We thank all the members of the Shonai Regional Industry Promotion Center for their help. This work was supported in part by research funds from Yamagata Prefecture and Tsuruoka City, by the National Cancer Center Research and Development Fund [grant number 28-A-9], and by the Japan Society for the Promotion of Science (JSPS) KAKENHI [grant number 17K07189] to HM.

1. Cell culture on day 1
NOTE: Each sample for metabolite extraction should be prepared from a single 100 mm tissue culture dish that is moderately but not fully confluent (containing approximately 2&#8211;5 million cells). Calculate the number of dishes needed for the assay and prepare them accordingly.
<ol>
	<li>Culture HCC827 and PC-9 cells in 5% CO2 at 37 &#176;C in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS).</li>
	<li>Aspirate the cell culture media from the 100 mm culture dishes.</li>
	<li>Wash cells on each dish using 2 mL of phosphate buffered saline (PBS) solution without calcium and magnesium. Gently rock each dish so that the PBS solution completely covers the surface of the dish.</li>
	<li>Aspirate the wash buffer from the culture dishes.</li>
	<li>Warm 0.25% trypsin-EDTA solution to 37 &#176;C and add 2 mL of trypsin-EDTA solution with a 5 mL serological pipette. Gently rock each dish so that the trypsin completely covers the surface of the dish.</li>
	<li>Incubate the culture dishes at 37 &#176;C for approximately 5 min.</li>
	<li>Add 4 mL of pre-warmed complete growth medium per dish. Resuspend the cells in medium by gently pipetting several times.</li>
	<li>Transfer each cell suspension to a separate 15 mL conical tube and centrifuge at 800 &#215; g for 5 min.</li>
	<li>Resuspend each cell pellet in 2 mL of pre-warmed complete growth medium.</li>
	<li>Determine the total number and percent viability of the cells using the automated cell counter and 0.4% trypan blue solution.
	<ol>
		<li>Mix 10 &#956;L of cell suspension and 10 &#956;L of 0.4% trypan blue solution.</li>
		<li>Load 10 &#956;L of sample into the cell counting chamber slide through capillary action.</li>
		<li>Insert the chamber slide into the automated cell counter. The transmitted light automatically illuminates and the instrument auto focuses on the cell.</li>
		<li>Press the capture button to capture the image and display the results.</li>
		<li>If necessary, add further growth medium to obtain the desired cell concentration.</li>
	</ol>
	</li>
	<li>Seed approximately 1&#8211;2.5 million cells per 100 mm cell culture dish. 
	NOTE: Metabolite concentrations determined by CE-MS analysis will be normalized based on the number of viable cells. For the purpose of cell counting, it is necessary to prepare at least one extra seeded culture dish for each group.</li>
	<li>Incubate the culture dishes in 5% CO2 at 37 &#176;C for 18 h.</li>
</ol>
2. Preparation of reagents
<ol>
	<li>Dilute a commercial internal standard solution including L-methionine sulfone and d-camphor-10-sulfonic acid 1000-fold in ultrapure water. 
	NOTE: For fewer than 80 samples, simply mix 50 &#956;L of the Internal Standard Solution 1 and 45 mL of ultrapure water in a 50 mL volumetric flask, then bring the solution up to 50 mL with ultrapure water.</li>
	<li>Prepare a 0.05 g/mL mannitol solution in ultrapure water as wash buffer. 
	NOTE: For fewer than 30 samples, simply dissolve 25 g of mannitol in 500 mL of ultrapure water. Approximately 15 mL of wash buffer is required per 100 mm culture dish, so prepare a sufficient volume of wash buffer according to the number of samples.</li>
</ol>
3. Pre-washing centrifugal filter units
<ol>
	<li>Pipette 250 &#956;L of ultrapure water into the filter cup of each centrifugal filter unit (see the Table of Materials). 
	NOTE: Two filter units per sample are required.</li>
	<li>Cap the filter units tightly and centrifuge at 9,100 &#215; g at 4 &#176;C for 5 min.</li>
	<li>Check the volume of each filtrate&#8212;if significant filtrate has accumulated during the first short spin, the filter unit may be defective. In this case, discard the filter unit and use a new filter unit instead.</li>
	<li>Close the lids of the filter units tightly and centrifuge again at 9,100 &#215; g at 4 &#176;C for 30 min.</li>
	<li>Ensure that no ultrapure water remains in any of the filter cups; remove the filtered ultrapure water in each collection tube with a pipette and discard. 
	NOTE: Do not try to remove residual water in a filter cup with a pipette as it may damage the filter.</li>
	<li>Replace the filter cups into their collection tubes. 
	NOTE: Use the centrifugal filter units within an hour since the filters may become damaged upon drying.</li>
</ol>
4. Cell culture on day 2
<ol>
	<li>Take out the 100 mm culture dishes from the incubator. 
	NOTE: The recommended cell culture duration is 18 h.</li>
	<li>Aspirate the cell culture medium from each 100 mm culture dish.</li>
	<li>Add 10 mL of cell culture medium that includes the appropriate concentrations of compounds or drugs to each dish, taking care to not disturb the cell layer. 
	NOTE: For demonstration purposes, we added 10 &#956;L of 250 mM diamide dissolved in PBS (final concentration of 250 &#956;M) in this experiment.</li>
	<li>Incubate the culture dishes at 37 &#176;C for 30 min in the presence of diamide or PBS as a control.</li>
	<li>Aspirate the cell culture medium from each 100 mm culture dish.</li>
	<li>Wash cells by gently adding 2 mL of 5% mannitol solution to the edge of each dish, taking care to not to disturb the cell layer, then slightly tilt the dish. 
	NOTE: PBS or saline solution interferes with CE-MS-based metabolomic analysis and adversely affects measurement results, and thus should not be used as wash buffer.</li>
	<li>Aspirate the wash buffer from each culture dish, then wash cells again by gently adding 10 mL of wash buffer per dish and slightly tilting the dish.</li>
	<li>Completely aspirate the wash buffer from the edge of each culture dish. 
	NOTE: Aspirate as much wash buffer as possible, while paying attention to not aspirate the cells. Residual mannitol may interfere with CE-MS analysis; aspiration of cells will decrease the number of cells and thus become a source of error in data normalization.</li>
</ol>
5. Extraction of metabolites from cultured cells
<ol>
	<li>Add 800 &#956;L of 99.7% methanol per culture dish. Gently rock each culture dish back and forth to cover its entire surface. Leave the dishes at room temperature for 30 s.</li>
	<li>Slowly add 550 &#956;L of the diluted internal standard solution per dish by immersing the tip of the pipette into the methanol and gently pipetting up and down several times.</li>
	<li>Gently rock each culture dish back and forth to cover its entire surface.</li>
	<li>Leave the dishes at room temperature for 30 s.</li>
</ol>
6. Ultrafiltration of cell extracts
<ol>
	<li>Transfer the extracted solution from each culture dish to a separate 1.5 mL microcentrifuge tube.</li>
	<li>Centrifuge the tubes at 2,300 &#215; g at 4 &#176;C for 5 min.</li>
	<li>Transfer 350 &#956;L of each supernatant into two centrifugal filter units per sample. 
	NOTE: From each culture dish, a total of 700 &#956;L of the extracted solution is transferred into two filter tubes (350 &#956;L/tube).</li>
	<li>Centrifuge the filter tubes at 9,100 &#215; g at 4 &#176;C for approximately 2 h until no liquid remains in the filter cups.</li>
	<li>Remove the filter cups and tightly close the lids of the collection tubes.</li>
</ol>
7. Sample evaporation
<ol>
	<li>Prepare a centrifugal evaporator&#8212;typically, this consists of an evaporator, a cold trap, and a vacuum pump.</li>
	<li>Place the collection tubes in the centrifugal evaporator. 
	NOTE: Leave the lids of the tubes open.</li>
	<li>Evaporate the extracted sample solutions under vacuum conditions at room temperature. 
	NOTE: Typical configurations for the number of rotations and pressure are 1,500 rpm and 1,000 Pa, respectively, and it usually takes approximately 3 h to completely evaporate the samples.</li>
	<li>Confirm that no liquid remains in any of the collection tubes and close the lids of the tubes tightly.</li>
	<li>Store the collection tubes in an ultra-low temperature (&#8722;80 &#176;C) deep freezer until metabolomic analysis.</li>
</ol>
8. Metabolomic analysis by CE-MS
<ol>
	<li>Resuspend the filtrate in 50 &#956;L of ultrapure water immediately before CE-MS analysis.</li>
	<li>Perform CE-MS analysis by methods described previously12,13 using capillary electrophoresis system and time-of-flight mass spectrometer system equipped with an isocratic pump, a CE-MS adapter, and a CE-ESI-MS sprayer. 
	NOTE: Both systems can be controlled by the software of the system vendors and are connected by a fused silica capillary (50 &#956;m internal diameter &#215; 80 cm total length).
	<ol>
		<li>Set up instruments and sample vials, prepare the capillary with a capillary cassette, replenish sheath liquids and appropriate electrophoresis buffers depending on anion or cation analysis mode, and then apply voltages. 
		NOTE: Instrumentation and analytical conditions are described in detail elsewhere12,13.</li>
		<li>Open the software and prepare a worklist containing data acquisition methods and sample information.</li>
		<li>Start a test run and check the data such as the signal intensity and the peak shape of internal standards and the peak resolution of other standard compounds.</li>
		<li>Fine-tune the analytical conditions if necessary.</li>
		<li>Inject the sample solutions at 50 mbar for 3 s and a voltage of 30 kV. 
		NOTE: CE-MS was conducted either in the positive or negative ion mode. Set the spectrometer to scan the mass range m/z 50&#8211;1000. The capillary voltage was set at 4 kV; the flow rate of nitrogen gas (heater temperature 300&#176;C). For positive mode, the fragmentor, skimmer and OCT RFV voltage were set at 75, 50 and 125 V, respectively. For negative ion mode, the fragmentor, skimmer, and OCT RFV voltage was set at 100, 50, and 200 V, respectively.</li>
	</ol>
	</li>
	<li>Analyze spectrum data.
	<ol>
		<li>Extract peaks from the mass spectral data using automatic integration software in order to obtain peak information including m/z, peak area, and migration time (MT). 
		NOTE: The method is described in detail elsewhere14.</li>
		<li>Exclude signal peaks corresponding to isotopomers, adduct ions, and other product ions of known metabolites.</li>
		<li>Annotate remaining peaks with information from the HMT metabolite database according to m/z values and MTs.</li>
		<li>Normalize areas of the annotated peaks to internal standard levels and numbers of cells per sample.</li>
		<li>Evaluate the concentration of each metabolite in the cultured cells (pmol/106 cells) using standard curves prepared for each metabolite.</li>
		<li>Use the quantified metabolite concentrations for subsequent statistical analyses and biological interpretations14.</li>
	</ol>
	</li>
</ol>

