Abstract 4973: The immunosuppressive tumor microenvironment of metastatic osteosarcoma inhibits the cytotoxic effect of tumor-infiltrating lymphocytes
Authors: Ligon John A. , Oke Teniola F. , Choi Woonyoung , Fong Megan H. , Levin Adam , Rhee Daniel S. , Morris Carol D. , Siegel Nicholas , Hsieu Emily H. , Meyer Christian F. , McConkey David J. , Anders Robert A. , Pardoll Drew M. , Llosa Nicolas J. 
Published: August 2019

Abstract
Background: Patients with metastatic osteosarcoma (OS) have a 5 year overall survival of &lt;25%. We aim to understand the immune architecture of the tumor microenvironment (TME) of OS, with the goal of harnessing the immune system as a major therapeutic strategy for the treatment of patients with OS.
Methods: Immunohistochemistry (IHC) slides from 66 formalin-fixed paraffin-embedded OS tissue blocks were analyzed. Laser capture microdissection and RNA extraction was performed on 13 OS specimens, and gene expression profiles of the tumor interior vs. tumor interface region were compared utilizing RNA seq. Tumor infiltrating lymphocytes (TILs) were isolated from 25 freshly obtained OS specimens and analyzed by multiparameter flow cytometry (MFC).
Results: Digital image analysis of IHC specimens revealed significantly higher immune cell infiltration (CD3+ and CD8+ cells) in the pulmonary metastases compared to primary bone tumors, particularly at the interface between the OS lesion and normal lung tissue. There is increased expression at the interface of the immune checkpoint molecules programmed cell death 1 (PD-1), T cell immunoglobulin mucin-3 (TIM-3) and lymphocyte activation gene 3 (LAG-3). Gene expression profiling showed increased CD8 T cell and resting CD4 memory T cell signature at the interface compared to the tumor interior, and a strong M2 and M0 macrophage signature in both regions. TILs isolated from pulmonary metastases and analyzed by MFC had higher expression of PD-1 and other immune checkpoint molecules, and these TILs were more capable of producing the effector molecules IFN-gamma and granzyme B.
Conclusions: Pulmonary metastatic OS lesions are more highly infiltrated with immune cells than primary bone lesions, particularly at the interface. The fact that these immune cells are shown via MFC to be capable of producing cytotoxic cytokines and proteases suggests that these TILs may be tumor-specific lymphocytes capable of acting against the tumor if they were not being inhibited by multiple immune checkpoint molecules. The inability of these immune cells to penetrate further into the tumor interior may represent an adaptive immune response by the tumor through a combination of repellant cytokines and inhibition of TILs via upregulation of PD-1, TIM-3 and LAG-3. Treatment with a combination of anti-PD-1, anti-TIM-3 or anti-LAG-3 directed treatments may unleash these immune cells and allow them to destroy OS tumor cells.
Citation Format: John A. Ligon, Teniola F. Oke, Woonyoung Choi, Megan H. Fong, Adam Levin, Daniel S. Rhee, Carol D. Morris, Nicholas Siegel, Emily H. Hsieu, Christian F. Meyer, David J. McConkey, Robert A. Anders, Drew M. Pardoll, Nicolas J. Llosa. The immunosuppressive tumor microenvironment of metastatic osteosarcoma inhibits the cytotoxic effect of tumor-infiltrating lymphocytes [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 4973.