A. J. Hesketh1,2,3, C. A. Behr1,2,4, M. Edelman1,4, R. D. Glick1,4, Y. J. Al-Abed2,3, M. J. Symons2,3, B. M. Steinberg2,3,4, S. Z. Soffer1,2,4 1Cohen Children’s Medical Center,Division Of Pediatric Surgery,New Hyde Park, NY, USA 2Feinstein Institute For Medical Research,Manhasset, NY, USA 3Elmezzi Graduate School Of Molecular Medicine,Manhasset, NY, USA 4Hofstra North Shore-LIJ School Of Medicine,Hempstead, NY, USA
Introduction: Metastatic Ewing’s Sarcoma (ES) carries a poor prognosis. Recent evidence demonstrates that tumor-associated macrophages in ES are associated with more advanced disease. While some macrophage phenotypes (M1) exhibit anti-tumor activity, distinct phenotypes (M2) may contribute to malignant progression and metastasis. Preliminary in vitro and animal model studies demonstrate that the macrophage inhibitor CNI-1493 prevents ES tumor cell invasion and lung metastases. We hypothesized that CNI-1493 inhibits M2 macrophage-stimulated ES tumor cell extravasation into the lung parenchyma, a key step in the establishment of viable metastatic foci.
Methods: To simulate the architecture of the lung extravasation process, in vitro extravasation assays were constructed using basement membrane-coated cell culture inserts featuring an opaque polyethylene membrane containing 8μm pores. A monolayer of primary human pulmonary microvascular endothelial cells (HPMECs) was plated on the surface of the basement membrane and impermeability was verified with Evans blue. Primary macrophages isolated from human blood were polarized to express the M2 phenotype by treatment with M-CSF followed by activation with IL-4. Fluorescently tagged human ES cells (SK-NEP) were grown in monoculture or in co-culture with M2 macrophages in the upper chamber of the cell culture insert. Alternatively, M2 macrophages were plated on the undersurface of the cell culture insert with SK-NEP cells in the upper chamber. After 48 hours, tumor cells that had invaded through the endothelial monolayer, basement membrane and culture insert into the lower chamber were quantified using fluorescence microscopy.
Results: Tumor cell extravasation was increased 3.4-fold in the presence of M2 macrophages (p<0.05) with no significant difference between SK-NEP/M2 co-cultures and cultures separated by the assay insert (Table 1). CNI-1493 significantly decreased the quantity of invading tumor cells stimulated by M2 macrophages (p<0.01), and the degree of this inhibition was similar whether an HPMEC monolayer was present or not (54% and 58% reduction, respectively).
Conclusion: M2 macrophages promote tumor cell extravasation in vitro. This interaction is independent of direct tumor cell contact, suggesting soluble mediators. CNI-1493 decreases M2-induced tumor cell extravasation independent of the presence of an endothelium, indicating a direct effect on macrophages expressing pro-metastatic phenotypes. CNI-1493 may be useful as a novel adjunct in the treatment or prevention of metastatic ES.
