Hypoxic Areas Research Articles

Hypoxia, metabolism, and long-lived plasma cells (LYM6P.765)

Abstract Evidence from multiple laboratories agrees that long-lived humoral immunity derives from long-lived plasma cells (LLPC) that reside in the bone marrow (BM), and existing literature makes a compelling case explaining that short-lived PC die due to high metabolic stress. However, the mechanism by which LLPC escape the same fate as SLPC is unexplored. We tested the hypothesis that glycolysis is used by BM LLPC, which might be triggered by hypoxic areas of the bone marrow. Hypoxyprobe™-1 (PIM) was used as an indicator of BM LLPC residing in areas of low O2 tension. PIM positive stain in PC suggests that most BM LLPC reside in hypoxic niches. In addition, data obtained from both ELISPOT and 24h ELISA suggest that Ig secretion from BM cultured in a hypoxia chamber (3% O2) is slightly enhanced in hypoxic versus normoxic conditions. Since BM PC appear to reside in hypoxic niches, we assessed whether glycolysis is the predominant metabolic strategy employed by PC. Using the MitoTracker probe that fluoresces upon oxidation in respiring mitochondria, we found BM PC in both MitoTracker-hi and -lo populations, suggesting PC may differ in their metabolic strategies, using aerobic respiration and glycolysis, respectively. BM LLPC in both Mitotracker subsets are functional as determined by ELISA and ELISPOT. Future studies will assess the role of the plasma cell “niche” in promoting and sustaining a metabolic phenotype, which likely governs long-term immunity.

Individual, population, and ecosystem effects of hypoxia on a dominant benthic bivalve in Chesapeake Bay

Hypoxia is an environmental stressor that affects abundance, biomass, diversity, and ecosystem function of benthic assemblages worldwide, yet its collective impact at individual, population, and ecosystem levels has rarely been investigated. We examined the effects of hypoxia on the biomass‐dominant clam, Macoma balthica, in the York and Rappahannock Rivers (Chesapeake Bay, USA). We (1) surveyed the M. balthica populations in both rivers in 2003 and 2004, (2) determined the effects of low dissolved oxygen (DO) on M. balthica fecundity in a laboratory experiment, and (3) employed a predator‐exclusion field experiment to establish the effects of hypoxia and prey density on predation upon M. balthica. The resultant data were used to parameterize a matrix model, which was analyzed to define potential effects of hypoxia at the population level. In both rivers, hypoxia decreased individual clam growth and caused local extinction of populations. Hypoxia reduced egg production of M. balthica by 40% and increased protein investment per egg. In the predator‐exclusion field experiment, hypoxia magnified predation rates threefold and altered the functional response of predators to M. balthica from a stabilizing type III functional response to a destabilizing type II functional response. In a density‐independent matrix model, hypoxia resulted in coupled source–sink metapopulation dynamics, with hypoxic areas acting as black‐hole sinks. Increases in the spatial and temporal extent of hypoxia caused the populations to decline toward extinction. In a second model that incorporated density dependence, under mild hypoxic conditions trophic transfer from M. balthica to predators increased, but at increased spatial or temporal extent of hypoxia trophic transfer decreased. The major decline in trophic transfer to predators under severe hypoxia resulted from diversion of M. balthica biomass into the microbial loop. Our model predicted that there are multiple stable states for M. balthica populations (high and very low densities), such that the saddle point (threshold at which the population switches from one state to the other) increased and resilience decreased with the spatial extent of hypoxia. We underscore how effects of a stressor at the individual level can combine to have substantial population and ecosystem‐level effects.

Effect of hypoxia on the expression of αB-crystallin in head and neck squamous cell carcinoma.

