Pimonidazole Binding Research Articles

Correlation of hypoxia PET tracer uptake with hypoxic radioresistance in cancer cells: PET biomarkers of resistance to stereotactic radiation therapy?

PurposeThe pO2 threshold of an ideal PET hypoxia tracer for radiotherapy planning in cancer would match those observed in clinically and biologically relevant processes such as radioresistance and HIF1α expression. To identify such tracers, we directly compared uptake in vitro of hypoxia PET tracers ([18F]FMISO, [64Cu]CuATSM, and analogues [64Cu]CuATS, [64Cu]CuATSE, [64Cu]CuCTS, [64Cu]CuDTS, [64Cu]CuDTSE, [64Cu]CuDTSM) with levels of radioresistance and HIF1α expression in cultured cancer cells under identical hypoxic conditions ranging from extreme hypoxia to normoxia. Pimonidazole uptake was also compared as a marker of hypoxia. MethodsA custom-built hypoxia apparatus enabled all experiments to be performed under identical hypoxic conditions with constant measurement of pO2 in media using an OxyLab pO2™ probe. HCT116 human colonic carcinoma and MCF-7 human Caucasian breast adenocarcinoma cells were irradiated using a cobalt teletherapy unit. Clonogenic assays were used to assess survival. HIF1α expression was determined by western blotting, tracer uptake by gamma counting and pimonidazole binding by flow cytometry. ResultsRadioresistance, pimonidazole binding and HIF1α expression increased gradually as pO2 decreased between 25 mmHg and 0 mmHg. In contrast, all the PET hypoxia tracers showed a sharp increase in uptake only when pO2 levels fell below 1 mmHg. Above this threshold, tracer uptake was not elevated above that in normoxic cells. ConclusionThis study highlights an important mismatch in pO2 thresholds between these PET tracers and other markers of hypoxia: tracer uptake only occurred at oxygen levels that were well below levels that induced radioresistance, pimonidazole uptake and HIF1α expression. Although their pO2 thresholds do not match the threshold for resistance to conventionally fractionated radiotherapy (pO2 2.5–10 mmHg), their specificity for extreme hypoxia (pO2 ≪ 1 mmHg) suggests these PET tracers may be of particular use to predict outcomes in stereotactic radiation therapy where these maximally resistant cells play a key role in determining the biological effect.

P16.09 Regulation of chromatin accessibility in the hypoxic tumor microenvironment of glioblastoma

Abstract BACKGROUND Chromatin structure is often dysregulated in cancers, including glioblastoma (GBM), the most aggressive type of primary brain tumor. GBM has the poorest prognosis with no efficient cure to date due to diffusive growth into the brain, resistance to treatments and the immunosuppressive tumor microenvironment (TME). The growth and invasiveness of GBM is supported by the heterogeneous TME including local microglia and bone-marrow-derived macrophages (collectively known as glioma-associated microglia and macrophages, GAMs). In addition, tumor hypoxia is a key factor in the progression of GBM, as it can globally and rapidly alter gene expression, induce cancer cell invasiveness, stemness and lead to therapy resistance. Hypoxia can influence the pro-tumorigenic function of GAMs by inducing the expression of cytokines and cell surface receptors. However, little is known on the hypoxia-imposed chromatin changes of GAMs and GBM cells, which can in turn impact the interaction between these cell populations. Here we analyze these changes using a single-cell method, which preserves in situ hypoxia within the TME of GBM. MATERIAL AND METHODS Single-cell Pi-ATAC-seq (Protein-indexed Assay of Transposase Accessible Chromatin with sequencing) method in a GL261 murine glioma model was used to simultaneously assess genome-wide chromatin accessibility and expression of intracellular protein markers in single cells, enabling accurate selection of hypoxic and non-hypoxic tumor cells and GAMs. Pi-ATAC-seq is used on paraformaldehyde-perfused tumors and therefore allows capturing unaltered hypoxia-dependent cellular states, that often become distorted during dissociation and preparation of fresh material in most common single-cell methods. RESULTS We optimized Pi-ATAC method in a GL261 GBM mouse model, with specific sorting of GAMs using CD11b+ immunosorting followed by separation of microglia and macrophages, based on intensity of CD45 staining. HIF-1α induction and binding of pimonidazole were used to mark hypoxic populations. Currently, we are investigating the chromatin accessibility profiles of cancer cells and GAMs within the hypoxic tumor microenvironment of GBM. Exploring open chromatin profiles in GAMs and glioma-microglia co-cultures will allow to unravel the mechanisms of chromatin accessibility modulation in the oxygen-dependent manner. CONCLUSION In summary, we optimized the Pi-ATAC method in a mouse GBM model to characterize the chromatin openness changes in GAMs and cancer cells in response to hypoxic stress. Further validation of these results will provide the potential to identify novel markers for GAMs/glioma interactions in hypoxic GBMs and develop novel therapeutic targets.

The Effect of Carbogen Breathing on 18F-FDG Uptake in Non-Small-Cell Lung Cancer.

