Design and optimization of small animal non-invasive imaging approaches for evaluating the effects of innovative treatments of Primary Central Nervous System Lymphomas

par Jérémie Cosette

Thèse de doctorat en Biologie interdisciplinaire

Sous la direction de Sylvain Fisson et de Patrice Flaud.

Soutenue le 11-07-2014

à Paris 5 , dans le cadre de École doctorale Frontières du vivant .

Le président du jury était Andràs Pàldi.

Le jury était composé de Sylvain Fisson, Patrice Flaud, Andràs Pàldi, Sharon Lori Bridal, Peter Kiesel, Georges Azar.

Les rapporteurs étaient Andràs Pàldi, Sharon Lori Bridal.

  • Résumé

    Primary central nervous system lymphomas (PCNSL) are very aggressive malignancies with poor survival rate even with treatments (survival median is 44 months). This disease affects immune cells (lymphocytes) and forms diffuse and non-surgically removable tumor in the central nervous system. High-dose chemotherapy and radiotherapy are the common treatments with severe side effects. New therapeutic approaches are required for increasing treatment efficiency. We focused on primary intraocular lymphomas (PIOL) and primary cerebral lymphomas (PCL), which are subtypes of PCNSL. PIOL and PCL cells have a high propensity to migrate and form metastases in the brain and in the controlateral eye in the case of PIOL, and in the eye in the PCL case. However, metastatic dissemation mechanisms remain unclear. The objective of the present work was to study the effects of innovative treatments of B-cell lymphoma on primary tumor, on metastases, and on circulating tumor cells in PIOL and PCL immunocompetent syngeneic murine models of lymphomas using non-invasive in vivo imaging methods. We studied the effects of Ublituximab, a glycoengineered anti-CD20 monoclonal antibody (mAb), and CpG-ODN, a TLR-9 agonist, in mouse models. We showed that Ublituximab exhibits significant anti-tumor effect in PIOL and PCL, while CpG showed significant anti-tumor effect in PCL. We monitored the tumor burden and metastases using innovating non-invasive optical imaging or cell detection methods: bioluminescence imaging (BLI) and in vivo flow cytometer (IVFC). BLI was used to locate metastasis and to quantify tumor burden. We indeed developed a bioluminescence-based tumor burden quantification method that reduces user-dependence, allows comparisons between experiments, reveals statistical relevance, and which is easy to use. An IVFC device was set up to investigate the role of circulating tumor cells (CTCs) in PIOL and PCL. This fluorescence-based technique allows detection of CTCs by analyzing the cells flowing in blood vessels. However we had to overcome the problem of autofluorescence and tissue absorption. Two approaches were studied in parallel: a elaborating new cell line expressing far red fluorescent proteins, modulating the excitation light of an IVFC device to give the cell a unique signature therefore enhancing sensitivity, increasing signal to noise ratio. The modulated excitation IVFC allowed us to calculate the velocity of cells, and infer their position in blood vessel phantoms. The analysis of treatment effects on tumor burden, metastases and CTCs in PIOL and PCL could help understanding lymphoma metastatic dissemination and contribute to treatment follow-up, thus allowing design of new therapeutic approaches with increased efficacy.

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