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Noninvasive imaging is a powerful tool for understanding the in vivo behavior of drug delivery systems and successfully translating promising platforms into the clinic. Extracellular vesicles (EVs), nano-sized vesicles with a lipid bilayer produced by nearly all cell types, are emerging platforms for drug delivery. To date, the biodistribution of EVs has been insufficiently investigated, particularly using nuclear imaging-based modalities such as positron emission tomography (PET). Herein, we developed positron-emitting radiotracers to investigate the biodistribution of EVs isolated from various human cell sources using PET imaging. Chelator conjugation did not impact EVs size and subsequent radiolabeling was found to be highly efficient and stable with Zr-89 (t1/2 = 78.4 h). In vivo tracking of EVs isolated from bone marrow-derived mesenchymal stromal cells (BMSCs EVs), primary human macrophages (Mϕ EVs), and a melanoma cell line (A375 EVs) were performed in immunocompetent ICR mice. Imaging studies revealed excellent in vivo circulation for all EVs, with a half-life of approximately 12 h. Significantly higher liver uptake was observed for Mϕ EVs, evidencing the tissue tropism of EV and highlighting the importance of carefully choosing EVs cell sources for drug delivery applications. Conversely, the liver, spleen, and lung uptake of the BMSC EVs and A375 EVs was relatively low. We also investigated the impact of immunodeficiency on the biodistribution of BMSC EVs using NSG mice. The spleen uptake drastically increased in NSG mice, which could confound results of therapeutic studies employing this mouse models. Lastly, PET imaging studies in a melanoma tumor model demonstrated efficient tumor uptake of BMSC EVs following intravenous injection. Overall, these imaging studies evidenced the potential of EVs as carriers to treat a variety of diseases, such as cancer or in regenerative medicine applications, and the necessity to understand EVs tropism to optimize their therapeutic deployment.

AbstractDevelopment of animal models that accurately recapitulate human cancer is an ongoing challenge. This is particularly relevant in the study of osteosarcoma (OS), a highly malignant bone tumor diagnosed in approximately 1000 pediatric/adolescent patients each year. Metastasis is the leading cause of patient death underscoring the need for relevant animal models of metastatic OS. In this study, we describe how existing OS mouse models can be interrogated in a time-course context to determine the kinetics of spontaneous metastasis from an orthotopically implanted primary tumor. We evaluated four highly metastatic OS cell lines (3 human, 1 mouse) to establish a timeline for metastatic progression in immune deficient NSG mice. To discern the effects of therapy on tumor development and metastasis in these models, we investigated cisplatin therapy and surgical limb amputation at early and late timepoints. These data help define the appropriate observational periods for studies of metastatic progression in OS and further our understanding of existing mouse models. Efforts to advance the study of metastatic OS are critical for facilitating the identification of novel therapeutics and for improving patient survival.

A numerous number of positive and negative signals via various molecules modulate T-cell activation. Within the various transmembrane proteins, SIRPγ is of interest since it is not expressed in rodents. SIRPγ interaction with CD47 is reevaluated in this study. Indeed, we show that the anti-SIRPγ mAb clone LSB2.20 previously used by others has not been appropriately characterized. We reveal that the anti-SIRPα clone KWAR23 is a Pan anti-SIRP mAb which efficiently blocks SIRPα and SIRPγ interactions with CD47. We show that SIRPγ expression on T cells varies with their differentiation and while being expressed on Tregs, is not implicated in their suppressive functions. SIRPγ spatial reorganization at the immune synapse is independent of its interaction with CD47. In vitro SIRPα-γ/CD47 blockade with KWAR23 impairs IFN-γ secretion by chronically activated T cells. In vivo in a xeno-GvHD model in NSG mice, the SIRPγ/CD47 blockade with the KWAR23 significantly delays the onset of the xeno-GvHD and deeply impairs human chimerism. In conclusion, we have shown that T-cell interaction with CD47 is of importance notably in chronic stimulation.

