Jamie Naden PhD Scientist Veterinary Science Research and Support Envigo A new tool for oncology researchers The RAG2 IL2R g double knockout R2G2 mouse 1 Overview of the of the R2G2 B6129 ID: 763184
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Jamie Naden, PhDScientistVeterinary Science, Research and SupportEnvigo A new tool for oncology researchers: The RAG2-/-IL2Rg-/- double knockout (R2G2) mouse 1
Overview of the of the R2G2 (B6;129-Rag2tm1Fwa II2rgtm1Rsky/DwlHsd ) model characteristics Comparative analysis of the R2G2 and NSG immune cell subsets in spleenComparative analysis of the R2G2 and NSG radiosensitivityEstrogen supplementation toleranceChemotherapy tolerance Tumor growth Summary Presentation overview 2
R2G2 Model: Background3 R2G2 = “Rag2” “IL2Rg” double knockout mouseModel licensed from Fox Chase Cancer Center (Temple) Created by backcrossing the IL2R g (common gamma chain) knockout mutation on to a Rag2 knockout mouseBackground is a mixture of C57-129 Nomenclature: B6;129- Rag2 tm1Fwa II2rg tm1Rsky /DwlHsd
Ultra (triple) immunodeficient mouse modelDeficient in TDeficient in B cells Deficient in NK cells No leakiness, compared to other SCID mice (varies with background) Less radiosensitiveLess sensitive to radiation as compared to traditional SCID modelsR2G2 Model: Characteristics
Flow results: B and T lymphocytes diminished CD3 B220 Wild type NSG R2G2- F R2G2- M Gating set up based on C57bl/6 spleen Diminished B cells and T cells in NSG and R2G2
Flow results: Th and Tc lymphocytes diminished CD4 CD8 Gating set up based on C57bl/6 spleen Wild type NSG RAG2 -/- IL2R g -/- - F RAG2 -/- IL2R g -/- - M Diminished helper T cells and cytotoxic T cells in NSG and R2G2
Flow results: NK lymphocytes diminished CD335 CD49b Gating set up based on C57bl/6 spleen Wild type NSG RAG2 -/- IL2R g -/- - F RAG2 -/- IL2R g -/- - M Diminished helper natural killer cells in NSG and R2G2
R2G2 Model: Growth curve8 = NOG/NSG Male = NOG/NSG Female R2G2 has a more robust growth curve than NSG
Survival analysis of R2G2 and NSG mice in response to a single dose of radiation at 4 Gy (n=10/group) 9At 4 Gy, NSG survivability began to drop at 6 days post radiation and all NSG died by day 8 At 4 Gy, all R2G2 survived
Survival analysis of R2G2 mice in response to differing doses of estrogen (n=10/group) 10 Dose response as anticipated
Survival analysis of R2G2 mice in response to chemotherapy treatment (n=10/group) 11 Dox at 5mg/kg was detrimental
Growth of PNX0225 Burkitt Lymphoma PDX12R2G2 had double the tumor volume vs. C.B-17 SCID at 19 days post implantation
Results – head and neck PDX13 Head and neck PDX 626 was transplanted (2.2 mm2 tissues) into 4 sections of each of two R2G2 mice each (n=2/PDX). Data are represented as individual tumors Head and neck PDX 635 was transplanted (2.2 mm 2 tissues) into 4 sections of each of two R2G2 mice each (n=2/PDX).
Results – human esophageal adenocarcinoma (OE33) 14Human esophageal adenocarcinoma OE33 cells (5x106 cells per mouse) were injected into the left and right flanks of SCID (n=3), Athymic Nude (n=3) and R2G2 (n=2) mice.
Results – human esophageal adenocarcinoma (FLO1) 15FLO1 cells (5x106 cells per mouse) were injected into both flanks of three each of Athymic Nude and SCID mice, and two R2G2 mice.
Flow cytometryComparable in Cytotoxic T, Th cells and NK cells to NSGGrowth CurveAdult M/F ~ 5-10g larger than NOG/NSG at similar ageRadiosensitivity R2G2 can tolerate double the single radiation dose than NSG miceBurkitt PDX xenograftR2G2 had approximately double the tumor volume at Day 20 compared to C.B-17-Prkdcscid miceHead and Neck PDXDifficult to grow in Athymic Nude and SCID mice; 100% take rate in R2G2 miceEsophageal Adenocarcinoma100% take rate was seen in R2G2 mice, whereas there was no growth in Athymic Nude or SCID mice R2G2 Model: Summary 16 Estrogen supplementation tolerance Estrogen supplementation tolerance
Gating Strategy: Lymphocyte panel FSC-W FSC-A Dead CD45 SSC-A Gating set up based on C57bl/6 spleen Live CD45+ (WBC) Single cells
Gating strategy: Lymphocyte panel FSC-W FSC-A CD4 CD8 B220+ (B) CD8+ (CTL) Lymphocytes CD3+ (T) CD4+ ( Th ) CD3 B220 Gating set up based on C57bl/6 spleen
Gating strategy: Lymphocyte panel FSC-W FSC-A Lymphocytes CD335 CD49b CD335+/CD49b+ (NK) Gating set up based on C57bl/6 spleen