Figure 4 from Exploration of Immune-Modulatory Effects of Amivantamab in Combination with Pembrolizumab in Lung and Head and Neck Squamous Cell Carcinoma

Single-cell RNA sequencing analysis of EGFRHIGH/METHIGH and EGFRLOW/METLOW subclusters in the tumor of humanized HNSCC (YHIM-3003) PDX mice showing relative increase in EGFR and MET expressing subcluster after treatment of pembrolizumab (n = 5 in each treatment group). Top 50 genes were analyzed by log2 fold change (FC) against P-values and immune related genes were divided into three categories as following: immunomodulation, metastasis potential and cancer progression (drug resistance/cancer stemness). A, Heatmap of tumor indicating a region of tumor subcluster with elevated expression of both EGFR and MET. B, Density plot illustrating EGFRHIGH/METHIGH and EGFRLOW/METLOW subclusters in treatment groups. Expression of EGFR and MET was relatively higher in the pembrolizumab treated group compared to the other treatment groups (C). D, Tumor subcluster with elevated dual expression of EGFR and MET (EMHIGH) was define and analyzed for DEGs. E, EGFR and MET in EMHIGH and EMLOW, showing increased expression of both markers in EMHIGH tumor cluster. F, DEG analysis of the top genes in the EGFRHIGH/METHIGH tumor subcluster showing genes related to immunomodulation, tumor metastasis, drug resistance and cancer stemness in volcano plot. G, Multiplex IHC of the tumor tissue showing reduced expression of EGFR in tumor treated with combination treatment. EGFR+ site, colored green, represents tumor regions that were stained positive for EGFR and does not directly translate to the level of expression. Tumor with damaged or indistinct regions of tumor nest was treated as an outlier and was removed from each group. H, Fluorescence intensity of EGFR in randomized region of interest sites converted into average H-scores. H-scores ranged from 0 (H-0, blue) to 3 (H-3, red), with H-3 representing the highest intensity as shown in the top. Average H-score of EGFR expression in pembrolizumab treated mice was significantly higher than the other groups (P