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>

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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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Antibodies</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>APC anti-human &#945;-SMA</td>
			<td>R&#38;D systems</td>
			<td>Cat# IC1420A</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-fibroblast microbeads</td>
			<td>Miltenyi Biotec</td>
			<td>Cat# 130-050-601</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell lines</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>A549 lung adenocarcinoma cells</td>
			<td>NCCS Pune</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>MRC-5 fetal lung fibroblasts</td>
			<td>ATCC</td>
			<td>CCL-171</td>
			<td></td>
		</tr>
		<tr>
			<td>THP-1 Human monocytes</td>
			<td>NCCS Pune</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Chemicals</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Himedia</td>
			<td>Cat# 9048-46-8</td>
			<td></td>
		</tr>
		<tr>
			<td>2,6-dichloroindophenol (DCPIP)</td>
			<td>SRL</td>
			<td>Cat# 55287</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcein-AM</td>
			<td>Thermo Fisher Scientific</td>
			<td>Cat# C3099</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Thermo Fisher Scientific</td>
			<td>Cat# D1306</td>
			<td></td>
		</tr>
		<tr>
			<td>DCFDA</td>
			<td>Sigma</td>
			<td>Cat# D6883</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Gibco</td>
			<td>Cat# 11995073</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS</td>
			<td>Gibco</td>
			<td>Cat# 14190-144</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Thermo fisher scientific</td>
			<td>Cat# 17892</td>
			<td></td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>SRL</td>
			<td>Cat# 62858</td>
			<td></td>
		</tr>
		<tr>
			<td>EZcoun Lactate Dehydrogenase Cell Assay Kit</td>
			<td>HiMedia</td>
			<td>Cat# CCK036</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Gibco</td>
			<td>Cat# 10082147</td>
			<td></td>
		</tr>
		<tr>
			<td>Halt Protease and Phosphatase Inhibitor Cocktail (100X)</td>
			<td>Thermo Fisher Scientific</td>
			<td>Cat# 87786</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Thermo Fisher Scientific</td>
			<td>Cat# 15630080</td>
			<td></td>
		</tr>
		<tr>
			<td>Horse heart Cytochrome c</td>
			<td>SRL</td>
			<td>Cat# 81551</td>
			<td></td>
		</tr>
		<tr>
			<td>Image-iT Red hypoxia reagent</td>
			<td>Thermo Fisher Scientific</td>
			<td>Cat# H10498</td>
			<td></td>
		</tr>
		<tr>
			<td>JC-1 Dye</td>
			<td>Thermo Fisher Scientific</td>
			<td>Cat# T3168</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Merck</td>
			<td>Cat# P9541</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Merck</td>
			<td>Cat# M8266</td>
			<td></td>
		</tr>
		<tr>
			<td>MOPS</td>
			<td>Thermo Fisher Scientific</td>
			<td>Cat# 69824</td>
			<td></td>
		</tr>
		<tr>
			<td>Nacl</td>
			<td>Sigma-Aldrich</td>
			<td>Cat# S9888</td>
			<td></td>
		</tr>
		<tr>
			<td>NADH MB Grade</td>
			<td>SRL</td>
			<td>Cat# 54941</td>
			<td></td>
		</tr>
		<tr>
			<td>NP-40</td>
			<td>Thermo Fisher Scientific</td>
			<td>Cat# 85124</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>Gibco</td>
			<td>Cat# 15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>Phenazine methosulfate (PMS)</td>
			<td>SRL</td>
			<td>Cat# 55782</td>
			<td></td>
		</tr>
		<tr>
			<td>Propidium iodide</td>
			<td>Thermo fisher scientific</td>
			<td>Cat# P1304MP</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI 1640</td>
			<td>Gibco</td>
			<td>Cat# 11875093</td>
			<td></td>
		</tr>
		<tr>
			<td>Single Cell Lysis Kit</td>
			<td>Thermo Fisher Scientific</td>
			<td>Cat# 4458235</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium ascorbate</td>
			<td>Merck</td>
			<td>Cat# A7631</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium cyanide</td>
			<td>Sigma</td>
			<td>Cat# 205222</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Deoxycholate</td>
			<td>Thermo Fisher Scientific</td>
			<td>Cat# 89904</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium dodecyl sulphate</td>
			<td>Sigma-Aldrich</td>
			<td>Cat# L3771</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium succinate hexahydrate</td>
			<td>SRL</td>
			<td>Cat# 36313</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma</td>
			<td>Cat# S0389</td>
			<td></td>
		</tr>
		<tr>
			<td>SuperScript VILO cDNA synthesis kit</td>
			<td>Thermo Fisher Scientific</td>
			<td>Cat# 11754-050</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma</td>
			<td>Cat# T8787</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin 0.25% EDTA</td>
			<td>Gibco</td>
			<td>Cat# 25200072</td>
			<td></td>
		</tr>
		<tr>
			<td>Universal SYBR Green Supermix</td>
			<td>BIO-RAD</td>
			<td>Cat# 172-5124</td>
			<td></td>
		</tr>
		<tr>
			<td>Plasticware</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MACS LS Columns</td>
			<td>Miltenyi Biotec</td>
			<td>Cat# 130-042-401</td>
			<td></td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Countess II FL Automated Cell Counter</td>
			<td>Thermo Fisher Scientific</td>
			<td>Cat# AMQAF1000</td>
			<td></td>
		</tr>
		<tr>
			<td>EVOS XL core imaging system</td>
			<td>Thermo Fisher Scientific</td>
			<td>Serial Number F0518-1727-0191</td>
			<td></td>
		</tr>
		<tr>
			<td>LAS X software</td>
			<td>Leica Microsystems</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Leica fluorescent inverted microscope</td>
			<td>s</td>
			<td>DMi8 automated S/N 424150)</td>
			<td></td>
		</tr>
		<tr>
			<td>Midi MACS separator</td>
			<td>Miltenyi Biotec</td>
			<td>Cat# 130-042-302</td>
			<td></td>
		</tr>
	</tbody>
</table>

