1. Chemotherapeutic tolerability and Estrogen dose response in
B6;129-Rag2tm1FwaIL2rgtm1Rsky/DwlHsd (R2G2) mice
Jamie Naden, PhD
Scientist
Veterinary Science, Research and Support
Envigo

2. Outline Background on the R2G2® Methods Overview Estrogen Study
Body Weights and Survival Analysis
Hematology and Clinical Chemistry Results
Overview Chemotherapy Study
Summary and Conclusion

3. B6;129-Rag2tm1FwaIL2rgtm1Rsky/DwlHsd (R2G2®)
Double knockout of Rag2 and IL2rg
Rag2 (recombination activating gene) deletion causes mature T and B cell deficiency
IL2rg deletion leads to a lack of functional receptors for IL-2, IL-4, IL-7, IL-9 and IL-15
Lacks NK cells
Decreased macrophages
Decreased dendritic cells
Decreased neutrophils
Immunodeficient mouse models are helping to advance the field of oncology research. The B6;129-Rag2tm1FwaIL2rgtm1Rsky/DwlHsd (R2G2) was developed at the Fox Chase Cancer Center (Philadelphia, PA) to provide an additional immunodeficient option for the oncology and immunology fields. This model was developed by backcrossing an IL2rg (Interleukin-2 receptor common gamma chain) knockout model to a RAG2 (recombinase activating gene) knockout model. Interleukin-2 receptor gamma is required to mediate the effects of multiple cytokines including IL-2, IL-4, IL-7, IL-9 and IL-15. Disruption of the IL2rg gene leads to major defects in lymphocyte and lymphoid tissue development. Recombinase activating gene 2 knockout animals were engineered to harbor a germline deletion of Rag2. Since the enzyme product encoded by Rag2 serves to ensure proper VDJ recombination, its deletion leads to B- and T-cell deficiencies. The B- and T-cell deficiency associated with the Rag2 mutation is similar to that associated with the scid mutation, however, Rag2 knockout mice do not become “leaky” like SCID mice. The combination of the Rag2 and IL2rg mutations in R2G2 mice results in a mouse that lacks various cytokines including IL-2, IL-4, IL-7, IL-9 and IL-15. The R2G2 also lacks B cells, T cells, NK cells and has a deficit in dendritic cells, macrophages and lymphocyte development.
A paucity of literature is available on the R2G2 as it is newly licensed for commercial sale. We wanted to establish data on the use of estrogen supplementation in this strain for use in estrogen-dependent tumor or cell lines. Additionally, R2G2 mice should be less susceptible to adverse effects of DNA damaging compounds compared to SCID mouse strains. For this reason we wanted to establish optimal dose ranges for three standard of care chemotherapeutic agents.

4. Methods
120 female R2G2® mice at 7-8 weeks of age were sourced from Envigo (Indianapolis, IN) to Horizon Discovery (St. Louis, MO)
Ten untreated control mice were added for comparison.
Animal numbers and study design were approved by the Horizon Discovery Institutional Animal Care and Use Committee. Horizon Discovery is fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC).
R2G2® mice were maintained on a 12:12 h light:dark cycle (22°C, 50% humidity) and provided standard diet (Envigo Teklad 2918, 18% protein, 6% fat) and water ad libitum
Animal numbers and study design were approved by the Horizon Discovery Institutional Animal Care and Use Committee. Horizon Discovery is fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC).
One hundred twenty, female B6;129-Rag2tm1FwaIL2rgtm1Rsky/DwlHsd (R2G2) mice at 7-8 weeks of age were sourced from Envigo (Indianapolis, IN) to Horizon Discovery (St. Louis, MO). Ten untreated control mice were added for comparison.
R2G2 mice were maintained on a 12:12 h light:dark cycle (22°C, 50% humidity) and provided standard diet (Envigo Teklad 2918, 18% protein, 6% fat) and water ad libitum.

5. Overview - Estrogen Study
Naïve R2G2® mice each received one 60-day release 17β-estradiol pellet (Innovative Research of America) at the following doses: 0.18 mg, 0.36 mg, 0.72 mg and 1.7 mg (n=10/group)
Body weight and survival were monitored for 60 days post-implantation
When animals became moribund (>20% body weight loss) or at the end of 60 days, non-fasted whole blood was collected for analysis of complete blood count (CBC) and clinical chemistry
All samples were used in final analysis
Naïve R2G2 mice each received one 60-day release 17β-estradiol pellet (Innovative Research of America) at the following doses: 0.18 mg, 0.36 mg, 0.72 mg and 1.7 mg (n=10/group). Body weight and survival were monitored for 60 days post-implantation. When animals became moribund (>20% body weight loss) or at the end of 60 days, non-fasted whole blood was collected for analysis of complete blood count (CBC) and clinical chemistry. All samples were used in final analysis.

