Mitomycin c

Transwell assays were performed by using transwell chambers (8 μm pore size, Corning) to examine the migration and invasion of ESCC cells as previously described (30 (link)). Briefly, tumor cells were pretreated with 10 μg/mL mitomycin C (Selleck, Shanghai, China) for 2 h. For the migration experiment, Approximately 5 × 10⁴ cells were placed into the upper chamber with 200 μl serum-free DMEM medium, The lower compartment filled with DMEM medium supplement with 20% FBS was used as the chemoattractant. For the invasion assay, the upper surface of chamber membrane were coated with 50 μL Matrigel (Cat. #354234, BD Biosciences, CA) to form a matrix barrier, and then 1 × 10⁵ tumor cells were added to the upper chamber. After incubation for 24 h in a humidified incubator containing 5% CO₂ at 37°C, the cells on the lower surface of the chamber membrane were fixed with 4.0% paraformaldehyde prior to 0.1% crystal violet staining. Pictures were then captured under a light microscope at 10 × and the mean number of migrated or invaded cells were counted in five random fields.

The human liver cancer cell line HCCLM3 was obtained from the Liver Cancer Institute, Zhongshan Hospital, Fudan University (Shanghai, China). The human liver cancer cell line Hep3B and the human embryonic kidney cell line HEK293T were obtained from American Type Culture Collection (ATCC). All cells were cultured in Dulbecco's Modified Eagle's medium (DMEM) (Corning) supplemented with 10% fetal bovine serum (FBS) (VISTECH), 100 U/ml of penicillin and 100 µg/ml of streptomycin (P/S) (Hyclone) in a humidified incubator at 37°C with 5% CO₂. Commercially available antibodies used in this study included anti-Rictor (Bethyl, A500-002A), anti-ABLIM1 (Abcam, ab222824 and Proteintech, 15129-1-AP), anti-MKL1 (Proteintech, 21166-1-AP), anti-phosphoserine (Abcam, ab9332), anti-phospho-AKT (Ser473) (Cell Signaling Technology, 4060T), anti-AKT (Cell Signaling Technology, 4685S), anti-Flag (Sigma, F3165-2MG), anti-β-Actin (Santa Cruz, sc-69879) and anti-GAPDH (Santa Cruz, sc-47724), anti-c-Fos (Cell Signaling Technology, 2250S), anti-Arp3 (Proteintech, 13822-1-AP). Mitomycin C was from Selleck (S8146).

Mixed lymphocyte reaction (MLR) was utilized to determine T cell activation induced by HS636 and BR102. For HS636, monocytes were isolated from PBMCs using CD14 microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany) and grown in Mo-DC differentiation medium (Miltenyi Biotec) and Mo-DC maturation medium (Miltenyi Biotec) to generate immature dendritic cells (DCs). Human CD4 + T cells isolated from a different PBMC donor were co-cultured with the DCs in a 96-well plate, and indicated antibodies were added. Alternatively, DCs were stimulated with Mitomycin C (50 μg/mL, Selleck, Houston, TX, USA) at 37 °C for 30 min. Subsequently, DCs (5 × 10³ cells) were co-cultured with allogeneic PBMCs (1 × 10⁵) in the presence of BR102 or HS636. After 3 days of culture, IFN-γ or IL-2 secretion in cell supernatants were analyzed by ELISA.

Transwell migration and invasion assays were undertaken to evaluate cell migration and invasion, respectively. Briefly, 3 × 10⁴ cells resuspended in serum-free medium added with 5 µg/ml mitomycin C (Selleck, Houston, TX, USA) to block cell proliferation were placed into the upper chamber of transwell filter inserts (8-μm pore size, Corning, NY, USA) with or without precoated Matrigel (Corning). Complete medium added with 10% FBS was loaded into the lower chambers. After culture for another 36 h, the cells remaining on the upper chamber were removed. The cells migrating or invading into the bottom side of the inserts were fixed, stained, and quantified under a microscope by counting at least five random high-power fields.

Dendritic cells (AllCells) were treated with 50 µg/mL Mitomycin C (Selleck) at 37°C for 30 min and then plated onto a 96-well plate at 5×10³ cells per well. Allogeneic PBMCs (AllCells) from different donors were added at a dendritic cell:PBMCs ratio of 1:20. Indicated mAbs were added at a concentration of 10 µg/mL immediately, and the cells were incubated at 37°C in 5% CO₂ for 5 days. Interferon (IFN)γ secretion were evaluated in the supernatant by ELISA (R&D Systems).