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. Here, CA9 is linked to head and neck squamous cell carcinoma.