A new algorithm for a better characterization and timing of the anti-VEGF vascular effect named "normalization".

Antiangiogenics are widely used in cancer treatment in combination with chemotherapy and radiotherapy for their vascular effects. Antiangiogenics are supposed to induce morphological and functional changes in the chaotic tumor vasculature that would help enhance the therapeutic efficacy of chemotherapy and radiotherapy through the amelioration of the drug delivery or the oxygenation in the tumor, respectively. However, finding the best treatment sequence is not an easy task to achieve and no consensus has yet been established because of the lack of knowledge regarding when and for how long the vascular network is ameliorated. The aim of this work was to develop a dedicated image processing algorithm able to analyze the vascular structures on optical microscopy images of the vascular network and to follow its fine modifications in vivo, over time. We applied this algorithm to follow the evolution of the vascular parameters (vascularized tissue surface, branches, sprouts and length), in response or not to anti-VEGF therapy (10 mg/kg/day) and determine precisely whether there is really a vascular "normalization" with anti-VEGF therapy in comparison with the parameters extracted from healthy vascular networks. We found that for this determination, the choice of region of interest to analyze is critical as it is important to compare only microcirculation areas and avoid areas with arteriole-venule-capillary hierarchy. The algorithm analysis allowed us to define a vascular "normalization" in treated tumors, between 8 and 12 days of bevacizumab treatment that was confirmed by standard immunohistochemical analysis, microvascular permeability assessment and immunohistological blood perfusion assessment.

Modulation of endothelial cell network formation in vitro by molecular signaling of head and neck squamous cell carcinoma (HNSCC) exposed to cetuximab.

Overexpression of EGFR plays a key-role in head and neck squamous cell carcinoma (HNSCC) and justifies the extensive use of cetuximab, a monoclonal anti-EGFR antibody, as well as EGFR-tyrosine kinase inhibitors (EGFR-TKI), which have been reported to inhibit tumor cell growth and the secretion of pro-angiogenic factors by tumor cells, such as VEGF and IL-8. Moreover, vessel normalization in tumors, suggesting a more complex mediation of endothelial cell growth control has also been observed in vivo. The present study was designed to investigate the angiogenic consequences of exposure of HNSCC tumor cell lines to cetuximab and intercellular signaling between tumor and endothelial cells by secretion of pro- and anti-angiogenic mediators in the conditioned media (CM). The results achieved showed that cetuximab decreased the secretion of VEGF by HNSCC cells and that exposure of human umbilical vein endothelial cells (HUVEC) to CM from HNSCC cells exposed to cetuximab induced an increase in endothelial cell network formation. Angiogenesis proteome profiling showed that cetuximab induced a complex alteration of the secretion of pro- and anti-angiogenic factors by HNSCC cells without enabling to identify a unique molecular marker. Expression of endothelial membrane receptors (VEGFR-2, EGFR, PECAM-1 and Notch-4) was investigated and only EGFR expression was found influenced when HUVEC were exposed to CM from cetuximab-exposed HNSCC cells. These results showed that the decrease in the secretion of pro-angiogenic agents like VEGF by HNSCC cells exposed to cetuximab could not be sufficient to justify its anti-angiogenic activity in vitro.

Neuropilin-1 targeting photosensitization-induced early stages of thrombosis via tissue factor release.

PURPOSE: This article characterizes the vascular effects following vascular-targeted photodynamic therapy with a photosensitizer which actively targets endothelial cells. METHODS: This strategy was considered by coupling a chlorin to a heptapeptide targeting neuropilin-1 in human malignant glioma-bearing nude mice. A laser Doppler microvascular perfusion monitor was used to monitor microvascular blood perfusion in tumor tissue. Endothelial cells' ultra structural integrity was observed by transmission electron microscopy. The consequences of photosensitization on tumor vessels, tissue factor expression, fibrinogen consumption, and thrombogenic effects were studied by immunohistochemical staining. RESULTS: Treatment of glioma-bearing mice with the conjugate showed a statistically significant tumor growth delay. Vascular effect was characterized by a decrease in tumor tissue blood flow at about 50% baseline during treatment not related to variations in temperature. This vascular shutdown was mediated by tumor blood vessels' congestion. A pro-thrombotic behavior of targeted endothelial cells in the absence of ultra structural changes led to the induction of tissue factor expression from the earliest times post-treatment. Expression of tissue factor-initiated thrombi formation was also related to an increase in fibrinogen consumption. CONCLUSION: Using a peptide-conjugated photosensitizer targeting neuropilin-1, induction of tissue factor expression immediately post-treatment, led to the establishment of thrombogenic effects within the vessel lumen.

