Female nod scid mice

Female NOD/SCID mice, aged 4 weeks and weighing between 16 and 18 grams, were procured from GemPharmatech Co., Ltd. and maintained under specific pathogen-free conditions. For each group, A431 cells, sh-normal control (NC) A431 cells, TGM3-shA A431 cells, and TGM3-shC A431 cells were injected subcutaneously into the left armpit of NOD/SCID mice (1 × 10⁷ cells in 500 μL of PBS). Once the tumor xenografts became apparent, the minimum width (W) and maximum length (L) of tumor size were measured every 6 days using a vernier caliper. Tumor volumes (V) were calculated using the formula V = LW²/2. Mice were sacrificed 50 days after tumor injection, and tumor weights were determined. The tumors were stored in liquid nitrogen or embedded in paraffin for hematoxylin-eosin (H&E) and IHC staining.

All animal experiments were approved by the Animal Protection and Use Committee of Nanjing Medical University (no. IACUC‐2105048). Female NOD/SCID mice at 6 weeks of age (Gem Pharmatech, China) were used to explore the effect of CSE‐induced macrophages on subcutaneous tumorigenesis of NSCLC cells. Female Balb/c nude mice at 4 weeks of age were obtained from the Experimental Animal Center of Nanjing Medical University, housed under specific pathogen‐free conditions, and provided with standard food. A549 cells (5 × 10⁶) were subcutaneously transplanted into nude mice. Tumor size was assessed every 7 days. EVs (10 µg) were injected subcutaneously into mice every 3 days. The mice were sacrificed, tumors were removed, and their weights were recorded in the fourth week. In addition, the nude mice were injected with A549 cells (2 × 10⁶) through the tail vein, and EVs (10 µg) were injected into mice via the tail vein every 3 days. The bilateral lung tissues were resected and stained with hematoxylin‐eosin.