Hypoxic Areas Research Articles

ACRIN 6684 assessment of tumor hypoxia in glioblastoma using 18F-fluoromisonidazole with PET and MRI.

TPS10635 Background: Glioblastoma (GBM) is an aggressive type of primary malignant brain tumor and despite treatment with surgery, radiation, and temozolomide (TMZ) chemotherapy, median overall survival (OS) remains poor. A pathologic hallmark of GBM is tumor necrosis, a suspected result from endogenous tumor hypoxia. Angiogenesis is stimulated by hypoxia-driven signaling cascades and is required for tumor proliferation. The inefficient blood supply of these hypoxic tumors also limits the efficacy of chemotherapy and radiotherapy. Surviving hypoxic tumor cells may be selected out and proliferate as a more aggressive tumor subtype, therefore hindering OS. 18F-Fluoromisonidazole (FMISO) is a PET radiotracer whose uptake in hypoxic tissues can be measured radiographically. The degree of tumor hypoxia has been negatively associated with time to tumor progression and survival (Spence et al, 2008). Knowledge of the degree/distribution of tumor hypoxia by PET uptake and perfusion MRI parameters may provide prognostic information and help guide therapy for patients with GBM. Methods: In this phase II prospective single arm multi-institution study, patients will undergo baseline FMISO PET and MR imaging two weeks prior to chemoradiotherapy (CRT). A subset of patients will receive a second FMISO PET one week prior to CRT to assess reproducibility. Clinical outcomes of OS and 6-month progression-free survival (PFS-6) will be correlated to PET and MRI parameters. Eligibility: Pathologically confirmed GBM with residual tumor after surgery (including T2/FLAIR hyperintensity consistent with tumor) scheduled to receive standard fractionated radiation therapy and temozolomide alone or with an anti-VEGF agent or PARP inhibitor. Current enrollment: 22 patients of 50 sample size Contact information: Please contact the PI, Elizabeth R. Gerstner, MD ( egerstner@partners.org ) for additional information. Significance: With a better understanding of the extent of tumor hypoxia and changes in hypoxia levels from treatment, more effective therapies could be developed to inhibit GBM growth, target hypoxic areas and individualize patient care.

The role of hypoxia-inducible factor 1 in atherosclerosis

Atherosclerosis is by far the most frequent underlying cause of coronary artery disease, carotid artery disease and peripheral arterial disease, and is associated with high morbidity and mortality. Hypoxic areas...

Physiological biomarkers of hypoxic stress in red swamp crayfish Procambarus clarkii from field and laboratory experiments

The crayfish industry in Louisiana is the largest in the United States, with crayfish frequently harvested from waters that experience episodic or chronic hypoxia (dissolved oxygen [DO]≤2mg/l). We examined physiological biomarkers (hemolymph lactate, glucose, and protein concentrations) of hypoxic stress in the red swamp crayfish Procambarus clarkii from chronically hypoxic natural habitats and laboratory hypoxia experiments. P. clarkii from normoxic and hypoxic areas in the Atchafalaya River Basin were sampled monthly from April to July 2010. Laboratory experiments subjected P. clarkii to severe hypoxia (1mg/l DO), moderate hypoxia (2mg/l DO), or normoxic conditions (control: DO>7.5mg/l) for 12, 24, and 48h. P. clarkii from normoxic and hypoxic natural habitats did not display significantly different hemolymph lactate or glucose concentrations; however, mean hemolymph protein concentration was significantly lower in crayfish from hypoxic areas. P. clarkii exposed to severe hypoxia in laboratory experiments had significantly higher hemolymph lactate and glucose concentrations for all three exposure times, whereas large differences in protein concentrations were not observed. These results suggest that elevated hemolymph lactate and glucose concentrations are responses to acute hypoxia in P. clarkii, while differences in protein concentrations are the result of chronic hypoxic exposure.

Abstract 2889: Development of a novel nanoconstruct for tumor hypoxia photothermal therapy

