[Establishment of a humanized mouse model of HIV-1 infection and quantification of integrated HIV proviral DNA in vivo].

In recent years, virological, pathological, and immunological studies need to be carried out for the emerging anti-human immunodeficiency virus (HIV) therapies such as gene therapy, broadly neutralizing antibodies, and the derived chimeric antigen receptor (CAR)-T immunotherapy, which necessitates suitable, simple, and inexpensive small-animal models and methods for accurate quantification of the viral genome in the HIV-1 infected. In our research, the HIV-∆ENV-Jurkat-EGFP-mCherry cell line was engineered through the infection with a dual-labelled HIV pseudovirus. A nested quantitative PCR (nested-qPCR) method with the cellular genome as the integrated standard was established for the quantification of HIV proviral copies. We administered intravenous injections of healthy human peripheral blood mononuclear cell (PBMC) into NOD/Prkdcscid/IL2rgnull (NPG) mice. To verify engraftment kinetics, we analyzed the percentages of hCD45+, hCD3+, hCD4+, and hCD8+ cells in the peripheral blood of hu-PBMC-NPG mice. To evaluate HIV-1 infection in hu-PBMC-NPG mice, we inoculated these mice with HIV NL4-3-NanoLuc by intraperitoneal (IP) injection. We then monitored the luciferase expression by the small animal imaging system and measured the viral load in the spleen by qPCR. The infiltration of human PBMCs in mice was detected 3-5 weeks after intravenous injection, and the percentage of hCD45 in humanized mouse PBMCs were more than 25% five weeks after IP inoculation. The expression of the virus-associated luciferase protein was detected by luciferase imaging 27 days post infection. Moreover, the viral total DNA, RNA, and proviral DNA copies reached 18 000 copies/106 cells, 15 000 copies/µg RNA, and 15 000 copies/106 cells, respectively, in the mouse spleen. Taken together, we reported a convenient method for building a simple humanized mouse model of HuPBMC-NPG/severe combined immunodeficiency (SCID) by intravenous injection with hu-PBMCs without advanced surgical skills and irradiation. Furthermore, we established a convenient method for the efficient determination of proviral DNA to assess HIV replication in vivo, viral reservoir sizes, and efficacy of novel anti-HIV therapies including CAR-T immunotherapy and gene therapy.

Human CD34+ Hematopoietic Stem Cell-Engrafted NSG Mice: Morphological and Immunophenotypic Features.

Immunodeficient mice engrafted with human immune cells represent an innovative tool to improve translatability of animal models for the study of human diseases. Immunophenotyping in these mice focuses on engraftment rates and cellular differentiation in blood and secondary lymphoid organs, and is predominantly carried out by FACS (fluorescent activated cell sorting) analysis; information on the morphological aspects of engraftment and the prevalence of histologic lesions is limited. We histologically examined 3- to 6-month-old NSG mice, naïve or engrafted with CD34+ human hemopoietic stem cells (HSC), and employed a quantitative immunohistochemical approach to identify human and murine cell compartments, comparing the results with the FACS data. NSG mice mainly exhibited incidental findings in lungs, kidneys, testes, and adrenal glands. A 6-month-old NSG mouse had a mediastinal lymphoblastic lymphoma. The lymphoid organs of NSG mice lacked typical lymphoid tissue architecture but frequently exhibited small periarteriolar leukocyte clusters in the spleen. Mice engrafted with human HSC frequently showed nephropathy, ovarian atrophy, cataract, and abnormal retinal development, lesions considered secondary to irradiation. In addition, 20% exhibited multisystemic granulomatous inflammatory infiltrates, dominated by human macrophages and T cells, leading to the observed 7% mortality and morbidity. Immunophenotypic data revealed variable repopulation of lymphoid organs with hCD45+ human cells, which did not always parallel the engraftment levels measured via FACS. The study describes the most common pathological features in young NSG mice after human HSC engraftment. As some of these lesions contribute to morbidity, morphological assessment of the engraftment at tissue level might help improve immunophenotypic evaluations of this animal model.

Mouse model recapitulates the phenotypic heterogeneity of human adult T-cell leukemia/lymphoma in bone.

Adult T-cell leukemia/lymphoma has a unique relationship to bone including latency in the marrow, and development of bone invasion, osteolytic tumors and humoral hypercalcemia of malignancy. To study these conditions, we established and characterized a novel mouse model of ATL bone metastasis. Patient-derived ATL cell lines including three that do not express HTLV-1 oncoprotein Tax (ATL-ED, RV-ATL, TL-Om1), an in vitro transformed human T-cell line with high Tax expression (HT-1RV), and an HTLV-1 negative T-cell lymphoma (Jurkat) were injected intratibially into NSG mice, and were capable of proliferating and modifying the bone microenvironment. Radiography, µCT, histopathology, immunohistochemistry, plasma calcium concentrations, and qRT-PCR for several tumor-bone signaling mRNAs were performed. Luciferase-positive ATL-ED bone tumors allowed for in vivo imaging and visualization of bone tumor growth and metastasis over time. ATL-ED and HT-1RV cells caused mixed osteolytic/osteoblastic bone tumors, TL-Om1 cells exhibited minimal bone involvement and aggressive local invasion into the adjacent soft tissues, Jurkat cells proliferated within bone marrow and induced minimal bone cell response, and RV-ATL cells caused marked osteolysis. This mouse model revealed important mechanisms of human ATL bone neoplasms and will be useful to investigate biological interactions, potential therapeutic targets, and new bone-targeted agents for the prevention of ATL metastases to bone.

Development and characteristics of preclinical experimental models for the research of rare neuroendocrine bladder cancer.

PURPOSE: For rare cancers such as neuroendocrine bladder cancer treatment options are limited due partly to the lack of preclinical models. Techniques to amplify rare primary neuroendocrine bladder cancer cells could provide novel tools for the discovery of drug and diagnostic targets. We developed preclinical experimental models for neuroendocrine bladder cancer. MATERIALS AND METHODS: Fresh tumor tissue from 2 patients with neuroendocrine bladder cancer was used to establish in vitro and in vivo models. We analyzed additional archived tissues in the National Center of Tumor Diseases tissue bank from patients with neuroendocrine bladder cancer. Primary tumor samples were collected during radical cystectomy. PHA-665752 was used to inhibit MET in animal models and cell cultures. The expression of markers and drug targets in neuroendocrine bladder cancer was determined by flow cytometry. The growth of neuroendocrine bladder cancer in vitro was determined by counting live cells. Tumor growth in mice was assessed by measuring tumor volume. Groups were compared using the nonparametric Kruskal-Wallis test. RESULTS: Xenograft models and serum-free cultures of neuroendocrine bladder cancer cells allowed screening for cell surface markers and drug targets. We found expression of the HGF receptor MET in neuroendocrine bladder cancer cultures, xenograft models and primary patient sections. The growth of neuroendocrine bladder cancer spheroids in vitro depended critically on HGF. Treatment of neuroendocrine bladder cancer bearing mice with a MET inhibitor significantly decreased tumor growth compared to that in control treated mice. CONCLUSIONS: Neuroendocrine bladder cancer xenografts and serum-free cultures provided suitable models in which to identify diagnostic markers and therapeutic targets. Using the models, we noted HGF dependent growth of human neuroendocrine bladder cancer and identified MET as a new treatment target for neuroendocrine bladder cancer.