Ex-vivo evaluation of gene therapy vectors in human pancreatic (cancer) tissue slices

Abstract

Aim: To culture human pancreatic tissue obtained from small resection specimens as a pre-clinical model for examining virus-host interactions.

Methods: Human pancreatic tissue samples (malignant and normal) were obtained from surgical specimens and processed immediately to tissue slices. Tissue slices were cultured ex vivo for 1-6 d in an incubator using 95% O(2). Slices were subsequently analyzed for viability and morphology. In addition the slices were incubated with different viral vectors expressing the reporter genes GFP or DsRed. Expression of these reporter genes was measured at 72 h after infection.

Results: With the Krumdieck tissue slicer, uniform slices could be generated from pancreatic tissue but only upon embedding the tissue in 3% low melting agarose. Immunohistological examination showed the presence of all pancreatic cell types. Pancreatic normal and cancer tissue slices could be cultured for up to 6 d, while retaining viability and a moderate to good morphology. Reporter gene expression indicated that the slices could be infected and transduced efficiently by adenoviral vectors and by adeno associated viral vectors, whereas transduction with lentiviral vectors was limited. For the adenoviral vector, the transduction seemed limited to the peripheral layers of the explants.

Conclusion: The presented system allows reproducible processing of minimal amounts of pancreatic tissue into slices uniform in size, suitable for pre-clinical evaluation of gene therapy vectors.

Figure 1

Histological staining of pancreatic tissue slices. Slices derived from normal pancreas and pancreatic cancer were cultured for 3 d. A, B: Normal pancreas of good viability; C: Normal pancreas of poor viability showing massive tissue slice necrosis; D: Poorly differentiated adenocarcinoma of good viability. Hematoxylin/eosin staining. Original magnification of all tissues (100 ×).

Figure 2

Viability of pancreatic (cancer) slices cultured for 3 d. All slices were cultured for 3 d and then stained with hematoxylin/eosin. The morphology of normal pancreas (n = 10) and pancreatic adenocarcinoma (n = 6) was studied microscopically and scored by an experienced pathologist.

Figure 3

Viability of human pancreas tissue slices cultured ex-vivo for up to 6 d. A: Viability of explants cultured in 1 mL DMEM (black line) or in DMEM supplemented with ITS growth factors (dashed line) was determined at day 1, 3, 4 and 6 of culturing a WST assay. All data given as the means of at least five slices ± SD; B: Normal pancreas with good viability at day 6; C: Normal pancreas of good viability at day 6 with Langerhans and nerve cells; D: Normal pancreas at day 6 with necrotic areas. Original magnification (100 ×).

Figure 4

Amylase secretion by human pancreas tissue slices. Human pancreatic cancer tissues were cultured ex-vivo for up to 3 d. Amount of amylase present in medium was determined using a colorimetric enzyme based assay. Data are the means ± SD (n = 12).

Figure 5

Transduction of pancreatic tissue slices with different viral vectors. A: Normal human pancreatic tissue slices are incubated with 1.0 × 10⁸ Ad5.GFP and 1.0 × 10⁸ Ad5.dsRed. After 48 h presence of GFP and ds.Red was detected using a fluorescent microscope; B: Normal pancreas slices were infected with 1.0 × 10⁸ genomic copies per slice of Ad5.GFP and Ad5.dsRED, or 1.2 × 10¹⁰ viral genomes AAV2-GFP. At 48 h after infection slices were lysed, GFP and ds.Red were quantified using the Novostar; C: Pancreatic adenocarcinoma explants were transduced with Ad-GFP. At 72 h after virus addition expression of GFP was detected using an anti-GFP antibody and counterstained with hematoxylin; D: GFP expression in normal pancreas at 72 h after incubation with lentivirus-GFP.