Raport stiintific privind implementarea proiectului in perioada ianuarie – decembrie 2013

Clinical and Biomathematical Modeling of Vascular Changes Following

Anti-Angiogenic Therapy in Advanced Colorectal Carcinoma

Activity report for January - September 2013

Project Director: Dr. Gabriel Gruionu

ABSTRACT: Colorectal carcinoma (CRC) represents an important health burden, being the third leading cause of cancer death in the world. The cancer-stromal cell interaction contributes directly to tumor growth and metastasis by creating an imbalance of positive and negative growth factors and increased microvascular density via angiogenesis. Recently, advances in chemotherapy and biological therapies targeting the angiogenic growth factors offered additional hope for treatment but the exact biological mechanisms are still not clear. Therefore, the long-term goal of the proposed research is to gain improved understanding of the mechanisms by which tumor microvascular networks (MVN) respond to chemotherapy and/or anti-angiogenic therapy. To address this, the first objective is to map the MVN and cellular components of normal colorectal tissue and CRC tumors before and after chemotherapy and/or anti-angiogenic treatment in CRC patients. Changes in morphometric and hemodynamic parameters of MVN will be observed with a novel combination of minimally invasive ultrasound and confocal imaging techniques (CE-EUS and CLE) correlated with the type and dosage of therapy. The second objective is to develop computer simulations of blood flow and structural adaptation of the MVN in CRC tumors. The predictions of the model will be compared with the observed changes in the MVN structure following chemo- and anti-angiogenic treatment. The project will have a significant scientific and social impact leading to a better categorization and prediction of patient’s response to treatment before exposure to chemotherapy or surgery. Specific activity calendar:

Activity A 1.1 Set-up of a structured database and definition of the experimental models Progress: A complex database was set up on a dedicated computer and back up to an external hard drive to include all the identifying information (age, ID, location, sex), imaging

M1-3 M4-6 M7-9 M10-11 M12-14 M15-17 M18-20 M21-23 M24-26 M27-29 M30-32 M33-35

A 3.1 Development of the mathematical model of normal and untreated CNC MVN

A 4.2 Forecasting angiogenesis during and after antiangiogenic treatment

A 1.1 Set-up of a structured database and definition of the experimental models

A 1.2 Protocol-based inclusion of patients (CE-EUS & CLE), tissue sampling

A 2.1 Pathological and immunohistochemical analysis of biopsy samples

A 2.3 Mathematical model of the anti-angiogenic treated MVN

procedure, biopsy location, diagnosis, treatment dosage and final evaluation (see attached database). A1.2. Protocol based inclusion of patients (CE-EUS and CLE) and data sampling. Progress: Up to date, over 40 patients were enrolled in the study (age 49-76 years). The consent forms that every patient has to sign before the beginning of the study were attached to the previous report. For CLE examination we use the dedicated system (EC-3870 CIFK, Pentax, Tokyo, Japan) which has a miniature confocal microscope integrated into the distal tip of a conventional flexible endoscope. The endomicroscope uses a laser bean with an excitation wavelength of 488 nm and a maximum laser power output at the surface of the targeted tissue of ≤1mW. This results in optical sections of 475x475µm2 with a lateral resolution of 0.7 µm for a 7 µm thick slice (z-axis). CLE imaging is performed on fresh biopsies of both normal mucosa and colorectal cancer tissue obtained during lower GI endoscopic procedures. The biopsies undergo a staining protocol, including incubation (1h, 37°C, 1:10 dilution) with Alexa-Fluor 488 labeled anti-CD31 antibodies. Scanning is performed with the biopsies on histology glass slides, placed in direct and gentle contact with the distal tip of the confocal laser endomicroscope. Consecutive images are captured and digitally stored on the system’s hard drive as grey-scale images (250-300 for each biopsy sample) for later download and processing. The imaging procedures are backed by immunohistochemical studies of corresponding biopsies for evaluating tumor vascular patterns and performing a thorough morphometric analysis. Compared to the previous article publishes in PLoS One, the new article introduced new morphometric parameters and demonstrated for the first time that tumor vessels are not actually more tortuous than the normal tissue vessels. The results were presented recently at the United European Gastroenterology Week (UEGW) international conference conference and submitted for publication in the PLoS ONE journal on November 12th, 2013 (see conference reference [1], manuscript abstract bellow and full manuscript in attachement). Evaluation of new morphometric parameters of neoangiogenesis in human colorectal cancer using confocal laser microscopy (CLE) and targeted panendothelial markers The tumor microcirculation is characterized by an abnormal vascular network with dilated, tortuous, and saccular vessels. Therefore, imaging the tumor vasculature and determining its morphometric characteristics represent a critical goal for optimizing the cancer treatment that targets the blood vessels (i.e. antiangiogenesis therapy). The aim of this study was to evaluate new vascular morphometric parameters in colorectal cancer, difficult to achieve through conventional immunohistochemistry, by using the confocal laser endomicroscopy method. Fresh biopsies from tumor and normal tissue were collected during colonoscopy from five patients with T3 colorectal carcinoma without metastasis and were marked with fluorescently labeled anti-CD31 antibodies (Figure 1). A series of optical slices spanning 250 µm inside the tissue were immediately collected for each sample using a confocal laser endomicroscope. All measurements were expressed as the mean ± standard error. The mean diameter of tumor vessels was significantly larger than the normal vessels (9.46±0.4 µm vs. 7.60±0.3 µm p=0.0166). The vessel density was also significantly higher in the cancer vs. normal tissue samples (5541.05±262.81 vs.3755.79 ±194.96 vessels/mm3, p=0.0006). There results were

