Name: generation and expansion of primary, malignant pleural mesothelioma tumor lines
Uploaded: 2022-04-21T12:40:00
Description: Watch this Scientific Journal Video about Generation and Expansion of Primary, Malignant Pleural Mesothelioma Tumor Lines at JoVE.com

We would like to acknowledge Raquel Laza-Briviesca for her contribution to starting this protocol and Drs. Boris Sepesi, Reza Mehran, and David Rice for collaboration on tissue collections. There is no additional funding associated with this work.

All methods described here have been approved by the Institution Review Board (IRB) of the University of Texas MD Anderson Cancer Center. This pertains to standard-of-care, surgically resected MPM tumors removed following informed consent.
1. Preparation of tumor digestion media and other associated media
<ol>
	<li>To prepare tumor digestion media, add 5 mL of 100% Pen/Strep (Penicillin/Streptomycin) to 500 mL of sterile Roswell Park Memorial Institute (RPMI) 1640 medium in ...

<ol>
	<li>Guzman-Casta, 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=Prognostic+factors+for+progression-free+and+overall+survival+in+malignant+pleural+mesothelioma.">Prognostic factors for progression-free and overall survival in malignant pleural mesothelioma.</a> Thoracic Cancer. 12 (7), 1014-1022 (2021).</li><li>Baas, P., 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=First-line+nivolumab+plus+ipilimumab+in+unresectable+malignant+pleural+mesothelioma+(CheckMate+743):+a+multicentre,+randomised,+open-label,+phase+3+trial.">First-line nivolumab plus ipilimumab in unresectable malignant pleural mesothelioma (CheckMate 743): a multicentre, randomised, open-label, phase 3 trial.</a> Lancet. 397 (10272), 375-386 (2021).</li><li>Testa, J. R., Berns, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preclinical+models+of+malignant+mesothelioma.">Preclinical models of malignant mesothelioma.</a> Frontiers in Oncology. 10, 101 (2020).</li><li>Usami, N., 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+and+characterization+of+four+malignant+pleural+mesothelioma+cell+lines+from+Japanese+patients.">Establishment and characterization of four malignant pleural mesothelioma cell lines from Japanese patients.</a> Cancer Science. 97 (5), 387-394 (2006).</li><li>Kobayashi, M., Takeuchi, T., Ohtsuki, Y. <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+three+novel+human+malignant+pleural+mesothelioma+cell+lines:+morphological+and+cytogenetical+studies+and+EGFR+mutation+status.">Establishment of three novel human malignant pleural mesothelioma cell lines: morphological and cytogenetical studies and EGFR mutation status.</a> Anticancer Research. 28 (1), 197-208 (2008).</li><li>Chernova, 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=Molecular+profiling+reveals+primary+mesothelioma+cell+lines+recapitulate+human+disease.">Molecular profiling reveals primary mesothelioma cell lines recapitulate human disease.</a> Cell Death and Differentiation. 23 (7), 1152-1164 (2016).</li><li>Boj, S. 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=Organoid+models+of+human+and+mouse+ductal+pancreatic+cancer.">Organoid models of human and mouse ductal pancreatic cancer.</a> Cell. 160 (1-2), 324-338 (2015).</li><li>Han, A. 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=Differential+expression+of+N-cadherin+in+pleural+mesotheliomas+and+E-cadherin+in+lung+adenocarcinomas+in+formalin-fixed,+paraffin-embedded+tissues.">Differential expression of N-cadherin in pleural mesotheliomas and E-cadherin in lung adenocarcinomas in formalin-fixed, paraffin-embedded tissues.</a> Human Pathology. 28 (6), 641-645 (1997).</li><li>Tachibana, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=N-cadherin-mediated+aggregate+formation;+cell+detachment+by+Trypsin-EDTA+loses+N-cadherin+and+delays+aggregate+formation.">N-cadherin-mediated aggregate formation; cell detachment by Trypsin-EDTA loses N-cadherin and delays aggregate formation.</a> Biochemical and Biophysical Research Communications. 516 (2), 414-418 (2019).</li><li>Donnenberg, V. S., Corselli, M., Normolle, D. P., Meyer, E. M., Donnenberg, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+cytometric+detection+of+most+proteins+in+the+cell+surface+proteome+is+unaffected+by+trypsin+treatment.">Flow cytometric detection of most proteins in the cell surface proteome is unaffected by trypsin treatment.</a> Cytometry A. 93 (8), 803-810 (2018).</li><li>Pham, K., 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=Isolation+of+pancreatic+cancer+cells+from+a+patient-derived+xenograft+model+allows+for+practical+expansion+and+preserved+heterogeneity+in+culture.">Isolation of pancreatic cancer cells from a patient-derived xenograft model allows for practical expansion and preserved heterogeneity in culture.</a> American Journal of Pathology. 186 (6), 1537-1546 (2016).</li><li>Derycke, L. D., Bracke, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=N-cadherin+in+the+spotlight+of+cell-cell+adhesion,+differentiation,+embryogenesis,+invasion+and+signalling.">N-cadherin in the spotlight of cell-cell adhesion, differentiation, embryogenesis, invasion and signalling.</a> International Journal of Developmental Biology. 48 (5-6), 463-476 (2004).</li></ol>