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 ...

assessment-mitochondrial-health-cancer-associated-fibroblasts

Research

JoVE Journal

Cancer Research

1.8K Views.  Indian Institute of Technology Ropar. 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.

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

In Vivo EPR Assessment of pH, pO2, Redox Status, and Concentrations of Phosphate and Glutathione in the Tumor Microenvironment

This protocol demonstrates the capability of low-field electron paramagnetic resonance (EPR)-based techniques in combination with functional paramagnetic probes to provide quantitative information on the chemical tumor microenvironment (TME), including pO2, pH, redox status, concentrations of interstitial inorganic phosphate (Pi), and intracellular glutathione (GSH). In particular, an application of a recently developed soluble multifunctional trityl probe provides unsurpassed opportunity for in vivo concurrent measurements of pH, pO2 and Pi in Extracellular space (HOPE probe). The measurements of three parameters using a single probe allow for their correlation analyses independent of probe distribution and time of the measurements.

In Vivo EPR Assessment of pH, pO2, Redox Status, and Concentrations of Phosphate and Glutathione in the Tumor Microenvironment

Low-field (L-band, 1.2 GHz) electron paramagnetic resonance using soluble nitroxyl and trityl probes is demonstrated for assessment of physiologically important parameters in the tumor microenvironment in mouse models of breast cancer.

This protocol demonstrates the capability of low-field electron paramagnetic resonance (EPR)-based techniques in combination with functional paramagnetic ...

The Establishment of a Lung Colonization Assay for Circulating Tumor Cell Visualization in Lung Tissues

Metastasis is the major cause of cancer death. The role of circulating tumor cells (CTCs) in promoting cancer metastasis, in which lung colonization by CTCs critically contributes to early lung metastatic processes, has been vigorously investigated. As such, animal models are the only approach that captures the full systemic process of metastasis. Given that problems occur in previous experimental designs for examining the contributions of CTCs to blood vessel extravasation, we established an in vivo lung colonization assay in which a long-term-fluorescence cell-tracer, carboxyfluorescein succinimidyl ester (CFSE), was used to label suspended tumor cells and lung perfusion was performed to clear non-specifically trapped CTCs prior to lung removal, confocal imaging, and quantification. Polymeric fibronectin (polyFN) assembled on CTC surfaces has been found to mediate lung colonization in the final establishment of metastatic tumor tissues. Here, to specifically test the requirement of polyFN assembly on CTCs for lung colonization and extravasation, we performed short term lung colonization assays in which suspended Lewis lung carcinoma cells (LLCs) stably expressing FN-shRNA (shFN) or scramble-shRNA (shScr) and pre-labeled with 20 &#956;M of CFSE were intravenously inoculated into C57BL/6 mice. We successfully demonstrated that the abilities of shFN LLC cells to colonize the mouse lungs were significantly diminished in comparison to shScr LLC cells. Therefore, this short-term methodology may be widely applied to specifically demonstrate the ability of CTCs within the circulation to colonize the lungs.

