Aspirin

B16F10, B16F10-NR, B16F10-R, and B16F10-R-knockout ptgs2 cells (1 × 10⁵ cells) were resuspended in phosphate-buffered saline PBS (Cytiva, Marlborough, MA, USA) and injected subcutaneously into the flanks of 6–8-week-old female human Pdcd1 transgenic mice. Mice were randomised into groups, each comprising 6–8 mice. When tumours grew to 50 mm³, mice were administered pembrolizumab (10 mg/kg; MSD, USA) or the control isotype for ophthalmic intravenous injection twice a week, and aspirin (10 mg/kg; Selleck, Shanghai, China), SC560 (5 mg/kg; Selleck), celecoxib (5 mg/kg; Selleck), and E7046 (10 mg/kg; Selleck) for intraperitoneal injection three times a week. The longest dimension (length) and longest perpendicular dimension (width) were measured every two days using a calliper. Tumour volume (mm³) = (length × width × width)/2. For in vivo B16F10-R cell selection, B16F10 tumours were digested with trypsin and collagenase until reaching 1500 mm³, which was the humane endpoint. CO₂ inhalation was used to euthanise the mice. The cells were resuspended in DMEM and cultured for two weeks. The tumour cells were re-injected subcutaneously into another mouse for subsequent rounds of in vivo selection.

Tissue microarrays were constructed by Shanghai Biochip Co., Ltd. (Shanghai, China). Paraffin-embedded tissue sections (4 μm) were prepared according to established methods^{30 (link)}. Immunohistochemistry, immunofluorescence, and immunoblotting were performed as previously described^{12 (link)}. CCN3, OPN, and TF expression levels were evaluated in tissue microarrays and cell lines. MEK1/2 inhibitor U0126, NFκB inhibitor EVP4593, aspirin, and sorafenib were obtained from Selleckchem (Houston, TX, USA). Primary antibodies included CCN3, E-cadherin, TF, p-C-RAF, MEK, ERK1/2, and p-ERK1/2 (Abcam, Cambridge, MA, USA); OPN, thrombin, and C-RAF (Cell Signaling, Beverly, MA, USA); p-MEK (Epitomics, Burlingame, CA, USA); and Actin (Jackson Labs, Bar Harbor, ME, USA).