While this protocol is straightforward, there are a few critical steps that must be closely followed. The early-passage freeze is important to preserve the ability to repeat the tumor-cell enrichment process if initially unsuccessful. The ability to assess fibroblastic contamination by eye to decide the correct splitting and media starvation technique is vital to preventing fibroblast overgrowth in the culture. In addition, the differential trypsinization method requires careful observation of the cells during incubation...

Malignant pleural mesothelioma (MPM) is a rare tumor highly associated with asbestos exposure. Although immunotherapy-based approaches have shown encouraging results, there is a paucity of treatment options available to patients that develop this disease, and the overall 5-year survival rate is low1,2. Efforts are underway at multiple institutions to better understand this disease and identify novel therapeutic targets that may improve patient outcomes. While there are multiple mesothelioma mouse models, access to primary mesothelioma tumor cells is more limited3. In vitro expansion of primary mesothelioma tumor cells would provide a valuable model system that can be utilized to study tumor cells directly and their interaction with autologous immune cells such as tumor-infiltrating lymphocytes. While there have been reports on the expansion of primary mesothelioma tumor cells lines, these are few and do not provide a detailed standard operation procedure (SOP). Furthermore, few cell lines are available from commercial sources such as American Type Culture Collection (ATCC). While the availability of primary tumor lines is limited, it has been demonstrated that tumor cells can be expanded from pleural effusions and directly from the tumor tissue4,5. In addition, expanded tumor cell lines have been shown to preserve the molecular profile of the original tumor4,5,6.
Our laboratory is studying the tumor-immune microenvironment of MPM and has developed a method for expanding primary MPM tumor cells lines from surgically resected cases. This method is adapted from our experience in establishing primary metastatic melanoma tumor cell lines. The goal of this work is to provide a detailed, practical approach to primary mesothelioma tumor line expansion using a 2D model system and subsequent phenotypic profiling. Given the recent success of checkpoint blockade strategies targeting CTLA4 and PD-1 in the first-line setting2, the ability to generate many primary tumor lines could further the understanding of both tumor intrinsic mechanisms of resistance as well as provide an important model system to assess T-cell recognition, thus deepening our understanding of the immune response in MPM.
The major limitation of this protocol is that every tumor contains a different microenvironment, and there is a high degree of variability of expansion success. In addition, this method selects for tumor cells that can expand in a 2D culture system. Other methods that involve the production of a 3D spheroid or organoid culture could provide an alternative approach that may allow for a higher success rate of expansion or result in the ability to derive cell lines that are unable to expand in a traditional 2D system. Such 3D cultures have been demonstrated to be useful for the generation of tumor types that are difficult to expand, for example, pancreatic cancer7.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>10 mL serological pipettes</td>
			<td>BD Falcon</td>
			<td>357551</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL conical tubes</td>
			<td>BD Falcon</td>
			<td>352097</td>
			<td></td>
		</tr>
		<tr>
			<td>2 mL aspirating pipettes</td>
			<td>BD Falcon</td>
			<td>357558</td>
			<td></td>
		</tr>
		<tr>
			<td>5 mL serological pipettes</td>
			<td>BD Falcon</td>
			<td>357543</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL conical tubes</td>
			<td>BD Falcon</td>
			<td>352098</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well microplates, tissue culture treated</td>
			<td>Corning</td>
			<td>3516</td>
			<td></td>
		</tr>
		<tr>
			<td>Accutase</td>
			<td>Innovative Cell Technologies</td>
			<td>AT104</td>
			<td>A protease-collagenase mixture considered to be more effective in preserving epitopes for flow cytometry.</td>
		</tr>
		<tr>
			<td>anti-CD325 (N-Cadherin) PE</td>
			<td>Invitrogen</td>
			<td>12-3259-42</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-CD90 PE-Cy7</td>
			<td>BD Biosciences</td>
			<td>561558</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-Mesothelin APC</td>
			<td>Miltenyi</td>
			<td>130-118-096</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin 30%</td>
			<td>Sigma</td>
			<td>A8577-1L</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Dissociation buffer, enzyme-free</td>
			<td>Thermo Fisher Scientific</td>
			<td>13151014</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell strainer (70 &#181;m)</td>
			<td>Greiner Bio-One</td>
