Name: automatic separation and collection of cancer-related substances from clinical samples
Uploaded: 2023-01-13T14:40:00
Description: Watch this Scientific Journal Video about Automatic Separation and Collection of Cancer-Related Substances from Clinical Samples at JoVE.com

Dette manuskriptet ble delvis støttet av Korea Medical Device Development Fund (KMDF, Grant No. RS-2020-KD000019) og Korea Health Industry Development Institute (KHIDI, Grant No. HI19C0521020020).

Alle fullblodsprøver ble tatt fra lungekreftpasienter. Forskningen og analysen ved Clinomics utføres av Cancer Genomics Research Institute, og IRB-forskningsgodkjenning av regjeringen ledes av Asan Medical Center Institutional Review Committee (IRB NO. 2021-0802) med IRB-nummeret registrert for forskning ved Clinomics.
1. Prøvepreparering
<ol><li>Samle 9 ml fullblod i et EDTA- eller cfDNA-stabilt blodinnsamlingsrør.</li>
	<li>Bland godt ved å snu røret opp og ned omtre...

<ol>
	<li>Babayan, A., Pantel, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+liquid+biopsy+approaches+for+early+detection+and+monitoring+of+cancer.">Advances in liquid biopsy approaches for early detection and monitoring of cancer.</a> Genome Medicine. 10 (1), 21 (2018).</li><li>Crowley, E., Di Nicolantonio, F., Loupakis, F., Bardelli, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liquid+biopsy:+monitoring+cancer-genetics+in+the+blood.">Liquid biopsy: monitoring cancer-genetics in the blood.</a> Nature Reviews Clinical Oncology. 10 (8), 472-484 (2013).</li><li>Bardelli, A., Pantel, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liquid+biopsies,+what+we+do+not+know+(yet).">Liquid biopsies, what we do not know (yet).</a> Cancer Cell. 31 (2), 172-179 (2017).</li><li>Mattox, A. K., 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=Applications+of+liquid+biopsies+for+cancer.">Applications of liquid biopsies for cancer.</a> Science Translational Medicine. 11 (507), (2019).</li><li>Heitzer, E., Perakis, S., Geigl, J. B., Speicher, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+potential+of+liquid+biopsies+for+the+early+detection+of+cancer.">The potential of liquid biopsies for the early detection of cancer.</a> NPJ Precision Oncology. 1 (1), 36 (2017).</li><li>Scudellari, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myths+that+will+not+die.">Myths that will not die.</a> Nature. 582 (7582), 322-326 (2015).</li><li>Prasad, V., Fojo, T., Brada, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Precision+oncology:+origins,+optimism,+and+potential.">Precision oncology: origins, optimism, and potential.</a> The Lancet Oncology. 17 (2), 81-86 (2016).</li><li>Prasad, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perspective:+The+precision-oncology+illusion.">Perspective: The precision-oncology illusion.</a> Nature. 537 (7619), 63 (2016).</li><li>Siravegna, G., Marsoni, S., Siena, S., Bardelli, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrating+liquid+biopsies+into+the+management+of+cancer.">Integrating liquid biopsies into the management of cancer.</a> Nature Reviews Clinical Oncology. 14 (9), 531-548 (2017).</li><li>Bettegowda, C., 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=Detection+of+circulating+tumor+DNA+in+early-and+late-stage+human+malignancies.">Detection of circulating tumor DNA in early-and late-stage human malignancies.</a> Science Translational Medicine. 6 (224), 24 (2014).</li><li>Udomruk, S., Orrapin, S., Pruksakorn, D., Chaiyawat, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Size+distribution+of+cell-free+DNA+in+oncology.">Size distribution of cell-free DNA in oncology.</a> Critical Reviews in Oncology/Hematology. 166, 103455 (2021).</li><li>Paterlini-Brechot, P., Benali, N. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circulating+tumor+cells+(CTC)+detection:+clinical+impact+and+future+directions.">Circulating tumor cells (CTC) detection: clinical impact and future directions.</a> Cancer Letters. 253 (2), 180-204 (2007).</li><li>Loeian, M. 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=Liquid+biopsy+using+the+nanotube-CTC-chip:+capture+of+invasive+CTCs+with+high+purity+using+preferential+adherence+in+breast+cancer+patients.">Liquid biopsy using the nanotube-CTC-chip: capture of invasive CTCs with high purity using preferential adherence in breast cancer patients.</a> Lab on a Chip. 19 (11), 1899-1915 (2019).</li><li>Rikkert, L. G., Van Der Pol, E., Van Leeuwen, T. G., Nieuwland, R., Coumans, F. A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Centrifugation+affects+the+purity+of+liquid+biopsy-based+tumor+biomarkers.">Centrifugation affects the purity of liquid biopsy-based tumor biomarkers.</a> Cytometry Part A. 93 (12), 1207-1212 (2018).</li><li>Sharma, 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=Circulating+tumor+cell+isolation,+culture,+and+downstream+molecular+analysis.">Circulating tumor cell isolation, culture, and downstream molecular analysis.</a> Biotechnology advances. 36 (4), 1063-1078 (2018).</li><li>Bennett, C. W., Berchem, G., Kim, Y. J., El-Khoury, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-free+DNA+and+next-generation+sequencing+in+the+service+of+personalized+medicine+for+lung+cancer.">Cell-free DNA and next-generation sequencing in the service of personalized medicine for lung cancer.</a> Oncotarget. 7 (43), 71013 (2016).</li><li>Lowes, L. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circulating+tumor+cells+(CTC)+and+cell-free+DNA+(cfDNA)+workshop+2016:+scientific+opportunities+and+logistics+for+cancer+clinical+trial+incorporation.">Circulating tumor cells (CTC) and cell-free DNA (cfDNA) workshop 2016: scientific opportunities and logistics for cancer clinical trial incorporation.</a> International Journal of Molecular Sciences. 17 (9), 1505 (2016).</li><li>Bryzgunova, O. E., Konoshenko, M. Y., Laktionov, P. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Concentration+of+cell-free+DNA+in+different+tumor+types.">Concentration of cell-free DNA in different tumor types.</a> Expert Review of Molecular Diagnostics. 21 (1), 63-75 (2021).</li><li>Park, 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=Circulating+tumour+cells+as+an+indicator+of+early+and+systemic+recurrence+after+surgical+resection+in+pancreatic+ductal+adenocarcinoma.">Circulating tumour cells as an indicator of early and systemic recurrence after surgical resection in pancreatic ductal adenocarcinoma.</a> Scientific Reports. 11 (1), 1-12 (2021).</li><li>Heidrich, I., A&#269;kar, L., Mossahebi Mohammadi, P., Pantel, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liquid+biopsies:+Potential+and+challenges.">Liquid biopsies: Potential and challenges.</a> International Journal of Cancer. 148 (3), 528-545 (2021).</li><li>Celec, P., Vlkov&#225;, B., Laukov&#225;, L., B&#225;b&#237;&#269;kov&#225;, J., Boor, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-free+DNA:+the+role+in+pathophysiology+and+as+a+biomarker+in+kidney+diseases.">Cell-free DNA: the role in pathophysiology and as a biomarker in kidney diseases.</a> Expert Reviews in Molecular Medicine. 20, 1 (2018).</li><li>Thierry, A. R., 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=Origin+and+quantification+of+circulating+DNA+in+mice+with+human+colorectal+cancer+xenografts.">Origin and quantification of circulating DNA in mice with human colorectal cancer xenografts.</a> Nucleic Acids Research. 38 (18), 6159-6175 (2010).</li><li>Moreira, V. G., de la Cera Mart&#237;nez, T., Gonzalez, E. G., Garcia, B. P., Menendez, F. V. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increase+in+and+clearance+of+cell-free+plasma+DNA+in+hemodialysis+quantified+by+real-time+PCR.">Increase in and clearance of cell-free plasma DNA in hemodialysis quantified by real-time PCR.</a> Clinical Chemistry and Laboratory Medicine (CCLM). 44 (12), 1410-1415 (2006).</li><li>Gauthier, V. J., Tyler, L. N., Mannik, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood+clearance+kinetics+and+liver+uptake+of+mononucleosomes+in+mice.">Blood clearance kinetics and liver uptake of mononucleosomes in mice.</a> Journal of Immunology. 156 (3), 1151-1156 (1996).</li><li>Meng, 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=Circulating+tumor+cells+in+patients+with+breast+cancer+dormancy.">Circulating tumor cells in patients with breast cancer dormancy.</a> Clinical Cancer Research. 10 (24), 8152-8162 (2004).</li><li>Alix-Panabi&#232;res, C., Pantel, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Challenges+in+circulating+tumour+cell+research.">Challenges in circulating tumour cell research.</a> Nature Reviews Cancer. 14 (9), 623-631 (2014).</li><li>Zhou, J., 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=Isolation+of+circulating+tumor+cells+in+non-small-cell-lung-cancer+patients+using+a+multi-flow+microfluidic+channel.">Isolation of circulating tumor cells in non-small-cell-lung-cancer patients using a multi-flow microfluidic channel.</a> Microsystems &amp; Nanoengineering. 5 (1), 8 (2019).</li><li>Sajay, B. N. 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=Towards+an+optimal+and+unbiased+approach+for+tumor+cell+isolation.">Towards an optimal and unbiased approach for tumor cell isolation.</a> Biomedical Microdevices. 15 (4), 699-709 (2013).</li><li>Bailey, P. C., Martin, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insights+on+CTC+biology+and+clinical+impact+emerging+from+advances+in+capture+technology.">Insights on CTC biology and clinical impact emerging from advances in capture technology.</a> Cells. 8 (6), 553 (2019).</li><li>Ahn, S. M., Simpson, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Body+fluid+proteomics:+Prospects+for+biomarker+discovery.">Body fluid proteomics: Prospects for biomarker discovery.</a> Proteomics-Clinical Applications. 1 (9), 1004-1015 (2007).</li></ol>

