## 3 Results
### Macrophages Secrete Mediators That May Drive the Metastatic Outbreak  
Macrophages have been suggested to contribute to the EMT process and to metastasis, but their contribution may strongly depend on their mode of activation [39]. TAMs have often been shown to be activated mostly as M2, and we show here, at least partially, that this holds true also for our in vitro setting. First, we show that the co-culturing of RAW 264.7 macrophages with CT26-WT tumor cells was sufficient to increase their expression of the typical M2 activation marker CD206 at the mRNA level (Figure S1A). To understand whether the macrophage mode of activation changes the effects they exert on the tumor cells, we activated RAW 264.7 macrophages for 24 h as M0 (no treatment), M1 (incubation with 100 U/mL IFN\(\gamma\) and 100 ng/mL LPS) or M2 (incubation with IL-4 and IL-13, 20 ng/mL each). We then collected the respective supernatants, diluted them 1:1 with fresh serum-starvation medium and added them to CT26-WT or CT26-KD cells. Relative to CT26-WT cells that were incubated with M1 supernatants, the proliferation of the same cells incubated with M0 or M2 supernatants was increased, whereas the CT26-KD cells exhibited reduced proliferation in the M0 and M2 groups and increased proliferation in the M1 group, relative to the CT26-WT cells (Figure S1B). A similar pattern was observed for the secretion of VEGF, with unchanged VEGF levels in the group receiving M1 supernatants (Figure S1C). Secretion of EMMPRIN was actually increased when CT26-WT cells were incubated with supernatants from M1-activated macrophages relative to M0- or M2-activated macrophages, in agreement with the ability of EMMPRIN to function as a pro-inflammatory protein under certain clinical settings (Figure S1D). EMMPRIN secretion was naturally reduced in all CT26-KD cells. These results suggest that the presence of tumor cells activates RAW 264.7 macrophages as M2 macrophages, and, in turn, these macrophages promote proliferation and angiogenesis in the co-cultured cells.  
We now asked whether the co-culturing of CT26-WT and CT26-KD tumor cells with RAW 264.7 macrophages enhanced the production of cytokines associated with the metastatic outbreak. In our experimental system, the macrophages and CT26 tumor cells were separated by inserts, so that cell-cell contacts were avoided, allowing only soluble mediators to drive the change. IL-6 and TNF\(\alpha\) are two of the inflammatory cytokines linked with the exit from dormancy [40; 41; 42]. We show that when both CT26-WT and CT26-KD cells were cultured alone, they produced minute amounts of these two cytokines (Figure 2C,D), but when they were co-cultured with RAW 264.7 macrophages, the secretion of both TNF\(\alpha\) and IL-6 was markedly enhanced, suggesting that they do not depend on EMMPRIN-specific signals (Figure 2C,D). In contrast, the concentrations of both TGF\(\beta\) and EMMPRIN itself in the supernatants of single cultures of CT26-KD cells were reduced compared to the single cultures of CT26-WT cells (Figure 2A,B). When CT26-KD cells were co-cultured with RAW 264.7 macrophages, these concentrations were markedly elevated compared to single cultures of CT26-KD, but were substantially lower compared to those of the CT26-WT co-cultures. While the reduced accumulation of EMMPRIN in the CT26-KD supernatants could be explained by its reduced expression in these cells, the reduced accumulation of TGF\(\beta\) strongly implicates EMMPRIN in its regulation. Since we were interested in the effects of the co-culture mediated by EMMPRIN, we simulated the effect of the co-culture in the following experiments by adding either recombinant TGF\(\beta\) or recombinant EMMPRIN to the tumor cells.