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	<li>Lunt, S. Y., Vander Heiden, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aerobic+glycolysis:+meeting+the+metabolic+requirements+of+cell+proliferation.">Aerobic glycolysis: meeting the metabolic requirements of cell proliferation.</a> Annual Review Cell and Developmental Biology. 27, 441-464 (2011).</li><li>Soga, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+metabolism:+key+players+in+metabolic+reprogramming.">Cancer metabolism: key players in metabolic reprogramming.</a> Cancer Science. 104 (3), 275-281 (2013).</li><li>Zong, W. X., Rabinowitz, J. D., White, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondria+and+Cancer.">Mitochondria and Cancer.</a> Molecular Cell. 61 (5), 667-676 (2016).</li><li>Fukuda, H., 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=Experimental+study+for+cancer+diagnosis+with+positron-labeled+fluorinated+glucose+analogs:+[18F]-2-fluoro-2-deoxy-D-mannose:+a+new+tracer+for+cancer+detection.">Experimental study for cancer diagnosis with positron-labeled fluorinated glucose analogs: [18F]-2-fluoro-2-deoxy-D-mannose: a new tracer for cancer detection.</a> European Journal of Nuclear Meddicine and Molecular Imaging. 7 (7), 294-297 (1982).</li><li>Miles, K. A., Williams, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Warburg+revisited:+imaging+tumour+blood+flow+and+metabolism.">Warburg revisited: imaging tumour blood flow and metabolism.</a> Cancer Imaging. 8, 81-86 (2008).</li><li>Hanahan, D., Weinberg, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hallmarks+of+cancer:+the+next+generation.">Hallmarks of cancer: the next generation.</a> Cell. 144 (5), 646-674 (2011).</li><li>Levine, A. J., Puzio-Kuter, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+control+of+the+metabolic+switch+in+cancers+by+oncogenes+and+tumor+suppressor+genes.">The control of the metabolic switch in cancers by oncogenes and tumor suppressor genes.</a> Science. 330 (6009), 1340-1344 (2010).</li><li>Makinoshima, H., 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=Epidermal+growth+factor+receptor+(EGFR)+signaling+regulates+global+metabolic+pathways+in+EGFR-mutated+lung+adenocarcinoma.">Epidermal growth factor receptor (EGFR) signaling regulates global metabolic pathways in EGFR-mutated lung adenocarcinoma.</a> The Journal of Biological Chemistry. 289 (30), 20813-20823 (2014).</li><li>Makinoshima, H., 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=Signaling+through+the+Phosphatidylinositol+3-Kinase+(PI3K)/Mammalian+Target+of+Rapamycin+(mTOR)+Axis+Is+Responsible+for+Aerobic+Glycolysis+mediated+by+Glucose+Transporter+in+Epidermal+Growth+Factor+Receptor+(EGFR)-mutated+Lung+Adenocarcinoma.">Signaling through the Phosphatidylinositol 3-Kinase (PI3K)/Mammalian Target of Rapamycin (mTOR) Axis Is Responsible for Aerobic Glycolysis mediated by Glucose Transporter in Epidermal Growth Factor Receptor (EGFR)-mutated Lung Adenocarcinoma.</a> The Journal of Biological Chemistry. 290 (28), 17495-17504 (2015).</li><li>Makinoshima, H., 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+Determinants+of+Sensitivity+to+Phosphatidylinositol+3-Kinase+Pathway+Inhibitor+in+Small-Cell+Lung+Carcinoma.">Metabolic Determinants of Sensitivity to Phosphatidylinositol 3-Kinase Pathway Inhibitor in Small-Cell Lung Carcinoma.</a> Cancer Research. 78 (9), 2179-2190 (2018).</li><li>Sato, 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=Metabolic+Characterization+of+Antifolate+Responsiveness+and+Non-responsiveness+in+Malignant+Pleural+Mesothelioma+Cells.">Metabolic Characterization of Antifolate Responsiveness and Non-responsiveness in Malignant Pleural Mesothelioma Cells.</a> Frontiers in Pharmacology. 9, 1129 (2018).</li><li>Ohashi, 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=Depiction+of+metabolome+changes+in+histidine-starved+Escherichia+coli+by+CE-TOFMS.">Depiction of metabolome changes in histidine-starved Escherichia coli by CE-TOFMS.</a> Molecular BioSystems. 4 (2), 135-147 (2008).</li><li>Ooga, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolomic+anatomy+of+an+animal+model+revealing+homeostatic+imbalances+in+dyslipidaemia.">Metabolomic anatomy of an animal model revealing homeostatic imbalances in dyslipidaemia.</a> Molecular BioSystems. 7 (4), 1217-1223 (2011).</li><li>Sugimoto, M., Wong, D. T., Hirayama, A., Soga, T., Tomita, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Capillary+electrophoresis+mass+spectrometry-based+saliva+metabolomics+identified+oral,+breast+and+pancreatic+cancer-specific+profiles.">Capillary electrophoresis mass spectrometry-based saliva metabolomics identified oral, breast and pancreatic cancer-specific profiles.</a> Metabolomics. 6 (1), 78-95 (2010).</li><li>Patra, K. C., Hay, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+pentose+phosphate+pathway+and+cancer.">The pentose phosphate pathway and cancer.</a> Trends in Biochemical Sciences. 39 (8), 347-354 (2014).</li></ol>