Mengden og konsentrasjonen av cfDNA og CTC avhenger av individ, stadium og type kreft. Det avhenger også av tilstanden til pasienten 2,4,5,10,20. Spesielt i de tidlige eller forstadier av kreft er konsentrasjonene av kreftrelaterte stoffer svært lave, så det er stor mulighet for at det ikke kan oppdages. Likevel har tidlig påvisning en svært positiv effek...

Tidlig og nøyaktig påvisning av ulike sykdommer, inkludert kreft, er den viktigste faktoren for å etablere en behandlingsstrategi 1,2,3,4. Spesielt er tidlig påvisning av kreft nært knyttet til økte overlevelsessjanser for pasienten 5,6,7,8. Nylig har flytende biopsier vært i søkelyset for tidlig påvisning av kreft. Solide svulster gjennomgår angiogenese og frigjør ulike stoffer i blodet. Spesielt har sirkulerende DNA (ctDNA), sirkulerende RNA (ctRNA), proteiner, vesikler som eksosomer og sirkulerende tumorceller (CTC) blitt funnet i blodet til kreftpasienter 2,9. Selv om det er forskjeller i mengden av disse stoffene, blir de konsekvent observert ikke bare i de tidlige stadiene, men også i de senere stadiene 6,10. Disse individuelle forskjellene er imidlertid svært høye; For eksempel er mengden cellefritt DNA (cfDNA) som inneholder ctDNA mindre enn 1000 ng, og antall CTC er mindre enn 100 i 10 ml fullblod fra kreftpasienter11,12,13. Mange studier har karakterisert kreft ved hjelp av disse stoffene som er tilstede i mindre mengder (dvs. cfDNA, ctDNA og CTC). For å oppnå nøyaktige resultater er det viktig å nøyaktig skille små mengder stoffer med høy renhet13,14. Konvensjonelle sentrifugeringsmetoder brukes ofte, men de er vanskelige å håndtere og har lav renhet avhengig av brukerens dyktighet. Siden oppdagelsen av CTC har flere separasjonsteknikker blitt utviklet, for eksempel sentrifugering eller tetthetsgradseparasjon, immunobead og mikrofluidiske metoder. Flere inneslutningsteknikker har blitt utviklet siden oppdagelsen av CTC. Imidlertid er disse teknikkene ofte begrenset når det er nødvendig å isolere celler fra de forskjellige sjetongene og membranene som brukes til å isolere dem15. Merkingsmetodene krever også utstyr som FACS, og det er grenser for nedstrømsprosessen på grunn av merking av forurensning.
Nylig har bruken av flytende biopsier økt, og ulike studier utføres for tidlig påvisning av kreft. Selv om denne metoden er enkel, er det fortsatt vanskeligheter med nedstrømsanalyse, og ulike studier forsøker å overvinne disse vanskelighetene16,17. I tillegg krever mange steder, inkludert sykehus, automatiserte, reproduserbare og høyrene metoder som er praktiske å bruke. Her har vi utviklet en lab-on-a-disc for automatisert separering av stoffer fra blodprøver etter flytende biopsi. Disse enhetene er basert på prinsippet om sentrifugering, mikrofluidikk og porestørrelsescellefangst. Det finnes tre typer plater: LBx-1 kan skaffe seg plasma og buffy frakk, mens LBx-2 kan skaffe plasma og PBMC fra fullblod med et volum på mindre enn 10 ml; FAST-auto kan også skaffe CTC-er ved hjelp av en membran som kan fjernes fra platen. Opptil fire av hver plate kan brukes i ett løp. Fremfor alt er fordelen med denne enheten og metoden at den kan oppnå en rekke kreftavledede stoffer fra samme prøve ved hjelp av en liten mengde blod. Dette betyr at pasientens blod bare trenger å bli trukket en gang. I tillegg har den fordelen av å utelukke feil på grunn av forskjeller i blodprøveperioden. Denne plattformen er enkel å bruke og gir nøyaktige resultater for flytende biopsier og nedstrøms applikasjoner. I denne protokollen introduseres bruken av enheten og kassetten.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1% BSA (Bovine Serum Albumin)</td>
			<td>Sigma-Aldrich</td>
			<td>A3059</td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 mL Microcentrifuge Tube</td>
			<td>Axygen</td>
			<td>MCT-150-C-S</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL Conical Tube</td>
			<td>SPL</td>
			<td>50015</td>
			<td></td>
		</tr>
		<tr>
			<td>4150 TapeStation System</td>
			<td>Agilent</td>
			<td>G2992AA</td>
			<td>Cell-free DNA Screen Tape (Agilent, 5067-5630), Cell-free DNA Sample Buffer (Agilent, 5067-5633)</td>
		</tr>
		<tr>
			<td>Apostle MiniMax High Efficiency Cell-Free DNA Isolation Kit&#160;</td>
			<td>Apostle</td>
			<td>A17622-250</td>
			<td>5 mL X 50 preps version</td>
		</tr>
		<tr>
			<td>BD Vacutainer blood collection tubes</td>
			<td>BD</td>
			<td>367525</td>
			<td>EDTA Blood Collection Tube (10 mL)</td>
		</tr>
		<tr>
			<td>BioViewCCBS</td>
			<td>Clinomics</td>
			<td></td>
			<td>BioView Clinomics-Customized Bioview System. Allegro Plus microscope-based customization equipment</td>
		</tr>
		<tr>
