Fig 2: The CEACAM6 expression in the cancer tissues and dysplastic gastric mucosa. (A) Immunohistochemical staining (IHC) was performed to examine the expression of CEACAM6 in tumor and tumor-adjacent tissues using tissue chips. The tissues expressed CEACAM6 were stained in brown. Scale bars = 500 μm. (B) The histograms showed the quantification of IHC staining. Bar represents the Mean (± SEM) of staining H-Score (n=89). ***P<0.001. (C) The survival curve was drawn according to the CEACAM6 expression data got from the IHC analysis above. (D) HE staining was performed on the ESD specimen, the normal mucosa had the regular glands (green box), the mucosa with dysplastic changes had the disordered arrangement of glands and large hyperchromatic nuclei (red box), Scale bars = 2.5 mm. The fields in the green or red boxes were magnified and shown on the upper panel, Scale bars = 250 μm. (E) IHC was performed to examine the expression of CEACAM6 in the dysplastic gastric mucosa got from ESD operation, the tissues expressed CEACAM6 were stained in brown, Scale bars = 2.5 mm, and the fields in the red or green box were magnified and shown on the upper panel, Scale bars = 250 μm. (F) The histograms showed the quantification of IHC staining, the data are expressed as the mean (± SEM) of staining H-Score (n = 15, mild dysplasia=5, moderate dysplasia=5, severe dysplasia=5). ***P<0.001. All immunohistochemical sections were scanned by 3D HISTECH (pannoramic MIDI) and 10 randomly selected fields were checked under pannoramic viewer, the percentage of positive cells was analyzed by the densito quant software and the staining H-Score was calculated.

Fig 3: CEACAM6 promoted the GC cells invasion, proliferation and metastasis. (A) AGS and MKN-45 cells were infected with CEACAM6 lentivirus (Lenti-CEACAM6) or negative control lentivirus (Lenti-NC), the cells infected with lentivirus were showed in green color when checked with fluorescent microscopy, and the infection efficiency was also checked with western-blot. (B) After 48 h of infection, 2.5x104 cells were trypsinized and seeded in 24-well plates with matrigel-coated membranes for the invasion assays. The upper panel represented the AGS cells and the down panel represented the MKN-45 cells. The number of cells in 10 randomly selected fields was counted at 48 h after incubation. Data represents the mean value of three independent experiments. Bars represent the mean (± SEM) number of invaded cells per field. **P < 0.01, ***P < 0.001. (C) 3x104 cells/ml were seeded in 96-well plates and grown for 24 h and then infected with Lenti-CEACAM6 and Lenti-NC for 48 h. Cells were stained with EdU-Apollo 567 and DAPI; DAPI blue fluorescence represents all live cells and the EdU-Apollo567 red fluorescence represents the proliferating cells. The number of cells in 10 randomly selected fields were counted. The proliferation ratio was calculated as number of proliferating cells/total number of cells x 100%. Mean (± SEM) values of three independent experiments are presented.*p<0.05, ***p<0.001. (D) Western-blot assays were performed to detect the expression of PI3K, p-PI3K Akt, p-Akt, Src, p-Src and MMP9. ß-Tubulin was used as the internal control for total proteins, while total protein was used as the internal control for the phosphorylated protein. Mean (± SEM) values of three independent experiments are presented, *p<0.05, **P<0.01, ***p<0.001. Control: Src inhibitor control, Lentivirus: CEACAM6 over expression lentivirus (Lenti-CEACAM6), Srci:Src inhibitor. (E) The possible pattern graph of CEACAM6 to Src/PI3K/Akt signaling pathway.

Fig 4: (A) The MKN−45 cells infected with Lenti-CEACAM6 or CEACAM6 RNAi were used to derive the subcutaneous tumor mice model, for the in vivo imaging study, the CEAACM6-mAb-IRDye800CW probe with the concentration of 2.5, 5 and 10 mg/kg was injected in the caudal vein of the mice respectively, the fluorescence intensity were observed at 2 h until 7 days, and the tissues which were expressed the CEACAM6 highly were labeled by the probe and showed the red signals. (B) After imaging, the mice were euthanasia the tumor, heart, liver, spleen, lung, kidney, stomach and muscle were removed and imaged, the tissues which were expressed the CEACAM6 highly were labeled by the probe and showed the red signals. (C) The surface change of the liver got above, the HE staining was applied to study the histology of liver, the metastases was showed in the white box, and the cancer cells were indicated with white arrows.

Fig 5: The effect of CEACAM6 on cell derived tumorigenesis. (A) Approximately 4 × 107 MKN-45 cells infected with Lenti-CEACAM6 or Lenti-NC were inoculated subcutaneously into the mice, the MKN-45 cell derived subcutaneous tumors in a xenograft mouse model was constructed. (B) 30 days after the inoculation, the width and length of tumor were measured and the volume was calculated in vivo every 5 days till day 50. (C) At the day 50, the mice were euthanasia, the subcutaneous tumors were harvested for IHC staining of p−Akt, p−PI3K p−Src, and MMP9. The images in the left panel were magnified 100 folds (x10), and images in the right panel are a magnification of the indicated black boxes, at 400 folds magnification (x40), the positive staining was showed in the brown. All IHC sections were scanned by 3D HISTECH (pannoramic MIDI) and 10 randomly selected fields were checked under pannoramic viewer, the percentage of positive cells was analyzed by the densito quant software and the staining H-Score was calculated and showed with histograms (right panel). The data are expressed as the mean (± SEM) of staining H-Score from three independent experiments.