Abstract 2932: Immuno-inhibitory pathway, TIM-3/Galectin-9, in lung adenocarcinoma: clinical and in vitro analysis
Authors: Oka Mikio , Ohue Yoshihiro , Kurose Koji , Nishio Yumi , Isobe Midori , Nakayama Eiichi 
Published: July 2017

Abstract
Introduction and Purpose:
Recently, immune checkpoint therapy has been incorporated into treatment of solid cancers including lung cancer, resulted in prolonged survival benefit. Checkpoint inhibitors target on PD-1/PD-L1 or CTLA-4 immuno-inhibitory molecules in immune or cancer cells. However, checkpoint therapy in lung adenocarcinoma (LAD) showed less than 20% response rate, suggesting existence of other immuno-inhibitory pathways.
Therefore, we focused on T cell immunoglobulin and mucin domain 3 (TIM-3) on the CD4 and CD8 T-cell surface and its ligand Galectin-9 in tumor cells, and analyzed the expression and function in LAD cells and tissues.
Materials and Methods:
TILs and PBMCs were obtained from 11 patients with LAD, and 194 LAD tissues resected surgically were analyzed. Expression of immuno-inhibitory molecules (PD-1, TIM-3, BTLA, LAG-3) was examined by flow cytometry using FACS on TILs and PBMCs, and PD-L1 and Galectin-9 expressions in LAD tissues was analyzed by immunohistochemistry using tissue microarray.
To investigate soluble Galectin-9 released from LAD cells, EGFR-mutated PC-9 and EGFR-wild-type OU-LC-SK cells were treated with EGFR-TKI afatinib at a concentration of 10 nM for 3 days. Then, the amount of Galectin-9 in culture supernatant was measured by ELISA. To investigate T-cell apoptosis induction by Galectin-9, established XAGE1-specifiic CD8 cloned T-cells were incubated with Galectin-9 protein for 8 hrs.
Results:
Increased expression in TILs compared to PBMCs was observed on PD-1 and TIM-3, but not on BTLA or LAG-3 in CD4 and CD8 T-cells. In LAD tissues, the frequencies of high PD-L1 and Galectin-9 expression in the tumor cell membrane or cytoplasm were 49% and 31%, respectively (in squamous cell carcinoma, the frequencies were 32% and 16%, respectively). Furthermore, correlated expression of PD-L1, Galectin-9 and CD3 (T-cell infiltration) was observed at the periphery of the tumor nest. Those findings suggest the relevance of the PD-1/PD-L1 and TIM-3/Galectin-9 pathways in the tumor microenvironment of LAD.
Next, soluble Galectin-9 released from the LAD cells was measured. Galectin-9 was detected only in the medium of EGFR-mutated LAD cells following treatment with afatinib. Moreover, apoptosis was induced in TIM-3-positive CD8 T-cell clones following interaction with Galectin-9 protein and this was inhibited by the addition of anti-Galectin-9 or an anti-TIM-3 antibody. The findings suggested that a significant amount of Galectin-9 could be released from LAD cells and induced T-cell apoptosis in tumor microenvironment.
Conclusions:
Our results suggested the relevance of the PD-1/PD-L1 and TIM-3/Galectin-9 immuno-inhibitory pathways in the tumor microenvironment of LAD, and that release of soluble Galectin-9 from LAD cells could negatively regulate T-cell function. For successful immune checkpoint therapy in LAD, simultaneous inhibition of TIM-3/Galectin-9 pathway may be needed.
Citation Format: Mikio Oka, Yoshihiro Ohue, Koji Kurose, Yumi Nishio, Midori Isobe, Eiichi Nakayama. Immuno-inhibitory pathway, TIM-3/Galectin-9, in lung adenocarcinoma: clinical and in vitro analysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2932. doi:10.1158/1538-7445.AM2017-2932