This work was supported by the SERB-Women Excellence Award Project, India (SB/WEA-02/2017) and the SERB-Early Career Research Award Project, India (ECR/2017/000892) to DP. The authors, LA and SR acknowledge IIT Ropar and MHRD for their research fellowships. MK acknowledges ICMR for her research fellowship.

1. Cell culture
<ol>
	<li>Culture human lung adenocarcinoma cell line A549, and human monocytic cell line THP-1 in RPMI1640 media supplemented with 10% FBS and 1% penicillin-streptomycin at 37 &#176;C in a humidified chamber with 5% CO2.</li>
	<li>Culture MRC-5 human lung fibroblast cells in DMEM medium supplemented with 10% FBS and 1% penicillin-streptomycin solution at 37 &#176;C in a humidified chamber with 5% CO2.</li>
</ol>
2. Preparation of multicellular tumor spheroids using A549 lung adenocarcinoma cell line, MRC5 fibroblasts, and THP-1 monocytes
NOTE: The multicellular tumorigenic and non-tumorigenic 3D spheroids were prepared using the hanging drop method in a 90 mm cell culture dish. A detailed description of these spheroids&#39; development is given below. All cell culture reagents such as complete medium, PBS, and 0.25% trypsin-EDTA solution should be pre-warmed at 37 &#176;C before use unless stated otherwise.
<ol>
	<li>Preparation of cell suspension
	<ol>
		<li>Grow A549 and MRC5 adherent cells (5 x 106 cells each) in DMEM complete growth medium (Dulbecco&#39;s modified eagle medium [DMEM] + 10% fetal bovine serum [FBS] + 1% penicillin-streptomycin) in T25 flasks at 37 &#176;C in a humidified chamber with 5% CO2.</li>
		<li>For THP-1 cells, grow the cell suspension (5 x 106 cells) in complete growth medium (RPMI1640 + 10% FBS + 1% penicillin-streptomycin) in T25 flasks. For better growth, place the T25 flasks at 37 &#176;C in a humidified chamber with 5% CO2 in a standing position.</li>
		<li>After 3 days, at 80%-85% confluency, wash the A549 and MRC5 cells with calcium- and magnesium-free PBS (phosphate buffer saline) by adding 1 mL of PBS (25-30 &#176;C) in each flask for 1 min and aspirating it.</li>
		<li>Harvest the A549 and MRC5 cells from the flask by incubating them with 500 &#181;L of 0.25% trypsin-EDTA solution for 5 min at 37 &#176;C in a humidified incubator with 5% CO2. Immediately after, add 4 mL of complete growth media to inactivate the trypsin.</li>
		<li>Collect the cell suspension from the T25 flask into a 15 mL tube and pellet it down at 125 x g for 5 min. Remove the supernatant and resuspend the cells in 5 mL of complete growth medium.</li>
	</ol>
	</li>
	<li>Establishment of multicellular tumor spheroids 
	NOTE: All steps of the tumor spheroid formation should be performed inside the biosafety cabinet in order to maintain sterile conditions. The maximum volume of the cell suspension has been standardized as a drop of 25 &#181;L to prepare a droplet such that it will not fall down while inverting the lids.
	<ol>
		<li>Count A549, MRC5, and THP-1 cell numbers using a cell counter. For each spheroid droplet (25 &#181;L) maintain the following cell numbers: 5,000 A549 cells, 4,000 MRC5 cells, and 1,000 THP-1 cells, following the protocol described by Arora et al.7. Calculate the cell numbers accordingly for a 1 mL volume. 
		&#8203;NOTE: Spheroids were initially prepared with three different cell counts per spheroid (i.e., 5000, 8000, and 10,000). Also, different cell ratios of tumor cells/fibroblasts/monocytes (1:1:1, 2:2:1, 4:2:1, 5:2:1, and 5:4:1) were checked. Finally, a successful 3D multicellular spheroid formation was seen with a ratio of 5:4:1 and was used for the study. Fibroblast concentration was increased based on its proportion in the tumor microenvironment, which further enhanced the rigidity of the tumor spheroids. The detailed procedure was reported in a recent publication by Arora et al.7.</li>
		<li>Prepare the cell suspension at a ratio of 5 (A549, 2 x 105 cells/mL) to 4 (MRC5, 1.6 x 105 cells/mL) to 1 (THP-1, 5 x 104 cells/mL) and make up the volume to 1 mL with complete DMEM.</li>
		<li>Place a drop of 25 &#181;L of cell suspension mixture onto the cover of a 90 mm culture dish (approximately 50 drops/90 mm dish). Fill the bottom of the 90 mm culture dish with 10 mL of sterile water.</li>
		<li>Carefully invert the lid over the water-filled hydration chamber and place the dish in a cell culture incubator for 3 days.</li>
		<li>Monitor the spheroids under the microscope at 10x magnification on day 4. To acquire images, switch on the power switch, place the 60 mm dish carefully on the stage, and select the magnification (10x). Adjust the lenses and examine the cells to analyze the cell aggregation and proliferation. Press the Freeze and Save buttons provided on the microscope to capture the image.</li>
		<li>Change the growth media on day 4 by carefully aspirating 20 &#181;L of medium from each droplet and replacing it with a fresh complete growth medium.</li>
	</ol>
	</li>
</ol>
3. Live-dead analysis of tumor spheroids
<ol>
	<li>On day 7 and 10, carefully invert the 90 mm dish in the biosafety cabinet and use a 200 &#181;L pipette to collect spheroids from each droplet. Collect five spheroids each in a 1.5 mL tube.</li>
	<li>Add 500 &#181;L of 1x PBS to the 1.5 mL tube containing spheroids and centrifuge at 125 x g for 5 min. Discard the supernatant carefully and resuspend the spheroids in 200 &#181;L of 1x PBS. Do not pipette rigorously to avoid spheroid disintegration.</li>
	<li>Pipette out the spheroids using a 200 &#181;L pipette on a 60 mm dish for calcein-AM and propidium iodide staining.</li>
	<li>Put 5 &#181;L of 1 &#181;M calcein-AM solution and 5 &#181;L of 2 mg/mL propidium iodide solution on the spheroids. Incubate for 10 min. After completion of the incubation period, wash the spheroids gently with 1x PBS twice.</li>
	<li>Place the 60 mm dish containing spheroids under the fluorescent inverted microscope, observe, and capture images at 10x magnification by selecting the fluorescence option in the microscope software and selecting the FITC (green fluorescence channel; excitation 490 nm, emission 515 nm) for calcein and Texas red channel (TXR; excitation 535 nm, emission 617 nm).
	<ol>
		<li>For acquiring the image, switch on Ctr Adv, which is switch 1, followed by switching on the CPU. Wait for the software system to boot. When booted, place the 60 mm dish carefully on the stage and select the magnification (10x) and fluorescent channels (FITC, TXR). Adjust the lenses and scan for the image.</li>
