Moreover, key regulators of hypoxia showed the same trend in the EGFRHIGH/METHIGH subcluster and the treatment groups (HIF1A, HDAC1, KDM1A, KDM2A, CA9, VEGFA, and TWIST1; Fig. 5D), suggesting that the tumor in the HNSCC PDX mice treated with pembrolizumab feasibly had an acidic environment by favoring anaerobic metabolism of glucose in the TME. This evidence concerns the gene TWIST1 and head and neck squamous cell carcinoma.