The authors have nothing to disclose.

Here, we describe a widely accessible methodology to prepare metabolites from cultured cancer cells for CE-MS-based metabolomic analysis. One of the most critical points in this protocol is the proper preparation of cancer cells, because measured metabolite concentrations are normalized to the number of viable cells. For accurate estimation of cell number, it is necessary to prepare at least one additional culture dish per experimental group to count the number of viable cells in parallel with the extraction of metabolites for metabolomic analysis. In addition, the same number of cells should be seeded in each dish for the replicates and in the dish for counting; in the future, this would be aided by a quick and stressor-free (e.g., trypsin-free) cell counting protocol that allows the same dish to be used for both counting viable cells and extracting metabolites. Care should be taken during washes so that cells do not detach from the surface of the dishes. Severe cytotoxicity tests and other experiments that reduce cell adhesion may be unsuitable for this extraction protocol due to potential loss of cells during the washing procedure.
It is important to use a 5% mannitol solution as the wash buffer for extracting metabolites from cultured cells for CE-MS-based metabolomic analysis, because salt-based buffers, such as PBS, interfere with metabolomic analysis and adversely affect measurement.
Two or three dishes can be combined as a single sample by individually extracting metabolites from each dish and then pooling samples; however, combining multiple dishes often increases residual mannitol in the extracted metabolite solution. This may also interfere with metabolomic analysis by CE-MS. Hence, it is recommended to not use multiple dishes or wells as a single sample.
This metabolomic analysis method using CE-MS has been developed for comprehensive measurement of charged molecules with molecular weights between 50 and 1000 Da; thus, this protocol is optimized for extraction of aqueous, low molecular weight compounds. Therefore, this protocol is not suitable for extracting hydrophobic metabolites such as lipids or macromolecules such as proteins and nucleic acids. Since there is an increasing demand for comprehensive lipid analyses or lipidomics of cultured cell samples, the development of an easy and effective protocol for simultaneous extraction of both hydrophilic and hydrophobic metabolites is needed.
The first step of metabolite extraction&#8212;aspirating medium and washing cells with mannitol&#8212;should be conducted as quickly as possible to minimize changes to the metabolic profile of the cells. Treatment of cells with methanol after washing with mannitol is assumed to denature proteins and thereby prevent enzymes from catalyzing further metabolic reactions. However, even after methanol treatment, non-enzymatic chemical reactions&#8212;such as redox reactions, some decarboxylation processes, and thiol linkages&#8212;may take place. As such, any concentrations of metabolites involved in these reactions measured by this protocol should be interpreted with caution. In contrast to the genome or transcriptome, the metabolome consists of molecules with a wide variety of chemical properties; hence, no single protocol can extract all metabolites without any loss or disturbance. For more accurate measurements of such highly reactive metabolites, a protocol specifically designed to extract certain groups of metabolites, which requires fractionations and derivatizations, should be consulted. The protocol presented here, however, describes a simple and quick extraction of aqueous metabolites from cultured cell samples for metabolomic analysis by CE-MS. In this paper we could not describe how to set up CE-MS in detail because the focus of the present manuscript is different, however, describing detailed steps to set up CE-MS may require a separate dedicated article.

