S1166

BALB/c or Rag^2–/– mice were inoculated subcutaneously with 5 × 10⁴ CT26 cells. C57BL/6J mice were inoculated subcutaneously with 5 × 10⁴ MC38 cells. Eight days later, mice were randomized into different treatment groups and treated with anti-TIGIT (10 mg/kg; purified in-house from 13G6 cell supernatants), oxaliplatin (1.5 or 6 mg/kg; S1224, Selleck), cisplatin (0.25 mg/kg; S1166, Selleck) or isotype-matched control antibody (10 mg/kg; purified in-house from ratserum) by intraperitoneal injection. Tumors were measured every two days by caliper, and tumor volume was calculated as 0.5 × length × width × width.

Xenograft assays were performed by injecting organoids (huTGO2) subcutaneously in the right flank of NSG mice. Tumor dimensions were measured every 7 days. In mice transplanted with gastric cancer-derived organoids, once tumor volume reached 100 cubic millimeters (within 14 days of transplantation), animals were treated with either vehicle (PBS, i.p.), or GANT61 (50 mg/kg in PBS, i.p. every alternate days), or cisplatin (3 mg/kg in saline, i.p. 2 days/week, Selleckchem, S1166), or cisplatin plus GANT61 for 30 days. Tumor height (h), length (l) and width (w) were measured once/week using a caliper. Tumor volume was calculated using a published equation [38 (link)].

The cisplatin resistance of ESCC cell lines was detected by MTS. The cells were plated in 96-well plates at 7000 cells per well for 12h and then were exposed to the indicated concentrations of cisplatin (S1166, Selleck). Cell viability was detected at the indicated time by adding 20 μL of the MTS reagent per well and the quantity of formazan product was measured by absorbance at 490nm after 2 hours of culture. For signaling pathways analysis, the cells were plated in 12-well plates at 2×10⁵ cells per well for 12h and then were exposed to 4 μM cisplatin or normal medium for 24h. The cell lysates were collected by RIPA lysis buffer (P0110, Maygene) for Western blot.

GATA4 gRNAs were designed, validated, and inserted in pSECC vector (generously provided by F.J. Sanchez-Rivera and T. Jacks, Koch Institute for Integrative Cancer Research at MIT). Lentivirus virus was packaged from pSECC-sgGATA4-infected 293T cells, validated through cell infection and administered nasally into lsl-Kras^G12D mice. After tumor burden was confirmed by MRI imaging 12 weeks after virus infection, mice were treated with vehicle solution, SB525334 (S1476, Selleckchem) 10 mg/kg/day, Trametinib (a gift from Qingsong Liu at High Magnetic Field Laboratory, Chinese Academy of Sciences) 10 mg/kg/day and the combination of two drugs. Lung tumor was documented again 2 weeks after drug dosing. The ratio of tumor size to chest size was calculated to quantify tumor burden. The tumor burden is calculated by area of tumors per area of lung.
For PDX assay, SH3166 or SH3a PDX was embedded to nude mice. Mice were treated with TGFBR inhibitor (SB525334, 10 mg/kg/day), Cisplatin (S1166, Selleckchem) (5 mg/kg, once a week) or combination as the size of xenograft between 100 and 200 mm³. Two weeks after treatment, mice sacrificed. The tumor volume is calculated by the formula: volume = (width)²*length*0.5.

The cell lines were analyzed for apoptosis, senescence and necrosis during treatment with pemetrexed and/or Cisplatin [76 (link)]. These agents was supplied by Selleckchem (Cisplatin: S1166, Pemetrexed: S1135, Houston, USA). Cisplatin was solubilized by Dimethyl sulfoxide (DMSO, Sigma-Aldrich, Missouri, USA) and pemetrexed was solubilized by dH2O.
For the treatment of cells, 5,000 cells (50 μl) were seeded into microplates 96/U (Eppendorf, Hamburg, Germany) that are suitable for luminescence detection. The cells could attach overnight at 37°C and 5% CO2. At the next day, 50 μl of fresh medium either containing one of the treatment agents or without additive was applied to each well. The concentrations of the agents were 0.25 μM for pemetrexed and 10 μM for Cisplatin. Cisplatin was combined with pemetrexed, representing the approved chemotherapeutics for MPM.

HK2 cells were seeded in a 96-well plate with 8000 cells per well and incubated overnight. Then, cells were treated with different doses of cisplatin (S1166, Selleck) or baicalein (S2268, Selleck) as indicated for 24, 48, or 72 h. After that, the medium was removed and replaced by a fresh medium containing CCK8 reagent (K1018, APExBIO, TX, US). The plate was incubated for 1 h and OD450 was measured by microplate reader MultiskanTM FC (Thermo Fisher Scientific).

The indicated cultures were grown in 4 ml SC medium (pH 5.8) for cisplatin exposure or YPD for MMC exposure overnight at 30°C, and subsequently diluted to 0.2 OD₆₀₀ and grown for 2.5 hours until the cultures reached logarithm phase. The cultures were five-fold serially diluted starting at 0.2 OD₆₀₀ and 5 μl were spotted on the indicated plates and grown for 2 days at 30°C before being photographed. Photoshop was used to crop and enhance the contrast of the images, and subsequently converted to black and white images in the Figs 1–8.
Cisplatin (SelleckChem S1166) was dissolved to 100 mg/ml stock solution in dimethyl sulfoxide (DMSO). Synthetic complete (SC) plates containing the indicated doses of cisplatin, were made by diluting the stock solution. Cisplatin was handled in the dark and plates were stored in the dark and used within 24 hours.
MMC (SelleckChem S8146) was dissolved to 50 mg/ml stock solution in dimethyl sulfoxide (DMSO). YPD plates containing the indicated doses of MMC, were made by diluting the stock solution. MMC was handled in the dark and plates were stored in the dark and used within 24 hours.