Name: organotypic slice cultures as preclinical models of tumor microenvironment in primary pancreatic cancer and metastasis
Uploaded: 2021-06-22T14:00:00
Description: Watch this Scientific Journal Video about Organotypic Slice Cultures as Preclinical Models of Tumor Microenvironment in Primary Pancreatic Cancer and Metastasis at JoVE.com

R. Braun was supported by the Clinician Scientist School L&#252;beck (DFG #413535489) and the Junior Funding Program of the University of L&#252;beck.

Tissue specimens were collected and processed after approval by the local ethics committee of the University of L&#252;beck (# 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...

<ol>
	<li>Nevala-Plagemann, C., Hidalgo, M., Garrido-Laguna, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+state-of-the-art+treatments+to+novel+therapies+for+advanced-stage+pancreatic+cancer.">From state-of-the-art treatments to novel therapies for advanced-stage pancreatic cancer.</a> Nature Reviews. Clinical Oncology. 17 (2), 108-123 (2020).</li><li>Tiriac, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organoid+profiling+identifies+common+responders+to+chemotherapy+in+pancreatic+cancer.">Organoid profiling identifies common responders to chemotherapy in pancreatic cancer.</a> Cancer Discovery. 8 (9), 1112-1129 (2018).</li><li>Waghray, M., Yalamanchili, M., di Magliano, M. P., Simeone, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deciphering+the+role+of+stroma+in+pancreatic+cancer.">Deciphering the role of stroma in pancreatic cancer.</a> Current Opinion in Gastroenterology. 29 (5), 537-543 (2013).</li><li>Hwang, R. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer-associated+stromal+fibroblasts+promote+pancreatic+tumor+progression.">Cancer-associated stromal fibroblasts promote pancreatic tumor progression.</a> Cancer Research. 68 (3), 918-926 (2008).</li><li>Jiang, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-lived+pancreatic+ductal+adenocarcinoma+slice+cultures+enable+precise+study+of+the+immune+microenvironment.">Long-lived pancreatic ductal adenocarcinoma slice cultures enable precise study of the immune microenvironment.</a> Oncoimmunology. 6 (7), 1333210 (2017).</li><li>Hessmann, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibroblast+drug+scavenging+increases+intratumoural+gemcitabine+accumulation+in+murine+pancreas+cancer.">Fibroblast drug scavenging increases intratumoural gemcitabine accumulation in murine pancreas cancer.</a> Gut. 67 (3), 497-507 (2018).</li><li>Feig, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+pancreas+cancer+microenvironment.">The pancreas cancer microenvironment.</a> Clinical Cancer Research. 18 (16), 4266-4276 (2012).</li><li>Koerfer, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organotypic+slice+cultures+of+human+gastric+and+esophagogastric+junction+cancer.">Organotypic slice cultures of human gastric and esophagogastric junction cancer.</a> Cancer Medicine. 5 (7), 1444-1453 (2016).</li><li>Gerlach, M. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Slice+cultures+from+head+and+neck+squamous+cell+carcinoma:+a+novel+test+system+for+drug+susceptibility+and+mechanisms+of+resistance.">Slice cultures from head and neck squamous cell carcinoma: a novel test system for drug susceptibility and mechanisms of resistance.</a> British Journal of Cancer. 110 (2), 479-488 (2014).</li><li>Holliday, D. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+practicalities+of+using+tissue+slices+as+preclinical+organotypic+breast+cancer+models.">The practicalities of using tissue slices as preclinical organotypic breast cancer models.</a> Journal of Clinical Pathology. 66 (3), 253-255 (2013).</li><li>Vaira, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preclinical+model+of+organotypic+culture+for+pharmacodynamic+profiling+of+human+tumors.">Preclinical model of organotypic culture for pharmacodynamic profiling of human tumors.</a> Proceedings of the National Academy of Sciences of the United States of America. 107 (18), 8352-8356 (2010).</li><li>Lim, C. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organotypic+slice+cultures+of+pancreatic+ductal+adenocarcinoma+preserve+the+tumor+microenvironment+and+provide+a+platform+for+drug+response.">Organotypic slice cultures of pancreatic ductal adenocarcinoma preserve the tumor microenvironment and provide a platform for drug response.</a> Pancreatology. 18 (8), 913-927 (2018).</li><li>Brandenburger, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organotypic+slice+culture+from+human+adult+ventricular+myocardium.">Organotypic slice culture from human adult ventricular myocardium.</a> Cardiovascular Research. 93 (1), 50-59 (2012).</li><li>Misra, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ex+vivo+organotypic+culture+system+of+precision-cut+slices+of+human+pancreatic+ductal+adenocarcinoma.">Ex vivo organotypic culture system of precision-cut slices of human pancreatic ductal adenocarcinoma.</a> Science Reports. 9 (1), 2133 (2019).</li><li>O'Brien, J., Wilson, I., Orton, T., Pognan, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Investigation+of+the+Alamar+Blue+(resazurin)+fluorescent+dye+for+the+assessment+of+mammalian+cell+cytotoxicity.">Investigation of the Alamar Blue (resazurin) fluorescent dye for the assessment of mammalian cell cytotoxicity.</a> European Journal of Biochemistry. 267 (17), 5421-5426 (2000).</li><li>Beutler, B., Cerami, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+necrosis,+cachexia,+shock,+and+inflammation:+a+common+mediator.">Tumor necrosis, cachexia, shock, and inflammation: a common mediator.</a> Annual Review of Biochemisty. 57, 505-518 (1988).</li></ol>

