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.

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Medicine

In vivo Bioluminescence Imaging of Tumor Hypoxia Dynamics of Breast Cancer Brain Metastasis in a Mouse Model

It is well recognized that tumor hypoxia plays an important role in promoting malignant progression and affecting therapeutic response negatively. There is little knowledge about in situ, in vivo, tumor hypoxia during intracranial development of malignant brain tumors because of lack of efficient means to monitor it in these deep-seated orthotopic tumors. Bioluminescence imaging (BLI), based on the detection of light emitted by living cells expressing a luciferase gene, has been rapidly adopted for cancer research, in particular, to evaluate tumor growth or tumor size changes in response to treatment in preclinical animal studies. Moreover, by expressing a reporter gene under the control of a promoter sequence, the specific gene expression can be monitored non-invasively by BLI. Under hypoxic stress, signaling responses are mediated mainly via the hypoxia inducible factor-1&alpha; (HIF-1&alpha;) to drive transcription of various genes. Therefore, we have used a HIF-1&alpha; reporter construct, 5HRE-ODD-luc, stably transfected into human breast cancer MDA-MB231 cells (MDA-MB231/5HRE-ODD-luc). In vitro HIF-1&alpha; bioluminescence assay is performed by incubating the transfected cells in a hypoxic chamber (0.1% O2) for 24 hr before BLI, while the cells in normoxia (21% O2) serve as a control. Significantly higher photon flux observed for the cells under hypoxia suggests an increased HIF-1&alpha; binding to its promoter (HRE elements), as compared to those in normoxia. Cells are injected directly into the mouse brain to establish a breast cancer brain metastasis model. In vivo bioluminescence imaging of tumor hypoxia dynamics is initiated 2 wks after implantation and repeated once a week. BLI reveals increasing light signals from the brain as the tumor progresses, indicating increased intracranial tumor hypoxia. Histological and immunohistochemical studies are used to confirm the in vivo imaging results. Here, we will introduce approaches of in vitro HIF-1&alpha; bioluminescence assay, surgical establishment of a breast cancer brain metastasis in a nude mouse and application of in vivo bioluminescence imaging to monitor intracranial tumor hypoxia.

In vivo Bioluminescence Imaging of Tumor Hypoxia Dynamics of Breast Cancer Brain Metastasis in a Mouse Model

Bioluminescence imaging of hypoxia inducible factor-1&alpha; activity is applied to monitor intracranial tumor hypoxia development in a breast cancer brain metastasis mouse model.

It is well recognized that tumor hypoxia plays an important role in promoting malignant progression and affecting therapeutic response negatively. There ...

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 real-time of cell:cell interactions. Our overall goal was to develop models that recapitulate the architecture of preinvasive breast lesions to study their progression to an invasive phenotype. Specifically, we developed models to analyze interactions among pre-malignant breast epithelial cell variants and other cell types of the tumor microenvironment that have been implicated in enhancing or reducing the progression of preinvasive breast epithelial cells to invasive ductal carcinomas. Other cell types studied to date are myoepithelial cells, fibroblasts, macrophages and blood and lymphatic microvascular endothelial cells. In addition to the MAME models, which are designed to recapitulate the cellular interactions within the breast during cancer progression, we have developed comparable models for the progression of prostate cancers. 
Here we illustrate the procedures for establishing the 3D cocultures along with the use of live-cell imaging and a functional proteolysis assay to follow the transition of cocultures of breast ductal carcinoma in situ (DCIS) cells and fibroblasts to an invasive phenotype over time, in this case over twenty-three days in culture. The MAME cocultures consist of multiple layers. Fibroblasts are embedded in the bottom layer of type I collagen. On that is placed a layer of reconstituted basement membrane (rBM) on which DCIS cells are seeded. A final top layer of 2% rBM is included and replenished with every change of media. To image proteolysis associated with the progression to an invasive phenotype, we use dye-quenched (DQ) fluorescent matrix proteins (DQ-collagen I mixed with the layer of collagen I and DQ-collagen IV mixed with the middle layer of rBM) and observe live cultures using confocal microscopy. Optical sections are captured, processed and reconstructed in 3D with Volocity visualization software. Over the course of 23 days in MAME cocultures, the DCIS cells proliferate and coalesce into large invasive structures. Fibroblasts migrate and become incorporated into these invasive structures. Fluorescent proteolytic fragments of the collagens are found in association with the surface of DCIS structures, intracellularly, and also dispersed throughout the surrounding matrix. Drugs that target proteolytic, chemokine/cytokine and kinase pathways or modifications in the cellular composition of the cocultures can reduce the invasiveness, suggesting that MAME models can be used as preclinical screens for novel therapeutic approaches.