It has been reported that 18F-FDG uptake is higher in hypoxic cancer cells than in well-oxygenated cells. We demonstrated that 18F-FDG uptake in lung cancer would be affected by high concentration oxygen breathing. Methods. Overnight fasted non-small-cell lung cancer A549 subcutaneous (s.c.) xenografts bearing mice (n = 10) underwent 18F-FDG micro-PET scans, animals breathed room air on day 1, and same animals breathed carbogen (95% O2 + 5% CO2) on the subsequent day. In separated studies, autoradiography and immunohistochemical staining visualization of frozen section of A549 s.c. tumors were applied, and to compare between carbogen-breathing mice and those with air breathing, a combination of 18F-FDG and hypoxia marker pimonidazole was injected 1 h before animal sacrifice, and 18F-FDG accumulation was compared with pimonidazole binding and glucose transporter 1 (GLUT-1) expression. Results. PET studies revealed that tumor 18F-FDG uptake was significantly decreased in carbogen-breathing mice than those with air breathing (P < 0.05). Ex vivo studies confirmed that carbogen breathing significantly decreased hypoxic fraction detected by pimonidazole staining, referring to GLUT-1 expression, and significantly decreased 18F-FDG accumulation in tumors. Conclusions. High concentration of O2 breathing during 18F-FDG uptake phase significantly decreases 18F-FDG uptake in non-small-cell lung cancer A549 xenografts growing in mice.

Comparison of dynamic contrast-enhanced magnetic resonance imaging and contrast-enhanced ultrasound for evaluation of the effects of sorafenib in a rat model of hepatocellular carcinoma

ObjectivesTo compare the accuracy of contrast-enhanced ultrasound (CEUS) and Dynamic contrast-enhanced (DCE) magnetic resonance imaging (MRI) for the assessment of changes in tissue vascularization as result of sorafenib treatment in a rat model of hepatocellular carcinoma (HCC). MethodsMale Buffalo rats with orthotopic liver tumors treated daily with 7.5 mg/kg sorafenib via oral gavage for 2 weeks (n = 9) were subject to DCE-MRI and CEUS 2 weeks after tumor implantation - right before treatment initiation - and also after treatment completion - right before tumor harvest. Untreated animals (n = 10) were used as control. Tumor tissue sections were stained for hematoxylin-eosin, pimonidazole, and CD34 for quantitative assessment of necrosis, hypoxia, and microvessel density (MVD), respectively. ResultsOf all the DCE-MRI parameters that were evaluated, only volume transfer constant (Ktrans) measurements were significantly lower in sorafenib-treated tumors (0.18 vs 0.33 min−1, p < 0.01), indicating a substantial decrease in vascular permeability caused by the therapy. This reduction was associated with decreased MVD (3.9 vs 10.8% CD34+ cells, p < 0.01), higher tumor necrosis (31.9 vs 21.8%, p < 0.001) and hypoxia (19.7 vs 10.5% pimonidazole binding, p < 0.01). Moreover, statistical analysis demonstrate significant correlation of DCE-MRI Ktrans with histopathologic tissue necrosis (r = −0.537, p < 0.05) and MVD (r = 0.599, p < 0.05). Interestingly, none of the CEUS measurements were significantly different between the control and treatment groups, and did not show statistical correlation with any of the histopathological parameters assessed (p > 0.05). ConclusionsSorafenib-induced reduction in vascular permeability in this preclinical model of HCC is detected more accurately through DCE-MRI than CEUS, and DCE-MRI parameters strongly correlate with histopathological changes in tissue vascularization and tissue necrosis.

Galectin-1 Inhibitor OTX008 Induces Tumor Vessel Normalization and Tumor Growth Inhibition in Human Head and Neck Squamous Cell Carcinoma Models.

Galectin-1 is a hypoxia-regulated protein and a prognostic marker in head and neck squamous cell carcinomas (HNSCC). Here we assessed the ability of non-peptidic galectin-1 inhibitor OTX008 to improve tumor oxygenation levels via tumor vessel normalization as well as tumor growth inhibition in two human HNSCC tumor models, the human laryngeal squamous carcinoma SQ20B and the human epithelial type 2 HEp-2. Tumor-bearing mice were treated with OTX008, Anginex, or Avastin and oxygen levels were determined by fiber-optics and molecular marker pimonidazole binding. Immuno-fluorescence was used to determine vessel normalization status. Continued OTX008 treatment caused a transient reoxygenation in SQ20B tumors peaking on day 14, while a steady increase in tumor oxygenation was observed over 21 days in the HEp-2 model. A >50% decrease in immunohistochemical staining for tumor hypoxia verified the oxygenation data measured using a partial pressure of oxygen (pO2) probe. Additionally, OTX008 induced tumor vessel normalization as tumor pericyte coverage increased by approximately 40% without inducing any toxicity. Moreover, OTX008 inhibited tumor growth as effectively as Anginex and Avastin, except in the HEp-2 model where Avastin was found to suspend tumor growth. Galectin-1 inhibitor OTX008 transiently increased overall tumor oxygenation via vessel normalization to various degrees in both HNSCC models. These findings suggest that targeting galectin-1—e.g., by OTX008—may be an effective approach to treat cancer patients as stand-alone therapy or in combination with other standards of care.