Abstract PURPOSE: Hypoxia is a common feature of solid tumors that occurs across a wide variety of malignancies and significantly reduces tumor sensitivity to radiation and chemotherapy. Carbonic anhydrase IX (CAIX), an extracellular protein that is overexpressed in hypoxic zones within many human tumors, has emerged as a promising biomarker. To pursue focal targeted therapy of tumor hypoxia, we prepared and characterized a novel nanoconstruct composed of gold nanorods (GNR), which show superior capability to assist tumor photothermal ablation, and anti-CAIX antibody (CAIX-Ab), which binds specifically to CAIX. EXPERIMENTS: GNR with aspect ratio (length 24 nm to width 8nm) of 3 were conjugated to CAIX-Ab through bifunctional polyethylene glycol (PEG). The conjugates were characterized by monitoring the absorption peak shift as well as size and zeta potential changes for each step of the preparation. The stabilities of the GNR, GNR-PEG and GNR/CAIX-Ab were challenged by 1X PBS. The binding affinity and specificity of the GNR/CAIX-Ab were evaluated with ELISA using CAIX protein and cell adhesion assay using HT29 human colorectal cells (overexpresing CAIX) and NIH-3T3 cells (no expressing CAIX, control). The photothermal cell ablation was performed on HT29 cells treated with medium (control), GNR-PEG (control) and GNR/CAIX-Ab. RESULTS: Obvious shifts of the absorption peaks of GNR at the NIR region and particle size and surface charge changes suggested the success of the conjugation. After challenging by 1XPBS, bare GNR formed aggregations and lost the peak at 745 nm while GNR-PEG and GNR/CAIX-Ab remained stable as their absorption peaks around 750 nm were preserved. This is an important finding suggesting that our conjugated GNR will be stable for in vivo work. From the sandwich ELISA study, we obtained a standard linear curve of absorbance vs. CAIX-Ab concentration with R2 of 0.997 and linear curve of absorbance vs. GNR/CAIX-Ab concentration with R2 of 0.999. And we found that the negative control GNR-PEG had no binding to the CAIX protein. We calculated the number of CAIX-Ab per GNR to be 1.47 ± 0.2. In cell adhesion assay, neither GNR-PEG nor GNR/CAIX-Ab had detectable binding to 3T3 cells; but with HT29 cells, GNR-PEG still showed no binding while GNR/CAIX-Ab bound specifically and avidly to the cells. Photothermal ablation of HT29 cells revealed complete cell death after treatment of GNR/CAIX-Ab while the cells remained alive when treated with medium or GNR-PEG. CONCLUSION: These findings suggested that GNR/CAIX-Ab is a stable and effective nanoconstruct appropriate for focal targeting and photothermal ablation of tumor hypoxia. This new photothermal therapy has the great potential as an alternative or complementary approach for standard cancer treatment to eliminate residual or recurrent tumor cells, especially within the hypoxic areas that are resistant to traditional therapies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2889. doi:1538-7445.AM2012-2889

Abstract 1894: Antitumor effects of everolimus, sorafenib, and axitinib in human renal cells carcinoma xenografts resistant to first line treatment by sunitinib

Abstract Background. Acquired resistance to sunitinib stands as a major clinical challenge in renal cell carcinoma. This study aimed to evaluate the antitumor effects of everolimus, sorafenib, and axitinib as second line treatment in renal cancer xenografts acquiring resistance to sunitinib. Materials and Methods: CAKI1 renal carcinoma cells (VHL+) were engrafted in nude mice and treated continuously with tumor-static doses of oral sunitinib (60 mg/kg) until progression (define as 100% increase tumor volume on 3 subsequent measurements). Mice that progressed were randomized to receive second line treatment with oral everolimus (2.5, 5, 10 and 20 mg/kg), sorafenib (60mg/kg), or axitinib (60mg/kg) for 3 weeks. Tumor responses and progression-free survival were calculated using Kaplan-Meier analysis. Tumors were analyzed by immunohistochemistry (IHC), Western blot, and q-RT-PCR. Results. First line sunitinib induced significant tumor growth delay compared with placebo (medians PFS: 50 and 17 days, respectively, p<0.0001) without toxicity. Tumors that progressed under sunitinib treatment showed higher mRNA levels of HIF-1α, VEGFA, CXCR4, and CA-IX as compared to tumors displaying response to sunitinib. An increase in CXCR4, vimentin, and HER3 protein expression was associated with sunitinib-resistance. No change in mouse CD31 mRNA was observed contrasting with an increased human CD31 mRNA expression in tumors developing resistance to sunitinib compared to sensitive tumors, suggesting that human cancer stem cells may dedifferentiate to gain angiogenic capacities at progression. Second line treatment with everolimus, sorafenib, and axitinib were well tolerated as defined by mouse weight, mortality, and behavior. Median PFS for 20 mg/kg everolimus was significantly higher than that of control group (13 and 5 days, respectively, p<0.001). Growth inhibition using 5 mg/kg/day everolimus was similar to that of axitinib. Doses >10mg/kg everolimus display higher effects on tumor growth than sorafenib and axitinib. Phospho-S6 expression was reduced in tumors treated with everolimus and axitinib but not with sorafenib, indicating that everolimus and axitinib inactivate the PI3k/AKT/mTOR signaling. IHC analysis of everolimus- and sorafenib-treated tumors showed no apoptosis, large hypoxic (by CA-IX staining) and necrotic areas associated, and a decreased number of tumor vessels (CD31 staining) compared to controls. Conversely, axitinib neither induce hypoxia nor necrosis in this model. Conclusions. This study shows that, using an appropriate tumor progression methodology, human tumor models can be used to evaluate first and second line treatment with targeted drugs. This setting also shows that xenograft models may allow testing molecular markers of response and resistance prior to clinical trials. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1894. doi:1538-7445.AM2012-1894

