Vesicular Secretion Of Suppressor Of Cytokine Signaling 3 By Alveolar Macrophages Is Dysregulated In Nsclc And Its Provision Inhibits Tumor Cell Function

Rationale: Inadequate expression of suppressor of cytokine signaling 3 (SOCS3) with subsequent activation of its target, the transcription factor STAT3, has been implicated in tumorigenesis and cancer progression in the lung and other organs. Our lab has recently reported the novel capability of alveolar macrophages (AMs) to secrete SOCS3 within microvesicles (MVs). While AM delivery of MV-encapsulated SOCS3 was shown to suppress inflammatory signaling in recipient lung epithelial cells, the potential significance of this process in restraining the development of lung cancer has not been studied. Methods: A KRAS G12D mutant mouse model was utilized to determine dysfunction of AM SOCS3 secretion in lung cancer. Mice were administered adenoviral Cre recombinase via intratracheal instillation, resulting in formation of lung tumors after 16 weeks. Bronchoalveolar lavage (BAL) fluid and AMs were isolated from the lungs of KRAS and wild-type (WT) mice, and analysis of SOCS3 secretion in BAL or AM cell culture medium was done via ELISA after sonication to disrupt vesicles. In vitro experiments utilized human adenocarcinoma cells (A549) or KRAS mutant rat lung epithelial cells (RLE-G12V). Proliferation, apoptosis and transformation were assessed by Cyquant assay, Annexin V staining, and soft agar assay, respectively. For SOCS3 provision studies, natural AM-derived MVs (isolated by ultracentrifugation) or synthetic liposomes containing recombinant SOCS3 were utilized. Results: Levels of secreted SOCS3 were ~50% lower in KRAS mice BAL than in WT BAL fluid. Additionally, although AMs isolated from KRAS mice contained similar amounts of intracellular SOCS3 and released similar numbers of MVs as those from WT mice, their ex vivo capacity for SOCS3 secretion was significantly lower than that of WT AMs. To determine whether provision of exogenous SOCS3 could inhibit tumorigenesis, synthetic SOCS3 liposomes were administered to RLE-G12V cells prior to chemical transformation with N-Methyl-N′-nitro-N-nitrosoguanidine (MNNG). Addition of exogenous SOCS3 had the capacity to significantly inhibit colony formation in soft agar. To investigate effects of exogenous SOCS3 on established tumor cell function, A549 cells were exposed to both natural AM-derived MVs and SOCS3-containing liposomes and effects on proliferation and apoptosis were measured. Both liposomes and natural MVs significantly induced apoptosis and inhibited proliferation. Finally, the reduction in secreted SOCS3 observed in the mouse model was confirmed in BAL samples of a cohort of NSCLC lung cancer patients compared to healthy volunteers. Conclusion: We report a novel dysregulation of immune surveillance in the form of decreased SOCS3 secretion by AMs that is elicited by the tumor microenvironment, and that may promote tumorigenesis via sustained STAT3 activation. Future studies will focus on the mechanism underlying this defect and whether rescuing SOCS3 secretion can inhibit cancer progression in vivo. Citation Format: Jennifer M. Speth, Loka R. Penke, Joseph Bazzill, Daniel J. Schneider, Douglas A. Arenberg, James J. Moon, Venkateshwar G. Keshamouni, Marc Peters-Golden. Vesicular secretion of suppressor of cytokine signaling 3 by alveolar macrophages is dysregulated in NSCLC and its provision inhibits tumor cell function [abstract]. In: Proceedings of the Fifth AACR-IASLC International Joint Conference: Lung Cancer Translational Science from the Bench to the Clinic; Jan 8-11, 2018; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(17_Suppl):Abstract nr A35.