Antiangiogenic therapy: Markers of response, "normalization" and resistance.

Currently in cancer treatment, one premise is to use antiangiogenic therapies in association with chemotherapy or radiotherapy to augment their efficacy by benefiting from the vascular "normalization" induced by antiangiogenic therapy. This concept defines the time during which the tumor blood vessels adopt normal-like morphology and functionality, i.e. the blood vessels become more mature, the perfusion augments and hypoxia decreases. To date, there is such a diversity of treatment protocols where the type of antiangiogenic to adopt, its dose and duration of administration are different, that knowing when and how to treat is problematic. In this review, we analyzed thoroughly preclinical and clinical studies that use antiangiogenic treatments to benefit from the "normalization" and showed that the effects depend on the type of antiangiogenic administrated (anti-VEGF, anti-VEGFR, Multi-Kinase Inhibitor) and on the duration of treatment. Finally, biomarkers of "normalization" and resistance that could be used in the clinic are presented.

Pharmacological and physicochemical factors in the pressor effects of conjugated haemoglobin-based oxygen carriers in vivo.

BACKGROUND: The hypertension induced by haemoglobin-based oxygen carriers could be a result of different pharmacological and physicochemical factors. OBJECTIVE: To investigate whether production of superoxide anion (O2*-) and release of endothelin could be the factors responsible. METHODS: We studied the variation in mean arterial pressure (MAP) in guinea pigs by carrying out a 50% isovolaemic exchange transfusion with conjugated oxyhaemoglobin (non-oxidized form) or conjugated methaemoglobin (fully oxidized form) in the presence or absence of BQ-788 (5 nmol/l), an endothelin receptor type B (ETR-B) antagonist. At key timepoints of variation in MAP, the plasma concentrations of O2*- were measured. The presence of conjugated oxyhaemoglobin and increases in ETR-B concentrations inside the vascular wall were investigated in different vessels, using western blotting. RESULTS: We found that the administration of conjugated oxyhaemoglobin induced a significant increase in MAP, whereas conjugated methaemoglobin had no significant haemodynamic effect. Pretreatment with BQ-788 attenuated the increase in MAP induced by conjugated oxyhaemoglobin. This haemoglobin induced the production of high concentrations of O2*- that declined towards control values after 120 min and decreased in the presence of BQ-788. Western blot analysis showed that the presence of conjugated oxyhaemoglobin inside the vascular wall was time-dependent and correlated with increased ETR-B. CONCLUSION: These results show that the release of O2*- during auto-oxidation of conjugated oxyhaemoglobin is associated with the observed increase in MAP, which may be a result of the vasoconstriction caused by an increase in activation of ETR-B. This activation may be caused by the massive release of endothelin induced by the production of O2*-.

Ascorbate removes key precursors to oxidative damage by cell-free haemoglobin in vitro and in vivo.

Haemoglobin initiates free radical chemistry. In particular, the interactions of peroxides with the ferric (met) species of haemoglobin generate two strong oxidants: ferryl iron and a protein-bound free radical. We have studied the endogenous defences to this reactive chemistry in a rabbit model following 20% exchange transfusion with cell-free haemoglobin stabilized in tetrameric form [via cross-linking with bis-(3,5-dibromosalicyl)fumarate]. The transfusate contained 95% oxyhaemoglobin, 5% methaemoglobin and 25 microM free iron. EPR spectroscopy revealed that the free iron in the transfusate was rendered redox inactive by rapid binding to transferrin. Methaemoglobin was reduced to oxyhaemoglobin by a slower process (t(1/2) = 1 h). No globin-bound free radicals were detected in the plasma. These redox defences could be fully attributed to a novel multifunctional role of plasma ascorbate in removing key precursors of oxidative damage. Ascorbate is able to effectively reduce plasma methaemoglobin, ferryl haemoglobin and globin radicals. The ascorbyl free radicals formed are efficiently re-reduced by the erythrocyte membrane-bound reductase (which itself uses intra-erythrocyte ascorbate as an electron donor). As well as relating to the toxicity of haemoglobin-based oxygen carriers, these findings have implications for situations where haem proteins exist outside the protective cell environment, e.g. haemolytic anaemias, subarachnoid haemorrhage, rhabdomyolysis.

[Blood substitutes: state of the art and technical setbacks and why we are still disappointed]. / Les substituts érythrocytaires: état de l'art et raisons d'un espoir retardé.

