Non-Hodgkin Lymphoma ARTICLE A CXCR4-targeted nanocarrier achieves highly Ferrata Storti Foundation selective tumor uptake in diffuse large B-cell lymphoma mouse models Aïda Falgàs,1,2,* Victor Pallarès,1,3,* Ugutz Unzueta,1,2 María Virtudes Céspedes,1,2 Irene Arroyo-Solera,1,2 María José Moreno,1 Alberto Gallardo,1,4 María Antonia Mangues,5 Jorge Sierra,3,6 Antonio Villaverde,2,7,8 Esther Vázquez,2,7,8* Ramon Mangues1,2,6,* and Isolda Casanova1,2,6 1 Biomedical Research Institute Sant Pau (IIB-Sant Pau), Hospital de la Santa Creu i Sant Haematologica 2018 Pau; 2CIBER en Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN); 3Department Volume 105(3):741-753 of Hematology, Hospital de la Santa Creu i Sant Pau; 4Department of Pathology, Hospital de la Santa Creu i Sant Pau; 5Department of Pharmacy, Hospital de la Santa Creu i Sant Pau; 6Josep Carreras Research Institute; 7Institut de Biotecnologia i de Biomedicina, Universitat Autònoma de Barcelona and 8Departament de Genètica i de Microbiologia, Universitat Autònoma de Barcelona, Barcelona, Spain *AF and VP contributed equally to this work. ABSTRACT O ne-third of diffuse large B-cell lymphoma patients are refractory to initial treatment or relapse after rituximab plus cyclophosphamide, doxorubicin, vincristine and prednisone chemotherapy. In these patients, CXCR4 overexpression (CXCR4+) associates with lower overall and disease-free survival. Nanomedicine pursues active targeting to selec- tively deliver antitumor agents to cancer cells; a novel approach that prom- Correspondence: ises to revolutionize therapy by dramatically increasing drug concentration in target tumor cells. In this study, we intravenously administered a ligand- RAMON MANGUES mangues@santpau.cat ed protein nanocarrier (T22-GFP-H6) targeting CXCR4+ lymphoma cells in mouse models to assess its selectivity as a nanocarrier by measuring its tis- ESTHER VÁZQUEZ sue biodistribution in cancer and normal cells. No previous protein-based esther.vazquez@uab.cat nanocarrier has been described as specifically targeting lymphoma cells. T22-GFP-H6 achieved a highly selective tumor uptake in a CXCR4+ lym- Received: November 19, 2018. phoma subcutaneous model, as detected by fluorescent emission. We Accepted: June 26, 2019. demonstrated that tumor uptake was CXCR4-dependent because pretreat- Pre-published: June 27 2019. ment with AMD3100, a CXCR4 antagonist, significantly reduced tumor uptake. Moreover, in contrast to CXCR4+ subcutaneous models, CXCR4– tumors did not accumulate the nanocarrier. Most importantly, after intra- doi:10.3324/haematol.2018.211490 venous injection in a disseminated model, the nanocarrier accumulated and internalized in all clinically relevant organs affected by lymphoma cells with Check the online version for the most updated negligible distribution to unaffected tissues. Finally, we obtained antitumor information on this article, online supplements, and information on authorship & disclosures: effect without toxicity in a CXCR4+ lymphoma model by administration of www.haematologica.org/content/105/3/741 T22-DITOX-H6, a nanoparticle incorporating a toxin with the same struc- ture 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 ©2020 Ferrata Storti Foundation CXCR4-mediated refractory or relapsed diffuse large B-cell lymphoma Material published in Haematologica is covered by copyright. without systemic toxicity. All rights are reserved to the Ferrata Storti Foundation. Use of published material is allowed under the following terms and conditions: https://creativecommons.org/licenses/by-nc/4.0/legalcode. Copies of published material are allowed for personal or inter- Introduction nal use. Sharing published material for non-commercial pur- poses is subject to the following conditions: https://creativecommons.org/licenses/by-nc/4.0/legalcode, Diffuse large B-cell lymphoma (DLBCL) represents 30-33% of all non-Hodgkin sect. 3. Reproducing and sharing published material for com- lymphomas (NHL).1 Management of DLBCL has been improved by the addition of mercial purposes is not allowed without permission in writing rituximab to CHOP (cyclophosphamide, doxorubicin, vincristine and prednisone) from the publisher. chemotherapy. However, despite this advancement, R-CHOP treatment is still associated with high toxicity, relapse and an unacceptably high treatment failure haematologica | 2020; 105(3) 741 A. Falgàs et al. rate.2 Relapse after R-CHOP therapy occurs in 40% of phoma-affected organs (LN and BM) and its capacity to patients;3,4 this is currently managed with salvage internalize in CXCR4+ lymphoma cells within these chemotherapy. This is followed by high-dose chemother- organs. Moreover, we evaluated whether T22-DITOX- apy and autologous bone marrow transplant in patients H6, a nanoparticle incorporating a diphtheria toxin with chemosensitive disease, which, however, leads to domain that maintains the same structure as the nanocar- long-term disease control in only half of the patients.5 rier, can selectively eliminate CXCR4+ DLBCL cells in SC Moreover, less than 20% of patients treated with an R- tumors. The study goal was to determine whether we CHOP front-line regimen who relapse within one year could use the nanocarrier to selectively deliver drugs to benefit from salvage autologous