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• W2885295219 abstract "•We provide a comparison of engraftment efficiencies in unconditioned NRG (NOD/RAG1/2−/−IL2Rγ−/−) and NRGS (NRG-SGM3) mice.•NRGS mice do not develop clinically accurate disease with cell line xenografts.•NRG mice do not efficiently engraft patient normal or leukemic cells.•NRG and NRGS mice tolerate high doses of induction therapeutics. Cell-line-derived xenografts (CDXs) or patient-derived xenografts (PDXs) in immune-deficient mice have revolutionized our understanding of normal and malignant human hematopoiesis. Transgenic approaches further improved in vivo hematological research, allowing the development of human-cytokine-producing mice, which show superior human cell engraftment. The most popular mouse strains used in research, the NOG (NOD.Cg-Prkdcscid Il2rγtm1Sug/Jic) and the NSG (NOD/SCID-IL2Rγ−/−, NOD.Cg-PrkdcscidIl2rγtm1Wjl/SzJ) mouse, and their human-cytokine-producing (interleukin-3, granulocyte-macrophage colony-stimulating factor, and stem cell factor) counterparts (huNOG and NSGS), rely partly on a mutation in the DNA repair protein PRKDC, causing a severe combined immune deficiency (SCID) phenotype and rendering the mice less tolerant to DNA-damaging therapeutics, thereby limiting their usefulness in the investigation of novel acute myeloid leukemia (AML) therapeutics. NRG (NOD/RAG1/2−/−IL2Rγ−/−) mice show equivalent immune ablation through a defective recombination activation gene (RAG), leaving DNA damage repair intact, and human-cytokine-producing NRGS (NRG-SGM3) mice were generated, improving myeloid engraftment. Our findings indicate that unconditioned NRG and NRGS mice can harbor established AML CDXs and can tolerate aggressive induction chemotherapy at higher doses than NSG mice without overt toxicity. However, unconditioned NRGS mice developed less clinically relevant disease, with CDXs forming solid tumors throughout the body, whereas unconditioned NRG mice were incapable of efficiently supporting PDX or human hematopoietic stem cell engraftment. These findings emphasize the contextually dependent utility of each of these powerful new strains in the study of normal and malignant human hematopoiesis. Therefore, the choice of mouse strain cannot be random, but must be based on the experimental outcomes and questions to be addressed. Cell-line-derived xenografts (CDXs) or patient-derived xenografts (PDXs) in immune-deficient mice have revolutionized our understanding of normal and malignant human hematopoiesis. Transgenic approaches further improved in vivo hematological research, allowing the development of human-cytokine-producing mice, which show superior human cell engraftment. The most popular mouse strains used in research, the NOG (NOD.Cg-Prkdcscid Il2rγtm1Sug/Jic) and the NSG (NOD/SCID-IL2Rγ−/−, NOD.Cg-PrkdcscidIl2rγtm1Wjl/SzJ) mouse, and their human-cytokine-producing (interleukin-3, granulocyte-macrophage colony-stimulating factor, and stem cell factor) counterparts (huNOG and NSGS), rely partly on a mutation in the DNA repair protein PRKDC, causing a severe combined immune deficiency (SCID) phenotype and rendering the mice less tolerant to DNA-damaging therapeutics, thereby limiting their usefulness in the investigation of novel acute myeloid leukemia (AML) therapeutics. NRG (NOD/RAG1/2−/−IL2Rγ−/−) mice show equivalent immune ablation through a defective recombination activation gene (RAG), leaving DNA damage repair intact, and human-cytokine-producing NRGS (NRG-SGM3) mice were generated, improving myeloid engraftment. Our findings indicate that unconditioned NRG and NRGS mice can harbor established AML CDXs and can tolerate aggressive induction chemotherapy at higher doses than NSG mice without overt toxicity. However, unconditioned NRGS mice developed less clinically relevant disease, with CDXs forming solid tumors throughout the body, whereas unconditioned NRG mice were incapable of efficiently supporting PDX or human hematopoietic stem cell engraftment. These findings emphasize the contextually dependent utility of each of these powerful new strains in the study of normal and malignant human hematopoiesis. Therefore, the choice of mouse strain cannot be random, but must be based on the experimental outcomes and questions to be addressed. Immune-deficient mice have revolutionized biomedical research, including the study of both normal human hematopoiesis and leukemogenesis [1Sykes SM Scadden DT Modeling human hematopoietic stem cell biology in the mouse.Semin Hematol. 