			<td>CD45 Monoclonal Antibody (HI30), PE-Alexa Fluor 610</td>
			<td>Invitrogen</td>
			<td>MHCD4522</td>
			<td></td>
		</tr>
		<tr>
			<td>FAST Auto cartridge</td>
			<td>Clinomics</td>
			<td>CLX-M3001</td>
			<td></td>
		</tr>
		<tr>
			<td>LBx-1 cartridge</td>
			<td>Clinomics</td>
			<td>CLX-M4101</td>
			<td></td>
		</tr>
		<tr>
			<td>LBx-2 cartridge</td>
			<td>Clinomics</td>
			<td>CLX-M4201</td>
			<td></td>
		</tr>
		<tr>
			<td>OPR-2000 instrument</td>
			<td>Clinomics</td>
			<td>CLX-I2001</td>
			<td></td>
		</tr>
		<tr>
			<td>Cover Glass</td>
			<td>Marienfeld Superior</td>
			<td>HSU-0101040</td>
			<td></td>
		</tr>
		<tr>
			<td>DynaMag 2 Magnet Stand</td>
			<td>Thermo Fisher Scientific</td>
			<td>12321D</td>
			<td></td>
		</tr>
		<tr>
			<td>Ficoll Paque Solution</td>
			<td>GE healthcare</td>
			<td>17-1440-03</td>
			<td>density gradient solution</td>
		</tr>
		<tr>
			<td>Filter Tip, 10 &#181;L</td>
			<td>Axygen</td>
			<td>AX-TF-10</td>
			<td>Pipette tips with aerosol barriers are recommended to help prevent cross contamination.</td>
		</tr>
		<tr>
			<td>Filter Tip, 200 &#181;L</td>
			<td>Axygen</td>
			<td>AX-TF-200</td>
			<td>Pipette tips with aerosol barriers are recommended to help prevent cross contamination.</td>
		</tr>
		<tr>
			<td>Filter Tip, 100 &#181;L</td>
			<td>Axygen</td>
			<td>AX-TF-100</td>
			<td>Pipette tips with aerosol barriers are recommended to help prevent cross contamination.</td>
		</tr>
		<tr>
			<td>Filter Tip, 1000 &#181;L</td>
			<td>Axygen</td>
			<td>AX-TF-1000</td>
			<td>Pipette tips with aerosol barriers are recommended to help prevent cross contamination.</td>
		</tr>
		<tr>
			<td>FITC anti-human CD326 (EpCAM) Antibody</td>
			<td>BioLegend</td>
			<td>324204</td>
			<td></td>
		</tr>
		<tr>
			<td>FITC Mouse Anti-Human Cytokeratin</td>
			<td>BD Biosciences</td>
			<td>347653</td>
			<td></td>
		</tr>
		<tr>
			<td>Formaldehyde solution (35 wt. % in H2O)</td>
			<td>Sigma Aldrich</td>
			<td>433284</td>
			<td></td>
		</tr>
		<tr>
			<td>Kimtech Science Wipers</td>
			<td>Yuhan-Kimberly</td>
			<td>41117</td>
			<td></td>
		</tr>
		<tr>
			<td>Latex glove</td>
			<td>Microflex</td>
			<td>63-754</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnetic Bead Separation Rack</td>
			<td>V&#38;P Scientific</td>
			<td>VP 772F2M-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Manual Pipetting&#160; (0.5-10 &#181;L)</td>
			<td>Eppendorf</td>
			<td>3120000020</td>
			<td></td>
		</tr>
		<tr>
			<td>Manual Pipetting&#160; (2-20 &#181;L)</td>
			<td>Eppendorf</td>
			<td>3120000038</td>
			<td></td>
		</tr>
		<tr>
			<td>Manual Pipetting&#160; (10-100 &#181;L)</td>
			<td>Eppendorf</td>
			<td>3120000046</td>
			<td></td>
		</tr>
		<tr>
			<td>Manual Pipetting&#160; (20-200 &#181;L)</td>
			<td>Eppendorf</td>
			<td>3120000054</td>
			<td></td>
		</tr>
		<tr>
			<td>Manual Pipetting&#160; (100-1000 &#181;L)</td>
			<td>Eppendorf</td>
			<td>3120000062</td>
			<td></td>
		</tr>
		<tr>
			<td>Mounting Medium With DAPI&#160;- Aqueous, Fluoroshield</td>
			<td>abcam</td>
			<td>ab104139</td>
			<td></td>
		</tr>
		<tr>
			<td>Normal Human IgG Control</td>
			<td>R&#38;D Systems</td>
			<td>1-001-A</td>
			<td></td>
		</tr>
		<tr>
			<td>OLYMPUS BX-UCB</td>
			<td>Olympus</td>
			<td>9217316</td>
			<td></td>
		</tr>
		<tr>
			<td>Pan Cytokeratin Monoclonal Antibody (AE1/AE3), Alexa Fluor 488</td>
			<td>Invitrogen</td>
			<td>53-9003-82</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS (Phosphate Buffered Saline Solution)</td>
			<td>Corning</td>
			<td>21-040CVC</td>
			<td></td>
		</tr>
		<tr>
			<td>Portable Pipet Aid</td>
			<td>Drummond</td>
			<td>4-000-201</td>
			<td></td>
		</tr>
		<tr>
			<td>Slide Glass</td>
			<td>Marienfeld Superior</td>
			<td>HSU-1000612</td>
			<td></td>
		</tr>
		<tr>
			<td>StainTray Staining box</td>
			<td>Simport</td>
			<td>M920</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile Serological Pipette (10 mL)</td>
			<td>SPL</td>
			<td>91010</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton&#160;X-100 solution</td>
			<td>Sigma Aldrich</td>
			<td>93443</td>
			<td></td>
		</tr>
		<tr>
			<td>TWEEN&#160;20</td>
			<td>Sigma Aldrich</td>
			<td>P7949</td>
			<td></td>
		</tr>
		<tr>
			<td>Whole Blood</td>
			<td></td>
			<td></td>
			<td>Stored at 4-8 &#176;C by collecting in EDTA or cfDNA stable tube : If the whole blood is insufficient in 9 mL, add PBS (phosphate buffered saline) as much as necessary.</td>
		</tr>
		<tr>
			<td>X-Cite 120Q (Fluorescence Lamp Illuminator)</td>
			<td>Excelitas</td>
			<td>010-00157</td>
			<td></td>
		</tr>
	</tbody>
</table>

