Name: preparation of human tissues embedded in optimal cutting temperature compound for mass spectrometry analysis
Uploaded: 2021-04-27T15:00:00
Description: Watch this Scientific Journal Video about Preparation of Human Tissues Embedded in Optimal Cutting Temperature Compound for Mass Spectrometry Analysis at JoVE.com

Services and support of the research project were provided by the VCU Massey Cancer Center Tissue and Data Acquisition and Analysis Core and the VCU Lipidomics and Metabolomics Core, which are supported in part with funding from NIH-NCI Cancer Center Support Grant P30CA016059. This work was supported by National Institutes of Health Grants R21CA232234 (Santiago Lima).

De-identified human lung tissues were obtained from the Virginia Commonwealth University (VCU) Tissue and Data Acquisition and Analysis Core under an internal review board (IRB) approved protocol (#HM2471). The use of mice for research and harvesting of mice tissues was approved by the VCU institutional animal care and use committee (IACUC).
1. Preparation of materials
NOTE: These steps should be performed a day prior to the tissue washing.
<ol>
	<li>Pre-label and...

<ol>
	<li>Maceyka, M., Spiegel, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sphingolipid+metabolites+in+inflammatory+disease.">Sphingolipid metabolites in inflammatory disease.</a> Nature. 510, 58-67 (2014).</li><li>Ogretmen, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sphingolipid+metabolism+in+cancer+signalling+and+therapy.">Sphingolipid metabolism in cancer signalling and therapy.</a> Nature Reviews. Cancer. 18 (1), 33-50 (2018).</li><li>Hannun, Y. A., Obeid, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sphingolipids+and+their+metabolism+in+physiology+and+disease.">Sphingolipids and their metabolism in physiology and disease.</a> Nature Reviews Molecular Cell Biology. 19 (3), 175-191 (2018).</li><li>Hla, T., Dannenberg, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sphingolipid+signaling+in+metabolic+disorders.">Sphingolipid signaling in metabolic disorders.</a> Cell Metabolism. 16 (4), 420-434 (2012).</li><li>Lima, S., Milstien, S., Spiegel, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sphingosine+and+Sphingosine+Kinase+1+involvement+in+endocytic+membrane+Trafficking.">Sphingosine and Sphingosine Kinase 1 involvement in endocytic membrane Trafficking.</a> The Journal of Biological Chemistry. 292 (8), 3074-3088 (2017).</li><li>Lima, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TP53+is+required+for+BECN1-+and+ATG5-dependent+cell+death+induced+by+sphingosine+kinase+1+inhibition.">TP53 is required for BECN1- and ATG5-dependent cell death induced by sphingosine kinase 1 inhibition.</a> Autophagy. , 1-50 (2018).</li><li>Young, M. M., 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=Sphingosine+Kinase+1+cooperates+with+autophagy+to+maintain+endocytic+membrane+trafficking.">Sphingosine Kinase 1 cooperates with autophagy to maintain endocytic membrane trafficking.</a> Cell Reports. 17 (6), 1532-1545 (2016).</li><li>Young, M. M., Wang, H. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sphingolipids+as+regulators+of+autophagy+and+endocytic+trafficking.">Sphingolipids as regulators of autophagy and endocytic trafficking.</a> Advances in Cancer Research. 140, 27-60 (2018).</li><li>Shen, 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=Coupling+between+endocytosis+and+sphingosine+kinase+1+recruitment.">Coupling between endocytosis and sphingosine kinase 1 recruitment.</a> Nature Cell Biology. 16 (7), 652-662 (2014).</li><li>Morad, S. A. F., Cabot, M. C., Chalfant, C. E., Fisher, P. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Advances in Cancer Research. 140, 235-263 (2018).</li><li>Rohrbach, T. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+method+for+sphingolipid+analysis+of+tissues+embedded+in+optimal+cutting+temperature+compound.">A simple method for sphingolipid analysis of tissues embedded in optimal cutting temperature compound.</a> Journal of Lipid Research. 61 (6), 953-967 (2020).</li><li>Weston, L. A., Hummon, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+LC-MS/MS+analysis+of+optimal+cutting+temperature+(OCT)+compound+removal+for+the+study+of+mammalian+proteomes.">Comparative LC-MS/MS analysis of optimal cutting temperature (OCT) compound removal for the study of mammalian proteomes.</a> Analyst. 138 (21), 6380-6384 (2013).</li><li>Holfeld, A., Vald&#233;s, A., Malmstr&#246;m, P. -. U., Segersten, U., Lind, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parallel+proteomic+workflow+for+mass+spectrometric+analysis+of+tissue+samples+preserved+by+different+methods.">Parallel proteomic workflow for mass spectrometric analysis of tissue samples preserved by different methods.</a> Analytical Chemistry. 90 (9), 5841-5849 (2018).</li><li>Zhang, W., Sakashita, S., Taylor, P., Tsao, M. S., Moran, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comprehensive+proteome+analysis+of+fresh+frozen+and+optimal+cutting+temperature+(OCT)+embedded+primary+non-small+cell+lung+carcinoma+by+LC-MS/MS.">Comprehensive proteome analysis of fresh frozen and optimal cutting temperature (OCT) embedded primary non-small cell lung carcinoma by LC-MS/MS.</a> Methods. 81, 50-55 (2015).</li><li>Shah, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+proteomics+using+chemical+immobilization+and+mass+spectrometry.">Tissue proteomics using chemical immobilization and mass spectrometry.</a> Analytical Biochemistry. 469, 27-33 (2015).</li></ol>

