Section 1: Leukemia transplantation to create AML PDX models

Timing: 4 Weeks

Mice were injected with human AML cells to create PDX models for experimentation and analysis.

Note: If not using a primary patient sample, a large repository of viably frozen PDX cells can be obtained from cBioportal (Townsend et al., 2016) (https://www.cbioportal.org). The time between tumor transplantation and leukemia development varies depending on PDX model. We were generally able to detect disease burden as circulating myeloblasts after 3 weeks of engraftment using FACS analysis.

Obtain human leukemia cells from a bone marrow (BM) biopsy.

Resuspend mononuclear cells in 200 μL of 20°C–22°C PBS.

Pause point: We used immunocompromised NSG mice without prior pre-conditioning. We acclimated mice for 5-7 days before serial transplantation.

Place the mice under a lamp light to briefly warm them so that tail veins become more visible. Sterilize the mouse’s tail with an alcohol wipe.

Inject 0.6–1.0 × 10⁶ human leukemia cells in 200 μL of PBS intravenously (IV) (Passage 1).

Using FACS analysis, determine the percentage of human myeloblasts circulating in the blood by gating on SSC^lowhCD45^dim cells (Figure 1).

To prepare cells for leukemia transplantation, resuspend 0.6–1.0 × 10⁶ spleen myeloblasts in 200 μL of PBS. Cells can also be viability frozen using freezing media at this stage for future transplantations.

Gating strategy for identification of circulating human myeloblasts in AML PDXs

Representative flow cytometry plots for circulating human myeloblasts in the peripheral blood of NSG mice.

(A) Healthy NSG mice (non-transplanted).

(B) NSG mice at 3 weeks post leukemia engraftment.

(C) NSG mice after leukemia progression.