Co-targeting TMEM16A with a novel monoclonal antibody and EGFR with Cetuximab inhibits the growth and metastasis of esophageal squamous cell carcinoma

Fig. 1. TMEM16A promotes ESCC cell motility and lymph node metastasis. A TMEM16A was more highly expressed in ESCC cells with high metastatic ability. Western blot (left) and grayscale scanning analysis (right) of TMEM16A level in ESCC cell lines. GAPDH was used as a loading control. B, C TMEM16A promotes ESCC cell migration and invasion. B Transwell chamber assays and Matrigel invasion assays for KYSE30lm3 and KYSE450lm2 cells after TMEM16A knockdown. C Transwell chamber assays and Matrigel invasion assays for KYSE450luc and KYSE410 cells overexpressing TMEM16A. D Fluorescence immunohistochemistry staining showed that the level of TMEM16A in ESCC tissues was significantly correlated with the degree of lymph node metastasis. The right figure shows the statistical graph of TMEM16A level and the number of lymph node metastasis in patients. The fluorescence values (MFI) of all samples were sorted numerically from smallest to largest based on the relative values of fluorescence intensity. High/low TMEM16A expression was distinguished according to MFI quartiles. Lower quartile Q1 was 31.145, denoted as " + ". Median Q2 was 38.506, and upper quartile Q3 was 43.821, denoted as " + + ". Samples…

Fig. 2. Inhibition of cell migration, invasion and chloride channel activity by TMEM16A-specific antibody mT16A#5. A Transwell chamber assays were employed to compared the ability of 6 anti-TMEM16A monoclonal antibodies to inhibit migration of ESCC cell lines. B MQAE probe fluorescence quenching assay were compared the inhibitory ability of 6 anti-TMEM16A monoclonal antibodies on chloride channel activity of ESCC cell lines. C Transwell chamber assays and Matrigel invasion assay demonstrated the mT16A#5 inhibited the cell motility in a concentration-dependent manner. D MQAE probe detected significant up-regulation of chloride channel activity with KYSE30luc cells overexpressing TMEM16A. E MQAE probe detected the concentration-dependent inhibitory effect of mT16A#5 on chloride channel activity in KYSE30lm3 and KYSE450lm2 cells. F The halide-sensitive cytoplasmic fluorescent sensor EYFP-H148Q/I152L detected the inhibitory effect of TMEM16A inhibitors CaCCinh-A01 and T16Ainh-A01 on cellular chloride ion activity at a concentration of 10 nM. G EYFP-H148Q/I152L system detected the concentration-dependent inhibitory effect of mT16A#5 on cellular chloride channel activity of KYSE30lm3 and…

Fig. 3. Mutual regulation between TMEM16A and EGFR. A, B Detection of TMEM16A and EGFR levels in tumors and adjacent normal tissues of 98 ESCC patients. A mIHC staining of tissues from patients with ESCC patients with fluorescent dye coupled to excitation light of 520 nm for the EGFR antibody, fluorescent dye coupled to excitation light of 650 nm for the TMEM16A antibody, and DAPI was used for staining nuclei. B The expression of TMEM16A and EGFR in ESCC tissues was positively correlated with each other. The staining results were statistically analyzed, and the spectral splitting and fluorescence semi-quantification were carried out after removing the background value of autofluorescence in the tissues. C Pull-down assay detected the interaction of TMEM16A with EGFR extracellular segment protein (aa 668–1210) in 293 T cells overexpressing TMEM16A. EGFR extracellular segment was used as Input. D Knockdown of TMEM16A down-regulated EGFR protein levels. E The inhibitory effect of mT16A#5 on migration and invasion of KYSE30lm3 cells was dependent on EGFR expression. F MQAE system assay showed that knockdown of EGFR was able to down-regulate the chloride channel activity of KYSE30lm3 cells.…

Fig. 4. Synergistic effects of mT16A#5 and Cetuximab on the inhibition of ESCC metastasis and growth. A, B Combination of mT16A#5 and Cetuximab inhibited migration and invasion of KYSE30lm3 cells. A Transwell chamber assays. B Matrigel invasion assays. C–E mT16A#5 and Cetuximab have synergistic effect on inhibiting footpad metastasis of ESCC in mice. C KYSE30lm3 cells were injected subcutaneously into the footpad of the right hindfoot of mice, and whole-body imaging was performed on each group of mice after four weeks of consecutive treatment to observe the metastasis of the footpad tumor. D Statistical graph of fluorescence intensity of each mouse foot. E Statistical graph of body weight of mice in each group at the end of treatment. F–H The mT16A#5 and Cetuximab had a synergistic effect on the inhibition of subcutaneous ESCC tumor growth in mice. F KYSE30lm3 cells were injected into the subcutaneous part of the right coeliac region of mice. The mice were executed after four weeks of treatment, and the tumors were stripped and observed for growth. G Statistical graph of subcutaneous tumor mass of mice in each group at the end of treatment. H Statistical graph of body weight of mice in…

Fig. 5. mT16A#5 inhibits phosphorylation of PI3K-AKT/JNK signaling pathway. A Western Blot detected the effect of antibody mT16A#5 on the levels of EGFR and its phosphorylated protein level in ESCC cells. B Western Blot detected the effect of antibody mT16A#5 on the total and phosphorylation protein level of PI3K-AKT/JNK signaling pathway related proteins in ESCC cells. C Western Blot detected the effect of antibody mT16A#5 on the LC3B and BCL-2 protein in KYSE30lm3