Name: a combined 3d tissue engineered in vitro/in silico lung tumor model for predicting drug effectiveness in specific mutational backgrounds
Uploaded: 2016-04-06T14:00:00
Description: Watch this Scientific Journal Video about A Combined 3D Tissue Engineered In Vitro/In Silico Lung Tumor Model for Predicting Drug Effectiveness in Specific Mutational Backgrounds at JoVE.com

This research was sponsored by the Center for Interdisciplinary Clinical Research (IZKF, grant BD247) of the University Hospital of Wuerzburg and the Bayern Fit program (granted to Heike Walles).

1. Two-dimensional (2D) Cell Culture
<ol>
	<li>Commercially obtain tumor cell line HCC827 (DSMZ). Culture the lung adenocarcinoma cell line HCC827 (EGFR mutated, KRAS wild-type) in RPMI-1640 supplemented with 20% FCS. Change medium every 2 - 3 days. Split the cells twice a week. Cells are used until passage 20 is reached.</li>
	<li>Commercially obtain tumor cell line A549 (DSMZ). Culture the lung carcinoma cell line A549 (EGFR wild-type, KRAS mutated) in RPMI-1640 supplemented with 1...

<ol>
	<li>Bhattacharjee, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomedicine+Pharma+firms+push+for+sharing+of+cancer+trial+data.">Biomedicine Pharma firms push for sharing of cancer trial data.</a> Science. 338, 29 (2012).</li><li>Kola, I., Landis, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Can+the+pharmaceutical+industry+reduce+attrition+rates?.">Can the pharmaceutical industry reduce attrition rates?.</a> Nat Rev Drug Discov. 3, 711-715 (2004).</li><li>Arrowsmith, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trial+watch:+Phase+II+failures:+2008-2010.">Trial watch: Phase II failures: 2008-2010.</a> Nat Rev Drug Discov. 10, 328-329 (2011).</li><li>Arrowsmith, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trial+watch:+phase+III+and+submission+failures:+2007-2010.">Trial watch: phase III and submission failures: 2007-2010.</a> Nat Rev Drug Discov. 10, 87 (2011).</li><li>Arrowsmith, J., Miller, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trial+watch:+phase+II+and+phase+III+attrition+rates+2011-2012.">Trial watch: phase II and phase III attrition rates 2011-2012.</a> Nat Rev Drug Discov. 12, 569 (2013).</li><li>Pampaloni, F., Reynaud, E. G., Stelzer, E. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+third+dimension+bridges+the+gap+between+cell+culture+and+live+tissue.">The third dimension bridges the gap between cell culture and live tissue.</a> Nat Rev Mol Cell Biol. 8, 839-845 (2007).</li><li>Hartung, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toxicology+for+the+twenty-first+century.">Toxicology for the twenty-first century.</a> Nature. 460, 208-212 (2009).</li><li>Stratmann, A. T., Dandekar, G., Nietzer, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+in+vitro+tumor+Models+as+an+Alternative+for+Animal+Models+in+Preclinical+Studies.">Three-dimensional in vitro tumor Models as an Alternative for Animal Models in Preclinical Studies.</a> Pharm Ind. 75, 485-489 (2013).</li><li>Stratmann, A. T., Dandekar, G., Nietzer, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+in+vitro+tumor+Models+as+an+Alternative+for+Animal+Models+in+Preclinical+Studies.">Three-dimensional in vitro tumor Models as an Alternative for Animal Models in Preclinical Studies.</a> Pharm Ind. 75, 675-680 (2013).</li><li>Gudjonsson, T., Ronnov-Jessen, L., Villadsen, R., Bissell, M. J., Petersen, O. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=To+create+the+correct+microenvironment:+three-dimensional+heterotypic+collagen+assays+for+human+breast+epithelial+morphogenesis+and+neoplasia.">To create the correct microenvironment: three-dimensional heterotypic collagen assays for human breast epithelial morphogenesis and neoplasia.</a> Methods. 30, 247-255 (2003).</li><li>Weaver, V. M., Fischer, A. H., Peterson, O. W., Bissell, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+importance+of+the+microenvironment+in+breast+cancer+progression:+recapitulation+of+mammary+tumorigenesis+using+a+unique+human+mammary+epithelial+cell+model+and+a+three-dimensional+culture+assay.">The importance of the microenvironment in breast cancer progression: recapitulation of mammary tumorigenesis using a unique human mammary epithelial cell model and a three-dimensional culture assay.</a> Biochem Cell Biol. 74, 833-851 (1996).</li><li>Antoni, D., Burckel, H., Josset, E., Noel, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+cell+culture:+a+breakthrough+in+vivo.">Three-dimensional cell culture: a breakthrough in vivo.</a> Int J Mol Sci. 16, 5517-5527 (2015).</li><li>Kim, J., Tanner, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recapitulating+the+Tumor+Ecosystem+Along+the+Metastatic+Cascade+Using+3D+Culture+Models.">Recapitulating the Tumor Ecosystem Along the Metastatic Cascade Using 3D Culture Models.</a> Front Oncol. 5, 170 (2015).</li><li>Worthington, P., Pochan, D. J., Langhans, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peptide+Hydrogels+-+Versatile+Matrices+for+3D+Cell+Culture+in+Cancer+Medicine.">Peptide Hydrogels - Versatile Matrices for 3D Cell Culture in Cancer Medicine.</a> Front Oncol. 5, 92 (2015).</li><li>Tanner, K., Gottesman, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Beyond+3D+culture+models+of+cancer.">Beyond 3D culture models of cancer.</a> Sci Transl Med. 7, 283ps9 (2015).