		<li>To view the image in the system, select the option Live and view the image. Adjust the intensity of fluorescence for appropriate optimization. Save the image by clicking the Save button. 
		&#8203;NOTE: At this stage, spheroids will be visible through the naked eye. Under the light microscope, spheroids will appear as round, rigid spheres at 10x magnification. Multiple spheroids can be collected at once using a 200 &#181;L pipette.</li>
	</ol>
	</li>
</ol>
4. Disintegration and cell suspension of tumor spheroids
<ol>
	<li>Collect 200 tumor spheroids each on day 7 and 10, using a 1 mL pipette in a 15 mL tube. 
	NOTE: Before collection, carefully check the shape and form of a single spheroid after transferring it onto a microscopic slide or on a 30 mm dish and observe under the microscope.</li>
	<li>Pellet the spheroids by centrifugation at 125 x g for 5 min. Aspirate the supernatant carefully without disturbing the tumor spheroids.</li>
	<li>Carefully wash the spheroids with 200 &#181;L of PBS, centrifuge at 125 x g for 5 min, and cautiously discard the supernatant.</li>
	<li>For the spheroid disintegration, add 400 &#181;L of 0.25% trypsin-EDTA solution and keep at 37 &#176;C for 10 min. Perform vigorous pipetting for complete disintegration of the spheroids.</li>
	<li>Neutralize trypsin by adding 1 mL of complete DMEM growth medium. Centrifuge at 125 x g for 5 min and discard the supernatant carefully.</li>
	<li>Resuspend the pellet in 1 mL of complete DMEM medium and count the total number of cells.</li>
</ol>
5. Cancer-associated fibroblast (CAF) isolation through microbeads
<ol>
	<li>For the isolation of CAFs from the tumor spheroids, resuspend 1 x 107 cells in 80 &#181;L of cold magnetic activated cell sorting (MACS) buffer (PBS at pH 7.2 containing 0.5% bovine serum albumin [BSA] and 2 mM ethylenediamine tetraacetic acid [EDTA]).</li>
	<li>Incubate the cell suspension (containing 1 x 107 cells) with 20 &#181;L of anti-fibroblast microbeads. Mix well by gently tapping the tube and incubate for 30 min at room temperature.</li>
	<li>Wash the cells with 1 mL of cold MACS buffer, centrifuge at 125 x g for 5 min, and aspirate the supernatant. Resuspend the cells in 500 &#181;L of MACS buffer.</li>
	<li>For magnetic bead-based cell separation, prepare the MACS column by rinsing it with 3 mL of MACS buffer.</li>
	<li>Place the cell suspension into the column followed by the collection of flow-through containing the unlabeled cell population.</li>
	<li>Wash the column three times with 3 mL of MACS buffer. Remove the column from the separator and place it on the collection tube.</li>
	<li>Collect the anti-fibroblast microbead-labeled cells by adding 5 mL of MACS buffer and firmly pushing the plunger into the column.</li>
	<li>Centrifuge the labeled cells at 125 x g for 5 min. Proceed with the isolated fibroblasts for downstream applications.</li>
</ol>
6. Flow cytometry-based analysis of ACTA2 expression in isolated CAFs
<ol>
	<li>Count the number of isolated CAFs and use approximately 6 x 104 cells. Wash the cells once with PBS, centrifuge at 125 x g for 5 min, and aspirate the supernatant.</li>
	<li>Add 100 &#181;L of cell permeabilization buffer (PBS + 0.5% BSA + 0.3% v/v Triton X-100) and incubate the cells at 4 &#176;C for 30 min.</li>
	<li>Vortex the cells intermittently to maintain a single cell suspension. Centrifuge and resuspend the cells in 100 &#181;L of cell permeabilization buffer.</li>
	<li>Add 2 &#181;L of APC conjugated anti-human &#945;-SMA antibody and incubate at 4 &#176;C for 45 min. Following incubation, add 1 mL of permeabilization buffer and centrifuge at 125 x g for 5 min to remove excess antibody.</li>
	<li>Resuspend the pellet in 400 &#181;L of permeabilization buffer for flow cytometric analysis. Acquire a total of 10,000 events of each sample in a flow cytometer. Select ACTA2 positive cells after distinguishing the populations of cells based on their forward and side scatter, selecting the singlet population followed by selecting the ACTA2 positive cells which appear as a single peak on the single parameter histogram.</li>
</ol>
7. JC-1 staining to determine mitochondrial membrane potential
<ol>
	<li>Count the number of isolated CAFs and use approximately 6 x 104 cells for 5,5,6,6&#39;-tetrachloro-1,1&#39;,3,3&#39; tetraethylbenzimi-dazoylcarbocyanine iodide (JC-1) staining using flow cytometry.</li>
	<li>Wash the cells thoroughly with PBS, centrifuge at 125 x g for 5 min, aspirate the supernatant, and add 100 &#181;L of cell staining buffer.</li>
	<li>Add JC-1 dye at a working concentration of 2 &#181;M and incubate at room temperature for 30 min. On termination of the incubation, wash the cells with PBS, centrifuge at 125 x g for 5 min, and resuspend in a final volume of 400 &#181;L.</li>
	<li>Acquire a total of 20,000 events of each sample in a flow cytometer. Quantify the mitochondrial membrane potential by measuring the red-shifted JC-1 aggregates on the FL-2 channel and the green-shifted monomers on the FL-1 channel.</li>
</ol>
8. DCFDA staining to estimate cellular reactive oxygen species (ROS) levels
<ol>
	<li>Use the whole spheroids (50 numbers) as well as the isolated CAFs (6 x 104 cells) from the spheroids for 2&#39;,7&#39;-dichlorodihydrofluorescein diacetate (DCFDA) staining.</li>
	<li>Wash the cells thoroughly with PBS, centrifuge at 125 x g for 5 min, aspirate the supernatant, and add 100 &#181;L of cell staining buffer.</li>
	<li>Add DCFDA dye at a working concentration of 1 &#181;M and incubate at room temperature for 30 min. Wash the cells twice with PBS, centrifuge at 125 x g for 5 min, and then resuspend in a final volume of 400 &#181;L.</li>
	<li>Acquire a total of 20,000 events of each sample in a flow cytometer. Assess the ROS levels by measuring the fluorescence at day 7 and day 10 for the spheroids as well as the isolated CAFs.</li>
</ol>
9. RT-qPCR analysis of CAF markers and glycolytic genes
<ol>
	<li>Extract RNA from the sorted CAFs using a single-cell lysis kit following the manufacturer&#39;s protocol. Prepare cDNA from 100 ng of RNA using a cDNA synthesis kit following the manufacturer&#39;s guidelines.</li>
