Figure 2. HC4 PERK inhibition decreases metastatic disease in lungs and bone marrow through elimination of quiescent DCCs.

(a) MMTV-HER2 females (24-week-old) were injected daily with vehicle or HC4 (50 mpk) for 2 weeks. IHC of pancreas and mammary gland sections with antibodies to P-PERK and P-EIF2α. Inserts show higher magnifications. Scale bars, 100 μm (left panel). Quantitation of the percentage area in the pancreas islets that show high intensity P-PERK staining (threshold method) (vehicle N=8, HC4 N=11) (right panel). (b) MMTV-HER2 tumors from control and HC4 treated mice were analyzed for P-PERK as well as total PERK levels by Western blot. Representative blot of three is shown. (c) Macro-metastases (>100 cells) were detected by H&E staining and quantified in 5 lung sections/animal ± s.d (vehicle N=22, HC4 N=17). Scale bar, 100 μm. P by Mann-Whitney test. (d) Micro-metastases (2-100 cells) were detected by IF staining using an anti-HER2 antibody and quantified per lung section/animal ± s.d. (vehicle N=6, HC4 N=6). Scale bar, 25 μm. P by Mann-Whitney test. (e) Solitary DCCs were detected by IF staining for HER2, classified as P-Rb+ or P-Rb− and quantified per lung section ± s.d. (vehicle N=5, HC4 N=6). Scale bar, 25 μm. P by Mann-Whitney test. (f) DCCs in bone marrow were detected by IF staining for HER2 in cytospins from mature hematopoietic cell-depleted bone marrow tissue (N=8 per condition). Scale bar, 25 μm. P by Mann-Whitney test. Mice were injected with MMTV-HER2 cells from primary tumor lesions via the tail vein, allowed them to expand into established metastatic lesions for 3 weeks and treated with vehicle or HC4 (50 mg/kg) for two additional weeks. (g) Metastasis were detected by H&E or IF (for HER2 detection) staining in 5 lung section/animal ± s.d (vehicle N=6, HC4 N=5) and total metastatic burden was calculated. P by Mann-Whitney test.