Evaluation of the photodynamic therapy effect using a tumor model in chorioallantoic membrane with melanoma cells

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1 Universidade de São Paulo Biblioteca Digital da Produção Intelectual - BDPI Departamento de Física e Ciências Materiais - IFSC/FCM Artigos e Materiais de Revistas Científicas - IFSC/FCM 2014 Evaluation of the photodynamic therapy effect using a tumor model in chorioallantoic membrane with melanoma cells Proceedings of SPIE,Bellingham : International Society for Optical Engineering - SPIE,v. 8931, p B B-7, Downloaded from: Biblioteca Digital da Produção Intelectual - BDPI, Universidade de São Paulo

2 Evaluation of the Photodynamic Therapy effect using a tumor model in Chorioallantoic Membrane with Melanoma Cells. Hilde Harb Buzzá a *; Layla Pires a ; Vanderlei S. Bagnato a ; Cristina Kurachi a a São Carlos Institute of Physics, University of São Paulo, Avenida Trabalhador São Carlense, 400, CEP: , Caixa Postal: 369, São Carlos, SP, Brazil *Corresponding author: Phone: adress: hilde.buzza@usp.br ABSTRACT Photodynamic Therapy (PDT) is a type of cancer treatment that is based on the interaction of light (with specific wavelength), a photosensitizing agent and molecular oxygen. The photosensitizer (PS) is activated by light and reacts with oxygen resulting in the production of singlet oxygen that is highly reactive and responsible for the cell death. The Chick Chorioallantoic Membrane (CAM) model is a transparent membrane that allows visualization and evaluation of blood vessels and structural changes, where a tumor model was developed. Two induction tumor models were investigated: tumor biopsy or cell culture. It was used a murine melanoma cell B16F10 in culture and a biopsy from a xenograft tumor in hairless mouse. Two PS were tested: Photodithazine and Photogem, a chlorine and porphyrin compounds, respectively. Using intravenous administration, the light-drug interval was of 30 minutes, 1 and 3 hours. Illumination was performed at 630 nm and 660 nm, and the vascular and tumor response was monitored and analyzed. The PS distribution was checked with confocal microscopy. This model can be useful to study several parameters of PDT and the effect of this therapy in the cancer treatment since it allows direct visualization of its effects. Keywords: Photodynamic Therapy, Chorioallantoic Membrane, Tumor model, Vascular Effect, Photosensitizers 1 INTRODUCTION Cancer is the term used to characterize more than 100 types of diseases that can affect several parts of the human body. The cell inability of differentiation and the cell s disorderly growth are common features of diseases classified, and, by his aggressiveness, form tumors such as malignant tumors. 1 Among the major current challenges today are diagnostic and control of such neoplasm, including the development and improvement of therapeutic modalities, as photodynamic therapy. Angiogenesis is a phenomenon characterized by the formation of new blood vessels and is closely related to several diseases, such as cancer, rheumatoid arthritis and psoriasis. Folkman [1971] described the relationship between this phenomenon and the cancer, showing that a process of tumor numbness occurs in the absence of blood vessels. The solid tumor growth and metastasis formation are closely related to the mechanisms of angiogenesis. 2-4 There has been a growing clinical interest in techniques that aim at the destruction of tumor cells associated with the blockage of neovascularization 3. Optical Methods for Tumor Treatment and Detection: Mechanisms and Techniques in Photodynamic Therapy XXIII, edited by David H. Kessel, Tayyaba Hasan, Proc. of SPIE Vol. 8931, 89310B 2014 SPIE CCC code: /14/$18 doi: / Proc. of SPIE Vol B-1

3 The chorioallantoic membrane (CAM) model with chicken eggs is probably the most widely used in vivo model for the study of both angiogenesis and activity of compounds and drugs in the vascular endothelium. It is a well established model that has facilitated the access to blood vessels and the embryo and is simple, inexpensive and of easy installation in laboratory. 5-9 It involves no pain, since pain receptors are developed from 14th day of embryo development and this is the great advantage of this model. The studies are made between the 11th and 14th of development, during which the blood vessels are in proper size without causing any distress to the embryo. Therefore, it is considered an alternative method for the animal use. 10 The chorioallantoic membrane model enables the introduction of several cell lines, including tumor cells, because it has a favorable immune system and provides a source of nutrition for these cells. The tumor microvasculature obtained must have been formed around it and allows the study of angiogenesis and the processes involved when the tumor becomes viable. Therefore, it is an excellent model for the study of anti-angiogenic and anti-tumor activities and enables the evaluation of the response directly and in real time. 11 The Photodynamic Therapy (PDT) requires the interaction among the light (with a specific wavelength), the nontoxic compound (Photosensitizer) and the molecular oxygen, which results in reactive species able to induce infeasibility of cells. There are several types of photosensitizers, including porphyrin and chlorine compounds. When the tumor cells with the photosensitizer are illuminated, the photosensitizer absorbs light and reacts with the cell oxygen, producing radical species (singlet oxygen) that lead to the cell death The improvement in this technical in the treatment of cancer involves the knowledge about cell death mechanisms, angiogenesis and destruction of blood vessels that nourish the tumor. Therefore a model with direct access to the whole tumor and blood vessels enables the analysis of therapy efficiency and the improvement in therapeutic modalities. 2 MATERIAL AND METHODS 2.1 Chorioallantoic Membrane Model (CAM Model) A local producer provided the eggs for the experiments (AD ORO S.A., SãoCarlos/SP, Brazil). On the first day of development the eggs were cleaned with alcohol 70% and were kept at 37.7 C in an incubator with a slow rotation. On day 3 the rotation was stopped and a window of approximately 2 cm² was opened using by a tweezers and was sealed with adhesive tape. They returned to the incubator until the introduction of tumor cells. All procedures were conducted inside the laminar flow to avoid contamination. 2.2 Tumor introduction in the CAM model The tumor was introduced in the CAM on the 4 or 5 day of the embryo development using two different ways: tumor cell in culture and solid tumor. For the culture cells, 35 µl of culture medium with melanoma cells were used totaling 3x10 5 cells per egg after counting in a chamber Neubauer. The cells were placed on the membrane and among the layers using an insulin syringe with 30G gauge. The cells can be introduced in hairless mice and a tumor solid is developed on their skin. With the aid of a scalpel, the tumor was removed and sliced into pieces of 2-3 mm diameter, which were placed on the membrane, near the small vessels. 2.3 Photodynamic Therapy Two photosensitizers, Photogem and Photodithazine (chlorine and porphyrin compounds, respectively) were administrated intravenously with an insulin syringe with a 29G gauge needle. The concentration of PS was 0.2 µl/cm² and the volume injected was 500 µl. The light sources employed were diode lasers (EagleEaron, Quantum Tech, Proc. of SPIE Vol B-2