	<li>Perform RT-qPCR to analyze the relative CAF markers (ACTA28 and COL1A29) and glycolytic gene (GLUT110 and MCT411) expression. Perform melt curve analysis after the final extension to ensure the specificity of the products. Normalize the data using the expression of GAPDH as the reference gene. The primer sequences used for RT-qPCR are listed in Supplementary Table 1.</li>
</ol>
10. Extraction and quantification of the cellular protein from CAFs
NOTE: Perform all steps of protein extraction on ice to avoid protein degradation.
<ol>
	<li>Resuspend approximately 4 x 106 CAF cells in 100 &#181;L of ice-cold RIPA lysis buffer (containing 30 mM HEPES, 150 mM NaCl, 1% NP-40, 0.1% SDS, and 0.5% sodium deoxycholate with 5 mM of Halt protease and phosphatase inhibitor cocktail, and 5 mM of EDTA at pH 7.4).</li>
	<li>Vortex rigorously for proper cell lysis. Sonicate the cell suspension at 20 Hz frequency, 20% amplitude for 15 s, and 3x pulse.</li>
	<li>After sonication, centrifuge the protein extract at 13,000 x g for 15 min at 4 &#176;C. Transfer the supernatant to a pre-chilled 1.5 mL tube. Perform the BCA protein assay to quantify protein.</li>
</ol>
11. Spectrophotometric analysis of enzymatic activities in CAFs
NOTE: The following enzyme activities are analyzed in tumor spheroid derived CAFs.
<ol>
	<li>Measurement of succinate dehydrogenase activity
	<ol>
		<li>To evaluate the activity of succinate dehydrogenase (SDH) in CAFs, prepare SHE buffer with 250 mM sucrose, 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), and 1 mM ethylene glycol-bis(&#946;-aminoethyl ether)-N,N,N&#8242;,N&#8242;-tetraacetic acid(EGTA) and adjust the pH to 7.3. Add 0.4 mM phenazine methosulfate (PMS), 0.2 mM 2,6-dichloroindophenol (DCPIP), 50 mM MgCl2, 0.02% Triton X-100, and 1 mM cyanide in the SHE buffer and keep at 37 &#176;C.</li>
		<li>Extract the cellular protein from the CAFs as described in step 10 (2 x 106 cells). In each well of a 96-well plate, incubate 100 &#181;L of 0.1 mg cellular protein with 100 &#181;L of SHE reaction mixture at 37 &#176;C for 15 min.</li>
		<li>To start the enzymatic activity, add 10 mM succinate12. Calculate the enzyme activity in nM/min/mL by measuring the changes in absorbance at 600 nm.</li>
		<li>The conversion factor for DCIP is 0.0215 A/nM based on its molar extinction coefficient12. Calculate the relative activity of SDH by using the mentioned formula: 
		Relative activity (nM/min/mL/enzyme) =&#160;&#160;<img alt="Equation 1" src="/files/ftp_upload/64315/64315eq01.jpg" style="margin: auto;" />&#160; X&#160;&#160;<img alt="Equation 2" src="/files/ftp_upload/64315/64315eq02.jpg" style="margin: auto;" />&#160; X V 
		Where &#916;A/min = rate of enzymatic reaction (Ainitial- Afinal)/(timefinal- A/mininitial), Ve = volume of sample, and V = volume of the reaction.</li>
	</ol>
	</li>
	<li>Estimation of cytochrome c oxidase activity
	<ol>
		<li>To evaluate the cytochrome c oxidase (COX) activity in CAFs, add 0.2 mg/mL of cellular protein in a KME reaction buffer solution containing KME buffer (125 mM KCl, 20 mM 3-(N-morpholino)propane sulfonic acid [MOPS], and 1 mM EGTA, pH 7.4), 0.02 % Triton X-100, and 5 mM sodium ascorbate.</li>
		<li>In a separate tube, mix 50 &#181;M horse heart cytochrome c with 5 mM sodium ascorbate and incubate at 37 &#176;C for 5 min13. After the incubation, add 20 &#181;L of reduced cytochrome c to 500 &#181;L of KME reaction buffer solution containing cellular protein.</li>
		<li>Calculate the enzyme activity in units/&#181;L by measuring the changes in absorbance at 550 nm. The absorption of cytochrome c at 550 nm changes with its oxidation state. The difference in extinction coefficients (mM) between reduced and oxidized cytochrome c is 21.84 at 550 nm14. Calculate the amount of enzyme activity in the cellular lysate as: 
		Units/&#181;L =&#160;&#160;<img alt="Equation 3" src="/files/ftp_upload/64315/64315eq03.jpg" style="margin: auto;" /> 
		Where &#916;A/min = A/minsample-A/minblank and 21.84 = &#916;&#1297;mM between oxidized cytochrome c and reduced cytochrome c at 550 nm</li>
	</ol>
	</li>
	<li>Assessment of lactate dehydrogenase activity
	<ol>
		<li>Perform a lactate dehydrogenase (LDH) assay using a lactate dehydrogenase cell assay kit following the manufacturer&#39;s instructions.</li>
		<li>Add LDH test reagent (10 &#181;L) to 0.5 mg/mL of cellular proteins and incubate for 5 min at 37 &#176;C. On termination of the incubation, measure the absorbance at 45 nm every 1 min for 8 min.</li>
		<li>Prepare the NADH standards for colorimetric detection using 0 (blank), 2.5, 5, 7.5, 10, and 12.5 nM/well of NADH followed by the addition of LDH test reagent to a final volume of 50 &#181;L.</li>
		<li>Plot the differences in absorbance between Tinitial and Tfinal to the NADH standard curve to determine the amount of NADH generation. Assess the activity of LDH by using the mentioned formula: 
		LDH activity =&#160;&#160;<img alt="Equation 4" src="/files/ftp_upload/64315/64315eq04.jpg" style="margin: auto;" />&#160; X sample dilution factor 
		Where B = amount (nmole) of NADH generated between Tinitial and Tfinal, reaction time = Tfinal - Tinitial (min), and Ve = sample volume (mL) added to well.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Kim, 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=Single-cell+RNA+sequencing+demonstrates+the+molecular+and+cellular+reprogramming+of+metastatic+lung+adenocarcinoma.">Single-cell RNA sequencing demonstrates the molecular and cellular reprogramming of metastatic lung adenocarcinoma.</a> Nature Communications. 11 (1), 1-5 (2020).</li><li>Davidson, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+RNA+sequencing+reveals+a+dynamic+stromal+niche+that+supports+tumor+growth.">Single-cell RNA sequencing reveals a dynamic stromal niche that supports tumor growth.</a> Cell Reports. 31 (7), 107628 (2020).</li><li>Zhang, Y., 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=Single-cell+analyses+of+renal+cell+cancers+reveal+insights+into+tumor+microenvironment,+cell+of+origin,+and+therapy+response.">Single-cell analyses of renal cell cancers reveal insights into tumor microenvironment, cell of origin, and therapy response.</a> Proceedings of the National Academy of Sciences. 118 (24), (2021).