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 a laminar flow hood.
	<ol>
		<li>Transfer the medium to a 500 mL, 0.22 &#181;m polyethersulfone (PES) sterile filter for vacuum filtration. 
		NOTE: Filtration and aspiration steps should be performed with a standard vacuum pressure of 75-150 torr.</li>
		<li>Label and date the tumor digestion medium and store it at 4 &#176;C. 
		NOTE: The medium can be stored for 1 month at 4 &#176;C.</li>
	</ol>
	</li>
	<li>To prepare the tumor-digesting enzymatic cocktail, add 2.5 mL of 3% collagenase type 1, 1 mL of 1.5 mg/mL hyaluronidase, 20 &#181;L of 250,000 units/mL DNAse 1 to 16.5 mL of digestion medium in a 50 mL conical tube in a laminar flow hood. 
	NOTE: The total volume of the tumor-digesting enzymatic cocktail is 20 mL; however, only 10 mL is needed per tumor sample. As such, the remaining cocktail can be stored at -20 &#176;C until needed. Do not refreeze aliquots.</li>
	<li>To prepare complete tumor medium, add 5 mL of 100% Pen/Strep and 50 mL of fetal bovine serum (FBS) to 500 mL of sterile RPMI 1640 in a laminar flow hood.
	<ol>
		<li>Transfer the medium to a 500 mL, 0.22 &#181;m PES sterile filter for vacuum filtration.</li>
		<li>Label and date the complete tumor medium and store it at 4 &#176;C. 
		NOTE: The medium can be stored for 1 month at 4 &#176;C.</li>
	</ol>
	</li>
	<li>To prepare reduced-serum medium for starvation, add 5 mL of 100% Pen/Strep and 5 mL of FBS to 500 mL of sterile RPMI 1640 in a laminar flow hood.
	<ol>
		<li>Transfer the medium to a 500 mL, 0.22 &#181;m PES sterile filter for vacuum filtration.</li>
		<li>Label and date the reduced-serum medium and store it at 4 &#176;C. 
		NOTE: The medium can be stored for 1 month at 4 &#176;C.</li>
	</ol>
	</li>
	<li>To prepare freeze medium, add 5 mL of dimethyl sulfoxide (DMSO) to 45 mL of FBS in a laminar flow hood.
	<ol>
		<li>Transfer the medium to a 50 mL, 0.22 &#181;m PES sterile filter for vacuum filtration.</li>
		<li>Label and date five 15 mL conical tubes.</li>
		<li>Transfer 10 mL of freeze medium to each tube and store the tubes at -20 &#176;C.</li>
	</ol>
	</li>
	<li>To prepare antibiotic-free medium for mycoplasma testing, add 50 mL of FBS to 500 mL of sterile RPMI 1640 in a laminar flow hood.
	<ol>
		<li>Transfer the medium to a 50 mL, 0.22 &#181;m PES sterile filter for vacuum filtration.</li>
		<li>Label and date the antibiotic-free medium and store it at 4 &#176;C. 
		NOTE: The medium can be stored for 1 month at 4 &#176;C.</li>
	</ol>
	</li>
	<li>To prepare fluorescence-activated cell sorting (FACS) buffer for phenotypic analysis, add 16.6 mL of sterile bovine serum albumin to 500 mL of sterile 1x phosphate-buffered saline (PBS) in a laminar flow hood.
	<ol>
		<li>Label and date the FACS buffer and store it at 4 &#176;C. 
		&#8203;NOTE: FACS buffer does not require filter sterilization as long as both components are stored sterile. FACS buffer can be stored prior to opening for 6 months at 4 &#176;C.</li>
	</ol>
	</li>
</ol>
2. Digestion of tumor tissue
<ol>
	<li>Transfer 10 mL of tumor-digesting enzymatic cocktail from step 1.2 to a dissociation tube.</li>
	<li>Transfer the piece of tumor tissue to a sterile 6-well plate lid using a sterile scalpel. 
	NOTE: The amount of medium that is transferred with the tumor tissue is sufficient for processing.
	<ol>
		<li>Remove all fatty tissue, necrotic tissue, and/or blood clots using a new sterile scalpel and forceps. 
		NOTE: Fatty tissue is normally yellowish in appearance and semisolid. Necrotic tissue is darker than the surrounding tissue in appearance and very soft; both fatty and necrotic tissue will break apart easily. Bloody tissue appears bright red and may release blood into the medium when manipulated. Once removed, the resulting tumor tissue should hold shape and be pale or pink, depending on the tissue type.</li>
