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Summary

Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Medicine

Organotypic Slice Cultures as Preclinical Models of Tumor Microenvironment in Primary Pancreatic Cancer and Metastasis

Published: June 22nd, 2021

DOI:

10.3791/62541

1Department of Surgery, University Medical Center Schleswig-Holstein, 2Institute of Medical and Marine Biotechnology, University of Lübeck, 3Fraunhofer Research and Development Center for Marine and Cellular Biotechnology, 4Institute for Surgical Pathology, Medical Center - University of Freiburg, Faculty of Medicine, University of Freiburg, 5Tumorbank Comprehensive Cancer Center Freiburg, Medical Center - University of Freiburg, 6Core Facility Histopathology and Digital Pathology Freiburg, Medical Center - University of Freiburg

This protocol describes the preparation of organotypic slice cultures (OTSCs). This technique facilitates the ex vivo cultivation of intact multicellular tissue. OTSCs can be used immediately to test for their respective response to drugs in a multicellular environment.

Realistic preclinical models of primary pancreatic cancer and metastasis are urgently needed to test the therapy response ex vivo and facilitate personalized patient treatment. However, the absence of tumor-specific microenvironment in currently used models, e.g., patient-derived cell lines and xenografts, only allows limited predictive insights. Organotypic slice cultures (OTSCs) comprise intact multicellular tissue, which can be rapidly used for the spatially resolved drug response testing.

This protocol describes the generation and cultivation of viable tumor slices of pancreatic cancer and its metastasis. Briefly, tissue is casted in low melt agarose and stored in cold isotonic buffer. Next, tissue slices of 300 µm thickness are generated with a vibratome. After preparation, slices are cultured at an air-liquid interface using cell culture inserts and an appropriate cultivation medium. During cultivation, changes in cell differentiation and viability can be monitored. Additionally, this technique enables the application of treatment to viable human tumor tissue ex vivo and subsequent downstream analyses, such as transcriptome and proteome profiling.

OTSCs provide a unique opportunity to test the individual treatment response ex vivo and identify individual transcriptomic and proteomic profiles associated with the respective response of distinct slices of a tumor. OTSCs can be further explored to identify therapeutic strategies to personalize treatment of primary pancreatic cancer and metastasis.

Existing preclinical models of pancreatic ductal adenocarcinoma (PDAC) and respective metastases are poor predictors of response to treatment in patients which is a major drawback in drug development and the identification of predictive biomarkers1. Although models such as patient-derived organoids and patient-derived xenografts are promising, their use remains limited2. Major limitations of these in vitro models are the lack of the tumor microenvironment and xenografting in non-human immunocompromised species. Especially in PDAC and its metastases, the tumor microenvironment has considerably gained interest ove....

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Tissue specimens were collected and processed after approval by the local ethics committee of the University of Lübeck (# 16-281).

1. Fresh tissue collection and handling

NOTE: Every unfixed human tissue specimen should be handled with caution to prevent the risk of infection from blood-borne pathogens. All patients should be tested to be negative for HIV, HBV, and HCV prior to tissue processing. Wear a protective coat and handle human tissue specimens with glove.......

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Figure 1 provides an overview of the workflow to culture OTSCs from fresh, unfrozen tumor tissue. Specimens of primary PDACs and metastases were collected directly after surgical resection and stored overnight on wet ice at 4 °C in the tissue storage solution. The specimens were processed, and slices were cultured as described in the protocol. The macroscopic morphology of each OTSC did not change grossly during cultivation. However, the size of the surface area of the OTSCs decrea.......

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OTSCs of fresh tumor samples are a close approximation of the tumor in situ. They maintain their baseline morphology, proliferative activity, and microenvironment during the cultivation for a defined, tissue-dependent period11,12,13. This technique enables the immediate application of treatment to viable human tumor tissue ex vivo and subsequent downstream analyses, such as profiling of the transcriptome and pr.......

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R. Braun was supported by the Clinician Scientist School Lübeck (DFG #413535489) and the Junior Funding Program of the University of Lübeck.

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Name Company Catalog Number Comments
Advanced DMEM/F-12 Medium Gibco 12634028
Agarose Low Melt Roth 6351.2 8% in Ringer solution
Antibody Diluent, Background Reducing Dako S3022
AquaTex Merck 108562
Bioethanol (99%, denatured) CHEMSolute 2,21,19,010
Citric Acid monohydrate Sigma Aldrich C7129
Cleaved Caspase-3 (Asp175) (5A1E) Rabbit mAb Cell Signalling Technology 9664 1:400 dilution
Derby Extra Double Edge Safety Razor Blades Derby Tokai
Embedding cassettes Roth H579.1
Eosin Y-solution 0,5% aqueous Merck 10,98,44,100
Eukitt Quick hardening mounting medium Sigma-Aldrich 3989
Fetal bovine serum Gibco 10270106
Formaldehyde solution 4,5%, buffered Büfa Chemikalien B211101000
Hem alum solution acid acc. to Mayer Roth T865
Human EGF Milteniy Biotec 130-097-794
Hydrocortisone Sigma Aldrich (Merck) H0888
Hydrogen peroxide 30% Merck 1,08,59,71,000
Insulin human Sigma Aldrich (Merck) 12643
Liquid DAB+ Substrate Chromogen System Dako K3468
MACS Tissue Storage Solution Milteniy Biotech 130-100-008
Methanol Merck ############
Microscope Slides Superfrost Plus Thermo Scientific J1800AMNZ
Millicell Cell Culture Insert, 30 mm, hydrophilic PTFE, 0.4 µm Millipore (Merck) PICM0RG50
Monoclonal mouse anti-human  Cytokeratin 7 (Clone OV-TL 12/30) Dako M7018 1:200 dilution
Monoclonal mouse anti-human Ki67 Clone MIB-1 Dako M7240 1:200 dilution
Monoclonal mouse Anti-vimentin (Clone V9) Dako M0725 1:200 dilution
Negative control Mouse IgG2a Dako X0943 1:200 dilution
Negative control Mouse IgG1 Dako X093101-2 1:200 dilution
Paraffin (melting temperature 56°- 58°) Merck 10,73,37,100
Penicillin-Streptomycin (10.000 U/ml) Gibco 15140122
PBS pH 7,4 (1x) Flow Cytometry Grade Gibco A12860301
Resazurin sodium salt; 10 mg/ml in PBS Sigma Aldrich R7017 1:250 dilution
Ringer's solution Fresenius Kabi 2610813
RPMI-1640 Medium Sigma Aldrich (Merck) R8758
Tissue culture testplate 6 TPP 92006
Triton X-100 Sigma Aldrich 9002-93-1
VECTASTAIN Elite ABC-Peroxidase Kit Vector Laboratories PK-6200
Xylene (extra pure) J.T.Baker 8,11,85,000
Equipment
ClarioStar Microplate Reader BMG Labtech
Paraffin Embedding Center E61110 Leica
Rotary Microtome Microm HM355S Thermo Scientific
Section Transfer System Microm STS Thermo Scientific
VT 1200S Vibratom Leica

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