Optimized Xenograft Protocol for Chronic Lymphocytic Leukemia Results in High Engraftment Efficiency for All CLL Subgroups.

Sarah Decker,Konrad Aumann,Christine Dierks,Shifa Khaja Saleem,Anabel Zwick,Sandra Kissel,Andres Rettig

doi:10.3390/ijms20246277

Abstract

Preclinical drug development for human chronic lymphocytic leukemia (CLL) requires robust xenograft models recapitulating the entire spectrum of the disease, including all prognostic subgroups. Current CLL xenograft models are hampered by inefficient engraftment of good prognostic CLLs, overgrowth with co-transplanted T cells, and the need for allogeneic humanization or irradiation. Therefore, we aimed to establish an effective and reproducible xenograft protocol which allows engraftment of all CLL subtypes without the need of humanization or irradiation. Unmanipulated NOD.Cg-PrkdcscidIl2rgtm1Sug/JicTac (NOG) mice in contrast to C.Cg-Rag2tm1Fwa-/-Il2rgtm1Sug/JicTac (BRG) mice allowed engraftment of all tested CLL subgroups with 100% success rate, if CLL cells were fresh, injected simultaneously intra-peritoneally and intravenously, and co-transferred with low fractions of autologous T cells (2%–4%). CLL transplanted NOG mice (24 different patients) developed CLL pseudofollicles in the spleen, which increased over 4–6 weeks, and were then limited by the expanding autologous T cells. Ibrutinib treatment studies were performed to validate our model, and recapitulated treatment responses seen in patients. In conclusion, we developed an easy-to-use CLL xenograft protocol which allows reliable engraftment for all CLL subgroups without humanization or irradiation of mice. This protocol can be widely used to study CLL biology and to explore novel drug candidates.

Highlights

Chronic lymphocytic leukemia (CLL) is characterized by a progressive accumulation of functionally incompetent CD5+ B-lymphocytes in the peripheral blood (PB), bone marrow (BM), and lymphoid organs [1,2]
Previous publications have shown huge advantages in CLL cell engraftment for late stage CLLs (Rai stage III/IV) [23,24,31] and CLL subsets with poor prognosis [21], but at least half of the patients seen in the clinic present with genetic and expression features of good prognosis and early stage CLL
Many xenograft models rely on complicated conditioning regimens or allogeneic settings

Summary

Introduction

Chronic lymphocytic leukemia (CLL) is characterized by a progressive accumulation of functionally incompetent CD5+ B-lymphocytes in the peripheral blood (PB), bone marrow (BM), and lymphoid organs [1,2]. Some patients die from the disease within a few months, whereas others live for 20 years or more even without treatment [5]. Several prognostic markers such as Rai and Binet staging systems, immunoglobulin VH gene mutational status [6], ζ-associated protein 70 (ZAP70) expression [7,8], cytogenetic abnormalities [9], and gene mutations can be used to predict the survival outcome and the need for treatment of patients with CLL [10,11]. In vitro monocultures of CLL cells are limited, and can only be used for drug screens based on short term readouts [12]. Co-culture of CLL cells with nurse-like cells or BM-stroma derived cell lines can maintain CLL survival for several days [13,14], but none of the available in vitro culture systems allow long-term culture or proliferation of CLL cells, which limits the relevance of such models for testing novel drugs [15]

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Conclusion