		<li>Cut the entire tumor tissue into 1 mm3 small fragments. To ensure the tissue does not dry out, add 500 &#181;L of sterile 1x Hank&#39;s Balanced Salt Solution (HBSS) to the tumor tissue.</li>
	</ol>
	</li>
	<li>Transfer tumor fragments into the dissociation tube containing 10 mL of tumor-digesting enzymatic cocktail (step 2.1). Close the tube tightly, place it cap-side down on the tissue dissociator, and add the heater apparatus by applying it like a sleeve over the dissociation tube and pressing to click it in place. 
	NOTE: The maximum capacity for each tube is 4 g of tumor and 10 mL volume.</li>
	<li>Select the preprogrammed setting on the dissociator 37C_h_TDK_1. Check the status after 10 min to ensure there is no clogging error as indicated by the flashing red light. 
	NOTE: This program processes at 1,865 rpm for 1 h with temperature on at 37 &#176;C. 
	&#8203;NOTE: If clogging occurs, remove the dissociation tube and manually swirl it to dislodge any tissue caught in the lid; then replace the tube onto the dissociator and resume the program.</li>
	<li>Following the 1 h digestion, remove the dissociation tube and place it in a laminar flow hood. Filter the digested tumor by placing a 70 &#181;m cell strainer on top of a 50 mL conical tube and pipetting the digested tumor using a 10 mL pipette onto the filter. If the filter clogs, switch to a new filter.
	<ol>
		<li>Rinse the dissociation tube with 10 mL of fresh tumor digestion media and pass the wash through the filter.</li>
		<li>Remove the 70 &#181;m filter and discard it.</li>
		<li>Bring the total volume to 40 mL with tumor digestion medium.</li>
	</ol>
	</li>
	<li>Centrifuge at 500 &#215; g for 5 min at room temperature.</li>
	<li>Using a 2 mL aspirating pipette attached to a vacuum source, aspirate the supernatant without disturbing the pellet, and resuspend the cells using 10 mL of sterile complete tumor medium.</li>
	<li>Repeat step 2.6.</li>
	<li>Aspirate the supernatant as described in step 2.7. Resuspend the cells in 3 mL of sterile complete tumor medium and transfer the cell suspension to one well of a sterile 6-well plate.</li>
	<li>Keep the plate in an incubator at 37 &#176;C with 5% CO2.</li>
	<li>After 24 h, transfer the used medium from the digested tumor in well 1 to a new well (well 2) in the same 6-well plate. Add 3 mL of sterile complete tumor medium to well 1.</li>
	<li>Transfer the used medium from wells 1 and 2 and combine into well 3 in the same 6-well plate 24 h after step 2.11. Add 3 mL of sterile complete tumor medium into wells 1 and 2.</li>
	<li>Aspirate the medium from all 3 wells and pipette 3 mL of complete tumor medium into each well 48 h after step 2.12.</li>
</ol>
3. Generation of primary tumor cell line
<ol>
	<li>Using an inverted phase microscope, determine the percentage of fibroblast contamination in the early passage culture. 
	NOTE: Fibroblasts are generally long and irregular and have an ill-defined cellular membrane. They appear thin or flat on a Z scale.
	<ol>
		<li>If fibroblast contamination is greater than 20% of the cells in the early passage culture, continue culturing after replacing the complete medium with reduced-serum medium.</li>
		<li>Repeat the replacement with reduced-serum medium in step 3.1.1 twice per week for 3-4 weeks.</li>
		<li>When the culture reaches 80% confluency, passage the cells by splitting 50:50 into 2 new wells of a 6-well plate. Continue passaging 50:50 in reduced-serum media once the cells reach 80% confluency.</li>
		<li>Repeat step 3.1.3 for up to 4 weeks, passaging in larger culture flasks as needed by splitting 50:50 once the culture reaches 80% confluency. If the fibroblast population is not reduced to 10% or less, continue to step 3.2 to attempt the differential trypsinization method to remove fibroblast contamination. Otherwise, proceed to step 3.3.</li>
	</ol>
	</li>
	<li>To enrich for tumor cells, gently rinse the well with 1x PBS to remove the medium containing serum.
	<ol>