</li><li>Stadler, 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=Increased+complexity+in+carcinomas:+Analyzing+and+modeling+the+interaction+of+human+cancer+cells+with+their+microenvironment.">Increased complexity in carcinomas: Analyzing and modeling the interaction of human cancer cells with their microenvironment.</a> Semin Cancer Biol. , (2015).</li><li>Stratmann, A. T., 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=Establishment+of+a+human+3D+lung+cancer+model+based+on+a+biological+tissue+matrix+combined+with+a+Boolean+in+silico+model.">Establishment of a human 3D lung cancer model based on a biological tissue matrix combined with a Boolean in silico model.</a> Mol Oncol. 8, 351-365 (2014).</li><li>Mok, T. 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=Gefitinib+or+carboplatin-paclitaxel+in+pulmonary+adenocarcinoma.">Gefitinib or carboplatin-paclitaxel in pulmonary adenocarcinoma.</a> N Engl J Med. 361, 947-957 (2009).</li><li>Maemondo, 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=Gefitinib+or+chemotherapy+for+non-small-cell+lung+cancer+with+mutated+EGFR.">Gefitinib or chemotherapy for non-small-cell lung cancer with mutated EGFR.</a> N Engl J Med. 362, 2380-2388 (2010).</li><li>Rosell, R., 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=Erlotinib+versus+standard+chemotherapy+as+first-line+treatment+for+European+patients+with+advanced+EGFR+mutation-positive+non-small-cell+lung+cancer+(EURTAC):+a+multicentre,+open-label,+randomised+phase+3+trial.">Erlotinib versus standard chemotherapy as first-line treatment for European patients with advanced EGFR mutation-positive non-small-cell lung cancer (EURTAC): a multicentre, open-label, randomised phase 3 trial.</a> Lancet Oncol. 13, 239-246 (2012).</li><li>Sequist, L. 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=Phase+III+study+of+afatinib+or+cisplatin+plus+pemetrexed+in+patients+with+metastatic+lung+adenocarcinoma+with+EGFR+mutations.">Phase III study of afatinib or cisplatin plus pemetrexed in patients with metastatic lung adenocarcinoma with EGFR mutations.</a> J Clin Oncol. 31, 3327-3334 (2013).</li><li>Lee, J. M., Dedhar, S., Kalluri, R., Thompson, E. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+epithelial-mesenchymal+transition:+new+insights+in+signaling,+development,+and+disease.">The epithelial-mesenchymal transition: new insights in signaling, development, and disease.</a> J Cell Biol. 172, 973-981 (2006).</li><li>Wells, A., Yates, C., Shepard, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=E-cadherin+as+an+indicator+of+mesenchymal+to+epithelial+reverting+transitions+during+the+metastatic+seeding+of+disseminated+carcinomas.">E-cadherin as an indicator of mesenchymal to epithelial reverting transitions during the metastatic seeding of disseminated carcinomas.</a> Clin Exp Metastasis. 25, 621-628 (2008).</li><li>Janne, P. A., 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=AZD9291+in+EGFR+inhibitor-resistant+non-small-cell+lung+cancer.">AZD9291 in EGFR inhibitor-resistant non-small-cell lung cancer.</a> N Engl J Med. 372, 1689-1699 (2015).</li><li>Moll, 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=Tissue+engineering+of+a+human+3D+in+vitro+tumor+test+system.">Tissue engineering of a human 3D in vitro tumor test system.</a> J Vis Exp. , (2013).</li><li>Funahashi, A., 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=CellDesigner+3.5:+A+Versatile+Modeling+Tool+for+Biochemical+Networks.">CellDesigner 3.5: A Versatile Modeling Tool for Biochemical Networks.</a> Proceedings of the IEEE. 96, 1254-1265 (2008).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Auto Threshold(ImageJ)v.v1.15. , (2013).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> BioVoxxel Toolbox (ImageJ / Fiji). , (2015).</li><li>Buettner, R., Wolf, J., Thomas, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lessons+learned+from+lung+cancer+genomics:+the+emerging+concept+of+individualized+diagnostics+and+treatment.">Lessons learned from lung cancer genomics: the emerging concept of individualized diagnostics and treatment.</a> J Clin Oncol. 31, 1858-1865 (2013).</li><li>Engelman, J. A., 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=MET+amplification+leads+to+gefitinib+resistance+in+lung+cancer+by+activating+ERBB3+signaling.">MET amplification leads to gefitinib resistance in lung cancer by activating ERBB3 signaling.</a> Science. 316, 1039-1043 (2007).</li><li>Mukohara, T., 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=Differential+effects+of+gefitinib+and+cetuximab+on+non-small-cell+lung+cancers+bearing+epidermal+growth+factor+receptor+mutations.">Differential effects of gefitinib and cetuximab on non-small-cell lung cancers bearing epidermal growth factor receptor mutations.</a> J Natl Cancer Inst. 97, 1185-1194 (2005).</li><li>Noro, R., 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=Gefitinib+(IRESSA)+sensitive+lung+cancer+cell+lines+show+phosphorylation+of+Akt+without+ligand+stimulation.">Gefitinib (IRESSA) sensitive lung cancer cell lines show phosphorylation of Akt without ligand stimulation.</a> BMC Cancer. 6, 277 (2006).</li><li>Gill, B. 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=A+synthetic+matrix+with+independently+tunable+biochemistry+and+mechanical+properties+to+study+epithelial+morphogenesis+and+EMT+in+a+lung+adenocarcinoma+model.">A synthetic matrix with independently tunable biochemistry and mechanical properties to study epithelial morphogenesis and EMT in a lung adenocarcinoma model.</a> Cancer Res. 72, 6013-6023 (2012).</li></ol>

