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

Abstract

Author contributions: van Geer MA generated the Adenovirus preparations and performed the transduction experiments in the slices; Kuhlmann KFD provided the resection specimens and performed the immune histological stainings; Bakker CT produced the Lentivirus and part of the adenovirus preparations and performed the slicing of the resection specimens; ten Kate FJW scored the viability of the slices after ex-vivo culture; Oude Elferink RPJ designed the study and obtained the grant required for performing this study and corrected the paper; Bosma PJ designed the study, generated the AAV vector and wrote the paper. METHODS: H u m a n p a n c re a t i c t i s s u e s a m p l e s (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% O2. Slices were subsequently analyzed for viability and morphology. In addition the slices were incubated with different viral vectors e x p r e s s i n g t h e r e p o r t e r g e n e s GFP o r 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:T h e p r e s e n t e d s y s t e m a l l o w s reproducible processing of minimal amounts of pancreatic tissue into slices uniform in size, suitable for pre-clinical evaluation of gene therapy vectors.

Ex Vivo Testing of Patient-Derived Xenografts Mirrors the Clinical Outcome of Patients with Pancreatic Ductal Adenocarcinoma

“…Organotypic cultures using 3D cultured tumor cells or live tissue slice have also been developed previously in evaluating clinical therapeutics (33-35). Most of tissue slice-based cultures and drug testing are in the format of 6-well or 24-well plate (36, 37), and tissue slice viability was measured with labor and time-intensive methods such as immunohistochemistry staining or western blotting, which are not applicable for large-scale drug testing (19, 27). In our system, the LTSA can be performed in a 96-well-plate format and allows testing drug sensitivity in 3-5 days objectively with a commercially available reagent, which will significantly reduce the time and resources compared to the test in animal models.…”

Sensitization of Pancreatic Cancer to Chemoradiation by the Chk1 Inhibitor MK8776

“…Thus, the development of pharmacodynamic biomarkers of Chk1 inhibition is important and has the potential to be predictive of the degree of tumor sensitization. To begin to develop a system which would permit pharmacodynamic assessment of a biomarker from a one-time biopsy in pancreatic cancer, we have integrated methodology which utilizes thinly sliced fresh tissue (21) into an ex vivo treatment platform. It will be important in future studies to determine if ex vivo biomarker analyses are feasible on patient pancreatic tumor biopsies and whether they are predictive of therapeutic efficacy (35).…”

“…Two hundred μm slices were prepared with a Leica VT 1200S microtome with vibrating blade (Leica Microsystems) while submerged in oxygenated, ice cold Krebs buffer, according to the previously described methodology (21). Slices were incubated in 12-well dishes containing DMEM (ATCC) supplemented with 2mM glutamine, penicillin, streptomycin, antibiotic-antimycotic (Gibco), and 20mM HEPES (pH 7.4) in a carbogen (95% O 2 , 5% CO 2 ) purged chamber inside of at a 37°C, humidified, 5% CO 2 incubator for up to 30 h, after which they were processed for biochemical or immunohistochemical analysis using the same methods as for intact tumors.…”

Hypoxia Pathways and Cellular Stress Activate Pancreatic Stellate Cells: Development of an Organotypic Culture Model of Thick Slices of Normal Human Pancreas

“…Only one study reported pancreatic precision slices techniques for examining virus-host interactions in pancreatic normal and adenocarcinoma tissue. Pancreatic normal and cancer tissue slices were cultured for up to 6 days, while retaining viability and moderate to good morphological features[18]. …”