OCT is a common long-term cryo-preservation agent used in biorepositories. However, OCT can result in ion suppression when tissues are analyzed by various mass spectrometry platforms12,13,14,15, or result in loss of signal when samples are analyzed by LC-ESI-MS/MS11. OCT in cryopreserved tissues can also interfere with tissue normalization methods such as weighing and pr...

Sphingolipids are bioactive metabolites known for their roles in human metabolism and disease1,2. They regulate complex cellular processes such as cell migration, cell survival and death, cell movement, vesicular trafficking, cellular invasion and metastasis, angiogenesis, and the production of cytokines1,2,3,4,5,6,7,8,9. Defects in the regulation of sphingolipid metabolism contribute to the initiation and progression of cancers, determine how aggressive cancers are, and how cancers respond to and develop resistance to therapy3,10. Therefore, because of these broad impacts on the etiology of disease, analytical methods that can precisely establish disease-specific sphingolipid alterations are important tools. Mass spectrometry (MS) is the most accurate and reliable method to analyze sphingolipid alterations.
Human specimens that can be used for the analysis of sphingolipid alterations can be obtained prospectively from the surgical suite or retrospectively from tissue biorepositories. Fresh tissues from surgery are advantageous because they can be directly analyzed by MS or other analytical methods. However, acquiring tissues prospectively has administrative, technical, and logistical hurdles, and collecting sufficient specimens to reach statistical power can be challenging. Obtaining tissues from biorepositories is advantageous because they can be acquired retrospectively, in large numbers, and biorepositories confirm histology and pathology, use standard operating procedures to cryogenically preserve and store tissues, and can provide clinicopathological data that can be used for correlation analyses. However, to preserve molecular and structural features, biorepositories may cryopreserve tissues by embedding them in optimal cutting temperature (OCT) compound, which we have shown interferes with data normalization assays and the quantification of sphingolipids by liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS)11. It has also been shown that polyvinyl alcohol and polyethylene glycol, the primary components in OCT compound, result in ion suppression in other MS analysis platforms12,13,14,15. Therefore, OCT compound must be removed from tissues prior to sphingolipidomic analysis by MS.
In a previous report, we have validated a protocol for the removal of OCT compound from human specimens for LC-ESI-MS/MS analysis11 and the methodology used for weighing tissues for data normalization11. Here, we detail the steps of the sphingolipidomic OCT compound-removal protocol (sOCTrP) and show representative data from human lung adenocarcinoma tumors and normal adjacent uninvolved tissues.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1 mL polypropylene pipette tips</td>
			<td>NA</td>
			<td>NA</td>
			<td>Used to retrieve specimens</td>
		</tr>
		<tr>
			<td>1.5 mL polypropylene centrifuge tubes</td>
			<td>NA</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>10 mL Erlenmeyer flask</td>
			<td>VWR</td>
			<td>89091-116</td>
			<td>Used for tube and tissue weighing</td>
		</tr>
		<tr>
			<td>AB Sciex Analyst 1.6.2</td>
			<td>Sciex</td>
			<td>NA</td>
			<td>Software to analyze and integrate MS data</td>
		</tr>
		<tr>
			<td>Ammonium formate</td>
			<td>Fisher Scientific</td>
			<td>A11550</td>
			<td>For LC mobile phases</td>
		</tr>
		<tr>
			<td>Analytical scale</td>
			<td>NA</td>
			<td>NA</td>
			<td>Scale that is accurate to 0.1 mg</td>
		</tr>
		<tr>
			<td>Bottle top dispenser</td>
			<td>Sartorius</td>
			<td>LH-723071</td>
			<td>Used for dispensing solvents</td>
		</tr>
		<tr>
			<td>C12-Ceramide (d18:1/C12:0); N-(dodecanoyl)-sphing-4-enine</td>
			<td>Avanti Polar Lipids</td>
			<td>LM2212</td>
			<td>Internal standard</td>
		</tr>
		<tr>
			<td>C12-glucosylceramide (d18:1/12:0); N-(dodecanoyl)-1-&#946;-glucosyl-sphing-4-eine</td>
			<td>Avanti Polar Lipids</td>