automatic separation and collection of cancer-related substances from clinical samples

Nylig har flytende biopsier blitt brukt til å diagnostisere ulike sykdommer, inkludert kreft. Kroppsvæsker inneholder mange stoffer, inkludert celler, proteiner og nukleinsyrer som stammer fra normalt vev, men noen av disse stoffene stammer også fra det syke området. Undersøkelsen og analysen av disse stoffene i kroppsvæskene spiller en sentral rolle i diagnosen av ulike sykdommer. Derfor er det viktig å nøyaktig skille de nødvendige stoffene, og flere teknikker er utviklet for å brukes til dette formålet.
Vi har utviklet en lab-on-a-disc type enhet og plattform kalt CD-PRIME. Denne enheten er automatisert og har gode resultater for prøvekontaminering og prøvestabilitet. Videre har det fordeler med et godt oppkjøpsutbytte, kort driftstid og høy reproduserbarhet. I tillegg, avhengig av hvilken type plate som skal monteres, kan plasma som inneholder cellefritt DNA, sirkulerende tumorceller, perifere mononukleære celler i blodet eller buffy strøk skilles. Dermed kan oppkjøpet av en rekke materialer som er tilstede i kroppsvæskene gjøres for en rekke nedstrøms applikasjoner, inkludert studiet av omics.