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...

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&#39; 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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Advanced DMEM/F-12 Medium</td>
			<td>Gibco</td>
			<td>12634028</td>
			<td></td>
		</tr>
		<tr>
			<td>Agarose Low Melt</td>
			<td>Roth</td>
			<td>6351.2</td>
			<td>8% in Ringer solution</td>
		</tr>
		<tr>
			<td>Antibody Diluent, Background Reducing</td>
			<td>Dako</td>
			<td>S3022</td>
			<td></td>
		</tr>
		<tr>
			<td>AquaTex</td>
			<td>Merck</td>
			<td>108562</td>
			<td></td>
		</tr>
		<tr>
			<td>Bioethanol (99%, denatured)</td>
			<td>CHEMSolute</td>
			<td>2,21,19,010</td>
			<td></td>
		</tr>
		<tr>
			<td>Citric Acid monohydrate</td>
			<td>Sigma Aldrich</td>
			<td>C7129</td>
			<td></td>
		</tr>
		<tr>
			<td>Cleaved Caspase-3 (Asp175) (5A1E) Rabbit mAb</td>
			<td>Cell Signalling Technology</td>
			<td>9664</td>
			<td>1:400 dilution</td>
		</tr>
		<tr>
			<td>Derby Extra Double Edge Safety Razor Blades</td>
			<td>Derby Tokai</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Embedding cassettes</td>
			<td>Roth</td>
			<td>H579.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Eosin Y-solution 0,5% aqueous</td>
			<td>Merck</td>
			<td>10,98,44,100</td>
			<td></td>
		</tr>
		<tr>
			<td>Eukitt Quick hardening mounting medium</td>
			<td>Sigma-Aldrich</td>
			<td>3989</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Gibco</td>
			<td>10270106</td>
			<td></td>
		</tr>
		<tr>
			<td>Formaldehyde solution 4,5%, buffered</td>
			<td>B&#252;fa Chemikalien</td>
			<td>B211101000</td>
			<td></td>
		</tr>
		<tr>
			<td>Hem alum solution acid acc. to Mayer</td>
			<td>Roth</td>
			<td>T865</td>
			<td></td>
		</tr>
		<tr>
			<td>Human EGF</td>
			<td>Milteniy Biotec</td>
			<td>130-097-794</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrocortisone</td>
			<td>Sigma Aldrich (Merck)</td>
			<td>H0888</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrogen peroxide 30%</td>
			<td>Merck</td>
			<td>1,08,59,71,000</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin human</td>
			<td>Sigma Aldrich (Merck)</td>
			<td>12643</td>
			<td></td>
		</tr>
		<tr>
			<td>Liquid DAB+ Substrate Chromogen System</td>
			<td>Dako</td>
			<td>K3468</td>
			<td></td>
		</tr>
		<tr>
			<td>MACS Tissue Storage Solution</td>
			<td>Milteniy Biotech</td>
			<td>130-100-008</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Merck</td>
			<td>############</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope Slides Superfrost Plus</td>
			<td>Thermo Scientific</td>
			<td>J1800AMNZ</td>
			<td></td>
		</tr>
		<tr>
			<td>Millicell Cell Culture Insert, 30 mm, hydrophilic PTFE, 0.4 &#181;m</td>
			<td>Millipore (Merck)</td>
			<td>PICM0RG50</td>
			<td></td>
		</tr>
		<tr>
			<td>Monoclonal mouse anti-human&#160; Cytokeratin 7 (Clone OV-TL 12/30)</td>