confirmed by immunohistochemistry. In addition, the tortuosity index and vessel lengths were not significantly different (1.06±0.016 and 28.78±3.27 µm in normal tissue, vs. 1.07±0.008 and 24.65±3.18 µm in tumor tissue respectively, p=0.4673 and p=0.7033). The daughter/mother ratio (ratio of the sum of the squares of daughter vessel radia over the square of the mother vessel radius) was 1.11±0.09 in normal tissue, and 1.10±0.08 in tumor tissue (p=0.6497) (Table 1). The confocal laser endomicroscopy is feasible for measuring more vascular parameters from fresh tumor biopsies than conventional immunohistochemistry alone. Provided new contrast agents will be clinically available, future in vivo use of CLE could lead to identification of novel biomarkers based on the morphometric characteristics of tumor vasculature.

Figure 1. Ex-vivo CLE Vascular measurements of Vessel Length and Tortuosity Index. The Z projection of the image stack for normal (A) and tumor (B) microvasculature, apparently with more tortuous branched vessels. The vessel length was measured using segmented line tool and reported in micrometres. The tortuosity index was calculated as the ratio of segmented and straight line lengths for both normal (C) and malignant (D) human colorectal tissue. *The scale bar is 50 µm.

Morphometric Parameters   Normal Mucosa Colorectal Tumor P Value

Vessel Diameter (µm) 7.60±0.3 9.46±0.4 0.0166

Vessel Density (vessels/mm3) 3755.79 ±194.96 5541.05±262.81 0.0006

Vessel Length (µm) 28.78±3.27 24.65±3.18 0.7033 Tortuosity Index 1.06±0.02 1.07±0.01 0.4673 Daughter/Mother

Ratio 1.11±0.09 1.10±0.08 0.6497

Table 1. Average values of the morphometric parameters measured with CLE. The values are expressed as the mean ± standard error. CE-EUS study We have enrolled 35 patients in the CE-EUS study and continue to analyze the data. A preliminary report was presented at the recent UEGW conference by Dr. Tatiana Cartana [2]. Title: Quantitative assessment of tumour perfusion of colorectal cancer patients by using Contrast-enhanced endoscopic ultrasonography: a feasibility study Introduction: Contrast enhanced endoscopic ultrasonography (CE-EUS) is a high resolution technique enabling minimally invasive assessment of tumour perfusion. Despite recent technology advancements, the use of CE-EUS in the evaluation of colorectal cancer (CRC) has not been previously reported. Aims & Methods: Therefore the aim of our study was to evaluate tumour vascularity in CRC by using CE-EUS and time intensity curve analysis in correlation with pathology parameters of angiogenesis. We included 35 patients with CRC that were examined by low mechanical index CE-EUS prior to any therapy, using 4.8 ml of Sonovue (Bracco, Italy) administered in bolus injection as contrast agent. Time intensity curve (TIC) parameters were determined by offline analysis of recorded video sequences with specific software (Fig. 2). Immunohistochemical assessment of tumour vascularization included microvascular density calculation using CD31 and CD105 specific staining, which was available for 18 of the patients.

Figure 2. Offline video analysis - Image-Pro Plus (Media Cybernetics, Bethesda, SUA Results: Most tumours were well vascularized at CE-EUS examination, demonstrating either homogenous uptake of the contrast agent or inhomogeneous enhancement, with stronger peripheral uptake and avascular areas towards the intestinal lumen. The mean values (± SD) for TIC parameters were: 10.08 ± 3.85 s for arrival time (AT), 24.03 ± 10.94 s for time to peak (TTP), 41.43 ± 19.24 a.u. for peak intensity (PI) and 5477.45 ± 2922.68 a.u.*s for the area under the curve (AUC) (Table 1). An inverse correlation was noted between AT and CD31 MVD, but without reaching statistical significance (Spearman r = -0.55, p= 0.1328) and also between TIC parameters Imax and AUC and lymph nodes involvement (r = -0.439, p = 0.0683).

