(D) Hematoxylin & Eosin (H&E) staining, anti-CD20 immunohistochemistry (IHC) for B-cell detection, and anti-CXCR4 IHC in bone marrow (BM) (cranium) and LN (cervical). organs affected by lymphoma cells with negligible distribution to unaffected tissues. Finally, we obtained antitumor effect without toxicity in a CXCR4+ lymphoma model by administration of T22-DITOX-H6, a nanoparticle incorporating a toxin with the same structure as the nanocarrier. Hence, the use of the T22-GFP-H6 nanocarrier could be a good strategy to load and deliver drugs or toxins to treat specifically CXCR4-mediated refractory or relapsed diffuse large MK-8745 B-cell lymphoma without systemic toxicity. Introduction Diffuse large B-cell lymphoma (DLBCL) represents 30-33% of all non-Hodgkin lymphomas (NHL).1 Management of DLBCL has been improved by the addition of rituximab to CHOP (cyclophosphamide, doxorubicin, vincristine and prednisone) chemotherapy. However, despite this advancement, R-CHOP treatment is still associated with high toxicity, relapse and an unacceptably high treatment failure rate.2 Relapse after R-CHOP therapy occurs in 40% of patients;3,4 this is currently managed with salvage chemotherapy. This is followed by high-dose chemotherapy and autologous bone marrow transplant in patients with chemosensitive disease, which, however, leads to long-term MK-8745 disease control in only half of the patients.5 Moreover, less than 20% of patients treated with an R-CHOP front-line regimen who relapse within one year benefit from salvage autologous hematopoietic cell transplant.2,6 Thus, novel therapeutic strategies that reduce relapse rates and enhance DLBCL Rabbit Polyclonal to Mst1/2 patient survival are urgently needed. Novel approaches based on selective-drug delivery to cancer cells promise to increase patient benefit by offering both higher cure rates and lower side effects in DLBCL patients. In this regard, we evaluated a previously developed protein nanocarrier as a possible drug carrier to pursue the selective elimination of DLBCL cells over-expressing CXCR4 (CXCR4+), which are responsible for DLBCL relapse and disease progression.7C9 Thus, the CXCR4-CXCL12 axis is involved in tumor pathogenesis, cancer cell survival, stem cell phenotype, and resistance to chemotherapy.10,11 In addition, CXCR4 is constitutively over-expressed in NHL cell lines,12,13 and also in approximately 50% of malignant B-cell lymphocytes derived from DLBCL patients.8 Interestingly, CXCR4+ DLBCL cell lines show resistance to rituximab but are sensitive to the combination of rituximab with a CXCR4 antagonist.14,15 Most importantly, we as well as others reported that CXCR4 overexpression associates with poor progression-free and overall survival in DLBCL patients treated with R-CHOP.7,8,14 Our group has developed T22-GFP-H6, a self-assembling protein nanocarrier, which uses the peptidic T22 ligand to target the CXCR4 receptor.16 This carrier displays a high recirculation time in blood and selectively biodistributes to tumor tissues in solid tumor models, internalizing selectively in CXCR4+ cancer cells, while increasing its tumor uptake compared MK-8745 to the untargeted GFP-H6 counterpart.17 This nanocarrier is also able to incorporate toxins (e.g. diphtheria toxin catalytic domain) leading to selective elimination of CXCR4+ colorectal cancer cells.18,19 Nevertheless, no previous protein-based nanocarrier has been described to specifically target cancer cells in hematologic neoplasias. Critical differences between solid cancers and hematologic neoplasias may raise doubts about its use to target CXCR4+ cancer cells in DLBCL models. Thus, the enhanced permeability/retention (EPR) effect, due to abnormal fenestrated vessels and limited lymphatic drainage, allows nanocarrier accumulation in solid tumors. In contrast, DLBCL is usually a disseminated disease that displays freely circulating lymphoma cells in blood concomitantly with their confinement at specific tumor niches, such as lymph nodes (LN) and bone marrow (BM), in which the EPR effect is unlikely to be present.20 Here, we studied whether active targeting of the T22-GFP-H6 nanocarrier leads to its selective uptake in CXCR4+ subcutaneous (SC) DLBCL tumors. We also assessed if this increased uptake associates with specific nanocarrier internalization in CXCR4+ lymphoma cells; issues still be to settled in nanomedicine.21,22 Importantly, we used a disseminated CXCR4+ DLBCL model (which replicates the organ involvement observed in DLBCL patients8) to study nanocarrier accumulation in lym phoma-affected organs (LN and BM) and its capacity to internalize in CXCR4+ lymphoma cells within these organs. Moreover, we evaluated whether T22-DITOX-H6, a nanoparticle incorporating a diphtheria toxin domain name that maintains the same structure as the nanocar-rier, can selectively eliminate CXCR4+ DLBCL cells in SC tumors. The study goal was to determine whether we could use the nanocarrier to selectively deliver drugs to target CXCR4+ DLBCL cells. Methods experiments Four-week aged female NOD/SCID mice were obtained from Charles River Laboratories. Mice were maintained in specific pathogen-free (SPF) conditions with sterile food and water at.