We have established a combined in vitro/in silico tumor test system for biomarker-guided treatment predictions. The in vitro model evaluates different important aspects of compound actions such as changes of tumor cell proliferation and apoptosis on a specific mutational background that can also be simulated in silico17. Here, we present the standardized protocol for 3D tumor model generation and compound testing including quantification of proliferation and apoptosis and the establi...

The pharmaceutical industry is facing high attrition rates of up to 95% in the field of cancer treatment in the clinical phase causing enormous costs 1-5. One reason for this deficit is the fact that currently efficacy of potential new compounds is assessed in large-scale screenings on 2D cell cultures of cancer cell lines or in animal models. Animal models have a higher complexity but there are crucial differences between mice and men 6,7. In the last decade, 3D cancer models using different approaches have been generated to bridge the gap between 2D culture of cancer cell lines and a complex in vivo tumor 6,8,9. The impact of 3D environment on cell differentiation and also on signaling has been shown in several studies years ago (e.g., by Mina Bissell) 10,11. Today, many 3D cell culture models are available such as spheroid cultures, hydrogels or microfluidic chips 12-16. Even though these models enhance complexity compared to conventional 2D culture systems, they mostly lack a tissue microenvironment that is known to have tumor-supporting effects and also impacts drug efficacy.
To address this issue, we generated a 3D tumor model based on a biological scaffold called SISmuc (small-intestine-submucosa + mucosa) that is derived from a decellularized porcine jejunum. Thereby, the tissue architecture and important components of the ECM such as different collagens as well as the basement membrane structure are preserved 17. This unique feature is crucial for tumor model generation of carcinomas that arise from epithelia and comprise about 80% of solid tumors. Furthermore, the proliferation rate in our tissue-engineered tumor model is reduced compared to the artificially high rates achieved in 2D culture. As proliferation is an important parameter in assessing drug efficacy, drug testing is enabled in our model in more similar conditions to in vivo tumors 17.
In order to evaluate the potential of our model to predict biomarker-dependent drug efficacy correctly, we here present data for two different lung cancer cell lines that differ in their EGFR-biomarker status. This mutational status has started to be determined routinely in NSCLC patients. Targeted treatments with TKIs such as the EGFR-inhibitor gefitinib against tumors bearing an activating EGFR mutation show superior outcomes compared to those with platinum-based chemotherapy 18-21.
We established several read-out techniques that are relevant for evaluating compound efficacy. Furthermore, after TGF-beta-1 stimulation we are able to investigate compound actions in tumor cells that started the EMT process, which is thought to be an important step in malignant transformation 22,23 and which is connected to drug resistance 24.
The 3D tumor model allow monitoring cell-specific responses to targeted treatments, chemotherapy, or drug combinations with good contrasts. To further enhance and speed up drug-screening and to encounter resistance, this is complemented by an in silico simulation. Based on a few experiments, the tumor response can be predicted in silico regarding the outcome for a full range of drugs and their combinations.

