A Xenograft Mouse Model to Assess Efficacy of Therapeutic Agents for Human Acute Leukemia

Summary

Mouse (Mus Musculus) models are being widely used to develop xenografts using human leukemia cells. These models provide a comparable biological system to study drug efficacy, pharmacodynamics, and pharmacokinetics. Modeling acute myeloid leukemia in immunocompromised mice is described in detail using the U937 cell line xenograft as an example.

Abstract

Preclinical evaluation of therapeutic agents using an appropriate animal model is a critical step and a requirement for selecting drugs worth testing in humans. Therapeutic agents such as small molecule inhibitors, biological agents, immune checkpoint inhibitors, and immunotherapy each have unique mechanisms of action and call for careful selection of in vivo systems in which their efficacy can be tested. The purpose of this article is to describe in detail development of one such leukemia xenograft model for testing the therapeutic efficacy of novel agents. Using an immunocompromised (NRG) murine model that lacks B, T, and NK cells helps engraftment of transplanted leukemia cells and provides an acceptable microenvironment to study the therapeutic efficacy of small molecule inhibitors and some biological agents. This article describes the development of leukemia murine xenografts for in vivo drug testing using an acute myeloid leukemia (AML) cell line murine model treated with the cytotoxic drugs daunorubicin and cytarabine as an example. Treatment response can be assessed during therapy using several noninvasive and minimally invasive methods. Bioluminescence imaging can be used to measure leukemia burden over time when luciferase prelabeled leukemia cells are used to develop xenografts. Peripheral blood count analysis provides vital information about side effects such as myelosuppression (e.g., cytopenia) and therapeutic effect (e.g., blast count or differentiation). These techniques help track differences in the development of leukemia or decrease in tumor burden at various time points during the drug treatment without scarifying the study animals. Secondary methods such as immunophenotyping using flow cytometry are applied to confirm differences in the leukemia burden among treated and untreated groups. The methods described here can be tailored and used for developing xenografts of other types of leukemia (e.g., acute lymphoblastic leukemia).

Introduction

Acute myeloid leukemia (AML) is a clonal disorder arising from a malignantly transformed multipotent hematopoietic stem cell that acquires consecutive genomic alterations, eventually advancing into clinically overt disease. It is a highly complex disease with significant genetic, epigenetic, and phenotypic heterogeneity¹. The uncontrolled proliferation and impaired differentiation of myeloid precursor cells (i.e., blasts) is one of the hallmarks of AML, leading to anemia, thrombocytopenia, and eventually death². According to the American Cancer Society, in 2019, ~21,450 new cases of AML will be diagnosed, and ~10,920 peo....

Protocol

All methods described here have been approved by the Institutional Animal Care and Use Committee (IACUC) of the Pennsylvania State University College of Medicine.

1. Selection of animals for the study

1. Maintain 18 healthy 10−12-week old male NOD.Cg-Rag1^tm1Mom Il2rg^tm1Wjl/SzJ (NRG) mice in a barrier environment under pathogen-free conditions.
   NOTE: The common source for the right mouse strain for the study is peer-reviewed pub.......

Discussion

Critical steps in the protocol
Characterization of the mouse model: Each cell line and primary cell-derived murine xenograft has a unique disease and host-specific factors that influence the time and characteristics of engraftment. Time to engraftment is usually defined as time taken for the bone marrow to have 25% blast cells or have bioluminescence signaling at least two logs higher than background or non-tumor bearing mice. It is critical to characterize the mouse model prior to starting an expe.......