			<td>LM2511</td>
			<td>Internal standard</td>
		</tr>
		<tr>
			<td>C12-lactosylceramide (d18:1/12:0); N-(dodecanoyl)-1-&#223;-lactosyl-sphing-4-ene</td>
			<td>Avanti Polar Lipids</td>
			<td>LM2512</td>
			<td>Internal standard</td>
		</tr>
		<tr>
			<td>C12-Sphingomyelin&#160; (d18:1/C12:0), N-(dodecanoyl)-sphing-4-enine-1-phosphocholine</td>
			<td>Avanti Polar Lipids</td>
			<td>LM2312</td>
			<td>Internal standard</td>
		</tr>
		<tr>
			<td>CHLOROFORM OMNISOLV 4L</td>
			<td>VWR</td>
			<td>EM-CX1054-1</td>
			<td></td>
		</tr>
		<tr>
			<td>ClickSeal Biocontainment Lids</td>
			<td>Thermo Scientific</td>
			<td>75007309</td>
			<td>To prevent biohazard aeresols during centrifugation</td>
		</tr>
		<tr>
			<td>Conflikt</td>
			<td>Decon Labs</td>
			<td>4101</td>
			<td>Decontaminant</td>
		</tr>
		<tr>
			<td>CTO-20A/20AC Column Oven</td>
			<td>Shimadzu</td>
			<td>NA</td>
			<td>For LC</td>
		</tr>
		<tr>
			<td>d17:1-Sphingosine;&#160; (2S,3R,4E)-2-aminoheptadec-4-ene-1,3-diol</td>
			<td>Avanti Polar Lipids</td>
			<td>LM2000</td>
			<td>Internal standard</td>
		</tr>
		<tr>
			<td>d17:1-Sphingosine-1-phosphate; heptadecasphing-4-enine-1-phosphate</td>
			<td>Avanti Polar Lipids</td>
			<td>LM2144</td>
			<td>Internal standard</td>
		</tr>
		<tr>
			<td>DGU20A5R degasser</td>
			<td>Shimadzu</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable Culture Tubes 13x100mm</td>
			<td>VWR</td>
			<td>53283-800</td>
			<td>13x100 mm screw top tubes</td>
		</tr>
		<tr>
			<td>Heated water bath</td>
			<td>NA</td>
			<td>NA</td>
			<td>For overnight lipid extraction</td>
		</tr>
		<tr>
			<td>Homogenizer 150</td>
			<td>Fisher Scientific</td>
			<td>15-340-167</td>
			<td>triturate tissues</td>
		</tr>
		<tr>
			<td>Homogenizer Plastic Disposable Generator Probe</td>
			<td>Fisher Scientific</td>
			<td>15-340-177</td>
			<td>for homogenization</td>
		</tr>
		<tr>
			<td>Kimwipes</td>
			<td>Kimtech</td>
			<td>34120</td>
			<td>Laboratory grade tissue used to make wicks</td>
		</tr>
		<tr>
			<td>Methanol LC-MS Grade 4L</td>
			<td>VWR</td>
			<td>EM-MX0486-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Nexera LC-30 AD binary pump system</td>
			<td>Shimadzu</td>
			<td>NA</td>
			<td>For LC-MS</td>
		</tr>
		<tr>
			<td>Permanent marker</td>
			<td>VWR</td>
			<td>52877-310</td>
			<td></td>
		</tr>
		<tr>
			<td>Phenolic Screw Thread Closure, Kimble Chase (caps for disposable culture tubes)</td>
			<td>VWR</td>
			<td>89001-502</td>
			<td>13x100 mm screw top tube caps</td>
		</tr>
		<tr>
			<td>Phosphate bufffered saline</td>
			<td>Thermo Scientific</td>
			<td>10010023</td>
			<td>To retrieve specimens from tubes after washing</td>
		</tr>
		<tr>
			<td>Repeater pipette</td>
			<td>Eppendorf</td>
			<td>4987000118</td>
			<td>To dispense LC-MS internal standards</td>
		</tr>
		<tr>
			<td>Screw Caps, Blue, Red PTFE/White Silicone</td>
			<td>VWR</td>
			<td>89239-020</td>
			<td>Autoinjector vial caps</td>
		</tr>
		<tr>
			<td>Screw Thread Glass Vials with ID Patch</td>
			<td>VWR</td>
			<td>46610-724</td>
			<td>Autoinjector vials</td>
		</tr>
		<tr>
			<td>SIL-30AC autoinjector</td>
			<td>Shimadzu</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>SpeedVac</td>
			<td>Thermo Scientific</td>
			<td>SPD2030P1220</td>
			<td>For drying solvents</td>
		</tr>
		<tr>
			<td>Supelco 2.1 (i.d.) x 50 mm Ascentis Express C18 column</td>
			<td>Sigma Aldrich</td>
			<td>53822-U</td>
			<td>For LC-MS</td>
		</tr>
		<tr>
			<td>Triple Quad 5500+ LC-MS/MS System</td>
			<td>Sciex</td>
			<td>NA</td>
			<td>For LC-ESI-MS/MS</td>
		</tr>
		<tr>
			<td>Ultrasonic water bath</td>
			<td>Branson</td>
			<td>Model 2800</td>
			<td>for homogenization and resuspension of extracted and dried lipids</td>
		</tr>
		<tr>
			<td>Vortexer</td>
			<td>NA</td>
			<td>NA</td>
			<td>For sOCTrP and resuspending dried lipids</td>
		</tr>
		<tr>
			<td>VWR Culture Tubes Disposable Borosilicate Glass</td>
			<td>VWR</td>
			<td>47729572</td>
			<td>13x100 glass culture tubes</td>
		</tr>
		<tr>
			<td>Water Hipersolve Chromanorm LC-MS</td>
			<td>VWR</td>
			<td>BDH83645.400</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

