Bioluminescence in Vivo Imaging Improves the Model of Individual Patients' AML Cells Growing in Mice for Sensitive and Reliable Preclinical Treatment Trials on Various Genetic Subgroups
Authors: Jeremias Irmela , Vick Binje , Rothenberg Maya , Sandhöfer Nadine , Carlet Michela , Krupka Christina , Trumpp Andreas , Corbacioglu Selim , Ebinger Martin , Andre Maya Caroline , Hiddemann Wolfgang , Schneider Stephanie , Subklewe Marion , Metzeler Klaus H , Spiekermann Karsten 
Published: October 2019

Abstract
We introduced bioluminescence in vivo imaging as a novel, sensitive and reliable readout parameter for preclinical treatment trials in the individualized model of patients' primary AML cells growing in mice.
Novel treatment approaches require preclinical in vivo evaluation. While the individualized xenograft mouse model of individual patients AML cells growing in mice inherits the advantage of mimicking the broad genetic heterogeneity of AML, disease monitoring remained challenging so far due to the lack of appropriate readout parameters. In the individualized mouse model of AML, primary patients' AML cells are xenotransplanted into immuno-compromised mice.
Here, we aimed at increasing sensitivity and reliability of disease monitoring in the individualized mouse model of patient-derived AML.
Towards this aim, we engrafted primary tumor cells from 16 adult patients with AML. 8/16 (50%) samples allowed serial transplantation and thereby generation of stable patient-derived xenograft (PDX) cells with constant characteristics regarding growth and immunophenotype. PDX cells were derived from genetically distinct patient samples, mimicking the known heterogeneity of AML. Targeted re-sequencing of 43 genes important for AML leukemogenesis revealed identical mutations in primary and PDX cells after initial or serial transplantation, except the loss of two minor subclones within two samples.
Lentiviral transduction was established to genetically manipulate PDX cells and introduce stable expression of transgenes which was feasible in 7/8 PDX AML samples tested. Transgenic PDX cells were enriched by flow cytometry gating on a co-expressed fluorochrome. Recombinant expression of luciferase enabled bioluminescence in vivo imaging for reliable follow up of PDX cell leukemia growth in mice. Imaging was highly sensitive and detected a single PDX cell within 10,000 normal mouse bone marrow cells covering the clinically important situation of minimal disease. Furthermore, imaging facilitated reliable analysis of preclinical treatment trials, visualizing drug effects in single mice over time.
Novel treatment approaches aim at eliminating AML propagating cells, and the limiting dilution transplantation assay represents the gold standard for determining frequency of AML propagating cells. Bioluminescence in vivo imaging facilitated quantifying AML propagating cells by determining engraftment as early as 5 weeks after cell transplantation.
Taken together, we advanced the individualized mouse model of AML by introducing serial transplantation, lentiviral transduction and in vivo imaging. These improvements now allow sensitive and reliable preclinical treatment trials in patient-derived AML cells of various different genetic subgroups including AML propagating cells.

Disclosures
No relevant conflicts of interest to declare.