Cutting Edge
Human Disease
Modeling Using
The NSG Mouse
Dominique Kagele, Ph.D.
Technical Information Services
Cutting Edge Human Disease Modeling
The NSG Mouse
NSG Basics
Enhanced disease modeling
o Solid and hematological cancers
o Human immunity and inflammation
Advanced and future disease
modeling
Additional information and
resources
JAX® Mice |
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Cutting Edge Human Disease Modeling
The NSG Mouse
NSG Basics
Enhanced disease modeling
o Solid and hematological cancers
o Human immunity and inflammation
Advanced and future disease
modeling
Additional information and
resources
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The Immune System in Mice
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NOD scid gamma (NSG)
Strain nomenclature: NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ
Stock Number: 005557
Additional resources: NSG Web Resources
www.jax.org/jaxmice/research/immunology/005557.html
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NSG
A Highly Immunodeficient Mouse
NOD background
Absence of hemolytic complement
Reduced dendritic and NK cell function
Defective macrophages
Optimal human hematopoietic stem cell engraftment (Sirpa allele)
scid mutation
prevents development of mature T and B cells
gamma chain (Il2rg knockout)
Eliminates signaling from 6 distinct interleukins and blocks NK cell
development
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NSG
A Highly Immunodeficient Mouse
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Gamma Chain Knockout (Il2rgtm1Wjl)
Complete deficiency of the interleukin 2 receptor, gamma chain
Common subunit found in multiple cytokine cell signaling
receptors
IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21 are key signaling molecules
in function and maturation of T, B, NK, neutrophils and dendritic
cells
Rochman Y et al. 2009. Nat Rev Immunol 9(7):480-90. PMID:19543225
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NSG
A Highly Immunodeficient Mouse
Longer lifespan (> 16 months) than NOD-scid (~ 9
months)
scid side effects: Radiation sensitivity; genotoxic drugs
can have higher toxicity
A platform for developing refined models (transgenic
and knockout)
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The Most Useful and Versatile
Immunodeficient Mouse
Research applications:
Primary tumor engraftment
Cancer stem cells
Human hematopoiesis
Humanized mice
Infectious disease
Transplantation research
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Cutting Edge Human Disease Modeling
The NSG Mouse
NSG Basics
Enhanced disease modeling
o Solid and hematological cancers
o Human immunity and inflammation
Advanced and future disease
modeling
Additional information and
resources
JAX® Mice |
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Classical Cancer Xenograft Modeling
Nude Mice
Before treatment
After treatment
Gulliya et al. 1994. Cancer 74:1725-1732. PMID:8082074
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Patient Tumor Characteristics
NSG vs scid
Cancer
Patient’s Lung
Tumor
Fragment
NSG
H&E
C.B-17
scid
H&E
Human cells
(anti-HLA)
Mouse blood cells
(anti-mCD45)
Simpson-Abelson MR et al. 2008.J Immunol 180(10):7009-18. PMID:18453623
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JAX® Patient-Derived Xenograft (PDX)
Program
Patient-derived tumor bank in NSG mice
Clinical information
o Tumor type, grade and markers (if known)
o Treatment history
Histology
Gene expression and CNV analysis
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JAX® Patient-Derived Xenograft (PDX)
Program
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JAX® PDX Tumor Model Bank (>350)
Tumor type
Number of models
Appendiceal
1
Bladder
15
Brain
33
Breast
16
Colorectal
32
Endometrial
4
Gastric/GIST
2
Hematology
4
Hepatocellular
1
Kidney
9
Lung
49
Ovary
10
Pancreas
18
Prostate
2
Sarcoma
14
Skin
8
To see all of our PDX models, visit www.tumor.informatics.jax.org
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PDX modeling in NSG
Estrogen Receptor-Positive Breast Carcinoma
TM00386 (ER+/PR+/Her2-)
TM00386-PT
TM00386-P0
TM00386-P1
PT= Patient Tumor; P0 = first patient derived xenograft (PDX) generation in mouse; P1 = 2nd generation
• NSG supports growth of ER+ breast tumors
• Tumors retain “organoid” growth pattern
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Orthotopic PDX Modeling in NSG
Bladder Cancer
Fidelity during tumor passaging
(subcutaneous)
Orthotopic model
Lin TY et al. 2012. Nanomedicine (Lond).[Epub ahead of print] PMID:23199207
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PDX Modeling in NSG mice
Bladder Cancer
Tumor-Targeting Micelles Prevent
Tumor Growth…
…and Improve Survival
Lin TY et al. 2012. Nanomedicine (Lond).[Epub ahead of print] PMID:23199207
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NSG
Improved Melanoma Metastasis Modeling
Cell line: A375 (RhoC, Luciferase)
injected I.V.
