In vivo selection and characterization of metastatic variants from human pancreatic adenocarcinoma by using orthotopic implantation in nude mice

Abstract

We determined whether the implantation of human pancreatic cancer cells into the pancreas of nude mice can be used to select variants with increasing metastatic potential. COLO 357 line fast-growing cells were injected into the spleen or pancreas of nude mice. Hepatic metastases were harvested, and tumor cells were reinjected into the spleen or pancreas. This cycle was repeated several times to yield cell lines L3.6sl (spleen to liver) and L3.6pl (pancreas to liver). The variant cells produced significantly higher incidence and number of lymph node and liver metastases than the parental cells. Their increased metastatic potential was associated with increased expression (mRNA and protein) of the proangiogenic molecules basic fibroblast growth factor, vascular endothelial growth factor, and interleukin-8. The metastatic cells also exhibited increased motility and invasiveness, which were associated with increased expression of collagenase type IV (MMP-9) and decreased expression of E-cadherin. Collectively, the data show that the orthotopic implantation of human pancreatic cancer cells in nude mice is a relevant model with which to study the biology of pancreatic cancer metastasis and to select variant cell lines with enhanced metastatic potential.

Figure 1

In vivo selection of metastatic human pancreatic cancer cells. L3.3 cells (derived from the FG line) were injected into the spleen or the pancreas of nude mice. Experimental liver metastases (spleen injection) or spontaneous liver metastases (pancreas injection) were harvested, established in culture, and designated L3.4sl (spleen-liver) and L3.4pl (pancreas-liver). The cultures were harvested and injected into the spleen or pancreas of another set of nude mice. The cycles were repeated twice more to yield lines L3.6sl and L3.6pl.

Figure 2

Percent cumulative survival. The pancreases of nude mice were injected with 1 x 10⁶ viable FG, L3.6sl, or L3.6pl cells. Moribund mice were killed and necropsied. Survival analysis was computed by the Kaplan-Meier method, and the unpaired Student t test was used to compare the results among the three different cell lines. FG versus L3.6sl, P < .002; FG versus L3.6pl, P < .0001; L3.6sl versus L3.6pl, P < 0.02.

Figure 3

Representative IHC staining of FG, L3.6sl, and L3.6pl human pancreatic cells growing in the pancreas of nude mice. Tissue sections were stained for expression of PCNA (to show cell division), MMP-9, IL-8, bFGF, VEGF/VPF, and CD31 (to show endothelial cells).

Figure 4

Representative ISH of FG, L3.6sl, and L3.6pl human pancreatic cells growing in the pancreas of nude mice. Metastatic cells (L3.6sl, L3.6pl) concurrently expressed high levels of MMP-2, MMP-9, bFGF, VEGF/VPF, IL-8, and low levels of E-cadherin. The low metastatic cells expressed high levels of E-cadherin.

Figure 5

Gelatin zymography. Lysates from FG L3.6sl and L3.6pl pancreatic tumors were prepared. Protein (1 µg) was loaded on a 7.5% polyacrylamide slab gel-impregnated with 1 mg/mL gelatin under nonreducing conditions.