Resumen de Antineoplastic effect of therapeutic nanoparticles targeted to CXCR4+ diffuse large b-cell lymphoma cells
Nowadays, novel therapeutic strategies are urgently needed to reduce relapse rates after R-CHOP treatment and to enhance the survival of diffuse large B-cell lymphoma (DLBCL) patients. Selective delivery of antineoplastic agents to cancer cells, using actively-targeted nanoparticles, is a novel approach that promises to revolutionize therapy by dramatically increasing the tumor uptake of the drug and tumor cell killing while reducing the off-target toxicity. Our approach is based on the sue of targeted protein nanoparticles towards the cell surface CXCR4 receptor. The CXCR4 overexpression (CXCR4+ ) in 30-50% of DLBCL patients increasesthe capacity of targeted nanoparticlesto internalize in target cells by endocytosis. Relevantly, CXCR4+ DLBCL cells are responsible for lymphoma cell dissemination, relapse and resistance to R-CHOP in DLBCL patients. Firstly, this thesis aims to evaluate the ability of the T22-GFP-H6 protein nanocarrier in selectively deliver drugs or toxins specifically to CXCR4+ DLBCL cells. Moreover, the second aim of this thesis is to determine the in vitro and in vivo antineoplastic effect of two therapeutic nanomedicines targeting CXCR4+ DLBCL cells, known as T22-PE24-H6 and T22-AUR, which transport the Pseudomonas aeruginosa exotoxin A and the microtubule targeting-agent monomethyl auristatin E, respectively. Hence, regarding our results, the T22-GFP-H6 nanocarrier achieved efficient in vitro CXCR4-dependent internalization in DLBCL cells without cytotoxic effect. Subsequently, T22-GFP-H6 showed a high tumor uptake in subcutaneous CXCR4+ DLBCL tumors that represented 86.1% of the total injected dose, whereas the rest of non-DLBCL affected organs presented negligible nanocarrier accumulation. Moreover, performing in vivo competition assays with the antagonist of CXCR4 AMD3100 as well as using subcutaneous mouse models bearing CXCR4 negative DLBCL cells or the same cell line transfected with the CXCR4 receptor, T22-GFP-H6 demonstrated an in vivo CXCR4-dependent tumor uptake.
Importantly, using a bioluminescent disseminated mouse model, we observed T22-GFP-H6 biodistribution to all clinically relevant sites affected by CXCR4+ DLBCL cells (lymph nodes and bone marrow) and nanocarrier internalization within these cells. Furthermore, T22-GFP-H6 was able to deliver toxins to CXCR4+ DLBCL cells since the T22-DITOX-H6 polypeptidic nanoparticle, that displays the same structure as T22-GFP-H6 while replacing the GFP by diphtheria toxin domains, induced a potent antitumor effect in subcutaneous CXCR4+ DLBCL tumors without damaging normal organs. Additionally, the therapeutic nanoconjugate T22-AUR demonstrated also CXCR4 receptor-mediated internalization in vitro and high tumor uptake and internalization in subcutaneous CXCR4+ DLBCL tumors. Moreover, both therapeutic nanoparticles (T22-PE24-H6 and T22-AUR) exerted in vitro CXCR4-dependent cell death in CXCR4+ DLBCL cells. Whereas T22-PE24-H6 induced cell death mediated by apoptosis, the lymphoma cell death by T22-AUR was not only mediated by apoptosis, but also involved cell cycle arrest in G2/M phase and mitotic catastrophe. Finally, T22- PE24-H6 and T22-AUR selectively killed CXCR4+ lymphoma cells in CXCR4+ DLBCL nodal, and extranodal, disseminated mouse models, respectively, without toxicity in the non-DLBCL infiltrated organs. In conclusion, our CXCR4-targeted nanoapproaches could be an effective strategy to enhance the survival and cure rates observed in R-CHOP refractory or relapsed CXCR4+ DLBCL patients.