			<td>542-070</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge with 15 mL and 50 mL adaptors</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase Type 1</td>
			<td>Sigma-Aldrich</td>
			<td>C-0130</td>
			<td>Dissolve 1.5 g of Collagenase type I into 100 mL of sterile DMEM and aliquot in 5 mL volumes. Label well and store at -20 &#176;C.</td>
		</tr>
		<tr>
			<td>Controlled-rate freezing chamber</td>
			<td>Thermo Fisher Scientific</td>
			<td>15-350-50</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryovials</td>
			<td>Thermo Fisher Scientific</td>
			<td>5000-0020</td>
			<td></td>
		</tr>
		<tr>
			<td>CulturPlate-96</td>
			<td>Packard Instrument Company</td>
			<td>6005680</td>
			<td>White, opaque 96-well microplate.</td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide</td>
			<td>Thermo Fisher Scientific</td>
			<td>BP231-100</td>
			<td></td>
		</tr>
		<tr>
			<td>DNAse I (from bovine pancreas)</td>
			<td>Sigma-Aldrich</td>
			<td>D4527</td>
			<td>Aliquot at 50 &#956;L, label well and store at -20 &#176;C. After thawing, keep at 4 &#176;C for up to 1 month.</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Phosphate buffered saline solution 1x</td>
			<td>Corning</td>
			<td>21-031-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol 200 proof</td>
			<td>Thermo Fisher Scientific</td>
			<td>A4094</td>
			<td></td>
		</tr>
		<tr>
			<td>FACS tubes-filter top</td>
			<td>BD Falcon</td>
			<td>352235</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Gemini-Bio</td>
			<td>100-106</td>
			<td></td>
		</tr>
		<tr>
			<td>GentleMACS C-tubes</td>
			<td>Miltenyi</td>
			<td>130-093-237</td>
			<td></td>
		</tr>
		<tr>
			<td>GentleMACS Octo-dissociator with heater apparatus</td>
			<td>Miltenyi</td>
			<td>130-096-427</td>
			<td>Includes specialized heater apparatus sleeves used to apply heat to the C-tubes during dissocation.</td>
		</tr>
		<tr>
			<td>Goat serum</td>
			<td>Sigma-Aldrich</td>
			<td>G9023</td>
			<td>Aliquot and store at -20 &#176;C.</td>
		</tr>
		<tr>
			<td>Hank&#39;s Balanced Salt solution, 500 mL</td>
			<td>Corning</td>
			<td>MT21022CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Hyaluronidase</td>
			<td>Sigma-Aldrich</td>
			<td>H3506</td>
			<td>Dissolve 0.15 g of Hyaluronidase into 100 mlLof sterile DMEM and distribute into 1.0 mL aliquots. Label well and store at -20 &#176;C.</td>
		</tr>
		<tr>
			<td>Inverted-phase microscope</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Laminar flow hood</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Live/dead yellow dye</td>
			<td>Life Technologies</td>
			<td>L-34968</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipettor tips, 20 &#181;L</td>
			<td>ART</td>
			<td>2149P</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipettor, 0.5-20 &#181;L</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Mycoalert assay control set</td>
			<td>Lonza</td>
			<td>LT07-518</td>
			<td>Aliquot positive controls and store at -20 &#176;C.</td>
		</tr>
		<tr>
			<td>Mycoalert Plus mycoplasma detection kit</td>
			<td>Lonza</td>
			<td>LT07-710</td>
			<td>Aliquot reagent and substrate and store at -20 &#176;C. Save remaining buffer and aliquot for negative control and store at -20&#176;C.</td>
		</tr>
		<tr>
			<td>Paraformaldehyde 16%</td>
			<td>Electron Microscopy Sciences</td>
			<td>15710</td>
			<td>Prepare stock by filtering through a PVDF syringe filter (Millex cat. no. SLVV033RS) and aliquot under a fume hood. Store at -20 &#176;C.</td>
		</tr>
		<tr>
			<td>Penicillin-streptomycin 10,000 U/mL</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipet aid</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Plate reader with luminescent capabilities</td>
			<td>BioTek</td>
			<td>Synergy HT</td>
			<td>any plate reader with these specifications can be used</td>
		</tr>
		<tr>
			<td>RPMI 1640 media</td>
			<td>Corning</td>
			<td>10-040-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel</td>
			<td>Andwin</td>
			<td>2975#21</td>
			<td></td>
		</tr>
		<tr>
			<td>Stericup Quick Release-GP sterile vacuum filtration system, 0.22 &#181;m PES Express PLUS, 500 mL</td>
			<td>EMD Millipore</td>
			<td>S2GPU05RE</td>
			<td></td>
		</tr>
		<tr>
			<td>Steriflip-GP filter, 0.22 &#181;m PES Express PLUS, 50 mL</td>
			<td>EMD Millipore</td>
			<td>SCGP00525</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile forceps</td>
			<td>Thermo Fisher Scientific</td>
			<td>12-000-157</td>
			<td></td>
		</tr>
		<tr>
			<td>T25 flasks, tissue culture treated</td>
			<td>Corning</td>
			<td>430639</td>
			<td></td>
		</tr>
		<tr>
			<td>T75 flasks, tissue culture treated</td>
			<td>Corning</td>
			<td>430641U</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan blue solution 0.4%</td>
			<td>Gibco</td>
			<td>15250-061</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin EDTA 0.05%</td>
			<td>Corning</td>
			<td>25-052-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>ViaStain acridine orange/propidium iodide (AO/PI) solution</td>
			<td>Nexcelom</td>
			<td>CS2-0106-5ML</td>
			<td></td>
		</tr>
	</tbody>
</table>