Målet med denne teknikken er å enkelt og automatisk isolere kreftassosierte stoffer fra fullblod. Spesielt kan hvem som helst bruke denne teknikken i alle egnede forsknings- og analyseområder. Samtidig og reproduserbar separasjon av flere stoffer i en enkelt blodprøve er signifikant i flytende biopsier. LBx-1- og LBx-2-platene brukes til å isolere plasma og buffy-belegg eller PBMC fra fullblod. Figur 1 viser materialene atskilt ved bruk av denne enheten. Først ble plasmaet oppnådd fra...

Watch this Scientific Journal Video about Automatic Separation and Collection of Cancer-Related Substances from Clinical Samples at JoVE.com

Automatic Separation and Collection of Cancer-Related Substances from Clinical Samples

Dette papiret beskriver anvendelsen av automatisert utstyr for enkelt og effektivt å skille og samle stoffer, for eksempel cellefritt DNA og sirkulerende tumorceller, fra fullblod.

Automatisk separasjon og innsamling av kreftrelaterte stoffer fra kliniske prøver

Recently, liquid biopsies have been used to diagnose various diseases, including cancer. Body fluids contain many substances, including cells, proteins, ...

Protokollen vår er brukervennlig. Ved hjelp av denne teknikken kan hvem som helst enkelt isolere komponent fra blodet. Forurensning kan minimeres og materialer kan separeres nøyaktig.Med automatisering og etablering av denne protokollen kan forskere enkelt få tak i kreftrelaterte stoffer med høy renhet, noe som er nyttig for ulike nedstrømsstudier. Spesielt forventes det å gi viktige fordeler for sluttbrukerundersøkelser. Demonstrere sin prosedyre vil være Jay Jeong, seniorforsker og assisterende leder ved Cancer Genomics Research Institute.For å begynne, legg i LBx 1-kassetten ved å velge kassetttype på instrumentets berøringsskjermpanel. Trykk på pilknappen for å endre antall prøver. Deretter åpner du instrumentdøren og setter inn alle patronene som skal brukes i rekkefølge etter nummeret på kassettholderen.For totalt fire kassetter, plasser dummypatronen i det tomme rommet på kassettholderen. Bruk støttehjulet til å montere patronen og stram låsemutteren for å feste den. Lukk døren, og trykk på kjøreknappen på berøringsskjermen.Instrumentet lukker ventilene på patronene, noe som tar omtrent 30 sekunder. Følg meldingen for å åpne døren. Fjern støttehjulet og fjern den ventillukkede kassetten fra kassettholderen.Pipette maksimalt 10 milliliter av en fullblodprøve ved hjelp av en serologisk pipette. Sett pipettespissen dypt inn i prøveinnløpet på sylinderampullen, og injiser sakte fullblodprøven. Etter at du har satt patronene inn i instrumentet, lukker du døren til instrumentet og trykker på OK-knappen.Plasma og buffy strøk skilles automatisk fra fullblod, noe som tar omtrent 30 minutter. Når plasma- og buffy-pelsen er skilt, vises en melding på skjermen ledsaget av en alarmlyd. Stopp alarmen ved å åpne døren eller trykke på stoppknappen.Åpne døren. Fjern kassetten og legg den på bordet. Gjenopprett tre milliliter plasma fra plasmautløpet ved hjelp av en milliliter pipettespiss.Deretter gjenvinner du tre milliliter av buffycoaten fra buffycoatutløpet ved hjelp av en pipettespiss på én milliliter. Deretter legger du i LBx 2-kassetten ved å velge navnet på kassetten på instrumentets berøringsskjermpanel. Trykk på pilknappen for å endre antall prøver, og følg samme fremgangsmåte som vist for at LBx 1-kassetten skal sette inn kassetten i kassettholderen.Etter at du har satt inn kassetten og lukket døren, trykker du på run-knappen på berøringsskjermen. Følg fremgangsmåten som er vist tidligere for å fjerne den ventillukkede sylinderampullen fra sylinderampulleholderen og plassere den på bordet. Pipetter tetthetsgradientløsningen ved hjelp av en serologisk pipette.Sett pipettespissen dypt inn i sylinderampullens innløp og injiser oppløsningen langsomt. Deretter pipetter fullblodprøven og injiser den sakte inn i prøveinnløpet. Etter at du har satt inn og sikret kassetten, lukker du døren og trykker på OK-knappen.Når plasma og PBMC automatisk skilles fra fullblod, vises en melding ledsaget av en alarmlyd. Åpne døren og fjern kassetten. Gjenopprett tre milliliter plasma fra plasmautløpet ved hjelp av en milliliter pipettespiss, og gjenopprett deretter tre milliliter av PBMC fra PBMC-utløpet.På samme måte, for FAST-auto-patronen, etter å ha fjernet den ventillukkede patronen fra kassettholderen, injiserer du seks milliliter av PBS-løsningen i PBS-innløpet til kassetten. Deretter pipetter tre milliliter fullblod eller buffy frakk oppnådd fra LBx 1, eller PBMC prøve oppnådd fra LBx 2, og sakte injisere prøven inn i prøveinnløpet til patronen. Etter å ha satt inn patronen og sikret den, lukk døren og trykk på OK-knappen.Når den automatiske anrikningen av CTC-er er fullført, stopper du alarmen. Fjern kassetten og sett bakplatefjerneren, eller BPR, inn i de fire hullene på forsiden av kassetten. Trykk på den mørkeblå vingen med tommelen på begge hender, og trykk deretter på den lyseblå kroppen til den klikker.Fjern patronkroppen forsiktig ved å løfte den. Veldig forsiktig, plukk opp filtermembranen ved kanten ved hjelp av en pinsett, og legg membranen i et 1, 5 milliliter rør for nukleinsyrepreparasjon, eller på et lysbildeglass for fargingen. Om nødvendig, gjenopprett det filtrerte blodet til bloduttaket ved hjelp av en milliliter pipettespiss.De ekstraherte cfDNA-ene ble evaluert ved hjelp av Bioanalyzer, og cfDNA-skjermbånd ble brukt sammen med en elektronisk stige. Den grønne linjen ble brukt til å justere stigen og prøven. Trekantmerkene indikerer DNA-båndet.De fleste cfDNA har en lengde på 166 basepar, og noen finnes i heltallsmultipler av lengde. Vanligvis måler konsentrasjonen av cfDNA opp til størrelsen på 50 til 700 basepar. Selv om det er en forskjell i mengden oppnådd fra hver enkelt prøve, for eksempel 8, 47 nanogram per milliliter og 5, 2 nanogram per milliliter, var resultatene av cfDNA-ekstraksjon gode for både konsentrasjon og størrelsesfordeling i alle tilfeller.Disse resultatene indikerer at LBx 1-metoden nøyaktig separerer plasma som inneholder cfDNA. I FAST-auto ble membranen fjernet og farget med fluorescerende antistoffer for å påvise CTC og hvite blodlegemer. Den fargede membranen, plassert på glassglass, ble observert under et fluorescensmikroskop.Etter membranfarging viser celler DAPI- eller kjerne-positive signaler i blått, mens EpCAM og CK eller CTCs-positive er uthevet i grønt, og CD45- eller WBC-positive er uthevet i rødt. Det er ikke vanskelig å fjerne membranen. Det er imidlertid nødvendig å være forsiktig med tap og forurensning av CTC.Fullblod, PBMC og buffy frakk er alle tilgjengelige. I stedet trenger du bare å stille inn hele volumet. Andre væsker kan også brukes, selv om testing vil være nødvendig.Denne teknikken kan betjenes direkte på sykehus, og stoffet kan oppnås raskt.

