Chrysin Modulates Tumor-Associated Macrophages in Xenograft Model

All procedures involving mice were approved by the Animal Protection and Use Committee of Ruige Biotechnology (protocol code 20230615-27). Four-week-old male BALB/c-nu/nu mice were purchased from the Guangdong Medical Experimental Animal Center (Guangzhou, China) for the establishment of the xenograft model. TAMs were coinjected with A549 cancer cells (3 × 10⁶) in a 1:3 ratio subcutaneously into the right dorsal side of the mice. The mice were randomly divided into Ctrl group, TAMs group, and TAMs + Chrysin group, with six mice in each group. Chrysin (Meilunbio, Dalian, China) was orally administered to the mice at a dose of 30 mg/kg daily [51 (link)]. The mice were observed for growth status every other day, and body weight and tumor size (calculated using the formula: 0.52 × length × width²) were measured and recorded. After three weeks of feeding, according to humane euthanasia principles, the mice were euthanized, and tissues were collected. The collected tumor tissues were fixed in 4% paraformaldehyde for 12 h, dehydrated, embedded in paraffin, and then cut into 4 μm sections for immunohistochemistry (IHC) and hematoxylin and eosin (H&E) staining experiments. Following animal welfare principles, the experiment was terminated immediately if the animals lost 20% of their body weight, developed signs of cachexia, or if tumor size exceeded 2000 mm³.