In this protocol, we describe in detail a method to remove OCT from cryo-preserved human tissues and weigh the tissues for analysis by LC-ESI-MS/MS. The materials required for this procedure are listed in Table of Materials. Shown in Figure 1 are results of a typical experiment where 10 human lung adenocarcinoma tumors and 10 normal adjacent tissues were washed to remove OCT and analyzed by LC-ESI-MS/MS. Importantly, as we have previously shown11, the...

Watch this Scientific Journal Video about Preparation of Human Tissues Embedded in Optimal Cutting Temperature Compound for Mass Spectrometry Analysis at JoVE.com

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

OCT is a cryo-preservation agent that is incompatible with mass spectrometry analysis. Using this protocol, researchers can remove OCT from tissues and quantify sphingolipids with tandem liquid chromatography mass spectrometry. The preparation of tissue drying wicks, tissue washing, and tissue weighing will be demonstrated by April Boyd from Virginia Commonwealth University, and the lipid extraction will be demonstrated by Dr.Jeremy Allegood, director of the Virginia Commonwealth Metabolomics and Lipidomics Core.To begin, clean surgical scissors by immersing them in methanol for five minutes, followed by wiping them with a clean laboratory grade tissue. Using powder-free gloves, twirl the end of a laboratory grade tissue to create a finely tapered 30 to 40 millimeter long wick. Then using the methanol cleaned scissors, cut the wick at the thick end and place it in a clean cardboard box.Next, using the methanol cleaned scissors, cut four to five millimeters off the tip of a one milliliter pipette tip and place the tip back in the tip holder. To wash away the OCT from the tissues, add 10 milliliters of ice cold ultrapure deionized water to each tube containing human lung tissue and cap them tightly. Allow the tubes to incubate for 10 minutes.Vigorously vortex each tube until all the tissue deposited on the walls of the tube or the tissue pellet is completely resuspended. Then centrifuge the tubes in a pre-chilled swinging bucket centrifuge at 4, 000 to 5, 000 times G for 10 minutes at four degrees Celsius. Once the centrifugation is complete, uncap the tubes on ice.Carefully aspirate the wash solution, leaving approximately 0.5 milliliters of the supernatant in the tube, then recap the tube. After removing the supernatant from all tubes, wash the tissues twice more in the same manner. After the three OCT removal washes, use the prepared shortened pipette tip to add 500 microliters of ice cold PBS to the tube.Using the same tip, gently resuspend the pellet and transfer all of the resuspended tissue into a pre-labeled and pre-weighed 1.5 milliliter centrifuge tube. Keep the tube on ice until all the tissue samples are transferred. Pellet the tissues by centrifuging the tubes in a pre-chilled centrifuge, then carefully aspirate as much of the PBS as possible without disturbing the pellet and place the tubes back on ice.Using the tissue drying wicks prepared earlier, gently remove any remaining wash solution, then close the tube and place it on ice. To weigh the tubes, place one tube at a time in a recently calibrated tared and zeroed analytical balance and close the chamber door. Once the weight has stabilized, record it in an electronic spreadsheet.After weighing, place the tube back on ice and add 300 microliters of ice cold PBS. Using a shortened