Abstract 5227: Multimodality approaches to treat hypoxic NSCLC tumor microenvironment

Abstract In a previous study we found both in vitro and in an orthotropic NSCLC model in SCID mice that survival of NSCLC cells in hypoxic microenvironment is dependent upon Notch-1 signaling. Active Notch-1 was expressed in hypoxic tumor microenvironment only. Notch-1 provides survival stimuli through exacerbated Akt-1 activation due to suppression of PTEN transcription and positive regulation of the transcription of IGF-1 and its receptor. Hypoxic tumor environment is a problem for NSCLC treatment because it is responsible for cancer resistance to chemotherapy, tumor recurrence and metastasization. Also, hypoxic tumor microenvironment is quiescent and represses mTORC-1 activity. We show a number of experiments aimed at targeting hypoxic tumor tissue in the orthotropic NSCLC model. We attempted to target the Notch-1/IGF-1R/Akt-1 axis using three agents (a γ-secretase inhibitor, MRK-003; a fully humanized antibody against the human IGF-1R that promotes the receptor degradation, MK-0646; a pan-Akt inhibitor, MK-2206, all drugs provided by Merck Inc.) alone or in various combinations. We also used in combination studies drugs that are used for the treatment of advanced NSCLC, namely cisplatin and Erlotinib. The orthotropic model was established using two cell lines non-dependent on Notch-3 signaling. siRNA to Notch-3 in A549 and H1437 affects neither the growth rate nor the survival of these cells. All treatments (besides MK-2206) significantly prolonged the median survival of mice compared to controls (16 mice/experimental arm). MRK-003 treatment yielded specific death of hypoxic tumor areas. The tumors excised from the mice reaching an end-point displayed a significant reduction of markers of hypoxia as assessed by RT-qPCR using human specific primers. Moreover, qPCR measurements of the ratio between human/mouse GAPDH in DNA extracted from the whole brains and livers showed a significant reduction in metastasization in MRK-003 treated mice. MK-0646 was not specific to hypoxic tumor environment, but substantially increased the median survival of treated mice compared to controls. NSCLC cells evaded MK-0646 treatment by activating EGF-R and ErbB3 exclusively both in vivo and in five cell lines in vitro. This phenomenon is achieved at the level of protein stability. One outcome of MK-0646 treatment is that cells poorly or not responsive to Erlotinib become more responsive to it. Sequential treatment with MK-0646 (three weeks), then combination of MK-0646 plus Erlotinib prolonged median survival of mice dramatically. If the two drugs where administered in combination from day one no survival advantage was observed in median survival compared to mice treated with Erlotinib alone and was actually less effective than MK-0646 used as single agent. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5227. doi:1538-7445.AM2012-5227

Abstract 4736: N-myc downstream regulated gene 1: A novel mediator of hypoxia-driven chemoresistance in glioblastoma