BackgroundThe presence of hypoxia in head and neck squamous cell carcinoma (HNSCC) is associated with therapeutic resistance and increased risk of metastasis formation. αB-crystallin (HspB5) is a small heat shock protein, which is also associated with metastasis formation in HNSCC. In this study, we investigated whether αB-crystallin protein expression is increased in hypoxic areas of HNSCC biopsies and analyzed whether hypoxia induces αB-crystallin expression in vitro and in this way may confer hypoxic cell survival.MethodsIn 38 HNSCC biopsies, the overlap between immunohistochemically stained αB-crystallin and pimonidazole-adducts (hypoxiamarker) was determined. Moreover, expression levels of αB-crystallin were analyzed in HNSCC cell lines under hypoxia and reoxygenation conditions and after exposure to reactive oxygen species (ROS) and the ROS scavenger N-acetylcysteine (NAC). siRNA-mediated knockdown was used to determine the influence of αB-crystallin on cell survival under hypoxic conditions.ResultsIn all biopsies αB-crystallin was more abundantly present in hypoxic areas than in normoxic areas. Remarkably, hypoxia decreased αB-crystallin mRNA expression in the HNSCC cell lines. Only after reoxygenation, a condition that stimulates ROS formation, αB-crystallin expression was increased. αB-crystallin mRNA levels were also increased by extracellular ROS, and NAC abolished the reoxygenation-induced αB-crystallin upregulation. Moreover, it was found that decreased αB-crystallin levels reduced cell survival under hypoxic conditions.ConclusionsWe provide the first evidence that hypoxia stimulates upregulation of αB-crystallin in HNSCC. This upregulation was not caused by the low oxygen pressure, but more likely by ROS formation. The higher expression of αB-crystallin may lead to prolonged survival of these cells under hypoxic conditions.

Cardiac myofilament Caa2+ sensitization disturbs regulation of coronary flow and leads to focal myocardial ischemia and arrhythmias during stress (1079.25)

Myofilament Ca2+ sensitization has been linked to increased arrhythmia susceptibility, but the underlying mechanism is not completely understood. Mice with the Ca2+ sensitizing TnT‐I79N mutation develop arrhythmias during stress, despite the absence of an overt anatomical substrate. Stress by fast pacing of isolated perfused TnT‐I79N hearts for 16 min elicited arrhythmias (63%) and caused the formation of hypoxic regions (14.1% of total tissue in I79N vs 2.6% in control, Hypoxyprobe) associated with focal cellular energy depletion (Connexin isoforms P0 and pT172‐AMP kinase both increased). The cause for the local energetic supply‐and‐demand mismatch is unclear and we tested if insufficient perfusion is at least in part responsible. Global coronary flow was not decreased in isolated TnT‐I79N hearts. To test if flow distribution was altered, we infused fluorescent 15 µm microspheres and compared the fraction of microspheres inside hypoxic areas with the fraction of hypoxic tissue. Microspheres inside hypoxic regions were drastically reduced, suggesting regional ischemia. These results were reproduced in control hearts treated with the Ca2+ sensitizing drug EMD57033. Next we tested if hypoxia also occurs naturally in TnT‐I79N mice in vivo. Despite expecting low sensitivity due to localized effects, the mRNA expression levels of genes up‐regulated by hypoxia (GLUT1, TUBB3) were increased (0.3fold, 3fold).We conclude that myofilament Ca2+ sensitization disturbs regulation of coronary flow and leads to focal ischemia and arrhythmias during stress. This suggests that myofilament properties of cardiomyocytes importantly contribute to local regulation of coronary flow.Grant Funding Source: supported by AHA 10SDG2640109 and NIH HL71670

Increased metabolites of 5-lipoxygenase from hypoxic ovarian cancer cells promote tumor-associated macrophage infiltration

5-lipoxygenase (5-LOX), a member of the lipoxygenase gene family, is a key enzyme assisting in the conversion of arachidonic acid to 5-HETE and leukotrienes. Tumor-associated macrophages (TAMs) have a critical role in the progression and metastasis of many tumors, including ovarian tumors. Moreover, TAMs are often found in a high density in the hypoxic areas of tumors. However, the relevant mechanisms have not been studied explicitly until now. In this study, we found that the expression of 5-LOX strongly correlated with the density of TAMs in hypoxic areas of human ovarian tumor tissues. In cultured ovarian cancer cells, 5-LOX metabolites were increased under hypoxic conditons. Increased 5-LOX metabolites from hypoxic ovarian cancer cells promoted migration and invasion of macrophages, which was further demonstrated to be mediated by the upregulation of matrix metalloproteinase (MMP)-7 expression through the p38 pathway. Besides, we also showed that 5-LOX metabolites enhanced the release of tumor necrosis factor (TNF-α) and heparin-binding epidermal growth factor-like growth factor through upregulation of MMP-7. Furthermore, in animal models, Zileuton (a selective and specific 5-LOX inhibitor) reduced the MMP-7 expression and the number of macrophages infiltrating in the xenograft. Our findings suggest for the first time that increased metabolites of 5-LOX from hypoxic ovarian cancer cells promote TAM infiltration. These results of this study have immediate translational implications for the therapeutic exploitation of TAMs.

