NOD/SCID GAMMA XENOGRAFT MODEL OF B-CELL MALIGNANCIES USING TP53- AND/OR ATM-DEFICIENT CELL LINES

ABSSUB-3978

Background: Xenograft models represent very promising biological tools to study the molecular basis and pathogenesis of many hematological malignancies. To establish a reliable and appropriate in vivo model of human leukemia we used immunodeficient NOD/SCID gamma (NSG) mice. These mice lack a functional receptor for interleukins and do not have functional T, B and NK cells, in contrast to NOD/SCID mice that retain NK cells. Here we report a study concerning mouse xenograft models using TP53- or ATM-deficient B-cell lines MEC-1, SU-DHL-4, JEKO-1, GRANTA-519 and REC-1. 

Aims: The present study was designed to compare engraftments in NSG mice of TP53- or ATM-deficient B-cell lines. Our goal was to create a mouse xenograft model by transplanting cells of five cell lines, including chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL) into NSG mice. We would then analyze the engraftment potential of these cell lines. The current study shows that B-cell lines-engrafted NSG mice can be successfully used for investigating the pathogenesis of hematological malignancies with potential for application in tailored therapy. 

Methods: In total, 30 six to eight-week-old non-irradiated mice (n=6 per experimental group) were injected with 5x10e6 malignant cells in phosphate-buffered, saline (PBS). The injections were given intraperitoneally (IP, n=3) or subcutaneously (SC, n=3). Six control mice (one per group) were injected with PBS only. All animals were monitored individually throughout the experiment. After 4 weeks, all recipient mice were sacrificed by cervical dislocation and peripheral blood (PB), bone marrow (BM) and tissue samples (spleen, liver, tumor) were analyzed by histology, immunohistochemistry and flow cytometry analysis.

Results: All malignant cell lines except SU-DHL-4 injected IP or SC into NSG mice successfully engrafted. Three to four weeks after SC injection, four cell lines produced rapidly growing subcutaneous tumors in both male and female mice. Immunophenotype of all engrafted cell lines corresponded to human mature B cells expressing CD10-19+20+HLA-DR+ except for SU-DHL-4 and REC-1 cells which were moreover CD10+ and HLA-DR- respectively. In two lines, MEC-1 (CLL in transformation) and GRANTA-519 (MCL), we observed highly efficient engraftment of leukemic cells with very aggressive growth. MEC-1 IP injection led to significant weight loss (P=0.03), mild hepatomegaly and huge splenomegaly and death in one case. In GRANTA-519, there was huge tumor found in abdominal cavity or subcutaneously in all mice. Intraperitoneal tumor filled always the entire abdominal cavity. Immunohistological and flow cytometric analysis revealed a massive infiltration of liver, spleen and bone marrow by human CD19 and CD20 positive cells with high proliferative index. Noteworthy, only with GRANTA-519 it was observed that transplantation of human tumor cells resulted in prominently higher infiltration of the bone marrow regardless of administration route. It might be due to wild-type TP53 status in combination with mutated ATM. In the other cell lines tested, the mutation status of ATM is unknown while TP53 is mutated.

Summary/Conclusion: According to our results, GRANTA-519 and MEC-1 cell lines appears to be the most suitable for xenograft model of human B-cell malignancies and would be a criterion for future use of selected lymphoid xenotransplant for chemical agent-based treatment experiments.

Supported by research grants MSMT MSM0021622430, IGA-MZ-CR NT11218-6/2010, IGA-MZ-CR NT13493-4/2012, MSMT CZ.1.07/2.3.00/20.0045, VaVPI CEITEC CZ.1.05/1.1.00/02.0068 and Czech Leukemia Study Group - For Life.

Keywords: Hematological malignancy, Mouse model, Xenotransplantation

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