Figure 4 from Anti-HDGF Antibody Targets EGFR Tyrosine Kinase Inhibitor–Tolerant Cells in NSCLC Patient-Derived Xenografts

Effect of anti-HDGF antibody on EGFR signaling in osimertinib-treated TM00219 PDX tumor. Mice with established TM00219 PDX tumors were randomized into two arms (n = 6 each) to receive osimertinib or osimertinib plus anti-HDGF antibody H3. After 10 days of treatment, tumors were collected from euthanized animals and split for formalin-fixing and paraffin-embedding and Western blot analysis. A–D, IHC staining of tumor formalin-fixed, paraffin-embedded sections using (A) P-Erk1/2 (T202/Y204), (B) P-Akt1 (S473), (C) P-PRAS40 (T246), and (D) P-4EBP1 (T37/46). Representative fields of stained tumor at 4× view on OlyVIA (P-ERK and P-PRAS40) or 8× view on ImageScope (P-Akt and P-4EBP1) were shown. Tumors staining intensities in the two treatment arms were scored and plotted as the Cleveland dot graph. Naïve tumor staining is also shown for comparison. E, Western blots of protein extracts (10 μg/lane) from a matching set of tumors probed with indicated antibodies. The Western blot images were scanned and quantified using ImageJ. Mean density for each treatment group was plotted as a bar graph ± SD, with Y-axis showing image density relative to the naïve group. N, naive; O, osimertinib; OH, osimertinib plus anti-HDGF antibody H3. Asterisks mark the datasets in which there is a significant difference between the treatment arms.