also protect old liver from IRI is unknown. METHODS: RIPC to the femoral vascular bundle was compared against direct ischemic preconditioning (IPC) and the standard of care intermittent clamping (IC) using a model of partial hepatic ischemia in mice aged 20 to 24 months. Liver injury was measured 6 hours after reperfusion. Protective signaling (serotonin-Vegf-Il10/Mmp8 axis, Kupffer cell polarization) was assessed immediately after preconditioning. Neutralizing antibody was used to test the role of Vegf. Hepatic vasculature was examined by electron microscopy. RESULTS: RIPC was superior over other strategies in protecting old liver from IRI, with standard IPC approaches being ineffective. RIPC induced the strongest elevations in circulating Vegf, and Vegf inhibition dampened protective signaling and abrogated the protective effects. RIPC was further associated with improvements in vascular functionality. CONCLUSIONS: RIPC is highly effective in protecting old liver from ischemic insults, mainly owing to its ability to induce circulating Vegf. These findings warrant efforts toward clinical translation.

Hypoxia-driven Hif2a coordinates mouse liver regeneration by coupling parenchymal growth to vascular expansion.

Interaction between sinusoidal endothelial cells and hepatocytes is a prerequisite for liver function. Upon tissue loss, both liver cell populations need to be regenerated. Repopulation occurs in a coordinated pattern, first through the regeneration of parenchyme (hepatocytes), which then produces vascular endothelial growth factor (VEGF) to enable the subsequent angiogenic phase. The signals that instruct hepatocytes to induce timely VEGF remain unidentified. Given that liver is highly vascularized, we reasoned that fluctuations in oxygenation after tissue loss may contribute to the coordination between hepatocyte and sinusoidal endothelial cell proliferation. To prevent drops in oxygen after hepatectomy, mice were pretreated with inositol trispyrophosphate (ITPP), an allosteric effector of hemoglobin causing increased O2 release from heme under hypoxic conditions. ITPP treatment delayed liver weight gain after hepatectomy. Comparison with controls revealed the presence of a hypoxic period around the peak of hepatocyte mitosis. Inhibition of hypoxia led to deficient hepatocyte mitosis, suppressed the regenerative Vegf wave, and abrogated the subsequent reconstruction of the sinusoidal network. These ITPP effects were ongoing with the reduction in hepatocellular hypoxia inducible factor 2a (Hif2a). In contrast, Hif1a was unaffected by ITPP. Hif2a knockdown phenocopied all effects of ITPP, including the mitotic deficiencies, Vegf suppression, and angiogenic failure. CONCLUSIONS: Oxygen is a key regulator of liver regeneration. Hypoxia-inherent to the expansion of parenchyme-activates Hif2a to couple hepatocyte mitosis with the angiogenic phase. Hif2a acts as a safeguard to initiate sinusoidal reconstruction only upon successful hepatocyte mitosis, thereby enforcing a timely order onto cell type-specific regeneration patterns. These findings portray the hypoxia-driven Hif2a-Vegf axis as a prime node in coordinating sinusoidal endothelial cell-hepatocyte crosstalk during liver regeneration. (Hepatology 2016;64:2198-2209).

Antihypoxic Potentiation of Standard Therapy for Experimental Colorectal Liver Metastasis through Myo-Inositol Trispyrophosphate.