</li><li>Bray, L. J., Hutmacher, D. W., Bock, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Addressing+patient+specificity+in+the+engineering+of+tumor+models.">Addressing patient specificity in the engineering of tumor models.</a> Frontiers in Bioengineering and Biotechnology. 7, 217 (2019).</li><li>Timmins, N. E., Nielsen, L. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+multicellular+tumor+spheroids+by+the+hanging-drop+method.">Generation of multicellular tumor spheroids by the hanging-drop method.</a> Tissue Engineering. , 141-151 (2007).</li><li>Dituri, 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=Complex+tumor+spheroid+formation+and+one-step+cancer-associated+fibroblasts+purification+from+hepatocellular+carcinoma+tissue+promoted+by+inorganic+surface+topography.">Complex tumor spheroid formation and one-step cancer-associated fibroblasts purification from hepatocellular carcinoma tissue promoted by inorganic surface topography.</a> Nanomaterials. 11 (12), 3233 (2021).</li><li>Arora, L., 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=Development+of+a+multicellular+3D+tumor+model+to+study+cellular+heterogeneity+and+plasticity+in+NSCLC+tumor+microenvironment.">Development of a multicellular 3D tumor model to study cellular heterogeneity and plasticity in NSCLC tumor microenvironment.</a> Frontiers in Oncology. 12, 881207 (2022).</li><li>Nurmik, M., Ullmann, P., Rodriguez, F., Haan, S., Letellier, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+search+of+definitions:+Cancer-associated+fibroblasts+and+their+markers.">In search of definitions: Cancer-associated fibroblasts and their markers.</a> International Journal of Cancer. 146 (4), 895-905 (2020).</li><li>Zhang, Y., 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=HIF-1&#945;+is+necessary+for+activation+and+tumour-promotion+effect+of+cancer-associated+fibroblasts+in+lung+cancer.">HIF-1&#945; is necessary for activation and tumour-promotion effect of cancer-associated fibroblasts in lung cancer.</a> Journal of Cellular and Molecular Medicine. 25 (12), 5457-5469 (2021).</li><li>Bu, L., 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=Biological+heterogeneity+and+versatility+of+cancer-associated+fibroblasts+in+the+tumor+microenvironment.">Biological heterogeneity and versatility of cancer-associated fibroblasts in the tumor microenvironment.</a> Oncogene. 38 (25), 4887-4901 (2019).</li><li>Whitaker-Menezes, D., 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=Evidence+for+a+stromal-epithelial+&amp;#34;lactate+shuttle&amp;#34;+in+human+tumors:+MCT4+is+a+marker+of+oxidative+stress+in+cancer-associated+fibroblasts.">Evidence for a stromal-epithelial &amp;#34;lactate shuttle&amp;#34; in human tumors: MCT4 is a marker of oxidative stress in cancer-associated fibroblasts.</a> Cell cycle. 10 (11), 1772-1783 (2011).</li><li>Mandujano-Tinoco, E. A., Gallardo-P&#233;rez, J. C., Mar&#237;n-Hern&#225;ndez, A., Moreno-S&#225;nchez, R., Rodr&#237;guez-Enr&#237;quez, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anti-mitochondrial+therapy+in+human+breast+cancer+multi-cellular+spheroids.">Anti-mitochondrial therapy in human breast cancer multi-cellular spheroids.</a> Biochimica et Biophysica Acta (BBA)-Molecular Cell Research. 1833 (3), 541-551 (2013).</li><li>Bregman, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Laboratory Investigations in Cell and Molecular Biology. , (2002).</li><li>Berry, E. A., Trumpower, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+determination+of+hemes+a,+b,+and+c+from+pyridine+hemochrome+spectra.">Simultaneous determination of hemes a, b, and c from pyridine hemochrome spectra.</a> Analytical Biochemistry. 161 (1), 1-15 (1987).</li><li>Avagliano, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolic+reprogramming+of+cancer+associated+fibroblasts:+the+slavery+of+stromal+fibroblasts.">Metabolic reprogramming of cancer associated fibroblasts: the slavery of stromal fibroblasts.</a> BioMed Research International. , (2018).</li><li>Lorusso, G., R&#252;egg, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+tumor+microenvironment+and+its+contribution+to+tumor+evolution+toward+metastasis.">The tumor microenvironment and its contribution to tumor evolution toward metastasis.</a> Histochemistry and Cell Biology. 130 (6), 1091-1103 (2008).</li><li>Liu, T., Zhou, L., Li, D., Andl, T., Zhang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer-associated+fibroblasts+build+and+secure+the+tumor+microenvironment.">Cancer-associated fibroblasts build and secure the tumor microenvironment.</a> Frontiers in Cell and Developmental Biology. 7, 60 (2019).</li><li>Sebastian, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+transcriptomic+analysis+of+tumor-derived+fibroblasts+and+normal+tissue-resident+fibroblasts+reveals+fibroblast+heterogeneity+in+breast+cancer.">Single-cell transcriptomic analysis of tumor-derived fibroblasts and normal tissue-resident fibroblasts reveals fibroblast heterogeneity in breast cancer.</a> Cancers. 12 (5), 1307 (2020).</li><li>Elyada, E., 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=Cross-species+single-cell+analysis+of+pancreatic+ductal+adenocarcinoma+reveals+antigen-presenting+cancer-associated+fibroblasts.">Cross-species single-cell analysis of pancreatic ductal adenocarcinoma reveals antigen-presenting cancer-associated fibroblasts.</a> Cancer Discovery. 9 (8), 1102-1123 (2019).</li><li>Ganguly, D., 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=Cancer-associated+fibroblasts:+Versatile+players+in+the+tumor+microenvironment.">Cancer-associated fibroblasts: Versatile players in the tumor microenvironment.</a> Cancers. 12 (9), 2652 (2020).</li><li>Harryvan, T. J., Verdegaal, E. M., Hardwick, J. C., Hawinkels, L. J., vander Burg, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+of+the+cancer-associated+fibroblast-T-cell+axis+in+solid+malignancies.">Targeting of the cancer-associated fibroblast-T-cell axis in solid malignancies.</a> Journal of Clinical Medicine. 8 (11), 1989 (2019).</li><li>Santi, A., Kugeratski, F. G., Zanivan, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+associated+fibroblasts:+the+architects+of+stroma+remodeling.">Cancer associated fibroblasts: the architects of stroma remodeling.</a> Proteomics. 18 (5-6), 1700167 (2018).</li></ol>