Abstract 2873: Imaging tumor heterogeneity after multikinase inhibitor therapy in rat hepatocellular carcinoma

Abstract Purpose To assess tumor tissue perfusion and vascular permeability through multiparametric functional imaging, and to evaluate their correspondence with histopathologic parameters of necrosis and hypoxia in a rat model of hepatocellular carcinoma treated with a multikinase inhibitor. Materials and Methods Rat hepatoma McA-RH7777 cells were implanted in the left liver lobe of nineteen male Buffalo rats. Exactly 2 weeks after tumor inoculation, the animals were randomly assigned to remain untreated (n=10) or to receive a daily dose of 7.5 mg/kg sorafenib by oral gavage (n=9) for 2 additional weeks. T2-weighted spin-echo magnetic resonance imaging (MRI), dynamic contrast-enhanced (DCE) and contrast-enhanced ultrasound (CEUS) were performed weekly. All tumors were harvested 4 weeks post-implantation &amp;gt;90 minutes after injecting 60mg/kg pimonidazole. Tissue sections were stained for hematoxylin-eosin and pimonidazole for quantitative assessment of necrosis and hypoxia, respectively. Differences between treatment groups were assessed using the Mann-Whitney test, and the correlation of imaging and histopathology parameters was determined by Spearman correlation analysis. Results In spite of the relatively low dose and short treatment duration, the response to sorafenib therapy was characterized by a significantly higher median tumor necrosis (60 vs 15%, P &amp;lt; 0.0001) and increased tumor tissue hypoxia (35 vs 18.5% pimonidazole binding, P &amp;lt; 0.0001). Similarly, the sorafenib regimen caused a substantial decrease in vascular permeability, measured by DCE-MRI Ktrans (P = 0.002) as well as reduction in tumor perfusion evaluated by CEUS peak enhancement (PE) (P =0.043). Our findings show a strong correlation between the two histopathologic parameters we assessed, tumor tissue necrosis and hypoxia (r = 0.645, P=0.002). Furthermore, our results demonstrate significant correlation of functional imaging parameters of vascular permeability, namely DCE-MRI Ktrans and DCE-MRI normalized initial area under the curve (NIAUC), with histopathologic tissue hypoxia (r=-0.663, P= 0.002, and r= -0.512, P=0.029, respectively). In contrast, histopathologic tumor necrosis appears more strongly correlated with a functional imaging parameter of tumor perfusion, specifically, CEUS PE (r = -0.547, P = 0.028). Conclusion Tumors exhibited wide heterogeneity in vascular perfusion and hypoxia after treatment. Interestingly, functional MRI parameters appear to correlate more strongly with tissue oxygenation, whereas functional CEUS parameters correlate with tumor viability. Novel predictive imaging biomarkers of treatment response may be developed through further analyses of spatial tumor heterogeneity in our animal model. Citation Format: Nina M. Muñoz, Adeeb A. Minhaj, Kiersten L. Maldonado, Charles Kingsley, Hideyuki Nishiofuku, Keith A. Michel, Andrea C. Cortes, James A. Bankson, Asif Rashid, Rony Avritscher. Imaging tumor heterogeneity after multikinase inhibitor therapy in rat hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2873. doi:10.1158/1538-7445.AM2017-2873

Anemia tolerance during normo-, hypo-, and hypervolemia.

Restrictive intraoperative fluid management has been demonstrated to improve outcome of visceral and lung surgery in several studies. However, subsequent hypovolemia (HOV) may be accompanied by a decrease of anemia tolerance, resulting in increased transfusion needs. We therefore investigated the effect of volume status on anemia tolerance. Eighteen domestic pigs of either sex (mean weight, 23.5 ± 4.8 kg) were anesthetized, ventilated, and randomized into three experimental groups: normovolemia (no intervention), HOV (blood loss of 40% of blood volume), and hypervolemia (HEV; volume infusion of 40% of blood volume). The animals were then hemodiluted until their individual critical hemoglobin concentrations (Hbcrit ) were reached by the exchange of whole blood for hydroxyethyl starch (HES; 130:0.4). Subsequently, organ-specific hypoxia was assessed using pimonidazole tissue staining in relevant organs. Hemodynamic and metabolic variables were also investigated. Despite significant differences in exchangeable blood volume, Hbcrit was the same in all groups (2.3 g/dL, NS). During HOV, tissue hypoxia was aggravated in the myocardium, brain, and kidneys, whereas tissue oxygenation of the liver and intestine was not influenced by volume status. HEV increased tissue hypoxia in the lungs, but did not impact tissue oxygenation of other organs. The combination of hemorrhagic HOV with subsequent anemia leads to accentuated tissue hypoxia, revealed by a significant increase in pimonidazole binding at Hbcrit , in heart, lungs, brain, and kidney. The lungs were the only organ that showed increased tissue hypoxia after pretreatment of HES infusion and subsequent anemia by normovolemic hemodilution.

Abstract 3042: The novel Akt inhibitor AZD5363 following radiotherapy improves tumor control in mouse models of head and neck cancer