6. Body weight in R2G2® mice in response to differing doses of estrogen (n=10/group)
There was no difference in body weight due to estrogen dose concentration (Figures 1 and 2). Body weight remained stable throughout the study. The control group continued to gain weight throughout the study and weights were significantly higher than all estrogen dose groups.

7. Survival analysis of R2G2® mice in response to differing doses of estrogen (n=10/group)
Survival (Figure 3) was lowest at 70% (by day 26) in the group given 1.7 mg estrogen. Eighty percent of animals given the 0.36 mg and 0.72 mg pellets survived through day 60 and 90% of the animals given 0.18 mg of estrogen survived through day 60, as anticipated.

8. Results - Hematology Hematology Units Control 5-FU 30 mg/Kg
CTX 100 mg/Kg
CTX 140 mg/Kg
DOX 2 mg/Kg
DOX 5 mg/Kg
n=4
n=5
White Blood Cells
K/uL
0.84
0.46
3.25
0.7*
1.82
0.8
1.47
0.25
1.83
0.55
Monocytes %
5.12
1.75
10.15
3.61
9.94
2.84
17.33
9.24*
12.63
6.67
Red Blood Cells
M/uL
10.62
0.09
9.93
0.20
10.20
0.59
9.71
0.22*
10.28
0.07
Data represent as mean ± standard deviation
* Significantly different from control. P<0.05
^ Significantly different from 0.36 mg/Kg. P<0.05

9. Results - Hematology Hematology Units Control 5-FU 30 mg/Kg
CTX 100 mg/Kg
CTX 140 mg/Kg
DOX 2 mg/Kg
DOX 5 mg/Kg
n=4
n=5
White Blood Cells
K/uL
0.84
0.46
3.25
0.7*
1.82
0.8
1.47
0.25
1.83
0.55
Monocytes %
5.12
1.75
10.15
3.61
9.94
2.84
17.33
9.24*
12.63
6.67
Red Blood Cells
M/uL
10.62
0.09
9.93
0.20
10.20
0.59
9.71
0.22*
10.28
0.07
Data represent as mean ± standard deviation
* Significantly different from control. P<0.05
^ Significantly different from 0.36 mg/Kg. P<0.05

10. Results - Hematology Hematology Units Control 5-FU 30 mg/Kg
CTX 100 mg/Kg
CTX 140 mg/Kg
DOX 2 mg/Kg
DOX 5 mg/Kg
n=4
n=5
White Blood Cells
K/uL
0.84
0.46
3.25
0.7*
1.82
0.8
1.47
0.25
1.83
0.55
Monocytes %
5.12
1.75
10.15
3.61
9.94
2.84
17.33
9.24*
12.63
6.67
Red Blood Cells
M/uL
10.62
0.09
9.93
0.20
10.20
0.59
9.71
0.22*
10.28
0.07
Data represent as mean ± standard deviation
* Significantly different from control. P<0.05
^ Significantly different from 0.36 mg/Kg. P<0.05

11. Results - Hematology Hematology Units Control 5-FU 30 mg/Kg
CTX 100 mg/Kg
CTX 140 mg/Kg
DOX 2 mg/Kg
DOX 5 mg/Kg
n=4
n=5
White Blood Cells
K/uL
0.84
0.46
3.25
0.7*
1.82
0.8
1.47
0.25
1.83
0.55
Monocytes %
5.12
1.75
10.15
3.61
9.94
2.84
17.33
9.24*
12.63
6.67
Red Blood Cells
M/uL
10.62
0.09
9.93
0.20
10.20
0.59
9.71
0.22*
10.28
0.07
Data represent as mean ± standard deviation
* Significantly different from control. P<0.05
^ Significantly different from 0.36 mg/Kg. P<0.05

12. Results - Clinical chemistry
Units
Control
0.18 mg/Kg
0.36 mg/Kg
0.72 mg/Kg
1.7 mg/Kg
n=4
n=5
Alkaline Phosphatase (ALP)
U/L
68.00
11.52
119.20
20.51*^
83.75
30.00
131.75
29.02*^
150.20
24.48*^
Cholesterol
mg/dL
148.50
31.65
133.80
18.05
114.25
8.77*
108.25
12.58*
104.00
14.87*
Data represent as mean ± standard deviation
* Significantly different from control. P<0.05
^ Significantly different from 0.36 mg/Kg. P<0.05