The authors have no conflicts of interest to disclose.

The present study introduces the development of multicellular tumor spheroids comprising tumor cells, stromal cell population (i.e., fibroblasts), and immune cell population (i.e., monocytes) using a modified hanging drop method. Fibroblasts and monocytes/macrophages are among the most significant populations that constitute the tumor microenvironment (TME), and their presence is often linked with poor patient prognosis16. When present in the TME, fibroblasts transform, exhibiting a specific cance...

Solid tumors are composed of heterogeneous cell populations which are guided by the tumor microenvironment (TME), however, the origin of most of the cells has yet to be discovered. Mainly stromal and immune cells (fibroblasts, endothelial cells, monocytes, macrophages, dendritic cells, B cells, T cells, and their subsets) reflect the tumor heterogeneity in lung, breast, renal, and other solid cancers1,2,3. Understanding the origin of each subtype and their trans-differentiation potential is of utmost need for developing advanced therapies against these cancers. The analysis of this diverse cell population in human biopsies presents itself with several challenges because of the tumor type, site, stage, limitation of sample amount, and patient-specific variabilities4. Thus, an experimental model is needed, which is not only reliable but can also simulate the in vivo tumor condition, proving itself to be ideal for studying tumor-stroma crosstalk and its involvement in disease pathophysiology.
Three-dimensional (3D) multicellular tumor spheroid (MCTS) cultures are an advantageous in vitro model system of tumors due to their resemblance to natural counterparts. MCTS can better replicate aspects of solid tumors than 2D cell culture models, including their spatial architecture, physiological responses, the release of soluble mediators, gene expression patterns, and drug resistance mechanisms. Moreover, one main advantage of MCTS is that it can be used to study tumor heterogeneity and the tumor microenvironment (TME). The hanging-drop method is the most commonly employed tool for developing and analyzing MCTS5. In this method, the different cells with media are suspended in the form of droplets, which allows its growth in a coherent 3D aggregate fashion and is simple to access for examination. The technique is straightforward; it doesn&#39;t require many cells and eliminates the requirement of a specialized substrate like agarose for spheroid development6. An additional advantage of this method lies in the reproducibility of its technique. Furthermore, this method has also been used to co-culture mixed cell populations, such as endothelial cells and tumor cells, to simulate early tumor angiogenesis7.
In this study, multicellular 3D lung tumor spheroids were prepared with lung adenocarcinoma cells, fibroblasts, and monocytes using the hanging drop method which mimics the lung tumor microenvironment. Then the cancer-associated fibroblast (CAF) population was isolated to investigate mitochondrial health. The main idea behind developing these spheroids is to isolate the CAFs as the crosstalk among the cells in spheroids could transform the fibroblasts into a myo-fibroblast-like activated CAF state. Secondly, this study may also depict how aberrant ROS production and mitochondrial dysfunction drives the normal fibroblasts toward the more aggressive CAF phenotype. It was found that fibroblasts assembled within tumor spheroids gained myofibroblastic characteristics with increased ROS activity and the induction of metabolic gene expression. This protocol highlights the importance of the tumor microenvironment in activating CAF and could be an excellent model for in vitro generation and study of CAF phenotypic characteristics.

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assessment of mitochondrial health in cancer-associated fibroblasts isolated from 3d multicellular lung tumor spheroids

Cancer-associated fibroblasts (CAFs) are among the most abundant stromal cells present in the tumor microenvironment, facilitating tumor growth and progression. Complexity within the tumor microenvironment, including tumor secretome, low-grade inflammation, hypoxia, and redox imbalance, fosters heterotypic interaction and allows the transformation of inactive resident fibroblasts to become active CAFs. CAFs are metabolically distinguished from normal fibroblasts (NFs) as they are more glycolytically active, produce higher levels of reactive oxygen species (ROS), and overexpress lactate exporter MCT-4, leading to the opening of the mitochondrial permeability transition pore (MPTP). Here a method has been described to analyze the mitochondrial health of activated CAFs isolated from the multicellular 3D tumor spheroids comprising of human lung adenocarcinoma cells (A549), human monocytes (THP-1), and human lung fibroblast cells (MRC5). Tumor spheroids were disintegrated at different time intervals and through magnetic-activated cell sorting, CAFs were isolated. The mitochondrial membrane potential of CAFs was assessed using JC-1 dye, ROS production by 2',7'-dichlorodihydrofluorescein diacetate (DCFDA) staining, and enzyme activity in the isolated CAFs. Analyzing the mitochondrial health of isolated CAFs provides a better understanding of the reverse Warburg effect and can also be applied to study the consequences of CAF mitochondrial changes, such as metabolic fluxes and the corresponding regulatory mechanisms on lung cancer heterogeneity. Thus, the present study advocates an understanding of tumor-stroma interactions on mitochondrial health. It would provide a platform to check mitochondrial-specific drug candidates for their efficacies against CAFs as potential therapeutics in the tumor microenvironment, thereby preventing CAF involvement in lung cancer progression.

