Syngeneic leukemia models using lentiviral transgenics

Abstract

Animal models are necessary to study cancer and develop treatments. After decades of intensive research, effective treatments are available for only a few types of leukemia, while others are currently incurable. Our goal was to generate novel leukemia models in immunocompetent mice. We had achieved abilities for overexpression of multiple driving oncogenes simultaneously in normal primary cells, which can be transplanted and followed in vivo. Our experiments demonstrated the induction of primary malignant growth. Leukemia lines that model various types of leukemia, such as acute myeloid leukemia (AML) or chronic lymphocytic leukemia (CLL), were passaged robustly in congenic wild-type immunocompetent mice. These novel leukemia lines, which may complement previous models, offer the flexibility to generate tailored models of defined oncogenes of interest. The characterization of our leukemia models in immunocompetent animals can uncover the mechanisms of malignancy progression and offer a unique opportunity to stringently test anti-cancer chemotherapies.

Fig. 1. Overexpression of various oncogenes in HSPCs induces malignant growth in vivo.

a Schematic representation of the experimental procedure. BM of CD45.2 rtTA mice sorted to obtain HSPCs. Candidate oncogenes were introduced in batches. Cells were transplanted into congenic mouse. FACS analysis of peripheral blood (PB) revealed the appearance and progression of leukemia. Passage into secondary and tertiary recipients established these models. b–f Representative FACS profiles of cells from the PB of ML23. Increased fraction of CD45.2⁺ZsGreen⁺ (b, c). Increase in Mac1⁺ (d), not in CD3e or B220 or e, f Same donor cells transduced with control-LV shown to yield no malignant growth (g). Data shown from one out of at least three independent experiments.

Fig. 2. Leukemia lines can passage into WT mice.

Representative PB FACS taken from secondary recipient 30 days after passage of ML23 (a), tertiary ML23 recipient (b), or ML21 (c). One million fresh cells were used for these passages. Plots show frequencies of the transplanted CD45.2 cells, expression of ZsGreen reporter, Mac1, CD3e, and B220 staining. Data shown from one out of at least three independent experiments. d Representative PB FACS of recipient mice after transplantation with previously frozen ML23 cells. Data shown from one out of at least three independent experiments.

Fig. 3. Using polymerase chain reaction (PCR) analysis to identify the driving oncogenes in the leukemic lines.

a Control PCR w/o leukemia-line DNA, or diluted plasmids (1 ng). b PCR of ML23 detecting HoxB5, HoxA9, and Meis1 oncogenes. The 100-bp ladder is on the left lanes. Data are from one of at least three independent experiments (n = 3).

Fig. 4. Leukemia pathophysiology and microscopic examination.

a Enlarged spleen and lymph-nodes of an ML23 mouse, alongside bones lacking the normal reddish-hematopoiesis marrow are shown, normal spleen and bone marrow of a control-healthy mouse are presented for comparison (representative of n = 7 mice). b Giemsa-stained ML23 or ML21 cells at ×20–40 magnification. c Spleens sections from WT, or ML23 mice, with nuclear-staining with Dapi. Green fluorescence is detected from ZsGreen-tagged spleen cells in the ML23 mice only.

Fig. 5. Response of leukemia models to conventional chemotherapy.

Four-week survival curves for untreated control mice, and chemo-treated mice (Fludarabine 25 mg/kg). a ML21 mice with lymphoid-leukemia; b ML23 mice with myeloid-leukemia. Per experiment n = 7–8 mice per treatment. Data shown are from one of three independent experiments.

Fig. 6. Leukemia cells contain a subpopulation with distinct stem-cell markers.

Representative FACS plots of ML23 expression of stem cell markers cKit, Sca1, CD150, and CD48, showing subpopulations of cKit^−/+Sca1⁺CD150⁺CD48^−/+. n = 10 mice; Data are from one of at least three independent experiments.

Fig. 7. Generation of a multiple leukemia line in vivo.

Schematic model demonstrating that overexpression of a mix of oncogenes in immunocompetent mice can induce malignant leukemia. Upper arrow: no leukemia develops. Middle arrow: initial growth suspected but no further passage. Lower arrow: leukemia is generated and passaged. Established leukemia-lines can easily transfer such models.