Abstract Like other solid tumors, glioblastomas (GB) feature vast hypoxic areas that are thought to promote tumor growth, angiogenesis and even drug resistance and hence facilitate an aggressive growth and recurrence after treatment. Elucidation of hypoxia-signalling and identification of the major regulators of hypoxia-dependent chemoprotection seems logical for the optimization of current treatment regimens. Using comparative 2D gel electrophoresis and mass spectrometry analysis, we here identify N-myc downstream regulated gene 1 (NDRG1) as upregulated by chronic sublethal hypoxia in GB cell lines. Immunohistochemical analysis of human astrocytoma specimens shows a marked increase in expression from WHO °II to °IV and a preferential perinecrotic localization of NDRG1 in GB. In vitro, besides the hypoxia-driven induction of NDRG1, which is mediated by Hif1α and Hif2α, NDRG1 is also induced by irradiation. NDRG1 limits glioma cell invasiveness in vitro and reduces proliferation in vitro and in vivo. Furthermore NDRG1 acts antiangiogenic with reduced endothelial cell migration and sprouting, and interferes with the expression of multiple angiogenesis-related signalling molecules. Most interestingly NDRG1 protects from temozolomide (TMZ)-induced G2/M-arrest and subsequent reduction of tumor cell proliferation. Analyzing pairs of GB tissue samples of relapsed glioblastoma patients reveals a dramatic increase of NDRG1-positive cells suggesting a treatment-related selection for NDRG1 expressing populations and a potential role for NDRG1 in the recurrence of GB. In line with these findings, quantifying the NDRG1 status in tissue specimens of the UKT05 GB trial, which looked at a dose-intensified TMZ therapy plus standard radiotherapy, reveals a negative correlation between NDRG1 status and overall survival. In the present work we identify hypoxia-driven NDRG1 as a modulator of tumor growth, invasion, angiogenesis and as a novel mediator of chemoresistance towards TMZ in GB. Our findings shed light on the role of hypoxia in adaption to chemotherapeutic treatment and introduce NDRG1 as a promising candidate for targeted therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4736. doi:1538-7445.AM2012-4736

Abstract 3950: Differential functions of neuropeptide Y in Ewing's sarcoma - hypoxia as a switch

Abstract Ewing's sarcoma (ES) is a group of aggressive pediatric malignancies triggered by a fusion protein, EWS-FLI1, acting as an aberrant transcription factor. A sympathetic neurotransmitter neuropeptide Y (NPY) and two of its receptors (Rs), Y1 and Y5, have been identified as EWS-FLI1 target genes up-regulated in ES. Paradoxically, we have shown that this EWS-FLI1-driven Y1R/Y5R/NPY autocrine loop stimulates ES cell death. On the other hand, however, microarray data associated high expression of Y2Rs, which are not detectable in ES cells in vitro, with a metastatic phenotype of the disease. These seemingly contradictory observations raised the question as to the localization and functions of Y2Rs in ES tumors. Previously, we have shown that aside from being an apoptotic factor for ES cells, NPY also stimulates ES tumor vascularization via Y2Rs present on endothelial cells (ECs). This effect is further enhanced by dipeptidyl peptidase IV (DPPIV), an enzyme that converts NPY to the Y2/Y5R-selective agonist, NPY3-36. Here, we have shown that this Y2R/NPY3-36/DPPIV growth promoting system is up-regulated by hypoxia and may contribute to ES progression. In Y2R-negative ES cells, exposure to 0.1% oxygen induced expression of Y2Rs and significantly up-regulated Y5Rs, while levels of Y1Rs remained unchanged. This shift in R pattern was accompanied by an increase in the expression of DPPIV and NPY itself, leading to the elevated release of the peptide, most likely as a Y2/Y5R-agonist, NPY3-36. These changes were observed at both mRNA and protein levels and confirmed by accumulation of Y2R- and DPPIV-positive ES cells in hypoxic areas of ES xenografts. Importantly, the induction of Y2R expression was particularly apparent in ES cancer stem cells (CSCs), which were identified based on the high activity of aldehyde dehydrogenase (ALDH). This shift in NPY R expression pattern was accompanied by changes in functions of NPY, which under hypoxic conditions stimulated ES CSC proliferation and migration in a Y2/Y5R-dependent manner. In addition to its effect on ES cells, NPY's angiogenic actions are augmented by hypoxia via up-regulation of Y2 and Y5 Rs in ECs, thereby sensitizing these cells to NPY. Consequently, the proliferative effect of ES conditioned media on hypoxic ECs was increased and this effect was NPY-dependent. Moreover, simultaneous increases in NPY release and DPPIV activity in hypoxic ES cells enhanced the angiogenic potential of conditioned media derived from these cells. The clinical relevance of our findings was confirmed by Y2R expression in ES and ECs cells in human tumors and by elevated NPY in sera of ES patients. In summary, hypoxia shifts the activity of NPY in ES from Y1/Y5-mediated tumor cell death to Y2/Y5R-driven stimulatory effects on ES CSCs and ECs. These hypoxia-driven activities of NPY may contribute to ES progression and explain the clinical association between high Y2R expression and metastatic phenotype of the disease. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3950. doi:1538-7445.AM2012-3950

NADPH Oxidase 2 Regulates Bone Marrow Microenvironment Following Hindlimb Ischemia: Role in Reparative Mobilization of Progenitor Cells