Figure 1 shows the development of multicellular tumor spheroids using three different cell populations-A549 (lung adenocarcinoma), MRC-5 (fibroblasts), and THP-1 (monocytes)-by the hanging drop method as observed on day 7 and day 10 under the microscope. On day 7, spheroids were compact and rigid with a 260 &#177; 5.3 &#181;m diameter, and on day 10, spheroids were 480 &#177; 7.5 &#181;m in diameter&#160;(Figure 1A upper panel, Figur...

Watch this Scientific Journal Video about Assessment of Mitochondrial Health in Cancer-Associated Fibroblasts Isolated from 3D Multicellular Lung Tumor Spheroids at JoVE.com

Assessment of Mitochondrial Health in Cancer-Associated Fibroblasts Isolated from 3D Multicellular Lung Tumor Spheroids

Multicellular 3D tumor spheroids were prepared with lung adenocarcinoma cells, fibroblasts, and monocytes, followed by the isolation of cancer-associated fibroblasts (CAFs) from these spheroids. Isolated CAFs were compared with normal fibroblasts to assess mitochondrial health by studying the mitochondrial transmembrane potential, reactive oxygen species, and enzymatic activities.

Cancer-associated fibroblasts (CAFs) are among the most abundant stromal cells present in the tumor microenvironment, facilitating tumor growth and ...

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Cancer Research

1.9K Views.  Indian Institute of Technology Ropar. Dit protocol benadrukt het belang van de tumormicro-omgeving bij het activeren van de kanker-geassocieerde fibroblasten of CAF. Het zou een uitstekend in vitro model kunnen zijn om fenotypische kenmerken te bestuderen. De belangrijkste beperkingen in de CAF-biologie liggen in de beperkte beschikbaarheid van CAF uit primaire tumorbiopsiemonsters.Deze techniek kan dus worden gebruikt om verschillende aspecten van de CAF-biologie te bestuderen. Deze studie toont afwijkende kruisproductie ...

Video: Assessment of Mitochondrial Health in Cancer-Associated Fibroblasts Isolated from 3D Multicellular Lung Tumor Spheroids

Preparation and Metabolic Assay of 3-dimensional Spheroid Co-cultures of Pancreatic Cancer Cells and Fibroblasts

Many cancer types, including pancreatic cancer, have a dense fibrotic stroma that plays an important role in tumor progression and invasion. Activated cancer associated fibroblasts are a key component of the tumor stroma that interact with cancer cells and support their growth and survival. Models that recapitulate the interaction of cancer cells and activated fibroblasts are important tools for studying the stromal biology and for development of antitumor agents. Here, a method is described for the rapid generation of robust 3-dimensional (3D) spheroid co-culture of pancreatic cancer cells and activated pancreatic fibroblasts that can be used for subsequent biological studies. Additionally, described is the use of 3D spheroids in carrying out functional metabolic assays to probe cellular bioenergetics pathways using an extracellular flux analyzer paired with a spheroid microplate. Pancreatic cancer cells (Patu8902) and activated pancreatic fibroblast cells (PS1) were co-cultured and magnetized using a biocompatible nanoparticle assembly. Magnetized cells were rapidly bioprinted using magnetic drives in a 96 well format, in growth media to generate spheroids with a diameter ranging between 400-600 &#181;m within 5-7 days of culture. Functional metabolic assays using Patu8902-PS1 spheroids were then carried out using the extracellular flux technology to probe cellular energetic pathways. The method herein is simple, allows consistent generation of cancer cell-fibroblast spheroid co-cultures and can be potentially adapted to other cancer cell types upon optimization of the current described methodology.

Here, a method is described for the preparation of 3-dimensional (3D) spheroid co-culture of pancreatic cancer cells and fibroblasts, followed by measurement of metabolic functions using an extracellular flux analyzer.

Many cancer types, including pancreatic cancer, have a dense fibrotic stroma that plays an important role in tumor progression and invasion. Activated ...

Evaluating the Role of Mitochondrial Function in Cancer-related Fatigue

Fatigue is a common and debilitating condition that affects most cancer patients. To date, fatigue remains poorly characterized with no diagnostic test to objectively measure the severity of this condition. Here we describe an optimized method for assessing mitochondrial function of PBMCs collected from fatigued cancer patients. Using a compact extracellular flux system and sequential injection of respiratory inhibitors, we examined PBMC mitochondrial functional status by measuring basal mitochondrial respiration, spare respiratory capacity, and energy phenotype, which describes the preferred energy pathway to respond to stress. Fresh PBMCs are readily available in the clinical setting using standard phlebotomy. The entire assay described in this protocol can be completed in less than 4 hours without the involvement of complex biochemical techniques. Additionally, we describe a normalization method that is necessary for obtaining reproducible data. The simple procedure and normalization methods presented allow for repeated sample collection from the same patient and generation of reproducible data that can be compared between time points to evaluate potential treatment effects.

Our goal was to develop a practical protocol to evaluate mitochondrial dysfunction associated with fatigue in cancer patients. This innovative protocol is optimized for clinical use involving only standard phlebotomy and basic laboratory procedures.

Fatigue is a common and debilitating condition that affects most cancer patients. To date, fatigue remains poorly characterized with no diagnostic test to ...