4 Brazil) changing the wavelength according with each photosensitizer nm for Photogem and 660 nm for Photodithazine. The light dose was 100 mw/cm² totaling dose of 30 J/cm². The PDT was applied to the CAM without tumor for the analysis of the effect of the parameters on the blood vessels. After the parameters had been adjusted, PDT was applied to the tumor model. Using intravenous administration, the light-drug interval was of 30 minutes, 1 and 3 hours. The illumination of the vascular and tumor response was monitored and analyzed. Images were captured with USB Digital Microscope (AVANTGARDE, China). Fluorescence images over time after application of photosensitizer and light have been done using the Evince equipment (MMOptics, Brazil), emitting in 408 nm. 2.4 Preparation of microscopic sample 2 ml of formalin solution (formaldehyde 37%) were placed on the chorioallantoic membrane and 30 minutes later the embryo died. The membrane was removed by tweezers and scissors. The tweezers raised the membrane and the scissors cut the portion of the membrane containing the tumor and blood vessels for analysis on the confocal microscope. 3 RESULTS AND DISCUSSION 3.1 Tumor model The application of the tumor to the chorioallantoic membrane enabled the evaluation of tumor growth by the introduction of these various forms of cells and formation of new vessels in the tumor. A survival rate of approximately 20% was obtained after the introduction of tumor cells using both ways. Figure 1 shows white light photograph the tumor model with the introduction of cell culture and how tumors can develop in different ways. Figure 1-a shows a cell concentrated around the blood vessel, but no formation of tumor mass. Figure 2-b illustrates the formation of the tumor mass and the membrane cut for microscopic analysis. Figure 1. a) Melanoma cells concentrated around the vessel in the egg. b) Membrane cut with a tumor (in box) to analysis. Confocal microscopic images were obtained for the analysis of the interaction between blood vessels and tumor cells. Figure 2 shows the formation of blood vessels near the tumor developed from a piece of melanoma biopsy. This behavior is expected because the vessels are responsible for the tumor nutrition and, therefore, the cells induce the angiogenesis. Proc. of SPIE Vol B-3

5 Figure 2. Confocal microscopic of new blood vessels near the cells concentration. The analysis of the confocal microscope also possible to observe the interface between the blood vessel and the cells on the membrane (shown in Figure 3-a). Figure 3-b shows the cells inside the vessel Figure 3. Confocal microscope images a) Membrane image showing the vessel borderline. b) Tumor cell inside the vessel. From the established model, it was possible to apply photodynamic therapy. 3.2 Photodynamic Therapy Photosensitizer is an important factor in photodynamic therapy and its location in the target cells is essential for its efficiency. Therefore, understanding how it arrives in the target cell can contribute to the improvement in the PDT technique. Photogem exhibits a specific fluorescence when excited with UV-blue light fluorescence and images over time after administration of the compound were made with Evince. Figure 4 shows the sequence of fluorescence images. Immediately after the photosensitizer application it was possible to note the fluorescence at the point of injection. After 90 minutes, the entire vessel exhibited the characteristic fluorescence. The egg was monitored for 24 hours and the fluorescence of the vessel was kept but with lower contrast. Other vessels showed a little fluorescence. Proc. of SPIE Vol B-4

6 Before Omin after 90 min after Figure 4. Fluorescence image of CAM as a function of post injection of Photogem. For an individual verification of the vascular effect of photodynamic therapy, Photogem and Photoditazine were injected into the vessel followed by lighting. Both photosensitizers have had similar action at the vascular network showing marked vessel destruction. Figure 5 shows this reduction with Photgem 150 minutes after PDT. The reduction was accentuated and the larger diameter vessels remained. These vessels, however, suffered a reduction in this diameter. Before 150 min after Figure 5. Images of CAM as a function of post-pdt time with Photogem (0,2 mg/ml) showing the destruction of the vascular network. With the parameters determined in the CAM without tumor, PDT was applied to the tumor model, with death of all embryos in the waiting drug-light interval, which undermined the analysis of the PDT action. Therefore, the next step of this research is the analysis of PDT application in the tumor model with confocal microscopic. 4 CONCLUSION The tumor model using chorioallantoic membrane can be useful for the study of several parameters of PDT and the effect of this therapy on the treatment of cancer since it allows direct visualization of its effects. Confocal microscopy is a powerful tool for the analysis the interaction of new blood vessels and cells. Furthermore, monitoring the photosensitizer throughout the blood vessel enables the knowledge of its distribution and the improvement of PDT. 5 ACKNOWLEDGMENTS The authors acknowledge the financial support provided by FAPESP (CEPOF-CEPID Program), CAPES (HH Buzzá scholarship), and A DORO S.A. (donation of eggs). Proc. of SPIE Vol B-5

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