Name: evaluating the role of mitochondrial function in cancer-related fatigue
Uploaded: 2018-05-17T12:30:00
Description: Watch this Scientific Journal Video about Evaluating the Role of Mitochondrial Function in Cancer-related Fatigue at JoVE.com

Denne undersøgelse er fuldt understøttet af Division af murene forskning af det nationale Institut for sygepleje forskning af NIH, Bethesda, Maryland.

Den nuværende undersøgelse (NCT00852111) blev godkendt af det institutionelle Review Board (IRB) af National Institutes of Health (NIH), Bethesda, Maryland. Deltagerne i denne undersøgelse var euthymic mænd, 18 år eller ældre, der blev diagnosticeret med ikke-metastatisk prostatakræft med eller uden forudgående prostatektomi og var planlagt til at modtage ekstern strålebehandling strålebehandling (EBRT). Potentielle deltagere var udelukket, hvis de havde en fremadskridende sygdom, der kan forårsage betydelig t...

<ol>
	<li>Berger, A. 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=Cancer-Related+Fatigue,+Version+2.2015.">Cancer-Related Fatigue, Version 2.2015.</a> Journal of the National Comprehensive Cancer Network. 13 (8), 1012-1039 (2015).</li><li>Feng, L. R., Dickinson, K., Kline, N., Saligan, L. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Different+phenotyping+approaches+lead+to+dissimilar+biologic+profiles+in+men+with+chronic+fatigue+following+radiation+therapy.">Different phenotyping approaches lead to dissimilar biologic profiles in men with chronic fatigue following radiation therapy.</a> Journal of Pain and Symptom Management. 52 (6), 832-840 (2016).</li><li>Feng, L. 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E., McLaren-Howard, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+fatigue+syndrome+and+mitochondrial+dysfunction.">Chronic fatigue syndrome and mitochondrial dysfunction.</a> International Journal of Clinical and Experimental Medicine. 2 (1), 1-16 (2009).</li><li>Pernas, L., Scorrano, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mito-Morphosis:+Mitochondrial+Fusion,+Fission,+and+Cristae+Remodeling+as+Key+Mediators+of+Cellular+Function.">Mito-Morphosis: Mitochondrial Fusion, Fission, and Cristae Remodeling as Key Mediators of Cellular Function.</a> Annual Review of Physiology. 78 (1), 505-531 (2016).</li><li>Vecchiet, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensory+characterization+of+somatic+parietal+tissues+in+humans+with+chronic+fatigue+syndrome.">Sensory characterization of somatic parietal tissues in humans with chronic fatigue syndrome.</a> Neuroscience Letters. 208 (2), 117-120 (1996).</li><li>Jones, D. 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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+review+of+the+mitochondrial+and+glycolytic+metabolism+in+human+platelets+and+leukocytes:+Implications+for+their+use+as+bioenergetic+biomarkers.">A review of the mitochondrial and glycolytic metabolism in human platelets and leukocytes: Implications for their use as bioenergetic biomarkers.</a> Redox Biology. 2, 206-210 (2014).</li><li>Garrabou, G., 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=The+Effects+of+Sepsis+on+Mitochondria.">The Effects of Sepsis on Mitochondria.</a> The Journal of Infectious Diseases. 205 (3), 392-400 (2012).</li><li>Mittal, M., Siddiqui, M. R., Tran, K., Reddy, S. P., Malik, A. 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K., Akkaya, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+optimized+protocol+to+analyze+glycolysis+and+mitochondrial+respiration+in+lymphocytes.">An optimized protocol to analyze glycolysis and mitochondrial respiration in lymphocytes.</a> Journal of Visualized Experiments. (117), e54918 (2016).</li><li>Nicholls, D. G., 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=Bioenergetic+profile+experiment+using+C2C12+myoblast+cells.">Bioenergetic profile experiment using C2C12 myoblast cells.</a> Journal of Visualized Experiments. (46), e2511 (2010).</li><li>Van den Bossche, J., Baardman, J., de Winther, M. P. 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Træthed hos cancerpatienter er en invaliderende tilstand, der ikke er klart defineret eller præget1. Diagnosticering af træthed afhængig helt af subjektive rapportering og der er ingen aktuelle diagnostiske standard eller behandling for denne tilstand, hovedsagelig på grund af manglende forståelse i sin pathobiology2. Af de foreslåede mekanismer bag træthed hos cancerpatienter, er nedskrivninger i mitokondrie-funktion en af de mest terapeutisk markedssegment veje. D...