hematopoietic cell trans- target CXCR4+ DLBCL cells. plant.2,6 Thus, novel therapeutic strategies that reduce relapse rates and enhance DLBCL patient survival are Methods urgently needed. Novel approaches based on selective-drug delivery to In vivo experiments cancer cells promise to increase patient benefit by offering Four-week old female NOD/SCID mice were obtained from both higher cure rates and lower side effects in DLBCL Charles River Laboratories. Mice were maintained in specific patients. In this regard, we evaluated a previously devel- pathogen-free (SPF) conditions with sterile food and water oped protein nanocarrier as a possible drug carrier to pur- ad libitum. Mouse experiments were approved by the Hospital de sue the selective elimination of DLBCL cells over-express- la Santa Creu i Sant Pau Animal Ethics Committee. ing CXCR4 (CXCR4+), which are responsible for DLBCL For SC models, 10 million DLBCL cells were injected in both relapse and disease progression.7-9 Thus, the flanks. Tumor growth was monitored twice a week with a CXCR4-CXCL12 axis is involved in tumor pathogenesis, caliper (tumor volume=width2 x length/2). When tumors cancer cell survival, stem cell phenotype, and resistance to reached a volume of 600-800 mm3, mice received a single intra- chemotherapy.10,11 In addition, CXCR4 is constitutively venous (IV) dose of 200 mg T22-GFP-H6, which contains a fluo- over-expressed in NHL cell lines,12,13 and also in approxi- rescent domain, or buffer (20 mM Tris, 500mM NaCl, pH 8). mately 50% of malignant B-cell lymphocytes derived T22-GFP-H6 design and production have been described in pre- from DLBCL patients.8 Interestingly, CXCR4+ DLBCL cell vious studies.16 Fluorescence intensity (FLI) was measured ex vivo lines show resistance to rituximab but are sensitive to the at different time points in tumors, plasma, and all organs. A plas- combination of rituximab with a CXCR4 antagonist.14,15 ma pool was obtained by centrifugation of total blood, obtained Most importantly, we and others reported that CXCR4 by intracardiac puncture (25G), at 600g for ten minutes (min) at overexpression associates with poor progression-free and 4ºC. T22-GFP-H6 biodistribution in SC tumors over time was overall survival in DLBCL patients treated with R- measured using the area under the curve (AUC). AUC analysis CHOP.7,8,14 of tumors and normal organs was measured using the GraphPad Our group has developed T22-GFP-H6, a self-assem- Prism 6 program. We subcutaneously administered AMD3100 in bling protein nanocarrier, which uses the peptidic T22 mice to perform CXCR4 blocking experiments, giving a total of ligand to target the CXCR4 receptor.16 This carrier displays three AMD3100 doses at 10 mg/kg, 1 hour (h) before and 1h and a high recirculation time in blood and selectively 2h after IV T22-GFP-H6 injection. We used SC tumor models to biodistributes to tumor tissues in solid tumor models, evaluate the antitumor effect and associated toxicity of T22- internalizing selectively in CXCR4+ cancer cells, while DITOX-H6. Mice received a single 25 mg IV dose of T22- increasing its tumor uptake compared to the untargeted DITOX-H6 or buffer when tumors reached a volume of 400-600 GFP-H6 counterpart.17 This nanocarrier is also able to mm3. Animals were euthanized 24h post administration. T22- incorporate toxins (e.g. diphtheria toxin catalytic domain) DITOX-H6 nanoparticle characterization has been published leading to selective elimination of CXCR4+ colorectal can- previously.18 cer cells.18,19 Nevertheless, no previous protein-based To generate the disseminated lymphoma model, NOD/SCID nanocarrier has been described to specifically target cancer mice were intravenously injected with 20x106 luminescent cells in hematologic neoplasias. Critical differences Toledo cells (Toledo-Luci) in 200 mL physiological serum. between solid cancers and hematologic neoplasias may Dissemination was monitored capturing bioluminescence inten- raise doubts about its use to target CXCR4+ cancer cells in sity (BLI) twice a week after intraperitoneal injection of firefly DLBCL models. Thus, the enhanced permeability/reten- D-luciferin. After 27-30 days, animals received a single IV dose tion (EPR) effect, due to abnormal fenestrated vessels and of 400 mg T22-GFP-H6 nanocarrier or buffer. Five hours later, FLI limited lymphatic drainage, allows nanocarrier accumula- was measured ex vivo in all organs. tion in solid tumors. In contrast, DLBCL is a disseminated Fluorescence intensity correlates to the amount of accumulat- disease that displays freely circulating lymphoma cells in ed protein in each tissue and is expressed as average radiant effi- blood concomitantly with their confinement at specific ciency. FLI from experimental mice was calculated subtracting tumor niches, such as lymph nodes (LN) and bone mar- the FLI auto-fluorescence of control mice. The emitted FLI and row (BM), in which the EPR effect is unlikely to be pres- BLI were measured using the IVIS Spectrum 200 Imaging System ent.20 (Xenogen). Finally, tumors and all organs were collected, fixed Here, we studied whether active targeting of the T22- and paraffined to perform histological, immunohistochemical or GFP-H6 nanocarrier leads to its selective uptake in immunofluorescent evaluations, and were also directly cryopre- CXCR4+ subcutaneous (SC) DLBCL tumors. We also served in liquid nitrogen for protein extraction. assessed if this increased uptake associates with specific Details of methods for cell culture, transfection with nanocarrier internalization in CXCR4+ lymphoma cells; Luciferase and CXCR4 plasmids, cell proliferation, flow cytom- issues still be to settled in nanomedicine.21,22 Importantly, etry, western blot, histopathology, 4′,6-diamidino-2-phenylin- we used a disseminated CXCR4+ DLBCL model (which dole (DAPI) staining, immunohistochemistry (IHC) and replicates the organ involvement observed in DLBCL immunofluorescence (IF) analyses can be found in the Online patients8) to study nanocarrier accumulation in lym- Supplementary Appendix. 