1. Validation of St6GalNAc2 as a metastasis suppressor gene.
Validation of St6GalNAc2 as a metastasis suppressor gene. A, 0.5 × 106 4T1-Luc cells infected with NTC shRNA (shNTC-A) or St6galnac2 shRNAs (shST6-A, shST6-B; see Supplementary Fig. S2A for qPCR analysis) were inoculated into the tail veins of BALB/c mice. On day 21, lungs removed at necroscopy were subjected to ex vivo IVIS imaging and weighed. Data shown are from 5 or 6 mice per group ±SEM. Student t test was used to generate P values. There was no statistical difference between the shST6-A and shST6-B groups (P = 0.48, IVIS; P = 0.19, lung weight). Middle, ex vivo bioluminescent images of excised lungs. Right, representative histologic sections illustrating the increase in lung tumor burden in the shST6 mice and the absence of detectable lung edema (see Supplementary Fig. S2B). Scale bar, 1 mm. Equivalent results were obtained in three independent experiments; for example, see Fig. 7C. B, 1.5 × 105 4T1-Luc cells were inoculated in female BALB/c mice. Primary tumor volume was measured until tumors reached the maximum allowable size (day 39), P = ns (not significant) at all time points. Metastases were quantified as percentage of tumor area in the lung from hematoxylin and eosin (H&E) sections. Data shown are from 7 animals in each group ±SEM. Two-way ANOVA used to generate P values. C, 0.5 × 106 shNTC-A or shST6-A 4T1-Luc cells were inoculated into the spleen of BALB/c mice. On day 21, livers were weighed at necroscopy and subjected to ex vivo IVIS imaging. Data shown are from 5 or 6 mice per group ±SEM. Student t test was used to generate P values. Representative images of liver metastases are shown. Scale bar, 250 μm. D, 2 × 106 MDA-MB-231-Luc cells expressing ectopic St6galnac2 (231-ST6) or vector-alone infected (231-Vec) were inoculated into the tail veins of CB17 NOD.SCID mice. At 5 weeks, lungs were subjected to ex vivo IVIS imaging at necroscopy. Data shown are from 13 to 14 mice per group ±SEM. Student t test was used to generate P values. Representative histology images are shown. Scale bar, 1 mm. Equivalent results were obtained in two independent experiments.
Nirupa Murugaesu et al. Cancer Discovery 2014;4:
©2014 by American Association for Cancer Research