2013; 50: 92-100Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar, 2Manz MG Di Santo JP Renaissance for mouse models of human hematopoiesis and immunobiology.Nat Immunol. 2009; 10: 1039-1042Crossref PubMed Scopus (71) Google Scholar, 3Cook GJ Pardee TS Animal models of leukemia: any closer to the real thing?.Cancer Metastasis Rev. 2013; 32: 63-76Crossref PubMed Scopus (29) Google Scholar]. Capable of harboring both normal and malignant human xenografts without rejection, highly immune-deficient mice are indispensable in hematological research, allowing differentiation and proliferation of these cells in vivo [4Ito R Takahashi T Katano I Ito M Current advances in humanized mouse models.Cell Mol Immunol. 2012; 9: 208-214Crossref PubMed Scopus (243) Google Scholar]. Xenograft mouse models consistently better predict the success of experimental chemotherapeutics in clinical trials [5Zuber J Radtke I Pardee TS et al.Mouse models of human AML accurately predict chemotherapy response.Genes Dev. 2009; 23: 877-889Crossref PubMed Scopus (204) Google Scholar], likely because of the complex and dynamic interaction between the bone marrow (BM) microenvironment and heterogeneous populations of leukemic cells [6Francia G Cruz-Munoz W Man S Xu P Kerbel RS Mouse models of advanced spontaneous metastasis for experimental therapeutics.Nat Rev Cancer. 2011; 11: 135-141Crossref PubMed Scopus (276) Google Scholar]. Seminal xenograft studies showed that only the most primitive fraction of patient leukemic cells (Lin–CD34+), but not mature blasts, were capable of transferring disease to primary and secondary immune-deficient mice [7Lapidot T Sirard C Vormoor J et al.A cell initiating human acute myeloid leukaemia after transplantation into SCID mice.Nature. 1994; 367: 645-648Crossref PubMed Scopus (3571) Google Scholar, 8Bonnet D Dick JE Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell.Nat Med. 1997; 3: 730-737Crossref PubMed Scopus (5301) Google Scholar]. These cells, dubbed leukemic stem cells (LSCs), exist at the top of a hierarchy similar to normal hematopoiesis, in which a small population of slowly proliferating, self-renewing LSCs gives rise to a large population of clonally expanded blasts [8Bonnet D Dick JE Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell.Nat Med. 1997; 3: 730-737Crossref PubMed Scopus (5301) Google Scholar]. Furthermore, current evidence supports the notion that specific chemotherapeutic protected niches exist within the BM microenvironment, providing sanctuary for LSCs, eventually causing lethal refractory relapse [9Rashidi A Uy GL Targeting the microenvironment in acute myeloid leukemia.Curr Hematol Malig Rep. 2015; 10: 126-131Crossref PubMed Scopus (51) Google Scholar, 10Meads MB Hazlehurst LA Dalton WS The bone marrow microenvironment as a tumor sanctuary and contributor to drug resistance.Clin Cancer Res. 2008; 14: 2519-2526Crossref PubMed Scopus (412) Google Scholar]. Therefore, it becomes clear that xenograft models are the most reliable preclinical method with which to model the highly complex interplay among leukemic populations, the BM microenvironment, clinical treatment outcomes, and, by extension, pharmacological efficacy of novel investigational therapeutics [11Nervi B Ramirez P Rettig MP et al.Chemosensitization of acute myeloid leukemia (AML) following mobilization by the CXCR4 antagonist AMD3100.Blood. 2009; 113: 6206-6214Crossref PubMed Scopus (392) Google Scholar, 12Majeti R Chao MP Alizadeh AA et al.CD47 is an adverse prognostic factor and therapeutic antibody target on human acute myeloid leukemia stem cells.Cell. 2009; 138: 286-299Abstract Full Text Full Text PDF PubMed Scopus (999) Google Scholar, 13Colmone A Amorim M Pontier AL Wang S Jablonski E Sipkins DA Leukemic cells create bone marrow niches that disrupt the behavior of normal hematopoietic progenitor cells.Science. 