Quantifying the heterogeneity of hypoxic and anoxic areas in the Baltic Sea by a simplified coupled hydrodynamic-oxygen consumption model approach

The Baltic Sea deep waters suffer from extended areas of hypoxia and anoxia. Their intra- and inter-annual variability is mainly determined by saline inflows which transport oxygenated water to deeper layers. During the last decades, oxygen conditions in the Baltic Sea have generally worsened and thus, the extent of hypoxic as well as anoxic bottom water has increased considerably. Climate change may further increase hypoxia due to changes in the atmospheric forcing conditions resulting in less deep water renewal Baltic inflows, decreased oxygen solubility and increased respiration rates. Feedback from climate change can amplify effects from eutrophication. A decline in oxygen conditions has generally a negative impact on marine life in the Baltic Sea. Thus, a detailed description of the evolution of oxygenated, hypoxic and anoxic areas is particularly required when studying oxygen-related processes such as habitat utilization of spawning fish, survival rates of their eggs as well as settlement probability of juveniles. One of today's major challenges is still the modeling of deep water dissolved oxygen, especially for the Baltic Sea with its seasonal and quasi-permanent extended areas of oxygen deficiency. The detailed spatial and temporal evolution of the oxygen concentrations in the entire Baltic Sea have been simulated for the period 1970–2010 by utilizing a hydrodynamic Baltic Sea model coupled to a simple pelagic and benthic oxygen consumption model. Model results are in very good agreement with CTD/O2-profiles taken in different areas of the Baltic Sea. The model proved to be a useful tool to describe the detailed evolution of oxygenated, hypoxic and anoxic areas in the entire Baltic Sea. Model results are further applied to determine frequencies of the occurrence of areas of oxygen deficiency and cod reproduction volumes.

Factors influencing export of dissolved inorganic nitrogen by major rivers: A new, seasonal, spatially explicit, global model

AbstractSubstantial effort has focused on understanding spatial variation in dissolved inorganic nitrogen (DIN) export to the coastal zone and specific basins have been studied in depth. Much less is known, however, about seasonal patterns and controls of coastal DIN delivery across large spatial scales. Understanding seasonal patterns of DIN export is critical to efforts to predict impacts of coastal eutrophication, such as algal blooms and hypoxic areas, which are often seasonal phenomena. Here we describe, test, and apply a global model that predicts seasonal DIN export to coastal regions for >6000 rivers using the Nutrient Export from Watersheds (NEWS2) model. NEWS2‐DIN‐S used spatially explicit, seasonal N inputs and was calibrated with measured DIN yield (kg N km−2 season−1) for 77 rivers, distributed globally. Of the characteristics considered, DIN‐transport efficiency was positively related to runoff and negatively related to temperature (r2 = 0.34–0.60, depending on season p < 0.0001), likely due to flushing effects and increased retention by plants and soils, respectively. NEWS2‐DIN‐S incorporated these insights and performed well in predicting DIN yield (Nash‐Sutcliffe Efficiency = 0.54–0.65, depending on season). Catchments were effective in retaining DIN and average export rates were lower during the growing season (3–5% of total nitrogen inputs) compared to other seasons (6–10%) for major latitude bands. Model output was insensitive to changes in the magnitude of N inputs, suggesting that refinement of seasonal N input budgets will not substantially improve model performance. Rather, better representation of land‐to‐river N transfers could improve future models because of strong landscape N attenuation.

