Role of MT1-MMP in cancer dissemination : insights from the real time imaging of tumors

Abstract : Matrix metalloproteinases (MMPs) are thought to play a major role in the tumor dissemination process as they degrade all components of the extracellular matrix. However, failure of clinical trials testing broad MMP inhibitors in cancer led to the consensus that a better understanding of the MMP biology was required. Using intravital multiphoton laser scanning microscopy, we developed an in vivo model to observe tumor dissemination and extracellular matrix remodeling in real time. We show that the matrix-modifying hormone relaxin increases tumor associated fibroblast interaction with collagen fibers by inducing integrin beta-1 expression. This causes changes in the collagen network that are mediated by MMP-8 and MT1-MMP. Also, we show that MMP-mediated collagen remodeling in vivo requires a direct contact between stationary tumor associated fibroblasts (TAFs) and collagen fibers. As MMPs are expressed in the tumor and stromal compartment of breast cancers we determined the importance of Membrane-type 1 MMP (MT1-MMP) from each compartment for cancer progression. We find that tumor-MT1-MMP promotes the invasion of the blood vasculature and blood-borne metastasis in vivo by enhancing tumor cell migration and endothelial basement membrane degradation. Interestingly, stromal-MT1-MMP cannot compensate for the lack of tumor-MT1-MMP but promotes peritumor collagen I remodeling. Thus, the function of MT1-MMP is context dependent and we identify the different but complementary roles of tumor and stromal MT1-MMP for tumor dissemination. Finally, we translate our preclinical findings in to human breast cancer samples. We show that tumor-MT1-MMP expression correlates with tumor invasion of the blood vasculature in ER-PR-HER2- breast cancers and that MT1-MMP expression increases with cancer progression. MT1-MMP could thus represent an interesting therapeutic target for the prevention of blood vasculature invasion in these tumors. Resumé : Les matrix metalloproteinases (MMPs) semblent jouer un rôle majeur pour la dissémination tumorale en raison de leur capacité à dégrader l'ensemble des composants de la matrice extracellulaire (MEC). Néanmoins, les résultats décevants des études cliniques testant les inhibiteurs des MMP ont conduit à la notion qu'une compréhension plus précise de la biologie des MMP était requise. Dans ce travail de thèse, nous avons développé un modèle murin qui permet d'observer simultanément la dissémination tumorale ainsi que les modifications de la MEC en temps réel. Nous démontrons que le traitement de tumeurs par l'hormone relaxin augmente l'interaction des fibroblastes tumoraux avec les fibres de collagène via l'intégrine beta-1. Nous montrons que cette interaction favorise et est nécessaire à la dégradation des fibres de collagène par MMP-8 et MT1-MMP. Ensuite, étant donné que les MMPs sont exprimées dans les cellules tumorales et stromales des cancers du sein, nous nous sommes intéressés au rôle de la MMP membranaire type 1 (MT1-MMP) exprimée dans chacun de ces compartiments. Nous démontrons que MT1-MMP dérivant des cellules tumorales favorise leur invasion dans les vaisseaux sanguins par la dégradation de la membrane basale vasculaire. De manière inattendue, nous montrons que l'expression de MT1-MMP par le compartiment stromal ne peut compenser le manque de MT1-MMP dans le compartiment tumoral. Néanmoins, nos résultats prouvent que MT1-MMP dérivant du compartiment stromal est impliqué dans la dégradation de collagène peritumorale. La fonction de la protéine MT1-MMP varie donc selon le compartiment tumoral d'origine. Finalement, nous avons testé nos résultats pré cliniques chez l'humain. Dans des biopsies de cancer du sein nous montrons une corrélation entre l'expression de MT1-MMP dans les cellules tumorales et l'invasion de vaisseaux sanguins par des tumeurs ER-PR-HER2-. MT1-MMP pourrait donc être une cible intéressante pour la prévention de dissémination vasculaire de ces tumeurs

