Abstract B93: The influence of macrophage stimulation on lung cancer migration/invasion, proliferation, and treatment sensitivity.
Authors: Rohrbach Timothy D. , Deshane Jessy S. , Steele Chad , Willey Christopher D. 
Published: August 2014

Background: Each year lung cancer takes the lives of over one million people worldwide demonstrating strong proliferative capabilities, invasiveness, and resistance to chemotherapy and radiation. Epidemiological studies suggest that individuals with chronic inflammation have a higher risk for certain types of cancers, and lung cancer is no exception. Clinical studies have shown that an increase in macrophages in and around tumors correlates with an increase in microvascular density and poor prognosis in lung cancer patients. Macrophages represent a very plastic and heterogeneous population of immune cells. Macrophage stimulated by interferon-gamma (INF-γ); tumor necrosis factor-alpha (TNF-α); or lipopolysaccaride (LPS) are known as classically activated or M1 macrophage. Classically activated macrophage produce pro-inflammatory cytokines, such as, interleukin – 1, 6, and 23 (IL-1, IL-6, and IL-23 respectively) along with reactive oxygen species (ROS). These products aid in immune system activation and pathogen removal. However, when macrophages are stimulated with IL-4, IL-10, IL-13, or glucocorticoids they acquire what is labeled as “an alternatively activated” or M2 phenotype. This population produces enzymes to remodel the extracellular matrix, such as MMP - 7, 9, along with factors to promote angiogenesis, for example vascular endothelial growth factor (VEGF). Tumor-associated macrophage (TAMs) are believed to possess an alternative, M2, phenotype and aid in tumor growth and invasion. Therefore, we investigated how different macrophage populations could influence lung cancer migration, proliferation, and radiation sensitivity in in vitro models.

Methods: Human lung cancer cell lines A549, H1299, H1792, and H1975 were used along with the murine lung cancer cell line Lewis Lung Carcinoma (LLC). Bone marrow derived macrophage (M0) were stimulated with IFN-γ or IL-4 to achieve classically activated (M1) and alternatively activated (M2) populations, respectively. M1 and M2 stimulation was confirmed through qRT-PCR examination of inflammatory markers. Conditioned media was collected at 24hrs. Migration was assessed using BD 24 well FluoroBlok cell culture inserts or scratch assay. Proliferation was measured using Perkin Elmer's ATPlite assay with or without ionizing radiation.

Results: M0, M1, and M2 macrophage were analyzed for expression of inflammatory markers using qRT-PCR. Nos2 and IL-6 levels were significantly up-regulated in M1 stimulated cells, while Arginase-1 was significantly up-regulated in M2 stimulated cells consistent with differentiation into the M1 and M2 populations. Conditioned media stimulation of the various cancer cell lines displayed differential effects on migration, proliferation, and radiation sensitivity. In general, M2 condition media promoted increased migration, proliferation, and radiation resistance though, not all effects were significant. The A549 cells were the most responsive of the cell lines in terms of cell migration demonstrating a 20% increase (p&lt;0.0001) when cultured with M2 conditioned macrophage media compared to M0 conditioned media. M2 conditioned media promoted an increase in proliferation in all cell lines tested, reaching significance (p&lt;0.05) in all but the A549 cells (Increases of 55% in LLC, 69% in H1792, 36% in H1299, and 50% in H1975). A similar trend of radiation resistance was seen in M2 conditioned media-treated cells, particularly the LLC cell line which showed significant (p&lt;0.05) radiation resistance even at low radiation dose (3 Gy).

Conclusion: Our studies suggest that M2 macrophage condition media promotes lung cancer migration, proliferation and resistance to radiation.

Citation Format: Timothy D. Rohrbach, Jessy S. Deshane, Chad Steele, Christopher D. Willey. The influence of macrophage stimulation on lung cancer migration/invasion, proliferation, and treatment sensitivity. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr B93.