		<li>Add trypsin as follows: 1 mL per well if in a 6-well plate, 2 mL for a T25 flask, 3 mL for a T75 flask.</li>
		<li>Place the 6-well plate or flask in an incubator at 37 &#176;C for 1 min. Remove the plate or flask from the incubator and check the adhesion of the cells using an inverted microscope. If the cells are partially lifting or floating, gently remove the suspended cells and trypsin only. Place the cells in a 15 mL conical tube containing an equal or greater volume of complete tumor medium. 
		NOTE: This partial collection of cells based on adhesion properties is the first of multiple fractions.</li>
		<li>Repeat steps 3.2.1 and 3.2.2 until all the cells are lifted, or there are 4 fractions removed.</li>
		<li>Centrifuge all independent fractions at 500 &#215; g for 5 min at room temperature.</li>
		<li>Aspirate the supernatant as described in step 2.7, and resuspend the cells using 5 mL of sterile, reduced-serum medium.</li>
		<li>Plate the cells in a T25 flask for each fraction and place the flasks in an incubator at 37 &#176;C.</li>
		<li>Assess the culture after 48 h to determine which fraction is enriched for tumor cells versus fibroblasts. Discard fibroblast-rich flasks. If fibroblast contamination is &#60;10%, continue expansion in complete tumor medium and proceed to step 4. If fibroblast contamination is 10-30%, repeat steps 3.1.2-3.1.4. If fibroblast contamination is greater than 30%, repeat steps 3.2-3.2.7.</li>
	</ol>
	</li>
	<li>If few or no fibroblasts are detected within the early passage culture, continue passaging the cells in complete tumor medium by splitting 50:50 once the cells are 80% confluent in their 6-well plate or flask.</li>
</ol>
4. Expansion of early passage primary tumor cell line
<ol>
	<li>Once the primary tumor culture contains 10% or less of fibroblast contamination, aspirate the medium and replace it with sterile complete tumor medium twice per week. 
	NOTE: The optimal medium volume for a T25 is 5 mL; T75 is 12 mL; T150 is 25 mL.
	<ol>
		<li>Passage the culture 50:50 into larger flasks as needed once it reaches 80% confluency in the plate or flask. 
		NOTE: The culture may need to be passaged at a higher ratio depending on its growth. Adjust so the culture reaches 80% confluency every 3-5 days.</li>
	</ol>
	</li>
	<li>Passage until the culture is at passage 4 or above. Once the culture is 80% confluent in a minimum of two T75 flasks, continue to step 5.</li>
</ol>
5. Characterization and banking of established primary tumor cell line
<ol>
	<li>Cryopreserve one T75 flask as an early-passage freeze. For mycoplasma testing of the remaining T75 flasks, continue to step 5.2.
	<ol>
		<li>For cryopreservation of early-passage cells, thaw 1 tube of aliquoted freeze medium prepared in step 1.5.</li>
		<li>Collect the cells by trypsinization by first washing the plate with 1x PBS as described in step 3.2 and adding trypsin as described in step 3.2.1.</li>
		<li>Place the flask in an incubator at 37 &#176;C for 3 min. Remove the flask from the incubator and check the adhesion of the cells using an inverted microscope. If the cells are lifting or floating, gently remove the suspended cells and trypsin. Place the cells in a 15 mL conical tube containing an equal or greater volume of complete tumor medium. 
		NOTE: If the cells remain adherent after 3 min, place the flask back in the incubator for an additional 2 min.</li>
		<li>Mix the tube well by pipetting gently and remove 20 &#181;L for counting.</li>
		<li>Centrifuge the tube at 500 &#215; g for 5 min at room temperature.</li>
		<li>While the cells are spinning, count using a lab-specific counting protocol such as trypan blue or acridine orange/propidium iodide (AO/PI).</li>
		<li>Resuspend the cells following centrifugation in freeze medium with a minimum of 2 &#215; 106 cells/1 mL freeze medium.</li>
		<li>Transfer 1 mL of the cell suspension from step 5.1.7 to prelabeled 1.5 mL cryovials.</li>