TIC Parameter Mean value ± SD Min - Max AT (s) 10.08 ± 3.85 4.17 - 16.67 TTP (s) 24.03 ± 10.94 9.33 - 56.17 PI (a.u.) 41.43 ± 19.24 10.1 - 80.1 AUC (a.u.*s) 5477.45 ± 2922.68 830.1 - 11735 Table 1. TIC parameters. a.u. - arbitrary units; AT - arrival time;TTP - time to peak; PI - peak intensity (Imax ); AUC - area under the curve Conclusions: CE-EUS using low mechanical index examination and TIC analysis is feasible for the assessment of intratumoral perfusion in colorectal cancer. Further studies on larger groups of patients are necessary to improve the examination technique and define its role in the evaluation of CRC patients. A2.1. Pathological and immunohistological analysis of biopsies. Progress: The patho-imuno-histological analysis was performed for every patient in parallel with other imaging techniques. All patient biopsies were analysed for diagnostic purposes. The samples were fixed in neutral buffered formalin and were further processed for paraffin inclusion. Aside from ensuring the pathological diagnostic, slides from the paraffin blocks underwent an antigen retrieval procedure and were immunostained for CD31 and CD105. A species specific polymeric HRP secondary antibody was used to amplify the reaction, and the signal was finally visualised using a specific precipitating HRP substrate (DAB, 3,3'-Diaminobenzidine). After image acquisition under a light microscope, the microvessel density (MVD) was assessed based on direct counting of areas with the highest vascular populations, according to the classically hotspot method. Furthermore total vascular areas were also calculated on the captured images using a stylus design tablet in order to obtain a fine delimitation of the signal and vascular lumens. After area-normalisation, both parameters were compared with CLE and EUS measurements. The results are included in the individual studies for each imaging method. Activity 3.1. Development of the mathematical model for the normal and treated CRC MVN (colorectal cancer microvascular network). Aim of the study: To develop computer simulations of blood flow and structural adaptation of the MVN in CRC tumors. Progress: The first and second parts of the mathematical modeling are underway as described below. The results were presented at the Annual Meeting of the Biomedical Engineering Society and published in the abstract form in the Annals of Biomedical Engineering Journal. The extended version of the results will be submitted to the Annals of Biomedical Engineering Journal or similar. Algorithm for the three-dimensional visualization of the vascular network. The 3D model was developed from B&W serial sections using manual tracing of bifurcation points and Matlab and SolidWorks software packages as described before (Gruionu et al. Am. J. Physiol., 2005) (Fig. 3).

Numerical modeling of the vascular network. To present a detailed distribution of vascular flow and pressure on overall tumor network, a finite elements model and simulation was developed using the three-dimensional geometry and as inputs the previous inflows and outflows. The model was used to evaluate the interstitial pressure in the CRC tumor and the relationship with the anti-angiogenic treatment. Diffusion simulation was performed using ANSYS CFX module on a porous domain that involves one fluid and a solid. A full porous model formulation was used with a porosity that modifies all terms in the governing equations as well as the loss term (Figure 2). This method supports models for the interaction between the fluid and solid parts of the domain. The results were presented at the 2012 Annual Biomedical Engineering Society [5].

A more extensive mathematical model of a normal and tumor vascular network was presented at the Experimental Biology [3] and Biomedical Engineering Society Annual meetings [4] and prepared for publication in Vascular Research journal (see abstracts below). Experimental Biology Annual Meeting: Flow-Induced Structural Adaptation of Tumor Vasculature by Selective Micro-Laser Ablation Gabriel Gruionu1, Lucian Gruionu, Lance L. Munn

