Organotypic Slice Cultures as Preclinical Models of Tumor Microenvironment in Primary Pancreatic Cancer and Metastasis
Summary
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.
Abstract
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.
Introduction
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 over the last years because of its crucial functions in tumor biology. It comprises cellular and acellular components, such as (myo-)fibroblasts, pancreatic stellate cells, immune cells, blood vessels, extracellular matrix, cytokines, and growth factors3. This microenvironment is not a non-functional tumor component, but induces tumor progression and metastasis and seems to contribute substantially to radio- and chemotherapy resistance4. The PDAC microenvironment not only mechanically compromises drug delivery, but also possesses immune and drug-scavenging activity5,6,7. Thus, preclinical models which reflect the complex interaction of tumor cells and the tumor microenvironment are urgently needed to adequately test patients' treatment response ex vivo and guide individualized clinical treatment.
Ex vivo cultures of fresh tumor samples represent a close approximation of the tumor in situ. Organotypic slice cultures (OTSCs) have been recently developed and studied for several tumors, such as head, neck, breast, prostate, lung, colon, and pancreatic cancers8,9,10,11,12. It has been shown that OTSCs maintain their baseline morphology, proliferative activity, and microenvironment during the cultivation for a defined, tissue-dependent period11,12,13. OTSCs of PDACs maintained their viability, morphology, and most components of their tumor microenvironment for 4-9 days in several in vitro studies5,12,14. Perspectively, this technique enables an immediate application of the treatment to viable human tumor tissue ex vivo and subsequent downstream analyses, such as profiling of the transcriptome and proteome.
The establishment of OTSCs provides a unique opportunity to test the treatment response ex vivo promptly after surgery. Thus, OTSCs will prospectively allow to identify therapeutic strategies to personalize treatment of metastatic disease. This protocol describes the generation and cultivation of viable OTSCs of pancreatic cancer.
Protocol
Representative Results
Discussion
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…
Offenlegungen
The authors have nothing to disclose.
Acknowledgements
R. Braun was supported by the Clinician Scientist School Lübeck (DFG #413535489) and the Junior Funding Program of the University of Lübeck.
Materials
| 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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