## 3 Results
### Reduced EMMPRIN Expression Enhances Markers of Dorancy  
We hypothesized that the incubation of tumor cells in co-culture with macrophages would promote an exit from dormancy. To this end, we now measured several indicators of dormancy. CT26-WT and CT26-KD cells were incubated alone or in co-culture with RAW 264.7 macrophages. Alternatively, to simulate the effects of the co-culture and to explore as to whether they are mediated primarily by either TGF\(\beta\) or EMMPRIN, we added the recombinant proteins to the single cultures of CT26-WT and CT26-KD cells. First, we assessed the ratio between the activated form of ERK1/2 and p38 MAPKs. The p38 MAPK activates a stress-like program that endows the cell with drug resistance and inhibits proliferation through the induction of the cyclin p21 and p27, whereas the ERK1/2 MAPK promotes proliferation [43]. Therefore, low ERK1/2 phosphorylation and high p38 phosphorylation indicate cellular dormancy in many cell types [43; 44]. We show that, in the CT26-WT cells co-cultured with macrophages or with recombinant EMMPRIN, the pERK/pP38 ratio was unchanged relative to the single cultures, whereas the addition of TGF\(\beta\) reduced it, indicating that TGF\(\beta\) alone could increase p38 phosphorylation and potentially enhance dormancy, rather than exit from dormancy, in the CT26-WT cells. This was unexpected, and is only due to the increase in p38 phosphorylation, whereas ERK1/2 phosphorylation was unchanged. Since TGF\(\beta\) can potentially activate both ERKs and p38 MAPK [45; 46], we suggest that the assessment of the cellular dormancy state in these cells should be based on the cumulative results of all the parameters tested. In contrast, when EMMPRIN was knocked-down and its expression was reduced, CT26-KD cells exhibited a reduced pERK/pP38 ratio compared to CT26-WT, and co-culturing, or addition of TGF\(\beta\) or EMMPRIN, increased this ratio, but did not return the CT26-WT levels to baseline  
Figure 2: Co-culturing enhances the secretion of EMT-driver cytokines. CT26-WT or CT26-KD cells (80,000 cells each) were each cultured alone or co-cultured with RAW 264.7 cells that were seeded in the upper chamber of the inserts (0.4 μm pore size) at a ratio of 1:1, in serum-starvation medium (final volume 650 μmL) for 48 h. At the end of the incubation, supernatants were collected and the concentrations of (**A**) TGF\(\beta\) (\(n=5\)), (**B**) soluble EMMPRIN (\(n=6\)), (**C**) TNF\(\alpha\) (\(n=6\)), and (**D**) IL-6 (\(n=6\)) were determined by ELISA. Data are presented as means \(\pm\) SE, and analyzed using a two-way ANOVA followed by [PERSON]’s post-hoc test. (Figure 3A-C). Thus, the CT26-KD cells are more dormant than the CT26-WT cells, and the co-culture or its simulation (addition of TGF\(\beta\) or EMMPRIN) promotes an exit from this dormant state. Reduced E-cadherin and increased vimentin expression are the hallmarks of cells undergoing EMT and dormancy [47]. We demonstrate that neither CT26-WT nor CT6-KD cells express E-cadherin, regardless of the treatment they receive (Figures 3D and S2). This is an indication that the parental CT26 cells have a more mesenchymal phenotype, as also indicated by their elongated morphology (Figure 3D) and their aggressive and proliferative behavior. Quantitation of the fluorescent signal of the vimentin expression shows that its expression was unchanged, either by the co-culture or by the addition of either TGF\(\beta\) or EMMPRIN to the CT26-WT cells, (Figure 3E-G). In contrast, the CT26-KD cells cultured alone show a higher expression of vimentin, which was decreased upon co-culturing with macrophages, addition of TGF\(\beta\), or addition of a limited amount of EMMPRIN (25 ng/mL). Figure 3: CT26-KD cells exhibit reduced levels of ERK activation and enhanced expression of vimentin, which are reversed by the co-culture or its simulation.