742 haematologica | 2020; 105(3) CXCR4-targeted nanocarrier to DLBCL cells Statistical analysis most in Toledo cells, followed by U-2932 and RIVA, In vitro experiments were performed in biological triplicates whereas it did not internalize in SUDHL-2 (Figure 1B). while in vivo experiments were performed in triplicates/quadrupli- Moreover, T22-GFP-H6 nanocarrier internalization was cates. The data for all experiments were reported as mean CXCR4-dependent. So, after preincubation with CXCR4 ±Standard Error of Mean (SEM). All results were analyzed using antagonist AMD3100, T22-GFP-H6 internalization the Student t-test. P<0.05 was considered statistically significant. decreased significantly in Toledo, U-2932 and RIVA cells Statistical calculations were performed using SPSS software ver- (Figure 1C). As expected, T22-GFP-H6 did not internalize sion.21 in SUDHL-2 cells (only background FLI was detected), whereas high internalization was registered in CXCR4+ SUDHL-2 cells. Similarly, AMD3100 preincubation had no Results effect on nanocarrier internalization in SUDHL-2 cells but led to a significant decrease in CXCR4+ SUDHL-2 cells CXCR4-dependent internalization of T22-GFP-H6 in (Figure 1D). Thus, we showed specific in vitro entry of human CXCR4+ diffuse large B-cell lymphoma cell lines T22-GFP-H6 into CXCR4+ DLBCL cells through the CXCR4 membrane levels were evaluated in four human CXCR4 receptor. DLBCL cell lines by flow cytometry (Figure 1A) and IHC (Online Supplementary Figure S1). CXCR4 expression was Non-cytotoxic effect of T22-GFP-H6 in diffuse large highest in Toledo cells, followed by U-2932 and RIVA, B-cell lymphoma cell lines in vitro whereas CXCR4 expression in the SUDHL-2 cell line was After exposure to T22-GFP-H6 (50-500nM range), cell undetectable. CXCR4-transfected SUDHL-2 cells viability for all four evaluated DLBCL cell lines was (CXCR4+ SUDHL-2) showed average CXCR4 levels. approximately or above 100% (Figure 1E). Therefore, T22-GFP-H6 nanocarrier internalization correlated with T22-GFP-H6 nanocarrier has no in vitro antineoplastic CXCR4 expression. Thus, T22-GFP-H6 internalized the effect against these DLBCL cell lines. A B C D E Figure 1. In vitro T22-GFP-H6 nanocarrier internalization in CXCR4+ diffuse large B-cell lymphoma (DLBCL) cell lines and its dependence on the CXCR4 receptor. (A) CXCR4 membrane expression of different DLBCL cell lines (Toledo, U-2932, RIVA and SUDHL-2) and the SUDHL-2 cell line transfected with a CXCR4 plasmid (CXCR4+ SUDHL-2) measured by flow cytometry. (B) Levels of intracellular fluorescence quantified by flow cytometry in Toledo, U-2932, RIVA and SUDHL-2 cells after 1 hour (h) exposure to T22-GFP-H6 nanocarrier at different concentrations (range: 0.1nM-250nM). (C) T22-GFP-H6 internalization, measured by flow cytometry, in Toledo, U-2932 and RIVA cells after 1h pretreatment with the antagonist AMD3100 (50nM T22-GFP-H6:500nM AMD3100). (D) Competition assays with AMD3100 (250nM T22-GFP-H6: 2500nM AMD3100) in SUDHL-2 cells and CXCR4+ SUDHL-2 cells. (E) Lack of cytotoxicity (measured as percentage of cell viability) after 48h exposure to high concentrations of T22-GFP-H6 nanocarrier (range: 50nM-500nM) in Toledo, U-2932, RIVA and SUDHL-2 cells. *P<0.05; **P<0.01; ***P<0.001; ns: non-significant; MFI: mean fluorescence intensity. haematologica | 2020; 105(3) 743 A. Falgàs et al. A B C D E F Figure 2. In vivo biodistribution and toxicity assessment of T22-GFP-H6 in the CXCR4+ subcutaneous (SC) Toledo mouse model. (A) Fluorescence intensity (FLI) detection in SC Toledo tumors at 2, 5 or 24 hours (h) after intravenous (IV) injection of 200 mg of T22-GFP-H6. No fluorescence was detected in buffer-treated mice. (B) Quantification of emitted fluorescence (measured as FLI ratio) at different times (2, 5 and 24h) in SC tumors and normal tissues (liver, spleen, heart, lungs, kid- neys and bone marrow). (C) FLI emitted by the nanocarrier in normal tissues 5h after T22-GFP-H6 administration. (D) Hematoxylin & Eosin (H&E) stained tissue sec- tions of normal organs and SC tumors at the 5h FLI peak. (E) Representation of the area under the curve (AUC) of emitted FLI over time (2-24h) registered in tumors, liver and lungs in T22-GFP-H6-treated mice. Notice that the AUC in the organs with an uptake lower than 2% (spleen, kidney, heart and BM) is not visible in this graph- ic. (F) Percentage of nanocarrier uptake (as measured by the AUC of emitted FLI) registered in each organ studied in relation to the total emitted FLI (sum of AUC in tumors and all studied normal organs) during the 2-24h period and expressed as mean±Standard Error of Mean (SEM). FLI ratio for experimental mice was calculated subtracting the FLI auto-fluorescence of control mice and dividing the FLI signal of each tumor/tissue by the FLI signal of the lungs (organ chosen as reference). Original magnification x400. BM: bone marrow. 