Evaluation of a gene-directed enzyme-product therapy (GDEPT) in human pancreatic tumor cells and their use as in vivo models for pancreatic cancer.

BACKGROUND: Gene-directed enzyme prodrug therapy (GDEPT) is a two-step treatment protocol for solid tumors that involves the transfer of a gene encoding a prodrug-activating enzyme followed by administration of the inactive prodrug that is subsequently activated by the enzyme to its tumor toxic form. However, the establishment of such novel treatment regimes to combat pancreatic cancer requires defined and robust animal model systems. METHODS: Here, we comprehensively compared six human pancreatic cancer cell lines (PaCa-44, PANC-1, MIA PaCa-2, Hs-766T, Capan-2, and BxPc-3) in subcutaneous and orthotopical mouse models as well as in their susceptibility to different GDEPTs. RESULTS: Tumor uptake was 83% to 100% in the subcutaneous model and 60% to 100% in the orthotopical mouse model, except for Hs-766T cells, which did not grow orthotopically. Pathohistological analyses of the orthotopical models revealed an infiltrative growth of almost all tumors into the pancreas; however, the different cell lines gave rise to tumors with different morphological characteristics. All of the resultant tumors were positive for MUC-1 staining indicating their origin from glandular or ductal epithelium, but revealed scattered pan-cytokeratin staining. Transfer of the cytochrome P450 and cytosine deaminase suicide gene, respectively, into the pancreatic cancer cell lines using retroviral vector technology revealed high level infectibility of these cell lines and allowed the analysis of the sensitivity of these cells to the chemotherapeutic drugs ifosfamide and 5-fluorocytosine, respectively. CONCLUSION: These data qualify the cell lines as part of valuable in vitro and in vivo models for the use in defined preclinical studies for pancreas tumor therapy

Cytochrome 2B1 protein expression in PCCWmCMV-transduced and non-transduced parental cell lines.

<p>Total cellular lysates from 8×10⁵ cells were separated on a 10% polyacrylamide gel under denaturing conditions. After blotting, CYP2B1 protein was detected with a CYP-specific antibody.</p

Summary of orthotopical tumor growth.

<p>Number of tumors growing after i.p. injection into mice and percentage of tumors that infiltrated the pancreatic tissue.</p>a<p>infiltrative tumor growth rate into the tail of the pancreas; percentages indicate the proportion of mice with infiltrative tumor growth among those with orthotopic tumor growth.</p

Pathohistological analyses of tumor tissues.

<p>(<b>2A</b>) Sections of pancreatic tumors derived of the indicated pancreatic cell lines were stained with haematoxylin and eosin. The Hs-766T specimen was dissected from a subcutaneous tumor; all other samples were taken from orthotopically grown tumors. (<b>2B</b>) Different sections of a BxPC-3-derived tumor were subjected to immunohistological analysis with antibodies against Mucin-1 (MUC-1), pan-cytokeratin (Pan-CK), and α-smooth muscle actin (SMA). The black bar represents a length of 53 µm, except for anti-MUC-1 staining (27 µm).</p

Cytosine deaminase gene expression in PCCDWmCMVpuro-transduced and in parental cell lines.

<p>Total cellular RNA was isolated from indicated cell lines and mRNA was reverse transcribed using oligo(dT) primers. CD- as well as GAPH-specific gene fragments were amplified using specific primers and separated on an agarose gel showing the expected sizes of ∼480 bp for CD and ∼350 bp for GAPDH. The cDNA amount used for PCR amplification was adjusted to yield comparable amounts of the GAPDH gene fragment.</p

Tumor growth development of subcutaneously growing tumors.

<p>5×10⁶ cells of each pancreatic cell line were subcutaneously injected into the left flank of C.B-17/IcrHsd-<i>Prkcd^scid Lyst^bg</i> mice at day 0. Visible tumors were measured twice a week with a caliper and size was calculated by the formula “length×width×width/2”.</p

Molecular alterations of tumor-associated markers in human pancreatic tumor cell lines (based on [27], [28]).

a<p>mutated;</p>b<p>deleted,</p>c<p>wild type,</p>*<p>no consistent information available.</p

Sensitivity of human pancreatic cancer cell lines towards ifosfamide.

<p>Cells stably transduced with the CYP2B1 gene as well as non-transduced parental cells (mock) were cultured in increasing concentrations of ifosfamide for five days as described in Materials and Methods. LD₅₀ values were calculated using Excel Fit software. Each experiment was performed in quadruplicates. Data from three independent experiments are shown.</p

Sensitivity of human pancreatic cancer cell lines towards 5-FC and 5-FU.

<p>Cells stably transduced with the yeast CD gene as well as non-transduced parental cells (mock) were cultured in increasing concentrations of 5-FC or 5-FU for five days as described in Materials and Methods. LD₅₀ values were calculated using Excel Fit software. Each experiment was performed in quadruplicates. Shown are data from three independent experiments.</p