			<td>Dako</td>
			<td>M7018</td>
			<td>1:200 dilution</td>
		</tr>
		<tr>
			<td>Monoclonal mouse anti-human Ki67 Clone MIB-1</td>
			<td>Dako</td>
			<td>M7240</td>
			<td>1:200 dilution</td>
		</tr>
		<tr>
			<td>Monoclonal mouse Anti-vimentin (Clone V9)</td>
			<td>Dako</td>
			<td>M0725</td>
			<td>1:200 dilution</td>
		</tr>
		<tr>
			<td>Negative control Mouse IgG2a</td>
			<td>Dako</td>
			<td>X0943</td>
			<td>1:200 dilution</td>
		</tr>
		<tr>
			<td>Negative control Mouse IgG1</td>
			<td>Dako</td>
			<td>X093101-2</td>
			<td>1:200 dilution</td>
		</tr>
		<tr>
			<td>Paraffin (melting temperature 56&#176;- 58&#176;)</td>
			<td>Merck</td>
			<td>10,73,37,100</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin (10.000 U/ml)</td>
			<td>Gibco</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS pH 7,4 (1x) Flow Cytometry Grade</td>
			<td>Gibco</td>
			<td>A12860301</td>
			<td></td>
		</tr>
		<tr>
			<td>Resazurin sodium salt; 10 mg/ml in PBS</td>
			<td>Sigma Aldrich</td>
			<td>R7017</td>
			<td>1:250 dilution</td>
		</tr>
		<tr>
			<td>Ringer&#39;s solution</td>
			<td>Fresenius Kabi</td>
			<td>2610813</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI-1640 Medium</td>
			<td>Sigma Aldrich (Merck)</td>
			<td>R8758</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue culture testplate 6</td>
			<td>TPP</td>
			<td>92006</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma Aldrich</td>
			<td>9002-93-1</td>
			<td></td>
		</tr>
		<tr>
			<td>VECTASTAIN Elite ABC-Peroxidase Kit</td>
			<td>Vector Laboratories</td>
			<td>PK-6200</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylene (extra pure)</td>
			<td>J.T.Baker</td>
			<td>8,11,85,000</td>
			<td></td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ClarioStar Microplate Reader</td>
			<td>BMG Labtech</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Paraffin Embedding Center E61110</td>
			<td>Leica</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Rotary Microtome Microm HM355S</td>
			<td>Thermo Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Section Transfer System Microm STS</td>
			<td>Thermo Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>VT 1200S Vibratom</td>
			<td>Leica</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

organotypic slice cultures as preclinical models of tumor microenvironment in primary pancreatic cancer and metastasis

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 &#181;m 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.

Figure 1&#160;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 &#176;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...

Watch this Scientific Journal Video about Organotypic Slice Cultures as Preclinical Models of Tumor Microenvironment in Primary Pancreatic Cancer and Metastasis at JoVE.com

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

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 ...