Materials

Name	Company	Catalog Number	Comments
1 mL Syringe	Fisher Scientific	309659
1.5 inch short bevel 20 g needle	Fisher Scientific	305179
1.5 mL microcentrifuge tube	Fisher Scientific	02-682-002
13 mm Single Ring slide	Fisherbrand	22-037-241
15 mL Polypropylene Conical Tube	FALCON	352097
1X RBC Lysis Buffer	Fisher Scientific	501129751
2 mL microcentrifgue tube	Fisher Scientific	05-408-138
4.5 inch blunt/straight tip scissors	Fisher Scientific	28251
4.5 inch serated straight forceps	VWR	82027-440
5 mL, 12x75 mm round bottom test tubes (flow tubes)	Corning	352008
5/8 inch sterile 25 g needle	Fisher Scientific	305122
6-well Non Treated Cell Culture Plate	USA Scientific	CC7672-7506
7AAD	Biolegend	420404
Alcohol Prep	COVIDIEN	6818
Aluminum Foil	VWR	89107-726
AutoFlow IR Water-Jacketed CO2 Incubator	NUAIRE	Model no. NU-8700
Blood Collection	RAM Scientific	76011
Brilliant Stain Buffer	BD Biosciences	563794
BV421-human CD33	Biolegend	366622
BV-650 mouse CD45	BD Biosciences	563410
Cell Analyzer	EMD Millipore Corparation	N/A
Cell Strainer	FALCON	352350
Centrifuge machine	BECKMAN COULTER	605168-AC
Count & Viability Kit	EMD Millipore Corparation	MCH100102
Cytocentrifuge	Fisher Scientific	A78300003
Cytoclip Slide Clip	Fisher Scientific	59-910-052
Dimethyl Sulfoxide (DMSO)	EMD Millipore Corparation	67-68-5
Disposable Centrifuge Tube	Fisher Scientific	05-539-8
D-Luciferin-Sodium Salt	GoldBio	LUCNA-1G
FACS buffer (PBS with 2% FBS (Heat-inactivated))	N/A	N/A
Filter Cards for Cytospin	Fisher Scientific	22-030410
FITC-human CD45	Biolegend	304014
Fixable Viability Dye	Thermo Fischer	65-0864-14
Fixation Buffer	Biolegend	420801
Flow cytometer	BD Biosciences	N/A
Flow Cytometry analysis software	FlowJo, LLC	Version 10
Freezing Medium (90% FBS + 10% DMSO)	N/A	N/A
Graduated Tips	USASCIENTIFIC	10/20 µL (1110-3700), 200 µL (1111-1700), and 1000 µL (1111-2720)
Hank’s Balanced Salt Solution (HBSS)	Gibco	14025092
Heat Inactivated Fetal Bovine Serum	Atlanta Biologicals	H17112
In Vivo Imaging system	Perkin Elmer	CLS136331
Insulin Syringes	BD	329461
Inverted Microscope	Olympus	CKX31
Isoflurane	VEDCO	NDC 50989-150-15
LABGARD CLASS II TYPE A2 BIOLOGICAL SAFETY CABINET	NUAIRE	Model no. NU-425-400
Living Image Software – IVIS Lumina Series	Perkin Elmer	128110
Low Flow CO2 Regulator	E-Z Systems	EP-1305
MB-10 tablets, sterilant	Quip Laboratories	MBTAB75BX
Micro cover glass	VWR	48366 205
Mounting medium	Fisher Scientific	SP15-100
Mouse anti-human Fc receptor antibody	BD Biosciences	564220
Mouse cage lid for euthanasia	E-Z Systems	E-20028
NOD.Cg-Rag1tm1Mom Il2rgtm1Wjl/SzJ (NRG)	Jackson laboratory	7799
ntracellular Staining Permeabilization Wash Buffer (10X)	Biolegend	421002
PE-anti-BTK	Biolegend	558528
PE-anti-pSTAT3-tyr705	Biolegend	651004
Penicilllin Streptomycin Solution, 100X	CORING	30-002-CI
Phosphate-Buffered Saline (PBS)	CORNIG cellgro	21-040-CV
Pipet controller	DRUMMOND Scientific	109883
Pipette	Eppendord Research	2.5 (O24694B), 10 (O31418B), 20 (O24694B), 100 (O337778), 200 (O26279B) and 1000 (O40665B)
Rat anti-mouse CD16/32 antibody	BD Biosciences	553142
RBC Lysis Buffer (10X)	Biolegend	420301
Refrigerated Centrifuge	NuAire	NU-C200R
Reusable sample chamber	Fisher Scientific	5991040
RPMI-1640 medium	CORING	10-040-CV
Serological pipet	FALCON	5mL (357543), 10mL (357551), 25mL (357535)
Styptic Powder	Fisher Scientific	NC1577028
Tailveiner for mouse	Agnthos	TV-150
Trypan Blue Solution	Corning	25900051
Wipes	Fisher Scientific	34155
Wright-Giemsa Stain	VWR	10143-106