Biomarkers predicting tumor response and evasion to anti-angiogenic therapy

No fully validated biological markers currently exist to predict responsiveness to or the development of evasion to anti-angiogenic therapy of cancer. The identification of such biomarkers is vital to move these therapies forward, as failure to respond to these treatments is often associated with rapid tumor progression that could have been averted had the intrinsic or acquired evasion to anti-angiogenic therapy been identified in a timely fashion. Furthermore, the high cost of antiangiogenic therapies makes it important to avoid utilizing them in the setting of lack of response or developing evasion, making the identification of biomarkers even more important. A number of potential physiologic, circulating, tissue, and imaging biomarkers have emerged from recently completed preclinical animal studies and clinical trials. In this review, we define 5 different types of biomarkers (physiologic, circulating, intratumoral, genetic polymorphisms, and radiographic); discuss the challenges in establishing biomarkers of antiangiogenic therapy in animal models and in clinical trials; and discuss future strategies to identify and validate biomarkers of anti-angiogenic therapy.     

Biomarkers predicting tumor response and evasion to anti-angiogenic therapy

No fully validated biological markers currently exist to predict responsiveness to or the development of evasion to anti-angiogenic therapy of cancer. The identification of such biomarkers is vital to move these therapies forward, as failure to respond to these treatments is often associated with rapid tumor progression that could have been averted had the intrinsic or acquired evasion to anti-angiogenic therapy been identified in a timely fashion. Furthermore, the high cost of antiangiogenic therapies makes it important to avoid utilizing them in the setting of lack of response or developing evasion, making the identification of biomarkers even more important. A number of potential physiologic, circulating, tissue, and imaging biomarkers have emerged from recently completed preclinical animal studies and clinical trials. In this review, we define 5 different types of biomarkers (physiologic, circulating, intratumoral, genetic polymorphisms, and radiographic); discuss the challenges in establishing biomarkers of antiangiogenic therapy in animal models and in clinical trials; and discuss future strategies to identify and validate biomarkers of anti-angiogenic therapy.     

Betamethasone inhibits tumor development, microvessel density and prolongs survival in mice with a multiresistant adenocarcinoma TA3

Tumor resistance to traditional cancer treatments poses an important challenge to modern science. Thus, angiogenesis inhibition is an important emerging cancer treatment. Many drugs are tested and corticosteroids have shown interesting results. Herein we investigate the effect on microvessel density, survival time and tumoral volume of mice with TA3-MTX-R tumors. Twenty six mice were inoculated with l x l0? tumor cells; 4-5 days after injection, six mice were injected with PBS (group A) and twenty mice were treated with β-met (group B). All animals from Group A died on day 22. Group B was divided into Bl (treated discontinued) and B2 (treated daily) and observed until day 88. All mice were processed for histo-immunohistochemical analysis and the blood vessels were counted. A decrease in microvessel density and tumoral volume and longer survival times were observed in the treated group. We propose that the antiangiogenic β-met effect explains, at least partially, its tumor inhibitory properties. As an important perspective, we will experimentally combine these strategies with those recently described by us with regard to the important antiangiogenic-antitumor effects of Trypanosoma cruzi calreticulin. Since the molecular targets of these strategies are most likely different, additive or synergic effects are envisaged.     

Radiation-induced alterations in microvessel density as predictor of treatment efficiency in oropharyngeal cancer

In this prospective study we have evaluated the predictive role of intratumoral microvessel density (MVD) for the therapeutic response and progression of inoperable oropharyngeal cancer (OPCC) treated with radiotherapy. Thirty-five OPCC patients were enrolled in this study. Biopsies from the primary tumor were taken before and after 20 Gy irradiation. Pathological factors such as histological grade, mitotic activity index and MVD were determined in both samples. Correlations of these factors with response to therapy, progression-free and overall survival were analyzed after a follow-up period of a minimum of 50 months. Objective response and survival was independent of the pretreatment MVD of OPCC. On the other hand, objective response was significantly affected by stage and low posttreatment MVD. Response to irradiation, and therapy-induced low postirradiation MVD were significant indicators of better overall and progression-free survival. We have shown in this small exploratory study that the anti-angiogenic effect of irradiation has a predictive value of the success of radiotherapy in locally advanced OPCC and can be used to select a radioresistant patient population which might require a more aggressive protocol.     