We have developed 3D coculture models for live-cell imaging in real-time of interactions among breast tumor cells and other cells in their microenvironment that impact progression to an invasive phenotype. These models can serve as preclinical screens for drugs to target paracrine-induced proteolytic, chemokine/cytokine and kinase pathways implicated in invasiveness.

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

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 percent of women with ovarian cancer develop new metastases, and the recurrence is often fatal2. It is, therefore, necessary to understand how secondary metastases arise in order to develop better treatments for intermediate and late stage ovarian cancer. Ovarian cancer metastasis occurs when malignant cells detach from the primary tumor site and disseminate throughout the peritoneal cavity. The disseminated cells can form multicellular clusters, or spheroids, that will either remain unattached, or implant onto organs within the peritoneal cavity3 (Figure 1, Movie 1). 
All of the organs within the peritoneal cavity are lined with a single, continuous, layer of mesothelial cells4-6 (Figure 2). However, mesothelial cells are absent from underneath peritoneal tumor masses, as revealed by electron micrograph studies of excised human tumor tissue sections3,5-7 (Figure 2). This suggests that mesothelial cells are excluded from underneath the tumor mass by an unknown process. 
Previous in vitro experiments demonstrated that primary ovarian cancer cells attach more efficiently to extracellular matrix than to mesothelial cells8, and more recent studies showed that primary peritoneal mesothelial cells actually provide a barrier to ovarian cancer cell adhesion and invasion (as compared to adhesion and invasion on substrates that were not covered with mesothelial cells)9,10. This would suggest that mesothelial cells act as a barrier against ovarian cancer metastasis. The cellular and molecular mechanisms by which ovarian cancer cells breach this barrier, and exclude the mesothelium have, until recently, remained unknown. 
Here we describe the methodology for an in vitro assay that models the interaction between ovarian cancer cell spheroids and mesothelial cells in vivo (Figure 3, Movie 2). Our protocol was adapted from previously described methods for analyzing ovarian tumor cell interactions with mesothelial monolayers8-16, and was first described in a report showing that ovarian tumor cells utilize an integrin &#x2013;dependent activation of myosin and traction force to promote the exclusion of the mesothelial cells from under a tumor spheroid17. This model takes advantage of time-lapse fluorescence microscopy to monitor the two cell populations in real time, providing spatial and temporal information on the interaction. The ovarian cancer cells express red fluorescent protein (RFP) while the mesothelial cells express green fluorescent protein (GFP). RFP-expressing ovarian cancer cell spheroids attach to the GFP-expressing mesothelial monolayer. The spheroids spread, invade, and force the mesothelial cells aside creating a hole in the monolayer. This hole is visualized as the negative space (black) in the GFP image. The area of the hole can then be measured to quantitatively analyze differences in clearance activity between control and experimental populations of ovarian cancer and/ or mesothelial cells. This assay requires only a small number of ovarian cancer cells (100 cells per spheroid X 20-30 spheroids per condition), so it is feasible to perform this assay using precious primary tumor cell samples. Furthermore, this assay can be easily adapted for high throughput screening.

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

The mesothelial clearance assay described here takes advantage of fluorescently labeled cells and time-lapse video microscopy to visualize and quantitatively measure the interactions of ovarian cancer multicellular spheroids and mesothelial cell monolayers. This assay 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 for significant morbidity and mortality1. Thus, there is a significant need to understand the molecular mechanisms controlling the establishment, growth and activity of tumors in bone. Several in vivo models have been established to study these events and each has specific benefits and limitations. The most commonly used model utilizes intracardiac inoculation of tumor cells directly into the arterial blood supply of athymic (nude) BalbC mice. This procedure can be applied to many different tumor types (including PC-3 prostate cancer, lung carcinoma, and mouse mammary fat pad tumors); however, in this manuscript we will focus on the breast cancer model, MDA-MB-231. In this model we utilize a highly bone-selective clone, originally derived in Dr. Mundy's group in San Antonio2, that has since been transfected for GFP expression and re-cloned by our group3. This clone is a bone metastatic variant with a high rate of osteotropism and very little metastasis to lung, liver, or adrenal glands. While intracardiac injections are most commonly used for studies of bone metastasis2, in certain instances intratibial4 or mammary fat pad injections are more appropriate. Intracardiac injections are typically performed when using human tumor cells with the goal of monitoring later stages of metastasis, specifically the ability of cancer cells to arrest in bone, survive, proliferate, and establish tumors that develop into cancer-induced bone disease. Intratibial injections are performed if focusing on the relationship of cancer cells and bone after a tumor has metastasized to bone, which correlates roughly to established metastatic bone disease. Neither of these models recapitulates early steps in the metastatic process prior to embolism and entry of tumor cells into the circulation. If monitoring primary tumor growth or metastasis from the primary site to bone, then mammary fat pad inoculations are usually preferred; however, very few tumor cell lines will consistently metastasize to bone from the primary site, with 4T1 bone-preferential clones, a mouse mammary carcinoma, being the exception 5,6.
This manuscript details inoculation procedures and highlights key steps in post inoculation analyses. Specifically, it includes cell culture, tumor cell inoculation procedures for intracardiac and intratibial inoculations, as well as brief information regarding weekly monitoring by x-ray, fluorescence and histomorphometric analyses.

