Quantitative Bioluminescent Method Appropriate for Bimodality Analysis To Improve and Follow a Syngenic Murine Model of B-Cell Lymphoma Expressing Human CD20.

The anti-CD20 monoclonal antibody rituximab (C2B8) has shown promising results in the clinical treatment of patients with B-cell Non-Hodgkin’s Lymphoma (B-NHL). However, its therapeutic effect is variable whereas a better knowledge of factors affecting rituximab response could lead to improve its efficacy. It has been suggested that tumour burden could influence rituximab exposure and response in human. The lack of method to assess precisely tumour burden has however prevent to confirm such influence. Study of factors affecting antibody exposure requires bimodality analysis allowing to precise factors related to antibody from those related to tumour. We purposed to develop a quantitative method of tumour burden using bioluminescence imaging (BLI) and simultaneously scintigraphic study of monoclonal antibody biodistribution and exposure (indium-111 labelled ibritumomab, 2B8). Firstly, we developed a murine model of T-lymphoma cells transduced with human CD20 cDNA (EL4-hCD20) and transfected with luciferase plasmid (EL4-hCD20-luc). C57Bl6J mice and C57Bl6J TyrC2-J mice (albino phenotype) were injected with 8.10^3 EL4-hCD20-luc lymphoma cells by i.v. route. All mice died within 30 days with liver, spleen, bone marrow and lymph nodes involvement confirmed by immunohistochemistry and PCR analysis. In order to evaluate tumour growth and dissemination, potassium luciferin (2 or 4 mg) were injected bi-weekly intra peritoneally and tumour burden was analysed using Hamamatsu® CCD imaging system. Background noises and cosmic rays, which disturb the signal of bioluminescence, were eliminated from merging image before bioluminescent activity analysis. The sensitivity of analysis was below than 10^2 El4-hCD20-luc cells and there was proportionality between bioluminescent activity and cells number from 10^3 to 10^9 cells. The use of different binning allowed the follow-up of tumour burden evolution from 9 days after inoculation until the death of mice. We validated the used of black phenotype C57Bl6J compared to C57Bl6J TyrC2-J phenotype and demonstrated that the amount of luciferin could influence tumour growth. Regions of interest were delimited by segmentation method and we developed a reliable quantification method of bioluminescence activity taking into account absorption coefficients of the cells light emitted according to their localization. Such model and quantification method makes possible the follow-up of tumour growth and monoclonal antibody efficacy and exposure studied by bimodality analysis. D.D. is supported by a grant from Région Centre.