		<li>Place the cryovials in a controlled-rate freeze chamber and transfer them immediately to -80 &#176;C.</li>
		<li>Store the cells at -80 &#176;C for up to 1 week before transferring them to liquid nitrogen for long-term storage.</li>
	</ol>
	</li>
	<li>Split a T75 flask 50:50 for mycoplasma testing. Change the medium to antibiotic-free medium prepared in step 1.6.
	<ol>
		<li>After 72 h, thaw the mycoplasma detection reagent, substrate, and controls at room temperature for 15 min before testing.</li>
		<li>Collect 1 mL of the supernatant from each culture to be tested and place it in a 15 mL conical tube.</li>
		<li>Pellet any suspended cells by centrifuging the supernatant at 500 &#215; g for 5 min at room temperature.</li>
		<li>Load 100 &#181;L of sample supernatants and controls into a white 96-well plate. Add 100 &#181;L of mycoplasma detection reagent to each sample and control.</li>
		<li>Incubate for 5 min at room temperature.</li>
		<li>Place the plate in a plate reader and read the luminescence (Reading A, i.e., first Reading). 
		NOTE: The mycoplasma kit manufacturer&#39;s protocol indicates keeping the default settings on multifunctional plate readers. The read is set at Luminescence endpoint with an integration time of 1 s and gain of 135. The plate reader uses an Auto-scale routine to optimize the gain signal for each experiment. The 1 s integration time is standard default. Both settings are used to adjust the intensity of the luminescent signal interpreted by the plate reader and may need to be adjusted depending on the individual plate reader.</li>
		<li>Remove the plate from the plate reader and add 100 &#181;L of the mycoplasma detection substrate to each sample and the controls.</li>
		<li>Incubate for 10 min at room temperature.</li>
		<li>Place the plate in the plate reader and read the luminescence (Reading B, i.e., second Reading).</li>
		<li>To determine mycoplasma positivity, determine the ratio of Reading B to Reading A. 
		NOTE: Mycoplasma positivity ratios are described in the mycoplasma kit manufacturer&#39;s protocol. Readings A and B are acquired with the same settings and simply reflect the reading order.
		<ol>
			<li>Consider a ratio &#60; 1 to be mycoplasma-negative.</li>
			<li>Consider a ratio of 1-1.2 to be inconclusive and repeat step 5.2.</li>
			<li>Consider a ratio &#62; 1.2 to be mycoplasma-positive and monitor this culture; terminate the culture if necessary.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Flow cytometry characterization of mycoplasma-negative tumor cells
	<ol>
		<li>Dislodge the tumor cells using cell-dissociation buffer.</li>
		<li>Count the cells and resuspend the cells at 1 &#215; 106/mL in FACS buffer from step 1.7.</li>
		<li>Remove 100 &#181;L of cell suspension into individual tubes for surface staining.</li>
		<li>Centrifuge at 500 &#215; g for 5 min at room temperature.</li>
		<li>Aspirate the supernatant and block Fc receptors by incubating the cells in 500 &#181;L of 5% goat serum in FACS buffer at room temperature for 10 min.</li>
		<li>Add 2 mL of FACS buffer and centrifuge the suspension at 500 &#215; g for 5 min at room temperature.</li>
		<li>Aspirate the supernatant and add the surface stain mix of antibodies (Table 1) and FACS buffer so that the final volume is 100 &#181;L. Cover the samples and stain them on ice for 30 min.</li>
		<li>Repeat step 5.3.6.</li>
		<li>Aspirate the supernatant and fix the cells by resuspending in 200 &#181;L of 1% paraformaldehyde (PFA) plus 0.25% EtOH. Incubate at room temperature for 20 min with the samples protected from light.</li>
		<li>Repeat step 5.3.6. Resuspend the cells in 200 &#181;L of FACS buffer for acquisition using an appropriate flow cytometer. 
		NOTE: Mesothelioma tumor cells are expected to be positive for mesothelin and N-cadherin. Some may also express CD90.</li>
	</ol>
	</li>
	<li>Expand in complete tumor medium and bank as described in steps 4.1 and 5.1, respectively.</li>
</ol>