2008; 322: 1861-1865Crossref PubMed Scopus (461) Google Scholar]. Discovered in 1962, nude mice were the first immune-deficient mice capable of accepting human cells and tissues and, since then, there has been continued effort to further increase immune ablation and improve patient sample engraftment [3Cook GJ Pardee TS Animal models of leukemia: any closer to the real thing?.Cancer Metastasis Rev. 2013; 32: 63-76Crossref PubMed Scopus (29) Google Scholar, 14Flanagan SP 'Nude', a new hairless gene with pleiotropic effects in the mouse.Genet Res. 1966; 8: 295-309Crossref PubMed Scopus (605) Google Scholar]. Nude mice were followed by severe combined immune deficiency (SCID) mice [15Bosma GC Custer RP Bosma MJ A severe combined immunodeficiency mutation in the mouse.Nature. 1983; 301: 527-530Crossref PubMed Scopus (1770) Google Scholar], which lack the ability to mount and maintain adaptive immune responses because of a recessive mutation resulting in loss of enzyme DNA-dependent protein kinase catalytic subunit (Prkdc) activity, an enzyme critically important for DNA damage repair. This enzyme is required for V(D)J recombination, so T- and B-lymphocyte maturation is blocked [16Dvir A Peterson SR Knuth MW Lu H Dynan WS Ku autoantigen is the regulatory component of a template-associated protein kinase that phosphorylates RNA polymerase II.Proc Natl Acad Sci U S A. 1992; 89: 11920-11924Crossref PubMed Scopus (332) Google Scholar, 17Goodwin JF Knudsen KE Beyond DNA repair: DNA-PK function in cancer.Cancer Discov. 2014; 4: 1126-1139Crossref PubMed Scopus (142) Google Scholar]. Further immune ablation was achieved by cross-breeding with nonobese diabetic (NOD) mice [18Shultz LD Schweitzer PA Christianson SW et al.Multiple defects in innate and adaptive immunologic function in NOD/LtSz-scid mice.J Immunol. 1995; 154: 180-191PubMed Google Scholar] and backcrossing to introduce a mutated interleukin (IL)-2rγ, resulting in two of the most commonly used strains in hematopoietic research, NOG and NSG (NOD/SCID IL-2Rγnull) [19Fortier JM Graubert TA Murine models of human acute myeloid leukemia.Cancer Treat Res. 2010; 145: 183-196Crossref PubMed Scopus (12) Google Scholar]. In addition to lacking T and B lymphocytes, these mice also lack natural killer cells, exhibit decreased macrophage function, and have impaired eosinophil function, thereby allowing for efficient engraftment of human tissues [20Ito M Hiramatsu H Kobayashi K et al.NOD/SCID/gamma(c)(null) mouse: an excellent recipient mouse model for engraftment of human cells.Blood. 2002; 100: 3175-3182Crossref PubMed Scopus (1058) Google Scholar, 21Shultz LD Lyons BL Burzenski LM et al.Human lymphoid and myeloid cell development in NOD/LtSz-scid IL2R gamma null mice engrafted with mobilized human hemopoietic stem cells.J Immunol. 2005; 174: 6477-6489Crossref PubMed Scopus (1155) Google Scholar, 22Sugamura K Asao H Kondo M et al.The interleukin-2 receptor gamma chain: its role in the multiple cytokine receptor complexes and T cell development in XSCID.Annu Rev Immunol. 1996; 14: 179-205Crossref PubMed Scopus (344) Google Scholar]. Despite these advances in murine leukemic xenograft models, most samples of acute myeloid leukemia (AML) fail to sufficiently engraft, likely because of several factors including the following: (1) homing to BM, (2) lack of stromal support, (3) AML sample intrinsic factors, (4) absence of human cytokines, and (5) residual innate mouse immunity [23Wunderlich M Mizukawa B Chou FS et al.AML cells are differentially sensitive to chemotherapy treatment in a human xenograft model.Blood. 2013; 121: e90-e97Crossref PubMed Scopus (81) Google Scholar]. Transgenic approaches have allowed researchers to develop several iterations of NOD/SCID IL-2Rynull mice (NSGS, NSS, etc.) expressing several human myeloid cytokines (IL-3, granulocyte-macrophage colony-stimulating factor [GM-CSF], and stem cell factor [SCF]), thus dramatically increasing engraftment of human AML [20Ito M Hiramatsu H Kobayashi K et al.NOD/SCID/gamma(c)(null) mouse: an excellent recipient mouse model for engraftment of human cells.Blood. 2002; 100: 3175-3182Crossref PubMed Scopus (1058) Google Scholar, 21Shultz LD Lyons BL Burzenski LM et al.Human lymphoid and myeloid cell development in NOD/LtSz-scid IL2R gamma null mice engrafted with mobilized human hemopoietic stem cells.J Immunol. 