An animal model is needed to decipher the role of circulating tumor cells (CTCs) in promoting lung colonization during cancer metastasis. Here, we established and successfully performed an in vivo assay to specifically test the requirement of polymeric fibronectin (polyFN) assembly on CTCs for lung colonization.

Metastasis is the major cause of cancer death. The role of circulating tumor cells (CTCs) in promoting cancer metastasis, in which lung colonization by ...

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 ...

Establishment and Characterization of Small Bowel Neuroendocrine Tumor Spheroids

Small bowel neuroendocrine tumors (SBNETs) are rare cancers originating from enterochromaffin cells of the gut. Research in this field has been limited because very few patient derived SBNET cell lines have been generated. Well-differentiated SBNET cells are slow growing and are hard to propagate. The few cell lines that have been established are not readily available, and after time in culture may not continue to express characteristics of NET cells. Generating new cell lines could take many years since SBNET cells have a long doubling time and many enrichment steps are needed in order to eliminate the rapidly dividing cancer-associated fibroblasts. To overcome these limitations, we have developed a protocol to culture SBNET cells from surgically removed tumors as spheroids in extracellular matrix (ECM). The ECM forms a 3-dimensional matrix that encapsulates SBNET cells and mimics the tumor micro-environment for allowing SBNET cells to grow. Here, we characterized the growth rate of SBNET spheroids and described methods to identify SBNET markers using immunofluorescence microscopy and immunohistochemistry to confirm that the spheroids are neuroendocrine tumor cells. In addition, we used SBNET spheroids for testing the cytotoxicity of rapamycin.

Neuroendocrine tumors (NETs) originate from neuroendocrine cells of the neural crest. They are slow growing and challenging to culture. We present an alternative strategy to grow NETs from the small bowel by culturing them as spheroids. These spheroids have small bowel NET markers and can be used for drug testing.

Small bowel neuroendocrine tumors (SBNETs) are rare cancers originating from enterochromaffin cells of the gut. Research in this field has been limited ...

A Mouse Model to Investigate the Role of Cancer-Associated Fibroblasts in Tumor Growth

Cancer-associated fibroblasts (CAFs) can play an important role in tumor growth by creating a tumor-promoting microenvironment. Models to study the role of CAFs in the tumor microenvironment can be helpful for understanding the functional importance of fibroblasts, fibroblasts from different tissues, and specific genetic factors in fibroblasts. Mouse models are essential for understanding the contributors to tumor growth and progression in an in vivo context. Here, a protocol in which cancer cells are mixed with fibroblasts and introduced into mice to develop tumors is provided. Tumor sizes over time and final tumor weights are determined and compared among groups. The protocol described can provide more insight into the functional role of CAFs in tumor growth and progression.

A protocol to co-inject cancer cells and fibroblasts and monitor tumor growth over time is provided. This protocol can be used to understand the molecular basis for the role of fibroblasts as regulators of tumor growth.

Cancer-associated fibroblasts (CAFs) can play an important role in tumor growth by creating a tumor-promoting microenvironment. Models to study the role ...

Isolation of Primary Cancer-Associated Fibroblasts from a Syngeneic Murine Model of Breast Cancer for the Study of Targeted Nanoparticles

Cancer-associated fibroblasts (CAFs) are key actors in the context of the tumor microenvironment. Despite being reduced in number as compared to tumor cells, CAFs regulate tumor progression and provide protection from antitumor immunity. Emerging anticancer strategies aim to remodel the tumor microenvironment through the ablation of pro-tumorigenic CAFs or reprogramming of CAFs functions and their activation status. A promising approach is the development of nanosized delivery agents able to target CAFs, thus allowing the specific delivery of drugs and active molecules. In this context, a cellular model of CAFs may provide a useful tool for in vitro screening and preliminary investigation of such nanoformulations.
This study describes the isolation and culture of primary CAFs from the syngeneic 4T1 murine model of triple-negative breast cancer. Magnetic beads were used in a 2-step separation process to extract CAFs from dissociated tumors. Immunophenotyping control was performed using flow cytometry after each passage to verify the process yield. Isolated CAFs can be employed to study the targeting capability of different nanoformulations designed to tackle the tumor microenvironment. Fluorescently labeled H-ferritin nanocages were used as candidate nanoparticles to set up the method. Nanoparticles, either bare or conjugated with a targeting ligand, were analyzed for their binding to CAFs. The results suggest that ex vivo extraction of breast CAFs may be a useful system to test and validate nanoparticles for the specific targeting of tumorigenic CAFs.