Otto Warburg observed that cancer cells acquire the unusual ability to take up glucose and ferment it to produce lactate in the presence of adequate oxygen&#8212;a phenomenon termed as Warburg effect or aerobic glycolysis1,2. Mitochondrial respiration defects are speculated as the underlying basis for aerobic glycolysis in cancer cells3. Indeed, the Warburg effect is the basis for tumor imaging by fluorodeoxyglucose (FDG)-positron emission tomography (PET), which is widely used in clinical practice4,5. A high rate of aerobic glycolysis is considered a key feature of cancer and has been recently adopted as one of the well-known &#8220;hallmarks of cancer,&#8221; as described by D. Hanahan and B. Weinberg6. Somatic mutations in oncogenes and tumor suppressor genes&#8212;such as HRAS/KRAS/NRAS, EGFR, BRAF, MYC, TP53, isocitrate dehydrogenase (IDH), and fumarate hydratase (FH)&#8212;have been linked to specific metabolic changes in cancer cells, believed to be a result of the Warburg effect7.
Metabolomic analysis is a promising approach not only to understand metabolic regulation in cancer cells but also to identify early-stage cancer biomarkers and chemotherapy response prediction. Following treatment of sensitive or resistant cancer cells with anticancer compounds, tracking of their metabolic responses facilitates identification of metabolic biomarkers to predict efficacy of specific anticancer therapies in cancer patients8,9,10,11. In this article, cancer cell lines derived from a lung adenocarcinoma with an EGFR mutation treated with diamide&#8212;which causes oxidative stress&#8212;were used as models for metabolomic analysis. The advantage of this analytical method using capillary electrophoresis-mass spectrometry (CE-MS) is its comprehensive measurement of charged metabolites with the mass range m/z 50-100012,13. The purpose of this article is to provide novices a detailed stepwise visual protocol for preparation of aqueous metabolites from cultured cancer cells and subsequent metabolomic analysis, particularly by CE-MS.

<table>
	<tbody>
		<tr>
			<td>Automated cell counter</td>
			<td>Thermo Scientific</td>
			<td>AMQAX1000</td>
			<td>Countess II automated cell counter</td>
		</tr>
		<tr>
			<td>Automatic integration software</td>
			<td>Agilent Technologies</td>
			<td>MassHunter G3335-60041</td>
			<td>version B.02.00</td>
		</tr>
		<tr>
			<td>CE system</td>
			<td>Agilent Technologies</td>
			<td>Agilent 7100 CE system</td>
			<td></td>
		</tr>
		<tr>
			<td>CE/MS adapter kit</td>
			<td>Agilent Technologies</td>
			<td>G1603A</td>
			<td></td>
		</tr>
		<tr>
			<td>CE-ESI-MS Sprayer kit</td>
			<td>Agilent Technologies</td>
			<td>G1607A</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell counting chamber slide</td>
			<td>Thermo Scientific</td>
			<td>C10282</td>
			<td>Countess cell counting chamber slides</td>
		</tr>
		<tr>
			<td>Centrifugal filter device, 5 kDa</td>
			<td>Human Metabolome Technologies</td>
			<td>ULTRAFREE MC PLHCC, UFC3LCCNB-HMT</td>
			<td></td>
		</tr>
		<tr>
			<td>Conical sterile polypropylene tube, 15 ml</td>
			<td>Thermo Scientific</td>
			<td>N339651</td>
			<td></td>
		</tr>
		<tr>
			<td>Conical sterile polypropylene tube, 50 ml</td>
			<td>Thermo Scientific</td>
			<td>N339653</td>
			<td></td>
		</tr>
		<tr>
			<td>Costar stripette, 10 ml</td>
			<td>Corning</td>
			<td>4488</td>
			<td></td>
		</tr>
		<tr>
			<td>Costar stripette, 5 ml</td>
			<td>Corning</td>
			<td>4487</td>
			<td></td>
		</tr>
		<tr>
			<td>D(-)-Mannitol</td>
			<td>Wako</td>
			<td>133-00845</td>
			<td>500 g</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s phosphate buffered saline (DPBS)</td>
			<td>Sigma-Aldrich</td>
			<td>D8537-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Electrophoresis buffer</td>
			<td>Human Metabolome Technologies</td>
			<td>H3301-1001</td>
			<td>for cation analysis</td>
		</tr>
		<tr>
			<td>Electrophoresis buffer</td>
			<td>Human Metabolome Technologies</td>
			<td>H3302-1021</td>
			<td>for anion analysis</td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Biowest</td>
			<td>S1780</td>
			<td></td>
		</tr>
		<tr>
			<td>Filter tip, 1000 &#956;l</td>
			<td>Watson</td>
			<td>124P-1000S</td>
			<td></td>
		</tr>
		<tr>
			<td>Filter tip, 20 &#956;l</td>
			<td>Watson</td>
			<td>124P-20S</td>
			<td></td>
		</tr>
		<tr>
			<td>Filter tip, 200 &#956;l</td>
			<td>Watson</td>
			<td>1252-703CS</td>
			<td></td>
		</tr>
		<tr>
			<td>Fused silica capillary</td>
			<td>Polymicro Technologies</td>
			<td>TSP050375</td>
			<td>50 &#956;m i.d. &#215; 80 cm total length</td>
		</tr>
		<tr>
			<td>HCC827</td>
			<td>American Type Culture Collection</td>
			<td>CRL-2868</td>
			<td></td>
		</tr>
		<tr>
			<td>Internal standard solution</td>
			<td>Human Metabolome Technologies</td>
			<td>H3304-1002</td>
			<td></td>
		</tr>
		<tr>
			<td>Isocratic pump</td>
			<td>Agilent Technologies</td>
			<td>Agilent 1100 Series Isocratic Pump</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Wako</td>
			<td>138-14521</td>
			<td>1 L, LC/MS grade</td>
		</tr>
		<tr>
			<td>Microtube, 1.5 ml</td>
			<td>Watson</td>
			<td>131-415C</td>
			<td></td>
		</tr>
		<tr>
			<td>Operating Software</td>
			<td>Agilent Technologies</td>
			<td>ChemStation G2201AA</td>
			<td>version B.03.01 for CE</td>
		</tr>
		<tr>
			<td>PC-9</td>
			<td>RIKEN Bio Resource Center</td>
			<td>RCB4455</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI-1640</td>
			<td>Sigma-Aldrich</td>
			<td>R8758-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile tissue culture dish, 100 mm</td>
			<td>Corning</td>
			<td>430167</td>
			<td></td>
		</tr>
		<tr>
			<td>Time-of-flight mass spectrometer</td>
			<td>Agilent Technologies</td>
			<td>Agilent G1969A Time-of-Flight LC/MS</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan blue solution, 0.4%</td>
			<td>Thermo Scientific</td>
			<td>T10282</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA solution</td>
			<td>Sigma-Aldrich</td>
			<td>T4049-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultrapure water</td>
			<td>Merck</td>
			<td>Milli-Q water</td>
			<td>18.2 M&#937;&#12539;cm pure water</td>
		</tr>
		<tr>
			<td>Volumetric flask, 50 ml</td>
			<td>Iwaki</td>
			<td>5640FK50E</td>
			<td>TE-32</td>
		</tr>
	</tbody>
</table>

