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 manuskript blev delvist støttet af Korea Medical Device Development Fund (KMDF, bevillingsnr. RS-2020-KD000019) og Korea Health Industry Development Institute (KHIDI, bevillingsnr. HI19C0521020020).

Alle fuldblodprøver blev taget fra lungekræftpatienter. Forskningen og analysen på Clinomics udføres af Cancer Genomics Research Institute, og IRB-forskningsgodkendelse fra regeringen ledes af Asan Medical Center Institutional Review Committee (IRB NR. 2021-0802) med IRB-nummeret registreret til forskning hos Clinomics.
1. Forberedelse af prøver
<ol><li>Saml 9 ml fuldblod i et EDTA- eller cfDNA-stabilt blodopsamlingsrør.</li>
	<li>Bland godt ved at vende røret op og n...

<ol>
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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. 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Mængden og koncentrationen af cfDNA og CTC afhænger af individet, stadiet og typen af kræft. Det afhænger også af patientens tilstand 2,4,5,10,20. Især i de tidlige eller precancerøse stadier af kræft er koncentrationerne af kræftrelaterede stoffer meget lave, så der er stor mulighed for, at det ikke kan påvises. Ikke desto mindre har tidlig påvisn...

Tidlig og præcis påvisning af forskellige sygdomme, herunder kræft, er den vigtigste faktor i etableringen af en behandlingsstrategi 1,2,3,4. Især tidlig påvisning af kræft er tæt forbundet med øgede overlevelseschancer for patienten 5,6,7,8. For nylig har flydende biopsier været i søgelyset for tidlig påvisning af kræft. Faste tumorer gennemgår angiogenese og frigiver forskellige stoffer i blodet. Især er cirkulerende DNA'er (ctDNA'er), cirkulerende RNA'er (ctRNA'er), proteiner, vesikler såsom exosomer og cirkulerende tumorceller (CTC'er) fundet i blodet hos kræftpatienter 2,9. Selv om der er forskelle i mængden af disse stoffer, observeres de konsekvent ikke kun i de tidlige stadier, men også i de senere stadier 6,10. Disse individuelle forskelle er imidlertid meget høje; for eksempel er mængden af cellefrit DNA (cfDNA) indeholdende ctDNA mindre end 1.000 ng, og antallet af CTC'er er mindre end 100 ud af 10 ml fuldblod fra kræftpatienter11,12,13. Mange undersøgelser har karakteriseret kræft ved hjælp af disse stoffer, der er til stede i mindre mængder (dvs. cfDNA, ctDNA og CTC'er). For at opnå nøjagtige resultater er det vigtigt nøjagtigt at adskille små mængder stoffer med høj renhed13,14. Konventionelle centrifugeringsmetoder er almindeligt anvendt, men de er vanskelige at håndtere og har lav renhed afhængigt af brugerens færdigheder. Siden opdagelsen af CTC'er er der udviklet flere separationsteknikker, såsom centrifugering eller densitetsseparation, immunoperler og mikrofluidiske metoder. Flere indeslutningsteknikker er blevet udviklet siden opdagelsen af CTC'er. Disse teknikker er imidlertid ofte begrænsede, når det er nødvendigt at isolere celler fra de forskellige chips og membraner, der bruges til at isolere dem15. Mærkningsmetoderne kræver også udstyr såsom FACS, og der er grænser for downstream-processen på grund af mærkning af forurening.
For nylig er brugen af flydende biopsier steget, og der udføres forskellige undersøgelser til tidlig påvisning af kræft. Selv om denne metode er enkel, er der stadig vanskeligheder med downstream-analyse, og forskellige undersøgelser forsøger at overvinde disse vanskeligheder16,17. Derudover kræver mange steder, herunder hospitaler, automatiserede, reproducerbare og metoder med høj renhed, der er praktiske at bruge. Her har vi udviklet et lab-on-a-disc til automatiseret adskillelse af stoffer fra blodprøver efter en flydende biopsi. Disse enheder er baseret på princippet om centrifugering, mikrofluidik og celleindfangning i porestørrelse. Der er tre typer diske: LBx-1 kan erhverve plasma og buffy coat, mens LBx-2 kan erhverve plasma og PBMC fra fuldblod med et volumen på mindre end 10 ml; FAST-auto kan også erhverve CTC'er ved hjælp af en membran, der kan fjernes fra disken. Op til fire af hver disk kan bruges i én kørsel. Frem for alt er fordelen ved denne enhed og metode, at den kan opnå en række kræftafledte stoffer fra den samme prøve ved hjælp af en lille mængde blod. Det betyder, at patientens blod kun skal trækkes én gang. Derudover har det den fordel, at det udelukker fejl på grund af forskelle i blodprøvetagningsperioden. Denne platform er nem at bruge og giver nøjagtige resultater for flydende biopsier og downstream-applikationer. I denne protokol introduceres brugen af enheden og patronen.