In vivo imaging of macrophages during the early-stages of abdominal aortic aneurysm using high resolution MRI in ApoE mice

Background

Angiotensin II (ANG II) promotes vascular inflammation and induces abdominal aortic aneurysm (AAA) in hyperlipidemic apolipoprotein E knock-out (apoE^−/−) mice. The aim of the present study was to detect macrophage activities in an ANG II-induced early-stage AAA model using superparamagnetic iron oxide (SPIO) as a marker.

Methodology/Principal Findings

Twenty-six male apoE^−/− mice received saline or ANG II (1000 or 500 ng/kg/min) infusion for 14 days. All animals underwent MRI scanning following administration of SPIO with the exception of three mice in the 1000 ng ANG II group, which were scanned without SPIO administration. MR imaging was performed using black-blood T2 to proton density -weighted multi-spin multi-echo sequence. In vivo MRI measurement of SPIO uptake and abdominal aortic diameter were obtained. Prussian blue, CD68,α-SMC and MAC3 immunohistological stains were used for the detection of SPIO, macrophages and smooth muscle cells. ANG II infusion with 1000 ng/kg/min induced AAA in all of the apoE^−/− mice. ANG II infusion exhibited significantly higher degrees of SPIO uptake, which was detected using MRI as a distinct loss of signal intensity. The contrast-to-noise ratio value decreased in proportion to an increase in the number of iron-laden macrophages in the aneurysm. The aneurysmal vessel wall in both groups of ANG II treated mice contained more iron-positive macrophages than saline-treated mice. However, the presence of cells capable of phagocytosing haemosiderin in mural thrombi also induced low-signal-intensities via MRI imaging.

Conclusions/Significance

SPIO is taken up by macrophages in the shoulder and the outer layer of AAA. This alters the MRI signaling properties and can be used in imaging inflammation associated with AAA. It is important to compare images of the aorta before and after SPIO injection.     

In-vivo response of the Human Uterus to Orciprenaline in Early Labour

The effect of orciprenaline on uterine activity in 10 women in early induced labour at term with intact membranes was studied. Nine had uterine contractions in response to intravenous oxytocin and one to prostaglandin E₂. The inhibition of uterine contractility was dose-dependent. The effective dose varied between 10 and 20 μg/minute. Tachyphylaxis was not observed. The only significant effects noted in the mother were tachycardia and increased pulse pressure and in the fetus a smaller increase in heart rate.     

Angiogenesis and inflammation in skeletal muscle in response to ascites tumor in mice

This study addresses the interaction between Ehrlich ascites tumor and skeletal abdominal muscle, presenting quantitative analysis of ascites-induced angiogenesis and inflammation in this tissue of mice bearing-tumor. Time-dependent changes in the muscle (cellular activity, angiogenesis, inflammation and cytokines production) were assessed by morphometric, functional, and biochemical parameters at days 1, 4 and 8 after i.p. inoculation of Ehrlich tumor cells (2.5 x 10(7)). The number of cells stained with AgNOR technique (argyrophilic nucleolar organizer region) in the muscle, together with MTS assay used as markers of cellular activity increased progressively in parallel with the out flow rate of sodium fluorescein (blood flow index), hemoglobin content (vascular index) and VEGF production. Likewise, the inflammatory process in the muscle, as assessed by myeloperoxidase (MPO) and n-acethylglucosaminidase (NAG) activities and the levels of the chemokines, keratinocyte-derived chemokine (CXC1-3/KC) and macrophage-chemoattractant protein (CCL2/MCP-1) increased with tumor development. The combination of techniques used to describe angiogenesis and inflammation in a muscle model system has proved to be suited for quantitative measurements of microvascular changes and cellular infiltration occurring in the abdominal muscle wall of ascites-bearing mice. This study holds potential for investigating events and mechanisms associated with skeletal muscle response to neoplasic stimulus.     

