Fig 1: CEAACM6-mAbIRDye800CW probe labeled the gastric mucosa with dysplasia. (A) The white light endoscopy of the lesion in upper gastric body (left panel), the ESD was carried out and the specimen was got, the specimen was fixed on the board with pin on the edges, and flushed with running tap water to remove the mucus on the surface, then the CEAACM6-mAb-IRDye800CW probe was sprayed on the surface of the specimen evenly and incubated, then the detector of the wide field fluorescent endoscopy was used to check the fluorescent intensity, the mucosa binding the probe showed the green fluorescent signals, the intensity of the signals indicated how much the probe be binded. The depressed gastric mucosa at the center of the specimen showed the strongest green signals and was signed in the white box. (B) After fluorescent probe study, the specimen was fixed with formalin and the HE staining were investigated (upper panel), the depression was signed with red box (left panel) and was amplified (right panel), the glands were irregular and the nucleus were large and hyperchromatic. IHC staining of CEACAM6 in the specimen of ESD operation (down panel). The depressed gastric mucosa was showed in the red box (left panel) and was amplified (right panle), the positive of CEACAM6 expression was showed in the brown. Scale bars in left panel = 500 µm, Scale bars in right panel = 250 µm. (C) The mean fluorescence intensity in the gastric mucosa with different lesions was quantified by Image-Pro Plus software (fold change from normal), Data are expressed as the means ± SD, the normal mucosa=5, mucosa with mild dysplasia=5, mucosa with moderate dysplasia=5, mucosa with severe dysplasia =5 and mucosa with cancer=5, m-s dys: moderate or severe dys. ***P < 0.001. (D) The correlation between fluorescence intensity and CEACAM6 IHC staining index was studied by Pearson correlation coefficients. (E) The micro structure of gastric mucosa surface was observed by the fluorescent micro endoscopy detector. The surface micro structure was showed in upper panel and the pattern diagrams were drawn (low panel).
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 mRNA expression in the gastric mucosa from normal to cancer. (A) Searched the CEACAM6 mRNA expression in the normal and stomach adenocarcinoma tissues in TCGA dataset through UALCAN (http://ualcan.path.uab.edu) website, normal (n=34), primary tumor (n=415). (B) Searched the CEACAM6 mRNA expression in the stomach adenocarcinoma tissues with different clinic stage in TCGA dataset, normal (n=34), stage 1 (n=34), stage 2 (n=123), stage 3 (n=169), stage 4 (n=41). The volcano map showed the deregulated mRNAs in CAG+IM (C), Dys (D) and Cancer (E), the red triangles represented up regulated mRNAs and the green triangles represented down regulated mRNAs (P<0.05), the deregulated mRNAs had no significant differences were showed in the black triangles, the up or down regulated CEA family members were verified by RT-qPCR and labeled in the map. The histograms in the volcano map showed the verification of CEACAM6 mRNA expression. *P < 0.05, **P < 0.01, ***P < 0.001, NS, no significance. CSG, Chronic Superficial Gastritis; CAG+IM, Chronic Atrophic Gastritis (CAG) + Intestinal Metaplasia (IM); Dys, Dysplasia; Cancer, Gastric Cancer.
Fig 4: The affinity of CEACAM6-mAb-Alexa Fluo488 probe to GC cells and the CEAACM6-mAb-IRDye800CW probe tracked the tumor in mice model. (A) The AGS, MKN-45 and GES-1 cells were cultured and incubated with CEACAM6-mAb-Alexa Fluo488 probe (1:20, green signal), the nucleus were stained with DAPI (blue signal), 10 randomly selected fields were checked under confocal microscope. (B) Quantification of the mean fluorescence intensity in the cells by Image-Pro Plus software (fold change from GES-1), and the bar graph was drawn, data are expressed as the mean (± SEM) of values from three independent experiments. ***p < 0.001. (C) The chemicalmolecular modeldiagram of CEACAM6-mAb-Alexa Fluor488 was showed. (D) The chemicalmolecular model diagram of CEACAM6-mAb-IRDye800 was showed. (E) The activity of CEAACM6-mAb-IRDye800CW was evaluated by ELISA method, the optical intensity was measured by spectrophotometer (OD450nm). The Y-axis respresented the OD value, the X-axis represented the different concentration of antibody, the fitting curve was generated based on the OD value and the IC50 was calculated according to the fitting curve by GraphPad Prism 8.0.2. (F, G) The surface plasmon resonance (SPR) analysis was used to test affinity of CEACAM6 mAb and CEACAM6 mAb-IRDye800CW to CEACAM6 respectively, Y-axis respresented the fluorescence signal value, the X-axis represented the times, the different diluted concentration of antibody were showed in the different colors.
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.
Supplier Page from Sino Biological, Inc. for Human CEACAM6 / CD66c Protein (His Tag)