2005; 174: 6477-6489Crossref PubMed Scopus (1155) Google Scholar, 24Ishikawa F Yasukawa M Lyons B et al.Development of functional human blood and immune systems in NOD/SCID/IL2 receptor {gamma} chain(null) mice.Blood. 2005; 106: 1565-1573Crossref PubMed Scopus (683) Google Scholar, 25Feuring-Buske M Gerhard B Cashman J Humphries RK Eaves CJ Hogge DE Improved engraftment of human acute myeloid leukemia progenitor cells in beta 2-microglobulin-deficient NOD/SCID mice and in NOD/SCID mice transgenic for human growth factors.Leukemia. 2003; 17: 760-763Crossref PubMed Scopus (92) Google Scholar]. Human cytokine expression supports myelopoiesis and engraftment of a variety of patient leukemia cells, but also the engraftment and multilineage expansion of mature human hematopoietic cells from CD34+ hematopoietic stem cells (HSCs) [26Wunderlich M Chou FS Link KA et al.AML xenograft efficiency is significantly improved in NOD/SCID-IL2RG mice constitutively expressing human SCF, GM-CSF and IL-3.Leukemia. 2010; 24: 1785-1788Crossref PubMed Scopus (254) Google Scholar, 27Yahata T Ando K Nakamura Y et al.Functional human T lymphocyte development from cord blood CD34+ cells in nonobese diabetic/Shi-scid, IL-2 receptor gamma null mice.J Immunol. 2002; 169: 204-209Crossref PubMed Scopus (152) Google Scholar]. Although these advancements are indeed significant, SCID-mediated immune deficiency makes the mice much less tolerant to standard of care (SOC) DNA-damaging AML therapeutics such as anthracyclines and cytarabine [5Zuber J Radtke I Pardee TS et al.Mouse models of human AML accurately predict chemotherapy response.Genes Dev. 2009; 23: 877-889Crossref PubMed Scopus (204) Google Scholar, 23Wunderlich M Mizukawa B Chou FS et al.AML cells are differentially sensitive to chemotherapy treatment in a human xenograft model.Blood. 2013; 121: e90-e97Crossref PubMed Scopus (81) Google Scholar]. Therefore, it is difficult to model xenograft response to SOC therapy, a necessary component of a truly physiologically and clinically relevant preclinical model. In 2003, Shultz et al. developed another strain of mice called NRG (NOD/Rag1/2null), in which the lymphocyte developmental phenotype is maintained through a defective recombination activation gene (RAG), leaving DNA damage repair unimpaired, with comparable immune ablation [28Shultz LD Banuelos S Lyons B et al.NOD/LtSz-Rag1nullPfpnull mice: a new model system with increased levels of human peripheral leukocyte and hematopoietic stem-cell engraftment.Transplantation. 2003; 76: 1036-1042Crossref PubMed Scopus (50) Google Scholar]. In 2014, NRG mice were crossed with NSGS mice (NSG-hIL3, h-GM-CSF, hSCF) and the SCID mutation removed through selective cross-breeding with NRG mice [29Wunderlich M Brooks RA Panchal R Rhyasen GW Danet-Desnoyers G Mulloy JC OKT3 prevents xenogeneic GVHD and allows reliable xenograft initiation from unfractionated human hematopoietic tissues.Blood. 2014; 123: e134-e144Crossref PubMed Scopus (41) Google Scholar]. This new strain of mice, called NRGS (NRG-SGM3), have a comparable degree of immune ablation compared with SCID mice, express human myeloid cytokines, and can likely tolerate higher doses of SOC therapy in a dosing regimen more similar to clinical practice [23Wunderlich M Mizukawa B Chou FS et al.AML cells are differentially sensitive to chemotherapy treatment in a human xenograft model.Blood. 2013; 121: e90-e97Crossref PubMed Scopus (81) Google Scholar]. These mice show consistent highly efficient engraftment and model the influence of the BM microenvironment, human cytokine signaling, and therapeutic outcomes. Given the recent development of these strains and their relative lack of use in AML research, the goal of this study was to provide an in-depth analysis of the comparative utility of unconditioned NRG and NRGS mice in the investigation of cell-line-derived xenografts (CDXs), patient-derived xenografts (PDXs), response to SOC chemotherapeutics, and humanization potential. The established human AML cell lines K562, MV411, and U937 were obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA) and cultured in RPMI medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin at 37°C with 5% CO2. The PDX models (CCHMC-23, CCHMC-7, CCHMC-9, and CCHMC-35) were established at Cincinnati Children's