<table border="0" cellpadding="0" cellspacing="0" width="808">
	<tbody>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Bioreactors</td>
			<td style="width:261px;">Chair of Tissue Engineering and Regenerative Medicine, W&#252;rzburg (GER)</td>
			<td style="width:119px;"></td>
			<td style="width:212px;">Bioreactor setup</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">BioVoxxel Toolbox (ImageJ / Fiji)</td>
			<td style="width:261px;">Jan Brocher, Thorsten Wagner, https://github.com/biovoxxel/BioVoxxel_Toolbox</td>
			<td style="width:119px;"></td>
			<td style="width:212px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Cell crowns</td>
			<td style="width:261px;">Chair of Tissue Engineering and Regenerative Medicine, W&#252;rzburg (GER)</td>
			<td style="width:119px;"></td>
			<td style="width:212px;">for static 3D culture</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">CellDesigner</td>
			<td style="width:261px;">http://www.celldesigner.org/</td>
			<td style="width:119px;">-</td>
			<td style="width:212px;">This software was used for drawing the network.</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;">citrate buffer stock solution (10x)</td>
			<td style="width:261px;">in house production</td>
			<td style="width:119px;"></td>
			<td style="width:212px;">42 g/l Citric acid monohydrate, 17.,6 g/l Sodium hydroxide pellets in deionized water, pH 6,.0, stored at RT.&#160;</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;">citrate buffer working solution</td>
			<td style="width:261px;">in house production</td>
			<td style="width:119px;"></td>
			<td style="width:212px;">10 % Citrate buffer stock solution in demineralized water, stored at RT.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Citric acid monohydrate</td>
			<td style="width:261px;">VWR, Darmstadt (GER)</td>
			<td style="width:119px;">1002441000</td>
			<td style="width:212px;">used for the citrate buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Cover slips</td>
			<td style="width:261px;">VWR, Darmstadt (GER)</td>
			<td>631-1339</td>
			<td style="width:212px;"></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:216px;">DAPI Fluoromount-GTM</td>
			<td style="width:261px;">SouthernBiotech, Birmingham (USA)</td>
			<td style="width:119px;">SBA-0100-20</td>
			<td style="width:212px;"></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Databases such as KEGG, HPRD and QIAGEN (Genes &#38; Pathways)</td>
			<td style="width:261px;">http://www.genome.jp/kegg/pathway.html; http://www.hprd.org/; https://www.qiagen.com/de/geneglobe/</td>
			<td style="width:119px;">-</td>
			<td style="width:212px;">Different known literature databases were used for generating the network topology.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Female Luer Lug Style Tee</td>
			<td style="width:261px;">Mednet, M&#252;nster (GER)</td>
			<td style="width:119px;">FTLT-1</td>
			<td style="width:212px;">Bioreactor setup</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Female Luer Thread Style with 5/16&#34; Hex to 1/4-28 UNF Thread</td>
			<td style="width:261px;">Mednet, M&#252;nster (GER)</td>
			<td style="width:119px;">SFTLL-J1A&#160;</td>
			<td style="width:212px;">Bioreactor setup</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Fetal calf serum</td>
			<td style="width:261px;">Bio&#38;SELL, Feucht (GER)</td>
			<td style="width:119px;">FCS.ADD.0500</td>
			<td style="width:212px;">not heat-inactivated</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Gefitinib</td>
			<td style="width:261px;">Absource Diagnostics GmbH, M&#252;nchen (GER)</td>
			<td style="width:119px;">S1025-100 mg</td>
			<td style="width:212px;">100 mM stock solution with DMSO</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Glas flask (Schott, GER) provided with glas hose connection</td>
			<td style="width:261px;">Weckert, Kitzingen (GER)</td>
			<td style="width:119px;">custom made</td>
			<td style="width:212px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Histofix 4 % (Paraformaldehyd)</td>
			<td style="width:261px;">Carl Roth, Karlsruhe (GER)</td>
			<td style="width:119px;">P087.1</td>
			<td style="width:212px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Hose coupling</td>
			<td style="width:261px;">Mednet, M&#252;nster (GER)</td>
			<td>CC-9</td>
			<td style="width:212px;">Bioreactor setup</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Incubator for bioreactors</td>
			<td style="width:261px;">Chair of Tissue Engineering and Regenerative Medicine, W&#252;rzburg (GER)</td>
			<td style="width:119px;"></td>
			<td style="width:212px;">Bioreactor setup</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:216px;">M30 CytoDeathTM ELISA</td>
			<td style="width:261px;">Peviva, Bromma (SWE)</td>
			<td style="width:119px;">10900</td>
			<td style="width:212px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Male Luer Integral Lock Ring</td>
			<td style="width:261px;">Mednet, M&#252;nster (GER)</td>
			<td style="width:119px;">MTLL230-J1A</td>
			<td style="width:212px;">Bioreactor setup</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Moisture chamber</td>
