Figure 6
Evaluation of tumor growth, metastatic dissemination and cancer stem cell frequency. (a) LM38-LP cells were harvested from subconfluent monolayers and orthotopically inoculated into the fat pad of BALB/c mice. Five days later, animals receive the different treatments that consist in a silastic pellet containing ATRA (10 mg) or an empty pellet as control. Mice additionally received a peritumoral injection of Gö6976 (0.2 mg/kg) in physiological solution or physiological solution as control. Size of the two perpendicular diameters was recorded and used to calculate tumor volume. Each data point represents mean ± S.D. (n = 5), *p < 0.05 versus control, **p < 0.01 versus control, #p < 0.05 versus Gö6976 (ANOVA test). Two independent experiments were performed with similar results. (b) Representative photographs of LM38-LP tumors at necropsy are shown. (c) The number and size of surface lung nodules was determined under a dissecting microscope. Each data point represents the number of lung nodules per animal. Median and range are indicated in each experimental group. **p < 0.01 versus control (Kruskall-Wallis test). Figure shows the results of one experiment representative of three independent assays. (d) RNA from LM38-LP tumors harvested post-treatment was isolated. Nanog and Sox2 expression was analyzed by RT-qPCR. The fold of change of mRNA levels was calculated using the ΔΔCt method with GAPDH used as an internal control. Histograms represent mean ± S.D. *p < 0.05 versus control, **p < 0.01 versus control, ***p < 0.001 versus control, ##p < 0.01 versus Gö6976 (ANOVA test). Three independent experiments were performed. (e) CSC frequency estimates and p values calculated by using the ELDA software are shown.