<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. 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CH is a member of the SAB for Briacell Therapeutics and the Mesothelioma Applied Research Foundation. All other authors have nothing to disclose.

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><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 &amp;micro;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 &amp;deg;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 &amp;mu;L, label well and store at -20 &amp;deg;C. After thawing, keep at 4 &amp;deg;C for up to 1 month.</td></tr><tr><td>Dulbecco&#039;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 &amp;deg;C.</td></tr><tr><td>Hank&#039;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 &amp;deg;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 &amp;micro;L</td><td>ART</td><td>2149P</td><td></td></tr><tr><td>Micropipettor, 0.5-20 &amp;micro;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 &amp;deg;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 &amp;deg;C. Save remaining buffer and aliquot for negative control and store at -20&amp;deg;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 &amp;deg;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 &amp;micro;m PES Express PLUS, 500 mL</td><td>EMD Millipore</td><td>S2GPU05RE</td><td></td></tr><tr><td>Steriflip-GP filter, 0.22 &amp;micro;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 ...

generation-expansion-primary-malignant-pleural-mesothelioma-tumor

Research

JoVE Journal

Cancer Research

1.8K Views.  University of Texas MD Anderson Cancer Center. 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. 

Video: Generation and Expansion of Primary, Malignant Pleural Mesothelioma Tumor Lines

Establishment of a Primary Culture of Patient-derived Soft Tissue Sarcoma

Soft tissue sarcomas (STS) represent a spectrum of heterogeneous malignancies with a difficult diagnosis, classification, and management. To date, more than 50 histological subtypes of these rare solid tumors have been identified. Thus, due to their extraordinary diversity and low incidence, our understanding of the biology of these tumors is still limited. Patient-derived cultures represent the ideal platform to study STS pathophysiology and pharmacology. We thus developed a human preclinical model of STS starting from tumor specimens harvested from patients undergoing surgical resection. Patient-derived STS cell cultures were obtained from the surgical specimens by collagenase digestion and isolated by filtration. Cells were counted, seeded, and left for 14 days in standard monolayer cultures and then processed by downstream analysis. Before performing molecular or pharmaceutical analyses, the establishment of STS primary cultures was confirmed through the evaluation of cytomorphologic features and, when available, immunohistochemical markers. This method represents a useful tool 1) to study the natural history of these poorly explored malignancies and 2) to test the effects of different drugs in an effort to learn more about their mechanisms of action.

The following protocol focuses on the establishment of a primary culture of patient-derived soft tissue sarcoma (STS). This model could help us to better understand the molecular background and pharmacological profile of these rare malignancies and could represent a starting point for further research aimed at improving STS management.

Soft tissue sarcomas (STS) represent a spectrum of heterogeneous malignancies with a difficult diagnosis, classification, and management. To date, more ...

A 3D Organotypic Melanoma Spheroid Skin Model

Malignant transformation of melanocytes, the pigment cells of human skin, causes formation of melanoma, a highly aggressive cancer with increased metastatic potential. Recently, mono-chemotherapies continue to improve by melanoma specific combination therapies with targeted kinase inhibitors. Still, metastatic melanoma remains a life-threatening disease because tumors exhibit primary resistance or develop resistance to novel therapies, thereby regaining tumorigenic capacity. In order to improve the therapeutic success of malignant melanoma, the determination of molecular mechanisms conferring resistance against conventional treatment approaches is necessary; however, it requires innovative cellular in vitro models. Here, we introduce an in vitro three-dimensional (3D) organotypic melanoma spheroid model that can portray the in vivo architecture of malignant melanoma and may warrant new insights into intra-tumoral as well as tumor-host interactions. The model incorporates defined numbers of mature and differentiated melanoma spheroids in a 3D human full skin reconstruction model consisting of primary skin cells. The cellular composition and differentiation status of the embedded melanoma spheroids is similar to the one of cutaneous melanoma metastasis in vivo. Using this organotypic melanoma spheroid model as a drug screening platform may support the identification of responders to selected combination therapies, while sparing the unnecessary treatment burden for non-responders, thereby increasing the benefit of therapeutic interventions.

Here, we present a protocol to generate a 3D organotypic melanoma spheroid skin model that recapitulates both the architecture and multicellular complexity of an organ/tumor in vivo but at the same time accommodates systematic experimental intervention.