			<td style="width:261px;">custom made</td>
			<td style="width:119px;"></td>
			<td style="width:212px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Mouse anti Pan-Cytokeratin</td>
			<td style="width:261px;">Sigma-Aldrich, Munich (GER)&#160;&#160;</td>
			<td style="width:119px;">C2562-2ML</td>
			<td style="width:212px;">Clone C-11+PCK-26+CY-90+KS-1A3+M20+A53-B/A2, used 1/100 for immunofluorescence</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Needlefree Swabable Valve Female Luer</td>
			<td style="width:261px;">Mednet, M&#252;nster (GER)</td>
			<td>NVFMLLPC</td>
			<td style="width:212px;">Bioreactor setup, for sampling, gamma-sterilized</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">O-Ring MVQ 10 red 37*3 mm</td>
			<td style="width:261px;">Arcus Dichtelemente, Seevetal (GER)</td>
			<td style="width:119px;">21444</td>
			<td>O-ring large, Bioreactor setup</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">O-Ring MVQ 70 red 27*2.5 mm</td>
			<td style="width:261px;">Arcus Dichtelemente, Seevetal (GER)</td>
			<td style="width:119px;">19170</td>
			<td>O-ring small, Bioreactor setup</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">PAP pen</td>
			<td style="width:261px;">Dako, Hamburg (GER)</td>
			<td>S002</td>
			<td style="width:212px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Paraffin</td>
			<td style="width:261px;">Carl Roth, Karlsruhe (GER)</td>
			<td style="width:119px;">6642.6</td>
			<td style="width:212px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Peristaltic pump</td>
			<td style="width:261px;">Ismatec, Wertheim-Mondfeld (GER)</td>
			<td style="width:119px;"></td>
			<td style="width:212px;">Bioreactor setup</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Phosphate Buffered Saline</td>
			<td style="width:261px;">Sigma-Aldrich, Munich (GER)&#160;&#160;</td>
			<td style="width:119px;">D8537-6x500ml</td>
			<td style="width:212px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Pump tubing cassette</td>
			<td style="width:261px;">Ismatec, Wertheim (GER)</td>
			<td>IS 3710</td>
			<td style="width:212px;">Bioreactor setup</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Rabbit anti Ki67</td>
			<td style="width:261px;">Abcam, Cambridge (UK)</td>
			<td style="width:119px;">ab16667</td>
			<td style="width:212px;">Clone SP6, used for 1/100 for IF</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Rabbit anti Vimentin</td>
			<td style="width:261px;">Abcam, Cambridge (UK)</td>
			<td style="width:119px;">ab92547</td>
			<td style="width:212px;">used 1/100 for IF</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">RPMI-1640 medium</td>
			<td style="width:261px;">Life technologies, Darmstadt (GER)</td>
			<td style="width:119px;">61870-044</td>
			<td style="width:212px;">warm in 37&#176;C waterbath before use</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Silicone tube</td>
			<td style="width:261px;">Carl Roth GmbH, Karlsruhe (GER)</td>
			<td style="width:119px;">HC66.1</td>
			<td style="width:212px;">Bioreactor setup</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sodium Hydroxide</td>
			<td style="width:261px;">Sigma-Aldrich, M&#252;nchen (GER)</td>
			<td>30620-1KG-R</td>
			<td style="width:212px;">used for the citrate buffer</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">SQUAD</td>
			<td style="width:261px;">http://sbos.eu/docu/docu/SQUAD/doku.php.htm</td>
			<td style="width:119px;">-</td>
			<td style="width:212px;">This software was used for performing the semiquantitative simulations.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Sterile air filter, pore size 0.2 &#181;m</td>
			<td style="width:261px;">Sartorius Stedium Biotech, G&#246;ttlingen (GER)</td>
			<td style="width:119px;">16596-HYK</td>
			<td style="width:212px;">Bioreactor setup</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Syringe Luer Lok 5ml</td>
			<td>BD Biosciences, Heidelberg (GER)</td>
			<td>309649</td>
			<td style="width:212px;">for bioreactor sampling</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Tissue culture test plates: 6-,&#160;&#160;&#160;&#160;&#160; 12-, 24-, 96- well</td>
			<td style="width:261px;">TPP Techno Plastic Products AG, Trasadingen (GER)</td>
			<td style="width:119px;">92006, 92012, 92024, 92048&#160;</td>
			<td style="width:212px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Transforming growth factor-beta 1 (TGF-&#946;1) with carrier</td>
			<td style="width:261px;">Cell Signaling, Frankfurt (GER)</td>
			<td style="width:119px;">8915LC</td>
			<td style="width:212px;">stock solution in sterile citrate buffer pH 3.0</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Triton X-100</td>
			<td style="width:261px;">Sigma-Aldrich, M&#252;nchen (GER)</td>
			<td style="width:119px;">X100-1L</td>
			<td style="width:212px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Tween-20</td>
			<td style="width:261px;">Sigma-Aldrich, M&#252;nchen (GER)</td>
			<td style="width:119px;">P7949-500ml</td>
			<td style="width:212px;">for washing buffer of immunofluorescent staining</td>
		</tr>
	</tbody>
</table>