Real-time Imaging and Mosaicking of Planar Surfaces

Laajojen pintojen kuvaaminen rajoitetussa työskentelytilassa riittävällä kuvatarkkuudella voi olla vaikeaa. Kuvaaminen on suoritettava osissa ja osat koottava saumattomaksi kokonaisnäkymäksi eli mosaiikkikuvaksi. Kuvauslaitetta käsin siirtelevän käyttäjän on saatava välitöntä palautetta, jotta mosaiikkiin ei jäisi aukkoja ja työ olisi nopeaa. Työn tarkoituksena oli rakentaa pieni, kannettava ja tarkka kuvauslaite paperi- ja painoteollisuuden tarpeisiin sekä kehittää palautteen antamiseen menetelmä, joka koostaaja esittää karkeaa mosaiikkikuvaa tosiajassa. Työssä rakennettiin kaksi kuvauslaitetta: ensimmäinen kuluttajille ja toinen teollisuuteen tarkoitetuista osista. Kuvamateriaali käsiteltiin tavallisella pöytätietokoneella. Videokuvien välinen liike laskettiin yksinkertaisella seurantamenetelmällä ja mosaiikkikuvaa koottiin kameroiden kuvanopeudella. Laskennallista valaistuksenkorjausta tutkittiin ja kehitetty menetelmä otettiin käyttöön. Ensimmäisessä kuvauslaitteessa on ongelmia valaistuksen ja linssivääristymien kanssa tuottaen huonolaatuisia mosaiikkikuvia. Toisessa kuvauslaitteessa nämä ongelmat on korjattu. Seurantamenetelmä toimii hyvin ottaen huomioon sen yksinkertaisuuden ja siihen ehdotetaan monia parannuksia. Työn tulokset osoittavat, että tosiaikainen mosaiikkikuvan koostaminen megapikselin kuvamateriaalista on mahdollista kuluttajille tarkoitetulla tietokonelaitteistolla.

Bioeffects of albumin-encapsulated microbubbles and real-time myocardial contrast echocardiography in an experimental canine model

Myocardial contrast echocardiography has been used for assessing myocardial perfusion. Some concerns regarding its safety still remain, mainly regarding the induction of microvascular alterations. We sought to determine the bioeffects of microbubbles and real-time myocardial contrast echocardiography (RTMCE) in a closed-chest canine model. Eighteen mongrel dogs were randomly assigned to two groups. Nine were submitted to continuous intravenous infusion of perfluorocarbon-exposed sonicated dextrose albumin (PESDA) plus continuous imaging using power pulse inversion RTMCE for 180 min, associated with manually deflagrated high-mechanical index impulses. The control group consisted of 3 dogs submitted to continuous imaging using RTMCE without PESDA, 3 dogs received PESDA alone, and 3 dogs were sham-operated. Hemodynamics and cardiac rhythm were monitored continuously. Histological analysis was performed on cardiac and pulmonary tissues. No hemodynamic changes or cardiac arrhythmias were observed in any group. Normal left ventricular ejection fraction and myocardial perfusion were maintained throughout the protocol. Frequency of mild and focal microhemorrhage areas in myocardial and pulmonary tissue was similar in PESDA plus RTMCE and control groups. The percentages of positive microscopical fields in the myocardium were 0.4 and 0.7% (P = NS) in the PESDA plus RTMCE and control groups, respectively, and in the lungs they were 2.1 and 1.1%, respectively (P = NS). In this canine model, myocardial perfusion imaging obtained with PESDA and RTMCE was safe, with no alteration in cardiac rhythm or left ventricular function. Mild and focal myocardial and pulmonary microhemorrhages were observed in both groups, and may be attributed to surgical tissue manipulation.

Real time B-mode ultrasound in pacas pregnancy (Agouti paca, Linnaeus, 1766)

O objetivo deste estudo foi estabelecer o tempo de prenhez da paca por meio de ultra-sonografia. Nove pacas prenhes foram periodicamente acompanhadas com um transdutor eletrônico setorial bi-frequencial de 5,0 e 7,5 MHz, em modo B, desde a detecção ultra-sonográfica da vesícula embrionária ou do feto até o nascimento dos filhotes. Os animais foram colocados em uma gaiola de ferro de prensagem lateral e permaneceram em posição quadrupedal durante as sessões. Um pano escuro foi usado para cobrir a gaiola e frutas foram oferecidas durante o exame ultra-sonográfico para evitar reações agressivas. Quanto mais precocemente ocorreu a detecção de prenhez, maior foi o período de acompanhamento ultra-sonográfico até o nascimento dos filhotes. Apenas um filhote nasceu por parto, com 796,5 ± 74,36 gramas (valor médio ± desvio padrão da amostra) e 33,46 ± 0,60 centímetros (valor médio ± desvio padrão da amostra) de comprimento (entre a borda rostral do focinho e a extremidade distal da cauda). O período de prenhez da paca abrange 135 a 139 dias.