Volume loading solutions used therapeutically (albumin, dextrans, modified gelatins and hydroxylstarches) are simple plasma substitutes and cannot ensure oxygen transport. The search for erythrocyte substitutes initially seemed utopian, but this goal is now within our reach, in the form of hemoglobin-based oxygen carriers (HBOC) for example. The clinical development of HBOC has been slowed by adverse effects, including an increase in arterial pressure due to the vasoconstrictive effect of cell-free hemoglobin in plasma, haemoprotein autoxidation leading to methemoglobin formation, and free radical generation that creates oxidative stress.

Oxygen binding and oxidation reactions of human hemoglobin conjugated to carboxylate dextran.

Human hemoglobin (Hb) conjugated to benzene tetracarboxylate substituted dextran produces a polymeric Hb (Dex-BTC-Hb) with similar oxygen affinity to that of red blood cells (P(50)=28-29 mm Hg). Under physiological conditions, the oxygen affinity (P(50)) of Dex-BTC-Hb is 26 mm Hg, while that of native purified human HbA(0) is 14 mm Hg, but it exhibits a slight reduction in cooperativity (n(50)), Bohr effect, and lacks sensitivity to inositol hexaphosphate (IHP), when compared to HbA(0). Oxygen-binding kinetics, measured by rapid mixing stopped-flow method showed comparable oxygen dissociation and association rates for both HbA(0) and Dex-BTC-Hb. The rate constant for NO-mediated oxidation of the oxy form of Dex-BTC-Hb, which is governed by NO entry to the heme pocket, was reduced to half of the value obtained for HbA(0). Moreover, Dex-BTC-Hb is only slightly more sensitive to oxidative reactions than HbA(0), as shown by about 2-fold increase in autoxidation, and slightly higher H(2)O(2) reaction and heme degradation rates. Dextran-BTC-based modification of Hb produced an oxygen-carrying compound with increased oxygen release rates, decreased oxygen affinity and reduced nitric oxide scavenging, desirable properties for a viable blood substitute. However, the reduction in the allosteric function of this protein and the lack of apparent quaternary T-->R transition may hinder its physiological role as an oxygen transporter.

Sensitivity of glioma initiating cells to a monoclonal anti-EGFR antibody therapy under hypoxia.

AIMS: Glioma initiating cells (GICs) represent a subpopulation of tumor cells endowed with self-renewal and multilineage differentiation capacity but also with innate resistance to cytotoxic agents, a feature likely to pose major clinical challenges towards the complete eradication of minimal residual disease in glioma patients. MATERIALS AND METHODS: In this work, GICs were obtained from two patient-derived high-grade gliomas xenograft model, expressing differently EGFR. GICs were exposed to anti-EGFR monoclonal antibody cetuximab during 48h in 1% or 21% oxygen tension. Cell viability and self-renewal capacity were then evaluated as well as their angiogenic properties. KEY FINDINGS: GICs were sensitive to cetuximab only in normoxic condition whatever the EGFR status. Nevertheless, under hypoxia cetuximab was able to decrease the self-renewal capacity as well as the expression of CD133 while expression of GFAP increased. Moreover, cetuximab decreased the effect of GICs on endothelial cell migration under hypoxia. SIGNIFICANCE: Consequently, anti-EGFR therapy can be envisaged to target specifically GICs in order to limit the tumor recurrence.

PTEN expression is involved in the invasive properties of HNSCC: a key protein to consider in locoregional recurrence.

Specific phenotypic effects of PTEN in head and neck squamous cell carcinoma (HNSCC) remain poorly defined without a direct causal connection between the loss of PTEN function and the progression of cancer. Here, we describe a potential role for PTEN in cancer progression. Using an shRNA targeting PTEN in HNSCC cells, we show that the loss of PTEN expression is associated with a decrease of cell adhesion, a reduction in E-cadherin expression while cell migration is promoted. Together with the tissue organization and molecular markers expressed in tumors derived from shPTEN cells in vivo, this study indicates that HNSCC cells deficient in PTEN expression undergo an epithelial­mesenchymal transition (EMT). Additionally, our results suggest that both the low levels of expression and subcellular localization of PTEN are involved in the EMT phenotype, and ultimately in possible locoregional reccurences. We hypothesize that the loss of PTEN expression as well as the subcellular localization could be of interest as a predictive marker of recurrence in HNSCC.

Design, synthesis and biological evaluation of new classes of thieno[3,2-d]pyrimidinone and thieno[1,2,3]triazine as inhibitor of vascular endothelial growth factor receptor-2 (VEGFR-2).