Autometallographic enhancement of the Golgi-Cox staining enables high resolution visualization of dendrites and spines

We present a method for autometallographic (AMG) enhancement of the Golgi-Cox staining enabling high resolution visualization of dendrites and spines. The method is cheaper and more flexible than conventional enhancement procedures performed with commercial photographic developers. The staining procedure is thoroughly described and we demonstrate with qualitative and quantitative data, how histological tissue sectioning, Golgi-Cox immersion time and different AMG enhancement length may influence the staining of dendrites and spines in the rat hippocampus. The described method will be of value for future behavioural-anatomical studies, examining changes in dendrite branching and spine density caused by brain diseases and their subsequent treatment.     

Reversibility of increased microvessel permeability in response to VE-cadherin disassembly

We determined the role of vascular endothelial (VE)-cadherin complex in regulating the permeability of pulmonary microvessels. Studies were made in mouse lungs perfused with albumin-Krebs containing EDTA, a Ca(2+) chelator, added to study the VE-cadherin junctional disassembly. We then repleted the perfusate with Ca(2+) to restore VE-cadherin integrity. Confocal microscopy showed a disappearance of VE-cadherin immunostaining in a time- and dose-dependent manner after Ca(2+) chelation and reassembly of the VE-cadherin complex within 5 min after Ca(2+) repletion. We determined the (125)I-labeled albumin permeability-surface area product and capillary filtration coefficient (K(fc)) to quantify alterations in the pulmonary microvessel barrier. The addition of EDTA increased (125)I-albumin permeability-surface area product and K(fc) in a concentration-dependent manner within 5 min. The permeability response was reversed within 5 min after repletion of Ca(2+). An anti-VE-cadherin monoclonal antibody against epitopes responsible for homotypic adhesion augmented the increase in K(fc) induced by Ca(2+) chelation and prevented reversal of the response. We conclude that the disassembled VE-cadherins in endothelial cells are mobilized at the junctional plasmalemmal membrane such that VE-cadherins can rapidly form adhesive contact and restore microvessel permeability by reannealing the adherens junctions.     

Stress analysis of carotid plaque rupture based on in vivo high resolution MRI

Atheromatous carotid plaque rupture is responsible for the majority of ischaemic strokes in the developed world. Plaque rupture has been associated with plaque morphology, plaque components' properties, inflammation and local stress concentration. High resolution multi-spectral magnetic resonance imaging (MRI) has allowed the plaque components to be visualized in vivo. This study combined the recent advances in finite element analysis (FEA) and MRI, and performed stress analysis of five vulnerable carotid plaques based on the geometry derived from in vivo MRI. Image segmentation was based on multi-spectral MRI and co-registered with histology for plaque characterization. Plaque fibrous cap, lipid pool and vessel wall were modelled as isotropic, incompressible hyperelastic materials undergoing large deformation under pulse pressure loading. High stress concentrations were predicted at the shoulders and the thinnest fibrous cap regions of the plaque, and the mean maximal stresses were found to be higher in the ruptured plaques (683.3 kPa) than those in the unruptured plaques (226.9 kPa). The effect of the relative stiffness of fibrous cap to lipid pool on the stress within the cap itself was studied. It was shown that larger relative stiffness of fibrous cap to lipid pool resulted in higher stress within the cap. Thus, it is likely that high stress concentrations in vulnerable plaque may cause plaque rupture and lead to acute ischaemic sequelae. A combination of in vivo high resolution MRI and FEA could potentially act as a useful tool to assess plaque vulnerability and risk stratify patients with carotid atheroma.     

