Expression of Exogenous Cytokine in Patient-derived Xenografts via Injection with a Cytokine-transduced Stromal Cell Line

Summary

Described here is a method for producing exogenous cytokine in patient-derived xenograft (PDX) mice via weekly intraperitoneal injection of a cytokine-transduced stromal cell line. This method broadens the utility of PDX and provides the option for transient or sustained exogenous cytokine delivery in a multitude of PDX models.

Abstract

Patient-derived xenograft (PDX) mice are produced by transplanting human cells into immune deficient mice. These models are an important tool for studying the mechanisms of normal and malignant hematopoiesis and are the gold standard for identifying effective chemotherapies for many malignancies. PDX models are possible because many of the mouse cytokines also act on human cells. However, this is not the case for all cytokines, including many that are critical for studying normal and malignant hematopoiesis in human cells. Techniques that engineer mice to produce human cytokines (transgenic and knock-in models) require significant expense before the usefulness of the model has been demonstrated. Other techniques are labor intensive (injection of recombinant cytokine or lentivirus) and in some cases require high levels of technical expertise (hydrodynamic injection of DNA). This report describes a simple method for generating PDX mice that have exogenous human cytokine (TSLP, thymic stromal lymphopoietin) via weekly intraperitoneal injection of stroma that have been transduced to overexpress this cytokine. Use of this method provides an in vivo source of continuous cytokine production that achieves physiological levels of circulating human cytokine in the mouse. Plasma levels of human cytokine can be varied based on the number of stromal cells injected, and cytokine production can be initiated at any point in the experiment. This method also includes cytokine-negative control mice that are similarly produced, but through intraperitoneal injection of stroma transduced with a control vector. We have previously demonstrated that leukemia cells harvested from TSLP-expressing PDX, as compared to control PDX, exhibit a gene expression pattern more like the original patient sample. Together the cytokine-producing and cytokine-negative PDX mice produced by this method provide a model system that we have used successfully to study the role of TSLP in normal and malignant hematopoiesis.

Introduction

Patient-derived xenografts (PDX) are a powerful in vivo model for studying the production of normal and malignant hematopoietic cells in a 'native' mammalian environment. Most often, PDX are produced by injecting or transplanting human cells into immune deficient mice. The production of PDX using normal human hematopoietic stem cells allows in vivo studies of normal human blood and immune cell development. PDX produced from leukemia or other cancer cells make it possible to study oncogenic mechanisms and to identify effective therapies in context of the range of genetic landscapes and mutations present in the human population.¹

Protocol

Studies were conducted in accordance with Loma Linda University's Institutional Animal Care and Use Committee (IACUC) and Institutional Review Board (IRB) approved protocols and according to all federal guidelines.

CAUTION: PDX and human tissues should be handled in accordance with safety procedures to prevent the transmission of blood borne pathogens.

1. Culture and Expansion of Cytokine-Transduced Stromal Cells

NOTE: Each time stroma are passaged, harvest the culture supernatant (1 mL aliquot) and store at -80 °C for future quantification of cytokine level via ELISA a....

Results

An overview of the model is shown in Figure 1. Once cytokine-producing (TSLP+ stroma) and control stroma have been obtained they are expanded in culture. Prior to stroma injection into mice, stromal cell supernatant is assessed by ELISA to verify cytokine production (Figure 2) and phospho-flow cytometry (Protocol #3) is used to verify the activity of the cytokine produced by the stroma. Supernatant is collected from confluent stromal cell cultures (contro.......