<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

For nylig er flydende biopsier blevet brugt til at diagnosticere forskellige sygdomme, herunder kræft. Kropsvæsker indeholder mange stoffer, herunder celler, proteiner og nukleinsyrer, der stammer fra normalt væv, men nogle af disse stoffer stammer også fra det syge område. Undersøgelsen og analysen af disse stoffer i kropsvæskerne spiller en afgørende rolle i diagnosen af forskellige sygdomme. Derfor er det vigtigt at adskille de krævede stoffer nøjagtigt, og der udvikles flere teknikker til dette formål.
Vi har udviklet en lab-on-a-disc type enhed og platform ved navn CD-PRIME. Denne enhed er automatiseret og har gode resultater for prøvekontaminering og prøvestabilitet. Desuden har det fordele ved et godt erhvervelsesudbytte, en kort driftstid og høj reproducerbarhed. Afhængigt af hvilken type disk der skal monteres, kan plasma, der indeholder cellefrit DNA, cirkulerende tumorceller, mononukleære celler i perifert blod eller buffy coats, adskilles. Således kan erhvervelsen af en række forskellige materialer, der er til stede i kropsvæskerne, udføres til en række downstream-applikationer, herunder undersøgelse af omics.

Målet med denne teknik er nemt og automatisk at isolere kræftassocierede stoffer fra fuldblod. Især kan enhver bruge denne teknik inden for alle egnede forsknings- og analyseområder. Den samtidige og reproducerbare adskillelse af flere stoffer i en enkelt blodprøve er signifikant i flydende biopsier. LBx-1- og LBx-2-diskene bruges til isolering af plasma og buffy coat eller PBMC fra fuldblod. Figur 1 viser materialerne adskilt ved anvendelse af denne enhed. For det første blev plasmaet...

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 papir beskriver anvendelsen af automatiseret udstyr til nemt og effektivt at adskille og indsamle stoffer, såsom cellefrit DNA og cirkulerende tumorceller, fra fuldblod.

Automatisk adskillelse og indsamling af kræftrelaterede stoffer fra kliniske prøver

