Fig. 8

Crenigacestat inhibits the growth of CD90 positive xenograft tumors throughh the AKT signaling pathway A Four weeks' treatment with Crenigacestat significantly inhibited the growth of KKU-M213_scramble shRNA xenograft tumors compared with the same tumors treated with vehicle. On the contrary, no effect was observed in KKU-M213_THY1/CD90-shRNA following Crenigacestat treatment. B Growth of HuCCT1_THY1 +  + xenograft tumors was significantly reduced after 4 weeks of Crenigacestat treatment compared with the same tumors treated with vehicle. Crenigacestat did not display any effect on the Hucct1_empty vector cells. Each group consisted of 10 mice. *p < 0.05; ***p < 0.001 calculated with ANOVA test. C Venn diagram showing the number of common genes from the two comparisons. D PCR array analysis of 9 common genes in KKum213_scramble shRNA tumors (constitutively expressing high levels of CD90) treated with vehicle or Crenigacestat. PCR array analysis of 9 common genes in tumors originated from HuCCT1_CD90 +  + cells (transfected for overexpressing CD90) treated with vehicle or Crenigacestat. Histograms represent the mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001. E Quantification and representative IHC staining of KKU-M213_scramble shRNA xenograft tumor treated with vehicle showing intense diffuse cytoplasmic positivity and complete circumferential membrane staining for 50% of AKT and 30% of pAKT. Weak cytoplasmic and membrane staining for 5% of AKT and pAKT in KKU-M213_scramble shRNA xenograft tumors treated with Crenigacestat. Scale bar 50 µM, Magnification 20X. Summary of semiquantitative analysis based on membrane localized-AKT and cytosolic or membrane pAKT expression in xenograft tumor masses with IHC score of positively staining cells: 0%; 15%; 30%. Scatter plot of pAKT expression level in tumors treated with vehicle or Crenigacestat obtained by measuring five microscopic fields randomly chosen for each section. Staining was calculated as mean ± SEM of the integrated density of pAKT normalized by the mean ± SEM of the integrated density of nuclei by Image J software on n = 10 KKU-M213_scramble shRNA xenograft tumors treated with vehicle and n = 10 KKU-M213_scramble shRNA xenograft tumors treated with Crenigacestat. **p < 0.01. F Quantification and representative IHC staining of HuCCT1_THY1 +  + xenograft tumors treated with vehicle showing intense circumferential complete positivity for 30% of AKT and pAKT. Absence of antigenic expression of AKT and pAKT in HuCCT1_THY1 +  + xenograft tumors treated with Crenigacestat. Scale bar 50 µM, Magnification 10X. Summary of semiquantitative analysis based on membrane localized-AKT and cytosolic or membrane pAKT expression in xenograft tumor masses with IHC score of positively staining cells: 0%; 15%; 30%. Scatter plot of pAKT expression level in tumors treated with vehicle or Crenigacestat obtained by measuring five microscopic fields randomly chosen for each section. Staining was calculated as mean ± SEM of the integrated density of pAKT normalized by the mean ± SEM of the integrated density of nuclei by Image J software on n = 10 HuCCT1_THY1 +  + xenograft tumors treated with vehicle and n = 10 HuCCT1_THY1 +  + xenograft tumors treated with Crenigacestat ***p < 0.001