Blood velocity

Normal Tumor

Figure 2. Blood velocity and diffusion model

Normal Tumor

Figure 3. 3D network model

The effects of anti-angiogenesis therapy on tumor vasculature are transient, and the exact mechanisms of vessel remodeling are not known. Here we developed experimental and theoretical models of tumor vascular remodeling for customizing the dose and timing of vessel-targeted drugs. We used a multiphoton laser to interrupt vessel segments downstream from a bifurcation and observed remodeling in the adjacent branches in normal skin vasculature and AK4.4 pancreatic tumors implanted in the mouse dorsal skin fold chamber. We assessed blood flow and cellular dynamics using fluorescence stereomicroscopy and optical frequency-domain imaging. The theoretical simulation of the vascular network dynamics is based on a previous network model developed by Gruionu et al (Am J Physiol, 2005). A finite element model was used to simulate oxygen and nutrient delivery in the surrounding tissue. The laser microsurgery caused significant remodeling in both arterial (mean: 151%, max: 229%) and venous (mean: 153%, max: 581%) sides of the circulation. The predictions of the model showed good correlation with the observed changes in the dorsal skinfold chamber. The models will help us define the contribution of local stimuli to remodeling of tumor vascular beds and generate predictions for altering tumor blood flow and development of future therapies. Biomedical Engineering Society Annual Meeting: Title: Selective Vascular Blockage by Multiphoton Laser Ablation Causes Flow-Induced Remodeling in Tumor Blood Vessels Introduction Normal vasculature adapts in response to changes in flow in normal and pathological conditions [1]. Numerous studies show that tumor vasculature undergoes similar structural changes in response to anti-angiogenesis treatment, improving flow in tumors, but little is known about the mechanisms of flow-induced vascular remodeling in tumors. To address this issue, we have created a novel experimental model of flow induced remodeling by altering flow in selected tumor feeding vessels. Materials and Methods. We used a custom built multiphoton laser to selectively ablate small arterial and venous vessel segments (20-100mm) through the glass window of the dorsal skin fold chamber in a novel model of RAG1 double transgenic mice which express GFP in Tie-2-positive cells and Ds red in α-SMA-positive cells. An orthotropic tumor was induced by implanting a 30ml construct consisting of Mu89 human melanoma cells embedded in a fibrin gel in the center of the dorsal chamber. We assessed diameter, length, and blood flow changes before and after ablation using a new adaptation of Optical Frequency-Domain Imaging (OFDI) that provides blood flow measurements. The simulation of blood flow was based on the observed structure of the arterial network and a previous structural adaptation model developed by Gruionu et al. The model simulates the adaptation of vascular diameters resulting from the combined action of hemodynamic (wall shear stress and intravascular pressure) and metabolic signals (Fig. 4). In addition to the flow simulation, a finite element model was developed using the ANSYS and SolidWorks software packages to develop a 3D visualization of vascular elements and simulation of blood flow.

Figure 4. Experiental and theoretical model of the blood flow in a microcirculatory network. Results and Discussion. Arteries and veins were interrupted downstream from a bifurcation and the resulting vascular remodeling was observed in the adjacent branches and parent vessels. The laser microsurgery caused significant remodeling in both arterial and venous sides of the circulation with a more pronounced remodeling on the venous side. Morphometric parameters were imported into the mathematical model and the entire vascular network was reconstructed. The predictions of the model showed good correlation with the observed changes in the dorsal skinfold chamber. Conclusions. The present experimental and theoretical models show that flow induced remodeling plays a significant role in tumor vascular beds; this process may be a viable target for development of future therapies. Bibliography:

1. Adriana Ciocâlteu, Adrian Săftoiu, Tatiana Cârţână, Lucian Gheorghe Gruionu, Dan I. Gheonea, Daniel Pirici, Claudia- Valentina Georgescu, Gabriel Gruionu. Evaluation of new morphometric parameters of neoangiogenesis in human colorectal cancer using confocal laser microscopy (CLE) and targeted panendothelial markers. UEGW, Berlin, Germany, 2013 (submitted for publication to PLoS ONE journal).

2. T. Cartana , L. Brink, D. I. Gheonea, J. G. Karstensen, A. Ciocalteu, D. Pirici, C. V. Georgescu, M. L. Malmstrøm A. Saftoiu, P. Vilmann G. Gruionu. Quantitative assessment of tumour perfusion of colorectal cancer patients by using Contrast-enhanced endoscopic ultrasonography: a feasibility study. United European

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THEORETICAL SIMULATION OF ENGINEERED VASCULAR NETWORKS

Initial Flow Direction Simulated Preablation Flow Simulated Postblation Flow (red - changed flow direction)

Gastroenterology Week (UEGW), 2013, poster presentation Berlin, Germany. 3. Gabriel Gruionu, Lucian Gruionu, Lance L. Munn. Flow-Induced Structural Adaptation

of Tumor Vasculature by Selective Micro-Laser Ablation. Experimental Biology, 2013, Boston, MA, USA.

4. Gabriel Gruionu, Lucian Gruionu, Lance L. Munn. Flow-Induced Remodeling of Normal and Tumor Microvasculature. 2013 Annual Meeting of the Biomedical Engineering Society, Seattle, WA, USA.

Project Director:

Conf. Dr. Gabriel Gruionu