Combined therapies of antithrombotics and antioxidants delayin silicobrain tumour progression

Glioblastoma multiforme (GBM), the most frequent type of primary brain tumour, is a rapidly evolving and spatially heterogeneous high-grade astrocytoma that presents areas of necrosis, hypercellularity and microvascular hyperplasia. The aberrant vasculature leads to hypoxic areas and results in an increase in oxidative stress, selecting for more invasive tumour cell phenotypes. In our study, we assay in silico different therapeutic approaches which combine antithrombotics (ATs), antioxidants and standard radiotherapy (RT). To do so, we have developed a biocomputational model of GBM that incorporates the spatio-temporal interplay among two glioma cell phenotypes corresponding to oxygenated and hypoxic cells, a necrotic core and the local vasculature whose response evolves with tumour progression. Our numerical simulations predict that suitable combinations of ATs and antioxidants may diminish, in a synergistic way, oxidative stress and the subsequent hypoxic response. This novel therapeutical strategy, with potentially low or no toxicity, might reduce tumour invasion and further sensitize GBM to conventional RT or other cytotoxic agents, hopefully increasing median patient overall survival time.

Low-Dose Nitrite Alleviates Early Effects of an X-ray Contrast Medium on Renal Hemodynamics and Oxygenation in Rats

Renal tissue hypoperfusion and hypoxia are pivotal pathophysiological elements in contrast media (CM)-induced acute kidney injury (AKI). According to a new paradigm of hypoxic vasodilation, nitrite is reduced to vasodilatory nitric oxide by hemoglobin in hypoxic areas. Here, we study the potential of low-dose nitrite administration to alleviate CM-induced AKI. In anesthetized rats, arterial blood pressure, total renal blood flow, cortical and medullary erythrocyte flux and oxygen tension (pO2), hindquarter blood flow, and methemoglobin were measured. Nitrite was continuously infused (0.172 mg/h per kilogram of body mass for 10 minutes; thereafter, 0.057 mg/h per kilogram of body mass); control rats received isotonic saline. Hypoxic vasodilation was studied through a brief period of hypoxia (10% inspiratory oxygen fraction). Then, 1.5 mL of the CM, iodixanol of 320 mg I/mL, was injected into the thoracic aorta and the effects studied for 60 minutes. Nitrite infusion slightly increased methemoglobin, but it did not change per se any other parameter including arterial pressure. However, nitrite enhanced the hypoxic vasodilation in the kidney and hindquarter (by 100% and 70%, respectively) during the brief hypoxic challenge. In the control rats, CM resulted in renal tissue hypoxia mainly because of renal vasoconstriction. Medullary and cortical pO2 dropped upon CM injection and remained 30% to 40% below pre-CM level throughout the observation period. Nitrite infusion improved renal tissue oxygenation mainly by hindering CM-induced renal vasoconstriction. After a small transient drop (<25% for <5 minutes), medullary and cortical pO2 was restored to values indistinguishable from pre-CM levels. Low-dose nitrite infusion greatly alleviates early adverse effects of CM on renal tissue oxygenation in rats. Nitrite's beneficial effect probably relies on its reduction to nitric oxide in hypoxic tissue with ensuing vasodilation. Our nitrite dose resulted in a negligible increase in methemoglobin and did not induce hypotension. Thus, low-dose nitrite infusion might prove to be a reasonably specific measure to reduce the risk for CM-induced AKI.

Blocking Macrophage NRP1 Signaling Preserves Antitumor Functionality

Abstract Sema3A/NRP1 signaling promotes TAM infiltration and protumorigenic functions in hypoxic tumor areas.