Bone marrow (BM) microenvironment, which is regulated by hypoxia and proteolytic enzymes, is crucial for stem/progenitor cell function and mobilization involved in postnatal neovascularization. We demonstrated that NADPH oxidase 2 (Nox2)-derived reactive oxygen species (ROS) are involved in postischemic mobilization of BM cells and revascularization. However, role of Nox2 in regulating BM microenvironment in response to ischemic injury remains unknown. Here, we show that hindlimb ischemia of mice increases ROS production in both the endosteal and central region of BM tissue in situ, which is almost completely abolished in Nox2 knockout (KO) mice. This Nox2-dependent ROS production is mainly derived from Gr-1(+) myeloid cells in BM. In vivo injection of hypoxyprobe reveals that endosteum at the BM is hypoxic with high expression of hypoxia-inducible factor-1α in basal state. Following hindlimb ischemia, hypoxic areas and HIF-1α expression are expanded throughout the BM, which is inhibited in Nox2 KO mice. This ischemia-induced alteration of Nox2-dependent BM microenvironment is associated with an increase in vascular endothelial growth factor expression and Akt phosphorylation in BM tissue, thereby promoting Lin(-) progenitor cell survival and expansion, leading to their mobilization from BM. Furthermore, hindlimb ischemia increases proteolytic enzymes membrane type 1-matrix metalloproteinase (MMP) expression and MMP-9 activity in BM, which is inhibited in Nox2 KO mice. In summary, Nox2-dependent increase in ROS plays a critical role in regulating hypoxia expansion and proteolytic activities in BM microenvironment in response to tissue ischemia. This in turn promotes progenitor cell expansion and reparative mobilization from BM, leading to postischemic neovascularization and tissue repair.

Genotoxic therapy stimulates error-prone DNA repair in dormant hepatocellular cancer stem cells

Previous studies have described distinct dormant and proliferating populations of cancer stem cells in hepatocellular carcinoma. The CD13 protein is involved in the scavenging of reactive oxygen species through the glutathione reductase pathway and is associated with resistance to chemotherapy. Whereas CD13(-) proliferating cancer stem cells are sensitive to chemotherapy, CD13(+) dormant cancer stem cells are associated with the development of resistance to chemotherapy. CD13(+) cells in hypoxic areas of the tumour survive chemotherapy, leading to subsequent disease relapse and metastasis. Whether CD13(+) dormant cells simply resume proliferation following therapy or whether they also acquire greater malignant potential, remains unknown. The mechanisms involved also remain unclear. In the present study, we investigated the repair of DNA damage in CD13(+) dormant and CD13(-) proliferating cells. Total RNA was extracted from tissues, and quantitative real-time polymerase chain reaction (PCR) was performed for specific genes and GAPDH following PCR. Products were then subjected to a temperature gradient of 55-95°C with continuous fluorescence monitoring to generate a melting curve. Cells were incubated with primary antibodies, washed twice, incubated with fluorescent-labelled secondary antibodies for 30 min on ice and analyzed by flow cytometry. The results revealed that the repair of DNA damage in CD13(+) dormant cells occurs predominantly through non-homologous end-joining, a repair process that is error-prone, whereas CD13(-) proliferating cells primarily utilise high-fidelity homologous recombination for DNA repair. These data indicate that not only is dormancy a protective mechanism for cancer stem cells to survive therapy, but it also enhances the generation and accumulation of mutations following DNA damage. Therefore, the CD13(+) dormant cancer stem cells must be eradicated fully to achieve complete remission of cancer.

Oncolytic Herpes Simplex Virus Counteracts the Hypoxia-Induced Modulation of Glioblastoma Stem-Like Cells

Glioblastoma (GBM), a fatal malignant brain tumor, contains abundant hypoxic regions that provide a "niche" to promote both the maintenance and enrichment of glioblastoma stem-like cells (GSCs) and confer resistance to chemo- and radiotherapy. Since GSCs, with an ability to resist conventional therapies, may be responsible for tumor recurrence, targeting GSCs located in such a hypoxic environment may be critical to improving the therapeutic outcome for GBM patients. Oncolytic viral therapies have been tested in the clinic as a promising therapeutic approach for GBM. In this study, we analyzed and compared the therapeutic effects of oncolytic herpes simplex virus (oHSV) type 1 G47Δ (γ34.5(-)ICP6(-)LacZ(+)α47(-)) in patient-derived GSCs under normoxia (21% oxygen) and hypoxia (1% oxygen). GSCs cultured in hypoxia showed an increased ability to form neurospheres and expressed higher levels of the putative stem cell marker CD133 compared with GSCs cultured in normoxia. G47Δ exhibited a comparable ability to infect, replicate, and kill GSCs in normoxia and hypoxia in vitro. Importantly, G47Δ could counteract hypoxia-mediated enhancement of the stem-like properties of GSCs, inhibiting their self-renewal and stem cell marker expression. Using orthotopic human GSC xenografts in mice, we demonstrated that intratumoral injection of G47ΔUs11fluc, a newly developed G47Δ derivative that expresses firefly luciferase driven by a true late viral promoter, led to an equivalent frequency of viral infection and replication in hypoxic and nonhypoxic tumor areas. These findings suggest that oHSV G47Δ represents a promising therapeutic strategy to target and kill GSCs, not only in normoxic areas of GBM but also within the hypoxic niche.