Organotypic tissue slices have been generated from various tissues. Due to the preserved tissue integrity and the development of cultivation techniques, they have been suitable for modeling complex physiological mechanisms. Organotypic slice cultures from pancreatic carcinomas closely recalculate the multicellular architecture of the tumor and its microenvironment ex vivo.They can be used for various downstream applications, such as drug response testing. They can be cultured immediately after surgical resection of the tumor, and are inexpensive and less time-consuming compared to most other primary culture techniques. Demonstrating the procedure will be Olha Lapshyna, a technical assistant from the University of Lubeck.To begin, prepare 100 milliliters of low-melting 8%agarose by dissolving eight grams of agarose in 100 milliliters of pre-warmed ringer solution, and store it at four degrees Celsius until needed. Upon the announcement of tumor resection, melt the agarose in a microwave. Place the agarose in a preheated water bath, allowing it to cool to physiological temperatures prior to the preparation.Place a razor blade into the holder of the Vibratome and perform an automated angle adjustment according to the manufacturer's instructions. Cool down the jacket of the cutting chamber using a cooling unit or wet ice. Fill the cutting chamber with approximately 100 milliliters of ringer solution, then place the mounted razor blade into the pre-chilled cutting solution, allowing the razor blade to cool down.Wash the tissue specimen with cooled PBS and place it into a large 14-centimeter Petri dish on ice. Remove macroscopically visible excess connective tissue on ice using a scalpel. Place the tissue into a small Petri dish.Adjust the tissue orientation, so that the remaining macroscopically visible connective tissue has the same orientation as the plain of the bottom of the Petri dish. Pour the prepared low-melting agarose into the small Petri dish, readjusting the orientation of the tissue. If needed, using forceps, then place the Petri dish on wet ice for faster hardening of the agarose.Carefully cut the tissue using a scalpel, leaving at least five millimeters of surrounding agarose on each side of the tissue. Add glue to the sample holder, spread it, and transfer the embedded tissue and glue it on the sample holder using superglue. After a few seconds, place the sample holder into the cutting chamber and adjust the orientation of the tissue toward the razor blade if needed.Define the outer limits of the cutting range according to the size of the tissue specimen. Adjust the blade toward the top of the tissue block. Set the cutting speed to 0.04 millimeters per second, cutting amplitude to one millimeter and slice thickness to 300 micrometers.Carefully cut the first slices and transfer the slices to a separate container with pre-chilled ringer solution on wet ice. Prepare a six-well plate with one milliliter of the appropriate cultivation medium per well. Place the six-well plate with the medium into an incubator, allowing temperature and pH to adjust prior to cultivation.Place slices onto cell culture inserts using a gaze filter, then remove any excess ringer solution by placing the loaded filter onto a sterile cloth. Place the loaded filter into the prepared six-well plate without adding any additional medium to the insert, then place the six-well plate in an incubator and change the medium every two days. Place the cultivation filter with the mounted slice on a Petri dish.With a scalpel, carefully cut out the filter membrane with the mounted tissue slice. Transfer the filter membrane with the mounted slice into a biopsy nylon bag and place it in an embedding cassette. Subsequently, transfer plastic embedding cassettes in a container with pre-chilled 4.5%formalin for a minimum of 24 hours or until further use.Cautiously rinse the formalin fixed slice culture with running tap water for 1.5 hours. Dehydrate the formalin fixed tissue slice by incubation in 70%95%and absolute ethanol. Then, clear the formalin fixed tissue slice with two three-hour incubations in xylene.Immerse the tissue with paraffin at 60 degrees Celsius overnight and then again for two hours. Embed the tissue in a paraffin block in a tissue embedding mold and place it on ice to cool the sample faster. Section the paraffin-embedded tissue block at four-micrometer thickness with a Microtome and in a 40-degree-Celsius water bath containing distilled water.Transfer the sections onto glass slides. Incubate paraffin sections for one hour at 60 degrees Celsius to bond the tissue to the glass. Then, incubate the slides overnight at 37 degrees Celsius.Deparaffinize sections by incubation in xylene three times for five minutes each. Then, rehydrate by successive incubations in absolute alcohol, 95%alcohol, and 70%alcohol. Stain the sections in Mayer Hematoxylin solution for five minutes and rinse with running tap water for 10 minutes.Counterstain in 0.5%ESN solution for 40 seconds and rinse with distilled water, then repeat the dehydration in ethanol. Clear the tissue in three changes of xylene for few seconds each, then place a drop of mounting medium and cover the slides with a cover slip. The macroscopic morphology of each OTSC decreased during cultivation for six days.The in viability is saved for OTSCs from two representative primary tumors in two different media showed a decrease in viability after day zero due to the sectioning procedure and adjustment to culture conditions, as well as an increase in viability of the tumor specimen in the right panel. H&E stain sections showed that the overall structure of the tissue was preserved over the entire time of cultivation ex vivo, and that no gross changes of proliferation and apoptosis were detected during the culture period of six days. Microscopic histopathologic evaluation of H&E sections did not reveal a substantial increase in necrosis of all cultivated tissues during cultivation.There was an increase in cleaved Caspase-3-positive cells after cultivating a primary pancreatic ductal adenocarcinoma, or PDAC, for 15 days. The histopathology of a peritoneal metastasis of a PDAC demonstrated a high intratumoral heterogeneity between individual slices, as well as naturally-occurring apoptosis measured by cleaved Caspase-3 staining. H&E staining and immunohistological characterization for Cytokeratin 7 in a PDAC metastasis of the abdominal wall showed that the derived OTSCs did not contain any tumor cells, but only consisted of connective tissue, which was partially necrotic.Each cultured slice can be profiled individually, depending on the specific research question, for example, by RNA sequencing for transcriptomics or mild imaging for spatially resolved proteomics. In the future, the combination with non-destructive analytical methods has great potential for predicting patient-specific responses to different therapeutic regimes.