Malignant transformation of melanocytes, the pigment cells of human skin, causes formation of melanoma, a highly aggressive cancer with increased ...

Establishment of Gastric Cancer Patient-derived Xenograft Models and Primary Cell Lines

The use of preclinical models to advance our understanding of tumor biology and investigate the efficacy of therapeutic agents is key to cancer research. Although there are many established gastric cancer cell lines and many conventional transgenic mouse models for preclinical research, the disadvantages of these in vitro and in vivo models limit their applications. Because the characteristics of these models have changed in culture, they no longer model tumor heterogeneity, and their responses have not been able to predict responses in humans. Thus, alternative models that better represent tumor heterogeneity are being developed. Patient-derived xenograft (PDX) models preserve the histologic appearance of cancer cells, retain intratumoral heterogeneity, and better reflect the relevant human components of the tumor microenvironment. However, it usually takes 4-8 months to develop a PDX model, which is longer than the expected survival of many gastric patients. For this reason, establishing primary cancer cell lines may be an effective complementary method for drug response studies. The current protocol describes methods to establish PDX models and primary cancer cell lines from surgical gastric cancer samples. These methods provide a useful tool for drug development and cancer biology research.

The current protocol describes methods to establish patient-derived xenograft (PDX) models and primary cancer cell lines from surgical gastric cancer samples. The methods provide a useful tool for drug development and cancer biology research.

The use of preclinical models to advance our understanding of tumor biology and investigate the efficacy of therapeutic agents is key to cancer research. ...

Induction of Mesenchymal-Epithelial Transitions in Sarcoma Cells

Phenotypic plasticity refers to a phenomenon in which cells transiently gain traits of another lineage. During carcinoma progression, phenotypic plasticity drives invasion, dissemination and metastasis. Indeed, while most of the studies of phenotypic plasticity have been in the context of epithelial-derived carcinomas, it turns out sarcomas, which are mesenchymal in origin, also exhibit phenotypic plasticity, with a subset of sarcomas undergoing a phenomenon that resembles a mesenchymal-epithelial transition (MET). Here, we developed a method comprising the miR-200 family and grainyhead-like 2 (GRHL2) to mimic this MET-like phenomenon observed in sarcoma patient samples.We sequentially express GRHL2 and the miR-200 family using cell transduction and transfection, respectively, to better understand the molecular underpinnings of these phenotypic transitions in sarcoma cells. Sarcoma cells expressing miR-200s and GRHL2 demonstrated enhanced epithelial characteristics in cell morphology and alteration of epithelial and mesenchymal biomarkers. Future studies using these methods can be used to better understand the phenotypic consequences of MET-like processes on sarcoma cells, such as migration, invasion, metastatic propensity, and therapy resistance.

We present here a cell culture method for inducing mesenchymal-epithelial transitions (MET) in sarcoma cells based on combined ectopic expression of microRNA-200 family members and grainyhead-like 2 (GRHL2). This method is suitable for better understanding the biological impact of phenotypic plasticity on cancer aggressiveness and treatments.

Phenotypic plasticity refers to a phenomenon in which cells transiently gain traits of another lineage. During carcinoma progression, phenotypic ...

Developmental Biology

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for progression-free and overall survival. Therefore, translational research needs to provide further molecular evidence to improve targeted therapies for malignant melanomas. In the past, oncogenic mechanisms related to melanoma were extensively studied in established cell lines. On the way to more personalized treatment regimens based on individual genetic profiles, we propose to use patient-derived cell lines instead of generic cell lines. Together with high quality clinical data, especially on patient follow-up, these cells will be instrumental to better understand the molecular mechanisms behind melanoma progression.
Here, we report the establishment of primary melanoma cultures from dissected fresh tumor tissue. This procedure includes mincing and dissociation of the tissue into single cells, removal of contaminations with erythrocytes and fibroblasts as well as primary culture and reliable verification of the cells' melanoma origin.
Recent reports revealed that melanomas, like the majority of tumors, harbor a small subpopulation of cancer stem cells (CSCs), which seem to exclusively fuel tumor initiation and progression towards the metastatic state. One of the key markers for CSC identification and isolation in melanoma is CD133. To isolate CD133+ CSCs from primary melanoma cultures, we have modified and optimized the Magnetic-Activated Cell Sorting (MACS) procedure from Miltenyi resulting in high sorting purity and viability of CD133+ CSCs and CD133- bulk, which can be cultivated and functionally analyzed thereafter.