pipette tip, carefully transfer all tissue into two milliliters of ice cold methanol in a pre-labeled screw top glass tube. Before lipid extraction, allow the tissues and mass spectrometry internal lipid standards to equilibrate to room temperature for 20 minutes, then vortex and immerse the internal standard solutions in an ultrasonic water bath until the lipid standards have fully dissolved.Using a repeating pipette, add 250 picomoles of the internal mass spectrometry standards to each tube and lightly recap the tubes. To facilitate homogenization, adjust the volume of methanol to two milliliters, then triturate the tissue using a homogenizer by moving the homogenizer tip in a circular motion gently pressed against the tube wall. When clumps are no longer visible, recap the tube.Immerse the tube at a 45 degree angle in an ultrasonic water bath for 5 to 10 seconds. Then under a fume hood, add chloroform and methanol to each tube to achieve a methanol to chloroform to water ratio of two to one to 0.1. Seal the tubes tightly and leave them on the benchtop until the end of the day.Before leaving that evening, place the tightly capped tubes in a 48 degree Celsius water bath for an overnight incubation. On the following day, pellet any solvent and soluble debris by centrifugation. Then in a fume hood, carefully decant the supernatant into pre-labeled 13 into 100 millimeter borosilicate glass test tubes tilted at a 30 to 45 degree angle to facilitate the decanting.After decanting the supernatant from all tubes, use a vacuum concentrator at maximum vacuum with an initial one-hour heating step at 40 degrees Celsius to evaporate all solvent, then add 500 microliters of methanol to each tube. To resuspend the dried lipid extracts, vortex the tube for 5 to 10 seconds, then immerse the tube in an ultrasonic water bath at a 45 degree angle with rotation for two minutes. Re-vortex the tube for five seconds and re-immerse in the ultrasonic water bath for an additional minute.Remove any insoluble debris by centrifugation, then decant the supernatant into a pre-labeled autoinjector vial tilted at a 30 to 45 degree angle to facilitate the decanting. Cap the vials tightly before storing them at minus 80 degrees Celsius until analysis. Liquid chromatography electrospray ionization tandem mass spectrometry analysis following OCT removal confirms that various chain-length species of ceramides and monohexosyl-ceramides are significantly elevated in human lung adenocarcinoma tumors compared to normal adjacent uninvolved tissues.Sphingolipid standards elute sequentially in the following order:d17:1 sphingosine, d17:1 sphingosine-1-phosphate, C12 lactosyl-ceramide, C12 monohexosyl-ceramide, C12 sphingomyelin, and C12 ceramide. For each of these sphingolipid species, using the gradient and instrumentation settings in the text, there is an approximate positive 0.15 minute shift in the retention time for every two carbon increase in chain-length. However, longer chain-length lipids such as C24 and C26 may have larger retention time shifts.Furthermore, a double bond in the fatty acid often results in a negative 0.15 minute shift in retention time. Thus, C18:1 ceramide will have a retention time similar to C16 ceramide. Rather than relying almost exclusively on freshly collected tissues, this protocol expands the tissue sources that can be used for mass spectrometry analysis to include those cryogenically stored at biorepositories.