Animal models are frequently utilized to study cancer metastasis to bone. In this protocol we will describe two common methods of tumor inoculation for bone metastasis studies and briefly describe some of the analyses utilized to monitor and quantify these models.

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 co-culture systems are frequently used to explicate tumor-stroma interactions, including their role in drug responses. However, many of the interactions that occur in vivo in the intact microenvironment cannot be completely replicated in these in vitro settings. Thus, direct visualization of these processes in real-time has become an important tool in understanding tumor responses to therapies and identifying the interactions between cancer cells and the stroma that can influence these responses. Here we provide a method for using spinning disk confocal microscopy of live, anesthetized mice to directly observe drug distribution, cancer cell responses and changes in tumor-stroma interactions following administration of systemic therapy in breast cancer models. We describe procedures for labeling different tumor components, treatment of animals for observing therapeutic responses, and the surgical procedure for exposing tumor tissues for imaging up to 40 hours. The results obtained from this protocol are time-lapse movies, in which such processes as drug infiltration, cancer cell death and stromal cell migration can be evaluated using image analysis software.

We describe a method for imaging response to anti-cancer treatment in vivo and at single cell resolution.

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 exponential tumor growth and metastasis to distal tissues and the collapse of anti-tumor immunity. Many useful animal models exist to study breast cancer, but none completely recapitulate the disease progression that occurs in humans. In order to gain a better understanding of the cellular interactions that result in the formation of latent metastasis and decreased survival, we have generated an inducible transgenic mouse model of YFP-expressing ductal carcinoma that develops after sexual maturity in immune-competent mice and is driven by consistent, endocrine-independent oncogene expression. Activation of YFP, ablation of p53, and expression of an oncogenic form of K-ras was achieved by the delivery of an adenovirus expressing Cre-recombinase into the mammary duct of sexually mature, virgin female mice. Tumors begin to appear 6 weeks after the initiation of oncogenic events. After tumors become apparent, they progress slowly for approximately two weeks before they begin to grow exponentially. After 7-8 weeks post-adenovirus injection, vasculature is observed connecting the tumor mass to distal lymph nodes, with eventual lymphovascular invasion of YFP+ tumor cells to the distal axillary lymph nodes. Infiltrating leukocyte populations are similar to those found in human breast carcinomas, including the presence of &#945;&#946; and &#947;&#948; T cells, macrophages and MDSCs. This unique model will facilitate the study of cellular and immunological mechanisms involved in latent metastasis and dormancy in addition to being useful for designing novel immunotherapeutic interventions to treat invasive breast cancer.

Activation of latent mutations with adenovirus-Cre into the mammary ductal system results in a clinically relevant metastatic breast cancer. Incorporation of a YFP promoter allows tracking of distal metastatic tumor cells. This model is useful to study latent metastasis, anti-tumor immunity, and for designing novel immunotherapies to treat 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

Invading colorectal cancer (CRC) cells have acquired the capacity to break free from their sister cells, infiltrate the stroma, and remodel the extracellular matrix (ECM). Characterizing the biology of this phenotypically distinct group of cells could substantially improve our understanding of early events during the metastatic cascade.