13. Results - Clinical chemistry
Units
Control
0.18 mg/Kg
0.36 mg/Kg
0.72 mg/Kg
1.7 mg/Kg
n=4
n=5
Alkaline Phosphatase (ALP)
U/L
68.00
11.52
119.20
20.51*^
83.75
30.00
131.75
29.02*^
150.20
24.48*^
Cholesterol
mg/dL
148.50
31.65
133.80
18.05
114.25
8.77*
108.25
12.58*
104.00
14.87*
Data represent as mean ± standard deviation
* Significantly different from control. P<0.05
^ Significantly different from 0.36 mg/Kg. P<0.05

14. Results - Clinical chemistry
Units
Control
0.18 mg/Kg
0.36 mg/Kg
0.72 mg/Kg
1.7 mg/Kg
n=4
n=5
Alkaline Phosphatase (ALP)
U/L
68.00
11.52
119.20
20.51*^
83.75
30.00
131.75
29.02*^
150.20
24.48*^
Cholesterol
mg/dL
148.50
31.65
133.80
18.05
114.25
8.77*
108.25
12.58*
104.00
14.87*
Data represent as mean ± standard deviation
* Significantly different from control. P<0.05
^ Significantly different from 0.36 mg/Kg. P<0.05

15. Summary and conclusion
The estrogen dose response study provided important information about the use of estrogen pellet supplementation in the R2G2® strain and supports the use of this mouse strain in estrogen-dependent, estrogen receptor positive cancer studies.
These data will assist investigators utilizing R2G2® mice in choosing the appropriate exogenous estrogen dose for their study to achieve highest survival percentage.
The estrogen dose response study provided important information about the use of estrogen pellet supplementation in the R2G2 strain and supports the use of this mouse strain in estrogen-dependent, estrogen receptor positive cancer studies. Published literature reports the most commonly used dose in SCID immunodeficient strains at 0.72 mg/kg, which is shown to have adverse effects in SCID strains1-4. These data will assist investigators utilizing R2G2 mice in choosing the appropriate exogenous estrogen dose for their study to achieve highest survival percentage.

16. Chemotherapeutic tolerability study

17. Overview - Chemotherapy Study
Naïve R2G2® mice were dosed via intraperitoneal injection with either:
5-fluorouracil (5-FU; 30 mg/kg body weight, three times weekly, or 60 or 100 mg/kg body weight, twice weekly for five weeks)
doxorubicin (DOX; 2 or 5 mg/kg body weight, once weekly for three weeks)
cyclophosphamide (CTX; 100 or 140 mg/kg body weight, once weekly for three weeks) (n=10/group)
An untreated control group (n=10) was added for comparison
Naïve R2G2 mice were dosed via intraperitoneal injection with either 5-fluorouracil (5-FU; 30 mg/kg body weight, three times weekly, or 60 or 100 mg/kg body weight, twice weekly for five weeks), doxorubicin (DOX; 2 or 5 mg/kg body weight, once weekly for three weeks), or cyclophosphamide (CTX; 100 or 140 mg/kg body weight, once weekly for three weeks) (n=10/group). An untreated control group (n=10) was added for comparison. The lowest dose was chosen based on the average dose used in immunodeficient mouse models and the highest dose was chosen based on the average dose used in immunocompetent mouse models. Body weight and survival were monitored for 60 days from the first dose. When animals became moribund (>20% body weight loss) or at the end of 60 days, whole blood was collected for analysis of complete blood count (CBC) and clinical chemistry.
Data analysis was completed using commercially available software (SigmaStat version 4.0, Chicago, IL). A one way ANOVA was performed on body weight, CBC and clinical chemistry measures. Statistical significance was determined at P < Data are represented as mean ± one standard deviation.

18. Overview - Chemotherapy Study
Body weight and survival were monitored for 60 days from the first dose
When animals became moribund (>20% body weight loss) or at the end of 60 days, whole blood was collected for analysis of complete blood count (CBC) and clinical chemistry
All samples were used in final analysis
The lowest dose was chosen based on the average dose used in immunodeficient mouse models and the highest dose was chosen based on the average dose used in immunocompetent mouse models. Body weight and survival were monitored for 60 days from the first dose. When animals became moribund (>20% body weight loss) or at the end of 60 days, whole blood was collected for analysis of complete blood count (CBC) and clinical chemistry.
Data analysis was completed using commercially available software (SigmaStat version 4.0, Chicago, IL). A one way ANOVA was performed on body weight, CBC and clinical chemistry measures. Statistical significance was determined at P < Data are represented as mean ± one standard deviation.

19. Body weight in R2G2® mice in response to chemotherapy treatment (n=10/group)
5-Florouricil was highly toxic at 60 and 100 mg/kg and all mice were euthanized by study day 12. The 30 mg/kg dose was well tolerated and mice maintained a stable weight throughout the study (Figures 4 and 5).
Cyclophosphamide was also well tolerated at both 100 and 140 mg/kg. Mice gained on average 10% body weight by the end of the study (Figures 4 and 5).

