Fig. 7

Clinical Validation of the Correlation between cg09897064 Methylation, ZBP1 Expression, and Macrophage M2 Polarization. Twenty-one patients with lung adenocarcinoma were enrolled to assess the correlation between cg09897064 methylation, ZBP1 expression, and macrophage M2 polarization using Methylation-specific PCR (MSP), Western blot, and flow cytometry analysis. A/B. MSP was performed to detect cg09897064 site methylation in the patients' adenocarcinoma pathological tissues, while Western blot was utilized to measure ZBP1 protein expression. The results showed that 47.6% (10/21) of the adenocarcinoma patients' pathological tissues exhibited methylation at the cg09897064 site. Furthermore, Western blot analysis revealed significantly reduced expression of ZBP1 protein in patients with methylation at the cg09897064 site (P < 0.001). C/D. Flow cytometry analysis was conducted to evaluate the proportion of CD68⁺CD206⁺ cells (M2-polarized macrophages) in single-cell suspensions derived from the adenocarcinoma tissues. The findings demonstrated a significant increase in the proportion of M2 macrophages in the adenocarcinoma tissues of patients with cg09897064 site methylation compared to non-methylated patients (P < 0.001)