In-vivo phosphorylation of the cardiac L-type calcium channel beta-subunit in response to catecholamines

In canine myocardium, the -subunit of the L-type Ca²⁺ channel is phosphorylated by cAMP dependent protein kinase in vitro as well as in vivo (Haase et al. FEBS Lett 335: 217–222, 1993). We have assessed the identity of the -subunit as well as its in vivo phosphorylation in representative experimental groups of catecholamine-challenged canine hearts. Adrenergic stimulation by high doses of both noradrenaline and isoprenaline induced rapid (within 20 sec) and nearly complete phosphorylation of the Ca²⁺ channel -subunit. Phosphorylation in vivo was about 4-fold higher as compared to untreated controls. When related to catecholamine-depleted (reserpine-treated) hearts noradrenaline and isoprenaline increased the in vivo phosphorylation of the -subunit even 8-fold. This phosphorylation correlated positively with tissue levels of cAMP, endogenous particulated cAMP-dependent protein kinase (PKA) and the rate of contractile force development dP/dt_max. The results imply the involvement of a PKA-mediated phosphorylation of the Ca²⁺ channel -subunit in the adrenergic stimulation of intact canine myocardium.     

Prediction of glioblastoma multiform response to bevacizumab treatment using multi-parametric MRI

Glioblastoma multiform (GBM) is a highly malignant brain tumor. Bevacizumab is a recent therapy for stopping tumor growth and even shrinking tumor through inhibition of vascular development (angiogenesis). This paper presents a non-invasive approach based on image analysis of multi-parametric magnetic resonance images (MRI) to predict response of GBM to this treatment. The resulting prediction system has potential to be used by physicians to optimize treatment plans of the GBM patients. The proposed method applies signal decomposition and histogram analysis methods to extract statistical features from Gd-enhanced regions of tumor that quantify its microstructural characteristics. MRI studies of 12 patients at multiple time points before and up to four months after treatment are used in this work. Changes in the Gd-enhancement as well as necrosis and edema after treatment are used to evaluate the response. Leave-one-out cross validation method is applied to evaluate prediction quality of the models. Predictive models developed in this work have large regression coefficients (maximum R ² = 0.95) indicating their capability to predict response to therapy.     

Purification and characterization of bovine lipoproteins: resolution of high density and low density lipoproteins using heparin-Sepharose chromatography

The selective and reversible adsorption of bovine low density lipoproteins (LDL) by heparin-Sepharose has been exploited as the critical step in a procedure for the preparative isolation of very low density lipoproteins (VLDL)/chylomicrons, LDL, and high density lipoproteins (HDL) from bovine plasma. Molecular size exclusion chromatography and isopycnic density gradient separation steps are also involved in the method described. The resulting HDL and LDL fractions are free from contamination by one another as judged by electrophoretic mobility in agarose gels. The major lipid and apolipoprotein compositions of the three resolved lipoprotein classes have been determined.     

Cell lines cultured at high density are resistant to lysis by tumor necrosis factor and natural cytotoxic cells

It has been suggested that natural cytotoxic (NC) cell activity and tumor necrosis factor (TNF), the molecular mediator of NC activity, are capable of protecting individuals against the progression of incipient tumors or could be useful in cancer therapy regimens. Much of this speculation arises as a result of in vitro studies, on a variety of tumor cells, demonstrating the cytolytic and cytostatic properties of NC and TNF activities. Here, evidence is presented showing that certain mouse fibroblast cell lines, generally considered sensitive to NC and TNF lysis, are quite resistant to these lytic activities when cultured at high cell density. Although a soluble factor that renders these same target cells resistant to NC and TNF lysis has been described, no such factor is involved in this high density-induced resistance. Rather, it appears that cell to cell contact of the targets is critical. Moreover, the induced resistance to NC and TNF lysis does not result from loss of either NC recognition determinants or TNF receptors by the target cells, but is the consequence of increased expression of a protein synthesis-dependent resistance mechanism. These observations raise the issue of the in vivo phenotype of cells characterized in vitro as sensitive to NC and TNF lysis. It is entirely possible that certain cells which are considered sensitive to NC and TNF activities are, in fact, resistant to these cytolytic activities when growing as tumors (i.e., at high cell density). Should this be the so, NC and TNF cytolytic activities may not function in vivo or may function only via some indirect means.     