Overexpression of nuclear NHERF1 in advanced colorectal cancer: Association with hypoxic microenvironment and tumor invasive phenotype

Over 57% of colorectal cancer (CRC) patients have regional or distant spread of their disease at the time of diagnosis. Despite recent advances, there is a compelling need to better characterize prognostic markers for advanced CRC. The present study investigates protein expression of NHERF1, HIF-1α and TWIST1 and their relationship in distant normal mucosa (DNM), tumor (T) and adjacent normal mucosa (ANM), lymph node metastasis (LNM) and liver metastasis (LM), determining their role as potential markers in advanced stages of human CRC. Overexpression of nuclear NHERF1 was shown in 47% of tumors, which exhibited a significant association with poor histological grade (P=0.0346). Nuclear NHERF1 showed a higher expression in T, LNM and LM than both DNM (P<0.0001) and ANM (P<0.05). Nuclear HIF-1α was significantly higher in T, LNM and LM than DNM and ANM (P<0.05, P<0.001, P<0.0001, respectively). A positive correlation between nuclear NHERF1 and nuclear HIF-1α was found in LNM (r=0.331, P=0.020), where an extended co-localization of the two proteins was demonstrated. TWIST1 was more expressed in T than DNM and ANM (P<0.0001) and was higher in T than LNM and LM (P<0.0001). Moreover, nuclear NHERF1 was directly correlated to TWIST1 (r=0.339, P=0.015) in T samples, where a high co-expression of the two proteins was demonstrated both in no longer polarized epithelial cells and in invasive mesenchymal elements adjacent to hypoxic and perinecrotic colonic areas. Overall, nuclear NHERF1 expression was associated with poorer differentiation grade and with higher expression both of HIF-1α in lymphatic metastasis and TWIST1 in invasive front of tumor. Our results support the oncogenic role of NHERF1 and promote nuclear NHERF1 as a potential new biomarker of advanced CRC.

Imaging of Hypoxia Using PET and MRI

Tumor hypoxia is the result of an inadequate supply of oxygen to tumor cells which can be caused by multiple factors. It is associated with aggressive local tumor growth and invasion, increased risk of metastasis, higher resistance to radiotherapy (RT) and chemotherapy, overall resulting in a poor clinical prognosis. Many locally advanced solid tumors may exhibit hypoxic and/or anoxic tissue areas that are heterogeneously distributed within the tumor mass. As hypoxia is a negative prognostic factor concerning response to radiotherapy and chemotherapy, in vivo measurement of tumor hypoxia could be helpful to identify patients with worse prognosis or patients that could benefit from appropriate treatments such as intensity modulated radiotherapy (IMRT) that may accurately conform the dose distribution to small intratumoral regions showing differences in the oxygen level. A manifold of different methods to assess the oxygen tension (pO2) in tissues have been developed, each of them offering advantages as well as drawbacks. They range from invasive direct measurement techniques of the pO2 in tissue by using a polarographic electrode, to non-invasive imaging techniques such as positron emission tomography (PET) or magnetic resonance imaging (MRI). This article provides an overview over the various methods, with a particular emphasis on PET and MRI for imaging of hypoxia, and reviews their performance in preclinical and clinical studies.