Hospital Medical Center (CCHMC) from specimens acquired under an institutional review board-approved protocol (#2008-0021). These primary specimens were established and expanded in NSGS mice at CCHMC and BM aspirates obtained from leukemic mice were frozen at –80°C in RPMI with 10% fetal bovine serum and 10% dimethylsulfoxide until xenograft. Primary AML blasts were isolated from de-identified diluted whole BM aspirate acquired from the James Graham Brown Cancer Center biorepository with informed consent and under institutional review board guidelines (#13.0188), by Histopaque gradient and centrifugation at 500 g for 20 minutes at room temperature. Human CD34+ HSCs purified from umbilical cord blood (UCB) were obtained from AllCells (Alameda, CA, USA). NRG (NOD/RAG1/2−/−IL2Rγ−/−, #007799) and NRGS (NOD/RAG1/2−/−IL2Rγ−/−Tg [CMV-IL3,CSF2,KITLG]1Eav/J, #024099) mice producing 2–4 ng/mL of human IL-3, GM-CSF, and SCF [28Shultz LD Banuelos S Lyons B et al.NOD/LtSz-Rag1nullPfpnull mice: a new model system with increased levels of human peripheral leukocyte and hematopoietic stem-cell engraftment.Transplantation. 2003; 76: 1036-1042Crossref PubMed Scopus (50) Google Scholar] were obtained from The Jackson Laboratory and bred and maintained under standard conditions in the University of Louisville Rodent Research Facility on a 12-hour light/12-hour dark cycle with food and water provided ad libitum. Animal procedures were approved by the institutional animal care and use committee. For all cell line derived xenograft studies and the human HSC study, mice received 5 × 105 cells, and for all passaged PDX studies, 1.25 × 105 human cells suspended in 200 µL of phosphate-buffered saline (PBS) per mouse by bolus intravenous (IV) injection. Similarly, mice engrafted with the primary patient sample JGB-AML1 received 2 × 106 cells in 200 µL per mouse by IV bolus. Mouse health was monitored for characteristic signs of leukemia such as scruffiness and hind-limb paralysis, at which time they were euthanized. Analysis of human cell engraftment was conducted on a case-by-case basis when mice became moribund and humane endpoints were reached. Using previously published data, we established a similar 5+3 induction therapy regimen (5 consecutive days of cytarabine with doxorubicin given for the first 3 days) using IV bolus injection of doxorubicin (3 mg/kg, days 1–3) combined with cytarabine (75 mg/kg, days 1–5). Mice began treatment 7 (or 25) days after transplantation of human leukemic cells, with all mice receiving injections balanced to a final volume of 200 μL of PBS. Patients receiving standard 7+3 induction therapy (60 mg/m2 doxorubicin and 100 mg/m2 cytarabine) get approximately 1.5 mg/kg doxorubicin and 2.5 mg/kg cytarabine. Therefore, similar to previously published work, our dose of doxorubicin was doubled and the cytarabine concentration was increased by 20-fold in an attempt to achieve necessary plasma concentrations of the drugs using a bolus injection compared with continuous infusion given in clinical care [23Wunderlich M Mizukawa B Chou FS et al.AML cells are differentially sensitive to chemotherapy treatment in a human xenograft model.Blood. 2013; 121: e90-e97Crossref PubMed Scopus (81) Google Scholar]. Clinical formulations of both doxorubicin and cytarabine were obtained from the James Graham Brown Cancer Center as self-sealing vials containing 20 mg/10 mL or 2 g/20 mL, respectively dissolved in saline, manufactured by APP (a division of Fresenius Kabi USA LLC, Lake Zurich, IL, USA). The spleen and BM cells were analyzed by fluorescence-activated cell sorting (FACS) as described previously [31Othus M Appelbaum FR Petersdorf SH et al.Fate of patients with newly diagnosed acute myeloid leukemia who fail primary induction therapy.Biol Blood Marrow Transplant. 