Discussion

This manuscript describes a simple, quick, and relatively cost effective method for engineering PDX to express exogenous human cytokine. The strategy described here is based on weekly intraperitoneal injections of a stromal cell line transduced to express the human cytokine, TSLP. Prior to performing the methods described here, stroma engineered to express high levels of the cytokine of interest (TSLP) and similarly engineered control stroma were generated. In the protocols presented here, stroma are expanded in culture .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Wet lab reagents
T-25 Nunc Cell Culture Treated EasYFlasks, 7 mL capacity,  25cm² culture area	Thermo Scientific / Fisher	156367
T-75 Falcon Tissue Culture Treated Flask, 250 mL capacity, 75 cm² culture area & vented cap	Corning Inc. / Fisher	353136
T-150 Falcon Tissue Culture Treated Flask, 600 mL capacity, 150 cm² culture area & vented cap	Corning Inc. / Fisher	355001
10 mL serological pipettes	Falcon	357551
15 mL  polypropylene conical tubes	Fisher	05-539-12
5 mL round-bottom polystyrene tubes	Falcon	352054
50 mL polypropylene conical tubes	Falcon	352098
2.0 mL cryogenic vials, externally threaded	Corning Inc. / Fisher	4230659
1 mL pipette tips	Fisher	2707509
200 μL pipette tips	Denville Scientific	P3020-CPS
10 μL pipette tips	Denville Scientific	P-1096-CP
Sterile Disposable Filter Units with SFCA Membrane, Nalgene Rapid-Flow	Fisher	09-740-28D
2 NH2SO4	BioLegend	423001
Dulbecco’s phosphate-buffered saline (PBS) without calicium and magnesium, 1×	Corning cellgro/ Mediatech	21-031-CV
Human TSLP ELISA Max Deluxe Set	BioLegend	434204
3% Acetic Acid with Methylene Blue	Stemcell Technologies	7060
Trypan Blue	Corning	25-900-Cl
"Trypsin" 1x 0.25% Trypsin 2.21 mM EDTA in HBSS	ThermoFisher	25-053
TWEEN 20 BioXtra	Sigma-Aldrich	P-7949
“R10” cell culture medium, % of total volume (makes 565 mL)	-	-	Lab Recipe
RPMI, 88.5% (500 mL)	Mediatech	10-040-CV
FBS, 9.9% (56 mL)	Mediatech	35-011-CV
L-Glutamine, 0.99% (5.6 mL)	Mediatech	25-005-Cl
Penicillin-Streptomycin, 0.50% (2.8 mL)	Mediatech	30-002-Cl
2-Mercaptoethanol, 0.10% (560 µL)	MP	190242
“R20” cell culture medium, % of total volume (makes 195 mL)	-	-	Lab Recipe
RPMI, 76.84% (150  mL)	see above	see above
FBS, 20.49% (40 mL)	see above	see above
2mM L-glutamine, 1.02% (2 mL)	see above	see above
1 mM Na pyruvate, 1.02% (2 mL )	see above	see above
Penicillin-Streptomycin,  0.51% (1 mL)	see above	see above
2-Mercaptoethanol, 0.10% (0.1 M),  0.10% (200 µL)	see above	see above
“Freezing medium”, % of total volume	-	-	Lab Recipe
“R10” medium, 45%	see "R10" recipe	-
FBS, 20%	see above	35-011-CV
DMSO, 10%	Corning	25-950-CQC
D(-)(+)-Trehalose dihydrate, 5%	Fisher Scientific	BP2687-100
Name	Company	Catolog Number	Comments
Biologics
"HS-27" HS-27A human stroma line	ATCC	CRL-2496
"CALL-4" MHH-CALL-4 cell line, human B cell precursor leukemia	DSMZ	ACC 337
“NSG mice” NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ, male or female, age 6 – 8 weeks	JAX Mice	# 005557
MUTZ-5 cell line, human B cell precursor leukemia	DSMZ	ACC 490
Recombinant Human TSLP protein	R&D Systems	1398-TS-010
Name	Company	Catolog Number	Comments
Flow cytomery antibodies (clone)*
Anti-mouse CD45 FITC (30F11)	Miltenyi Biotec	130-102-778
CD127 PE (MB15-18C9) (alternate name is IL-7Rα)	Miltenyi Biotec	130-098-094
CD34 APC (8G12)	BD Biosciences	340667
CD45 PE-Cy7  (HI30)	eBioscience	25-0459
"FVD" Fixable viability dye  eFluor 780	eBioscience	65-0865
Ig κ light chain FITC  (G20-193)	BD Pharmingen	555791
Ig λ light chain FITC (JDC-12)	BD Pharmingen	561379
IgD PE (IA6-2)	BioLegend	348203
IgM PE-Cy5 (G20-127)	BD Biosciences	551079
pSTAT5 PE,  mouse anti-STAT5 (pY694)	BD PhosphoFlow	612567
Name	Company	Catolog Number	Comments
Other materials & equipment
Animal Implantable Nano Transponder with Canula	Trovan	ID-100B(1.25)
EMLA lidocaine anesthetic cream (obtain by presciption through animal care facility)	perscription	perscription
BD ½ cc LO-DOSE U-100 Insulin Syringe 28G½	BD	#329461
BD Microtainer Tubes with K2EDTA	BD	#365974
Centrifuge	Beckman Coulter	Alegra X-15R
FisherBrand Capillary Tubes (Heparinized)	Fisher Scientific	22-260-950
Hemocytometer	Fisher	02-671-6
MACSQuant Analyzer 10	Miltenyi Biotec	130-096-343
Mouse Pie Cage	Braintree Scientific, Inc.	MPC-1
Mouse Tail Illuminator Restrainer	Braintree Scientific, Inc.	MTI STD
Pistol Grip Implanter	Trovan	IM-300(1.25)
µQuant	Bio-Tek Instruments Inc.	MQX200
FlowJo flow cytometry analysis software	FlowJo, LLC	Version 10
*All antibodies are anti-human unless otherwise stated.