## 3 Results
### Reduced EMMPRIN Expression Inhibits Proliferation  
The most obvious manifestation of cellular dormancy is the rate of cell proliferation. To assess the proliferation of the CT26 tumor cells, we examined their viability through the CCK8 kit, that measures mitochondrial activity in viable cells, as well as the gene expression  
Figure 4: Expression of the EMT-TFs and dormancy markers is enhanced in the CT26-KD cells, but reduced in the co-culture or its simulation. CT26-WT or CT26-KD cells (8 \(\times\) 10\({}^{4}\) cells each) were incubated alone or in co-culture with RAW 264.7 cells as described before for 48 h. Alternatively, single cultures of CT26-WT or CT26-KD cells (8 \(\times\) 10\({}^{4}\) cells) were cultured with or without the addition of recombinant TGF\(\beta\) (10 ng/mL) or recombinant EMMPRIN (25 ng/mL or 250 ng/mL). Total RNA was extracted from the CT26 cells, cDNA was prepared, and the genes for the dormancy markers _NR2F1_ and _p21_ or the EMT-TFs _Slug_ and _Zeb1_ were amplified by qPCR as described in the methods. (**A–D**) cells incubated in co-cultures (_n_ = 5–6), (**E–H**) single cultures incubated with the addition of TGF\(\beta\) (10 ng/mL) (_n_ = 5–6), and (**I–L**) cells incubated with the addition of recombinant EMMPRIN (25 and 250 ng/mL) (_n_ = 5). Data are presented as means \(\pm\) SE, and analyzed using a two-way ANOVA followed by [PERSON]’s post-hoc test. of _cyclin D1_ or _Ki67_, which are associated with the cell cycle and cellular proliferation. CCK8 viability of the CT26-WT cells was not changed after co-culturing (Figure 5A), whereas the addition of TGF\(\beta\) (Figure 5D) or low levels of EMMPRIN (Figure 5G) increased it. The expression of the proliferation markers _cyclin D1_ and _Ki67_ was not changed in the co-cultured CT26-WT cells relative to CT26-WT cells cultured alone (Figure 5B,C) or when TGF\(\beta\) was added (Figure 5E,F). Interestingly, the addition of EMMPRIN enhanced the viability of the CT26-WT cells in a hormetic manner, peaking at 25 ng/mL, and then decreasing thereafter (Figure 5G), creating a bell-shaped curve. This hormetic response was also detected when incorporation of BrdU (Figure 5H) or the expression of cyclin D1 (Figure 5I) were used to detect proliferation. In contrast to the CT26-WT cells, the viability of the CT26-KD cells was reduced relative to the CT26-WT cells cultured alone, and co-culturing them with macrophages or adding TGF\(\beta\) increased it (Figure 5A,D) to the level observed when CT26-WT cells were cultured alone. Likewise, the expression levels of cyclin D1 and Ki67 were reduced in CT6-KD cells relative to the CT26-WT controls, and co-culturing or the addition of TGF\(\beta\) increased them (Figure 5B,C,E,F). However, addition of EMMPRIN at any concentration did not change the viability (Figure 5G), ability to incorporate BrdU (Figure 5H), or expression of _cyclin D1_ (Figure 5I) of the CT26-KD cells. Thus, TGF\(\beta\) and EMMPRIN can each enhance CT26-WT cell proliferation, whereas the co-culture probably delivers additional signals that mask their effect. In contrast, CT26-KD cells proliferate more slowly, and the macrophages or TGF\(\beta\), but not EMMPRIN, can increase the proliferation. Figure 5: CT26-KD exhibits reduced proliferation. CT26-WT or CT26-KD cells (\(8\times 10^{4}\) cells) were incubated under the same conditions as described in Figure 4. Cell proliferation was measured using (**A,D,G**) the CCK8 kit (\(n=8\)) or (**H**) the BrdU kit (\(n=7\)) as well as the expression of (**B,E,I**) the cyclin D1 mRNA (\(n=5\)) or (**C,F**), and the Ki67 mRNA (\(n=4\)). (**A–C**) Cells incubated in co-cultures, (**D,F**) single cultures incubated with the addition of TGF\(\beta\) (10 ng/mL), and (**G–I**) cells incubated with the addition of recombinant EMMPRIN (25 and 250 ng/mL). Data are presented as means \(\pm\) SE, and analyzed using a two-way ANOVA followed by [PERSON]’s post-hoc test. ***, \(p<0.001\) relative to the CT26-WT without addition of rec. EMMPRIN; §SS5, \(p<0.001\) relative to CT26-WT with 25 ng/ml rec.