Driven by a multidisciplinary approach combination (Structure-Based (SB) Three-Dimensional Quantitative Structure-Activity Relationships (3-D QSAR), molecular modeling, organic chemistry and various biological evaluations) here is reported the disclosure of new thienopyrimidines 1-3 as inhibitors of KDR activity and human umbilical vein endothelial cell (HUVEC) proliferation. More specifically, compound 2f represents a new lead compound that inhibits VEGFR-2 and HUVEC at µM concentration. Moreover by the mean of an endothelial cell tube formation in vitro model 2f tartaric acid salt proved to block angiogenesis of HUVEC at µM level.

Tumor vascular responses to antivascular and antiangiogenic strategies: looking for suitable models.

Antiangiogenic and vascular disrupting agents are in the current cancer therapeutic armamentarium. A better understanding of the intricate mechanisms ruling neovessel survival within tumors during or after treatment is needed. Refinement of imaging and a growing knowledge of molecular biology of tumor vascularization provide new insights. It is necessary to define suitable methods for monitoring tumor response and appropriate tools to analyze data. This review compares most commonly used preclinical models, considering their recent improvements, and describes promising new approaches such as microfluidics, real-time electrical impedance based technique and noninvasive imaging techniques. The advantages and limitations of the in vitro, ex vivo and in vivo models are discussed. This review also provides a critical summary of emerging approaches using mathematical modeling.

Cellular response to cetuximab in PTEN-silenced head and neck squamous cell carcinoma cell line.

The implication of loss of PTEN expression in resistance to targeted therapy has already been described in many tumor types. The absence of response to anti-EGFR agents in PTEN-deficient tumors relies on persistent activation of signaling pathways downstream of pEGFR. To investigate the role of PTEN loss of expression in head and neck squamous cell carcinoma (HNSCC) response to cetuximab, we used siRNA in Cal 27 cells and then evaluated key signaling protein activation (pAKT and pERK 1/2) as well as cell viability and proliferation. PTEN silencing in Cal 27 cells led to a constitutive activation of signaling pathways evidenced by a strong increase in pAKT and pERK 1/2 expression. Moreover, PTEN-silenced cells did not show any significant changes either in cell viability or proliferation, only slight modifications on cell cycle. Additionally and unpredictably, our results indicated that PTEN silencing, led to a drastic reduction in pEGFR expression whereas total EGFR level did not significantly vary. Strikingly, despite this overactivation of signaling pathways ruling cell survival and proliferation in siPTEN cells, cetuximab fully exerted pAKT and pERK 1/2 inhibition of expression, similarly to its effect in untransfected Cal 27 cells. In conclusion, our study established that in Cal 27 cells, cetuximab keeps full ability to inhibit EGFR-dependent mechanisms, as shown by a decreased pAKT and pERK 1/2 level of expression, despite a strong PTEN silencing-induced overactivation. In Cal 27 cells, loss of PTEN expression does not lead to a loss of cetuximab efficacy in inhibiting EGFR-downstream signaling pathways, contrarily to data shown in previous works conducted in other tumor types.

Hemoglobin-based oxygen carrier distribution inside vascular wall and arterial pressure evolution: is there a relationship?

The hemoglobin-based oxygen carriers (HBOC), like dextran-benzene-tetracarboxylate-hemoglobin (Dex-BTC-Hb), which are present at high concentrations in plasma disturb arterial pressure and induce hypertension. To study if the increase of mean arterial pressure (MAP) is due to the presence of cell-free hemoglobin (Hb) inside abdominal aortic wall, we followed on a model of 50% isovolemic exchange transfusion (IET) in anesthetized guinea pigs, the kinetic of Dex-BTC-Hb distribution inside abdominal aortic wall and we investigated the relationship between arterial pressure modifications and modified Hb distribution. The administration of Dex-BTC-Hb induced instantaneously an increase of MAP that reached its maximum (53% of hypertension from baseline) at 17 min after the end of the IET and was maintained maximally up to 30 min. A significantly decrease of MAP (45% of hypertension from baseline) was observed after 60 min and the baseline level was recovered at 180 min. The investigation of tissue at 17 min by confocal microscopy showed the presence of free Hb in or upon endothelial cells (EC) in intima and in vasa vasorum. At 180 min, the free Hb was found in or upon EC and inside all abdominal aortic wall meanwhile MAP recovered its basal value. These results suggest for the first time that Hb in intima seems to induce the hypertension observed upon IET but can not sustain it even if Hb stayed present in intima and in abdominal aortic wall.