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>
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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. 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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 ...

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 ...

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 ...

Tissue-Engineered Graft for Circumferential Esophageal Reconstruction in Rats

The use of biocompatible materials for circumferential esophageal reconstruction is a technically challenging task in rats and requires an optimal implant ...