C. Maloney1,2, M. P. Kallis1,2, M. C. Edelman4, M. Symons3, B. M. Steinberg1,3, S. Z. Soffer2,3 1Feinstein Institute Of Medical Research,Elmezzi School Of Molecular Medicine,Manhasset, NY, USA 2Hofstra Northwell School Of Medicine,Surgery,Manhasset, NY, USA 3Feinstein Institute Of Medical Research,Karches Center For Oncology And Cell Biology,Manhasset, NY, USA 4Hoftstra Northwell School Of Medicine,Pathology And Laboratory Medicine,Manhasset, NY, USA
Introduction: We have previously shown that macrophages promote osteosarcoma (OS) invasion in vitro which is inhibited by gefitinib, an epidermal growth factor receptor (EGFR) inhibitor (p<0.001). In vivo, gefitinib reduces both the incidence of gross lung metastasis and the metastatic outgrowth of pulmonary micro-metastases. However, EGFR is neither present on macrophages nor on OS cells, suggesting a non-EGFR mechanism for these findings. An alternative target of gefitinib, RIP2K, is expressed in antigen-presenting cells and plays a central role in NOD-mediated innate immune responses and NF-κB activation. The role of RIPK2 in tumor-associated macrophages has not been studied. We investigated the role of RIP2K in macrophage-promoted OS invasion in vitro and the effect of gefitinib on pulmonary macrophage phenotype in vivo
Methods: In vitro, mouse OS cells (K7M2) were incubated ± conditioned media from wild type or RIPK2 knock-out mouse bone marrow-derived macrophages (BMDMs) ± the RIP2K inhibitor OD36 (5.3nM). Invasive capacity was assessed utilizing 3D-invasion assays. In vivo, K7M2 cells were implanted into the tibia of BALB/c mice (n=6). Mice were treated with either control or gefitinib-impregnated chow beginning 1 week post-implantation. Non-tumor-bearing wild type BALB/c mice were used as controls. Four weeks post-implantation, lungs were harvested and homogenized into a single-cell suspension. The cells were washed, stained with antibodies targeting the macrophage markers CD45, CD11b and F4/80, and MHC II, which is upregulated on pro-inflammatory macrophages, and subjected to flow cytometry. Data was acquired on an LSRFortessa Flow Cytometer (BD Biosciences) and analyzed with FlowJo (FlowJo, LLC).
Results: In vitro, conditioned medium from wild-type BMDMs significantly promoted OS invasion (<0.001) while conditioned medium from RIPK2 knockout macrophages did not. Conditioned medium from macrophages pretreated with the RIPK2 inhibitor also inhibited macrophage-promoted invasion (p<0.01). Addition of the inhibitor to tumor cells in the absence of macrophages had no effect on OS invasion (p=0.876). In vivo, macrophages in the lungs of tumor-bearing mice showed decreased MHCII expression, a pro-inflammatory anti-tumor marker, when compared to non-tumor bearing control mice. Conversely, macrophages from gefitinib-treated tumor-bearing mice displayed an increase in MHCII expression compared to untreated tumor-bearing mice, suggesting that gefitinib suppressed or reversed the polarization of the macrophages.
Conclusions: Macrophage-promoted invasion of osteosarcoma requires RIPK2 activity. Gefitinib promotes a pro-inflammatory phenotype in pulmonary macrophages and prevents outgrowth of pulmonary metastases in vivo. Upfront treatment with gefitinib may limit metastatic progression of OS by modulating macrophages via RIP2K inhibition.