Value of adenosine infusion for infarct size determination using real-time myocardial contrast echocardiography

Abstract Background Myocardial contrast echocardiography has been used for determination of infarct size (IS) in experimental models. However, with intermittent harmonic imaging, IS seems to be underestimated immediately after reperfusion due to areas with preserved, yet dysfunctional, microvasculature. The use of exogenous vasodilators showed to be useful to unmask these infarcted areas with depressed coronary flow reserve. This study was undertaken to assess the value of adenosine for IS determination in an open-chest canine model of coronary occlusion and reperfusion, using real-time myocardial contrast echocardiography (RTMCE). Methods Nine dogs underwent 180 minutes of coronary occlusion followed by reperfusion. PESDA (Perfluorocarbon-Exposed Sonicated Dextrose Albumin) was used as contrast agent. IS was determined by RTMCE before and during adenosine infusion at a rate of 140 mcg·Kg-1·min-1. Post-mortem necrotic area was determined by triphenyl-tetrazolium chloride (TTC) staining. Results IS determined by RTMCE was 1.98 ± 1.30 cm2 and increased to 2.58 ± 1.53 cm2 during adenosine infusion (p = 0.004), with good correlation between measurements (r = 0.91; p < 0.01). The necrotic area determined by TTC was 2.29 ± 1.36 cm2 and showed no significant difference with IS determined by RTMCE before or during hyperemia. A slight better correlation between RTMCE and TTC measurements was observed during adenosine (r = 0.99; p < 0.001) then before it (r = 0.92; p = 0.0013). Conclusion RTMCE can accurately determine IS in immediate period after acute myocardial infarction. Adenosine infusion results in a slight better detection of actual size of myocardial damage.

Information Filtering for Ultrasound-based Real-time Registration

This paper presents methods based on Information Filters for solving matching problems with emphasis on real-time, or effectively real-time applications. Both applications discussed in this work deal with ultrasound-based rigid registration in computer-assisted orthopedic surgery. In the first application, the usual workflow of rigid registration is reformulated such that registration algorithms would iterate while the surgeon is acquiring ultrasound images of the anatomy to be operated. Using this effectively real-time approach to registration, the surgeon would then receive feedback in order to better gauge the quality of the final registration outcome. The second application considered in this paper circumvents the need to attach physical markers to bones for anatomical referencing. Experiments using anatomical objects immersed in water are performed in order to evaluate and compare the different methods presented herein, using both 2D as well as real-time 3D ultrasound.

Radio-guided surgery: advantages of a new portable γ-camera (Sentinella(®) ) for intraoperative real time imaging and detection of sentinel lymph nodes in cutaneous malignancies

The histological status of the sentinel lymph node (SLN) is one of the most relevant prognostic factors for the overall survival of patients with cutaneous malignancies, independent of tumour depth of the primary tumour.

Relative indexes of cutaneous blood perfusion measured by real-time laser Doppler imaging (LDI) in healthy volunteers.

We used real-time LDI to study regional variations in microcirculatory perfusion in healthy candidates to establish a new methodology for global perfusion body mapping that is based on intra-individual perfusion index ratios. Our study included 74 (37 female) healthy volunteers aged between 22 and 30 years (mean 24.49). Imaging was performed using a recent microcirculation-imaging camera (EasyLDI) for different body regions of each volunteer. The perfusion values were reported in Arbitrary Perfusion Units (APU). The relative perfusion indexes for each candidate's body region were then obtained by normalization with the perfusion value of the forehead. Basic parameters such as weight, height, and blood pressure were also measured and analyzed. The highest mean perfusion value was reported in the forehead area (259.21APU). Mean perfusion in the measured parts of the body correlated positively with mean forehead value, while there was no significant correlation between forehead blood perfusion values and room temperature, BMI, systolic blood pressure and diastolic blood pressure (p=0.420, 0.623, 0.488, 0.099, respectively). Analysis of the data showed that perfusion indexes were not significantly different between male and female volunteers except for the ventral upper arm area (p=.001). LDI is a non-invasive, fast technique that opens several avenues for clinical applications. The mean perfusion indexes are useful in clinical practice for monitoring patients before and after surgical interventions. Perfusion values can be predicted for different body parts for patients only by taking the forehead perfusion value and using the perfusion index ratios to obtain expected normative perfusion values.