Abstract Radiation therapy (RT) is an important anti-cancer treatment. Understanding the molecular pathways involved in the response of tumors to RT has led to interest in developing drugs which target these; one such target is Akt. Previous studies support the hypothesis that Akt inhibition might enhance the radiosensitivity of tumors through modification of cellular and micro-environmental factors. AZD5363 is a novel, orally bio-available, ATP competitive inhibitor of Akt and here we have evaluated whether AZD5363 will increase the efficacy of RT in preclinical models of head and neck (H&amp;N) cancer. The ability of AZD5363 to inhibit the Akt pathway was investigated in vitro in 5 H&amp;N cell lines and in vivo (FaDu xenografts) using western blot and ELISA techniques. The effect of AZD5363 on the radiosensitivity in vitro was assessed using clonogenic assays. The FaDu xenograft model was used to test the effect of AZD5363 on tumor growth when used as a sequential treatment (for 7 days before RT) or adjuvant therapy (continuously after RT). RT was given as a 6Gy single dose. The effect of these treatments on the tumor microenvironment prior to and after irradiation was also assessed using FAZA PET/CT, immunohistochemistry and ELISA techniques. AZD5363 effectively inhibited the Akt pathway in in vitro and in vivo. There was no effect on radiosensitivity in vitro when drug was administered in a variety of sequences and durations around RT. In vivo, there was no improvement in tumor control when AZD5363 was given for 7 days before RT. But survival was improved (p = 0.006) when AZD5363 was administered continuously after RT with reduced tumor growth (p = 0.0035, 7 days after RT). There was no effect of drug alone on tumor growth and no difference in the level of tumor hypoxia (measured using FAZA PET/CT or pimonidazole binding) after 7 days AZD5363. Human VEGF and HIF1α levels were reduced (p = 0.0104 and p = 0.002, respectively) after 7 days AZD5363. There was no difference in microvessel density (MVD) prior to RT, but 7 days after, MVD was significantly reduced if adjuvant AZD5363 had been given (p = 0.0317). AZD5363 was also found to effect the rate of proliferation of human umbilical vein endothelial (HUVEC) cells in vitro. AZD5363 effectively improves long term control of FaDu tumors when combined with RT. However, this only occurs when the drug is used in the adjuvant setting. AZD5363 has no effect on the radiosensitivity of H&amp;N cells in vitro, which suggests that the in vivo effects are dependent on the tumor microenvironment. AZD5363 reduces tumor VEGF and HIF1α in an oxygen independent manner and inhibits the rate of proliferation of vascular endothelial cells, effects that would reduce vascular recovery after RT. This is the first time a specific inhibitor of Akt has been reported to have this sequence dependent ability to enhance the effects of RT and has implications for how AZD5363 and potentially other Akt inhibitors are used in combination with RT in the clinic. Citation Format: Emma J. Searle, Brian Telfer, Duncan Forster, Kaye J. Williams, Barry Davies, Timothy Illidge, Ian Stratford. The novel Akt inhibitor AZD5363 following radiotherapy improves tumor control in mouse models of head and neck cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3042.

Placental growth factor inhibition modulates the interplay between hypoxia and unfolded protein response in hepatocellular carcinoma.

BackgroundHepatocellular carcinoma (HCC) is a leading cause of cancer-related mortality. We previously showed that the inhibition of placental growth factor (PlGF) exerts antitumour effects and induces vessel normalisation, possibly reducing hypoxia. However, the exact mechanism underlying these effects remains unclear. Because hypoxia and endoplasmic reticulum stress, which activates the unfolded protein response (UPR), have been implicated in HCC progression, we assessed the interactions between PlGF and these microenvironmental stresses.MethodsPlGF knockout mice and validated monoclonal anti-PlGF antibodies were used in a diethylnitrosamine-induced mouse model for HCC. We examined the interactions among hypoxia, UPR activation and PlGF induction in HCC cells.ResultsBoth the genetic and pharmacological inhibitions of PlGF reduced the chaperone levels and the activation of the PKR-like endoplasmic reticulum kinase (PERK) pathway of the UPR in diethylnitrosamine-induced HCC. Furthermore, we identified that tumour hypoxia was attenuated, as shown by reduced pimonidazole binding. Interestingly, hypoxic exposure markedly activated the PERK pathway in HCC cells in vitro, suggesting that PlGF inhibition may diminish PERK activation by improving oxygen delivery. We also found that PlGF expression is upregulated by different chemical UPR inducers via activation of the inositol-requiring enzyme 1 pathway in HCC cells.ConclusionsPlGF inhibition attenuates PERK activation, likely by tempering hypoxia in HCC via vessel normalisation. The UPR, in turn, is able to regulate PlGF expression, suggesting the existence of a feedback mechanism for hypoxia-mediated UPR that promotes the expression of the angiogenic factor PlGF. These findings have important implications for our understanding of the effect of therapies normalising tumour vasculature.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-015-1990-6) contains supplementary material, which is available to authorized users.

Abstract A66: Preclinical rationale for the ongoing Phase 2 study of the hypoxia-activated EGFR-TKI tarloxotinib bromide (TH-4000) in patients with advanced squamous cell carcinoma of the head and neck (SCCHN) or skin (SCCS)