Immunohistochemical Study of CD68 and CR3/43 in Astrocytic Gliomas

Diffuse and high-grade astrocytomas are invasive neoplasms which grow diffusely into the brain parenchyma. Microglia has been termed the brain's immune system, although its specific role remains uncertain. Objective of this study was to assess in a series of astrocytic neoplasms, the expression of a macrophage marker CD 68 and Major Histocompatibility Complex Class II CR3/43. We examined 10 pilocytic astrocytomas, 13 diffuse astrocytomas and 17 anaplastic astrocytomas. For macrophages we used the CD68 monoclonal mouse antibody. For assessing the presence of MHC Class II complexes we used the specific monoclonal antibody CR3/43. CD68-positive mononuclear cells were observed in perivascular and hypoxic areas, within neoplastic tissue, inside and contiguous to vessel wall. CR3/43 positive complexes were detected in mononuclear elongated elements with amoeboid extensions strictly attached to endothelial cells, or contiguous to perinecrotic areas within neoplastic tissue. We suggest an active involvement of macrophage/microglia infiltrates in neovascularization and malignancy in astrocytomas. Macrophage infiltration and major histocompatibility complex class II complexes reactivity in gliomas could also suggest the occurrence of immune surveillance with a preliminary host's immune response. In addition, macrophages could promote angiogenesis mechanisms and induction of tumor growth.

The “go or grow” potential of gliomas is linked to the neuropeptide processing enzyme carboxypeptidase E and mediated by metabolic stress

Glioblastoma (GBM), the most common malignant brain tumor, is among the most lethal neoplasms, with a median survival of approximately 1 year. Prognosis is poor since GBMs possess a strong migratory and highly invasive potential, making complete surgical resection impossible. Reduced expression of carboxypeptidase E (CPE), a neuropeptide-processing enzyme, in a cell death-resistant glioma cell line and lower CPE expression levels in the cohort of GBM samples of The Cancer Genome Atlas compared to normal brain control specimens prompted us to analyze the function of CPE as a putative tumor suppressor gene. In our samples, CPE was also reduced in GBM compared to normal brain with the strongest loss in cells surrounding hypoxic tumor areas as well as in most glioma cell lines and primary glioma cells. In our cohort of glioma patients, loss of CPE predominantly occurred in glioblastomas and was associated with worse prognosis. In glioma cells, CPE overexpression was significantly reduced, whereas knockdown or inhibition enhanced glioma cell migration and invasion. The decreased migratory potential following CPE overexpression was paralleled by altered cellular morphology, promoting a transition to focal adhesions and associated stress fibers. In contrast to the decreased migration, high CPE levels were associated with higher proliferative rates. As microenvironmental regulation cues, we identified CPE as being downregulated upon hypoxia or glucose deprivation. Our findings indicate an oxygen- and nutrition-dependent anti-migratory, but pro-proliferative role of CPE in gliomas with prognostic impact for patient survival, thereby contributing to the understanding of the "go or grow" hypothesis in gliomas.

Drug Resistance in Glioblastoma: A Mini Review

Glioblastoma multiforme (GBM) is recognized as the most common and lethal form of central nervous system cancer. Currently used surgical techniques, chemotherapeutic agents, and radiotherapy strategies have done very little in extending the life expectancies of patients diagnosed with GBM. The difficulty in treating this malignant disease lies both in its inherent complexity and numerous mechanisms of drug resistance. In this review, we summarize several of the primary mechanisms of drug resistance. We reviewed available published literature in the English language regarding drug resistance in glioblastoma. The reasons for drug resistance in glioblastoma include drug efflux, hypoxic areas of tumor cells, cancer stem cells, DNA damage repair, and miRNAs. Many potential therapies target these mechanisms, including a series of investigated alternative and plant-derived agents. Future research and clinical trials in glioblastoma patients should pursue combination of therapies to help combat drug resistance. The emerging new data on the potential of plant-derived therapeutics should also be closely considered and further investigated.

Tumor-secreted SDF-1 promotes glioma invasiveness and TAM tropism toward hypoxia in a murine astrocytoma model

A distinguishing feature of high-grade gliomas is the infiltration of neoplastic cells into adjacent brain tissues that mark most of these tumors surgically incurable. To study the factors associated with tumor invasion, we established a new murine brain tumor model, ALTS1C1 derived from SV40 large T antigen-transfected astrocytes. This new brain tumor model recapitulates several histopathological features of human high-grade glioma including increased cellularity, prominent cellular pleomorphism, geographic necrosis, active mitosis, and extensive invasion of tumor cells into adjacent brain tissues. More importantly, ALTS1C1 expressed a relatively high level of stromal-derived factor-1 (SDF-1/CXCL12) in vitro and in vivo and higher microvascular density (MVD) in vivo. To define the roles of SDF-1 in this tumor model, the expression of SDF-1 in ALTS1C1 cells was inhibited by specific siRNA. SDF-knockdown ALTS1C1 (SDFkd) cells took longer than parental ALTS1C1 cells to form tumors and in contrast to the wild-type tumors they had well-defined regular borders and lacked infiltration tracts. The SDFkd tumors were also associated with a lower MVD and more hypoxic areas. In contrast to parental tumors, the density of F4/80-positive tumor-associated macrophages (TAMs) in SDFkd tumor was higher in non-hypoxic than in hypoxic regions. SDF-1 production by tumor cells therefore seems critical for the aggregation of TAMs into areas of hypoxia and tumor invasiveness. This study not only provides new insight into the role of SDF-1 in brain tumor invasion and the relationship between TAMs and hypoxia, but also provides a new preclinical brain tumor model for designing new treatment options for invasive cases.