744 haematologica | 2020; 105(3) CXCR4-targeted nanocarrier to DLBCL cells A B C D E Figure 3. T22-GFP-H6 pharmacokinetics in plasma and clearance in CXCR4+ SC Toledo mouse model. (A) Representative images of fluorescence intensity (FLI) reg- istered in plasma (250 mL) of mice treated with buffer or 200 mg T22-GFP-H6, 10 minutes (min), 30 min, 2 hour (h), 5h, 15h, 24h and 48h after its administration. (B) Graphical representation of FLI quantification in plasma over time. (C and D) Western blot analysis of the fate of the T22-GFP-H6 protein in hepatic and renal tis- sue in mice treated with buffer or 10 min, 30 min, 2h, 5h, 15h, 24h and 48h after nanocarrier administration. (E) Western blot analysis of the fate and processing of the T22-GFP-H6 protein in subcutaneous (SC) Toledo tumors in mice treated with buffer or 10min, 30min, 2h, 5h, 15h, 24h and 48h after nanocarrier adminis- tration. Note the almost complete proteolysis of the full-length protein nanocarrier in tumor tissue, and the absence of proteolysis in liver and kidney. GAPDH was used as a loading control. T22-GFP-H6 was detected with an anti-GFP antibody. Highly selective T22-GFP-H6 tumor uptake in mice low accumulation in the expected non-tumor drug clear- bearing subcutaneous CXCR4+ diffuse large B-cell ance organs (i.e. liver and kidney) triggered the analysis of lymphoma tumors without toxicity the nanocarrier fate in these organs by western blot. After We evaluated T22-GFP-H6 biodistribution in the a single T22-GFP-H6 dose, we observed the full-length CXCR4+ SC Toledo mouse model, measuring the fluores- protein (≈30kDa) present in liver and kidney 10min post cence emitted by the nanocarrier GFP domain, after a sin- administration (Figure 3C and D), becoming undetectable gle 200 mg IV dose. T22-GFP-H6 accumulated in CXCR4+ over a period which ranged from 30min to 48h. In sharp SC tumors 2h after injection, reaching a FLI peak 5h post contrast, we detected full-length T22-GFP-H6 protein in injection, and decreasing considerably after 24h (Figure 2A Toledo SC tumors at 10min, 30min, 2h and 5h. and B). Tumor uptake 5h post injection was 35.85 times Interestingly, faint proteolytic bands appeared over a peri- higher than lung uptake, which was taken as a reference od which ranged from 30min to 2h, which became more among the normal mouse organs because, although intense at 5h. Over a period which ranged from 15h to almost negligible, it did show the most sustained FLI emis- 48h, the full-length protein decreased dramatically and the sion over time (Figure 2B and Online Supplementary Table nananocarrier was mostly proteolyzed (Figure 3E). These S1). Similar observations were made in all non-tumor results, together with the observed FLI AUC in tumor and organs analyzed (Figure 2B and C). Moreover, we did not normal organs, suggest that the proteolytic activity observe any histological alteration in Hematoxylin & observed in the tumor makes it the main nanocarrier clear- Eosin (H&E) stained normal organs (Figure 2D). ance organ. The quantification of the AUC of emitted FLI over the study period (Figure 2E and Online Supplementary Table S2) T22-GFP-H6 and CXCR4 receptor co-localization in showed that tumor tissue accumulated 86.13±4.04% of the cell membrane followed by its internalization in the total FLI detected in all organs, including tumor and CXCR4+ diffuse large B-cell lymphoma cells non-tumor tissues. In contrast, the liver, which was the At the FLI peak (5h) after a single 200µg injection, we non-tumor organ with higher AUC, reached only observed nanocarrier internalization in 56.2±12.0% of the 5.96±2.83% (Figure 2F). Therefore, T22-GFP-H6 displayed Toledo cells (green staining with anti-GFP IF) in tumors, a specific targeting of CXCR4+ SC DLBCL tumors with whereas all (100%) tumor cells over-expressed CXCR4 negligible nanocarrier accumulation in non-tumor bearing (red staining with anti-CXCR4 IF). In buffer-treated organs, which supports a highly selective tumor uptake as tumors, the CXCR4 receptor localized mainly at Toledo compared to normal cells. cell membrane, while a dot-like staining inside the cell After a single T22-GFP-H6 IV administration, measure- cytosol was observed in the nanocarrier-treated-tumors; a ment of circulating nanocarrier showed a fast biodistribu- finding consistent with receptor internalization within tion half-life (t1/2≈20min) in the blood compartment, fol- endocytic vesicles. Merged (yellow) images showed lowed by a slower elimination phase (t1/2≈75min), becom- nanocarrier and CXCR4 co-localization in the membrane ing undetectable in plasma at 2h (Figure 3A and B). of Toledo cells in T22-GFP-H6-treated tumors. Once into The highly unusual T22-GFP-H6 tumor uptake and its the cytosol, the CXCR4 and T22-GFP-H6 stained endoso- haematologica | 2020; 105(3) 745 A. Falgàs et al. Figure 4. Co-localization of T22-GFP-H6 and CXCR4 receptor in the cell membrane and nanocarrier internalization