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Cancer Research

Four-color Fluorescence Immunohistochemistry of T-cell Subpopulations in Archival Formalin-fixed, Paraffin-embedded Human Oropharyngeal Squamous Cell Carcinoma Samples

The four-color fluorescence immunohistochemistry (IHC) technique is a method to quantify cell populations of interest while taking into account their relative distribution and their localization in the tissue. This technique has been extensively applied to study the immune infiltrate in various tumor types. The tumor microenvironment is infiltrated by immune cells that are attracted to the tumor site. Different immune cell populations have been found to play different roles in the tumor microenvironment and to have a different impact on the outcome of disease. This manuscript describes the use of multiparameter fluorescence IHC on oropharyngeal squamous cell carcinoma (OPSCC) as an example. This technique can be extended to other tissue samples and cell types of interest. In the presented study, we analyzed the intraepithelial and stromal compartment of a large OPSCC cohort (n = 162). We focused on total T lymphocytes (CD3+), immunosuppressive regulatory T cells (Tregs; i.e., FoxP3+), and T helper 17 (Th17) cells (i.e., IL-17+CD3+) using a nuclear counterstain to distinguish tumor epithelium from stroma. A high number of T cells was found to be correlated with improved disease-free survival in patients with a low number of intratumoral IL-17+ non-T cells. This suggests that IL-17+ non-T cells may be correlated with a poor immune response in OPSCC, which is in agreement with the correlation described between IL-17 and poor survival in cancer patients. Currently, novel multiparameter fluorescence IHC techniques are being developed using up to 7 different fluorochromes and will enable the more precise characterization and localization of immune cells in the tumor microenvironment.

Multiparameter fluorescence immunohistochemistry can be used to assess the number, relative distribution, and localization of immune cell populations in the tumor microenvironment. This manuscript describes the use of this technique to analyze T-cell subpopulations in oropharyngeal cancer.

The four-color fluorescence immunohistochemistry (IHC) technique is a method to quantify cell populations of interest while taking into account their ...

Determining the Optimal Inhibitory Frequency for Cancerous Cells Using Tumor Treating Fields (TTFields)

Tumor Treating Fields (TTFields) are an effective treatment modality delivered via the continuous, noninvasive application of low-intensity (1-3 V/cm), alternating electric fields in the frequency range of several hundred kHz. The study of TTFields in tissue culture is carried out using the TTFields in vitro application system, which allows for the application of electric fields of varying frequencies and intensities to ceramic Petri dishes with a high dielectric constant (&#400; &#62; 5,000). Cancerous cell lines plated on coverslips at the bottom of the ceramic Petri dishes are subjected to TTFields delivered in two orthogonal directions at various frequencies to facilitate treatment outcome tests, such as cell counts and clonogenic assays. The results presented in this report demonstrate that the optimal frequency of the TTFields with respect to both cell counts and clonogenic assays is 200 kHz for both ovarian and glioma cells.