The impact of eutrophication and commercial fishing on molluscan communities in Long Island Sound, USA

Benthic communities in Long Island Sound (LIS) have experienced over 150years of commercial shellfishing and excess nutrient loading (eutrophication) which causes hypoxia. We established an ecological baseline using a combination of live, dead, archaeological, and fossil material to investigate the impacts of these stressors on the molluscan community. We expected that ecological change would increase with eutrophication-hypoxia west towards New York City. Instead we found that taxonomic similarity, rank-order abundance, and drilling frequency are more strongly controlled by commercial fishing pressure than by decreasing dissolved oxygen. Commercial fisherman collecting quahog clams (Mercenaria mercenaria), physically disrupt surface-dwelling organisms and also kill large numbers of predatory gastropods, including the channeled whelk, Busycotypus canaliculatus, and the drilling moonsnails Neverita duplicata and Euspira heros, to protect hard clam stocks. As a result, areas dredged by commercial fishermen yield fewer shells with drill-holes and fewer surface-dwelling organisms than unfished sites. In spite of recent reductions in lobster fishing, crushing predation by crabs and lobsters on clams has been suppressed below baseline levels throughout LIS, even in the well oxygenated east. The absence of a clear relationship between eutrophication-hypoxia and ecological change questions the effectiveness of nitrogen reduction alone as a restoration strategy. LIS fossils revealed a relatively ancient loss of those mollusks associated with seagrass and oyster habitats (e.g., oysters, Crassostrea virginica; jingle shells, Anomia simplex; scallops, Argopecten irradiens; and the gastropod Bittiolum alternatum) that predates the accumulation of dead shells and underscores the need for older material to reveal the shifting baseline. The interactive nature of multiple stressors means that past overfishing may have dampened the response of communities in LIS to eutrophication or inhibited their capactiy to recover. The unexpected role of hypoxic areas protected from commercial fishing as refuges highlights the utility of no-take marine preserves in eutrophied estuaries worldwide.

Mesothelioma and Hypoxia: Modulation of the Inflammation-Related Phenotype and Identification of Prognostic Markers

Objective: Malignant Mesothelioma is an aggressive tumor occurring in the context of chronic inflammation and characterized by hypoxic areas. This study explores how hypoxia impacts on the pro-inflammatory phenotype of MSTO-211H and MPP89 mesothelioma cells and investigates the role of HIF-1 alpha and NF-kB in this process. The prognostic values of two molecules upregulated by oxygen deprivation, HIF-3 alpha and CXCR4, is also analyzed. Methods: Hypoxic condition was obtained in a sealed modular incubator chamber flushed with 1% O2. mRNA and protein levels were evaluated by real-time PCR and western blot. Silencing of HIF-1alpha was achieved by specific shRNA and NF-kB inhibition by parthenolide treatment. HIF-3 alpha and CXCR4 expression in tumor tissues from mesothelioma patients was detected by immunohistochemistry. Results: The hypoxic stimulation of mesothelioma cells induced an early activation of HIF-1 alpha and NF-kB and a later increase of HIF-3 alpha expression. In addition, the upregulation of a set of inflammation-related genes was observed. Silencing of HIF-1 alpha and treatment with parthenolide highlighted that the observed increase in gene expression depends on both HIF-1 alpha and NF-kB transcriptional activity. A correlation between high expression of CXCR4 in human mesothelioma samples and poor survival was also observed and HIF3 alpha was suggested as a potential new prognostic marker. Conclusions: This study evidences a cross-talk between hypoxia adaptation and pro-inflammatory phenotype in mesothelioma accomplished through the combined transactivation activity of HIFs and NFkB. Immunohistochemistry analysis of tissue samples confirms CXCR4 and suggests HIF-3 alpha as potential prognostic markers for mesothelioma.

Interleukin-1β promotes hypoxia-induced apoptosis of glioblastoma cells by inhibiting hypoxia-inducible factor-1 mediated adrenomedullin production

Glioblastoma is the most common brain tumor in adults. Advanced glioblastomas normally contain hypoxic areas. The primary cellular responses to hypoxia are generally mediated by the transcription factor hypoxia-inducible factor 1 (HIF-1). Interleukin-1β (IL-1β) is a cytokine that is often present in the glioblastoma microenvironment and is known to be a modulator of glioblastoma progression. However, the role of IL-1β in regulating glioblastoma progression is still controversial. In this study, we found that in the human glioblastoma cell lines U87MG and U138MG, IL-1β inhibits the transactivation activity of HIF-1 by promoting the ubiquitin-independent proteasomal degradation of the oxygen-labile α-subunit of HIF-1 and downregulates the expression of the HIF-1 target gene adrenomedullin (AM). Apoptosis and MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assays showed that AM protects glioblastoma cells against hypoxia-induced apoptosis in a dose-dependent manner. Thus, in the presence of IL-1β more glioblastoma cells undergo hypoxia-induced cell death. Our findings suggest that when estimating the influence of IL-1β on the prognosis of glioblastoma patients, factors such as the degree of hypoxia, the expression levels of HIF-1 and AM should be taken into consideration. For the AM-producing glioblastoma cells, IL-1β represents a potent apoptosis inducer.