generation and expansion of primary, malignant pleural mesothelioma tumor lines

Current methodologies for the expansion of primary tumor cell lines from rare tumor types are lacking. This protocol describes methods to expand primary tumor cells from surgically resected, malignant pleural mesothelioma (MPM) by providing a complete overview of the process from digestion to enrichment, expansion, cryopreservation, and phenotypic characterization. In addition, this protocol introduces concepts for tumor generation that may be useful for multiple tumor types such as differential trypsinization and the impact of dissociation methods on the detection of cell surface markers for phenotypic characterization. The major limitation of this study is the selection of tumor cells that will expand in a two-dimensional (2D) culture system. Variations to this protocol, including three-dimensional (3D) culture systems, media supplements, plate coating to improve adhesion, and alternate disaggregation methods, could improve this technique and the overall success rate of establishing a tumor line. Overall, this protocol provides a base method for establishing and characterizing tumor cells from this rare tumor.

To determine fibroblast contamination of early-passage cultures, cells are assessed using an inverted phase microscope to identify the frequency of fibroblasts relative to the other adherent cells present. Figure 1 shows examples of increasing fibroblast contamination of 80% (Figure 1A), 50% (Figure 1B), and 30% (Figure 1C), compared to a culture with no fibroblast contamination (Fi...

Watch this Scientific Journal Video about Generation and Expansion of Primary, Malignant Pleural Mesothelioma Tumor Lines at JoVE.com

Generation and Expansion of Primary, Malignant Pleural Mesothelioma Tumor Lines

The goal of this method paper is to demonstrate a robust and reproducible methodology for the enrichment, generation, and expansion of primary tumor cell lines from surgically resected pleural mesothelioma.

Current methodologies for the expansion of primary tumor cell lines from rare tumor types are lacking. This protocol describes methods to expand primary ...

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