Fig. 4

Autophagy blockade with chloroquine improves lapatinib efficacy in vivo in zebrafish and mouse xenografts of GSDMB-expressing tumors. A-B Representative fluorescence stereomicroscope images (left panel) of GFP expressing control (shNTC), and GSDMB-silenced (shGB1) HCC1954 xenografts (A) or HCC1954 LR and control (C) tumors (B) treated with the LC50 of lapatinib and chloroquine (35,1 mM and 116,4 mM, respectively). Insets represent an augmented image of GFP-positive tumor. Tumor proliferation rate (right panel) of the different HCC1954 xenografts was analyzed by measuring the fluorescence intensity ratio (48 hpt/0 hpt). Statistical significance was determined by two-tailed unpaired t-test. At least, n = 20 per each indicated condition. C Experimental design of the mouse xenograft model (n = 5 per condition). Mice were inoculated with either HCC1954-mCherry-luc control (shNTC), or GSDMB-silenced cells (shGB1 and shGB2) and treated with lapatinib (100 mg/kg, orally, once daily), CQ (50 mg/kg, intraperitoneally, once daily), or a combination of both (lapatinib + CQ). An aqueous solution containing 0.1% Tween 80 and 0.5% Hypromellose was used as vehicle. The experiment was performed for 30 days, according to the approved protocol and conditions of animal research (detailed in Supplementary Methods). D-G Quantification of the tumor weight (D) tumor volume evolution of shNTC (E), shGB1 (F) and shGB2 (G) HCC1954 xenografts, treated with the indicated regimens. Statistical significance was determined by multiple unpaired t-test – comparing vehicle with each of the other conditions at every time point. H Representative images of GSDMB immunohistochemical analysis and hematoxylin and eosin staining in shNTC tumors, treated with the different therapeutic strategies indicated in (C). Immunohistochemical images were taken on 10X and 40X (insets) magnification. (*P < 0.05; **P < 0.01; ***P < 0.001; ns, nonsignificant). Data are shown as the mean ± s.e.m. LC50, 50% lethal concentration; hpt, hours post-treatment. NTC, non-targeting control. LR, Lapatinib resistant cells, CQ, chloroquine. Lap, lapatinib