Real-Time Imaging of the Axonal Transport of Granules Containing a Tissue Plasminogen Activator/Green Fluorescent Protein Hybrid

A hybrid protein, tPA/GFP, consisting of rat tissue plasminogen activator (tPA) and green fluorescent protein (GFP) was expressed in PC12 cells and used to study the distribution, secretory behavior, and dynamics of secretory granules containing tPA in living cells with a neuronal phenotype. High-resolution images demonstrate that tPA/GFP has a growth cone-biased distribution in differentiated cells and that tPA/GFP is transported in granules of the regulated secretory pathway that colocalize with granules containing secretogranin II. Time-lapse images of secretion reveal that secretagogues induce substantial loss of cellular tPA/GFP fluorescence, most importantly from growth cones. Time-lapse images of the axonal transport of granules containing tPA/GFP reveal a surprising complexity to granule dynamics. Some granules undergo canonical fast axonal transport; others move somewhat more slowly, especially in highly fluorescent neurites. Most strikingly, granules traffic bidirectionally along neurites to an extent that depends on granule accumulation, and individual granules can reverse their direction of motion. The retrograde component of this bidirectional transport may help to maintain cellular homeostasis by transporting excess tPA/GFP back toward the cell body. The results presented here provide a novel view of the axonal transport of secretory granules. In addition, the results suggest that tPA is targeted for regulated secretion from growth cones of differentiated cells, strategically positioning tPA to degrade extracellular barriers or to activate other barrier-degrading proteases during axonal elongation.

Comparison between real-time three-dimensional echocardiography, T1-201 single photon emission computed tomography and cardiac magnetic resonance imaging in the quantitative assessment of left ventricular volumes

Left ventricular (LV) volumes have important prognostic implications in patients with chronic ischemic heart disease. We sought to examine the accuracy and reproducibility of real-time 3D echo (RT-3DE) compared to TI-201 single photon emission computed tomography (SPECT) and cardiac magnetic resonance imaging (MRI). Thirty (n = 30) patients (age 62±9 years, 23 men) with chronic ischemic heart disease underwent LV volume assessment with RT-3DE, SPECT, and MRI. Ano vel semi-automated border detection algorithmwas used by RT-3DE. End diastolic volumes (EDV) and end systolic volumes (ESV) measured by RT3DE and SPECT were compared to MRI as the standard of reference. RT-3DE and SPECT volumes showed excellent correlation with MRI (Table). Both RT- 3DE and SPECT underestimated LV volumes compared to MRI (ESV, SPECT 74±58 ml versus RT-3DE 95±48 ml versus MRI 96±54 ml); (EDV, SPECT 121±61 ml versus RT-3DE 169±61 ml versus MRI 179±56 ml). The degree of ESV underestimation with RT-3DE was not significant.

Real-time imaging of human cortical activity evoked by painful esophageal stimulation

Background & Aims: Current models of visceral pain processing derived from metabolic brain imaging techniques fail to differentiate between exogenous (stimulus-dependent) and endogenous (non-stimulus-specific) neural activity. The aim of this study was to determine the spatiotemporal correlates of exogenous neural activity evoked by painful esophageal stimulation. Methods: In 16 healthy subjects (8 men; mean age, 30.2 ± 2.2 years), we recorded magnetoencephalographic responses to 2 runs of 50 painful esophageal electrical stimuli originating from 8 brain subregions. Subsequently, 11 subjects (6 men; mean age, 31.2 ± 1.8 years) had esophageal cortical evoked potentials recorded on a separate occasion by using similar experimental parameters. Results: Earliest cortical activity (P1) was recorded in parallel in the primary/secondary somatosensory cortex and posterior insula (∼85 ms). Significantly later activity was seen in the anterior insula (∼103 ms) and cingulate cortex (∼106 ms; P = .0001). There was no difference between the P1 latency for magnetoencephalography and cortical evoked potential (P = .16); however, neural activity recorded with cortical evoked potential was longer than with magnetoencephalography (P = .001). No sex differences were seen for psychophysical or neurophysiological measures. Conclusions: This study shows that exogenous cortical neural activity evoked by experimental esophageal pain is processed simultaneously in somatosensory and posterior insula regions. Activity in the anterior insula and cingulate - brain regions that process the affective aspects of esophageal pain - occurs significantly later than in the somatosensory regions, and no sex differences were observed with this experimental paradigm. Cortical evoked potential reflects the summation of cortical activity from these brain regions and has sufficient temporal resolution to separate exogenous and endogenous neural activity. © 2005 by the American Gastroenterological Association.