Utveckla en in vitro metod för att mäta penetration av antikropp-nuklidkonjugat i tumörvävnad: initiala försök på ovarialcancer

Abstract

Degree project, Programme in Medicine To develop an in vitro method to measure the penetration of antibody-nuclide conjugates in tumor tissue: initial attempts on ovarian cancer. Diana Abona, January 2017 Supervisor: Prof Ragnar Hultborn Department of Oncology. Institute of Clinical Sciences Background. Ovarian cancer is the fifth leading cause of cancer death and the most malignant of gynecological cancer. After standard treatment with surgery and intravenous chemotherapy, 70% of all women will relapse. Therefore it is important to develop new treatment methods for ovarian cancer. Aim. The Targeted Alpha Therapy Group in Gothenburg develops a radioimmunotherapy based on the use of an alpha particle emitting nuclide, 211- Astatine, conjugated with ovarian cancer specific monoclonal antibodies, e.g. MX35. Since the alpha particle track length is only 70 um it is essential to know how deep into tumour cell clusters the antibody-nuclide complex can reach. Thus we wanted to develop a simple in vitro method that enables measuring of how deep into an experimental “micrometastasis”, the vector of the alphaemitting nuclide, i.e. the antibody with various modifications with respect to size and affinity etc., can penetrate. Method. Hollow fibers with a diameter of 1 mm and permeable to molecules up to 500 kD are used. Cultured human ovarian cancer cells, Ovcar-3, in a dense suspension is added into the "fiber" with a pipette. The fiber containng Ovcar-3 is then centrifuged at approximately 3000 g for 15 minutes resulting in a densly compacted cell cylinder, simulating a micrometastsis. The present work describes the procedures used to reach this goal. The use of this highly artificial micrometastasis model will be: The fiber with compacted tumour cells is introduced into a solution containing the murine ovarian cancer specific antibody (MX35). After different exposure times, the fiber with the tumour cell cylinder is fixed in formalin. After usual dehydration, the fiber is embedded in paraffin and cut in 5-10 μm sections. After rehydration, immunohistochemistry is made using anti-mouse IgG antibody with fluorescence or peroxidase analysis to see how deep the antibody penetrated into the compacted cell cylinder. Results. We succeded, after many attempts with different approaches, to produce a relatively well filled fiber with tumour cells. Conclusion. The work has involved several methodological developments with the achievement of basic cell culture techniques and conventional histotechnology. We have not yet fully reached the goal of reproducibly establishing a fully-filled fiber with compacted cells. We will try with cells suspended in a medium of extracellular matrix, as well as to try different degrees of centrifugation, fixation as well as cryosection. Key words: Ovarian cancer, Alpharadioimmunotherapy, Astatine-211