Abstract A122: Non-small cell lung cancer cells induce PD-L1 expression in the tumor microenvironment in an orthotopic mouse model of lung cancer
Authors: Li Howard , McSharry Maria , Clambey Eric , Nguyen Teresa , McNamee Eoin , Weiser-Evans Mary , Nemenoff Raphael 
Published: May 2016

Abstract
Background: In many cancers, tumor cells induce local and systemic immunosuppression, inhibiting T-cell-mediated cytotoxic responses to tumor growth. Activation of the programmed cell death 1 (PD-1) pathway has been shown to contribute to immune escape in the setting of lung cancer. Antibody-mediated blockade of PD-1 and programmed death-ligand 1 (PD-L1) induces durable tumor regression and prolongs stabilization of disease in patients with non-small cell lung cancer (NSCLC), which has lead to FDA approval of an anti-PD-1 agent for the treatment of patients with advanced squamous NSCLC. However, patient selection criteria for immunotherapy targeting the PD-1 axis have not been established. Although recent studies have focused on the regulation of PD-L1 expression on lung cancer cells, the role of PD-L1 expression in the lung tumor microenvironment (TME) in mediating responses to anti-PD-1 or anti-PD-L1 therapy has not been defined. The purpose of this study was to determine the relative contributions of PD-L1 expression on cancer cells versus cells in the TME on lung cancer progression.
Methods: Two murine lung cancer cell lines derived from C57BL/6 mice were used in an orthotopic model of tumor progression: Lewis Lung Carcinoma (LLC) cells, which are a de-differentiated cell line in which driver mutations have not been identified; and CMT167 cells, which are an epithelial lung cancer cell line that express oncogenic K-Ras. Tumor cells were implanted in the left lungs of wild-type C57BL/6 mice. One week after tumor implantation, mice were treated with anti-PD-1 neutralizing antibody, anti-PD-L1 neutralizing antibody, or isotype control antibodies. The effect of pharmacologic blockade of PD-1 or PD-L1 on tumor progression was determined. In separate experiments, tumor cells were implanted into green fluorescent protein (GFP)-expressing mice, and lungs from tumor-bearing mice were analyzed by flow cytometry to evaluate the expression of PD-L1 on lung cancer cells (GFP-negative cells) and in the TME (GFP-positive cells).
Results: Antibody-mediated pharmacologic blockade of PD-1 or PD-L1 suppressed primary tumor growth and liver metastasis of both LLC and CMT167 tumors in our orthotopic model. Whereas there was a modest effect only on LLC tumors, PD-1/PD-L1 antibody blockade had a dramatic effect on CMT167 primary tumor size, with 40% of mice injected with CMT167 cells and treated with anti-PD-L1 antibody having no detectable lung tumors at the time of sacrifice. In contrast to previously published work that has shown epithelial-to-mesenchymal transition can upregulate PD-L1 expression on lung cancer cells, leading to CD8-positive T-cell immunosuppression (Chen et al, Nat Commun 2014), the well-differentiated CMT167 cells were more responsive than LLC cells to PD-L1 inhibition. Moreover, sensitivity to PD-1/PD-L1 antibody blockade did not correlate with expression of PD-L1 on tumor cells. Analysis of lungs from tumor-bearing mice revealed that both LLC cells and CMT167 cells induce PD-L1 expression in the TME (most likely tumor-associated macrophages), and that expression of PD-L1 is higher in the TME than on cancer cells in vivo. Interestingly, CMT167 tumors are associated with higher numbers of both PD-L1-positive cells and PD-1-positive cells in the TME than LLC tumors.
Conclusions: In an orthotopic mouse model, lung cancer cells upregulated PD-L1 expression in the TME, which may be an important mechanism whereby tumor cells induce T-cell immunosuppression. The ability to upregulate PD-L1 in the TME may also represent an important factor that predicts sensitivity to PD-1/PD-L1 antibody blockade.
Citation Format: Howard Li, Maria McSharry, Eric Clambey, Teresa Nguyen, Eoin McNamee, Mary Weiser-Evans, Raphael Nemenoff. Non-small cell lung cancer cells induce PD-L1 expression in the tumor microenvironment in an orthotopic mouse model of lung cancer. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A122.