in CXCR4+ subcutaneous (SC) tumors. Representative immunofluorescence images from SC Toledo tumors of mice treated with T22-GFP-H6 (200 mg, 5h) or buffer. T22-GFP-H6 and CXCR4 co-localization was mainly seen in the cell membrane (yellow dots), whereas internalized nanocarriers were observed in the cell cytosol (green dots) and the endocytic vesicles with the CXCR4 receptor (red dots). DAPI staining (blue), anti-GFP protein (green), anti-CXCR4 receptor (red) and merged images from the three stains. Scale bars=10 mm. mal vesicles were dissociated (Figure 4). These results sug- T22-GFP-H6 biodistributes to all diffuse large B-cell gest that T22-GFP-H6 interacts with the CXCR4 receptor lymphoma-infiltrated organs and internalizes in in the cell membrane, where both co-localize and, after lymphoma cells in a CXCR4+ diffuse large B-cell internalizing jointly within endosomal vesicles, they are lymphoma disseminated mouse model able to release the nanocarrier in the CXCR4+ DLBCL cell We evaluated the biodistribution of T22-GFP-H6 in vivo cytosol. in a CXCR4+ Toledo-Luci disseminated DLBCL mouse model, while monitoring lymphoma cell dissemination by Selective CXCR4-dependent T22-GFP-H6 tumor uptake measuring BLI levels emitted by the infiltrated organs in subcutaneous diffuse large B-cell lymphoma tumors in vivo (Figure 6A). In addition, we precisely identified the We also assessed the dependence of nanocarrier tumor organs showing infiltration by Toledo-Luci cells, BM (cra- uptake on CXCR4 receptor, performing in vivo competi- nium and hind limbs) and LN (cervical and renal). In some tion assays using the CXCR4 antagonist AMD3100 in mice (37.5%), we detected residual BLI levels in the spleen mice bearing CXCR4+ Toledo-derived SC tumors (Figure and no infiltration was observed in any other organ 5A). Five hours after T22-GFP-H6 administration, we reg- (Figure 6B). Macroscopic LN (cervical and renal) infiltra- istered a peak of nanocarrier accumulation in tumors that tion was identified in 100% of mice (Figure 6C). H&E reached 3.23±0.38E7. In contrast, the AMD3100 adminis- staining and anti-CD20 IHC confirmed Toledo-Luci cell tration prior and after nanocarrier injection blocked infiltration in BM and LN tissue sections. CXCR4 mem- nanocarrier uptake in tumors, since the emitted FLI was 10 brane expression was maintained in DLBCL cells located times lower (0.31±0.52E7) (Figure 5B). Differences in all infiltrated organs (Figure 6D). between the Toledo tumors treated with T22-GFP-H6 and We went on to study T22-GFP-H6 biodistribution after those treated with AMD3100 plus T22-GFP-H6 were IV injection (400 mg dose) or buffer in mice displaying highly significant (Figure 5C). This inhibition of nanocar- complete dissemination of Toledo cells (27-30 days post rier uptake by AMD3100 confirms that tumor uptake injection). Five hours after nanocarrier injection, we depends on the CXCR4-receptor. observed high FLI in BM (cranium and hind limbs) and LN Additional support for this selective uptake comes from (renal and cervical), whereas fluorescence was negligible additional biodistribution assays comparing CXCR4– or undetectable in non-infiltrated organs (Figure 7A and SUDHL-2 and CXCR4+ SUDHL-2 SC tumor-bearing mice. B). Indeed, T22-GFP-H6 was specifically delivered to the Five hours after 200 mg T22-GFP-H6 administration, FLI DLBCL infiltrated organs since FLI levels in BM and LN emission from CXCR4+ SUDHL-2 tumors was significant- were 31.05- and 12.98-fold higher, respectively, in com- ly higher (2.12±0.46E7) than from CXCR4– SUDHL-2 parison to lungs (the reference organ showing background tumors (0.04±0.21E7) (Figure 5C and D). FLI levels) (Figure 7B and Online Supplementary Table S3). Consistently, Toledo and CXCR4+ SUDHL-2 tumors Moreover, no histopathological alterations were observed showed CXCR4 membrane expression, as measured by in any tissue analyzed in nanocarrier-treated mice (data not IHC, whereas CXCR4– SUDHL-2 tumors did not (Figure shown). IF analysis using anti-GFP showed T22-GFP-H6 5E); a finding that confirms the specific directioning of (green) in Toledo-Luci cell cytosol in affected BM and LN. T22-GFP-H6 to tumors containing CXCR4+ DLBCL cells. In addition, CXCR4 dot-like (red) and nanocarrier (green) 746 haematologica | 2020; 105(3) CXCR4-targeted nanocarrier to DLBCL cells staining co-localized (yellow) on the cell membrane. T22-GFP-H6 internalization in CXCR4+ mouse cells Moreover, similar to findings in SC Toledo tumors, in the To support the relevance of our CXCR4+ DLBCL models disseminated model, we found a release of the nanocarrier for clinical translation of the tumor (human cells) and non- into CXCR4+ DLBCL cell cytosol separated from endocyt- tumor (mouse cells) biodistribution data, we assessed ic vesicles containing the CXCR4 receptor (Figure 7C). whether the nanocarrier internalized in mouse cells. A B C D E Figure 5. CXCR4-dependent uptake of T22-GFP-H6 nanocarrier in diffuse large B-cell lymphoma (DLBCL) subcutaneous (SC) tumors. (A) For the CXCR4 blocking experiment, mice were injected with a total of three SC doses of AMD3100 at 10 mg/kg. The time point of mice sacrifice was 2 hours (h) after the last AMD3100 SC injection, which corresponds to the 5h fluorescence intensity (FLI) peak after T22-GFP-H6 