extraction of aqueous metabolites from cultured adherent cells for metabolomic analysis by capillary electrophoresis-mass spectrometry

Metabolomic analysis is a promising omics approach to not only understand the specific metabolic regulation in cancer cells compared to normal cells but also to identify biomarkers for early-stage cancer detection and prediction of chemotherapy response in cancer patients. Preparation of uniform samples for metabolomic analysis is a critical issue that remains to be addressed. Here, we present an easy and reliable protocol for extracting aqueous metabolites from cultured adherent cells for metabolomic analysis using capillary electrophoresis-mass spectrometry (CE-MS). Aqueous metabolites from cultured cells are analyzed by culturing and washing cells, treating cells with methanol, extracting metabolites, and removing proteins and macromolecules with spin columns for CE-MS analysis. Representative results using lung cancer cell lines treated with diamide, an oxidative reagent, illustrate the clearly observable metabolic shift of cells under oxidative stress. This article would be especially valuable to students and investigators involved in metabolomics research, who are new to harvesting metabolites from cell lines for analysis by CE-MS.

Since metabolite concentrations in cancer cells (pmol/106 cells) are normalized to the number of viable cells, experimental conditions should be set up with care so as to minimize variation in the number of viable cells between conditions. For example, diamide treatment was at a relatively high concentration (250 &#956;m) but for a short time to allow all the cells to grow as equally as possible, thereby equalizing the number of viable cells analyzed. Under these experimental conditions, HCC827 and PC-9 cells grew equally for 3 h (Figure 1). CE-MS analysis of diamide-treated cells compared with PBS-treated (control) cells revealed 175 and 150 differential metabolites in HCC827 and PC-9 cells, respectively. Among these, several intermediates in the pentose phosphate pathway (PPP) and in upper glycolysis were significantly higher in the diamide-treated conditions in both cell lines, whereas a few tricarboxylic acid (TCA) cycle intermediates were lower in the treated conditions (Figure 2 and Figure 3).
The PPP generates reducing equivalents in the form of reduced nicotinamide adenine dinucleotide phosphate (NADPH), which is used for redox homeostasis maintenance and fatty acid biosynthesis15. Following diamide treatment, the level of gluconic acid&#8212;an oxidized glucose&#8212;increased 12-fold in HCC827 cells and 10-fold in PC-9 cells; similarly, following diamide treatment, the level of glucose 6-phosphate (G6P)&#8212;a phosphorylated glucose and the first hexokinase-catalyzed glycolysis product&#8212;also increased 6.3- and 3.5-fold in HCC827 and PC-9 cells, respectively (Figure 4). In addition, following diamide treatment, the levels of 6-phosphogluconate (6PG)&#8212;the first intermediate in PPP&#8212;dramatically increased 89-fold in HCC827 cells and 231-fold in PC-9 cells compared to the levels seen in the PBS controls (Figure 4). In contrast, levels of other glycolytic intermediates, such as fructose 6-phosphate (F6P) and fructose 1,6-bisphosphate (F1,6P), did not change in the diamide experimental condition (Figure 4). Total nicotinamide adenine dinucleotide phosphate (NADP+) levels were nearly equivalent between diamide treatment and PBS control conditions (Figure 4), suggesting that glucose was mainly catabolized via the PPP.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59551/59551fig1.jpg" /> 
Figure 1. Unchanged cell numbers upon diamide treatment. Cell growth responses to 250 &#956;m of diamide were measured using trypan blue staining. Cell numbers of (A) HCC827 and (B) PC-9 cells treated with PBS (blue) or diamide (red; 250 &#956;m) for 1 or 3 h are shown. Data are shown as the mean &#177; SD (n = 6). <a href="https://www.jove.com/files/ftp_upload/59551/59551fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/59551/59551fig2.jpg" /> 
Figure 2. Representative MS peaks of metabolites. Electropherograms annotated as (A) gluconic acid, (B) glucose 6-phosphate (G6P), (C) 6-phosphogluconate (6PG), and (D) nicotinamide adenine dinucleotide phosphate (NADP+) obtained by CE-MS analysis. Each line indicates the cell line (solid, HCC827; dotted, PC-9) and treatment (blue, PBS; red, diamide) used. <a href="https://www.jove.com/files/ftp_upload/59551/59551fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/59551/59551fig3.jpg" /> 
Figure 3. Metabolome profiles of intracellular metabolites. Fold changes of metabolites in (A) HCC827 and (B) PC-9 cells treated with diamide are shown as log2(diamide/PBS). In total, 175 and 150 metabolites were annotated in HCC827 and PC-9 cells, respectively. <a href="https://www.jove.com/files/ftp_upload/59551/59551fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/59551/59551fig4.jpg" /> 
Figure 4. Up-regulation of PPP upon diamide treatment. Intracellular concentrations (pmol/106 cells) of key metabolites involved in glycolysis and the pentose phosphate pathway (PPP) after treatment with diamide are shown. Metabolites were extracted from HCC827 and PC-9 cells treated with PBS (blue) or diamide (red, 250 &#956;m) for 30 min. Representative metabolites such as gluconic acid, glucose 6-phosphate (G6P), fructose 6-phosphate (F6P), 6-phosphogluconate (6PG), and nicotinamide adenine dinucleotide phosphate (NADP+) are shown. <a href="https://www.jove.com/files/ftp_upload/59551/59551fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Extraction of Aqueous Metabolites from Cultured Adherent Cells for Metabolomic Analysis by Capillary Electrophoresis-Mass Spectrometry at JoVE.com