Abstract Tarloxotinib bromide (T), a hypoxia-activated prodrug of a potent irreversible EGFR tyrosine kinase inhibitor (T-TKI), was designed to provide clinical benefit in patients with advanced SCCHN and SCCS. In these clinical settings excessive wild type (WT) EGFR overexpression may underlie the limited activity seen with currently available EGFR-TKI where inadequate tumor signal inhibition is evident at recommended doses (Annals Oncol 2007, 18:761). In contrast to the systemic delivery of EGFR-TKI, our prodrug approach selectively targets the hypoxic microenvironment of tumors, with the goal of increasing tumor dose-intensification and thus improving the therapeutic index. Preclinical studies were conducted to test the efficacy of clinically relevant doses of T and the approved EGFR-targeted agents cetuximab and afatinib in preclinical models of SCCHN (FaDu) and SCCS (A431) that express wild type EGFR. Under varying hypoxic conditions, EGFR expression and signal transduction was assessed by western blotting (WB). Pharmacodynamic (PD) endpoints (p-EGFR, p-MAPK, p-AKT) were evaluated by WB or immunohistochemistry (IHC). Under hypoxic conditions, T was 13- and 16-fold more active than in normoxic conditions against FaDu and A431 cells, respectively, with hypoxia-specific release of T-TKI from T by LC/MS (294 and 176 pmol/h/106 cells). Notably, hypoxic exposure of FaDu cells induced a marked 25-fold induction in EGFR phosphorylation (Y1092) that was stable upon reoxygenation (≥5 h), and EGFR signalling in A431 cells was upregulated 2.3-fold post-anoxia. Dose-dependent inhibition of EGFR signalling was observed and correlated with antiproliferative activity. The SCCHN FaDu tumor xenograft had a mean hypoxic fraction (HF) of 14% as determined by IHC detection of pimonidazole binding. Treatment with 48 mg/kg (qw) T (plasma exposure = 150 mg/m2 IV in human subjects) resulted in 100% (8/8) response rate (RR). In contrast, 8 mg/kg (qw) cetuximab treatment (saturating dose) failed to control growth with 8/10 (80%) tumors progressing on treatment and 2/10 (20%) with stable disease. The SCCS A431 tumor xenograft exhibited a mean HF of 31%. Treatment with 30 mg/kg (qw) T (plasma exposure equivalent to 104 mg/m2 IV in human subjects) resulted in 100% (6/6) RR. In contrast, 5 mg/kg daily oral afatinib (plasma exposure = 33 mg daily in human subjects) failed to regress tumors with best response of stable disease in 4/6 (66%) during treatment, while 2/6 (33%) progressed during treatment. T efficacy was accompanied by marked shutdown of total tumor p-EGFR (Try1092) in both xenograft models as well as p-AKT (Ser473) and p-MAPK (Thr202/Tyr204), in the FaDu and A431 xenografts, respectively, which was not evident with the comparator treatments of cetuximab and afatinib. In preclinical models, T exhibited superior efficacy with a 100% response rate compared to cetuximab in SCCHN and afatinib in SCCS with no responses and superior pharmacodynamic effects of T on EGFR target modulation. Based on these models performed with clinically relevant plasma PK levels T may possess a superior therapeutic index relative to approved EGFR-targeted agents. These preclinical data support the weekly dose of 150 mg/m2 of T in the ongoing phase 2 clinical trial in SCCHN and SCCS (NCT02449681). Citation Format: Victoria Jackson, Shevan Silva, Maria Abbattista, Christopher Guise, Matthew Bull, Amir Ashoorzadeh, Charles Hart, Tillman Pearce, Jeff Smaill, Adam V. Patterson. Preclinical rationale for the ongoing Phase 2 study of the hypoxia-activated EGFR-TKI tarloxotinib bromide (TH-4000) in patients with advanced squamous cell carcinoma of the head and neck (SCCHN) or skin (SCCS). [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A66.

Abstract 5214: Synergistic activity of aspirin, atenolol and metformin in the inhibition of angiogenesis, local and metastatic growth of breast cancer by targeting both neoplastic and microenvironment cells

Abstract We have recently described that the human white adipose tissue (WAT) contains progenitors with cooperative roles in breast cancer (BC) angiogenesis, local and metastatic progression (Martin-Padura et al, 2012; Orecchioni et al, 2013). The biguanide metformin (met), commonly used for type 2 diabetes, might have activity against BC and we found it able to inhibit angiogenesis in vivo (Dallaglio et al, 2014; Orecchioni et al, 2014). We studied met and another biguanide, phenformin (phe), in vitro and in vivo in orthotopic NSG murine models of local and metastatic BC. As met is frequently administered with aspirin or atenolol in diabetic/obese patients, we studied in vitro and vivo their association. In vitro, biguanides activated AMPK, inhibited complex 1 of the respiratory chain and induced apoptosis of BC and WAT endothelial cells. Aspirin was synergistic with met and phe in inducing apoptosis of estrogen receptor+ BC cells. This synergistic effect was less evident in triple negative BC cells. In co-culture, biguanides significantly inhibited the production of several angiogenic proteins. In vivo, biguanides inhibited local and metastatic growth of triple negative and HER2+ BC in immune-competent and immune-deficient mice orthotopically injected with BC. Biguanides also inhibited local and metastatic BC growth in a genetically engineered model of HER2+ BC. In vivo, biguanides increased pimonidazole binding (but not HIF-1 expression) of WAT progenitors, reduced tumor microvessel density and impaired of the vascular pericyte/endothelial cell ratio, so that cancer vessels displayed a dysplastic phenotype. This effect was significantly increased by the addition of aspirin or atenolol. This was evident also when AMPK activation was assessed in combination therapy regimens. AMPK phosphorylation was significantly increased in BC cells treated with met+aspirin or phe+atenolol as compared to met or phe alone. In WAT progenitors, AMPK activation was enhanced only under met+aspirin and met+atenolol combinations. In immune-competent mice, met effect in BC models was significantly enhanced by the addition of atenolol or of aspirin. Phe was significantly more active than met both in vitro and in vivo. Considering their safety profile, biguanides (alone or in combination with aspirin or atenolol) deserve to be further investigated for BC prevention in high-risk subjects, in combination with chemo and/or targeted therapy and/or as post-therapy consolidation or maintenance therapy for the prevention of BC recurrence. Citation Format: Giovanna Talarico, Francesca Reggiani, Stefania Orecchioni, Patrizia Mancuso, Angelica Calleri, Giuliana Gregato, Valentina Labanca, Douglas M. Noonan, Katiuscia Dallaglio, Adriana Albini, Francesco Bertolini. Synergistic activity of aspirin, atenolol and metformin in the inhibition of angiogenesis, local and metastatic growth of breast cancer by targeting both neoplastic and microenvironment cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5214. doi:10.1158/1538-7445.AM2015-5214

Severe blunt muscle trauma in rats: only marginal hypoxia in the injured area.