Træthed er en udbredt og sørgelig tilstand, som har en negativ indvirkning på livskvaliteten for kræft patienter1. Til denne dato, kræft træthed forbliver dårligt defineret og beror kun på subjektive rapportering af patienter2. Derfor er der et presserende behov for at identificere en let at tilpasse diagnoselaboratorium test for at objektivt karakterisere træthed i kliniske omgivelser3,4.
Flere underliggende mekanismer, herunder mitokondrier dysfunktion, er blevet foreslået til at forårsage træthed5. Mitokondrier er kraftcenter organeller, leverer 95% af cellulære energibehov via oxidativ fosforylering, og spiller en vigtig rolle i calcium signalering apoptose, immun signalering og regulering af andre intracellulær signalering begivenheder6 . Derfor kan nedsat mitokondriel bioenergetik og fejl i energiproduktion bidrage til træthed. Understøtter denne hypotese, har tidligere undersøgelser observeret mutationer i mitokondrie-DNA i patienter med kronisk træthed syndrom7. Mens det er uklart, hvorvidt træthed patofysiologiske oprindelse ligger inden for det centrale nervesystem eller perifere væv, såsom skeletmuskulatur8,9, er der i øjeblikket ingen direkte metode til præcist at vurdere mitokondriel dysfunktion relateret til træthed i live, respiring celler.
Ved hjælp af perifert blod mononukleære celler (PBMC) for at studere mitokondrie funktion giver flere fordele. Først, PBMC er let tilgængelige i de kliniske omgivelser ved hjælp af standard tapning og kan være isolerede hurtigt ved hjælp af grundlæggende laboratoriemetoder. Andet er blod samling mindre invasiv end indsamling af andre væv som en muskel biopsi. Således kan blodprøver indsamles fra den samme patient flere gange over tid, som letter langsgående vurdering af behandling effekter. Interessant, syntes mitokondrie funktion i PBMC at være godt korreleret med nyre mitokondrie status i en dyremodel10. Desuden har immun celle mitokondrier været brugt som en proxy for opdage systemændringer under forskellige sygdom betingelser11,12. Mitokondrier i cirkulerende immunceller er særligt følsomme over for ændringer i immun funktioner og immun signalering molekyler såsom cytokiner13,14,15. For eksempel, er blevet observeret, at PBMC fra patienter med akut inflammatoriske gigtsygdomme udviser høj baseline ilt forbrug14. Derimod blev iltforbrug reduceret i PBMC isoleret fra patienter med systemiske betændelsestilstande, herunder sepsis16. Under betændelsestilstande, kan frie radikaler produceret af dysfunktionelle mitochondrier yderligere bidrage til forhøjede oxidativ stress og langvarig betændelse17. Den centrale rolle af mitochondrier i energiproduktion såvel som i oxidativ stress antyder den potentielle nytte af ved hjælp af mitokondrie funktion som en proxy for at studere træthed i kræft patienter 13.
Tidligere studier undersøger mitokondrie funktion udnyttet biokemiske teknikker, mitokondrielle membran potentielle måling eller isolation af specifikke cellepopulationer, der ikke kan let kan tilpasses i de kliniske omgivelser5, 14,18. I de seneste år, udviklingen af ekstracellulære flux assays har tilladt forskere til let og undersøge præcist ændringer i ilt forbrugssats (OCR) i svar til automatiseret injektioner af respiratorisk hæmmere19,20 , 21 , 22. dog de fleste af disse undersøgelser er designet til specifikke celletyper og høj overførselshastighed storformat kan ikke anvendes i kliniske omgivelser. I dette manuskript beskriver vi en optimeret protokol for behandlingen af mitokondrie-funktion til klinisk brug.