Assessing Cell Viability and Death in 3D Spheroid Cultures of Cancer Cells

Three-dimensional spheroids of cancer cells are important tools for both cancer drug screens and for gaining mechanistic insight into cancer cell biology. The power of this preparation lies in its ability to mimic many aspects of the in vivo conditions of tumors while being fast, cheap, and versatile enough to allow relatively high-throughput screening. The spheroid culture conditions can recapitulate the physico-chemical gradients in a tumor, including the increasing extracellular acidity, increased lactate, and decreasing glucose and oxygen availability, from the spheroid periphery to its core. Also, the mechanical properties and cell-cell interactions of in vivo tumors are in part mimicked by this model. The specific properties and consequently the optimal growth conditions, of 3D spheroids, differ widely between different types of cancer cells. Furthermore, the assessment of cell viability and death in 3D spheroids requires methods that differ in part from those employed for 2D cultures. Here we describe several protocols for preparing 3D spheroids of cancer cells, and for using such cultures to assess cell viability and death in the context of evaluating the efficacy of anticancer drugs.

Here, we present several simple methods for evaluating viability and death in 3D cancer cell spheroids, which mimic the physico-chemical gradients of in vivo tumors much better than the 2D culture. The spheroid model, therefore, allows evaluation of the cancer drug efficacy with improved translation to in vivo conditions.

Three-dimensional spheroids of cancer cells are important tools for both cancer drug screens and for gaining mechanistic insight into cancer cell biology. ...

A 3D Spheroid Model as a More Physiological System for Cancer-Associated Fibroblasts Differentiation and Invasion In Vitro Studies

Defining the ideal model for an in vitro study is essential, mainly if studying physiological processes such as differentiation of cells. In the tumor stroma, host fibroblasts are stimulated by cancer cells to differentiate. Thus, they acquire a phenotype that contributes to the tumor microenvironment and supports tumor progression. By using the spheroid model, we have set up such a 3D in vitro model system, in which we analyzed the role of laminin-332 and its receptor integrin &#945;3&#946;1 in this differentiation process. This spheroid model system not only reproduces the tumor microenvironment conditions in a more accurate way, but also is a very versatile model since it allows different downstream studies, such as immunofluorescent staining of both intra- and extracellular markers, as well as deposited extracellular matrix proteins. Moreover, transcriptional analyses by qPCR, flow cytometry and cellular invasion can be studied with this model. Here, we describe a protocol of a spheroid model to assess the role of CAFs&#39; integrin &#945;3&#946;1 and its ectopically deposited ligand, laminin-332, in differentiation and in supporting the invasion of pancreatic cancer cells.

The goal of this protocol is to establish a 3D in vitro model to study the differentiation of cancer-associated fibroblasts (CAFs) in a tumor bulk-like environment, which can be addressed in different analysis systems, such as immunofluorescence, transcriptional analysis and life cell imaging.

Defining the ideal model for an in vitro study is essential, mainly if studying physiological processes such as differentiation of cells. In the tumor ...

A Novel Stromal Fibroblast-Modulated 3D Tumor Spheroid Model for Studying Tumor-Stroma Interaction and Drug Discovery

Tumor-stroma interactions play an important role in cancer progression. Three-dimensional (3D) tumor spheroid models are the most widely used in vitro model in the study of cancer stem/initiating cells, preclinical cancer research, and drug screening. The 3D spheroid models are superior to conventional tumor cell culture and reproduce some important characters of real solid tumors. However, conventional 3D tumor spheroids are made up exclusively of tumor cells. They lack the participation of tumor stromal cells and have insufficient extracellular matrix (ECM) deposition, thus only partially mimicking the in vivo conditions of tumor tissues. We established a new multicellular 3D spheroid model composed of tumor cells and stromal fibroblasts that better mimics the in vivo heterogeneous tumor microenvironment and its native desmoplasia. The formation of spheroids is strictly regulated by the tumor stromal fibroblasts and is determined by the activity of certain crucial intracellular signaling pathways (e.g., Notch signaling) in stromal fibroblasts. In this article, we present the techniques for coculture of tumor cells-stromal fibroblasts, time-lapse imaging to visualize cell-cell interactions, and confocal microscopy to display the 3D architectural features of the spheroids. We also show two examples of the practical application of this 3D spheroid model. This novel multicellular 3D spheroid model offers a useful platform for studying tumor-stroma interaction, elucidating how stromal fibroblasts regulate cancer stem/initiating cells, which determine tumor progression and aggressiveness, and exploring involvement of stromal reaction in cancer drug sensitivity and resistance. This platform can also be a pertinent in vitro model for drug discovery.

A novel three-dimensional spheroid model based on the heterotypic interaction of tumor cells and stromal fibroblasts is established. Here, we present coculture of tumor cells and stromal fibroblasts, time-lapse imaging, and confocal microscopy to visualize the formation of spheroids. This three-dimensional model offers a pertinent platform to study tumor-stroma interactions and test cancer therapeutics.

Tumor-stroma interactions play an important role in cancer progression. Three-dimensional (3D) tumor spheroid models are the most widely used in vitro ...

A Three-Dimensional Spheroid Model to Investigate the Tumor-Stromal Interaction in Hepatocellular Carcinoma

The aggressiveness and lack of well-tolerated and widely effective treatments for advanced hepatocellular carcinoma (HCC), the predominant form of liver cancer, rationalize its rank as the second most common cause of cancer-related death. Preclinical models need to be adapted to recapitulate the human conditions to select the best therapeutic candidates for clinical development and aid the delivery of personalized medicine. Three-dimensional (3D) cellular spheroid models show promise as an emerging in vitro alternative to two-dimensional (2D) monolayer cultures. Here, we describe a 3D tumor spheroid&#160;model which exploits the ability of individual cells to aggregate when maintained in hanging droplets, and is more representative of an in vivo environment than standard monolayers. Furthermore, 3D spheroids can be produced by combining homotypic or heterotypic cells, more reflective of the cellular heterogeneity in vivo, potentially enabling the study of environmental interactions that can influence progression and treatment responses. The current research optimized the cell density to form 3D homotypic and heterotypic tumor spheroids by immobilizing cell suspensions on the lids of standard 10 cm3 Petri dishes. Longitudinal analysis was performed to generate growth curves for homotypic versus heterotypic tumor/fibroblasts spheroids. Finally, the proliferative impact of fibroblasts (COS7 cells) and liver myofibroblasts (LX2) on homotypic tumor (Hep3B) spheroids was investigated. A seeding density of 3,000 cells (in 20 &#181;L media) successfully yielded Huh7/COS7 heterotypic spheroids, which displayed a steady increase in size up to culture day 8, followed by growth retardation. This finding was corroborated using Hep3B homotypic spheroids cultured in LX2 (human hepatic stellate cell line) conditioned medium (CM). LX2 CM triggered the proliferation of Hep3B spheroids compared to control tumor spheroids. In conclusion, this protocol has shown that 3D tumor spheroids can be used as a simple, economical, and prescreen in vitro tool to study tumor-stromal interactions more comprehensively.