Extraction of Aqueous Metabolites from Cultured Adherent Cells for Metabolomic Analysis by Capillary Electrophoresis-Mass Spectrometry

The purpose of this article is to describe a protocol for extraction of aqueous metabolites from cultured adherent cells for metabolomic analysis, particularly, capillary electrophoresis-mass spectrometry.

Metabolomic analysis is a promising omics approach to not only understand the specific metabolic regulation in cancer cells compared to normal cells but ...

extraction-aqueous-metabolites-from-cultured-adherent-cells-for

Research

JoVE Journal

Cancer Research

8.2K Views.  Shonai Regional Industry Promotion Center. The purpose of this article is to describe a protocol for extraction of aqueous metabolites from cultured adherent cells for metabolomic analysis, particularly, capillary electrophoresis-mass spectrometry.

Video: Extraction of Aqueous Metabolites from Cultured Adherent Cells for Metabolomic Analysis by Capillary Electrophoresis-Mass Spectrometry

Two-dimensional Gel Electrophoresis Coupled with Mass Spectrometry Methods for an Analysis of Human Pituitary Adenoma Tissue Proteome

Human pituitary adenoma (PA) is a common tumor that occurs in the human pituitary gland in the hypothalamus-pituitary-targeted organ axis systems, and may be classified as either clinically functional or nonfunctional PA (FPA and NFPA). NFPA is difficult for early stage diagnosis and therapy due to barely elevating hormones in the blood compared to FPA. Our long-term goal is to use proteomics methods to discover reliable biomarkers for clarification of PA molecular mechanisms and recognition of effective diagnostic, prognostic markers and therapeutic targets. Effective two-dimensional gel electrophoresis (2DE) coupled with mass spectrometry (MS) methods were presented here to analyze human PA proteomes, including preparation of samples, 2D gel electrophoresis, protein visualization, image analysis, in-gel trypsin digestion, peptide mass fingerprint (PMF), and tandem mass spectrometry (MS/MS). 2-Dimensional gel electrophoresis matrix-assisted laser desorption/ionization mass spectrometry PMF (2DE-MALDI MS PMF), 2DE-MALDI MS/MS, and 2DE-liquid chromatography (LC) MS/MS procedures have been successfully applied in an analysis of NFPA proteome. With the use of a high-sensitivity mass spectrometer, many proteins were identified with the 2DE-LC-MS/MS method in each 2D gel spot in an analysis of complex PA tissue to maximize the coverage of human PA proteome.

Here, we present a two-dimensional gel electrophoresis (2DE) coupled with mass spectrometry (MS) to separate and identify human pituitary adenoma tissue proteome, which presents a good and reproducible 2DE pattern. Many proteins are observed in each 2DE spot when analyzing complex cancer proteome with the use of high-sensitivity MS.

Human pituitary adenoma (PA) is a common tumor that occurs in the human pituitary gland in the hypothalamus-pituitary-targeted organ axis systems, and may ...

Simple and Rapid Method to Obtain High-quality Tumor DNA from Clinical-pathological Specimens Using Touch Imprint Cytology

It is critical to determine the mutational status in cancer before administration and treatment of specific molecular targeted drugs for cancer patients. In the clinical setting, formalin-fixed paraffin-embedded (FFPE) tissues are widely used for genetic testing. However, FFPE DNA is generally damaged and fragmented during the fixation process with formalin. Therefore, FFPE DNA is sometimes not adequate for genetic testing because of low quality and quantity of DNA. Here we present a method of touch imprint cytology (TIC) to obtain genomic DNA from cancer cells, which can be observed under a microscope. Cell morphology and cancer cell numbers can be evaluated using TIC specimens. Furthermore, the extraction of genomic DNA from TIC samples can be completed within two days. The total amount and quality of TIC DNA obtained using this method was higher than that of FFPE DNA. This rapid and simple method allows researchers to obtain high-quality DNA for genetic testing (e.g., next generation sequencing analysis, digital PCR, and quantitative real time PCR) and to shorten the turnaround time for reporting results.

Obtaining high-quality genomic DNA from tumor tissues is an essential first step for analyzing genetic alterations using next generation sequencing. In this article, we present a simple and rapid method to enrich tumor cells and obtain intact DNA from touch imprint cytology specimens.

It is critical to determine the mutational status in cancer before administration and treatment of specific molecular targeted drugs for cancer patients. ...

Determination of Protein Expression Level in Cultured Cells by Immunocytochemistry on Paraffin-embedded Cell Blocks

Immunofluorescent staining is currently the method of choice for determination of protein expression levels in cell-culture systems when morphological information is also necessary. The protocol of immunocytochemical staining on paraffin-embedded cell blocks, presented herein, is an excellent alternative to immunofluorescent staining on non-paraffin-embedded fixed cells. In this protocol, a paraffin cell block from HeLa cells was prepared using the thromboplastin-plasma method, and immunocytochemistry was performed for the evaluation of two proliferation markers, CKAP2 and Ki-67. The nuclei and cytoplasmic morphology of the HeLa cells were well preserved in the cell-block slides. At the same time, the CKAP2 and Ki-67 staining patterns in the immunocytochemistry were quite similar to those in immunohistochemical staining in paraffin cancer tissues. With modified cell-culture conditions, including pre-incubation of HeLa cells under serum-free conditions, the effect could be evaluated while preserving architectural information. In conclusion, immunocytochemistry on paraffin-embedded cell blocks is an excellent alternative to immunofluorescent staining.