20. Survival analysis of R2G2® mice in response to chemotherapy treatment (n=10/group)
Doxorubicin was tolerated well at the 2 mg/kg dose and body weights remained stable. At 5 mg/kg, mice continually lost weight. At 50% survival, body weights were no longer reported (Figure 6).
As mentioned earlier, 5-FU-30, CTX-100 and DOX-2 were well tolerated in the R2G2 mouse and 100% of these animals survived. Only 10% of animals in the DOX-5 group survived by day 40 (Figure 6).

21. Results – Hematology Hematology Units Control 5-FU 30 mg/Kg
CTX 100 mg/Kg
CTX 140 mg/Kg
DOX 2 mg/Kg
DOX 5 mg/Kg
n=4
n=5
Red Blood Cells
M/uL
10.62
0.09
9.24
0.35*
10.51
0.18
10.59
0.19
9.99
0.48
9.95
1.24
Mean Corpuscular Volume fL
46.40
0.55
52.40
0.55*
46.75
0.96
46.50
0.58
0.89
1.71
Mean Corpuscular Hemoglobin pg
14.78
0.16
15.98
0.04*
15.00
0.29
14.98
0.26
14.92
15.23
0.68 21
Data represented as mean ± standard deviation
* Significantly different from control. P<0.05

22. Results – Hematology Hematology Units Control 5-FU 30 mg/Kg
CTX 100 mg/Kg
CTX 140 mg/Kg
DOX 2 mg/Kg
DOX 5 mg/Kg
n=4
n=5
Red Blood Cells
M/uL
10.62
0.09
9.24
0.35*
10.51
0.18
10.59
0.19
9.99
0.48
9.95
1.24
Mean Corpuscular Volume fL
46.40
0.55
52.40
0.55*
46.75
0.96
46.50
0.58
0.89
1.71
Mean Corpuscular Hemoglobin pg
14.78
0.16
15.98
0.04*
15.00
0.29
14.98
0.26
14.92
15.23
0.68 22
Data represented as mean ± standard deviation
* Significantly different from control. P<0.05

23. Results – Hematology Hematology Units Control 5-FU 30 mg/Kg
CTX 100 mg/Kg
CTX 140 mg/Kg
DOX 2 mg/Kg
DOX 5 mg/Kg
n=4
n=5
Red Blood Cells
M/uL
10.62
0.09
9.24
0.35*
10.51
0.18
10.59
0.19
9.99
0.48
9.95
1.24
Mean Corpuscular Volume fL
46.40
0.55
52.40
0.55*
46.75
0.96
46.50
0.58
0.89
1.71
Mean Corpuscular Hemoglobin pg
14.78
0.16
15.98
0.04*
15.00
0.29
14.98
0.26
14.92
15.23
0.68 23
Data represented as mean ± standard deviation
* Significantly different from control. P<0.05

24. Results – Hematology Hematology Units Control 5-FU 30 mg/Kg
CTX 100 mg/Kg
CTX 140 mg/Kg
DOX 2 mg/Kg
DOX 5 mg/Kg
n=4
n=5
Red Blood Cells
M/uL
10.62
0.09
9.24
0.35*
10.51
0.18
10.59
0.19
9.99
0.48
9.95
1.24
Mean Corpuscular Volume fL
46.40
0.55
52.40
0.55*
46.75
0.96
46.50
0.58
0.89
1.71
Mean Corpuscular Hemoglobin pg
14.78
0.16
15.98
0.04*
15.00
0.29
14.98
0.26
14.92
15.23
0.68 24
Data represented as mean ± standard deviation
* Significantly different from control. P<0.05

25. Summary and Conclusion
Since the R2G2® strain lacks the SCID mutation, we wanted to establish tolerable doses of a few chemotherapeutic agents
These data can be used by investigators using R2G2® mice to determine the optimal dose of these specific standard of care treatments
Severe combined immunodeficiency (SCID) strains can be more sensitive to some chemotherapy agents due their inability to repair DNA damage5. Since the R2G2 strain has different mutations from SCID mice, we wanted to establish tolerable doses of a few chemotherapeutic agents. These data can be used by investigators using R2G2 mice to determine the optimal dose of these specific standard of care treatments.

26. Overall conclusion
Our goal is to add to the characterization data available for this model to better assist investigators when trying to choose a model for oncology research
These data support that the R2G2 mouse model may be a good alternative to SCID models when administering DNA damaging chemotherapies or when estrogen supplementation is required for xenograft growth