Abstract 3234: PD-1 is intrinsically expressed by lung cancer cells with stemness features inhibited by PD-1 blockade
Authors: Rotolo Ramona , Leuci Valeria , Donini Chiara , Sanlorenzo Martina , Vujic Igor , Medico Giovanni , Vita Francesca , Gammaitoni Loretta , Righi Luisella , Riganti Chiara , Vigna Elisa , D'Ambrosio Lorenzo , Grignani Giovanni , Scagliotti Giorgio V. , Novello Silvia , Aglietta Massimo , Sangiolo Dario 
Published: August 2019

Abstract
Purpose: Aim of this study is to explore the intrinsic expression, functional role and therapeutic modulation of PD-1 receptor in Non Small Cell Lung Cancer (NSCLC) cells. We previously reported (Sanlorenzo M et al. Clinical Cancer Res 2018) that PD-1+ melanoma cells may sustain disease relapse following treatment with BRAF/MEK inhibitors. We hypothesized that PD-1+ tumor cells may characterize a “stem-like” compartment also in NSCLC, sustaining chemo-resistance and disease relapse with potential therapeutic implications.
Experimental procedures: The expression of PD-1 by NSCLC cells was explored by flow cytometry, western blot (WB) and RT-PCR. Its presence and role in lung cancer stem cells (CSC) was explored by sphere formation essays. CSC were further visualized by tumor-engineering with a lentiviral CSC-detector vector (LV-CSC) encoding eGFP under control of the OCT4 stem gene promoter. Selective PD-1 blockade and PD-1 stimulation with soluble ligand (PD-L1) were used to study the functional role and therapeutic modulation of intrinsic PD-1 in NSCLC.
Results: PD-1 is intrinsically expressed by a small subset of NSCLC cells with stemness features. We found PD-1 consistently expressed on the membrane of a small tumor cell fraction (2% ± 0.3) within 6 NSCLC cell lines (H1975, EBC-1, H23, H820, HCC827), including a primary patient-derived NSCLC culture (SL1). Data were confirmed by RT-PCR and WB. Viable PD-1+ tumor cells were significantly enriched in NSCLC spheres generated in stem conditioned cultures, compared with the monolayer controls (10% [4-36] vs 2% [1-5] P&lt;0,0001, n=6). The levels of PD-1 and stem gene OCT4 RNA comparably increased in NSCLC spheres (4.5 vs 4 fold, n=5). The tumor-intrinsic expression of PD-1 was confirmed by data mining in 67 adenocarcinoma (Affimetryx RNA value 4.348 [3.882-6.361]) and 28 squamous lung carcinoma (RNA value 4.338 [3.995-5.178]) cell lines (CCLE). The formation of NSCLC spheres was significantly inhibited (-30%±2, n=6 P=0.0004) by anti-PD-1 blocking antibody (100μg/μl), while enhanced (25% ± 2, n=3, P= 0.007) by soluble PD-L1 (50 μg/μl). Similar results were confirmed by selective PD-1 RNA-interference that revoked the pro-tumorigenic effect of soluble PD-L1. In vitro treatment with Cisplatin (IC50 dose) led to a relative enrichment of PD-1+ (2.5±0.3 fold, n=4) cells and OCT4+ CSC (3.5±0.3 fold, n=4). The sequential PD-1 inhibition significantly delayed NSCLC cell recovery (-45%±9.8, n=4) and sphere formation after cisplatin.
Conclusions: PD-1 is intrinsically expressed by NSCLC cells with stemness features and mediates pro-tumorigenic activity. PD-1+ NSCLC cells are enriched after chemotherapy and may be inhibited by selective PD-1 blockade. We report a new, intrinsic, expression pattern of PD-1 in NSCLC, providing rationale to explore a lymphocyte-independent activity of anti-PD1 antibodies.
Citation Format: Ramona Rotolo, Valeria Leuci, Chiara Donini, Martina Sanlorenzo, Igor Vujic, Giovanni Medico, Francesca Vita, Loretta Gammaitoni, Luisella Righi, Chiara Riganti, Elisa Vigna, Lorenzo D'Ambrosio, Giovanni Grignani, Giorgio V. Scagliotti, Silvia Novello, Massimo Aglietta, Dario Sangiolo. PD-1 is intrinsically expressed by lung cancer cells with stemness features inhibited by PD-1 blockade [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3234.