*NSG also better platform for
cell line engraftment (data not
shown)
Carreno et al. 2008.
Clin Cancer Res 15(10):3277-86. PMID:19447870
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NEW
PDX Live™
NSG Mice
M
Readily Available from
Inventory and Pre-Engrafted with
Patient-Derived Tumors
Fast-track PDX Studies of 12 Human Tumor Types
Triple negative breast carcinoma (2), acute myeloid leukemia (4), non-small cell lung
cancer, B-cell lymphomas, metastatic colon adenocarcinoma, metastatic rectal
carcinoma, serous adenocarcinoma, squamous cell carcinoma
Save 6-12 Weeks of Valuable Research Time
Eliminate the need to cryo-recover & passage tumors between donor and study mice
Make Decisions Earlier during Drug Development
JAX® PDX Mice are available at lower passages than any other patient-derived
xenograft model provider. Low passage tumors…
o Preserve the heterogeneity of the original human cancers and the closest
possible response to primary human tumors
o Improve accuracy in assessing clinical efficacy of novel therapeutics
PDX Live™
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Cutting Edge Human Disease Modeling
The NSG Mouse
NSG Basics
Enhanced disease modeling
o Solid and hematological cancers
o Human immunity and inflammation
Advanced and future disease
modeling
Additional information and
resources
JAX® Mice |
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Effective Modeling of Patient-Derived
ALL (T & B Cell)
Cells: Unsorted patient-derived T or B cell ALL
Route: Tail vein
Age of mice: 6-8 weeks
*Patient samples
engrafted at lower doses
and with faster kinetics in
NSG
Diamanti P et al. 2011. Leukemia Feb;26(2):376-80. PMID:21860430
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NSG Efficiently Models Patient AML
Samples from different patients showed improved engraftment versus other models (NSB)
Sanchez PV et al. 2009. Leukemia 23(11):2109-2117. PMID:19626050
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Cutting Edge Human Disease Modeling
The NSG Mouse
NSG Basics
Enhanced disease modeling
o Solid and hematological cancers
o Human immunity and inflammation
Advanced and future disease
modeling
Additional information and
resources
JAX® Mice |
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NSG Supports a Human Immune
System
Most efficient engraftment of human HSCs to date
Permits differentiation of all major cell types:
o Myeloid lineage
Erythrocytes, megakaryocytes, platelets
Monocytes, macrophages, dendritic cells, granulocytes
o Lymphoid lineage
B cells
T cells (CD4+ and CD8+)
NK cells
Some mucosal and adaptive immune function
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NSG Supports a Human Immune
System
NSG is a superior host for human immune
cells when compared to other strains:
1. Any strain expressing the scid mutation alone (NOD-scid,
B6-scid, C.B17-scid)
2. “scid beige”
3. The same mutations—scid (or a Rag1 or Rag2 knockout)
and “gamma”—on other backgrounds
(for example, BALB/c)
Shultz LD et al. 2005. J Immunol 174(10):6477-89. PMID:15879151
Lepus CM et al. 2009. Hum Immunol Oct;70(10):790-802. PMID:19524633
Brehm MA et al. 2010. Clin Immunol 135(1):84-98. PMID:20096637
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Improved Engraftment of CD34+ HSCs
and PBMCs
*NSG also steadily engrafts human immune cell populations over time
(data not shown)
King M et al. 2008. Clin Immunol 126(3):303-14. PMID:18096436
Brehm MA et al. 2010. Clin Immunol 135(1):84-98 PMID:20096637
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NSG
The Most Efficient Humanized Model
Engraftment Efficiency of Bone Marrow
Strain
Exp 1
Exp 2
Combined
Percentage
NOD/Lt-scid
9/11
5/7
14/18
77.8
NOD/Shi-scid
12/12
3/11
15/23
65.2