<table border="0" cellpadding="0" cellspacing="0" style="width:876px;" width="875">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:331px;">CPT Mononuclear Cells Preparation Tube&#160;</td>
			<td style="width:123px;">BD Biosciences</td>
			<td style="width:165px;">362761</td>
			<td style="width:257px;">For isolating PBMCs following phlebotomy</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">RPMI-1640&#160;</td>
			<td>Corning</td>
			<td>10-040</td>
			<td>For making growth media for PBMCs</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Fetal bovine serum (FBS)</td>
			<td>Corning</td>
			<td>35-010-CV</td>
			<td>For making growth media for PBMCs</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Penicillin/Streptomycin</td>
			<td>ThermoFisher</td>
			<td>15140122</td>
			<td>For making growth media for PBMCs</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Cell-Tak</td>
			<td>Corning</td>
			<td>354240</td>
			<td>Cell and Tissue adhesive solution; allows suspension cells to adhere to the surface</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Seahorse XF Calibrant Solution</td>
			<td>Agilent</td>
			<td>103059-000</td>
			<td>For hydrating cartridges</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">XFp Fluxpak (miniplates and sensor cartridges)</td>
			<td>Agilent</td>
			<td>103022-100</td>
			<td>Contains XFp cell culture miniplates and sensor cartridges</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">XF base media</td>
			<td>Agilent</td>
			<td>103335-100</td>
			<td>For making XF assay media</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">45% cell culture D-(+)-Glucose solution</td>
			<td>Corning</td>
			<td>25-037-CI</td>
			<td>For making XF assay media</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Sodium pyruvate solution</td>
			<td>Corning&#160;</td>
			<td>25-000-CI</td>
			<td>For making XF assay media</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">L-glutamine solution</td>
			<td>ThermoFisher</td>
			<td>25030081</td>
			<td>For making XF assay media</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Seahorse XFp Mito Stress Test Kit</td>
			<td>Agilent</td>
			<td>103010-100</td>
			<td>Contains oligomycin, FCCP, antimycin A/rotenone</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">CyQUANT Direct Cell Proliferation Assay</td>
			<td>ThermoFisher</td>
			<td>C35011</td>
			<td>For quantification of live cells and data normalization</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Seahorse XFp Analyzer</td>
			<td>Agilent</td>
			<td>S7802AEA</td>
			<td>For measuring mitochondrial function in live cells</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Cytation 5 Cell Imaging Multi-Mode Reader (or any instrument that can quantify fluorescent cells in a plate)</td>
			<td>BioTek</td>
			<td>BTCYT5PV</td>
			<td>For quantification of live cells and data normalization</td>
		</tr>
	</tbody>
</table>

evaluating the role of mitochondrial function in cancer-related fatigue

Træthed er en fælles og invaliderende tilstand, der påvirker de fleste kræftpatienter. Til dato, test træthed forbliver dårligt karakteriseret med ingen diagnostiske til objektivt foranstaltning sværhedsgraden af denne tilstand. Her beskriver vi en optimeret metode til vurdering af mitokondrie funktion af PBMC indsamlet fra trætte kræftpatienter. Ved hjælp af en kompakt ekstracellulære flux system og sekventiel injektion af respiratorisk hæmmere, undersøgte vi PBMC mitokondrie funktionelle status af måling af basal mitokondrie respiration, respiratorisk overskudskapacitet og energy fænotype, som beskriver den foretrukne energi pathway til at reagere for at understrege. Frisk PBMC er let tilgængelige i de kliniske omgivelser ved hjælp af standard tapning. Hele analysen i denne protokol beskrevne kan være afsluttet i mindre end 4 timer uden inddragelse af komplekse biokemiske teknikker. Derudover beskriver vi en normalisering metode, der er nødvendige for at opnå reproducerbare data. De enkle procedure og normalisering metoder præsenteres giver mulighed for gentagen prøvetagning fra den samme patient og generation af reproducerbare data, der kan sammenlignes mellem tidspunkter at vurdere potentielle virkninger og behandling.