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

This article describes the preparation of freshly obtained melanoma tissue into primary cell cultures, and how to remove contaminations of erythrocytes and fibroblasts from the tumor cells. Finally, we describe how CD133+ putative melanoma stem cells are sorted from the CD133- bulk using Magnetic Activated Cell Sorting (MACS).

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for ...

Medicine

Creation and Maintenance of a Living Biobank - How We Do It

In light of the growing knowledge about the inter-individual properties and heterogeneity of cancers, the emerging field of personalized medicine requires a platform for preclinical research. Over recent years, we have established a biobank of colorectal and pancreatic cancers comprising of primary tumor tissue, normal tissue, sera, isolated peripheral blood lymphocytes (PBL), patient-derived xenografts (PDX), as well as primary and secondary cancer cell lines. Since original tumor tissue is limited and the establishment rate of primary cancer cell lines is still relatively low, PDX allow not only the preservation and extension of the biobank but also the generation of secondary cancer cell lines. Moreover, PDX-models have been proven to be the ideal in vivo model for preclinical drug testing. However, biobanking requires careful preparation, strict guidelines and a well attuned infrastructure. Colectomy, duodenopancreatectomy or resected metastases specimens are collected immediately after resection and transferred to the pathology department. Respecting priority of an unbiased histopathological report, at the discretion of the attending pathologist who carries out the dissections, small tumor pieces and non-tumor tissue are harvested. 
Necrotic parts are discarded and the remaining tumor tissue is cut into small, identical cubes and cryopreserved for later use. Additionally, a small portion of the tumor is minced and strained for primary cancer cell culture. Additionally, blood samples drawn from the patient pre- and postoperatively, are processed to obtain serum and PBLs. For PDX engraftment, the cryopreserved specimens are defrosted and implanted subcutaneously into the flanks of immunodeficient mice. The resulting PDX closely recapitulate the histology of the "donor" tumors and can be either used for subsequent xenografting or cryopreserved for later use. In the following work, we describe the individual steps of creation, maintenance and administration of a large biobank of colorectal and pancreatic cancer. Moreover, we highlight the crucial details and caveats associated with biobanking.

In the following work, we describe the consecutive steps necessary for the establishment of a large biobank of colorectal and pancreatic cancer.

In light of the growing knowledge about the inter-individual properties and heterogeneity of cancers, the emerging field of personalized medicine requires ...

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 ...

Orthotopic Implantation and Peripheral Immune Cell Monitoring in the II-45 Syngeneic Rat Mesothelioma Model

The enormous upsurge of interest in immune-based treatments for cancer such as vaccines and immune checkpoint inhibitors, and increased understanding of the role of the tumor microenvironment in treatment response, collectively point to the need for immune-competent orthotopic models for pre-clinical testing of these new therapies. This paper demonstrates how to establish an orthotopic immune-competent rat model of pleural malignant mesothelioma. Monitoring disease progression in orthotopic models is confounded by the internal location of the tumors. To longitudinally monitor disease progression and its effect on circulating immune cells in this and other rat models of cancer, a single tube flow cytometry assay requiring only 25 &#181;l whole blood is described. This provides accurate quantification of seven immune parameters: total lymphocytes, monocytes and neutrophils, as well as the T-cell subsets CD4 and CD8, B-cells and Natural Killer cells. Different subsets of these parameters are useful in different circumstances and models, with the neutrophil to lymphocyte ratio having the greatest utility for monitoring disease progression in the mesothelioma model. Analyzing circulating immune cell levels using this single tube method may also assist in monitoring the response to immune-based treatments and understanding the underlying mechanisms leading to success or failure of treatment.

The generation of an orthotopic rat model of pleural malignant mesothelioma by implantation of II-45 mesothelioma cells into the pleural cavity of immune competent rats is presented. A flow cytometric method to analyze seven immune cell subsets in these animals from a 25 &#181;l&#160;blood sample is also described.