Transplantable in vivo CRISPR/Cas9 knockout screens, in which cells are transduced in vitro and inoculated into mice to form tumours in vivo, offer the opportunity to evaluate gene function in a cancer model that incorporates the multicellular interactions of the tumour microenvironment. In this study, we sought to develop a head and neck squamous cell carcinoma (HNSCC) tumour xenograft model for whole-genome screens that could maintain high gRNA representation during tumour initiation and progression. To achieve this, we sought early-passage HNSCC cell lines with a high frequency of tumour initiation-cells, and identified the pseudodiploid UT-SCC-54C line as a suitable model from 23 HNSCC lines tested based on a low tumourigenic dose for 50% takes (TD50) of 1100 cells in NSG mice. On transduction with the GeCKOv2 whole-genome gRNA library (119,461 unique gRNAs), high (80-95%) gRNA representation was maintained in early (up to 14 d) UT-SCC-54C tumours in NSG mice, but not in UT-SCC-74B tumours (TD50=9200). However, loss of gRNA representation was observed in UT-SCC-54C tumours following growth for 38-43 days, which correlated with a large increase in bias among gRNA read counts due to stochastic expansion of clones in the tumours. Applying binomial thinning simulations revealed that the UT-SCC-54C model would have 40-90% statistical power to detect drug sensitivity genes with log2 fold change effect sizes of 1-2 in early tumours with gRNA libraries of up to 10,000 gRNAs and modest group sizes of 5 tumours. In large tumours, this model would have had 45% power to detect log2 fold change effect sizes of 2-3 with libraries of 2,000 gRNAs and 14 tumours per group. Based on our findings, we conclude that gRNA library size, sample size and tumour size are all parameters that can be individually optimised to ensure transplantable in vivo CRISPR screens can successfully evaluate gene function.

Collagen VI is distributed in the interstitium and is secreted mainly by mesenchymal stromal cells (MSCs) in skeletal muscle. Mutations in COL6A1-3 genes cause a spectrum of COL6-related myopathies. In this study, we performed a systemic transplantation study of human-induced pluripotent stem cell (iPSC)-derived MSCs (iMSCs) into neonatal immunodeficient COL6-related myopathy model (Col6a1KO/NSG) mice to validate the therapeutic potential. Engraftment of the donor cells and the resulting rescued collagen VI were observed at the quadriceps and diaphragm after intraperitoneal iMSC transplantation. Transplanted mice showed improvement in pathophysiological characteristics compared with untreated Col6a1KO/NSG mice. In detail, higher muscle regeneration in the transplanted mice resulted in increased muscle weight and enlarged myofibers. Eight-week-old mice showed increased muscle force and performed better in the grip and rotarod tests. Overall, these findings support the concept that systemic iMSC transplantation can be a therapeutic option for COL6-related myopathies.

Abstract Leukocyte adhesion deficiency type 1 (LAD-1) is an inherited primary immunodeficiency caused by loss-of-function mutation within the ITGB2 gene, which encodes the beta2 integrin subunit CD18. Individuals with LAD-1 experience significant loss of neutrophil-mediated innate cellular immune function, resulting in delayed wound healing, severe periodontitis, and life-long bouts of bacterial infection. LAD-1 is a prime candidate for lentiviral vector-mediated genetic intervention as i) it is an intractable, potentially life-threatening disease with limited treatment options, ii) it is amenable to current ex vivo gene therapy procedures, and iii) partial phenotypic correction would present a high likelihood of significant clinical benefit. Allogeneic stem cell transplant can be curative, but suffers from matched donor availability and the potential for graft-versus-host disease. Autologous ex vivo gene therapy may provide a viable alternative to allogeneic transplant in LAD-1 patients. We have evaluated the ability of a CD18-expressing lentiviral vector (LV-hCD18) to mediate ex vivo transduction of LAD-1 patient-derived CD34+ hematopoietic stem and progenitor cells (HSPCs) and subsequent long-term LAD-1 HSPC engraftment in immunodeficient NOD-scid IL2Rg null (NSG) mice. An open reading frame encoding human CD18 was placed under the transcriptional control of the MND promoter (a modified retroviral promoter associated with high levels of stable transgene expression) and packaged in VSV-G-pseudotyped lentiviral particles. After 1 day of pre-stimulation, LAD-1 HSPCs were transduced with LV-hCD18 (MOI = 10) in the presence or absence of transduction-enhancing adjuvants, poloxamer 407 (P407) and prostaglandin E2 (PGE 2), for 24 hours. Sublethally irradiated NSG mice (7 mice/group) were transplanted with either mock-transduced LAD-1 HSPCs, LAD-1 HSPCs transduced in the absence of adjuvants, or LAD-1 HSPCs transduced in the presence of P407/PGE 2. Bone marrow was harvested at ~5.5 months post-transplant for flow cytometric analyses of engraftment efficiency, transgene marking, and human blood cell lineage reconstitution. Bone marrow from mice that received mock-transduced LAD-1 HSPCs showed an average total of 6.45 ± 2.54% (mean ± SEM) CD45+ human cells. Mice that received LAD-1 