Ecm gel e1270

The 20(S)-Rg3 was provided by the Yiyan Biological Technology Company (Shanghai, China). Rg3 was dissolved in dimethyl sulfoxide (DMSO) in a 200mg/ml stock solution and stored at -20°C and diluted with fresh culture medium before use. An equal volume of DMSO (final concentration <0.1%) was added to the controls. The recombinant human TGF-β was purchased from PeproTech (Rocky Hill, NJ, USA). Primary monoclonal antibodies including rabbit anti-human MMP-2, MMP-9, E-cadherin, N-cadherin, Vimentin, Snail, Slug, Twist, Zinc Finger E-Box Binding Homeobox 1 (ZEB1), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). The horseradish peroxidase-conjugated secondary antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The ECM gel (E1270) was obtained from Sigma (St. Louis, MO, USA).

All animals were housed under pathogen-free conditions, and all animal procedures were approved by Ariel University Institutional Animal Care and Use Committee. Cell line suspensions were prepared in 1:1 matrigel (ECM Gel #E1270, Sigma-Aldrich) and approximately 1.5 × 10⁶ cells were subcutaneously injected into the flanks of 9-week-old female nude (Nu/Nu) mice (Envigo, Ness Ziona, Israel). Tumors were measured with calipers and the tumor volume was calculated according to the formula Vol = 0.52 × L × W2. When tumors reached the volume of approximately 200–300 mm³, mice were randomly assigned into either treatment group or control group. FX9847 (100 mg/kg) was administered intraperitoneally once daily and osimertinib (2.5 mg/kg) was administered by oral gavage once daily. Tumor volumes were measured, and body weights were monitored twice weekly for the entire span of the experiment.