Jamie Naden, PhD Scientist Veterinary Science, Research and Support - PowerPoint Presentation

Jamie Naden, PhDScientistVeterinary Science, Research and SupportEnvigo 1 Patient-derived and cell line xenograft growth in the B6;129-Rag2tm1FwaIL2rgtm1Rsky/DwlHsd (R2G2®) mouse model

Background on the R2G2 ® PurposeHead and neck PDXHuman head and neck squamous cell carcinomaHuman esophageal adenocarcinomaHuman gastric adenocarcinomaMouse colon carcinomaMouse B-cell lymphomaSummary and conclusionOutline2

Double knockout of Rag2 and IL2rgRag2 (recombination activating gene) deletion causes mature T and B cell deficiencyIL2rg deletion leads to a lack of functional receptors for IL-2, IL-4, IL-7, IL-9 and IL-15 Lacks NK cellsDecreased macrophagesD ecreased dendritic cellsDecreased neutrophilsB6;129-Rag2tm1FwaIL2rgtm1Rsky/DwlHsd (R2G2®)3

R2G2 was licensed from Fox Chase Cancer Center by Envigo in 2016Literature in lackingFirst set of compiled tumor growth data for public consumptionPurpose 4

HNSCC is the 6th leading cause of cancer worldwideCell line models have shown to be poor predictors of drug response in humansPDX models maintain similar histology, gene expression and heterogeneityHead and neck PDX engraftment rate has been shown to very low (17%) in Nude mice and only 45% in more immunodeficient mice Peng, S., Creighton, C. J., Zhang, Y., Sen, B., Mazumdar, T., Myers, J. N., ... & Johnson, F. M. (2013). Tumor grafts derived from patients with head and neck squamous carcinoma authentically maintain the molecular and histologic characteristics of human cancers.  Journal of translational medicine, 11(1), 198.Klinghammer, K., Raguse, J. D., Plath, T., Albers, A. E., Joehrens, K., Zakarneh, A., ... & Fichtner, I. (2015). A comprehensively characterized large panel of head and neck cancer patient‐derived xenografts identifies the mTOR inhibitor everolimus as potential new treatment option. International journal of cancer, 136(12), 2940-2948.Background – Head and neck PDX5

Results – Head and neck PDX (626)6 Head and neck PDX 626 was transplanted (2.2 mm2 tissues) into 4 sections of each of two R2G2 mice each (n=2/PDX).

Results – Head and neck PDX (635)7 Head and neck PDX 635 was transplanted (2.2 mm2 tissues) into 4 sections of each of two R2G2 mice each (n=2/PDX).

Results – Head and neck squamous cell carcinoma (SQ20b)8 Head and neck squamous cell carcinoma SQ20b cells were injected into one flank of ten R2G2 mice.

Esophageal adenocarcinoma is increasing in incidence in Western countries with generally poor prognosis and no widely accepted animal modelCell lines have proven difficult to grow in miceOne paper showed 18.7% take rate for xenograft growth (Nude)Contino , G., Eldridge, M. D., Secrier, M., Bower, L., Elliott, R. F., Weaver, J., ... & Fitzgerald, R. C. (2016). Whole-genome sequencing of nine esophageal adenocarcinoma cell lines.  F1000Research, 5.Melsens, E., De Vlieghere, E., Descamps, B., Vanhove, C., De Wever, O., Ceelen, W., & Pattyn, P. (2017). Improved xenograft efficiency of esophageal adenocarcinoma cell lines through in vivo selection. Oncology reports, 38(1), 71-81.De Both, N. J., Wijnhoven, B. P. L., Sleddens, H. F. B. M., Tilanus, H. W., & Dinjens, W. N. M. (2001). Establishment of cell lines from adenocarcinomas of the esophagus and gastric cardia growing in vivo and in vitro. Virchows Archiv, 438(5), 451-456. Background – Esophageal adenocarcinoma 9

Results – Human esophageal adenocarcinoma (OE33)10 Human esophageal adenocarcinoma OE33 cells (5x10 6 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)11 FLO1 cells (5x10 6 cells per mouse) were injected into both flanks of three each of Athymic Nude and SCID mice, and two R2G2 mice.

Results – Human esophageal adenocarcinoma (FLO1)12 FLO1 cells (10x10 6 cells per mouse) were injected into both flanks of SCID and R2G2 mice (n=4/strain).

With increasing incidence, a high death rate and low 5-year survival, more efficient therapeutic protocols for gastric cancer are neededChen, T., Yang, K., Yu, J., Meng, W., Yuan, D., Bi, F., ... & Wang, F. (2012). Identification and expansion of cancer stem cells in tumor tissues and peripheral blood derived from gastric adenocarcinoma patients. Cell research , 22(1), 248.There is a lack of models that mimic the invasiveness seen in humans Busuttil, R. A., Liu, D. S., Di Costanzo, N., Schröder, J., Mitchell, C., & Boussioutas, A. (2018). An orthotopic mouse model of gastric cancer invasion and metastasis. Scientific reports, 8(1), 825.Background – Gastric adenocarcinoma13

Results – Human gastric adenocarcinoma (AGS)14 Human gastric adenocarcinoma AGS cells (2x10 6 cell per mouse) were injected into both flanks of two each of R2G2 and SCID mice.

Syngeneic tumors can be grown in immunodeficient models to determine the role of the immune system in drug effectsthe role of a specific immune cell typesBerge, G., Eliassen, L. T., Camilio, K. A., Bartnes, K., Sveinbjørnsson , B., & Rekdal, Ø. (2010). Therapeutic vaccination against a murine lymphoma by intratumoral injection of a cationic anticancer peptide. Cancer immunology, immunotherapy, 59(8), 1285-1294.Adris, S. K., Klein, S., Jasnis, M. A., Chuluyan, E., Ledda, M. F., Bravo, A. I., ... & Podhajcer, O. L. (1999). IL-10 expression by CT26 colon carcinoma cells inhibits their malignant phenotype and induces a T cell-mediated tumor rejection in the context of a systemic Th2 response. Gene therapy, 6(10), 1705.Background – Syngeneic tumor growth 15

Results – Mouse colon carcinoma (CT26)16 The mouse colon carcinoma CT26 cells (1x105 cells per mouse) were injected subcutaneously into the flank of ten R2G2 mice.

Results – Mouse B-cell lymphoma (A20)17 Mouse B-cell lymphoma A20 cells (4x106 cells per mouse) were injected subcutaneously into the flank of ten R2G2 mice.

This is the first compiled data for tumor growth in the R2G2 model The R2G2 model can be a valuable tool for oncology programs, including PDX and cell line oncology researchhigh take rates and rapid growth of allogeneic modelsThis data will aide investigators in choosing the optimal mouse model for their oncology research programSummary and conclusion18