HSPCs transduced in the absence of adjuvants showed 7.99 ± 1.82% CD45+ human cells, whereas mice transplanted with LAD-1 HSPCs transduced in the presence of adjuvants showed 7.33 ± 1.90% CD45+ cells. A Kruskal-Wallis statistical test indicated no significant difference in the level of human cell engraftment among the recipient groups (P=0.72). Consistent with the LAD-1 phenotype, human myeloid cells from mice that received mock-transduced LAD-1 HSPCs displayed only background levels of CD18 marking (0.13 ± 0.06% CD45+CD13+CD18+ cells). Mice that received LAD-1 HSPCs transduced in the absence of adjuvants showed 4.05 ± 0.40% CD18+ human myeloid cells (range 2.19% to 5.50%), whereas mice that received LAD-1 HSPCs transduced in the presence of P407/PGE 2 showed 9.56 ± 0.96% CD18+ human myeloid cells (range 4.63% to 13.10%), thus representing a >2-fold increase in in vivo, vector-mediated transgene marking levels when adjuvant was used. Moreover, vector-mediated expression of CD18 rescued endogenous expression of a major CD18 heterodimerization partner in neutrophils, CD11b. In mock-transduced LAD-1 HSPC recipients, CD13+ human myeloid cells were devoid of cell surface CD11b expression (0.01 ± 0.01% CD45+CD13+CD11b+ cells). In contrast, CD13+ human myeloid cells in mice that received LAD-1 HSPCs transduced in the absence of adjuvant showed detectable levels of CD11b expression (2.62 ± 0.19% of CD18-expressing human myeloid cells), and CD11b levels were increased to 6.90 ± 0.98% in LAD-1 HSPCs transduced in the presence of P407/PGE 2. Multilineage engraftment, as evidenced by the presence of CD3+ T cells and CD20+ B cells, was noted within all groups; however, human myeloid cells represented the most prominent human blood cell compartment observed. Colony-forming-unit assays of transduced cells and non-transduced control cells pre-transplant showed similar clonogenic output and colony diversity. In sum, successful transduction, engraftment, transgene marking, CD11b rescue, and multilineage reconstitution supports further development of lentiviral vector-mediated gene therapy for LAD-1. Disclosures No relevant conflicts of interest to declare.

Abstract Checkpoint inhibition (CPI) has shown dramatic improvements in overall survival in many malignant diseases. However, in multiple myeloma (MM) the results were disappointing resulting in an early termination of clinical trials. Despite the advantages in therapy the disease remains incurable. Methods: We analyzed the efficiency and immunological mechanisms of PD-1/PD-L1 blockade using KaLwRij mice that develop MM upon injection of 5T33 myeloma cells. Treatment of mice started d19 post inoculation. Bone marrow (BM) and spleen cells were analyzed by flow cytometry for the phenotype of immune cells. Results: Comprehensive immunophenotyping including the analysis of T and NK cell subpopulations revealed no differences of early MM disease stage compared to healthy control groups. Treatment of mice with mAbs blocking PD-1 or PD-L1 had no effect on tumor growth and survival. It was demonstrated that HDAC inhibitors beside their direct effect on malignant cells may increase the immunogenicity of malignant cells by improving the presentation of tumor antigens and modulate the immunological composition of the tumor microenvironment (TME). The pan-HDAC inhibitor panobinostat that is approved for the treatment of myeloma patients inhibited the development of myeloma in treated mice. Surprisingly, the combined application of the anti-PD-1 blocking antibody with panobinostat reduced the anti-myeloma effect of the compound and resulted in decreased survival. By analyzing the phenotype of immune cells in the different populations, we found in the panobinostat treated animal group an increase in the CXCR4 expressing CD4+ NKT cells. Additionally, the CD8+ T cells expressing CD1d and CXCR4 decreased compared to the other groups in the spleen. CD1d is a MHC like receptor for glycolipids activating NKT cells, whereas CXCR4 is a BM homing receptor and linked to metastasis and tumor aggressiveness. We found a gradually increase of CXCR4+ NKT cells in the BM corresponding to MM disease progression. Interestingly, we observed a shift in the CD4+/CD4- NKT cell ratio during disease progression, whereby the CXCR4+ CD4- NKT cells seem to be associated with advanced tumor growth, while the increase of CXCR4+ CD4+ NKT are associated with prolonged survival as observed in in the panobinostat treated group. To further analyze the role of PD-L1 expression on myeloma cells we generated a PD-L1 KO of the 5T33 cell line using the CRISPR/Cas9 technology. We found no differences in the expression of surface molecules such as MHC class I and II, co-stimulatory or adhesion molecules, proliferation and migration of the genetically engineered cells in comparison to the mock control. Interestingly, mice inoculated with the 5T33 PD-L1 KO cells showed a significant longer survival compared with the 5T33 mock injected, indicating that blocking of the PD-L1 molecule on myeloma cells plays an important role in the pathogenesis of MM and its direct blocking on malignant cells rather than in the TME might have an impact on the clinical efficiency. When analyzing the spleen of the mock vs PD-L1 KO myeloma inoculated mice, we found the