a combined 3d tissue engineered in vitro/in silico lung tumor model for predicting drug effectiveness in specific mutational backgrounds

In the present study, we combined an in vitro 3D lung tumor model with an in silico model to optimize predictions of drug response based on a specific mutational background. The model is generated on a decellularized porcine scaffold that reproduces tissue-specific characteristics regarding extracellular matrix composition and architecture including the basement membrane. We standardized a protocol that allows artificial tumor tissue generation within 14 days including three days of drug treatment. Our article provides several detailed descriptions of 3D read-out screening techniques like the determination of the proliferation index Ki67 staining&#39;s, apoptosis from supernatants by M30-ELISA and assessment of epithelial to mesenchymal transition (EMT), which are helpful tools for evaluating the effectiveness of therapeutic compounds. We could show compared to 2D culture a reduction of proliferation in our 3D tumor model that is related to the clinical situation. Despite of this lower proliferation, the model predicted EGFR-targeted drug responses correctly according to the biomarker status as shown by comparison of the lung carcinoma cell lines HCC827 (EGFR -mutated, KRAS wild-type) and A549 (EGFR wild-type, KRAS-mutated) treated with the tyrosine-kinase inhibitor (TKI) gefitinib. To investigate drug responses of more advanced tumor cells, we induced EMT by long-term treatment with TGF-beta-1 as assessed by vimentin/pan-cytokeratin immunofluorescence staining. A flow-bioreactor was employed to adjust culture to physiological conditions, which improved tissue generation. Furthermore, we show the integration of drug responses upon gefitinib treatment or TGF-beta-1 stimulation - apoptosis, proliferation index and EMT - into a Boolean in silico model. Additionally, we explain how drug responses of tumor cells with a specific mutational background and counterstrategies against resistance can be predicted. We are confident that our 3D in vitro approach especially with its in silico expansion provides an additional value for preclinical drug testing in more realistic conditions than in 2D cell culture.

On the basis of the SISmuc scaffold (Figure 2A to C), we established a standardized operating protocol for the generation, stimulation and treatment of a 3D tumor test system (Figure 2D). This model enables the determination of the proliferation index and the quantification of apoptosis using M30-ELISA as shown in Figure 1 and Figure 3, respectively. Figure 3 shows representative H&#38;E staining of A549 ...

Watch this Scientific Journal Video about A Combined 3D Tissue Engineered In Vitro/In Silico Lung Tumor Model for Predicting Drug Effectiveness in Specific Mutational Backgrounds at JoVE.com

A Combined 3D Tissue Engineered In Vitro/In Silico Lung Tumor Model for Predicting Drug Effectiveness in Specific Mutational Backgrounds

We present a three-dimensional (3D) lung cancer model based on a biological collagen scaffold to study sensitivity towards non-small-cell-lung-cancer-(NSCLC)-targeted therapies. We demonstrate different read-out techniques to determine the proliferation index, apoptosis and epithelial-mesenchymal transition (EMT) status. Collected data are integrated into an in silico model for prediction of drug sensitivity.

