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 The Immune System in Mice
JAX® Mice | 4 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
JAX® Mice | 5 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
JAX® Mice | 6 NSG A Highly Immunodeficient Mouse
JAX® Mice | 7 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
JAX® Mice | 8 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)
JAX® Mice | 9 The Most Useful and Versatile Immunodeficient Mouse
Research applications:
Primary tumor engraftment
Cancer stem cells
Human hematopoiesis
Humanized mice
Infectious disease
Transplantation research
JAX® Mice | 10 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 Classical Cancer Xenograft Modeling Nude Mice
Before treatment After treatment
Gulliya et al. 1994. Cancer 74:1725-1732. PMID:8082074
® JAX Mice | 12 Patient Tumor Characteristics NSG vs scid
Cancer Patient’s Lung Tumor Fragment NSG
H&E C.B-17 scid
H&E Human cells Mouse blood cells (anti-HLA) (anti-mCD45)
Simpson-Abelson MR et al. 2008.J Immunol 180(10):7009-18. PMID:18453623
JAX® Mice | 13 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® Patient-Derived Xenograft (PDX) Program
In Vivo Pharmacology Services | 15 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
In Vivo Pharmacology Services | 16 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
In Vivo Pharmacology Services | 17 Orthotopic PDX Modeling in NSG Bladder Cancer
Fidelity during tumor passaging Orthotopic model (subcutaneous)
Lin TY et al. 2012. Nanomedicine (Lond).[Epub ahead of print] PMID:23199207
In Vivo Pharmacology Services | 18 PDX Modeling in NSG mice Bladder Cancer
Tumor-Targeting Micelles Prevent …and Improve Survival Tumor Growth…
Lin TY et al. 2012. Nanomedicine (Lond).[Epub ahead of print] PMID:23199207
In Vivo Pharmacology Services | 19 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
In Vivo Pharmacology Services | 20 NEW PDX Live™ NSG MiceM 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™ 21 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 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
JAX® Mice | 23 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
JAX® Mice | 24 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
JAX® Mice | 26 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
JAX® Mice | 27 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
In Vivo Pharmacology Services | 28 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
JAX® Mice | 29 Experimental Timeline for JAX® Hu-NSG
Whole body irradiation Human Human Tail vein injection B cells T cells appear appear
Mouse Age 3 weeks 12 weeks 15 weeks
CD34+ cells engrafted in 3 week old female mice
In Vivo Pharmacology Services | 30 Successful Engraftment in Peripheral Blood Human Mouse Engrafted NSG
Mostly neutrophils, monocytes Human CD45 Mouse CD45
Human white blood cells abundant in circulation
Mature human T (CD3+) and B (CD20+) cells differentiated in the NSG mouse Human CD3 Human CD20
In Vivo Pharmacology Services | 31 Multi-lineage Differentiation in Bone Marrow Human vs Mouse Human HSC 86.1% 88.6% 0.13% 11.2% SSC hCD34 hCD45 FSC mCD45 hCD38 Human Erythroid Human B cells 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
In Vivo Pharmacology Services | 33 PBMCs Transplanted with human islets, +/- STZ mice: NSGtreatedwithDiabetic Diabetes Transplant RejectionStudies:
JAX King M etal. 2008. ® Mice 32 days post-engraft. | No PBMC Clin Immunol 126(3):303-14. PMID: + PBMC 18096436 34
Insulin hCD45 H&E 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
35 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
Whole body irradiation Human Human Tail vein injection of B cells T cells human CD34+ HSCs appear appear and tumors
Mouse Age: 3 weeks 12 weeks 15 weeks
In Vivo Pharmacology Services | 38 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
In Vivo Pharmacology Services | 39 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
In Vivo Pharmacology Services | 40 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
In Vivo Pharmacology Services | 41 Humanized tumor-bearing NSG mice
Irradiation
CD34+ HSC NSG
Solid tumor fragment or tumor cell line Humanized NSG
Humanized tumor-bearing NSG mice
In Vivo Pharmacology Services | 42 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
JAX® Mice | 43 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.
JAX® Mice | 44 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 Online NSG Resources
www.jax.org/jaxmice/research/immunology/005557
JAX® Mice | 47 Categorized & Annotated NSG References List
IMMUNOLOGY STEM CELLS / REGENERATIVE MEDICINE Hematopoiesis and stem cell engraftment DIABETES T cell function / B cell function / Myeloerythroid lineages SOLID TUMORS NSG as a superior host for human immune system Breast cancer components Brain cancer NSG strains expressing human cytokine transgenes Cancer stem cells NSG strains expressing HLA transgenes Colon cancer Reviews and protocols Esophageal cancer Transplantation research / graft-versus-host disease (GVHD) / PBMC engraftment Head and neck cancer Vaccines Hepatoblastoma Autoimmunity Hepatocellular carcinoma INFECTIOUS DISEASE Lung cancer Malaria Immunotherapy Cytomegalovirus Melanoma / metastasis Dengue virus Ovarian cancer Epstein-Barr Prostate cancer HIV modeling & therapeutics Sarcoma Measles HEMATOLOGICAL CANCERS Sepsis AML Lymphoma Typhoid fever ALL Mastocytoma Varicella-zoster virus CML Multiple myeloma CLL Chronic lymphocytic leukemia
www.jax.org/jaxmice./research/immunology/005557
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