same downregulation of CXCR4 and CD1d on CD8+ T cells in the PD-L1 KO myeloma group as observed in the panobinostat treated group with extended survival. In addition, we used NOD. scid. Il2Rγc null (NSG) mice to proof that the survival prolongation is a result of the immunological response to PD-L1 and that the myeloma cells are not otherwise effected in their tumor cell properties in vivo. NSG mice experience the same tumor burden post 5T33 mock and PD-L1 KO challenge, assuming that the previous observed survival prolongation is exclusively dependent on the PD-L1 tumor- immune cell interaction. Conclusion: We found that PD-1 blockade might negatively affect and inhibit the therapeutic efficacy of HDAC inhibitors such as panobinostat. Genetic down regulation of PD-L1 on the myeloma cells enables a significant improvement and longer survival. These results give new insights into the complexity of the action of CPI in the treatment of malignant diseases which might help to develop combinatorial approaches of checkpoint inhibitors in clinical trials. Furthermore, the increase of CD4- CXCR4 expressing NKT cells in the BM might be used as biomarker to monitor MM disease progression, whereas the increase of the CD4+/CD4- NKT cell ratio in the BM might be associated with the shrinkage of MM tumor burden. Disclosures Brossart: BMS: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria; MSD: Honoraria.

Abstract Alpha granules in megakaryocytes contain a mixture of endogenously expressed proteins as well as proteins taken up from the intramedullar fluid. Both pools are thought to be found in all alpha granules in the megakaryocytes and released platelets. We have been studying the ectopic expression of urokinase (uPA) in platelets as a targeting strategy for fibrinolysis of nascent thrombi without causing fibrinolysis of established thrombi. These studies also demonstrated that there are two distinct pools of alpha granules, an endogenous cargo pool of granules and an exogenous uptake cargo pool of granules. Using in vitro grown megakaryocytes from two sources (1) CD34+-hematopoietic progenitor cells and (2) induced-pluripotent stem cell derived line imMKCL kindly provided by Dr. Koji Eto at Kyoto University, we demonstrated that urokinase can be localized within alpha granules in the megakaryocytes by either adding urokinase to the media or by ectopically expressing the protein using a lentiviral strategy. We observed that both a human single-chain uPA (scuPA) or a plasmin-insensitive but thrombin-activatable truncated human uPA mutant (uPA-T) in the media were internalized into granules distinct from granules containing ectopically expressed mouse scuPA following lentiviral transduction. Endocytosed uPA showed no co-localization with endogenous von Willebrand Factor (vWF), but significant colocalization with endocytosed Factor V or plasminogen (PLG) on confocal immunofluorescent microscopy. Further, Factor V competed with both uPA variants for uptake from the media. Uptake of these proteins was inhibited by the LRP1 antagonist receptor-associated protein (RAP) and by anti-LRP1 antibodies. This suggests that both proteins use the same endocytic receptor pathway and share this pathway with other proteins taken up from the media, including Factor V. We found that in vitro-generated CD34+ megakaryocytes pre-loaded with exogenously added PLG and co-incubated thereafter with recombinant scuPA and FV significantly degraded FV; however, no vWF degradation was observed in CD34+-derived megakaryocytes that had endocytosed or ectopically expressed scuPA with exogenously added PLG, suggesting that only the proteins which are endocytosed by in vitro-generated megakaryocytes are degraded by uPA-generated plasmin, whereas endogenous alpha-granular proteins remain intact. We then asked whether uPA localized in these two distinct pools can be released at sites of nascent thrombus formation and be effective in preventing nascent thrombus growth. We infused CD34+-derived MKs into NOD-scid IL2rγnull (NSG) mice homozygous for VWF R1326H (a mutation switching binding VWF specificity from mouse to human GPIb/IX). NSG/VWF R1326H mice have impaired clotting after vascular injury compared to NSG mice unless infused with human platelets or MKs . Significantly less post-injury clotting was seen upon infusion of either endogenous or exogenous scuPA-containing MK infusion. Further studies to define relative efficacy at the same levels of scuPA are being pursued. These studies show that there are two sets of alpha granules that remain separate during megakaryopoiesis in vitro: granules with endogenously expressed cargo and granules with endocytosed cargo with limited mixing between the two pools by confocal microscopy studies and following PLG uptake studies. The extent of mixing that occurs subsequently in released platelets was not studied nor has these finding been done with primary MKs not grown in culture; however, we believe that these studies extend our understanding of the nature of alpha granules and offer new insights into how to manipulate their cargo. Disclosures Cines: Dova: Consultancy; Rigel: Consultancy; Treeline: Consultancy; Arch Oncol: Consultancy; Jannsen: Consultancy; Taventa: Consultancy; Principia: Other: Data Safety Monitoring Board.