automatic-separation-collection-cancer-related-substances-from

Research

JoVE Journal

Cancer Research

Ultrasonographic Evaluation of Breast Cancer-related Lymphedema

Lymphedema is one of the most common complications after breast cancer surgery. There are many diagnostic tools for lymphedema, but no standard method yet exists. Progressive Resistance Exercise (PRE) is expected to improve lymphedema without additional swelling. This study showed the therapeutic effects of PRE on lymphedema by using ultrasonography to measure the change in thickness of the muscle and subcutaneous tissue. The thickness of subcutaneous tissue decreased more in the PRE group than in the non-PRE group. Ultrasonography is widely used in many clinics because of its easy accessibility, safety, and inexpensiveness. Ultrasound is one of the best tools for diagnosing and determining treatment efficacy on breast cancer-related lymphedema (BCRL).

This study utilized ultrasonography for diagnosis and follow-up tests to confirm treatment efficacy of Progressive Resistance Exercise (PRE) in breast cancer-related lymphedema (BCRL). Ultrasonography can be applied effectively to ensure diagnosis and treatment of lymphedema.

Ultralyd Evaluering av brystkreft relatert lymfødem

Lymphedema is one of the most common complications after breast cancer surgery. There are many diagnostic tools for lymphedema, but no standard method yet ...

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.

Enkel og rask metode å få høy kvalitet svulst DNA fra klinisk-patologisk prøver bruker Touch forlaget cytologi

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

Evaluating the Role of Mitochondrial Function in Cancer-related Fatigue

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

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

Vurdere rollen mitokondrie funksjon i kreft relatert tretthet

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

Using 22C3 Anti-PD-L1 Antibody Concentrate on Biopsy and Cytology Samples from Non-small Cell Lung Cancer Patients

Pembrolizumab monotherapy has been approved for the first- and second-line treatment of patients with PD-L1-expressing advanced non-small cell lung cancer (NSCLC). Testing for PD-L1 expression with the PD-L1 immunohistochemistry (IHC) 22C3 companion diagnostic assay, which gives a tumor proportion score (TPS), has been validated on tumor tissue. We developed an optimized laboratory-developed test (LDT) that uses the 22C3 antibody (Ab) concentrate on a widely available IHC autostainer for biopsy and cytology specimens. The PD-L1 TPS was evaluated with 120 paired whole-tumor tissue sections and biopsy samples and with 70 paired biopsy and cytology samples (bronchial washes, n = 40; pleural effusions, n = 30). The 22C3 Ab concentrate-based LDT showed a high concordance rate between biopsy (~100%) and cytology (~95%) specimens when compared to PD-L1 IHC expression determined using the PD-L1 IHC 22C3 companion assay at both TPS cut points (&#8805;1%, &#8805;50%). The optimized LDT presented here, using the 22C3 Ab concentrate to determine the PD-L1 expression in both tumor tissue and in cytology specimens, will expand the ability of laboratories worldwide to assess the eligibility of patients with NSCLC for treatment with pembrolizumab monotherapy in a reliable and reproducible manner.

