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 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 ...

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

Generation of High-Throughput Three-Dimensional Tumor Spheroids for Drug Screening

Cancer cells have routinely been cultured in two dimensions (2D) on a plastic surface. This technique, however, lacks the true environment a tumor mass is exposed to in vivo. Solid tumors grow not as a sheet attached to plastic, but instead as a collection of clonal cells in a three-dimensional (3D) space interacting with their neighbors, and with distinct spatial properties such as the disruption of normal cellular polarity. These interactions cause 3D-cultured cells to acquire morphological and cellular characteristics which are more relevant to in vivo tumors. Additionally, a tumor mass is in direct contact with other cell types such as stromal and immune cells, as well as the extracellular matrix from all other cell types. The matrix deposited is comprised of macromolecules such as collagen and fibronectin.
In an attempt to increase the translation of research findings in oncology from bench to bedside, many groups have started to investigate the use of 3D model systems in their drug development strategies. These systems are thought to be more physiologically relevant because they attempt to recapitulate the complex and heterogeneous environment of a tumor. These systems, however, can be quite complex, and, although amenable to growth in 96-well formats, and some now even in 384, they offer few choices for large-scale growth and screening. This observed gap has led to the development of the methods described here in detail to culture tumor spheroids in a high-throughput capacity in 1536-well plates. These methods represent a compromise to the highly complex matrix-based systems, which are difficult to screen, and conventional 2D assays. A variety of cancer cell lines harboring different genetic mutations are successfully screened, examining compound efficacy by using a curated library of compounds targeting the Mitogen-Activated Protein Kinase or MAPK pathway. The spheroid culture responses are then compared to the response of cells grown in 2D, and differential activities are reported. These methods provide a unique protocol for testing compound activity in a high-throughput 3D setting.

Across a wide variety of disease indications, more physiologically relevant models are being developed and implemented into drug discovery programs. The new model system described here demonstrates how three-dimensional tumor spheroids can be cultured and screened in a high-throughput 1536-well plate-based system to search for new oncology drugs.

Cancer cells have routinely been cultured in two dimensions (2D) on a plastic surface. This technique, however, lacks the true environment a tumor mass is ...

Rapid Generation of Primary Murine Melanocyte and Fibroblast Cultures

Defects in fibroblast or melanocyte function are associated with skin diseases, including poor barrier function, defective wound healing, pigmentation defects and cancer. Vital to the understanding and amelioration of these diseases are experiments in primary fibroblast and melanocyte cultures. Nevertheless, current protocols for melanocyte isolation require that the epidermal and dermal layers of the skin are trypsinized and manually disassociated. This process is time consuming, technically challenging and contributes to inconsistent yields. Furthermore, methods to simultaneously generate pure fibroblast cultures from the same tissue sample are not readily available. Here, we describe an improved protocol for isolating melanocytes and fibroblasts from the skin of mice on postnatal days 0-4. In this protocol, whole skin is mechanically homogenized using a tissue chopper and then briefly digested with collagenase and trypsin. Cell populations are then isolated through selective plating followed by G418 treatment. This procedure results in consistent melanocyte and fibroblast yields from a single mouse in less than 90 min. This protocol is also easily scalable, allowing researchers to process large cohorts of animals without a significant increase in hands-on time. We show through flow cytometric assessments that cultures established using this protocol are highly enriched for melanocytes or fibroblasts.

This protocol outlines a rapid method to simultaneously generate melanocyte and fibroblast cultures from the skin of 0-4 day old mice. These primary cultures can be maintained and manipulated in vitro to study a variety of physiologically relevant processes, including skin cell biology, pigmentation, wound healing and melanoma.

Defects in fibroblast or melanocyte function are associated with skin diseases, including poor barrier function, defective wound healing, pigmentation ...

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. ...

Analysis of Side Population in Solid Tumor Cell Lines

Cancer stem cells (CSCs) are an important cause of tumor growth, metastasis, and recurrence. Isolation and identification of CSCs are of great significance for tumor research. Currently, several techniques are used for the identification and purification of CSCs from tumor tissues and tumor cell lines. Separation and analysis of side population (SP) cells are two of the commonly used methods. The methods rely on the ability of CSCs to rapidly expel fluorescent dyes, such as Hoechst 33342. The efflux of the dye is associated with the ATP-binding cassette (ABC) transporters and can be inhibited by ABC transporter inhibitors. Methods for staining cultured tumor cells with Hoechst 33342 and analyzing the proportion of their SP cells by flow cytometry are described. This assay is convenient, fast, and cost-effective. Data generated in this assay can contribute to a better understanding of the effect of genes or other extracellular and intracellular signals on the stemness properties of tumor cells.

A convenient, fast, and cost-effective method to measure the proportion of side population cells in solid tumor cell lines is presented.

Cancer stem cells (CSCs) are an important cause of tumor growth, metastasis, and recurrence. Isolation and identification of CSCs are of great ...