Abstract Introduction: Multiple myeloma (MM) is a disease of malignant plasma cells, characterized by high CD38 expression. Although the CD38-targeting monoclonal antibodies are highly effective, resistance invariably arises. Tumor CD38 levels decrease after anti-CD38 therapy, but the expression is rarely permanently silenced. This suggests that CD38 expression may offer a tumor cell survival advantage, but the direct impact of CD38 loss on tumor dynamics has not been extensively characterized. Methods: CD38 knockout (KO) cell lines were generated by CRISPR-Cas9. Immunocompetent Balb/c and immunodeficient NSG mice were injected subcutaneously with either non-targeting (NT) or CD38 KO J558 cells. Stromal adhesion was compared using labeled NT and KO cells, with OP-9 murine stroma cells. Cellular NAD content was quantified using the Promega Glo Assay. Mitochondria were isolated with the Mitochondria Isolation Kit (Thermo Scientific). Oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were quantified using the Seahorse Assay. Response to hypoxia was evaluated using a modular hypoxic chamber. Cell cycle was quantified using propidium iodine staining. Results: To examine the role of CD38 in murine models, we utilized the CD38-expressing, murine plasmacytoma cell line J558. Strikingly, CD38 KO cells injected into Balb/c mice demonstrated significantly decreased tumor volume compared to NT (113 mm 3 (KO) vs. 1293 mm 3 (NT) at day 25, p <0.001). In contrast, in vitro cell proliferation and colony formation between KO and NT J558 cells were nearly identical, suggesting that the effects of CD38-loss were highly context dependent. Since tumoral CD38 expression may negatively modulate the immune response, we next compared CD38 KO and NT cells injected into immunodeficient NSG mice. CD38 KOs demonstrated an approximately 2.2-fold decreased tumor volume compared to the NT (708 mm 3 (KO) vs. 1592 mm 3 (NT), p=0.07). Further examination of the role of CD38 on the immune microenvironment are ongoing. Considering that some tumor growth impairment was maintained in immunodeficient mice, we next interrogated the effect of CD38 loss on other aspects of cell proliferation using J558 as well as human MM cell lines RPMI-8226 and NCI-H929. Daratumumab induced CD38 internalization has been shown to reduce stromal adhesion of MM cells. Similarly, CD38 KO cells demonstrated reduced stromal adhesion (2.5-fold decrease for J558, p<0.005 and 2-fold decrease for H929, p<0.005). Although stroma is a known promoter of cell survival and proliferation, we further questioned whether the NAD-metabolizing activity of CD38 modulates tumor growth. CD38 overexpression can drive down intracellular NAD and impair mitochondrial biogenesis. Accordingly, we found significantly higher NAD levels in the KO J558 tumor cells compared to NT (2-fold change, p <0.05). Additionally, CD38 KO cells demonstrated significantly higher levels of mitochondrial protein compared with the NTs (5-fold in J558 and 2-fold in H929). CD38 KO cell lines also showed markedly increased metabolic activity, with nearly 2-fold increase in basal OCR and ECAR, as well as in spare respiratory and glycolytic capacity. Given the contrast between in vivo and in vitro growth capacity, we questioned whether changes in mitochondrial content and metabolic function could confer an advantage for CD38-expressing cells under conditions of hypoxia, which is an important characteristic of the tumor microenvironment. Strikingly, under hypoxia, but not normoxia, CD38 KO MM cells demonstrated significantly more cell cycle arrest, defined by G0/G1 blockage (p=0.003 for H929 and p=0.004 for RPMI). Conclusion: We have shown that CD38 KO cells demonstrate decreased tumor growth in vivo but not in vitro. While the immune modulatory potential of CD38 is recognized, some of the growth impairment we observed may be explained by non-immune mediated mechanisms such as reduced stroma adherence as well as changes in cell metabolism. Loss of CD38 was associated with increased mitochondrial respiration, but also elevated ECAR and glycolytic rate. Higher reliance on mitochondrial respiration could explain impaired CD38 KO proliferation rates under hypoxia, possibly as a result of increased generation of reactive oxygen species. Disclosures Ghiaur: Menarini Richerche: Research Funding; Syros Pharmaceuticals: Consultancy.