Bone marrow-derived monocyte lineage cells recruited by MIP-1β promote physiological revascularization in mouse model of oxygen-induced retinopathy

Recent clinical observations have indicated that vascular endothelial growth factor (VEGF) is a key factor that stimulates the development of preretinal pathological neovascularization (NV). However, it has not been established how intraretinal physiological revascularization of hypoxic avascular areas is regulated. Our earlier study on the gene expression profile of hypoxic retinas in a mouse model of oxygen-induced retinopathy (OIR) showed that macrophage inflammatory protein-1β (MIP-1β) was the most upregulated protein. The purpose of this study was to investigate the role played by MIP-1β in recruiting bone marrow-derived monocyte lineage cells (BM-MLCs) in a mouse model of OIR. Our results showed that MIP-1β was upregulated, and its receptor, CCR5, was expressed in BM-MLCs in the hypoxic inner retina. Neutralizing Ab against MIP-1β reduced the infiltration of BM-MLCs into the OIR retinas and increased the avascular area and preretinal neovascular tufts. A very strong significant correlation was found between the area of the preretinal neovascular tufts and the avascular area, regardless of the extent of BM-MLC infiltration into the OIR retinas. Additional treatment with VEGF-A-neutralizing Ab showed that the MIP-1β-regulated pathological NV strongly depended on VEGF-A, which was probably secreted by the hypoxic avascular retinas. These results indicate that MIP-1β is involved in the recruitment of BM-MLCs, which have a significant role in the physiological revascularization of hypoxic avascular retinas. Overall, these findings indicate that the MIP-1β induction of BM-MLCs might possibly be used to promote intraretinal revascularization and thus prevent the abnormal NV in ischemic vision-threatening retinal diseases.

Clostridial Spores for Cancer Therapy: Targeting Solid Tumour Microenvironment

Solid tumour accounts for 90% of all cancers. The current treatment approach for most solid tumours is surgery, however it is limited to early stage tumours. Other treatment options such as chemotherapy and radiotherapy are non-selective, thus causing damage to both healthy and cancerous tissue. Past research has focused on understanding tumour cells themselves, and conventional wisdom has aimed at targeting these cells directly. Recent research has shifted towards understanding the tumour microenvironment and it's differences from that of healthy cells/tissues in the body and then to exploit these differences for treatmeat of the tumour. One such approach is utilizing anaerobic bacteria. Several strains of bacteria have been shown to selectively colonize in solid tumours, making them valuable tools for selective tumour targeting and destruction. Amongst them, the anaerobic Clostridium has shown great potential in penetration and colonization of the hypoxic and necrotic areas of the tumour microenvironment, causing significant oncolysis as well as enabling the delivery of therapeutics directly to the tumour in situ. Various strategies utilizing Clostridium are currently being investigated, and represent a novel area of emerging cancer therapy. This review provides an update review of tumour microenvironment as well as summary of the progresses and current status of Clostridial spore-based cancer therapies.

Frizzled receptor 6 marks rare, highly tumourigenic stem-like cells in mouse and human neuroblastomas

Wnt signalling is an important component of vertebrate development, required for specification of the neural crest. Ten Wnt receptors [Frizzled receptor 1-10 (Fzd1-10)] have been identified so far, some of which are expressed in the developing nervous system and the neural crest. Here we show that expression of one such receptors, Fzd6, predicts poor survival in neuroblastoma patients and marks rare, HIF1/2 α-positive cells in tumour hypoxic areas. Fzd6 positive neuroblastoma cells form neurospheres with high efficiency, are resistant to doxorubicin killing and express high levels of mesenchymal markers such as Twist1 and Notch1. Expression of Fzd6 is required for the expression of genes of the non-canonical Wnt pathway and the spheres forming activity. When transplanted into immunodeficient mice, neuroblastoma cells expressing the Fzd6 marker grow more aggressively than their Fzd6 negative counterparts. We conclude that Fzd6 is a new surface marker of aggressive neuroblastoma cells with stem cell-like features.