Differential molecular sieving permits visualization of hexokinase isoenzymes following electrophoretic separation in high resolution acrylamide gels

1. Differential molecular sieving is the concept applied to bring together isoenzymes of ATP:D-hexose-6-phosphotransferase (hexokinases) with glucose 6-phosphate dehydrogenase in acrylamide gels by utilization of their dissimilar electrophoretic mobilities. 2. The hexokinase isoenzymes migrate and separate in gels with pore sizes selected to entrap glucose 6-phosphate dehydrogenase in their interstices. The locations of the bands of specific activity are visualized by fluorescence of NADPH under long wave, ultraviolet radiation. 3. A new discontinuous electrochemical system has been devised to deliver protective thiol groups into the gel. Cysteine (trailing ion) and SO4(2-) (leading ion) form a sharp moving boundary. 4. The high resolution of the system has permitted visualization of a rapidly migrating, high Km hexokinase in murine spleen, fat, kidney and lymph nodes. Hexokinase Types I and II, were observed in all tissues tested, but Type IV was seen only in the liver. 5. The importance of glucose concentration effects on hexokinase activity is emphasized by inactivation of slowly migrating low Km hexokinase Types I and II following exposure to 200 mM glucose during preparation of extracts.     

Scavenger receptor CD36 mediates uptake of high density lipoproteins in mice and by cultured cells

The mechanisms of HDL-mediated cholesterol transport from peripheral tissues to the liver are incompletely defined. Here the function of scavenger receptor cluster of differentiation 36 (CD36) for HDL uptake by the liver was investigated. CD36 knockout (KO) mice, which were the model, have a 37% increase (P = 0.008) of plasma HDL cholesterol compared with wild-type (WT) littermates. To explore the mechanism of this increase, HDL metabolism was investigated with HDL radiolabeled in the apolipoprotein (¹²⁵I) and cholesteryl ester (CE, [³H]) moiety. Liver uptake of [³H] and ¹²⁵I from HDL decreased in CD36 KO mice and the difference, i. e. hepatic selective CE uptake ([³H]¹²⁵I), declined (–33%, P = 0.0003) in CD36 KO compared with WT mice. Hepatic HDL holo-particle uptake (¹²⁵I) decreased (–29%, P = 0.0038) in CD36 KO mice. In vitro, uptake of ¹²⁵I-/[³H]HDL by primary liver cells from WT or CD36 KO mice revealed a diminished HDL uptake in CD36-deficient hepatocytes. Adenovirus-mediated expression of CD36 in cells induced an increase in selective CE uptake from HDL and a stimulation of holo-particle internalization. In conclusion, CD36 plays a role in HDL uptake in mice and by cultured cells. A physiologic function of CD36 in HDL metabolism in vivo is suggested.     

Epi-illumination dark-field microscopy enables direct visualization of unlabeled small organisms with high spatial and temporal resolution

Dark-field microscopy is known to offer both high resolution and direct visualization of thin samples. However, its performance and optimization on thick samples is under-explored and so far, only meso-scale information from whole organisms has been demonstrated. In this work, we carefully investigate the difference between trans- and epi-illumination configurations. Our findings suggest that the epi-illumination configuration is superior in both contrast and fidelity compared to trans-illumination, while having the added advantage of experimental simplicity and an “open top” for experimental intervention. Guided by the theoretical analysis, we constructed an epi-illumination dark-field microscope with measured lateral and axial resolutions of 260 nm and 520 nm, respectively. Subcellular structures in whole organisms were directly visualized without the need for image reconstruction, and further confirmed via simultaneous fluorescence imaging. With an imaging speed of 20 to 50 fps, we visualize fast dynamic processes such as cell division and pharyngeal pumping in Caenorhabditis elegans.