Abstract B088: Elucidating the role of amphiregulin in the ovarian tumor immune microenvironment
Authors: Ebott Jasmine , McAdams Julia , De La Cruz Payton , Kim Chloe , James Nicole E. 
Published: March 2024

Objective: The goal of this study was to determine the effect that amphiregulin (AREG) has on the ovarian tumor immune microenvironment (TIME). Methods: OVCAR8WT cells were stimulated with recombinant AREG (rAREG) and submitted for NanoString nCounter® PanCancer IO360 analysis to identify genomic changes in over 770 genes related to tumor immunology. A commercially available phospho-kinase proteome profiler and western blot analysis was employed to determine signaling pathways changes affected by rAREG in OVCAR8WT, and the additional high grade serous ovarian cancer (HGSOC) cell line PEA1. OVCAR8WT and PEA1 cells were co-cultured with peripheral blood mononuclear cells (PBMCs) with and without the addition of rAREG and viability of the HGSOC cells was assessed. Furthermore, chemoresistant HGSOC cells PEA2 were co-treated with carboplatin and an AREG neutralizing antibody (AREG nab), and cell viability was determined. Finally, an in vivo study was performed utilizing an ID8p53-/- C57/BL6 model in which mice were treated with rAREG or saline daily for up to 6 days following tumor formation. Tumors, ascites, and serum from mice were collected at time of euthanasia, and serum and ascites fluid submitted for multiplex cytokine/chemokine analysis to Eve Technologies. Immunohistochemistry was employed to determine PD-L1 and CD8+ T cell levels in mouse tumors. Results: Nanostring analysis revealed that OVCAR8WT cells treated with rAREG led to numerous significant (p&lt;0.006) tumor-intrinsic immune increases, notably in DUSP5 (4.1- fold), DUSP1 (1.6-fold), IL6 (1.6-fold), CXCL8 (11.2-fold), CXCL2 (3.2-fold), and CXCL1 genes (5.4- fold), Phospho-kinase proteome profiler results revealed an increase in STAT3 expression following rAREG exposure in OVCAR8WT (2.9- fold) and PEA1(1.6- fold) cells. These results were validated via western blot. Furthermore, western blot analysis also revealed that phospho-AKT, phospho-ERK, and programmed death ligand 1 (PD-L1) were increased following rAREG treatment in HGSOC cells. OVCAR8WT and PEA1 cells co-treated with PBMCs stimulated with rAREG demonstrated a significantly(p&lt;0.05) higher cell viability than cells treated with PBMCs alone. Moreover, PEA2 cells co-treated with both carboplatin and an AREG nab synergistically and significantly (p&lt;0.05) decreased cell viability. Furthermore, mice treated with rAREG demonstrated significantly (p&lt;0.05) lower levels of IL-2, IL-5, and IL-11 in their ascites and higher serum levels of IL-20. Finally, intratumoral levels of PD-L1 were significantly (p&lt;0.05) upregulated in rAREG treated mouse tumors as well as significantly (p&lt;0.05) lower levels of CD8+ T cells. Conclusions: These findings demonstrate that increased AREG leads to tumor-intrinsic activation of key immune pathways and associated genes and promotes tumor immune evasion in an immunocompetent HGSOC mouse model. Further studies include determining the effects of targeting AREG alone and in combination with chemotherapy in vivo.

Citation Format: Jasmine Ebott, Julia McAdams, Payton De La Cruz, Chloe Kim, Nicole E. James. Elucidating the role of amphiregulin in the ovarian tumor immune microenvironment [abstract]. In: Proceedings of the AACR Special Conference on Ovarian Cancer; 2023 Oct 5-7; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_2):Abstract nr B088.