Mito Stress Test bygger på måling af ilt forbrugssats (OCR) efter sekventielle injektion af forskellige respiratorisk hæmmere tilknytte en komplet mitokondrie profil. OCR målinger efter hver enkelt drug injektion kan bruges til at beregne følgende parametre relateret til mitokondrie sundhed: Basal OCR måles først før enhver stof injektion til at vurdere iltforbrug skulle opfylde hvile niveau ATP efterspørgsel. Basal respiration er beregnet ved at fratrække ikke-...

Watch this Scientific Journal Video about Evaluating the Role of Mitochondrial Function in Cancer-related Fatigue at JoVE.com

Evaluating the Role of Mitochondrial Function in Cancer-related Fatigue

Vores mål var at udvikle en praktisk protokol for at vurdere mitokondriel dysfunktion associeret med træthed hos cancerpatienter. Denne innovative protokol er optimeret til klinisk brug der involverer kun standard tapning og grundlæggende laboratorieprocedurer.

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A Mouse Model of Fatigue Induced by Peripheral Irradiation

En mus Model af træthed induceret af perifere Bestråling

Cancer-related fatigue (CRF) is a distressing and costly condition that often affects patients receiving cancer treatments, including radiation therapy. ...

Evaluating In Vitro DNA Damage Using Comet Assay

Evaluating In Vitro DNA Damage Using Comet Assay

Evaluering af In Vitro DNA skader med Comet Assay

DNA damage is a common phenomenon for each cell during its lifespan, and is defined as an alteration of the chemical structure of genomic DNA. Cancer ...

Methods for Evaluating the Role of c-Fos and Dusp1 in Oncogene Dependence

Metoder til evaluering af c-Fos rolle og Dusp1 i onkogen afhængighed

The demonstration of tyrosine kinase inhibitors (TKIs) in treating chronic myeloid leukemia (CML) has heralded a new era in cancer therapeutics. However, ...

Three-Dimensional Bone Extracellular Matrix Model for Osteosarcoma

Tre-dimensionelle knogle ekstracellulære Matrix Model for osteosarkom

Osteosarcoma (OS) is the most common and a highly aggressive primary bone tumor. It is characterized with anatomic and histologic variations along with ...

Flow Cytometric Analysis of Mitochondrial Reactive Oxygen Species in Murine Hematopoietic Stem and Progenitor Cells and MLL-AF9 Driven Leukemia

Flow Cytometrisk analyse af mitokondrie reaktive oxygen arter i murine hæmatopoietiske stamceller og Progenitorcellerne og MLL-AF9 drevet leukæmi

We present a flow cytometric approach for analyzing mitochondrial ROS in various live bone marrow (BM)-derived stem and progenitor cell populations from ...

Integration of Bioinformatics Approaches and Experimental Validations to Understand the Role of Notch Signaling in Ovarian Cancer

Integrering af Bioinformatik tilgange og eksperimentelle valideringer for at forstå rollen af notch signalering i kræft i æggestokkene

Notch signaling is a highly conserved regulatory pathway involved in many cellular processes. Dysregulation of this signaling pathway often leads to ...

Measuring the Motor Aspect of Cancer-Related Fatigue using a Handheld Dynamometer

Måling af motoraspektet af kræftrelateret træthed ved hjælp af et håndholdt dynamometer

Cancer-related fatigue (CRF) is commonly reported by patients both during and after receiving treatment for cancer. Current CRF diagnoses rely on ...

Mapping the Structure-Function Relationships of Disordered Oncogenic Transcription Factors Using Transcriptomic Analysis

Kortlægning af strukturfunktionsforhold for uordnede onkogene transskriptionsfaktorer ved hjælp af transskriptomisk analyse

Many cancers are characterized by chromosomal translocations which result in the expression of oncogenic fusion transcription factors. Typically, these ...

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

En musemodel til at undersøge rollen som kræftassocierede fibroblaster i tumorvækst

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