A Combined 3D Tissue Engineered In Vitro/In Silico Lung Tumor Model for Predicting Drug Effectiveness in Specific Mutational Backgrounds

In the present study, we combined an in vitro 3D lung tumor model with an in silico model to optimize predictions of drug response based on a specific ...

The overall goals of this combined In Vitro/In Silico Lung Cancer Model are to study the sensitivity of non-small cell lung cancer targeted therapies and to predict the efficacy of new substances. This 3-D model system allows the monitoring of cell specific responses to targeted treatments or direct combinations. The main advantage of this technique is that the covered with in Silico predictions can facilitate pre-clinical drug testing in a more physiological relevant environment.The implications of this technique extend towards the therapy of different tumor entities, as it can be used to optimize drug response predictions based on specific mutational backgrounds. Demonstrating the procedure will be, Franziska Schmitt, a research assistant, from our laboratory. To set up a dynamic culture, begin by culturing the tumor model under static culture conditions, in a cell crown at 37 degrees Celsius and 5%CO2, in a conventional incubator.After three days, assemble an Autoclaved Bioreactor, and insert a seeded, biological small intestine submucoso plus mucosa scaffold, into the system. Next, add 45 milliliters of RPMI, supplemented with the appropriate amount of FCS to a flask and connect the needle free sampling device to the tubing system between the Bioreactor and the flask of medium. Place the Bioreactor into the incubator and connect it to the pump to culture the tumor model, for an additional 14 days, with a constant medium flow of three milliliters per minute.To treat a static tumor model with, Gefitinib, on day 11 of culture in the conventional incubator, first use a pasteur pipet to remove the old medium, from the wells. Then, add one milliliter of freshly prepared, RPMI, supplemented with, FCS and Gefitinib, to the inside of each cell crown and 1.5 milliliters to the bottom of each well. To collect Elisa's samples from a dynamic culture, on days 11 through 14 of culture, attach the syringe to the sampling device and remove one milliliter of medium from the Bioreactor system.Then store the samples at, minus 80 degrees Celsius, until the Elisa is performed. To determine the number of Ki67 positive nuclei, first use an inverted microscope to obtain five DAPI and five Ki67 staining fluorescent images of 2D cultures at a 20 fold magnification of each condition, from non-overlapping sections of the specimen. To manually count the Ki67 positive nuclei, open emerged image in Fiji and select, Plugins, and Cell Counter, then, click initialize, select a counter type and click on each Ki67 positive nucleus.The number of clicked cells will appear next to the selected counter type. For automatic cell counting of the total number of nuclei in the 2D images, start the macro recorder, open a DAPI image, and click image, type and 8-bit, then click enhance contrast and set the saturated parameter to one and normalize. To sharpen the image, set the unsharp mask, using a radius of one and a mask of 60.To binderize the image, click auto threshold and choose the, Reny/Entropy method with white objects on black background. Then, click Plugins, followed by BioVoxxel, and watershed irregular feature, with an erosion radius of five to separate the cells. Click analyze, and analyze particles and set the size to 02 infinity and check summarize.The number of cells will be shown in the summary table as counts. Then, click create, to save the macro to allow automatic counting of nuclei, in further 2D cell culture images. After using know databases to generate a tumor network, open the appropriate network analysis software, click file and new, and input jove tumor models.Click ok, to generate a new network, then click, square closeup North and ok, to visualize the receptor in a double membrane. To visualize the nodes as rectangles, click generic protein, then EGFR, then Ok.To label the nodes in the network, right-click to change color and shape, and set the node color codes as indicated. To visualize the readout parameters as hexagons, select, phenotype, enter proliferation, and click ok.Then, right-click and select change color and shape again to set the color code to 255, 204, 204. To visualize the edges of node interactions, click state transition for activation arrows, and inhibition for blunted inhibition arrows. Then, click file and save as, and save the generated signaling network, as jove tumor models.xml.For steady state analysis of the system equilibrium, open the SQUAD software, click load network, and upload the jove tumor models. xml file. Click, run analysis.To write a simulation protocol, click advanced, and pertubator, then click edit protocol, to simulate the anit-EGFR resistance. Next, click, pertubation and select the standard initial state equals steady state four. Click add, to add the new constant pulse, then select the state parameter and set the target to FLIP, the time to zero, and the value to 4.Click, ok, and set the states for c-MET and Gefitinib, in the same way. To set a state value to the active EGFR node, click active node, then, click edit. Set the state to 8 and click ok.Click ok, to save the protocol. To display the specific curves, under the options panel, select EGF-EGFR, star EGF-EGFR, star Mek, caspases, PI3K, Gegitinib, apoptosis, proliferation, MEk inhibitor and PI3K inhibitor. Then, click initialize and run to start a complete simulation of the systems response.To simulate a combined PI3K and Mek inhibitor treatment, click edit protocol. Next, using the same node parameter as just demonstrated, click pertubation, followed by add, to add a new constant pulse. Then, select a state parameter, and set the target to PI3K inhibitor, the time to zero and the value to one, and click ok.Click add, again, to add another new constant pulse and under the state parameter, set the target to Mek inhibitor, set the time to zero and the value to one, and click ok, followed by ok again, to save the protocol. Under the options panel, using the same nodes for the graphical representation as just demonstrated, click initialize, and run to start a complete simulation of the systems response. Then, to finish the simulation, click reset.This 3D tumor test system, facilitates the determination of the proliferation index. And the quantification of apoptosis using M30 Elisa. In this representative experiment, for example, only EGFR mutated HCC827 tumor cells, incubated under static culture conditions, responded to epidermal growth factor inhibition.By the Tyrosine-kinase inhibitor, Gefitinib, as evident by the increase in apoptosis observed in the HCC827 tumor model system. Here representative images of static 3D tumor models, with or without, Epithelial-Mesenchymal transition inducer, TGF-beta-1 simulation, are shown. Note the increase in vimentin expression, in the treated tumor cell cultures.Under dynamic conditions, Gefitinib, exerts a robust effect on, EGFR mutated, HCC827 cells, as evidenced by the decrease of tissue-like structures, in the presence of the Tyrosine-kinase inhibitor, compared to the untreated controlled culture. In this graph, the simulation of the known drive in mutation, EGFR and c-MET co-activation, and increased proliferation rate, and a decreased apoptosis rate in HCC827 cells, over time, are observed, reflecting a Gefitinib induced resistance that correlates with activation of MEK and PI3K. Indeed, modeling of the combined PI3K and MEK inhibitors results in a reversal of the anti-EGFR resistance effect, a reduction in proliferation and the induction of apoptosis.Following this procedure, other methods like, Western Blot or can be performed to investigate downstream signaling pathways. Moreover, co-culture systems with different cell types may also be used to analyze the cross-talk between various cells in the tumor micro environment. This technique paves the way for researchers in the field of drug development to explore drug efficacy in Silico.After watching this video, you should have a good understanding of how to predict drug efficacy in stratified patient groups, by simulating signaling changes and analyzing the resulting system responses in different mutation and backgrounds.