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

Vores protokol er brugervenlig. Ved hjælp af denne teknik kan enhver let isolere komponent fra blodet. Kontaminering kan minimeres, og materialer kan adskilles nøjagtigt.Med automatisering og etablering af denne protokol kan forskere let få kræftrelaterede stoffer med høj renhed, hvilket er nyttigt for forskellige nedstrømsundersøgelser. Det forventes især at give vigtige fordele for slutbrugerforskning. Demonstrere deres procedure vil være Jay Jeong, seniorforsker og assisterende leder ved Cancer Genomics Research Institute.For at begynde skal du indlæse LBx 1-patronen ved at vælge patrontypen på instrumentets berøringsskærmpanel. Tryk på pileknappen for at ændre antallet af prøver. Åbn derefter instrumentdækslet, og indsæt alle de patroner, der skal bruges, i rækkefølge efter nummeret på patronholderen.Hvis der er tale om i alt fire cylinderampulenter, skal prøvedukkepatronen placeres i det tomme rum på patronholderen. Brug støttehjulet til at montere patronen, og stram låsemøtrikken for at fastgøre den. Luk døren, og tryk på kør-knappen på touchpad-skærmen.Instrumentet lukker ventilerne på patronerne, hvilket tager ca. 30 sekunder. Følg meddelelsen for at åbne døren. Fjern støttehjulet, og fjern den ventillukkede patron fra patronholderen.Der pipetteres højst 10 ml fuldblodsprøve ved hjælp af en serologisk pipette. Sæt pipettespidsen dybt ind i cylinderampullens prøveindgang, og injicer langsomt hele blodprøven. Når du har indsat patronerne i instrumentet, skal du lukke døren til instrumentet og trykke på knappen OK.Plasma og buffy coats adskilles automatisk fra fuldblod, hvilket tager cirka 30 minutter. Når plasma og buffy coat er adskilt, vises en meddelelse på skærmen ledsaget af en alarmlyd. Stop alarmen ved at åbne døren eller trykke på stopknappen.Åbn døren. Fjern patronen, og læg den på bordet. Genvind tre milliliter plasma fra plasmaudløbet ved hjælp af en pipettespids på en milliliter.Derefter genvindes tre milliliter af buffy coaten fra buffy coat-udløbet ved hjælp af en pipettespids på en milliliter. Ilæg derefter LBx 2-patronen ved at vælge patronnavnet på instrumentets berøringsskærmpanel. Tryk på pileknappen for at ændre antallet af prøver, og følg den samme procedure, der blev vist for LBx 1-cylinderampullen, for at indsætte patronen i patronholderen.Når du har indsat patronen og lukket døren, skal du trykke på kør-knappen på touchpad-skærmen. Følg den tidligere viste procedure for at fjerne den ventillukkede patron fra patronholderen og placere den på bordet. Densitetsgradientopløsningen pipetteres ved hjælp af en serologisk pipette.Sæt pipettespidsen dybt ind i cylinderampullens indløb, og injicer langsomt opløsningen. Derefter pipetteres hele blodprøven, og den injiceres langsomt i prøveindløbet. Når cylinderampullen er iført og fastgjort, skal du lukke døren og trykke på knappen OK.Når plasma og PBMC automatisk adskilles fra fuldblod, vises en meddelelse ledsaget af en alarmlyd. Åbn døren, og fjern patronen. Gendan tre milliliter plasma fra plasmaudløbet ved hjælp af en pipettespids på en milliliter, og genvind derefter tre ml PBMC fra PBMC-udløbet.På samme måde injiceres seks ml PBS-opløsning i PBS-indgangen til cylinderampullen, efter at den ventillukkede patron er fjernet fra patronholderen. Derefter pipetteres tre ml fuldblod eller buffy coat opnået fra LBx 1 eller PBMC-prøve opnået fra LBx 2, og injicer langsomt prøven i prøveindgangen til cylinderampullen. Når du har indsat patronen og fastgjort den, skal du lukke døren og trykke på knappen OK.Når den automatiske berigelse af CTC'er er fuldført, skal du stoppe alarmen. Fjern patronen, og sæt bagpladefjerneren, eller BPR, i de fire huller på forsiden af patronen. Tryk på den mørkeblå vinge med tommelfingrene på begge hænder, og tryk derefter på den lyseblå krop, indtil den klikker.Fjern forsigtigt patronhuset ved at løfte det. Tag forsigtigt filtermembranen op ved kanten ved hjælp af en pincet, og læg membranen i et 1,5 ml rør til nukleinsyrepræparation eller på et glideglas til farvning. Genvind om nødvendigt det filtrerede blod til blodudløbet ved hjælp af en pipettespids på en milliliter.De ekstraherede cfDNA'er blev evalueret ved hjælp af Bioanalyzer, og cfDNA-skærmbånd blev brugt sammen med en elektronisk stige. Den grønne linje blev brugt til at justere stigen og prøven. Trekantmærkerne angiver DNA-båndet.De fleste cfDNA har en længde på 166 basepar, og nogle findes i heltalsmultipla af længde. Normalt måler koncentrationen af cfDNA op til størrelsen 50 til 700 basepar. Selvom der er en forskel i mængden opnået fra hver enkelt prøve, såsom 8,47 nanogram pr. milliliter og 5,2 nanogram pr. milliliter, var resultaterne af cfDNA-ekstraktion gode for både koncentration og størrelsesfordeling i alle tilfælde.Disse resultater indikerer, at LBx 1-metoden nøjagtigt adskiller plasma indeholdende cfDNA. I FAST-auto blev membranen fjernet og farvet med fluorescerende antistoffer til påvisning af CTC'er og hvide blodlegemer. Den farvede membran, anbragt på glasglas, blev observeret under et fluorescensmikroskop.Efter membranfarvning viser celler DAPI eller nukleare-positive signaler i blåt, mens EpCAM og CK eller CTC'er positive fremhæves med grønt, og CD45 eller WBC-positive fremhæves med rødt. Det er ikke svært at fjerne membranen. Det er dog nødvendigt at være forsigtig med tab og forurening af CTC'er.Hele blod, PBMC og buffy frakke er alle tilgængelige. I stedet skal du bare indstille hele lydstyrken. Andre væsker kan også anvendes, selvom test vil være påkrævet.Denne teknik kan betjenes direkte på hospitaler, og stoffet kan opnås hurtigt.

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.

Ultrasonografi af brystkræft-relaterede 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 ...

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 hurtig metode til at opnå høj kvalitet Tumor DNA fra klinisk-patologisk prøvemateriale ved hjælp af Touch aftryk 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.

Evaluering af rollen mitokondriel funktion i Cancer-relaterede træthed

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.

Ved hjælp af 22C 3 Anti-PD-L1 antistof koncentrere sig om biopsi og cytologi prøver fra patienter med ikke-småcellet lungecancer

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.

Den kliniske anvendelse af Tumor behandling af felter 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 optælling af cirkulerende tumor celler fra ikke-småcellet lunge cancer patienter ved immunofluorescens

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.

Opnåelse kræft stamceller kugler fra gynækologiske og brystkræft tumorer

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.

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 ...

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 funktionel vurdering af stamceller fra brystkræft fra celle- og vævsprøver

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 hurtig rullende cirkelforstærkningsbaseret påvisning af topoisomerase 1-aktivitet i rå biologiske prøver

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