Tumor invasion is a dynamic process facilitated by bidirectional interactions between malignant epithelium and the cancer associated stroma. In order to examine cell-specific responses at the tumor stroma-interface we have combined organotypic co-culture and laser micro-dissection techniques.
Organotypic models, in which key stromal constituents such as fibroblasts are 3-dimentioanally co-cultured with cancer epithelial cells, are highly manipulatable experimental tools which enable invasion and cancer-stroma interactions to be studied in near-physiological conditions.

Laser microdissection (LMD) is a technique which entails the surgical dissection and extraction of the various strata within tumor tissue, with micron level precision.
By combining these techniques with genomic, transcriptomic and epigenetic profiling we aim to develop a deeper understanding of the molecular characteristics of invading tumor cells and surrounding stromal tissue, and in doing so potentially reveal novel biomarkers and opportunities for drug development in CRC.&#160;&#160;&#160;

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

Molecular profiling of laser microdissected cells and stroma from synthetic, tissue-engineered colorectal cancer models represents a novel and manipulatable approach to characterize and dissect the distinctive biology at the interface between tumor cells at the invasive front and cancer associated stromal cells. &#160;

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

The molecular analysis of established cancer cell lines has been the mainstay of cancer research for the past several decades. Cell culture provides both direct and rapid analysis of therapeutic sensitivity and resistance. However, recent evidence suggests that therapeutic response is not exclusive to the inherent molecular composition of cancer cells but rather is greatly influenced by the tumor cell microenvironment, a feature that cannot be recapitulated by traditional culturing methods. Even implementation of tumor xenografts, though providing a wealth of information on drug delivery/efficacy, cannot capture the tumor cell/microenvironment crosstalk (i.e., soluble factors) that occurs within human tumors and greatly impacts tumor response. To this extent, we have developed an ex vivo (fresh tissue sectioning) technique which allows for the direct assessment of treatment response for preclinical and clinical therapeutics development. This technique maintains tissue integrity and cellular architecture within the tumor cell/microenvironment context throughout treatment response providing a more precise means to assess drug efficacy.

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

Established cancer cell lines and xenografts have been the mainstay of cancer research for the past several decades. However, recent evidence suggests that therapeutic response is greatly influenced by the tumor cell microenvironment. Therefore, we have developed an ex vivo analysis of primary tumor specimens for drug development purposes.

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 genotypes now possess the ability to invade and survive in other tissues. In this protocol, mouse mammary tumors are removed and primary cells are prepared from tumors. The cells isolated from this procedure are then available for gene profiling experiments. For successful metastasis, these cells must be able to intravasate, survive in circulation, extravasate to distant organs, and survive in that new organ system. The lungs are the typical target of breast cancer metastasis. A set of genes have been discovered that mediates the selectivity of metastasis to the lung. Here we describe a method of studying lung metastasis from a genetically engineered mouse model.. Furthermore, another protocol for analyzing mouse embryonic fibroblasts (MEFs) from the mouse embryo is included. MEF cells from the same animal type provide a clue of non-cancer cell gene expression. Together, these techniques are useful in studying mouse mammary tumorigenesis, its associated signaling mechanisms and pathways of the abnormalities in embryos.

The goal of this protocol is to study breast tumorigenesis. With this technique, mouse mammary tumors are removed and primary cells are prepared from tumors. A lung extraction protocol is included for studying lung metastasis. Furthermore, another protocol for analyzing mouse embryonic fibroblasts from the mouse embryo is included.

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

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 heterogeneity of patient-derived multiple myeloma (MM) cells. This approach consists in seeding primary MM cells freshly extracted from bone marrow aspirates into microfluidic chambers implemented in multi-well plates, each consisting of a reconstruction of the bone marrow microenvironment, including extracellular matrix (collagen or basement membrane matrix) and stroma (patient-derived mesenchymal stem cells) or human-derived endothelial cells (HUVECs). The chambers are drugged with different agents and concentrations, and are imaged sequentially for 96 hr through bright field microscopy, in a motorized microscope equipped with a digital camera. Digital image analysis software detects live and dead cells from presence or absence of membrane motion, and generates curves of change in viability as a function of drug concentration and exposure time. We use a computational model to determine the parameters of chemosensitivity of the tumor population to each drug, as well as the number of sub-populations present as a measure of tumor heterogeneity. These patient-tailored models can then be used to simulate therapeutic regimens and estimate clinical response.

We describe a high-throughput drug sensitivity assay for primary multiple myeloma cells. It consists of a reconstruction of the bone marrow microenvironment (including extracellular matrix and stroma) in multi-well plates, and a non-invasive method for longitudinal quantification of cell viability.

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