Determining the Optimal Inhibitory Frequency for Cancerous Cells Using Tumor Treating Fields TTFields

Tumor Treating Fields (TTFields) are an effective anti-tumor treatment modality delivered via the continuous, noninvasive application of low-intensity, intermediate-frequency, alternating electric fields. TTFields application to cell lines using a TTFields in vitro application system allows for the determination of the optimal frequency that leads to the highest reduction in cell counts.

Tumor Treating Fields (TTFields) are an effective treatment modality delivered via the continuous, noninvasive application of low-intensity (1-3 V/cm), ...

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 ...

Phosphopeptide Enrichment Coupled with Label-free Quantitative Mass Spectrometry to Investigate the Phosphoproteome in Prostate Cancer

Phosphoproteomics involves the large-scale study of phosphorylated proteins. Protein phosphorylation is a critical step in many signal transduction pathways and is tightly regulated by kinases and phosphatases. Therefore, characterizing the phosphoproteome may provide insights into identifying novel targets and biomarkers for oncologic therapy. Mass spectrometry provides a way to globally detect and quantify thousands of unique phosphorylation events. However, phosphopeptides are much less abundant than non-phosphopeptides, making biochemical analysis more challenging. To overcome this limitation, methods to enrich phosphopeptides prior to the mass spectrometry analysis are required. We describe a procedure to extract and digest proteins from tissue to yield peptides, followed by an enrichment for phosphotyrosine (pY) and phosphoserine/threonine (pST) peptides using an antibody-based and/or titanium dioxide (TiO2)-based enrichment method. After the sample preparation and mass spectrometry, we subsequently identify and quantify phosphopeptides using liquid chromatography-mass spectrometry and analysis software.

This protocol describes a procedure to extract and enrich phosphopeptides from prostate cancer cell lines or tissues for an analysis of the phosphoproteome via mass spectrometry-based proteomics.

Phosphoproteomics involves the large-scale study of phosphorylated proteins. Protein phosphorylation is a critical step in many signal transduction ...

Elastic Staining on Paraffin-embedded Slides of pT3N0M0 Gastric Cancer Tissue

The elastic lamina, which is usually located in the sub-mesothelial layer, adjacent to the peritoneum mesothelial cells, is amphiphilic on hematoxylin and eosin (H&amp;E) staining but can be visualized through elastic staining. One of the important benefits of elastic staining is that it makes it easy to determine whether tumor cells have invaded beyond the elastic lamina. This helps in examining the extent of peritoneal surface invasion, thereby distinguishing the pT3 and pT4 stages in gastrointestinal cancer. In this study, we present a protocol to identify elastic fibers in the formalin-fixed, paraffin-embedded pT3N0M0 gastric cancer tissue sections. We prepare 5-&#181;m paraffin sections fixed on slides, and then all the slides are deparaffinated and rehydrated during the staining procedure. Subsequently, all the sections are oxidized by potassium permanganate, bleached by oxalic acid, stained by elastic staining, and counterstained by Van Gieson's. Finally, we examine the effect of staining; elastic lamina is stained blue-black and collagen fibers are stained red, while the cancer cells are stained in varying shades of yellow. We also describe in detail the methods used for determining the positional relationship between cancer cells and elastic lamina. This method is simple, low-cost, and widely applicable in the identification of peritoneal surface invasion in gastrointestinal cancer because of its outstanding selectivity for elastic fibers and authentic results.

Here, we present a protocol of elastic staining to identify elastic fibers in pT3N0M0 gastric cancer tissues on formalin-fixed, paraffin-embedded sections. Subsequently, we describe a method to determine whether tumor cells invade beyond the elastic lamina.

The elastic lamina, which is usually located in the sub-mesothelial layer, adjacent to the peritoneum mesothelial cells, is amphiphilic on hematoxylin and ...

Digital Analysis of Immunostaining of ZW10 Interacting Protein in Human Lung Tissues

The purpose of this study is to introduce a methodology of the immunostaining of human lung tissues, followed by whole-slide digital scanning and image analysis. Digital scanning is a fast way to scan a stack of slides and produce digital images with high quality. It can produce concordant results with conventional light microscopy (CLM) by pathologists. Furthermore, the availability of digital images makes it possible that the same slide can be concurrently observed by multiple people. Moreover, digital images of slides can be stored in a database, which means the long-term deterioration of glass slides is avoided. The limitations of this technique are as follows. First, it needs high-quality prepared tissue and the original immunohistochemistry (IHC) slides without any damage or excess sealant residue. Second, tumor or nontumor areas should be specified by experienced pathologists before the analysis using software, in order to avoid any confusion about the tumor or nontumor areas during scoring. Third, the operator needs to control the color reproduction throughout the digitization process in whole-slide imaging.

