Figure 1.
Intra-tumoral heterogeneity in the ability to develop metastatic colonization.
(A) H&E section demonstrating normal colonic area and primary colon carcinoma (magnification 40×). (B) 4 to 6 week old male BALB/c nude mice were orthotopically implanted with HCT116 or HCT116b tumor xenografts. GFP imaging revealed primary tumor growth in both HCT116 and HCT116b animals. However, distant metastasis was only observed in HCT116 implanted animals by week 6.
Table 1.
Results of orthotopic implantation of HCT116 and HCT116b tumor xenografts demonstrating the primary invasion and metastases based on histologic evaluation.
Figure 2.
Increased cell survival associated with HCT116 cells.
Comparison of primary tumor sections of HCT116 and HCT116b mice by TUNEL staining as mentioned in Materials and Methods to determine their apoptotic rates. HCT116b tumor tissues had high TUNEL staining indicative of cell death. However, HCT116 tumor tissues showed negligible response to TUNEL staining indicating high rate of cell survival.
Figure 3.
Comparison of genes signatures associated with metastasis signaling between HCT116 and HCT116b microarray datasets by Ingenuity Pathway Analysis (IPA) tool.
The molecules highlighted in green are upregulated in the highly metastatic HCT116 cells and downregulated in poorly metastatic iso-clonal HCT116b cells. The molecules marked in red are upregulated in HCT116b cells compared to HCT116 cells.
Figure 4.
Cell death response under growth factor deprivation stress (GFDS).
(A) HCT116 cells are resistant to GFDS-induced cell death as determined by DNA fragmentation assay. However, HCT116b iso-clonal cells induce cell death under GFDS. (B) HCT116b cells induce cell death by time-dependent increase in PARP and caspase 3 cleavages and dephosphorylation of anti-apoptotic pBad protein.
Figure 5.
Cell survival signaling response under growth factor deprivation stress (GFDS).
HCT116 cells upregulated AKT, MAPK and PLC signaling under GFDS conditions to counter stress. In contrast, HCT116b cells downregulated these signaling molecules by dephosphorylating and thereby inactivating AKT, ERK and PLC gamma to induced cell death.
Figure 6.
Differential cell motility responses between HCT116 and HCT116b cells under growth factor deprivation stress (GFDS).
(A) HCT116 cells were observed to be highly motile in comparison to HCT116b cells as demonstrated by Transwell Assay. (B) HCT116 cells upregulated the invasion-motility associated proteins p-Fak, p-Paxillin, RhoA, Rac1 and Rock1 under GFDS conditions. In contrast, HCT116b cells downregulated RhoA, Rac1 and Rock1 proteins and showed a lower increase of p-Fak and p-Paxillin under similar conditions.