Drug Screening of Primary Patient Derived Tumor Xenografts in Zebrafish

Patient derived xenograft models are critical in defining how different cancers respond to drug treatment in an in vivo system. Mouse models are the standard in the field, but zebrafish have emerged as an alternative model with several advantages, including the ability for high-throughput and low-cost drug screening. Zebrafish also allow for in vivo drug screening with large replicate numbers that were previously only obtainable with in vitro systems. The ability to rapidly perform large scale drug screens may open up the possibility for personalized medicine with rapid translation of results back to clinic. Zebrafish xenograft models could also be used to rapidly screen for actionable mutations based on tumor response to targeted therapies or to identify new anti-cancer compounds from large libraries. The current major limitation in the field has been quantifying and automating the process so that drug screens can be done on a larger scale and be less labor-intensive. We have developed a workflow for xenografting primary patient samples into zebrafish larvae and performing large scale drug screens using a fluorescence microscope equipped imaging unit and automated sampler unit. This method allows for standardization and quantification of engrafted tumor area and response to drug treatment across large numbers of zebrafish larvae. Overall, this method is advantageous over traditional cell culture drug screening as it allows for growth of tumor cells in an in vivo environment throughout drug treatment, and is more practical and cost-effective than mice for large scale in vivo drug screens.

Zebrafish xenograft models allow for high-throughput drug screening and fluorescent imaging of human cancer cells in an in vivo microenvironment. We developed a workflow for large scale, automated drug screening on patient-derived leukemia samples in zebrafish using an automated fluorescence microscope equipped imaging unit.

Patient derived xenograft models are critical in defining how different cancers respond to drug treatment in an in vivo system. Mouse models are the ...

Circulating Tumor Cell Lines: an Innovative Tool for Fundamental and Translational Research

Metastasis is a leading cause of cancer death. Despite improvements in treatment strategies, metastatic cancer has a poor prognosis. We thus face an urgent need to understand the mechanisms behind metastasis development, and thus to propose efficient treatments for advanced cancer. Metastatic cancers are hard to treat, as biopsies are invasive and inaccessible. Recently, there has been considerable interest in liquid biopsies including both cell-free circulating deoxyribonucleic acid (DNA) and circulating tumor cells from peripheral blood and we have established several circulating tumor cell lines from metastatic colorectal cancer patients to participate in their characterization. Indeed, to functionally characterize these rare and poorly described cells, the crucial step is to expand them. Once established, circulating tumor cell (CTC) lines can then be cultured in suspension or adherent conditions. At the molecular level, CTC lines can be further used to assess the expression of specific markers of interest (such as differentiation, epithelial or cancer stem cells) by immunofluorescence or cytometry analysis. In addition, CTC lines can be used to assess drug sensitivity to gold-standard chemotherapies as well as to targeted therapies. The ability of CTC lines to initiate tumors can also be tested by subcutaneous injection of CTCs in immunodeficient mice.
Finally, it is possible to test the role of specific genes of interest that might be involved in cancer dissemination by editing CTC genes, by short hairpin ribonucleic acid (shRNA) or Crispr/Cas9. Modified CTCs can thus be injected into immunodeficient mouse spleens, to experimentally mimic part of the metastatic development process in vivo.
In conclusion, CTC lines are a precious tool for future research and for personalized medicine, where they will allow prediction of treatment efficiency using the very cells that are originally responsible for metastasis.

Culturing CTCs allows a deeper functional characterization of cancer, through assaying specific marker expression, and assessing drug resistance and the ability to colonize the liver among other possibilities. Overall, CTC culture could be a promising clinical tool for personalized medicine to improve patient outcome.

Metastasis is a leading cause of cancer death. Despite improvements in treatment strategies, metastatic cancer has a poor prognosis. We thus face an ...

Generation of Zebrafish Larval Xenografts and Tumor Behavior Analysis

Zebrafish larval xenografts are being widely used for cancer research to perform in vivo and real-time studies of human cancer. The possibility of rapidly visualizing the response to anti-cancer therapies (chemo, radiotherapy, and biologicals), angiogenesis and metastasis with single cell resolution, places the zebrafish xenograft model as a top choice to develop preclinical studies.

The zebrafish larval xenograft assay presents several experimental advantages compared to other models, but probably the most striking is the reduction of size scale and consequently time. This reduction of scale allows single cell imaging, the use of a relatively low number of human cells (compatible with biopsies), medium-high-throughput drug screenings, but most importantly enables a significant reduction of the time of the assay. All these advantages make the zebrafish xenograft assay extremely attractive for future personalized medicine applications.

Many zebrafish xenograft protocols have been developed with a wide diversity of human tumors; however, a general and standardized protocol to efficiently generate zebrafish larval xenografts is still lacking. Here we provide a step-by-step protocol, with tips to generate xenografts and guidelines for tumor behavior analysis, whole-mount immunofluorescence, and confocal imaging quantification.

Here, we provide a step-by-step protocol, with tips to generate xenografts and guidelines for tumor behavior analysis, whole-mount immunofluorescence, and confocal imaging quantification.

Zebrafish larval xenografts are being widely used for cancer research to perform in vivo and real-time studies of human cancer. The possibility of rapidly ...