CD44 Expressed on Cancer-Associated Fibroblasts Is a Functional Molecule Supporting the Stemness and Drug Resistance of Malignant Cancer Cells in the Tumor Microenvironment

Cells constituting the tumor microenvironment are attractive targets for developing new cancer therapies. Here we show that cancer-associated fibroblasts (CAFs) support tumor growth in vivo and maintain the stemness of cancer stem/initiating cells in an in vitro model using an established CAF cell line. We found that CD44 is abundantly expressed on CAFs. This molecule is a cancer stem cell marker in several tumors, but its role in tumorigenesis when expressed by CAFs has not been investigated. It is generally accepted that hypoxic and hyponutritional conditions are triggers of cancer malignancy. We found that CAFs strongly express CD44 in hypoxic and avascular areas in the tumor and that its expression on established CAFs is upregulated under hypoxic and hyponutritional conditions in vitro. In addition, CAF CD44-positivity in tumor tissues was increased after treatment with inhibitors of angiogenesis. Using cocultures and tumor sphere formation assays, CAFs from wild-type mice were found to sustain the stemness of cancer stem/initiating cells, while CD44-deficient CAFs did not. Furthermore, CD44 was involved in malignant cancer cell drug resistance mechanisms. In conclusion, our study suggests that CD44 on CAFs is a functional molecule contributing to the maintenance of cancer stem cell populations in the tumor microenvironment.

Cutting Edge: Hypoxia-Induced Nanog Favors the Intratumoral Infiltration of Regulatory T Cells and Macrophages via Direct Regulation of TGF-β1

Emerging evidence suggests a link between tumor hypoxia and immune suppression. In this study, we investigated the role of hypoxia-induced Nanog, a stemness-associated transcription factor, in immune suppression. We observed that hypoxia-induced Nanog correlated with the acquisition of stem cell-like properties in B16-F10 cells. We further show that Nanog was selectively induced in hypoxic areas of B16-F10 tumors. Stable short hairpin RNA-mediated depletion of Nanog, combined with melanocyte differentiation Ag tyrosinase-related protein-2 peptide-based vaccination, resulted in complete inhibition of B16-F10 tumor growth. Nanog targeting significantly reduced immunosuppressive cells (regulatory T cells and macrophages) and increased CD8(+) T effector cells in tumor bed in part by modulating TGF-β1 production. Additionally, Nanog regulated TGF-β1 under hypoxia by directly binding the TGF-β1 proximal promoter. Collectively, our data establish a novel functional link between hypoxia-induced Nanog and TGF-β1 regulation and point to a major role of Nanog in hypoxia-driven immunosuppression.

Hypoxic Tumor Environments Exhibit Disrupted Collagen I Fibers and Low Macromolecular Transport

Hypoxic tumor microenvironments result in an aggressive phenotype and resistance to therapy that lead to tumor progression, recurrence, and metastasis. While poor vascularization and the resultant inadequate drug delivery are known to contribute to drug resistance, the effect of hypoxia on molecular transport through the interstitium, and the role of the extracellular matrix (ECM) in mediating this transport are unexplored. The dense mesh of fibers present in the ECM can especially influence the movement of macromolecules. Collagen 1 (Col1) fibers form a key component of the ECM in breast cancers. Here we characterized the influence of hypoxia on macromolecular transport in tumors, and the role of Col1 fibers in mediating this transport using an MDA-MB-231 breast cancer xenograft model engineered to express red fluorescent protein under hypoxia. Magnetic resonance imaging of macromolecular transport was combined with second harmonic generation microscopy of Col1 fibers. Hypoxic tumor regions displayed significantly decreased Col1 fiber density and volume, as well as significantly lower macromolecular draining and pooling rates, than normoxic regions. Regions adjacent to severely hypoxic areas revealed higher deposition of Col1 fibers and increased macromolecular transport. These data suggest that Col1 fibers may facilitate macromolecular transport in tumors, and their reduction in hypoxic regions may reduce this transport. Decreased macromolecular transport in hypoxic regions may also contribute to poor drug delivery and tumor recurrence in hypoxic regions. High Col1 fiber density observed around hypoxic regions may facilitate the escape of aggressive cancer cells from hypoxic regions.