PURPOSE: Tumor hypoxia activates hypoxia-inducible factors (Hifs), which induce a range of malignant changes including vascular abnormalities. Here, we determine whether inhibition of the hypoxic tumor response through myo-inositol trispyrophosphate (ITPP), a compound with antihypoxic properties, is able to cause prolonged vascular normalization that can be exploited to improve standard-of-care treatment. EXPERIMENTAL DESIGN: We tested ITPP on two syngeneic orthotopic mouse models of lethal colorectal cancer liver metastasis. Tumors were monitored by MRI and analyzed for the hypoxic response and their malignant potential. A Hif activator and in vitro assays were used to define the working mode of ITPP. Hypoxic response and vasculature were re-evaluated 4 weeks after treatment. Finally, we determined survival following ITPP monotherapy, FOLFOX monotherapy, FOLFOX plus Vegf antibody, and FOLFOX plus ITPP, both overlapping and sequential. RESULTS: ITPP reduced tumor load, efficiently inhibited the hypoxic response, and improved survival. These effects were lost when mice were pretreated with a Hif activator. Its immediate effects on the hypoxic response, including an apparent normalization of tumor vasculature, persisted for at least 4 weeks after treatment cessation. Compared with FOLFOX alone, Vegf antibody combined with FOLFOX prolonged survival by <30%, whereas ITPP combined with FOLFOX extended survival by >140%, regardless of whether FOLFOX was given in overlap or after ITPP exposure. CONCLUSIONS: Our findings reveal a truly antihypoxic mechanism for ITPP and demonstrate the capacity of this nontoxic compound to potentiate the efficacy of existing anticancer treatment in a way amenable to clinical translation. Clin Cancer Res; 22(23); 5887-97. ©2016 AACR.

Constitutive androstane receptor (Car)-driven regeneration protects liver from failure following tissue loss.

BACKGROUND & AIMS: Liver can recover following resection. If tissue loss is too excessive, however, liver failure will develop as is known from the small-for-size-syndrome (SFSS). The molecular processes underlying liver failure are ill-understood. Here, we explored the role and the clinical potential of Nr1i3 (constitutive androstane receptor, Car) in liver failure following hepatectomy. METHODS: Activators of Car, various hepatectomies, Car(-/-) mice, humanized CAR mice, human tissue and ex vivo liver slice cultures were used to study Car in the SFSS. Pathways downstream of Car were investigated by in vivo siRNA knockdown. RESULTS: Excessive tissue loss causing liver failure is associated with deficient induction of Car. Reactivation of Car by an agonist normalizes all features associated with experimental SFSS. The beneficial effects of Car activation are relayed through Foxm1, an essential promoter of the hepatocyte cell cycle. Deficiency in the CAR-FOXM1 axis likewise is evident in human SFSS. Activation of human CAR mitigates SFSS in humanized CAR mice and improves the culture of human liver slices. CONCLUSIONS: Impaired hepatic Car-Foxm1 signaling provides a first molecular characterization of liver that fails to recover after tissue loss. Our findings place deficient regeneration as a principal cause behind the SFSS and suggest CAR agonists may bear clinical potential against liver failure. LAY SUMMARY: The unique regenerative capacity of liver has its natural limits. Following tissue loss that is too excessive, such as through extended resection in the clinic, liver failure may develop. This is known as small-for-size-syndrome (SFSS) and represents the most frequent cause of death due to liver surgery. Here we show that deficient induction of the protein Car, a central regulator of liver function and growth, is a cause of liver failure following extended resection; reactivation of Car through pharmacological means is sufficient to prevent or rescue the SFSS.

Selective portal vein injection for the design of syngeneic models of liver malignancy.

Liver metastases are the most frequent cause of death due to colorectal cancer (CRC). Syngeneic orthotopic animal models, based on the grafting of cancer cells or tissue in host liver, are efficient systems for studying liver tumors and their (patho)physiological environment. Here we describe selective portal vein injection as a novel tool to generate syngeneic orthotopic models of liver tumors that avoid most of the weaknesses of existing syngeneic models. By combining portal vein injection of cancer cells with the selective clamping of distal liver lobes, tumor growth is limited to specific lobes. When applied on MC-38 CRC cells and their mouse host C57BL6, selective portal vein injection leads with 100% penetrance to MRI-detectable tumors within 1 wk, followed by a steady growth until the time of death (survival ∼7 wk) in the absence of extrahepatic disease. Similar results were obtained using CT-26 cells and their syngeneic Balb/c hosts. As a proof of principle, lobe-restricted liver tumors were also generated using Hepa1-6 (C57BL6-syngeneic) and TIB-75 (Balb/c-syngeneic) hepatocellular cancer cells, demonstrating the general applicability of selective portal vein injection for the induction of malignant liver tumors. Selective portal vein injection is technically straightforward, enables liver invasion via anatomical routes, preserves liver function, and provides unaffected liver tissue. The tumor models are reproducible and highly penetrant, with survival mainly dependent on the growth of lobe-restricted liver malignancy. These models enable biological studies and preclinical testing within short periods of time.