To expand the ability of laboratories worldwide to assess the eligibility of patients with lung cancer for treatment with pembrolizumab, in a reliable and reproducible manner, we developed an assay that uses the 22C3 antibody concentrate on a widely available immunohistochemical autostainer, for both biopsy and cytology specimens.

Med 22C 3 Anti-PD-L1 antistoff konsentrere seg om biopsi og cytologi eksempler fra ikke-småcellet lungekreft pasienter

Pembrolizumab monotherapy has been approved for the first- and second-line treatment of patients with PD-L1-expressing advanced non-small cell lung cancer ...

The Clinical Application of Tumor Treating Fields Therapy in Glioblastoma

Glioblastoma is the most common and lethal form of brain cancer, with a median survival of 15 months after diagnosis and a 5 year survival rate of only 5% with current standard of care. Tumors often recur within 9 months following initial surgery, radiation and chemotherapy, at which point treatment options become limited. This highlights the pressing need for the development of better therapeutics to prolong survival and increase the quality of life for these patients.
Tumor Treating Fields (TTFields) therapy was developed to take advantage of the effect of low frequency alternating electrical fields on cells for cancer therapy. TTFields have been demonstrated to disrupt cells during mitosis and slow tumor growth. There is also growing evidence that they act through stimulating immune responses within exposed tumors. The advantages of TTFields therapy include its noninvasive approach and increased quality of life compared to other treatment modalities such as cytotoxic chemotherapies. The Food and Drug Administration approved TTFields therapy for the treatment of recurrent glioblastoma in 2011 and for newly diagnosed glioblastoma in 2015. We report on the effects of TTFields during mitosis, the results of electric fields modeling, and proper transducer array placement. Our protocol outlines the clinical application of TTFields on a patient post-surgery, using the second-generation device.

Glioblastoma is the most common and aggressive primary brain malignancy in adults, with most tumors recurring after initial treatment. Tumor Treating Fields (TTFields) therapy is the newest treatment modality for glioblastoma. Here, we describe the proper application of TTFields-transducer arrays on patients and discuss theory and aspects of treatment.

Klinisk anvendelse av svulst behandling av feltene terapi i glioblastom

Glioblastoma is the most common and lethal form of brain cancer, with a median survival of 15 months after diagnosis and a 5 year survival rate of only 5% ...

Semi-automatic PD-L1 Characterization and Enumeration of Circulating Tumor Cells from Non-small Cell Lung Cancer Patients by Immunofluorescence

Circulating tumor cells (CTCs) derived from the primary tumor are shed into the bloodstream or lymphatic system. These rare cells (1&#8722;10 cells per mL of blood) warrant a poor prognosis and are correlated with shorter overall survival in several cancers (e.g., breast, prostate and colorectal). Currently, the anti-EpCAM-coated magnetic bead-based CTC capturing system is the gold standard test approved by the U.S. Food and Drug Administration (FDA) for enumerating CTCs in the bloodstream. This test is based on the use of magnetic beads coated with anti-EpCAM markers, which specifically target epithelial cancer cells. Many studies have illustrated that EpCAM is not the optimal marker for CTC detection. Indeed, CTCs are a heterogeneous subpopulation of cancer cells and are able to undergo an epithelial-to-mesenchymal transition (EMT) associated with metastatic proliferation and invasion. These CTCs are able to reduce the expression of cell surface epithelial marker EpCAM, while increasing mesenchymal markers such as vimentin. To address this technical hurdle, other isolation methods based on physical properties of CTCs have been developed. Microfluidic technologies enable a label-free approach to CTC enrichment from whole blood samples. The spiral microfluidic technology uses the inertial and Dean drag forces with continuous flow in curved channels generated within a spiral microfluidic chip. The cells are separated based on the differences in size and plasticity between normal blood cells and tumoral cells. This protocol details the different steps to characterize the programmed death-ligand 1 (PD-L1) expression of CTCs, combining a spiral microfluidic device with customizable immunofluorescence (IF) marker set.

The characterization of circulating tumor cells (CTCs) is a popular topic in translational research. This protocol describes a semi-automatic immunofluorescence (IF) assay for PD-L1 characterization and enumeration of CTCs in non-small cell lung cancer (NSCLC) patient samples.

Semi-automatisk PD-L1 karakterisering og opplisting av sirkulerende tumor celler fra ikke-liten celle Lungekreftpasienter ved Immunofluorescence

Circulating tumor cells (CTCs) derived from the primary tumor are shed into the bloodstream or lymphatic system. These rare cells (1&#8722;10 cells per mL ...

Obtaining Cancer Stem Cell Spheres from Gynecological and Breast Cancer Tumors

Cancer stem cells (CSC) are a small population with self-renewal and plasticity which are responsible for tumorigenesis, resistance to treatment and recurrent disease. This population can be identified by surface markers, enzymatic activity and a functional profile. These approaches per se are limited, due to phenotypic heterogeneity and CSC plasticity. Here, we update the sphere-forming protocol to obtain CSC spheres from breast and gynecological cancers, assessing functional properties, CSC markers and protein expression. The spheres are obtained with single-cell seeding at low density in suspension culture, using a semi-solid methylcellulose medium to avoid migration and aggregates. This profitable protocol can be used in cancer cell lines but also in primary tumors. The tridimensional non-adherent suspension culture thought to mimic the tumor microenvironment, particularly the CSC-niche, is supplemented with epidermal growth factor and basic fibroblast growth factor to ensure CSC signaling. Aiming for robust identification of CSC, we propose a complementary approach, combining functional and phenotypic evaluation. Sphere-forming capacity, self-renewal and sphere projection area establish CSC functional properties. Additionally, characterization comprises flow cytometry evaluation of the markers, represented by CD44+/CD24- and CD133, and Western blot, considering ALDH. The presented protocol was also optimized for primary tumor samples, following a sample digestion procedure, useful for translational research.

