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 | 2
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 | 3
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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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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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)
NSG A Highly Immunodeficient Mouse
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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 | 11
Before treatment After treatment
Classical Cancer Xenograft Modeling Nude Mice
Gulliya et al. 1994. Cancer 74:1725-1732. PMID:8082074
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NSG
C.B-17 scid
H&E Human cells (anti-HLA)
Mouse blood cells (anti-mCD45)
H&E
Cancer Patient’s Lung
Tumor Fragment
Patient Tumor Characteristics NSG vs scid
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
In Vivo Pharmacology Services | 14
JAX® PDX Tumor Model Bank (>350)
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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 In Vivo Pharmacology Services |
PDX modeling in NSG Estrogen Receptor-Positive Breast Carcinoma
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TM00386-PT
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
TM00386 (ER+/PR+/Her2-)
TM00386-P0 TM00386-P1
Fidelity during tumor passaging (subcutaneous)
Orthotopic model
Orthotopic PDX Modeling in NSG Bladder Cancer
Lin TY et al. 2012. Nanomedicine (Lond).[Epub ahead of print] PMID:23199207
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Tumor-Targeting Micelles Prevent Tumor Growth…
…and Improve Survival
PDX Modeling in NSG mice Bladder Cancer
Lin TY et al. 2012. Nanomedicine (Lond).[Epub ahead of print] PMID:23199207
In Vivo Pharmacology Services | 19
Cell line: A375 (RhoC, Luciferase) injected I.V.
NSG Improved Melanoma Metastasis Modeling
Carreno et al. 2008. Clin Cancer Res 15(10):3277-86. PMID:19447870
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*NSG also better platform for cell line engraftment (data not shown)
PDX Live™ NSG Mice Readily Available from Inventory and Pre-Engrafted with
Patient-Derived Tumors
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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™
NSG M
NEW
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 | 22
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
Effective Modeling of Patient-Derived ALL (T & B Cell)
Diamanti P et al. 2011. Leukemia Feb;26(2):376-80. PMID:21860430
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Samples from different patients showed improved engraftment versus other models (NSB)
NSG Efficiently Models Patient AML
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 | 25
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
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 also steadily engrafts human immune cell populations over time (data not shown)
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
NSG The Most Efficient Humanized Model
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
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CD34+ cells engrafted in 3 week old female mice
Human T cells appear
Human B cells appear
Whole body irradiation Tail vein injection
3 weeks 12 weeks 15 weeks Mouse Age
In Vivo Pharmacology Services |
Human Mouse Engrafted NSG H
uman
CD
45
Mouse CD45
Hum
an C
D3
Human CD20
Human white blood cells abundant in circulation
Mature human T (CD3+) and B (CD20+) cells differentiated in the NSG mouse
Mostly neutrophils, monocytes
Successful Engraftment in Peripheral Blood
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mCD45 hC
D45
88.6%
FSC
SS
C
86.1% 0.13% 11.2%
hCD38
hCD
34
Human vs Mouse Human HSC
Human Erythroid Human B cells Human APCs
Multi-lineage Differentiation in Bone Marrow
Sensitization Sensitization Challenge +/- treatmentDay -1 Day 1 Day 2 Day 7 Day 8 Day 9 Day 14Ear thickness Ear thickness Ear thickness Ear thickness Ear thickness Ear thickness
Sensitized with DNFB
Naive
Sensitized with Olive Oil
DNFB + Hydrocortisone
DTH Response in Humanized NSG is Evidence for Intact T Cell Functions
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H&
E
hCD
45
Insu
lin
+ PBMC 32 days post-engraft. No PBMC
Transplant Rejection Studies: Diabetes Diabetic mice: NSG treated with STZ Transplanted with human islets, +/- PBMCs
King M et al. 2008. Clin Immunol 126(3):303-14. PMID:18096436
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Infectivity correlates with infiltration of human T cells into vaginal mucosa
NSG is Superior to NOD scid for Humanized HIV Models
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
In Vivo Pharmacology Services | 36
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 | 37
Humanized tumor-bearing NSG mice The next step in cancer modeling
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Human T cells appear
Human B cells appear
Whole body irradiation Tail vein injection of
human CD34+ HSCs and tumors
3 weeks 12 weeks 15 weeks Mouse Age:
In Vivo Pharmacology Services |
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
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Wege et.al., mAbs. 2014. PMID: 24870377
Humanized NSG mice Engraftment of human tumors
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Injected tumor cells grew, metastasized (data not shown), and retained Her2- positivity
In Vivo Pharmacology Services |
Wege et.al., mAbs. 2014. PMID: 24870377
Humanized tumor-bearing NSG mice Human immune cell engraftment and tumor infiltration
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T cells T cells Macrophages B cells
In Vivo Pharmacology Services |
Wege et.al., mAbs. 2014. PMID: 24870377
Humanized tumor-bearing NSG mice
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Humanized NSG
Solid tumor fragment or tumor cell line
NSG
Irradiation
Humanized tumor-bearing NSG mice
CD34+ HSC
In Vivo Pharmacology Services |
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 JAX® Mice | 45
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 | 46
Categorized & Annotated NSG References List
www.jax.org/jaxmice./research/immunology/005557
IMMUNOLOGY Hematopoiesis and stem cell engraftment T cell function / B cell function / Myeloerythroid lineages 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 engraftmentVaccines Autoimmunity INFECTIOUS DISEASE Malaria Cytomegalovirus Dengue virus Epstein-Barr HIV modeling & therapeutics Measles Sepsis Typhoid feverVaricella-zoster virus
STEM CELLS / REGENERATIVE MEDICINE DIABETES SOLID TUMORS Breast cancer Brain cancer Cancer stem cells Colon cancer Esophageal cancer Head and neck cancer Hepatoblastoma 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
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JAX® Mice, Clinical & Research Services
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