Endothelial Rictor is crucial for midgestational development and sustained and extensive FGF2-induced neovascularization in the adult.

To explore the general requirement of endothelial mTORC2 during embryonic and adolescent development, we knocked out the essential mTORC2 component Rictor in the mouse endothelium in the embryo, during adolescence and in endothelial cells in vitro. During embryonic development, Rictor knockout resulted in growth retardation and lethality around embryonic day 12. We detected reduced peripheral vascularization and delayed ossification of developing fingers, toes and vertebrae during this confined midgestational period. Rictor knockout did not affect viability, weight gain, and vascular development during further adolescence. However during this period, Rictor knockout prevented skin capillaries to gain larger and heterogeneously sized diameters and remodeling into tortuous vessels in response to FGF2. Rictor knockout strongly reduced extensive FGF2-induced neovascularization and prevented hemorrhage in FGF2-loaded matrigel plugs. Rictor knockout also disabled the formation of capillary-like networks by FGF2-stimulated mouse aortic endothelial cells in vitro. Low RICTOR expression was detected in quiescent, confluent mouse aortic endothelial cells, whereas high doses of FGF2 induced high RICTOR expression that was associated with strong mTORC2-specific protein kinase Cα and AKT phosphorylation. We demonstrate that the endothelial FGF-RICTOR axis is not required during endothelial quiescence, but crucial for midgestational development and sustained and extensive neovascularization in the adult.

"A randomized, double-blind study of the effects of omega-3 fatty acids (Omegaven) on outcome after major liver resection".

BACKGROUND: The body is dependent on the exogenous supply of omega-3 polyunsaturated fatty acids (n3-PUFA). These essential fatty acids are key players in regulating metabolic signaling but also exert anti-inflammatory and anti-carcinogenic properties. The liver is a major metabolic organ involved in fatty acid metabolism. Under experimental conditions, n3-PUFA exert beneficial effect on hepatic steatosis, regeneration and inflammatory insults such as ischemic injury after surgery. Some of these effects have also been observed in human subjects. However, it is unclear whether perioperative administration of n3-PUFA is sufficient to protect the liver from ischemic injury. Therefore, we designed a randomized controlled trial (RCT) assessing n3-PUFA (pre-) conditioning strategies in patients scheduled for liver surgery. METHODS/DESIGN: The Omegaven trial is a multi-centric, double-blind, randomized, placebo- controlled trial applying two single doses of Omegaven or placebo on 258 patients undergoing major liver resection. Primary endpoints are morbidity and mortality one month after hospital discharge, defined by the Clavien- Dindo classification of surgical complications (Ann Surg 240(2):205-13, 2004) as well as the Comprehensive Complication Index (CCI) (Ann Surg 258(1):1-7, 2013). Secondary outcome variables include length of Intensive Care Unit (ICU) and hospital stay, postoperative liver function tests, fatty acid and eicosanoid concentration, inflammatory markers in serum and in liver tissue. An interim analysis is scheduled after the first 30 patients per randomization group. DISCUSSION: Long-term administration of n3-PUFA have a beneficial effect on metabolism and hepatic injury. Patients often require surgery without much delay, thus long-term n3-PUFA uptake is not possible. Also, lack of compliance may lead to incomplete n3-PUFA substitution. Hence, perioperative Omegaven™ may provide an easy and controllable way to ensure hepaative application of tic protection. TRIAL REGISTRATION: ClinicalTrial.gov: ID: NCT01884948 , registered June 14, 2013; Institution Ethical Board Approval: KEK-ZH-Nr. 2010-0038; Swissmedic Notification: 2012DR3215.