Abstract
Introduction: Tumor angiogenesis is necessary for tumor growth and metastasis. Of the many known inducers that promote tumor angiogenesis, vascular endothelial growth factor (VEGF) plays the most important role. Increased VEGF expression has been found in many tumor types, including non-Hodgkin lymphoma (NHL). Bevacizumab is a humanized monoclonal antibody that recognizes all known isoforms of VEGF. This study aims to design a diagnostic molecular probe, Cy7-Bevacizumab, in which Cy7 is a near-infrared fluorescent dye designed to in-vivo non-invasively identify VEGF expression in NHL tumors.
Methodology: VEGF expression was confirmed using flow cytometry on NHL cell line Toledo. Cy7-Bevacizumab was synthesized through nucleophilic substitution reaction between monofunctional N-hydroxysuccinimide ester (Cy7-NHS) and Bevacizumab [1]. After purification, the conjugate was characterized by SDS-PAGE and spectrophotometry. For in-vivo imaging, Cy7-Bevacizumab (1 nmol) was injected intravenously in NHL tumor-bearing Balb/c nude mice (n=3) and was evaluated with near-infrared fluorescence (NIRF) at 2, 6, 24, 48, 72 and 96 h post-injection. After 96 h the lymph nodes accumulation was analyzed by ex-vivo NIRF images.
Results: Flow cytometry showed that NHL Toledo cell line expresses high levels of VEGF. Bevacizumab was successfully labeled with Cy7, as shown by SDS-PAGE and spectrophotometry. Non-invasively NIRF in-vivo imaging of NHL tumor-bearing Balb/c nude mice allowed identification of tumors up to 96 h post-injection. Ex-vivo NIRF images confirmed tracer deposition at tumors.
Conclusions: NHL Toledo cell line expresses VEGF. Cy7 labeled Bevacizumab has the potential to become an optical imaging agent for VEGF expressing tumors such as NHL, being possibly useful in guiding surgical excision of tumors and biopsies, which merits further evaluation.
Acknowledgments: Agencia Nacional de Innovación e Investigación - Uruguay (ANII), Roche Laboratories, Pro.In.Bio (Uruguay), PEDECIBA Química (Uruguay)) and Comisión Sectorial de Investigación Científica-Universidad de la República-Uruguay (CSIC, UdelaR) I+D Grupos Oncología Nuclear.
Disclosures: No relevant conflicts of interest to declare.
References:
1. Camacho, X; Machado, CL; García, MF; Gambini, JP; Banchero, A; Fernández, M; Oddone, N; Bertolini Zanata, D; Rosal, C; Buschpiguel, CA; Chammas, R; Riva, E; Cabral, P. "Technetium-99m- or Cy7-Labeled Rituximab as an Imaging Agent for Non -Hodgkin Lymphoma". Oncology, 15(92):229-42, 2017.
No relevant conflicts of interest to declare.
