Figure 1.
Establishment of HT29 derived cell lines with enhanced metastatic potential.
(A) Illustration of metastatic CRC in vivo selection process. Experimental liver metastases were harvested, established in culture and designated HT29 LM1, LM2, and LM3 cell lines. (B) Bioluminescent images of mice 4 wks after intrasplenic injection of parental HT29 and HT29 LM2 cell lines.
Figure 2.
The aggressive metastatic phenotype of the HT29 LM1, LM2, and LM3 cell lines is associated with the activation of the c-MET pathway.
HT29 LM1, LM2, LM3 and parental cell line were cultured in normal medium for 24(first 4 lanes). In another experiment, cells were cultured in serum free medium for 24 h and stimulated with complete medium for 10 min (remaining 4 lanes). Protein expression profiles were analyzed by Western blot. β-actin was used as a loading control.
Figure 3.
Expression of CD44 is significantly increased in the HT29 LM1, LM2, and LM3 cell lines.
(A) HT29 LM1, LM2, LM3 and parental cell line were cultured in normal medium for 24 h. Protein expression profiles were analyzed by Western blot. β-actin was used as a loading control. (B) IHC analysis of CD44 and p-MET expression in HT29 LM1 and HT29 LM3 liver metastasis tissue sections. (C) Flow cytometry analysis of CD44 geometric mean fluorescence intensity in parental HT29 and HT29 LM3 cells.
Figure 4.
CD44 independent c-MET activation in HT29 LM3 cells.
(A) Analysis of CD44, p-MET, c-MET, p-AKT and AKT expression in parental HT29 and HT29 LM3 after HGF stimulation (50 ng/mL; 5 min) by Western blot. β-actin was used as a loading control. (B) HT29 LM3 cells were transfected with CD44 siRNA and stimulated with HGF (50 ng/mL; 5 min) 24 h after siRNA transfection. (C) CD44 expression in HT29 LM3 and parental HT29 liver metastasis tissue sections were analyzed by IHC. (D) HT29 LM3 cells were sorted for high and low CD44 expression. Cells were gated and sorted to collect 10% of the cells with high CD44 expression and cells with low CD44 expression. (E) Analysis of CD44, p-MET, and c-MET expression in parental HT29, HT29 LM3 CD44−, and HT29 LM3 CD44+ cells. (F) Analysis of p-MET, c-MET, CD44, p-AKT, and AKT expression in parental HT29, HT29 LM3 CD44−, and HT29 LM3 CD44+ cells after HGF (50 ng/mL; 5 min) stimulation.
Figure 5.
Role of CD44 high and CD44 low expression in CRC liver metastasis.
(A) Adhesion of parental HT29 and HT29 LM3 cells to HMVEC-L cells was assessed as described in Materials and Methods. Data shown as mean fold changes in number of parental HT29 cells attached to HMVEC-L cells versus HT29 LM3 cells (*p<0.001). Representative images show adhesion of parental HT29 and HT29 LM3 cells to HMVEC-L cells. (B) Fluorescent GFP imaging of liver metastasis 4 wks after intrasplenic injection of HT29 LM3 CD44+ and HT29 LM3 CD44− cells (5×106; 100 µl of PBS). IHC analysis of CD44 expression in CD44 high and CD44 low CRC liver metastasis.