Comprehensive in vitro models that faithfully recapitulate the relevant human disease are lacking. The current study presents three-dimensional (3D) tumor spheroid creation and culture, a reliable in vitro tool to study the tumor-stromal interaction in human hepatocellular carcinoma.

The aggressiveness and lack of well-tolerated and widely effective treatments for advanced hepatocellular carcinoma (HCC), the predominant form of liver ...

Exploring Mitochondrial Energy Metabolism of Single 3D Microtissue Spheroids Using Extracellular Flux Analysis

Three-dimensional (3D) cellular aggregates, termed spheroids, have become the forefront of in vitro cell culture in recent years. In contrast to culturing cells as two-dimensional, single-cell monolayers (2D culture), spheroid cell culture promotes, regulates, and supports physiological cellular architecture and characteristics that exist in vivo, including the expression of extracellular matrix proteins, cell signaling, gene expression, protein production, differentiation, and proliferation. The importance of 3D culture has been recognized in many research fields, including oncology, diabetes, stem cell biology, and tissue engineering. Over the last decade, improved methods have been developed to produce spheroids and assess their metabolic function and fate.
Extracellular flux (XF) analyzers have been used to explore mitochondrial function in 3D microtissues such as spheroids using either an XF24 islet capture plate or an XFe96 spheroid microplate. However, distinct protocols and the optimization of probing mitochondrial energy metabolism in spheroids using XF technology have not been described in detail. This paper provides detailed protocols for probing mitochondrial energy metabolism in single 3D spheroids using spheroid microplates with the XFe96 XF analyzer. Using different cancer cell lines, XF technology is demonstrated to be capable of distinguishing between cellular respiration in 3D spheroids of not only different sizes but also different volumes, cell numbers,&#160;DNA content and type.
The optimal mitochondrial effector compound concentrations of oligomycin, BAM15, rotenone, and antimycin A are used to probe specific parameters of mitochondrial energy metabolism in 3D spheroids. This paper also discusses methods to normalize data obtained from spheroids and addresses many considerations that should be considered when exploring spheroid metabolism using XF technology. This protocol will help drive research in advanced in vitro spheroid models.

These protocols will help users probe mitochondrial energy metabolism in 3D cancer cell-line-derived spheroids using Seahorse extracellular flux analysis.

Three-dimensional (3D) cellular aggregates, termed spheroids, have become the forefront of in vitro cell culture in recent years. In contrast to culturing ...

Establishing 3-Dimensional Spheroids from Patient-Derived Tumor Samples and Evaluating their Sensitivity to Drugs

Despite remarkable advances in understanding tumor biology, the vast majority of oncology drug candidates entering clinical trials fail, often due to a lack of clinical efficacy. This high failure rate illuminates the inability of the current preclinical models to predict clinical efficacy, mainly due to their inadequacy in reflecting tumor heterogeneity and the tumor microenvironment. These limitations can be addressed with 3-dimensional (3D) culture models (spheroids) established from human tumor samples derived from individual patients. These 3D cultures represent real-world biology better than established cell lines that do not reflect tumor heterogeneity. Furthermore, 3D cultures are better than 2-dimensional (2D) culture models (monolayer structures) since they replicate elements of the tumor environment, such as hypoxia, necrosis, and cell adhesion, and preserve the natural cell shape and growth. In the present study, a method was developed for preparing primary cultures of cancer cells from individual patients that are 3D and grow in multicellular spheroids. The cells can be derived directly from patient tumors or patient-derived xenografts. The method is widely applicable to solid tumors (e.g., colon, breast, and lung) and is also cost-effective, as it can be performed in its entirety in a typical cancer research/cell biology lab without relying on specialized equipment. Herein, a protocol is presented for generating 3D tumor culture models (multicellular spheroids) from primary cancer cells and evaluating their sensitivity to drugs using two complementary approaches: a cell-viability assay (MTT) and microscopic examinations. These multicellular spheroids can be used to assess potential drug candidates, identify potential biomarkers or therapeutic targets, and investigate the mechanisms of response and resistance.

The present protocol describes generating 3D tumor culture models from primary cancer cells and evaluating their sensitivity to drugs using cell-viability assays and microscopic examinations.

Despite remarkable advances in understanding tumor biology, the vast majority of oncology drug candidates entering clinical trials fail, often due to a ...

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 ...

Three-dimensional Co-culture Model for Tumor-stromal Interaction

Cancer progression (initiation, growth, invasion and metastasis) occurs through interactions between malignant cells and the surrounding tumor stromal cells. The tumor microenvironment is comprised of a variety of cell types, such as fibroblasts, immune cells, vascular endothelial cells, pericytes and bone-marrow-derived cells, embedded in the extracellular matrix (ECM). Cancer-associated fibroblasts (CAFs) have a pro-tumorigenic role through the secretion of soluble factors, angiogenesis and ECM remodeling. The experimental models for cancer cell survival, proliferation, migration, and invasion have mostly relied on two-dimensional monocellular and monolayer tissue cultures or Boyden chamber assays. However, these experiments do not precisely reflect the physiological or pathological conditions in a diseased organ. To gain a better understanding of tumor stromal or tumor matrix interactions, multicellular and three-dimensional cultures provide more powerful tools for investigating intercellular communication and ECM-dependent modulation of cancer cell behavior. As a platform for this type of study, we present an experimental model in which cancer cells are cultured on collagen gels embedded with primary cultures of CAFs.

Here we present a protocol to co-culture in three-dimensions, which is useful for investigating multicellular interactions and extracellular matrix-dependent modulation of cancer cell behavior. In this experimental model, cancer cells are cultured on collagen gels embedded with human cancer-associated fibroblasts.

Cancer progression (initiation, growth, invasion and metastasis) occurs through interactions between malignant cells and the surrounding tumor stromal ...

Assessment of Mitochondrial Health in Cancer-Associated Fibroblasts Isolated from 3D Multicellular Lung Tumor Spheroids

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