injection. (B) Representative images of emitted FLI by SC Toledo tumors from buffer, T22-GFP-H6, AMD3100 or AMD3100+T22-GFP-H6 treated animals. (C) FLI levels of SC tumor-bearing-mice of Toledo cells administered with T22-GFP- H6 or AMD3100+T22-GFP-H6 and FLI levels of the tumors in T22-GFP-H6-treated bearing SC CXCR4+ SUDHL-2 tumors or SC CXCR4– SUDHL-2 tumors. (D) A repre- sentative image of the FLI in SC tumors of CXCR4– SUDHL-2 and CXCR4+ SUDHL-2 cells after 5h of T22-GFP-H6 or buffer administration. (E) Level of membrane CXCR4 expression detected by immunohistochemistry (IHC) in SC tumors derived from Toledo, CXCR4+ SUDHL-2 and CXCR4– SUDHL-2 cells. **P<0.01. Original magnification x1000. haematologica | 2020; 105(3) 747 A. Falgàs et al. Firstly, we evaluated CXCR4 expression in the mouse T22-DITOX-H6 antitumor effect and lack of toxicity in B-cell lymphoma WEHI-231 cell line that showed medium a CXCR4+ subcutaneous diffuse large B-cell lymphoma CXCR4 membrane expression by flow cytometry and mouse model IHC (Online Supplementary Figure S2A). Then, we demon- Finally, we evaluated whether the therapeutic nanoparti- strated intracellular nanocarrier uptake in mouse WEHI- cle T22-DITOX-H6, incorporating a toxin domain with 231 cells and its dependence on CXCR4 expression, since known antitumor activity, induced cell death of Toledo it was inhibited by AMD3100 (Online Supplementary Figure cells in SC tumors without damaging normal cells. T22- S2B). Therefore, T22-GFP-H6 internalizes in both DITOX-H6 caused apoptosis in lymphoma cells in these CXCR4+ human and CXCR4+ mouse lymphoma cells. tumors since a single IV 25 mg T22-DITOX-H6 dose signif- A B C D Figure 6. Pattern of organ infiltration in the Toledo-Luci diffuse large B-cell lymphoma (DLBCL) disseminated mouse model. (A) Bioluminescent intensity (BLI) follow up by IVIS Spectrum of mice intravenously injected with Toledo cells transfected with the Luciferase gene (Toledo-Luci cells). (B) Ex vivo representative images of the recorded BLI emission in different mouse organs: spleen, liver, kidneys, heart, lungs, hind limbs, cranium, cervical lymph nodes (LN) and renal LN. (C) Macroscopic images showing Toledo-Luci cell infiltration in cervical LN and renal LN. White arrows show the LN location. (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). Original magnification x400. Scale bars=50 mm. 748 haematologica | 2020; 105(3) CXCR4-targeted nanocarrier to DLBCL cells icantly increased the number of apoptotic bodies and 8C). A direct comparison showed that CXCR4 expression cleaved PARP level compared to buffer-treated mice (Figure in SC Toledo tumors was significantly (22.87 times) higher 8A and B). than CXCR4 in mouse BM hematopoietic cells (Online We then confirmed CXCR4 expression in hematopoietic Supplementary Figure S2C and D). No histopathological cells of the mouse BM (CXCR4+ CD20- staining) (Figure alterations (H&E) nor induction of cell death (DAPI stain- A B C Figure 7. T22-GFP-H6 nanocarrier biodistribution in the Toledo-Luci diffuse large B-cell lymphoma (DLBCL) disseminated mouse model. (A) Level of fluorescence intensity (FLI) emission in bone marrow (BM) (cranium and hind limbs) and lymph nodes (LN) (cervical and renal) 5 hours (h) after the administration of 400 mg T22- GFP-H6 or buffer in a Toledo-Luci disseminated mouse model. (B) Comparison of FLI emission by T22-GFP-H6 accumulated in infiltrated DLBCL organs (BM and LN) as compared to non-DLBCL infiltrated organs (spleen, liver, kidneys, heart and lungs). FLI ratio from experimental mice was calculated subtracting the FLI auto-fluo- rescence of control mice and dividing the FLI recorded for each tissue by the FLI emitted by the lungs. (C) Representative immunofluorescent images of BM (cranium) and LN (cervical) in nanocarrier-treated mice and buffer-treated mice. Notice that green dots depicting internalized nanocarrier in the cytosol are only observed in T22-GFP-H6 treated animals. DAPI staining (blue), anti-GFP protein (green), anti-CXCR4 receptor (red) and merged images from the three stains. Scale bars=10 mm. haematologica | 2020; 105(3) 749 A. Falgàs et al. ing) was observed in the BM of T22-DITOX-H6-treated of the nanocarrier under examination to efficiently deliver mice (Figure 8C). Lastly, we did not find any macroscopic antitumor agents to achieve the selective killing of CXCR4+ (data not shown) or microscopic (H&E staining) alteration in lymphoma cells without inducing toxicity on CXCR4+ liver and kidneys (Figure 8D). Our results support the use mouse hematopoietic cells or systemic organs. A B C D Figure 8. Evaluation of T22-DITOX-H6 antitumor effect and toxicity in a CXCR4+ subcutaneous (SC) Toledo mouse model. (A) Apoptosis detection by cleaved Poly(ADP-ribose) polymerase (PARP) immunohistochemistry (IHC) as well as nuclear condensation by 4′,6-diamidino-2-phenylindole (DAPI) staining. (B) Mean quan- tification of cleaved PARP stained area (above) and number of apoptotic bodies by DAPI staining (bottom) in SC Toledo tumors 24 hours (h) after treatment with buffer or a single intravenous (IV) injection of 25 mg T22-DITOX-H6. (C) Lack of T22-DITOX-H6-induced toxicity in mouse bone marrow (BM). Human anti-CD20 and mouse anti-CXCR4 IHC assays were used to identify CXCR4+ mouse cells resident in BM. Hematoxylin & Eosin (H&E) and DAPI staining samples were examined to detect possible alterations in cell morphology or cell death induction. (D) Lack of systemic toxicity in liver or kidney by histological analysis of tissue sections H&E 24h after treatment with buffer or 25 mg T22-DITOX-H6. All images were taken at x400 and inserts at x1000. *P<0.05. 