Currently, immunofluorescent staining on fixed cells is the method of choice for determination of protein expression levels when morphological information is also necessary. This protocol presented herein provides for an alternative method of immunocytochemistry on paraffin-embedded cell blocks.

Immunofluorescent staining is currently the method of choice for determination of protein expression levels in cell-culture systems when morphological ...

High-sensitivity Detection of Micrometastases Generated by GFP Lentivirus-transduced Organoids Cultured from a Patient-derived Colon Tumor

Despite current advances in human colorectal cancer (CRC) treatment, few radical therapies are effective for the late stages of CRC. To overcome this clinical challenge, tumor xenograft mouse models using long-established human carcinoma cell lines and many transgenic mouse models with tumors have been developed as preclinical models. They partially mimic the features of human carcinomas, but often fail to recapitulate the key aspects of human malignancies including invasion and metastasis. Thus, alternative models that better represent the malignant progression in human CRC have long been awaited.
We herein show generation of patient-derived tumor xenografts (PDXs) by subcutaneous implantation of small CRC fragments surgically dissected from a patient. The colon PDXs develop and histopathologically resemble the CRC in the patient. However, few spontaneous micrometastases are detectable in conventional cross-sections of affected distant organs in the PDX model. To facilitate the detection of metastatic dissemination into distant organs, we extracted the tumor organoid cells from the colon PDXs in culture and infected them with GFP lentivirus prior to injection into highly immunodeficient NOD/Shi-scid IL2R&#947;null (NOG) mice. Orthotopically injected PDX-derived CRC organoid cells consistently form primary tumors positive for GFP in recipient mice. Moreover, spontaneously developing micrometastatic colonies expressing GFP are notably detected in the lungs of these mice by fluorescence microscopy. Moreover, intrasplenic injection of CRC organoids frequently produces hepatic colonization. Taken together, these findings indicate GFP-labelled PDX-derived CRC organoid cells to be visually detectable during a multistep process termed the invasion-metastasis cascade. The described protocols include the establishment of PDXs of human CRC and 3D culture of the corresponding CRC organoid cells transduced by GFP lentiviral particles.

To allow highly sensitive detection of the disseminating human colorectal cancer (CRC) cells colonizing tissues, we herein show a protocol for efficient transduction of green fluorescent protein (GFP) lentiviral particles into PDX-derived CRC organoid cells prior to their injection into recipient mice, with stereo-fluorescence microscopic observation.

Despite current advances in human colorectal cancer (CRC) treatment, few radical therapies are effective for the late stages of CRC. To overcome this ...

Extraction of Extracellular Vesicles from Whole Tissue

Circulating and interstitial small membrane-bound extracellular vesicles (EVs) represent promising targets for the development of novel diagnostic or prognostic biomarker assays, and likely serve as important players in the progression of a vast spectrum of diseases. Current research is focused on the characterization of vesicles secreted from multiple cell and tissue types in order to better understand the role of EVs in the pathogenesis of conditions including neurodegeneration, inflammation, and cancer. However, globally consistent and reproducible techniques to isolate and purify vesicles remain in progress. Moreover, methods for extraction of EVs from solid tissue ex vivo are scarcely described. Here, we provide a detailed protocol for extracting small EVs of interest from whole fresh or frozen tissues, including brain and tumor specimens, for further characterization. We demonstrate the adaptability of this method for multiple downstream analyses, including electron microscopy and immunophenotypic characterization of vesicles, as well as quantitative mass spectrometry of EV proteins.

Here, we provide a detailed protocol to isolate small extracellular vesicles (EVs) from whole tissues, including brain and tumor specimens. This method offers a reproducible technique to extract EVs from solid tissue for further downstream analyses.

Circulating and interstitial small membrane-bound extracellular vesicles (EVs) represent promising targets for the development of novel diagnostic or ...

An Orthotopic Endometrial Cancer Model with Retroperitoneal Lymphadenopathy Made From In Vivo Propagated and Cultured VX2 Cells

Endometrial cancer is the most common gynecologic malignancy in North America and the incidence is rising worldwide. Treatment consists of surgery with or without adjuvant therapy depending on lymph node involvement as determined by lymphadenectomy. Lymphadenectomy is a morbid procedure, which has not been shown to have a therapeutic benefit in many patients, and thus a new method to diagnose lymph node metastases is required. To test novel imaging agents, a reliable model of endometrial cancer with retroperitoneal lymph node metastases is needed. The VX2 endometrial cancer model has been described frequently in the literature; however, significant variation exists with respect to the method of model establishment. Furthermore, no studies have reported on the use of cultured VX2 cells to create this model as only cells propagated in vivo have been previously used. Herein, we present a standardized surgical method and post-operative monitoring method for the establishment of the VX2 endometrial cancer model and report on the first use of cultured VX2 cells to create this model.

This protocol presents a standardized method to grow VX2 cells in culture and to create an orthotopic VX2 model of endometrial cancer with retroperitoneal lymph node metastases in rabbits. Orthotopic endometrial cancer models are important for the pre-clinical study of novel imaging modalities for the diagnosis of lymph node metastases.

Endometrial cancer is the most common gynecologic malignancy in North America and the incidence is rising worldwide. Treatment consists of surgery with or ...

Evaluating Cell Death Using Cell-Free Supernatant of Probiotics in Three-Dimensional Spheroid Cultures of Colorectal Cancer Cells

This manuscript describes a protocol to evaluate cancer cell deaths in three dimensional (3D) spheroids of multicellular types of cancer cells using supernatants from Lactobacillus fermentum cell culture, considered as probiotics cultures. The use of 3D cultures to test Lactobacillus cell-free supernatant (LCFS) are a better option than testing in 2D monolayers, especially as L. fermentum can produce anti-cancer effects within the gut. L. fermentum supernatant was identified to possess increased anti-proliferative effects against several colorectal cancer (CRC) cells in 3D culture conditions. Interestingly, these effects were strongly related to the culture model, demonstrating the notable ability of L. fermentum to induce cancer cell death. Stable spheroids were generated from diverse CRCs (colorectal cancer cells) using the protocol presented below. This protocol of generating 3D spheroid is time saving and cost effective. This system was developed to easily investigate the anti-cancer effects of LCFS in multiple types of CRC spheroids. As expected, CRC spheroids treated with LCFS strongly induced cell death during the experiment and expressed specific apoptosis molecular markers as analyzed by qRT-PCR, western blotting, and FACS analysis. Therefore, this method is valuable for exploring cell viability and evaluating the efficacy of anti-cancer drugs.