Simulated halocline variability in the Baltic Sea and its impact on hypoxia during 1961–2007

Salinity and halocline depth variations in the Baltic Sea during 1961–2007 are studied using a three‐dimensional ocean circulation model. Significant interannual and interdecadal variations in the halocline depth are found, together with distinct periods characterized either by shallow (1970–1975) or deep halocline (1990–1995). The model simulation indicates that the mean top layer salinity in the Baltic Sea is mainly controlled by the accumulated river runoff, while the mean below halocline salinity in the Baltic proper (which comprises Bornholm and Gotland basins) is more dependent on the low‐pass filtered zonal wind stress, with cutoff period of 4 years, henceforth called the mean zonal wind stress. The halocline depth and stratification strength in the Baltic Sea are significantly affected by the mean zonal wind stress, while the impact of runoff is smaller. The ventilation of the halocline from bottom layers is stronger during the shallow and from surface layers during the deep halocline period. Due to changes in ventilation variations in halocline depth systematically affect bottom oxygen concentrations on seasonal and decadal, but not on interannual time scales. For instance, a deeper halocline reduces hypoxic (oxygen concentration in bottom water below 2 mL/L) and anoxic (anoxic conditions in bottom water) areas and increases the bottom oxygen concentrations in the Gulf of Finland but decreases them in the deeper parts of the Baltic proper. Model results suggest that due to undersampling during 1961–2007 mean hypoxic and anoxic areas calculated from observed profiles are underestimated by 41% and 43%, respectively.

Impeding Macrophage Entry into Hypoxic Tumor Areas by Sema3A/Nrp1 Signaling Blockade Inhibits Angiogenesis and Restores Antitumor Immunity

Recruitment of tumor-associated macrophages (TAMs) into avascular areas sustains tumor progression; however, the underlying guidance mechanisms are unknown. Here, we report that hypoxia-induced Semaphorin 3A (Sema3A) acts as an attractant for TAMs by triggering vascular endothelial growth factor receptor 1 phosphorylation through the associated holoreceptor, composed of Neuropilin-1 (Nrp1) and PlexinA1/PlexinA4. Importantly, whereas Nrp1 levels are downregulated in the hypoxic environment, Sema3A continues to regulate TAMs in an Nrp1-independent manner by eliciting PlexinA1/PlexinA4-mediated stop signals, which retain them inside the hypoxic niche. Consistently, gene deletion of Nrp1 in macrophages favors TAMs' entrapment in normoxic tumor regions, which abates their pro-angiogenic and immunosuppressive functions, hence inhibiting tumor growth and metastasis. This study shows that TAMs' heterogeneity depends on their localization, which is tightly controlled by Sema3A/Nrp1 signaling.

A Phase 1 Study Of TH-302, An Investigational Hypoxia-Targeted Drug, In Patients With Advanced Leukemias