The aim of this methodology is to identify cancer stem cells (CSC) in cancer cell lines and primary human tumor samples with the sphere-forming protocol, in a robust manner, using functional assays and phenotypic characterization with flow cytometry and Western blot.

Få kreft stamcellesfærer fra gynekologiske og brystkreft svulster

Cancer stem cells (CSC) are a small population with self-renewal and plasticity which are responsible for tumorigenesis, resistance to treatment and ...

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

Cancer-related fatigue (CRF) is commonly reported by patients both during and after receiving treatment for cancer. Current CRF diagnoses rely on self-report questionnaires which are subject to report and recall biases. Objective measurements using a handheld dynamometer, or handgrip device, have been shown in recent studies to correlate significantly with subjective self-reported fatigue scores. However, variations of both the handgrip fatigue test and fatigue index calculations exist in the literature. The lack of standardized methods limits the utilization of the handgrip fatigue test in the clinical and research settings. In this study, we provide detailed methods for administering the physical fatigue test and calculating the fatigue index. These methods should supplement existing self-reported fatigue questionnaires and help clinicians assess fatigue symptom severity in an objective and quantitative manner.

Simple and accessible methods were developed to measure the motor aspect of cancer-related fatigue objectively and quantitatively. We describe, in detail, ways to administer the physical fatigue test using a simple handgrip device as well as methods to calculate fatigue indices.

Måle motoraspektet ved kreftrelatert tretthet ved hjelp av 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 ...

Isolation and Functional Assessment of Human Breast Cancer Stem Cells from Cell and Tissue Samples

Breast cancer stem cells (BCSCs) are cancer cells with inherited or acquired stem cell-like characteristics. Despite their low frequency, they are major contributors to breast cancer initiation, relapse, metastasis and therapy resistance. It is imperative to understand the biology of breast cancer stem cells in order to identify novel therapeutic targets to treat breast cancer. Breast cancer stem cells are isolated and characterized based on expression of unique cell surface markers such as CD44, CD24 and enzymatic activity of aldehyde dehydrogenase (ALDH). These ALDHhighCD44+CD24- cells constitute the BCSC population and can be isolated by fluorescence-activated cell sorting (FACS) for downstream functional studies. Depending on the scientific question, different in vitro and in vivo methods can be used to assess the functional characteristics of BCSCs. Here, we provide a detailed experimental protocol for isolation of human BCSCs from both heterogenous populations of breast cancer cells as well as primary tumor tissue obtained from breast cancer patients. In addition, we highlight downstream in vitro and in vivo functional assays including colony forming assays, mammosphere assays, 3D culture models and tumor xenograft assays that can be used to assess BCSC function.

This experimental protocol describes the isolation of BCSCs from breast cancer cell and tissue samples as well as the in vitro and in vivo assays that can be used to assess BCSC phenotype and function.

Isolering og funksjonell vurdering av humane brystkreftstamceller fra celle- og vevsprøver

Breast cancer stem cells (BCSCs) are cancer cells with inherited or acquired stem cell-like characteristics. Despite their low frequency, they are major ...

Simple and Fast Rolling Circle Amplification-Based Detection of Topoisomerase 1 Activity in Crude Biological Samples

Isothermal amplification-based techniques such as the rolling circle amplification have been successfully employed for the detection of nucleic acids, protein amounts, or other relevant molecules. These methods have shown to be substantial alternatives to PCR or ELISA for clinical and research applications. Moreover, the detection of protein amount (by Western blot or immunohistochemistry) is often insufficient to provide information for cancer diagnosis, whereas the measurement of enzyme activity represents a valuable biomarker. Measurement of enzyme activity also allows for the diagnosis and potential treatment of pathogen-borne diseases. In all eukaryotes, topoisomerases are the key DNA-binding enzymes involved in the control of the DNA topological state during important cellular processes and are among the important biomarkers for cancer prognosis and treatment.
Over the years, topoisomerases have been substantially investigated as a potential target of antiparasitic and anticancer drugs with libraries of natural and synthetic small-molecule compounds that are investigated every year. Here, the rolling circle amplification method, termed rolling circle enhanced enzyme activity detection (REEAD) assay that allows for the quantitative measurement of topoisomerase 1 (TOP1) activity in a simple, fast, and gel-free manner is presented.By cleaving and ligating a specially designed DNA substrate, TOP1 converts a DNA oligonucleotide into a closed circle, which becomes the template for rolling circle amplification, yielding ~103 tandem repeat rolling circle products. Depending on the nucleotide incorporation during the amplification, there is the possibility of different readout methods, from fluorescence to chemiluminescence to colorimetric. As each TOP1-mediated cleavage-ligation generates one closed DNA circle, the assay is highly sensitive and directly quantitative.

A protocol for the sensitive and quantitative detection of topoisomerase 1 activity using the rolling circle enhanced enzyme activity detection assay is described. The method allows detection of topoisomerase 1 activity from purified components or cell/tissue extracts. This protocol has wide-ranging applications in any field involving detection of enzymatic activity.

Enkel og rask rullende sirkelforsterkningsbasert påvisning av topoisomerase 1-aktivitet i biologiske råvarer

Isothermal amplification-based techniques such as the rolling circle amplification have been successfully employed for the detection of nucleic acids, ...