Research

JoVE Journal

Medicine

MAME Models for 4D Live-cell Imaging of Tumor: Microenvironment Interactions that Impact Malignant Progression

We have developed 3D coculture models, which we term MAME (mammary architecture and microenvironment engineering), and used them for live-cell imaging in ...

In vitro Mesothelial Clearance Assay that Models the Early Steps of Ovarian Cancer Metastasis

In vitro Mesothelial Clearance Assay that Models the Early Steps of Ovarian Cancer Metastasis

Ovarian cancer is the fifth leading cause of cancer related deaths in the United States1. Despite a positive initial response to therapies, 70 to 90 ...

Models of Bone Metastasis

Bone metastases are a common occurrence in several malignancies, including breast, prostate, and lung. Once established in bone, tumors are responsible ...

Live Imaging of Drug Responses in the Tumor Microenvironment in Mouse Models of Breast Cancer

The tumor microenvironment plays a pivotal role in tumor initiation, progression, metastasis, and the response to anti-cancer therapies. Three-dimensional ...

Initiation of Metastatic Breast Carcinoma by Targeting of the Ductal Epithelium with Adenovirus-Cre: A Novel Transgenic Mouse Model of Breast Cancer

Breast cancer is a heterogeneous disease involving complex cellular interactions between the developing tumor and immune system, eventually resulting in ...

Molecular Profiling of the Invasive Tumor Microenvironment in a 3-Dimensional Model of Colorectal Cancer Cells and Ex vivo Fibroblasts

Molecular Profiling of the Invasive Tumor Microenvironment in a 3-Dimensional Model of Colorectal Cancer Cells and Ex vivo Fibroblasts

Invading colorectal cancer (CRC) cells have acquired the capacity to break free from their sister cells, infiltrate the stroma, and remodel the ...

Ex Vivo Treatment Response of Primary Tumors and/or Associated Metastases for Preclinical and Clinical Development of Therapeutics

Ex Vivo Treatment Response of Primary Tumors and/or Associated Metastases for Preclinical and Clinical Development of Therapeutics

The molecular analysis of established cancer cell lines has been the mainstay of cancer research for the past several decades. Cell culture provides both ...

Primary Tumor and MEF Cell Isolation to Study Lung Metastasis

In breast tumorigenesis, the metastatic stage of the disease poses the greatest threat to the affected individual. Normal breast cells with altered ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor ...

An Organotypic High Throughput System for Characterization of Drug Sensitivity of Primary Multiple Myeloma Cells

In this work we describe a novel approach that combines ex vivo drug sensitivity assays and digital image analysis to estimate chemosensitivity and ...