O2138: Extracellular Vesicles from Osteosarcoma Change Macrophages in the Lung to Promote Metastatic Growth

Background: Osteosarcoma (OS) is a highly metastatic sarcoma affecting adolescents. Metastasis at diagnosis carries high mortality, and the most common site of metastasis is in the lungs. Macrophages in the tumor microenvironment are termed tumor associated macrophages, which have been shown to be responsible for metastatic growth in OS. Extracellular Vesicles (EVs) have been linked to tumor growth and metastases in many different cancers and we previously have shown EVs to increase metastasis in OS vivo and change macrophages to act more like tumor associated macrophages in vitro. Understanding mechanisms of metastases can help better target metastatic disease and catch micrometastases at an earlier stage, thus, decreasing mortality for these patients. The aim of this study is to elucidate the impact of extracellular vesicles on macrophages in the lung.

Methods: Two murine OS cell lines, K12, with low metastatic potential and K7M2, with high metastatic potential, were used for experiments. EVs from these cell lines were collected via ultracentrifugation and injected in equal amounts via the tail vein into mice a total of 10 times over three weeks for ‘education.’ Mice were euthanized and lungs collected for immunofluorescent staining. The lungs were stained with an antibody for CD86, a marker of M1 or CD206, a marker of M2 and with an antibody for F4/80 to confirm macrophage staining and nuclear counterstain with DAPI. Images were taken with EVOS fluorescent microscope and fluorescence measured with ImageJ. Amount of M1 or M2 polarization calculated as an average over five random images and reported as positive cells per mm2.

Results: Education with EVs from both K12 and K7M2 cell lines increased both M1 and M2 macrophage infiltration in the lungs (p= 0.007, p=0.013, respectively). EVs from K12, the less metastatic cell line, led to increased M1 polarized cells compared to baseline, a more inflammatory macrophage (Figure 1). EVs from K12 and K7M2 increased M2 polarized cells, more like tumor associated macrophages, compared to baseline, with K7M2 EVs, the more metastatic cell line, increasing M2 polarization more than K12 EVs (Figure 2).

Conclusion: Tumor associated macrophages can encourage survival, growth and continued spread of tumor cells in a metastatic environment. EVs from highly metastatic cells educate macrophages towards ‘M2’ polarization in the metastatic site, which may be a mechanism by which metastatic spread occurs. Understanding the interplay between EVs and metastasis is useful for creating targets for immunotherapies.