Abstract 269: PD-L1 dominated tumor-CAF-immune cell crosstalk of stromal microenvironment: Endometrial tumor vs. tumor-adjacent normal
Authors: Lin Xiaoqian , Aske Jennifer , Dale Adam , Koirala Nischal , Thompson Ethan , Fagerness Mary , Flier Natasha , Davies Paige , Kehoe Stacy , Ageton Cheryl , Gaster Kris , Espaillat Luis Rojas , Starks David , Sulaiman Raed , De Pradip , Dey Nandini 
Published: June 2022

Abstract
Background: In endometrial cancers (EC), the mechanistic contribution of PD-L1/L2 and PD-1 signaling of host’s stromal microenvironment (SME) [cancer-associated fibroblast (CAF) and immune cells] in relation to the tumor (T) cells is elusive.
Aim: To understand the tumor-stroma-immune crosstalk in EC, we studied the compartmental pattern of PD-L1, PD-L2, and PD-1 expression in EC tissues and their matched CAFs from both T and tumor-adjacent normal (N) tissues.
Method: Over 116 surgically resected T and N tissues were obtained from consented patients with EC at the Avera Cancer Institute (March ‘16 and September ’21). IHC expression was performed in T, N-epithelium, SME (mesenchyme) in the T and N tissues. The staining intensity and distribution pattern were evaluated by a pathologist using a standard scoring system (1% cut-off value). Since fibroblasts are the most abundant cells of SME, the PD-L1 was parallelly evaluated in the multiple passages of cultured T and N derived CAFs by the qRT-PCR, flow-cytometry, and ICC.
Result: The expression of 3 immune checkpoint proteins in the epithelial and mesenchyme of N was limited to only rare and focal positivity for PD-L1. The epithelial compartments of the T tissue, in contrast, had 70% positivity for PD-L1, which was independent of differentiation status, presence of TILs, or lymphovascular invasion. PD-L1 positivity was 90% of the SME of tested samples, identified predominantly in the (1) immune component, including macrophages and lymphocytes, and (2) EpCAM-/SMA+/FAP+/S100A4+ CAFs. Both PD-L1 and PD-L2 were identified by flow, ICC, and qRT-PCR in NCAF and TCAF. The overall expression of PD-L2 was found to be qualitatively and quantitatively lesser than that of PD-L1, primarily contributed by SME/stromal macrophages and lymphocytes. PD-1 expression was restricted to the SME/stromal immune components, predominantly lymphocytes. The expression for PD-L1 and PD-L2 mRNA was higher in TCAF than NCAF in successive passages, which ICC and flow cytometry confirmed.
Summary: The expression of immune checkpoint proteins, PD-L1, and PD-L2, are primarily tumor-driven and associated with the endometrioid (invasive) and serous adenocarcinomas of grades II and above with myometrial invasion. The expression of PD-L1 and PD-L2 are distributed in both tumor and SME/stromal components, while PD-1 expression is exclusively SME/stromal.
Citation Format: Xiaoqian Lin, Jennifer Aske, Adam Dale, Nischal Koirala, Ethan Thompson, Mary Fagerness, Natasha Flier, Paige Davies, Stacy Kehoe, Cheryl Ageton, Kris Gaster, Luis Rojas Espaillat, David Starks, Raed Sulaiman, Pradip De, Nandini Dey. PD-L1 dominated tumor-CAF-immune cell crosstalk of stromal microenvironment: Endometrial tumor vs. tumor-adjacent normal [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 269.