Research

JoVE Journal

Bioengineering

Tissue Engineering of a Human 3D in vitro Tumor Test System

Tissue Engineering of a Human 3D in vitro Tumor Test System

Cancer is one of the leading causes of death worldwide.  Current therapeutic strategies are predominantly developed in 2D culture  systems, which ...

Generation and Grafting of Tissue-engineered Vessels in a Mouse Model

The construction of vascular conduits is a fundamental strategy for surgical repair of damaged and injured vessels resulting from cardiovascular diseases. ...

Production, Characterization and Potential Uses of a 3D Tissue-engineered Human Esophageal Mucosal Model

The incidence of both esophageal adenocarcinoma and its precursor, Barrett&#8217;s Metaplasia, are rising rapidly in the western world. Furthermore ...

Engineering 3D Cellularized Collagen Gels for Vascular Tissue Regeneration

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. ...

Engineered 3D Silk-collagen-based Model of Polarized Neural Tissue

Despite huge efforts to decipher the anatomy, composition and function of the brain, it remains the least understood organ of the human body. To gain a ...

Engineered Vascularized Muscle Flap

One of the main factors limiting the thickness of a tissue construct and its consequential viability and applicability in vivo, is the control of oxygen ...

Automated Contraction Analysis of Human Engineered Heart Tissue for Cardiac Drug Safety Screening

Cardiac tissue engineering describes techniques to constitute three dimensional force-generating engineered tissues. For the implementation of these ...

Repressing Gene Transcription by Redirecting Cellular Machinery with Chemical Epigenetic Modifiers

Regulation of chromatin compaction is an important process that governs gene expression in higher eukaryotes. Although chromatin compaction and gene ...

Seeding and Implantation of a Biosynthetic Tissue-engineered Tracheal Graft in a Mouse Model

Treatment options for congenital or secondary long segment tracheal defects have historically been limited due to an inability to replace functional ...

Human Neural Organoids for Studying Brain Cancer and Neurodegenerative Diseases

The lack of relevant in vitro neural models is an important obstacle on medical progress for neuropathologies. Establishment of relevant cellular models ...