BackgroundTH-302 is a 2-nitroimidazole prodrug of the DNA alkylator bromo-isophosphoramide mustard designed to be selectively activated in hypoxic conditions. Preclinical data in mice with acute lymphoblastic leukemia (ALL) have demonstrated marked expansion of hypoxia in areas of marrow leukemia infiltrates (Benito et al., PLoS One 2011). TH-302 also exhibited specific hypoxia-dependent cytotoxicity when tested against primary ALL and acute myelogenous leukemia (AML) samples in vitro (Benito et al., ASH 2012). Based on these findings, a phase 1 study of TH-302 was designed for advanced leukemias. MethodsEligible patients had ECOG ≤ 3, relapsed/refractory leukemias for which no standard therapy options were available, and acceptable hepato-renal function. A standard 3+3 dose-escalation design was used with 40% dose increments. TH-302 was administered IV over 30-60 min daily, either by 30 min-bolus administration or as a continuous infusion on days 1-5 of a 21-day cycle. The objectives were to determine the maximum tolerated dose (MTD) and pharmacokinetic (PK) profile of TH-302 with these schedules and to assess preliminary clinical activity of TH-302. ResultsA total of 49 patients with previously treated AML (n=39), ALL (n=9) or CML in blast phase (n=1) received TH-302 at bolus doses of 120 (n=4), 170 (n=4), 240 (n=3), 330 (n=3), 460 (n=20), 550 (n=4) mg/m2 or continuous doses of 330 (n=8) or 460 (n=3) mg/m2. Two of 3 evaluable patients treated with bolus TH-302 (550 mg/m2) experienced DLTs of grade 3 esophagitis; bolus administration MTD was established at 460 mg/m2. Two of 3 patients treated with continuous infusion of TH-302 (460 mg/m2) experienced DLTs of grade 3 mucositis or grade 3 hyperbilirubinemia; continuous administration MTD was established at 330 mg/m2. Thirteen patients received greater than 1 cycle. Generally, a significant rapid cytoreduction was evident early in the cycle, but was not maintained prior to initiation of the next cycle. Two AML patients who received 550 mg/m2 bolus TH-302 had complete resolution of leukemia cutis. One AML patient at 550 mg/m2 bolus TH-302 had a complete response with incomplete platelet recovery (CRp), and one AML patient at 440 mg/m2 bolus TH-302 had a CR. ConclusionsIn patients with advanced leukemias, MTDs were established for daily bolus infusion and 5-day continuous infusion of TH-302 at 460 mg/m2 and 330 mg/m2, respectively. Increased incidence of skin and mucosal toxicity were observed at higher dose levels with both administration schedules. Clinical activity of single-agent TH-302 has been noted with a few objective responses, but the majority of cytoreductions were transient. Disclosures:Konopleva:Threshold Pharmaceuticals: Research Funding. Handisides:Threshold Pharmaceuticals: Employment, Equity Ownership. Kroll:Threshold Pharmaceuticals: Employment, Equity Ownership. Kantarjian:Ariad: Research Funding; Novartis: Research Funding; Pfizer: Research Funding; BMS: Research Funding.

Crizotinib Inhibits Metabolic Inactivation of Gemcitabine in c-Met–driven Pancreatic Carcinoma

Pancreatic ductal adenocarcinoma (PDAC) remains a major unsolved health problem. Most drugs that pass preclinical tests fail in these patients, emphasizing the need of improved preclinical models to test novel anticancer strategies. Here, we developed four orthotopic mouse models using primary human PDAC cells genetically engineered to express firefly- and Gaussia luciferase, simplifying the ability to monitor tumor growth and metastasis longitudinally in individual animals with MRI and high-frequency ultrasound. In these models, we conducted detailed histopathologic and immunohistochemical analyses on paraffin-embedded pancreatic tissues and metastatic lesions in liver, lungs, and lymph nodes. Genetic characteristics were compared with the originator tumor and primary tumor cells using array-based comparative genomic hybridization, using frozen specimens obtained by laser microdissection. Notably, the orthotopic human xenografts in these models recapitulated the phenotype of human PDACs, including hypovascular and hypoxic areas. Pursuing genomic and immunohistochemical evidence revealed an increased copy number and overexpression of c-Met in one of the models; we examined the preclinical efficacy of c-Met inhibitors in vitro and in vivo. In particular, we found that crizotinib decreased tumor dimension, prolonged survival, and increased blood and tissue concentrations of gemcitabine, synergizing with a cytidine deaminase-mediated mechanism of action. Together, these more readily imaged orthotopic PDAC models displayed genetic, histopathologic, and metastatic features similar to their human tumors of origin. Moreover, their use pointed to c-Met as a candidate therapeutic target in PDAC and highlighted crizotinib and gemcitabine as a synergistic combination of drugs warranting clinical evaluation for PDAC treatment.