ZW10 interacting protein (ZWINT) participates in the mitotic spindle checkpoint and the pathogenesis of carcinoma. Here, we introduce a methodology of the immunostaining of ZWINT in human lung cancer tissues, followed by the digital scanning of whole slides and image analysis. This methodology can provide high-quality digital images and reliable results.

The purpose of this study is to introduce a methodology of the immunostaining of human lung tissues, followed by whole-slide digital scanning and image ...

Capturing Small Molecule Communication Between Tissues and Cells Using Imaging Mass Spectrometry

Imaging mass spectrometry (IMS) has routinely been applied to three types of samples: tissue sections, spheroids, and microbial colonies. These sample types have been analyzed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) to visualize the distribution of proteins, lipids, and metabolites across the biological sample of interest. We have developed a novel sample preparation method that combines the strengths of the three previous applications to address an underexplored approach for identifying chemical communication in cancer, by seeding mammalian cell cultures into agarose in coculture with healthy tissues followed by desiccation of the sample. Mammalian tissue and cells are cocultured in close proximity allowing chemical communication via diffusion between the tissue and cells. At specific time points, the agarose-based sample is dried in the same manner as microbial colonies prepared for IMS analysis. Our method was developed to model the communication between high grade serous ovarian cancer derived from the fallopian tube as it interacts with the ovary during metastasis. Optimization of the sample preparation resulted in the identification of norepinephrine as a key chemical component in the ovarian microenvironment. This newly developed method can be applied to other biological systems that require an understanding of chemical communication between adjacent cells or tissues.

A novel method of sample preparation was developed to accommodate cell and tissue coculture to detect small molecule exchange using imaging mass spectrometry.

Imaging mass spectrometry (IMS) has routinely been applied to three types of samples: tissue sections, spheroids, and microbial colonies. These sample ...

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.

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 ...

Preparation of Mitochondria from Ovarian Cancer Tissues and Control Ovarian Tissues for Quantitative Proteomics Analysis

Ovarian cancer is a common gynecologic cancer with high mortality but unclear molecular mechanism. Most ovarian cancers are diagnosed in the advanced stage, which seriously hampers therapy. Mitochondrial changes are a hallmark of human ovarian cancers, and mitochondria are the centers of energy metabolism, cell signaling, and oxidative stress. In-depth insights into the changes of the mitochondrial proteome in ovarian cancers compared to control ovarian tissue will benefit in-depth understanding of the molecular mechanisms of ovarian cancer, and the discovery of effective and reliable biomarkers and therapeutic targets. An effective mitochondrial preparation method coupled with an isobaric tag for relative and absolute quantification (iTRAQ) quantitative proteomics are presented here to analyze human ovarian cancer and control mitochondrial proteomes, including differential-speed centrifugation, density gradient centrifugation, quality assessment of mitochondrial samples, protein digestion with trypsin, iTRAQ labeling, strong cation exchange fractionation (SCX), liquid chromatography (LC), tandem mass spectrometry (MS/MS), database analysis, and quantitative analysis of mitochondrial proteins. Many proteins have been successfully identified to maximize the coverage of the human ovarian cancer mitochondrial proteome and to achieve the differentially expressed mitochondrial protein profile in human ovarian cancers.

This article presents a protocol of differential-speed centrifugation in combination with density gradient centrifugation to separate mitochondria from human ovarian cancer tissues and control ovarian tissues for quantitative proteomics analysis, resulting in a high-quality mitochondrial sample and high-throughput and high-reproducibility quantitative proteomics analysis of a human ovarian cancer mitochondrial proteome.

Ovarian cancer is a common gynecologic cancer with high mortality but unclear molecular mechanism. Most ovarian cancers are diagnosed in the advanced ...

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 ...