## 4 Discussion  
This study demonstrates the importance of EMMPRIN as a gatekeeper that prevents tumor cells from entering a dormant state. First, we show that tumor cells alone can modulate the activation of untreated macrophages, and shift them towards M2 macrophages that enhance the expression of the characteristic M2 activation maker CD206. Moreover, incubation with the tumor cells for 48 h is sufficient to induce the secretion of macrophages derived factors that enhance tumor cell proliferation and secretion of VEGF. This suggests that the metastatic cells actively modulate their neighbouring macrophages to become M2-activated, and thereby gradually, and over time, change the immediate microenvironment  
Figure 7: The combination of recombinant TGF\(\beta\) and EMMPRIN has no effect on the addition of TGF\(\beta\) or EMMPRIN alone. CT26-WT or CT26-KD cells (2.5 \(\times\) 10\({}^{4}\) cells) were incubated in triplicates for 48 h in serum starvation medium, with or without the addition of recombinant TGF\(\beta\) (5 ng/mL), EMMPRIN (5 ng/mL), or their combination. The effect on the (**A**) proliferation, as measured by CCK (\(n=10\)), (**B**) the concentrations of secreted VEGF (\(n=5\)), as well as the mRNA expression of (**C**) _NR2F1_ (\(n=6\)), (**D**) _p21_ (\(n=6\)), (**E**) _Sug_ (\(n=6\)), and (**F**) _Zeb1_ (\(n=6\)). Data are presented as means \(\pm\) SE, and analyzed using a two-way ANOVA followed by [PERSON]’s post-hoc test. to accommodate their needs. Moreover, this is, at least partially, mediated by EMMPRIN, as CT26-KD cells exhibit a reduced response. Data also suggests that EMMPRIN is only partially required for mediating the interactions with macrophages that are needed to exit dormancy. We moreover show that several changes occur upon knocking-down EMMPRIN expression, including inhibition in TGF\(\beta\) production, reduction in the pERK/pP38 ratio, and the enhancement of vimentin expression, dormancy markers and EMT-TFs. Additionally, proliferation and the angiogenic potential of the cells are inhibited. Thus, the single intrinsic change of knocking down EMMPRIN expression is sufficient to drive cells into a dormant state, affecting both cellular and angiogenic dormancy. When macrophages are co-cultured with CT26-KD cells, all the above-mentioned indicators return to their parental WT values, even if this is not achieved completely. The pERK/pP38 ratio is increased, but does not reach the levels of co-cultured CT26-WT cells, the expression of vimentin, dormancy markers, and EMT-TFs is reduced almost to the levels of the co-cultured CT26-WT cells, and proliferation and angiogenic potential are increased, reaching the CT26-WT levels when cultured alone. Therefore, when EMMPRIN expression is reduced, co-culturing with macrophages can drive CT26-KD cells out of dormancy. This could be due to the residual expression of EMMPRIN in the CT26-KD cells, which may be sufficient to drive the exit from dormancy when extrinsic signals are received, or due to the influence of additional signaling pathways triggered by the co-culture, such as the pathways induced by the increased levels of IL-6 and TNF\(\alpha\) levels, which may be at play. Note that these two options can co-exist, and are not mutually exclusive. Therefore, the ability of macrophages to promote an exit from dormancy depends only partially on the presence of EMMPRIN. Macrophages are producers of inflammatory cytokines in the TME, and especially of cytokines such as TGF\(\beta\), IL-6 and TNF\(\alpha\) that have been recognized as drivers of the EMT process [51]. Moreover, inflammation has already been implicated in having a role in the exit from dormancy [51], in agreement with our findings. Here we show that the co-culture enhanced the secretion of TGF\(\beta\), IL-6 and TNF\(\alpha\), but only TGF\(\beta\), and not TNF\(\alpha\) or IL-6, was reduced in the CT26-KD cells by the reduced expression of EMMPRIN. This suggests that TGF\(\beta\) and EMMPRIN regulate each other.