This paper aims to provide a protocol for the isolation and culture of primary cancer-associated fibroblasts from a syngeneic murine model of triple-negative breast cancer and their application for the preclinical study of novel nanoparticles designed to target the tumor microenvironment.&#160;

Cancer-associated fibroblasts (CAFs) are key actors in the context of the tumor microenvironment. Despite being reduced in number as compared to tumor ...

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 ...

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 ...

Pancreatic Cancer-Derived Tumor Organoids and Fibroblasts from Fresh Tissue

Establishment of Pancreatic Cancer-Derived Tumor Organoids and Fibroblasts From Fresh Tissue

Tumor organoids are three-dimensional (3D) ex vivo tumor models that recapitulate the biological key features of the original primary tumor tissues. Patient-derived tumor organoids have been used in translational cancer research and can be applied to assess treatment sensitivity and resistance, cell-cell interactions, and tumor cell interactions with the tumor microenvironment. Tumor organoids are complex culture systems that require advanced cell culture techniques and culture media with specific growth factor cocktails and a biological basement membrane that mimics the extracellular environment. The ability to establish primary tumor cultures highly depends on the tissue of origin, the cellularity, and the clinical features of the tumor, such as the tumor grade. Furthermore, tissue sample collection, material quality and quantity, as well as correct biobanking and storage are crucial elements of this procedure. The technical capabilities of the laboratory are also crucial factors to consider. Here, we report a validated SOP/protocol that is technically and economically feasible for the culture of ex vivo tumor organoids from fresh tissue samples of pancreatic adenocarcinoma origin, either from fresh primary resected patient donor tissue or patient-derived xenografts (PDX). The technique described herein can be performed in laboratories with basic tissue culture and mouse facilities and is tailored for wide application in the translational oncology field.

Author Spotlight: Establishment of Pancreatic Cancer-Derived Tumor Organoids and Fibroblasts From Fresh Tissue  

Tumor organoids have revolutionized cancer research and the approach to personalized medicine. They represent a clinically relevant tumor model that allows researchers to stay one step ahead of the tumor in the clinic. This protocol establishes tumor organoids from fresh pancreatic tumor tissue samples and patient-derived xenografts of pancreatic adenocarcinoma origin.

Tumor organoids are three-dimensional (3D) ex vivo tumor models that recapitulate the biological key features of the original primary tumor tissues. ...

Examining Tumor Cell Dissemination from Lung Metastases

Tracking Tumor Cell Dissemination from Lung Metastases Using Photoconversion

Metastasis - the systemic spread of cancer - is the leading cause of cancer-related deaths. Although metastasis is commonly thought of as a unidirectional process wherein cells from the primary tumor disseminate and seed metastases, tumor cells in existing metastases can also redisseminate and give rise to new lesions in tertiary sites in a process known as &#34;metastasis-from-metastases&#34; or &#34;metastasis-to-metastasis seeding.&#34; Metastasis-to-metastasis seeding may increase the metastatic burden and decrease the patient&#39;s quality of life and survival. Therefore, understanding the processes behind this phenomenon is crucial to refining treatment strategies for patients with metastatic cancer.
Little is known about metastasis-to-metastasis seeding, due in part to logistical and technological limitations. Studies on metastasis-to-metastasis seeding rely primarily on sequencing methods, which may not be practical for researchers studying the exact timing of metastasis-to-metastasis seeding events or what promotes or prevents them. This highlights the lack of methodologies that facilitate the study of metastasis-to-metastasis seeding. To address this, we have developed - and describe herein - a murine surgical protocol for the selective photoconversion of lung metastases, allowing specific marking and fate tracking of tumor cells redisseminating from the lung to tertiary sites. To our knowledge, this is the only method for studying tumor cell redissemination and metastasis-to-metastasis seeding from the lungs that does not require genomic analysis.

Author Spotlight: Decoding Metastasis-to-Metastasis Seeding Using a New In Vivo Technique for Tracking Breast Cancer Spread 

We present a method for studying tumor cell redissemination from lung metastases involving a surgical protocol for the selective photoconversion of lung metastases, followed by the identification of redisseminated tumor cells in tertiary organs.

Metastasis - the systemic spread of cancer - is the leading cause of cancer-related deaths. Although metastasis is commonly thought of as a unidirectional ...