NSG
12/12
11/11
23/23
100
NOG
10/12
9/10
19/22
86.4
*NSG also had the greatest efficiency in spleen & thymus
Adapted from McDermott SP et al. 2010. Blood. Jul 15;116(2):193-200. PMID:20404133
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Experimental Timeline for JAX® Hu-NSG
Whole body irradiation
Tail vein injection
Mouse Age
3 weeks
Human
B cells
appear
Human
T cells
appear
12 weeks
15 weeks
CD34+ cells engrafted in 3 week old female mice
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Successful Engraftment in Peripheral
Blood
Human
Engrafted NSG
Mouse
Human CD45
Mostly
neutrophils,
monocytes
Mouse CD45
Mature human T (CD3+) and B (CD20+)
cells differentiated in the NSG mouse
Human CD3
Human white blood cells abundant in
circulation
Human CD20
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Multi-lineage Differentiation in Bone
Marrow
Human vs Mouse
Human HSC
Human Erythroid
11.2%
hCD34
hCD45
SSC
FSC
0.13%
88.6%
86.1%
mCD45
Human B cells
hCD38
Human APCs
DTH Response in Humanized NSG is
Evidence for Intact T Cell Functions
Sensitization Sensitization Challenge +/- treatment
Day -1
Day 1
Day 2
Day 7
Day 8
Day 9
Day 14
Ear thickness Ear thickness Ear thickness
Ear thickness Ear thickness Ear thickness
Naive
Sensitized with DNFB
Sensitized with Olive Oil
DNFB + Hydrocortisone
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Transplant Rejection Studies:
Diabetes
Insulin
hCD45
H&E
Diabetic mice: NSG treated with STZ
Transplanted with human islets, +/PBMCs
32 days post-engraft.
No PBMC
+ PBMC
King M et al. 2008. Clin Immunol 126(3):303-14. PMID:18096436
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NSG is Superior to NOD scid for
Humanized HIV Models
Infectivity correlates with infiltration of human T cells into vaginal mucosa
Stoddart CA et al. 2011. Virology Aug 15;417(1):154-60. PMID:21684569
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Research Ready Humanized NSG
Routinely engraft NSG mice with human CD34+ hematopoietic stem cells
BLT & PBMC engrafted mice available
Mice are delivered 12 weeks after engraftment with at least 25% hCD45+
cells in the peripheral blood
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Cutting Edge Human Disease Modeling
The NSG Mouse
NSG Basics
Enhanced disease modeling
o Solid and hematological cancers
o Human immunity and inflammation
Advanced and future disease
modeling
Additional information and
resources
JAX® Mice |
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Humanized tumor-bearing NSG mice
The next step in cancer modeling
Whole body irradiation
Tail vein injection of
human CD34+ HSCs
and tumors
Mouse Age:
3 weeks
Human
B cells
appear
Human
T cells
appear
12 weeks
15 weeks
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Humanized tumor-bearing NSG mice
Proof-of-concept validation
Study design:
o Human CD34+ HSCs were co-transplanted with human breast
cancer cell lines (BT474 or SK-BR-3)
o Tumor growth and metastasis were measured
o Human immune cell engraftment and function were assessed
Goal:
o Validate a preclinical model that supports both human tumors
and immune cells for the development of novel cancer
immunotherapies
Wege et.al., mAbs. 2014. PMID: 24870377
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Humanized NSG mice
Engraftment of human tumors
Injected tumor cells grew, metastasized (data not shown), and
retained Her2- positivity
Wege et.al., mAbs. 2014. PMID: 24870377
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Humanized tumor-bearing NSG mice
Human immune cell engraftment and tumor
infiltration
T cells
T cells
Macrophages
B cells
Wege et.al., mAbs. 2014. PMID: 24870377
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Humanized tumor-bearing NSG mice