2015; 21: 559-564Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar]. Briefly cells were isolated from tissues and red blood cells were lysed and blocked for 10 min at 4°C with Fc Block (#553142, BD Biosciences, Miami, FL, USA). The samples were then stained with the appropriate fluorophore-conjugated antihuman antibody (Alexa Fluor 700-CD45, APC-CD11b, APC-CD19, PE-CD3, PE-Cy.5-CD34, and PE-Cy.5-CD33) from BioLegend (San Diego, CA) for 20 min at 4°C and then analyzed on a Becton Dickinson FACScan with FlowJo software. All statistics were performed using GraphPad Prism 6 software. Unless specified below, significance was determined by one-way ANOVA followed by Tukey tests using a cutoff of p < 0.05. For all survival curves, the log–rank (Mantel–Cox) test was used, with a cutoff of p < 0.05. First, we wanted to examine differential engraftment efficiency of several established, commonly used human leukemic cell lines (K562, MV411, and U937) in NRG and NRGS mice. All cell lines showed high rates of engraftment in both strains, resulting in disease progression and eventual euthanasia upon achievement of humane endpoints (Figure 1A). However, the formation of large intraperitoneal (IP) tumors developed selectively in NRGS mice engrafted with all established cell lines tested, whereas NRG mice developed clinically similar diffuse disease with expected signs of disease progression such as splenomegaly. Necropsy revealed that most of the NRGS mice engrafted with MV411 cells had masses found both IP and lymphatically (Figure 1B, bottom right). K562 cells had a high propensity to form tumors in the kidneys (Figure 1B, bottom left), whereas U937 cells formed solid tumors in the spleens of only NRGS mice (Figure 1B, bottom middle). All mice engrafted with any of these cell lines showed pronounced leukemic infiltration of hematopoietic organs (Figures 2A and 2B), but NRG mice selectively failed to develop IP masses or solid tumors and showed a more clinically relevant disease presentation. Therefore, it is evident that NRG mice, but not NRGS mice, are well suited for studies involving CDXs.Figure 2H&E staining of spleen sections from NRG and NRGS mice xenografted with established human cell lines. (A) H&E-stained sections of representative formalin-fixed spleens from NRG mice IV xenografted with 5 × 105 K562, MV411, or U937 cells per mouse suspended in 200 μL of PBS. (B) Representative spleens from NRGS mice IV xenografted with 5 × 105 K562, MV411, or U937 cells per mouse suspended in 200 μL of PBS. PBS, phosphate-buffered saline.View Large Image Figure ViewerDownload Hi-res image Download (PPT) We next examined the differential engraftment efficiency of several patient-derived samples in both NRG and NRGS mice. All patient-derived samples were acquired from pediatric patients upon relapse and three of four displayed mixed-lineage leukemia translocations, whereas the final sample was cytogenetically normal (Figure 3A). Disease progression required euthanasia upon reaching humane endpoints in all of the NRGS mice irrespective of sample origin, whereas all NRG mice survived until the 100-day study endpoint without overt signs of disease (Figure 3B). As expected, efficient engraftment occurred with all four patient-derived samples in NRGS mice, leading to leukemic infiltration of the hematopoietic compartment, whereas three of the four patient samples tested (CCHMC-7, CCHMC-23, and CCHMC-35) showed virtually no engraftment of human cells (<5%) in NRG mice (Figure 3C). Furthermore, no IP or lymphatic solid tumors formed in the NRGS mice. Interestingly, large numbers of human cells were detected in the hematopoietic organs of NRG mice engrafted with CCHC-9 cells, but without symptomatic disease presentation Given the recent push for the development of humanized mice bearing functional human immunity, we next examined the engraftment potential and multilineage expansion of human CD34+ HSCs. Purified CD34+ cells from UCB (Figure 4A) were transplanted into NRG and NRGS mice, but only NRGS mice showed significant, durable engraftment of human hematopoietic cells 105 days later. Human cells were still detectable in the hematopoietic organs of NRG mice, but at an extremely low percentage. Hematoxylin and eosin (H&E) staining of spleen sections from NRGS revealed distinct patches of cells absent in NRG mice, likely human HSCs undergoing extramedullary hematopoiesis (Figure 4B). Multilineage expansion of human pre-B cells, myeloid cells, and T cells was observed only in NRGS mice, as characterized by expression of human CD19, CD33, and CD3, respectively (Figure 4C). In contrast to published work indicating that human cytokine expression is counterproductive to human HSC engraftment, we show that