Defining Gene Functions in Tumorigenesis by Ex vivo Ablation of Floxed Alleles in Malignant Peripheral Nerve Sheath Tumor Cells

The development of new drugs that precisely target key proteins in human cancers is fundamentally altering cancer therapeutics. However, before these drugs can be used, their target proteins must be validated as therapeutic targets in specific cancer types. This validation is often performed by knocking out the gene encoding the candidate therapeutic target in a genetically engineered mouse (GEM) model of cancer and determining what effect this has on tumor growth. Unfortunately, technical issues such as embryonic lethality in conventional knockouts and mosaicism in conditional knockouts often limit this approach. To overcome these limitations, an approach to ablating a floxed embryonic lethal gene of interest in short-term cultures of malignant peripheral nerve sheath tumors (MPNSTs) generated in a GEM model was developed.
This paper describes how to establish a mouse model with the appropriate genotype, derive short-term tumor cultures from these animals, and then ablate the floxed embryonic lethal gene using an adenoviral vector that expresses Cre recombinase and enhanced green fluorescent protein (eGFP). Purification of cells transduced with adenovirus using fluorescence-activated cell sorting (FACS) and the quantification of the effects that gene ablation exerts on cellular proliferation, viability, the transcriptome, and orthotopic allograft growth is then detailed. These methodologies provide an effective and generalizable approach to identifying and validating therapeutic targets in vitro and in vivo. These approaches also provide a renewable source of low-passage tumor-derived cells with reduced in vitro growth artifacts. This allows the biological role of the targeted gene to be studied in diverse biologic processes such as migration, invasion, metastasis, and intercellular communication mediated by the secretome.

Defining Gene Functions in Tumorigenesis by Ex vivo Ablation of Floxed Alleles in Malignant Peripheral Nerve Sheath Tumor Cells

Here, we present a protocol for performing gene knockouts that are embryonic lethal in vivo in genetically engineered mouse model-derived tumors and then assessing the effect that the knockout has on tumor growth, proliferation, survival, migration, invasion, and the transcriptome in vitro and in vivo.

The development of new drugs that precisely target key proteins in human cancers is fundamentally altering cancer therapeutics. However, before these ...

Transmitochondrial Cybrid Generation From Suspension-Growing Cancer Cells

Transmitochondrial Cybrid Generation Using Cancer Cell Lines

In recent years, the number of studies dedicated to ascertaining the connection between mitochondria and cancer has significantly risen. However, more efforts are still needed to fully understand the link involving alterations in mitochondria and tumorigenesis, as well as to identify tumor-associated mitochondrial phenotypes. For instance, to evaluate the contribution of mitochondria in tumorigenesis and metastasis processes, it is essential to understand the influence of mitochondria from tumor cells in different nuclear environments. For this purpose, one possible approach consists of transferring mitochondria into a different nuclear background to obtain the so-called cybrid cells. In the traditional cybridization techniques, a cell line lacking mtDNA (&#961;0, nuclear donor cell) is repopulated with mitochondria derived from either enucleated cells or platelets. However, the enucleation process requires good cell adhesion to the culture plate, a feature that is partially or completely lost in many cases in invasive cells. In addition, another difficulty found in the traditional methods is achieving complete removal of the endogenous mtDNA from the mitochondrial-recipient cell line to obtain pure nuclear and mitochondrial DNA backgrounds, avoiding the presence of two different mtDNA species in the generated cybrid. In this work, we present a mitochondrial exchange protocol applied to suspension-growing cancer cells based on the repopulation of rhodamine 6G-pretreated cells with isolated mitochondria. This methodology allows us to overcome the limitations of the traditional approaches, and thus can be used as a tool to expand the comprehension of the mitochondrial role in cancer progression and metastasis.

Author Spotlight: Transmitochondrial Cybrid Generation Using Cancer Cell Lines  

This protocol describes a technique for cybrid generation from suspension-growing cancer cells as a tool to study the role of mitochondria in the tumorigenic process.

In recent years, the number of studies dedicated to ascertaining the connection between mitochondria and cancer has significantly risen. However, more ...

Generation of 3D Tumor Spheroids for Drug Evaluation Studies

In recent decades, in addition to monolayer-cultured cells, three-dimensional tumor spheroids have been developed as a potentially powerful tool for the evaluation of anticancer drugs. However, the conventional culture methods lack the ability to manipulate the tumor spheroids in a homogeneous manner at the three-dimensional level. To address this limitation, in this paper, we present a convenient and effective method of constructing average-sized tumor spheroids. Additionally, we describe a method of image-based analysis using artificial intelligence-based analysis software that can scan the whole plate and obtain data on three-dimensional spheroids. Several parameters were studied. By using a standard method of tumor spheroid construction and a high-throughput imaging and analysis system, the effectiveness and accuracy of drug tests performed on three-dimensional spheroids can be dramatically increased.

This article demonstrates a standardized method for constructing three-dimensional tumor spheroids. A strategy for spheroid observation and image-based deep-learning analysis using an automated imaging system is also described.

In recent decades, in addition to monolayer-cultured cells, three-dimensional tumor spheroids have been developed as a potentially powerful tool for the ...