BackgroundAfter severe muscle trauma, hypoxia due to microvascular perfusion failure is generally believed to further increase local injury and to impair healing. However, detailed analysis of hypoxia at the cellular level is missing. Therefore, in the present work, spectroscopic measurements of microvascular blood flow and O2 supply were combined with immunological detection of hypoxic cells to estimate O2 conditions within the injured muscle area.Materials and MethodsSevere blunt muscle trauma was induced in the right Musculus gastrocnemius of male Wistar rats by a standardized “weight-drop” device. Microvascular blood flow, relative hemoglobin amount, and hemoglobin O2 saturation were determined by laser Doppler and white-light spectroscopy. Hypoxic cells were detected by histologic evaluation of covalent binding of pimonidazole and expression of HIF-1α.ResultsDirectly after trauma and until the end of experiment (480 minutes), microvascular blood flow and relative hemoglobin amount were clearly increased. In contrast to blood flow and relative hemoglobin amount, there was no immediate but a delayed increase of microvascular hemoglobin O2 saturation. Pimonidazole immunostaining revealed a hypoxic fraction (percentage area of pimonidazole-labelled muscle cells within the injured area) between 8 to 3%. There was almost no HIF-1α expression detectable in the muscle cells under each condition studied.ConclusionsIn the early phase (up to 8 hours) after severe blunt muscle trauma, the overall microvascular perfusion of the injured area and thus its O2 supply is clearly increased. This increased O2 supply is obviously sufficient to ensure normoxic (or even hyperoxic) conditions in the vast majority of the cells.

The biguanides metformin and phenformin inhibit angiogenesis, local and metastatic growth of breast cancer by targeting both neoplastic and microenvironment cells.

The human white adipose tissue (WAT) contains progenitors with cooperative roles in breast cancer (BC) angiogenesis, local and metastatic progression. The biguanide Metformin (Met), commonly used for Type 2 diabetes, might have activity against BC and was found to inhibit angiogenesis in vivo. We studied Met and another biguanide, phenformin (Phe), in vitro and in vivo in BC models. In vitro, biguanides activated AMPK, inhibited Complex 1 of the respiratory chain and induced apoptosis of BC and WAT endothelial cells. In coculture, biguanides inhibited the production of several angiogenic proteins. In vivo, biguanides inhibited local and metastatic growth of triple negative and HER2+ BC in immune-competent and immune-deficient mice orthotopically injected with BC. Biguanides inhibited local and metastatic BC growth in a genetically engineered murine model model of HER2+ BC. In vivo, biguanides increased pimonidazole binding (but not HIF-1 expression) of WAT progenitors, reduced tumor microvessel density and altered the vascular pericyte/endothelial cell ratio, so that cancer vessels displayed a dysplastic phenotype. Phe was significantly more active than Met both in vitro and in vivo. Considering their safety profile, biguanides deserve to be further investigated for BC prevention in high-risk subjects, in combination with chemo and/or targeted therapy and/or as post-therapy consolidation or maintenance therapy for the prevention of BC recurrence.

Detecting functional changes with [(18)F]FAZA in a renal cell carcinoma mouse model following sunitinib therapy.

BackgroundThe multitargeting tyrosine kinase inhibitor (TKI) sunitinib is currently the first-line drug therapy for metastasizing renal cell carcinoma (RCC). TKIs have profound effects on tumor angiogenesis, leading to modifications of the tumor microenvironment. The goal of this study was to determine whether these treatment-induced changes can be detected with [18F]FAZA.MethodsThe present study utilized positron emission tomography (PET) to analyze tumor oxygenation status during and after sunitinib therapy in the murine Caki-1 RCC tumor model. Dynamic and static scans were performed, as well as ex vivo biodistributions at 3 h post injection (p.i.). Immunohistochemical analysis of tumor tissue was carried out for the quantification of pimonidazole binding and the hypoxia-associated factors CD-31, Ki-67, and Von Willebrand factor (VWF). In addition, in vitro cellular uptake studies were done to analyze the direct effects of sunitinib on the Caki-1 cells.ResultsDuring therapy with sunitinib (40 mg/kg/day), uptake of [18F]FAZA into Caki-1 mice decreased by 46 ± 5% (n = 4; 5 days) at 3 h post injection (p.i.) during the first study and 22 ± 5% (n = 8; 9 days) during the long-term study, indicating a decrease in the tumor's hypoxia level. However, when drug therapy was stopped, this effect was reversed completely, and the tumor [18F]FAZA uptake increased to 126 ± 6% (n = 6) of the control tumor uptake, indicative of an even higher level of tumor hypoxia compared to the therapy starting point. Sunitinib had no direct effect on [18F]FAZA uptake into Caki-1 cells in vitro.Conclusion[18F]FAZA PET could be used to monitor drug response during sunitinib therapy in RCC and may guide combination therapies based on the tumor's hypoxia status.