The enormous upsurge of interest in immune-based treatments for cancer such as vaccines and immune checkpoint inhibitors, and increased understanding of ...

Pre-Conditioning the Airways of Mice with Bleomycin Increases the Efficiency of Orthotopic Lung Cancer Cell Engraftment

Lung cancer is a deadly treatment refractory disease that is biologically heterogeneous. To understand and effectively treat the full clinical spectrum of thoracic malignancies, additional animal models that can recapitulate diverse human lung cancer subtypes and stages are needed. Allograft or xenograft models are versatile and enable the quantification of tumorigenic capacity in vivo, using malignant cells of either murine or human origin. However, previously described methods of lung cancer cell engraftment have been performed in non-physiological sites, such as the flank of mice, due to the inefficiency of orthotopic transplantation of cells into the lungs. In this study, we describe a method to enhance orthotopic lung cancer cell engraftment by pre-conditioning the airways of mice with the fibrosis inducing agent bleomycin. As a proof-of-concept experiment, we applied this approach to engraft tumor cells of the lung adenocarcinoma subtype, obtained from either mouse or human sources, into various strains of mice. We demonstrate that injuring the airways with bleomycin prior to tumor cell injection increases the engraftment of tumor cells from 0-17% to 71-100%. Significantly, this method enhanced lung tumor incidence and subsequent outgrowth using different models and mouse strains. In addition, engrafted lung cancer cells disseminate from the lungs into relevant distant organs. Thus, we provide a protocol that can be used to establish and maintain new orthotopic models of lung cancer with limiting amounts of cells or biospecimen and to quantitatively assess the tumorigenic capacity of lung cancer cells in physiologically relevant settings.

We describe a method to significantly enhance orthotopic engraftment of lung cancer cells into the murine lungs by pre-conditioning the airways with injury. This approach may also be applied to study stromal interactions within the lung microenvironment, metastatic dissemination, lung cancer co-morbidities, and to more efficiently generate patient derived xenografts.

Lung cancer is a deadly treatment refractory disease that is biologically heterogeneous. To understand and effectively treat the full clinical spectrum of ...

Implantation and Monitoring by PET/CT of an Orthotopic Model of Human Pleural Mesothelioma in Athymic Mice

Malignant pleural mesothelioma (MPM) is a rare and aggressive tumor arising in the mesothelium that covers the lungs, the heart, and the thoracic cavity. MPM development is mainly associated with asbestos. Treatments provide only modest survival since the median survival average is 9&#8211;18 months from the time of diagnosis. Therefore, more effective treatments must be identified. Most data describing new therapeutic targets were obtained from in vitro experiments and need to be validated in reliable in vivo preclinical models. This article describes one such reliable MPM orthotopic model obtained after injection of a human MPM cell line H2052/484 into the pleural cavity of immunodeficient athymic mice. Transplantation in the orthotopic site allows studying the progression of tumor in the natural in vivo environment. Positron emission tomography/computed tomography (PET/CT) molecular imaging using the clinical [18F]-2-fluoro-2-deoxy-D-glucose ([18F]FDG) radiotracer is the diagnosis method of choice for examining patients with MPM. Accordingly, [18F]FDG-PET/CT was used to longitudinally monitor the disease progression of the H2052/484 orthotopic model. This technique has a high 3R potential (Reduce the number of animals, Refine to lessen pain and discomfort, and Replace animal experimentation with alternatives) since the tumor development can be monitored non-invasively and the number of animals required could be significantly reduced.
This model displays a high development rate, a rapid tumor growth, is cost-efficient and allows for rapid clinical translation. By using this orthotopic xenograft MPM model, researchers can assess biological responses of a reliable MPM model following therapeutic interventions.

This article describes the generation of an orthotopic mouse model of human pleural mesothelioma by implantation of H2052/484 mesothelioma cells into the pleural cavity of immunocompromised athymic mice. The longitudinal monitoring of the development of intrapleural tumors was assessed by non-invasive multimodal [18F]-2-fluoro-2-deoxy-D-glucose positron emission tomography and computed tomography imaging.

Malignant pleural mesothelioma (MPM) is a rare and aggressive tumor arising in the mesothelium that covers the lungs, the heart, and the thoracic cavity. ...