Protocol for the development of a bioluminescent AML-PDX mouse model for the evaluation of CAR T cell therapy

Abstract

Patient-derived xenograft (PDX) models of acute myeloid leukemia (AML-PDX) offer advantages over cell line models by capturing the complexity and heterogeneity of patient-derived samples. Here, we present a protocol for developing a bioluminescent AML-PDX model in mice to evaluate chimeric antigen receptor (CAR) T cell therapy. We describe steps for transducing, engrafting, expanding, and enriching AML-PDX cells. We then detail procedures for in vitro and in vivo validation of the AML-PDX model for the evaluation of CAR T cell immunotherapy. For complete details on the use and execution of this protocol, please refer to Caulier et al.¹.

Keywords: cancer; cell biology; cell culture; flow cytometry; immunology; model organisms; molecular biology.

Graphical abstract

Figure 1

Key steps for expansion and enrichment of GFP-LUC+ AML-PDX cells via FACS and in vivo engraftment in NSG mice p = passage. (A) Following low transduction efficiency (1.41%), all 3 × 10⁵ AML-PDX cells exposed to lentiviral particles are injected into a recipient NSG mouse to ensure engraftment and facilitate expansion of the GFP positive population. (B) The GFP+ cell population (1.44%) remains stable in P0 mice, allowing for sorting and enrichment for further engraftment (purple). (C) 51.8% of AML-PDX cells isolated from the spleen of P1 are GFP+. The top 20% highest GFP-expressing cells are FACS sorted and injected into P2 mice (blue). (D) Spleen cells from P2 mice are 96.6% GFP+ and cryopreserved for future use.

Figure 2

In vitro bioluminescence imaging and quantification of AML-PDX^LUC cells (A) Black walled 96-well plate containing dilution series of AML-PDX^LUC cells in triplicates. ROIs are indicated by red circles. (B) Regression curve of bioluminescence intensity plotted as total flux (p/sec) versus cell number. The coefficient of determination for the regression curve is 0.9878.

Figure 3

Flow cytometry plots showing the percentage of GFP positive cells, CD37 positive cells, and IgG isotype control positive cells for AML-PDX untransduced cells (upper row) and for AML-PDX successfully transduced cells (lower row)

Figure 4

In vivo bioluminescence imaging and quantification of AML-PDX^LUC mice (A) Dorsal and ventral BLI images acquired weekly for 3 NSG mice inoculated with AML-PDX^LUC cells. Minimum and maximum values for bioluminescence intensity are adjusted for each time point. (B) Quantification of bioluminescence intensity over time.

Figure 5

Weekly dorsal and ventral BLI imaging of AML-PDX mice representative of four different CAR T cell therapy groups: CD37CAR, CD33CAR, CD19CAR, and Mock Red rectangles indicate weeks when CAR T cells were administered. Minimum and maximum values for bioluminescence intensity are adjusted for each time point.