A34 Session 10: Adjunctive Therapeutic Approaches: Vascular targeting approaches to enhance thermal therapy applications

The physiological reaction of tumor tissue and the progression of normal vascular and cellular repair processes in the body after thermal ablation suggest specific ways that adjuvant therapies may improve tumor control. This is a topic of growing importance as thermal ablation techniques are being used more than ever yet continue to have significant and unpredictable recurrence rates in clinical settings. We hypothesized that incomplete ablation of tumor tissue near the margin and around major blood vessels allows re-oxygenation and repopulation of the tumor. Microvessel functionality after partial thermal ablation of experimental murine tumors has been investigated after both high and low temperature thermal ablations. Interestingly, the oxygenation in the tumor margin increased from 1.2 ± 0.98 mmHg to 10.4 ± 6.3 mmHg during and for the first 20–30 min after a typical hot or cold ablation session, suggesting that the vascular response is a common feature to all of ablative medicine. Vessels with active blood flow in the peripheral regions of the tumor were seen to persist at varying levels from 24–72 h post-ablation. The level of hypoxia detected by pimonidazole binding was inversely related to the number of functional vessels detected. Our results suggest that simultaneous or tightly sequenced ablation and ionizing radiation therapy may have merit as an efficient means to completely sterilize tumor volume since radiation is most cytotoxic in areas of improved oxygenation and ablation most effectively debulks volumes with low or impaired perfusion. Furthermore, nanomedicine or other vascular-targeted drugs that can selectively impair the vasculature of the tumor may significantly increase the volume of complete ablation obtained and improve reproducibility of the ablation zone from tumor to tumor. A vascular sensitizing/damaging agent may also increase the overall effect of radiotherapy and thus improve tumor control. We have also documented a contribution of progenitor cells in repairing sublethally damaged tissue after ablation and promoting establishment of a vascular niche capable of regenerating the tumor mass which suggests agents that block the mobilization of bone marrow derived cells could also serve to improve the results of thermal ablative therapy for a variety of tumor types. These aspects of combined treatments will be discussed for both high temperature ablations and cryosurgery. Supported by NCI CA44114, Arkansas Breast Cancer Research Program and the Winthrop P. Rockefeller Cancer Institute at UAMS.

18F-Fluorodeoxyglucose Uptake and Tumor Hypoxia: Revisit 18F-Fluorodeoxyglucose in Oncology Application

This study revisited 18F-fluorodeoxyglucose (18F-FDG) uptake and its relationship to hypoxia in various tumor models. METHODS: We generated peritoneal carcinomatosis and subcutaneous xenografts of colorectal cancer HT29, breast cancer MDA-MB-231, and non–small cell lung cancer A549 cell lines in nude mice. The partial oxygen pressure (pO2) of ascites fluid was measured. 18F-FDG accumulation detected by digital autoradiography was related to tumor hypoxia visualized by pimonidazole binding and glucose transporter-1 (GLUT-1) in frozen tumor sections. RESULTS: Ascites pO2 was 0.90 ± 0.53 mm Hg. Single cancer cells and clusters suspended in ascites fluid as well as submillimeter serosal tumors stained positive for pimonidazole and GLUT-1 and had high 18F-FDG uptake. In contrast, 18F-FDG uptake was significantly lower in normoxic portion (little pimonidazole binding or GLUT-1 expression) of larger serosal tumors or subcutaneous xenografts, which was not statistically different from that in the liver. CONCLUSIONS: Glucose demand (18F-FDG uptake) in severely hypoxic ascites carcinomas and hypoxic portion of larger tumors is significantly higher than in normoxic cancer cells. Warburg effect originally obtained from Ehrlich ascites carcinoma may not apply to normoxic cancer cells. Our findings may benefit the better understanding of 18F-FDG PET in oncology application.

Hypoxia and Differentiation: Contrast Between Pimonidazole Binding and CK19 Expression in Tumors Derived From Stratified and Simple Epithelia

The objective is to compare pimonidazole binding and CK19 expression in breast cancer and squamous cell carcinoma. The purpose is to examine the general claim that hypoxia induces dedifferentiation in human tumors.

Remote post-conditioning reduces hypoxic damage early after experimental stroke

Objectives: Given that reliable markers for early ischemic brain damage are lacking, we set out to test whether pimonidazole can be used as a reliable tool in the quantification of hypoxic insults, at early time points following experimental stroke.Methods: We have used semi-quantitative Western blotting detection of pimonidazole adducts in a rat model of reversible middle cerebral artery occlusion (MCAO), treated with remote post-conditioning.Results: First, we demonstrated that a linear relationship exist between pimonidazole binding in the ischemic hemisphere and duration of ischemia, in animals subjected to 5, 15, 30, or 60 minutes of occlusion followed by 120 minutes of reflow. Then we showed a significant reduction in pimonidazole binding in the infarcted hemisphere, when rats with 60 minutes of MCAO, immediately after establishment of cerebral reflow, had 3×15 minutes intermittent hind limb ischemia followed by 24-hour survival. We analysed the middle cerebral arteries from animals with 60 minutes of MCAO and early remote post-conditioning, followed by 30 minutes, 24, or 48 hours of reflow. At 24 hours of reflow increases in phosphorylated protein kinase C-alpha with concomitantly increased levels of p38 phosphorylation were observed.Conclusions: Our investigation demonstrates that pimonidazole can be used for quantifying ischemic impact in stroke, even after very short survival times. It furthermore shows that early remote post-conditioning reduces ischemic damage, probably through hyperpolarization and reduced reflow vasospasm in the conduit middle cerebral arteries.

