Testing Cancer Immunotherapeutics in a Humanized Mouse Model Bearing Human Tumors

Summary

This protocol outlines the generation of human immune system (HIS) mice for immuno-oncology studies. Instructions and considerations in the use of this model for testing human immunotherapeutics on human tumors implanted in this model are presented with an emphasis on characterizing the response of the human immune system to the tumor.

Abstract

Reversing the immunosuppressive nature of the tumor microenvironment is critical for the successful treatment of cancers with immunotherapy drugs. Murine cancer models are extremely limited in their diversity and suffer from poor translation to the clinic. To serve as a more physiological preclinical model for immunotherapy studies, this protocol has been developed to evaluate the treatment of human tumors in a mouse reconstituted with a human immune system. This unique protocol demonstrates the development of human immune system (HIS, "humanized") mice, followed by implantation of a human tumor, either a cell-line derived xenograft (CDX) or a patient derived xenograft (PDX). HIS mice are generated by injecting CD34+ human hematopoietic stem cells isolated from umbilical cord blood into neonatal BRGS (BALB/c Rag2^-/- IL2RγC^-/- NOD^SIRPα) highly immunodeficient mice that are also capable of accepting a xenogeneic tumor. The importance of the kinetics and characteristics of the human immune system development and tumor implantation is emphasized. Finally, an in-depth evaluation of the tumor microenvironment using flow cytometry is described. In numerous studies using this protocol, it was found that the tumor microenvironment of individual tumors is recapitulated in HIS-PDX mice; "hot" tumors exhibit large immune infiltration while "cold" tumors do not. This model serves as a testing ground for combination immunotherapies for a wide range of human tumors and represents an important tool in the quest for personalized medicine.

Introduction

Mouse cancer models are important for establishing basic mechanisms of tumor growth and immune escape. However, cancer treatment studies in mouse models have yielded finite translation to the clinic due to limited syngeneic models and species-specific differences^1,2. The emergence of immune therapies as a dominant approach to control tumors has reiterated the need for an in vivo model with a functional human immune system. Advancements in human immune system mice (HIS mice) over the past decade have made it possible to study immuno-oncology in vivo in a wide variety of cancer types and immuno....

Protocol

All animal work was performed under animal protocols approved by the University of Colorado Denver Institutional Animal Care and Use Committee (IACUC Protocols #00593 and #00021). All animal work was performed in accordance with the Office of Laboratory Animal Resources (OLAR), an accredited facility by the American Association for Laboratory Animal Care, at the University of Colorado Denver Anschutz Medical Campus. All human cord blood samples were obtained as donations from de-identified donors and are thus not subject to approval by the human research ethics committee.

NOTE: Compositions of all media and solutions mentioned in the protoc....

Results

Following the flank tumor protocol and experimental timeline (Figure 1), the tumor growth and immune response to a targeted tyrosine kinase inhibitor (TKI) therapy and nivolumab combination treatment was studied in two distinct human colorectal cancer (CRC) PDXs. The TKI drugs have been studied in immunodeficient hosts to evaluate tumor growth only²⁹. This model enabled the study of changes in the immune response of the TKI alone, and more importantly, in combination .......

Discussion

Over the past 6 years, using our expertise in both immunology and humanized mice, our research team has developed a much needed preclinical model to test immunotherapies on a variety of human tumors^3,7,30,31. This protocol emphasizes the consideration of the variability of the model, with special attention to the immunotherapy-centric human T cell populations. In this protocol, the generation o.......

Materials

Name	Company	Catalog Number	Comments
1 mL syringe w/needles	McKesson	1031815
15 mL tubes	Grenier Bio-One	188271
2-mercaptoethanol	Sigma	M6250
50 mL tubes	Grenier Bio-One	227261
AutoMACS Pro Separator	Miltenyi	130-092-545
BD Golgi Stop Protein Transport Inhibitor with monensin	BD Bioscience	BDB563792
BSA	Fisher Scientific	BP1600100
Cell Stim Cocktail	Life Technologies	509305
Chill 15 Rack	Miltenyi	130-092-952
Cotton-plugged glass pipettes	Fisher Scientific	13-678-8B
Cultrex Basement membrane extract	R&D Systems	363200502
Cytek Aurora	Cytek
DNase	Sigma	9003-98-9
eBioscience FoxP3/Transcription Factor Staining Buffer Set	Invitrogen	00-5523-00
Embryonic Stemcell FCS	Gibco	10439001
Eppendorf Tubes; 1.5 mL volume	Grenier Bio-One	616201
Excel	Microsoft
FBS	Benchmark	100-106 500mL
Ficoll Hypaque	GE Healthcare	45001752
FlowJo Software	BD Biosciences
Forceps - fine	Roboz Surgical	RS5045
Forceps normal	Dumont	RS4919
Formaldehyde	Fisher	F75P1GAL
Frosted Glass Slides	Corning	1255310
Gentlemacs C-Tubes	Miltenyi	130-096-334
GentleMACS Dissociator	Miltenyi	130-093-235
glass pipettes	DWK Life Sciences	63A53
Glutamax	Gibco	11140050
HBSS w/ Ca & Mg	Sigma	55037C
HEPES	Corning	MT25060CI
IgG standard	Sigma	I2511
IgM standard	Sigma	401108
IMDM	Gibco	12440053
Liberase DL	Roche	5466202001
LIVE/DEAD Fixable Blue	Thermo	L23105
MDA-MB-231	ATCC	HTB-26
MEM	Gibco	1140050
mouse anti-human IgG-AP	Southern Biotech	JDC-10
mouse anti-human IgG-unabeled	Southern Biotech	H2
mouse anti-human IgM-AP	Southern Biotech	UHB
mouse anti-human IgM-unlabeled	Southern Biotech	SA-DA4
MultiRad 350	Precision X-Ray
PBS	Corning	45000-446
Pen Strep	Gibco	15140122
Petri Dishes	Fisher Scientific	FB0875713A
p-nitrophenyl substrate	Thermo	34045
PRISM	Graphpad
Rec Hu FLT3L	R&D systems	308-FK-005/CF
Rec Hu IL6	R&D systems	206-IL-010/CF
Rec Hu SCF	R&D systems	255SC010
RPMI 1640	Corning	45000-39
Saponin	Sigma	8047-15-2
Scissors	McKesson	862945
Serological pipettes 25 mL	Fisher Scientific	1367811
Sterile filter	Nalgene	567-0020
Sterile molecular water	Sigma	7732-18-5
Yeti Cell Analyzer	Bio-Rad	12004279
Zombie Green	biolegend	423112