unconditioned NRGS mice retain high levels of human HSCs (huCD45+/huCD34+) in both the spleen (Figure 5A) and the BM (Figure 5B) even 105 days after xenotransplantation.Figure 5Comparison of long-term engraftment of mature and stem human hematopoietic cells in unconditioned NRG and NRGS mice. (A) Representative flow plots and relative percentages of human stem (huCD45+/huCD34+) and mature (huCD45+/huCD34-) hematopoietic cells in the spleens of unconditioned NRG (n = 5) and NRGS (n = 5) mice 105 days after transplantation of 5 × 105 human CD34+ UBC cells, indicating significantly higher engraftment of both stem and mature human hematopoietic cells. (B) Representative flow plots and relative percentages of mature and stem human hematopoietic cells detected in the BM of unconditioned NRG and NRGS mice showing highly significant enrichment of both stem and mature human hematopoietic cells selectively in NRGS mice. BM, bone marrow.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Useful AML xenograft mouse models also require the ability to model SOC therapeutic outcomes, which in current clinical practice rely heavily on the cytotoxic combination of an anthracycline and cytarabine. Therefore, we wanted to examine both the tolerability and the efficacy of an adapted 5+3 induction regimen in both NRG and NRGS mice xenografted with human leukemia cells. The established human cell lines K562 and MV411 formed large solid tumors IP and lymphatically selectively in NRGS mice upon xenotransplantation. Mice injected with U937 cells show similar survival trajectories and engraftment efficiency in both strains and NRGS solid tumor formation was restricted to the spleen. Further, given the finding that PDX samples fail to engraft efficiently in NRG mice, we chose U937 cells to examine comparative SOC therapeutic outcomes between NRG and NRGS mice. Mice from both strains were able to tolerate our adapted 5+3 bolus IV induction regimen and all treated mice showed a highly significant enhancement in survival compared with vehicle-injected controls (Figure 6A). These findings indicate that both strains are tolerant of a clinically similar induction regimen, and therapy is efficacious in treating disease (Figure 6B). Comparing patient xenograft induction sensitivity in each strain proved a challenge because none of the four patient samples (CCHC-7, CCHC-9, CCHC-23, and CCHC-35) gave rise to leukemia in NRG mice at the time points examined. Although the inherent limitations of NRG mice prevented us from examining comparative SOC therapeutic outcomes with patient-derived AML xenografts, we still felt that it was important to examine the efficacy of our regimen against patient-derived AML. Therefore, NRG and NRGS mice were engrafted with the patient sample CCHC-23, and 5+3 induction therapy was initiated 7 days after xenograft. Again, we were able significantly prolong the survival of all treated NRGS mice compared with vehicle-injected controls (Figure 6C) and therapy reduced the leukemic burden (Figure 6C). NRG mice in this study failed to develop disease and survived until the experimental endpoint. Similarly, our induction therapy regimen was efficacious in significantly prolonging the survival of NRGS mice harboring either CCHC-7 or CCHC-9 PDX (Figure 6D). These findings indicate that our 5+3 induction regimen is efficacious against several patient-derived AML of varied etiology at rates comparable to those observed in the aforementioned study with the established U937 cell line. These data are crucial to establishing the feasibility and efficacy of modeling SOC treatment outcomes in mice xenografted with patient-derived AML. Once established that our model strains were tolerant of our aggressive 5+3 induction regimen, we wanted to test the limi" @default.
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• W2885295219 date "2018-11-01" @default.
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• W2885295219 title "Comparative utility of NRG and NRGS mice for the study of normal hematopoiesis, leukemogenesis, and therapeutic response" @default.
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• W2885295219 doi "https://doi.org/10.1016/j.exphem.2018.08.004" @default.
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