750 haematologica | 2020; 105(3) CXCR4-targeted nanocarrier to DLBCL cells Discussion the tumor;33 thus, although ADC are standard treatment in some neoplasias, protein nanocarriers could offer an enor- A huge limitation for the clinical translation of mous opportunity to improve drug delivery to tumors. nanomedicines in oncology is the fact that only 0.7-5.0% Our results on nanocarrier biodistribution in the SC of the administered dose reaches the tumor.23,24 In contrast, tumor model demonstrate a specific co-localization of the our biodistribution studies show a very high level of T22- nanocarrier together with the CXCR4 receptor in the cell GFP-H6 uptake in tumor tissue (86.1% of the total emitted membrane followed by their internalization, via endocy- fluorescence) compared to the combined fluorescence tosis, to reach the cytosol of CXCR4+ DLBCL cells. Once emitted by all normal tissues (13.9% of total tumor+non- inside the cytosol, the structure of the nanocarrier elicits tumor fluorescence), including the spleen, liver, kidney, endosomal escape and delivery of the materials into the heart, lung and BM. We have recently reported a similar cytoplasm, before its ultimate intracellular proteolysis.16 finding for the same nanocarrier in a SC colorectal cancer Furthermore, the efficacy of a T22-GFP-H6 nanocarrier (CRC) model.25 These data are consistent with the fast that targets CXCR4+ DLBCL cells appears to be exclusive- biodistribution half-life for the nanocarrier in blood ly dependent on the overexpression of CXCR4 receptor in (approx. 20 min) and the detection of the full length pro- the membrane of tumor cells. This notion is currently sup- tein in the 10min-5h period in SC CXCR4+ DLBCL ported by two main findings: on the one hand, T22-GFP- tumors. Unexpectedly, we found that most of the prote- H6 displays a tumor uptake significantly higher than that olytic metabolism of T22-GFP-H6 occurs in tumor tissues, achieved in the same SC tumor when CXCR4 is inhibited whereas clearance in liver or kidney is negligible, being by AMD3100 in the competition assay. On the other detectable in these organs at 10 min, probably by access- hand, T22-GFP-H6 administration to mice bearing ing the fenestrated vessels during a short time period, but CXCR4+ SC SUDHL-2 tumors shows significantly higher being unable to reach their parenchyma. Our data are in uptake than CXCR4– SC SUDHL-2 tumors. Moreover, we dramatic contrast to the reported biodistribution of most confirmed the capacity of T22-GFP-H6 to internalize in nanocarriers studied so far, regardless of whether this was CXCR4+ mouse cells, similar to our findings in CXCR4+ targeted actively or passively. human cells. Thus, the high T22-GFP-H6 tumor uptake, Nowadays, most nanocarriers that transport medicinal and its low uptake in non-tumor organs, is necessarily drugs in clinical trials, or that are available on the market, related to the huge CXCR4 overexpression in DLBCL use passive targeting (e.g. liposomal doxorubicin or albu- lymphoma cells and the negligible or low CXCR4 expres- min-paclitaxel). They enhance the drug antitumor effect sion in normal organs, including BM mouse hematopoietic because its particulate size increases its permeability and cells. retention in the tumor (EPR effect). Nevertheless, 50-80% Importantly, in the disseminated CXCR4+ DLBCL of these nanocarriers accumulate in the liver.26 Although mouse model, involving BM and LN, this nanocarrier also still at an initial stage, active nanocarrier targeting is being shows a high tumor uptake in the organs affected by developed to selectively deliver antitumor drugs to tumor CXCR4+ lymphoma cells, while displaying low biodistrib- cells through specific surface receptors.27 Regarding B-cell ution to normal tissues (with low or null CXCR4 expres- lymphoma therapy, the use of doxorubicin-loaded meso- sion). Unlike low molecular weight drugs that passively porous silica nanoparticles bound to rituximab, for target- diffuse to all cells in the body, the biodistribution of the ing CD20+ B cells, demonstrated a significant increase in nanocarrier, or drug-loaded nanocarriers, is limited by doxorubicin tumor uptake and higher inhibition of tumor their size; thus, it becomes highly dependent on the phys- growth than free doxorubicin.28 Moreover, additional tar- iology and anatomy of specific organs in the body. geted and non-targeted therapeutic nanoparticles are cur- Nanocarriers are unable to access organs irrigated by ves- rently being evaluated for treatment of B-cell malignancies; sels with continuous endothelia and unable to penetrate however, no efficacy data are available yet because Phase I membranes, unless they are actively targeted for endocy- clinical assays to test their tolerability are still ongoing.29 tosis.34 Our protein nanocarrier can accumulate in the sinu- The strategy we have used here with the actively target- soids of BM and LN infiltrated with