Abstract 5426: Severe hypoxia drives the increased glucose demand in ascites carcinomas.

Abstract Warburg reported his discovery in Ehrlich ascites carcinoma model, that in the presence of ample oxygen (21% or 150 mmHg O2), cancer cells utilize glucose by “aerobic glycolysis” (Warburg effect), apparently, ascites carcinoma had been assumed to be oxic. However, during past decade, growing evidences indicated that hypoxic cancer cells had evaluated radiolabeled glucose analog18F-FDG uptake than oxic cancer cell in vitro and in vivo. The objective of this study was to observe the oxygen status of ascites fluid and ascites carcinoma in mouse model of ascites carcinoma and its effect on glucose metabolism. Methods: Ascites carcinoma (floating single cells or clusters) and serosa tumors (attached to serosa) were induced by HT29, A549 or MDA-MB-231 cancer cells in the mouse peritoneal cavity. Ascites pO2 was measured using OxyLite systems in ketamine/xylazene anesthetized mice. Hypoxia status in ascites carcinoma and serosa tumors was tested by immunohistochemical visualization of exogenous hypoxia marker pimonidazole binding and HIF1alpha induced glucose transporter-1 expression. Glucose metabolism of was examine by 18F-FDG autoradiography. Effect of carbogen breathing (95% O2 &amp; 5% CO2) on 18F-FDG uptake was investigated. Results: The pO2 of ascites was measured as low as 0.90mmHg (or 0.5% O2) in mice with HT29 ascites carcinomas. In all cell lines we tested, ascites carcinomas was evident 4-6 wk after cell inoculation, all ascites tumors were stained positive for pimonidazole and glucose transporter-1 (GLUT-1), indicating severe hypoxia. Hypoxia status in serosa tumors was heterogeneous and dependent on tumor size. 18F-FDG uptake was significantly higher in ascites and sub-millimeter serosa tumors than in larger ones (generally greater than 1 mm), the former was stained positive for pimonidazole and GLUT-1, but the latter was not. Carbogen breathing (2hrs) significantly reduced 18F-FDG uptake and pimonidazole binding in ascites carcinoma and submillimeter serosa tumors. GLUT1 expression was not affected. Conclusions: In mouse model of ascites carcinoma, ascites and floating ascites carcinoma are severely hypoxic which is in contradict with ample oxygen condition of Ehrlich ascites carcinoma model where “Warburg effect” derived from. Glucose utilization measured by 18F-FDG uptake is oxygen dependent in both ascites carcinoma and serosa tumors, this is unexplained sole by “aerobic glycolysis” of glucose metabolism, thus challenging Warburg effect theory. This study was supported by Kentucky Lung Cancer Research Program Award Citation Format: Xiao-Feng Li. Severe hypoxia drives the increased glucose demand in ascites carcinomas. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5426. doi:10.1158/1538-7445.AM2013-5426

Determination of Organ-Specific Anemia Tolerance*

Utilization of anemia tolerance reduces the need for and risks of perioperative transfusion. Recent publications indicate that the critical limit for oxygen supply might not be the same for each organ system. Therefore, we investigated the effects of acute dilutional anemia on heart, brain, kidneys, liver, small intestine, and skeletal muscle to quantify organ-specific tolerance of different levels of acute anemic hypoxia. We hypothesized that, in some organs, tissue hypoxia occurs before the critical limits of systemic oxygen supply are reached. Laboratory animal experiments. Animal research laboratory at university medical school. A total of 18 domestic pigs of either sex (average weight: 19.6 kg). Animals were anesthetized, ventilated, and randomized into three groups and then hemodiluted by exchange of 6% hydroxyethyl starch (130,000:0.4) for whole blood to the group-specific endpoint: Sham (no hemodilution), Hb4 (hemoglobin 4.3 g/dL), Hbcrit (2.7 g/dL). Subsequently, 10 mg/kg pimonidazole (which forms protein adducts in hypoxic cells) was injected. One hour after injection, tissue samples were collected and analyzed for pimonidazole-protein adduct quantification (dot blot) and as a surrogate for transcriptional activation during hypoxia the expression of vascular endothelial growth factor messenger RNA. Relevant hemodynamic and metabolic parameters were collected. Hemodynamics, metabolic parameters, or oxygen consumption did not indicate that tissue oxygenation was restricted before reaching Hbcrit. However, kidneys and skeletal muscle showed enhanced pimonidazole binding and vascular endothelial growth factor expression at Hb4. By contrast, liver oxygenation was actually improved at Hb4. Heart, brain, and liver showed no signs of tissue hypoxia at Hb4. Heart, brain, kidneys, liver, small intestine, and skeletal muscle experience tissue hypoxia at different degrees of acute anemia, as assessed by the pimonidazole method and vascular endothelial growth factor expression. Further studies are needed to elucidate the mechanisms that determine organ-specific anemia tolerance.