Irradiation
NSG
CD34+ HSC
Humanized
NSG
Solid tumor fragment or
tumor cell line
Humanized tumor-bearing NSG mice
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NOD Rag1 Gamma (NRG)
A Radiation Resistant Alternative to NSG
Nomenclature: NOD.Cg-Rag1tm1Mom Il2rgtm1Wjl/SzJ
Stock Number: (007799)
Webpage: www.jax.org/jaxmice/strain/007799
Severe defects in both innate and adaptive immunity
Hematopoietic stem cell engraftment comparable to NSG
Radiation resistant
Emerging model
Pearson T et al. 2008. Clin Exp Immunol 154(2): 270-84. PMID:18785974
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New NSG Model Innovations
HLA Class I Transgenic
NOD.Cg-Prkdcscid Il2rgtm1Wjl Tg(HLA/H2-D/B2M)1Dvs/SzJ (014570)
o HLA Class I heavy and light chains (A2.1 haplotype)
NOD.Cg-Prkdcscid Il2rgtm1Wjl Tg(HLA-A2.1)1Enge/SzJ (009617)
o Expresses HLA Class I transgene (A2.1 haplotype)
HLA Class II Transgenic
NOD.Cg-Prkdcscid Il2rgtm1Wjl H2-Ab1tm1Gru Tg(HLA-DRB1)31Dmz/SzJ (017637)
o Lacks mouse MHC class II, expresses chimeric HLA class II DR4 transgene
NOD.Cg-Prkdcscid Il2rgtm1Wjl Tg(HLA-DRA*0101,HLA DRB1*0101)1Dmz/GckRoly
(012479)
o NSG strain expresses HLA class II transgene (DR1 haplotype)
NOD.Cg-Rag1tm1Mom Il2rgtm1Wjl Tg(HLA-DRA,HLA-DRB1*0401)39-2Kito/ScasJ
(017914)
o “DRAG” strain with HLA class II transgene (DR4; HLA-DRA/HLA-DRB1*0401)
For all of our NSG-derived strains, please visit our NSG mouse model portfolio.
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New NSG Model Innovations
PBMC Engraftment
NOD.Cg-Prkdcscid B2mtm1Unc Il2rgtm1Wjl/SzJ (010636)
o MHC class I deficient NSG strain with enhanced PBMC engraftment
Cytokine Transgenic
NOD.Cg-Prkdcscid Il2rgtm1Wjl Tg(PGK1-KITLG*220)441Daw/SzJ (017830)
o Kit ligand (SCF) transgenic that does not require preconditioning
irradiation
NOD.Cg-Prkdcscid Il2rgtm1Wjl
Tg(CMV-IL3,CSF2,KITLG)1Eav/MloySzJ (013062)
o Expresses human IL-3, CSF2, Kit ligand
o Supports human myeloid cells (CD34-humanized immune system;
acute myeloid leukemia)
Cancer Research
NOD.Cg-Prkdcscid Il2rgtm1Wjl Hprtb-m3/EshJ (012480)
o Hprt-null NSG mouse
o Addition of HAT medium depletes mouse cells to enable isolation of
human cells
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Cutting Edge Human Disease Modeling
The NSG Mouse
NSG Basics
Enhanced disease modeling
o Solid and hematological cancers
o Human immunity and inflammation
Advanced and future disease
modeling
Additional information and
resources
JAX® Mice |
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Online NSG Resources
www.jax.org/jaxmice/research/immunology/005557
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Categorized & Annotated NSG
References List
IMMUNOLOGY
Hematopoiesis and stem cell engraftment
T cell function / B cell function / Myeloerythroid lineages
STEM CELLS / REGENERATIVE MEDICINE
DIABETES
SOLID TUMORS
NSG as a superior host for human immune system
components
NSG strains expressing human cytokine transgenes
NSG strains expressing HLA transgenes
Reviews and protocols
Transplantation research / graft-versus-host disease
(GVHD) / PBMC engraftment
Breast cancer
Brain cancer
Cancer stem cells
Colon cancer
Esophageal cancer
Head and neck cancer
Hepatoblastoma
Vaccines
Autoimmunity
INFECTIOUS DISEASE
Malaria
Cytomegalovirus
Dengue virus
Epstein-Barr
HIV modeling & therapeutics
Measles
Sepsis
Typhoid fever
Varicella-zoster virus
Hepatocellular carcinoma
Lung cancer
Immunotherapy
Melanoma / metastasis
Ovarian cancer
Prostate cancer
Sarcoma
HEMATOLOGICAL CANCERS
AML
Lymphoma
ALL
Mastocytoma
CML
Multiple myeloma
CLL
Chronic lymphocytic leukemia
www.jax.org/jaxmice./research/immunology/005557
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