tumor cells because ed T22-GFP-H6 nanocarrier achieves selective and they display vascular beds with discontinuous endotheli- enhanced biodistribution to tumor tissue with no toxicity um and 100-200nm fenestrations that allow the transport in the non-tumor organs. One possible explanation for the of macromolecules, including nanocarriers.35-37 Moreover, enhanced T22-GFP-H6 tumor uptake relates to the nature as we have showed in the SC mouse model, T22-GFP-H6 of the nanocarrier material. While our nanocarrier is made also has the capacity to internalize specifically in the of self-assembled proteins, most, if not all, nanocarriers CXCR4+ DLBCL cells, here localized in BM and LN in the showing limited biodistribution to tumor are either inor- DLBCL disseminated model. Even though there is no con- ganic (gold, silica, iron oxide, quantum dots) or organic sistent EPR effect in hematologic neoplasias,20 the struc- (dendrimers, liposomes polymers, hydrogels) rather than ture of the vessels in the sinusoids of the DLBCL niches protein-based.23,24 Once administered in blood, non-pro- and the active targeting to CXCR4 allow T22-GFP-H6 tein-based nanocarriers are covered by a protein corona accumulation and internalization in the tumor niches that that changes the conformation of the nanocarrier surface30 are infiltrated by CXCR4+ DLBCL cells. and undergo intensive phagocytosis by resident Given the high selectivity that T22-GFP-H6 achieves in macrophages in clearance organs.31 A completely different targeting CXCR4+ DLBCL cells within the tumor, we used protein drug delivery system is represented by the targeted the SC CXCR4+ Toledo model to test the antitumor activ- antibody-drug conjugates (ADC), which have lower load- ity of T22-DITOX-H6, a therapeutic nanoparticle derived ing capacity and flexibility for encapsulating various cargos from this nanocarrier that incorporates the diphtheria and display a less controllable drug release kinetics com- cytotoxic domain. This therapeutic nanoparticle induced a pared to nanocarriers.32 Consequently, in clinical studies, high level of apoptotic cell death in tumor tissue without only 0.001-0.01% of the injected antibody dose reaches toxicity, since it did not induce any macroscopic or histo- haematologica | 2020; 105(3) 751 A. Falgàs et al. logical alteration in normal organs, including the BM. The Sant Pau, Barcelona) and Lola Mulero Pérez (histology unit from higher levels of CXCR4 expression in DLBCL cells, as CMRB, Barcelona) for their technical support. compared to normal hematopoietic cells in the BM, were likely responsible for the cytotoxic activity, observed Funding exclusively in tumor cells. These data confirm the capacity This work was supported by Instituto de Salud Carlos III of the studied protein nanocarrier to be used as a platform (ISCIII, Co-funding from FEDER) [PI18/00650, PIE15/00028, for the delivery of antitumor agents to DLBCL cells. We PI15/00378 and EU COST Action CA 17140 to RM, FIS have previously described also the potential use of T22- PI17/01246, RD12/0036/0071 and FIS PI14/00450 to JS; GFP-H6 as an antitumor drug delivery agent for the treat- CP15/00163 to MVC; FIS PI15/00272 to EV]; Agencia Estatal ment of colorectal cancer and leukemia.18,38,39 To our de Investigación (AEI) and Fondo Europeo de Desarrollo knowledge, no protein-based therapeutic nanoparticle has Regional (FEDER) (grant BIO2016-76063-R, AEI/FEDER, been previously reported as a possible drug carrier for UE) to AV; CIBER-BBN [CB06/01/1031 and 4NanoMets to lymphoma therapy. RM, and VENOM4CANCER to AV]; AGAUR [2017 FI_B So far, most research studies for DLBCL therapy target- 00680 to AF; 2017-SGR-865 to RM, 2017-SGR-1395 to JS ing CXCR4 are performed with CXCR4 antagonists (e.g. and 2017SGR-229 to AV]; Josep Carreras Leukemia Research plerixafor or BKT140)8,10,15 or inverse agonists (e.g. IQS- Institute [P/AG to RM]; a grant from the Cellex Foundation, 01.01RS).40 Our approach differs from these studies since Barcelona [to JS]; a grant from La Generalitat de Catalunya it is not focused on inhibiting signaling downstream of the (PERIS) [SLT002/16/00433 to JS]; a grant from Fundacion CXCR4 receptor, but, instead, in delivering high concen- MMA [AP166942017 to MVC] and the Generalitat de trations of potent therapeutic agents to specifically kill Catalunya CERCA Programme. The work was also funded by CXCR4+ lymphoma cells. The active delivery of the drug- Grants PERIS SLT006/17/00093 [to UU], Fundación Española loaded nanocarriers only to CXCR4+ cells should increase de Hematología y Hemoterapia (FEHH) [to VP] and a Miguel the therapeutic index compared to low molecular weight Servet contract from ISCIII to MVC. The bioluminescent follow- CXCR4 inhibitors, which biodistribute to all tissues inde- up of cancer cells and nanoparticle biodistribution and toxicity pendently of their CXCR4 expression.41,42 In conclusion, studies have been performed in the ICTS-141007 Nanbiosis specifically eliminating CXCR4+ DLBCL cells could be an Platform, using its CIBER-BBN Nanotoxicology Unit effective strategy to enhance the survival and cure rates (http://www.nanbiosis.es/portfolio/u18-nanotoxicology-unit/). observed in R-CHOP refractory or relapsed patients. 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