Fig 2: Spontaneous OH-1 metastases express CEA.Primary OH-1 tumor as well as spontaneous OH-1 lung and bone metastases were positively stained for CD44, CEA, EpCAM and PGP9.5 (red = positive cells). Metastases are indicated by red asterisks. Scale bar: 50 µm for all panels.

Fig 3: ELK1 promotes CRC progression through increasing CEA expression. A, Knockdown of CEA in LS174T cells decreases cell adhesion, indicated by the reduced numbers of crystal violet‐stained cells. Left: Representative images for crystal violet staining for 15 min. Right: Statistical analysis on 6 repeats. B, Migratory ability of LS174T cells is inhibited when CEA is knocked down. Left: Recorded image by RTCA for a 80 h period. Right: Statistical analysis on 4 repeats. C, The invasive ability is equally well inhibited in CEA knocked‐down LS174T cells. Left: Recorded image by RTCA for 80 h. Right: Satistical analysis on 4 repeats. D, The number of adhesive cells stained by crystal violet is decreased in ELK1 overexpressed Caco‐2 cells (Caco‐2ELK1) upon introduction of shCEA. Left: Representative images for crystal violet staining for 15 min. Right: Statistical analysis on 6 repeats. E, F, CEA knocked‐down Caco‐2ELK1 cells display impaired migratory (E) and invasive (F) abilities compared with Caco‐2ELK1 cells. Left: Recorded image by RTCA for 70 h. Right: Statistical analysis on 4 repeats. *P < .05, **P < .01, ***P < .001

Fig 4: Inoculation of human cancer cells into immunodeficient mice and in vivo macroscopic imaging using a fluorescence zoom microscope. (a) Schema of the sites of inoculation of human cancer cells. The cells were inoculated s.c. into the back skin of nude mice at the rostral–ventral site (HT1080-GFP cells), the caudal–ventral site (HT1080-GFP-CEA cells) or the dorsal site (MKN45-GFP cells). (b) Schema of preparation of skin flaps. Seven or eight days after the inoculation, the inoculation sites were exposed by the skin-flap method. (c–e) In vivo macro imaging of tumors. In vivo macro imaging of the tumor masses was performed using a fluorescence zoom microscope 24 h after injection of Alexa Fluor 594-conjugated anti-CEA antibody (50 µg/mouse). Exposure times for the GFP and Alexa Fluor 594 fluorescence images were 30 and 100 ms, respectively. These experiments were repeated three times and similar results were obtained.

Fig 5: C-KIT-ERK1/2 signaling activates ELK1 and upregulates CEA expression in CRC. A, c-KIT mRNA levels from 30 patients diagnosed with CRC at the Beijing Friendship Hospital, Capital Medical University were increased compared with the corresponding adjacent normal tissues. B, c-KIT protein is also clearly increased in the tissue samples from 30 patients with CRC compared with adjacent normal tissues. Left: Representative western blot bands for c-KIT with ß-actin as control. Right: Statistical analysis on the 30 samples. C, c-KIT is overexpressed in LS174T cells following lentiviral mediation. Upon additional treatment with 50 ng/mL of rhSCF for 15 min, c-KIT and its downstream ELK1/2 are hyperactivated in c-KIT overexpressed LS174T cells, with activation of ELK1 and increased expression of CEA. Left: Representative western blot bands with ß-actin as control. Right: Statistical analysis on 3 repeats. D, c-KIT overexpressed LS174T cells (LS174TKIT) are treated with 10 µmol/L U0126, 2 µmol/L Imatinib or 30 µmol/L ISCK03 for 2.5 h and 50 ng/mL of rhSCF for another 15 min. Compared with the DMSO control group, cells in the U0126 groups have decreased p-ERK1/2, p-ELK1, and CEA expression levels. In addition, both Imatinib and ISCK03 could significantly inhibit c-KIT phosphorylation which inhibits downstream p-ERK1/2 and p-ELK1 as well as CEA expression. Left: Representative western blot bands with ß-actin as control. Right: Statistical analysis on 3 repeats. E, c-kit loss-of-functional Wadsm/m mice are used to further verify the role of c-KIT signaling in ELK1 expression in vivo. Compared with their WT littermates, the Wadsm/m mice had decreased c-KIT, ERK1/2 and ELK1 activities. Left: Representative western blot bands with ß-actin as control. Right: Statistical analysis on 5 mice. *P < .05, **P < .01, ***P < .001