Here methods are presented to understand anti-cancer effects of Lactobacillus cell-free supernatant (LCFS). Colorectal cancer cell lines show cell deaths when treated with LCFS in 3D cultures. The process of generating spheroids can be optimized depending on the scaffold and the analysis methods presented are useful for evaluating the involved signaling pathways.

This manuscript describes a protocol to evaluate cancer cell deaths in three dimensional (3D) spheroids of multicellular types of cancer cells using ...

Preparation of Human Tissues Embedded in Optimal Cutting Temperature Compound for Mass Spectrometry Analysis

Sphingolipids are cellular components that have well-established roles in human metabolism and disease. Mass spectrometry can be used to determine whether sphingolipids are altered in a disease and investigate whether sphingolipids can be targeted clinically. However, properly powered prospective studies that acquire tissues directly from the surgical suite can be time consuming, and technically, logistically, and administratively challenging. In contrast, retrospective studies can take advantage of cryopreserved human specimens already available, usually in large numbers, at tissue biorepositories. Other advantages of procuring tissues from biorepositories include access to information associated with the tissue specimens including histology, pathology, and in some instances clinicopathological variables, all of which can be used to examine correlations with lipidomics data. However, technical limitations related to the incompatibility of optimal cutting temperature compound (OCT) used in the cryopreservation and mass spectrometry is a technical barrier for the analysis of lipids. However, we have previously shown that OCT can be easily removed from human biorepository specimens through cycles of washes and centrifugation without altering their sphingolipid content. We have also previously established that sphingolipids in human tissues cryopreserved in OCT are stable for up to 16 years. In this report, we outline the steps and workflow to analyze sphingolipids in human tissue specimens that are embedded in OCT, including washing tissues, weighing tissues for data normalization, the extraction of lipids, preparation of samples for analysis by liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS), mass spectrometry data integration, data normalization, and data analysis.

Sphingolipids are bioactive metabolites with well-established roles in human disease. Characterizing alterations in tissues with mass-spectrometry can reveal roles in disease etiology or identify therapeutic targets. However, the OCT-compound used for cryopreservation in biorepositories interferes with mass-spectrometry. We outline methods to analyze sphingolipids in human tissues embedded in OCT with LC-ESI-MS/MS.

Sphingolipids are cellular components that have well-established roles in human metabolism and disease. Mass spectrometry can be used to determine whether ...

Modeling Brain Metastasis by Internal Carotid Artery Injection of Cancer Cells

Brain metastasis is a cause of severe morbidity and mortality in cancer patients. Critical aspects of metastatic diseases, such as the complex neural microenvironment and stromal cell interaction, cannot be entirely replicated with in vitro assays; thus, animal models are critical for investigating and understanding the effects of therapeutic intervention. However, most brain tumor xenografting methods do not produce brain metastases consistently in terms of the time frame and tumor burden. Brain metastasis models generated by intracardiac injection of cancer cells can result in unintended extracranial tumor burden and lead to non-brain metastatic morbidity and mortality. Although intracranial injection of cancer cells can limit extracranial tumor formation, it has several caveats, such as the injected cells frequently form a singular tumor mass at the injection site, high leptomeningeal involvement, and damage to brain vasculature during needle penetration. This protocol describes a mouse model of brain metastasis generated by internal carotid artery injection. This method produces intracranial tumors consistently without the involvement of other organs, enabling the evaluation of therapeutic agents for brain metastasis.

Brain metastasis is a cause of severe morbidity and mortality in cancer patients. Most brain metastasis mouse models are complicated by systemic metastases confounding analysis of mortality and therapeutic intervention outcomes. Presented here is a protocol for internal carotid injection of cancer cells that produces consistent intracranial tumors with minimal systemic tumors.

Brain metastasis is a cause of severe morbidity and mortality in cancer patients. Critical aspects of metastatic diseases, such as the complex neural ...

Preparation of Cytoplasmic and Nuclear Long RNAs from Primary and Cultured Cells

The separation of intracellular components has been a key tool in cellular biology for many years now and has been able to provide useful insight into how their location can impact their function. In particular, the separation of nuclear and cytoplasmic RNA has become important in the context of cancer cells and the quest to find new targets for drugs. Purchasing kits for nuclear-cytoplasmic RNA extraction can be costly when many of the required materials can be found within a typical lab setting. Using the present method, which can replace more expensive kits or other time-consuming processes, only a homemade lysis buffer, a benchtop centrifuge, and RNA isolation purification columns are needed to isolate nuclear and cytoplasmic RNA. Lysis buffer is used to gently lyse the cell's outer membrane without affecting the integrity of the nuclear envelope, allowing for releasing its intracellular components. Then, the nuclei can be isolated by a simple centrifugation step since they possess a higher density than the lysis solution. Centrifugation is utilized to separate these areas based on their density differences to isolate subcellular elements in the nucleus from those in the cytoplasm. Once the centrifugation has isolated the different components, an RNA clean-up kit is utilized to purify the RNA content, and qPCR is performed to validate the separation quality, quantified by the amount of nuclear and cytoplasmic RNA in the different fractions. Statistically significant levels of separation were achieved, illustrating the protocol's effectiveness. In addition, this system can be adapted for the isolation of different types of RNA (total, small RNA, etc.), which allows for targeted studying of cytoplasm-nucleus interactions, and aids in understanding the differences in the function of RNA that reside in the nucleus and cytoplasm.

The present protocol offers an efficient and flexible method to isolate RNA from nuclear and cytoplasmic fractions using cultured cells, and then validate using qPCR. This effectively serves as a replacement for other RNA preparation kits.

The separation of intracellular components has been a key tool in cellular biology for many years now and has been able to provide useful insight into how ...