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

이 원고는 한국의료기기개발기금(KMDF, 보조금 번호 RS-2020-KD000019)과 한국보건산업진흥원(KHIDI, 보조금 번호. HI19C0521020020)의 일부 지원을 받았다.

모든 전혈 샘플은 폐암 환자로부터 수득하였다. 클라이노믹스의 연구 및 분석은 암유전체학연구소에서 수행하며, 정부의 IRB 연구 승인은 서울아산의료원 기관심의위원회(IRB NO. 2021-0802)가 주도하고 있으며, IRB 번호는 클라이노믹스에 등록되어 있습니다.
1. 샘플 준비
<ol><li>9mL의 전혈을 EDTA 또는 cfDNA 안정 혈액 수집 튜브에 수집합니다.</li>
	<li>튜브를 위아래로 ?...

<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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cfDNA와 CTC의 양과 농도는 암의 개인, 단계 및 유형에 따라 다릅니다. 또한 환자 2,4,5,10,20의 상태에 따라 다릅니다. 특히 암의 초기 또는 전암기에서는 암 관련 물질의 농도가 매우 낮아 검출이 불가능할 가능성이 높다. 그럼에도 불구하고 조기 발견은 환자의 생존 및 치료 전?...

암을 포함한 다양한 질병의 조기에 정확한 발견은 치료 전략 1,2,3,4를 수립하는 데 가장 중요한 요소입니다. 특히, 암의 조기 발견은 환자 5,6,7,8의 생존 기회 증가와 밀접한 관련이 있습니다. 최근에는 암의 조기 발견을 위해 액체 생검이 각광을 받고 있습니다. 고형 종양은 혈관 신생을 겪고 다양한 물질을 혈액으로 방출합니다. 특히, 암 환자의 혈액에서 순환 DNA (ctDNA), 순환 RNA (ctRNA), 단백질, 엑소 좀과 같은 소포 및 순환 종양 세포 (CTC)가 발견되었습니다 2,9. 이러한 물질의 양에는 차이가 있지만 초기 단계뿐만 아니라 후기 단계 6,10에서도 일관되게 관찰됩니다. 그러나 이러한 개인차는 매우 높습니다. 예를 들어, ctDNA를 함유하는 무세포 DNA (cfDNA)의 양은 1,000 ng 미만이고, CTC의 수는 암 환자 11,12,13의 전혈 10 mL 중 100 개 미만이다. 많은 연구에서 적은 양으로 존재하는 이러한 물질(즉, cfDNA, ctDNA 및 CTC)을 사용하여 암을 특성화했습니다. 정확한 결과를 얻으려면 고순도13,14로 소량의 물질을 정확하게 분리하는 것이 중요합니다. 종래의 원심분리 방법이 일반적으로 사용되지만 사용자의 기술에 따라 취급이 어렵고 순도가 낮습니다. CTC의 발견 이후, 원심분리 또는 밀도 등급 분리, 면역비드 및 미세유체 방법과 같은 여러 분리 기술이 개발되었습니다. CTC가 발견된 이후 몇 가지 격납 기술이 개발되었습니다. 그러나, 이들 기술은 세포를 단리하는데 사용되는 다양한 칩 및 멤브레인으로부터 세포를 단리할 필요가 있을 때 종종 제한된다(15). 또한 태깅 방식에는 FACS와 같은 장비가 필요하며, 태깅 오염으로 인해 다운스트림 공정에 한계가 있습니다.
최근에는 액체 생검의 사용이 증가하고 암의 조기 발견을위한 다양한 연구가 진행되고 있습니다. 이 방법은 간단하지만 다운 스트림 분석에는 여전히 어려움이 있으며 이러한 어려움을 극복하기 위해 다양한 연구가 시도되고 있습니다16,17. 또한 병원을 포함한 많은 현장에서는 사용하기 편리한 자동화되고 재현 가능한 고순도 분석법이 필요합니다. 여기에서 우리는 액체 생검 후 혈액 샘플에서 물질을 자동으로 분리하기 위한 랩-온-어-디스크를 개발했습니다. 이러한 장치는 원심분리, 미세유체공학 및 기공 크기의 세포 포획 원리를 기반으로 합니다. 디스크에는 세 가지 유형이 있습니다 : LBx-1은 혈장 및 버피 코트를 얻을 수 있고 LBx-2는 10mL 미만의 부피로 전혈에서 혈장 및 PBMC를 얻을 수 있습니다. FAST-auto는 디스크에서 제거 가능한 멤브레인을 사용하여 CTC를 획득 할 수도 있습니다. 한 번에 최대 4개의 디스크를 사용할 수 있습니다. 무엇보다도 이 장치 및 방법의 장점은 소량의 혈액을 이용하여 동일한 시료로부터 다양한 암 유래 물질을 얻을 수 있다는 점이다. 이것은 환자의 혈액을 한 번만 채취하면된다는 것을 의미합니다. 또한 혈액 채취 기간의 차이로 인한 오류를 배제할 수 있는 장점이 있습니다. 이 플랫폼은 사용하기 쉽고 액체 생검 및 다운스트림 응용 분야에 대한 정확한 결과를 제공합니다. 이 프로토콜에서는 장치 및 카트리지의 사용법이 소개됩니다.

<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

최근에는 암을 비롯한 다양한 질병을 진단하기 위해 액체 생검이 사용되었습니다. 체액에는 정상 조직에서 유래한 세포, 단백질 및 핵산을 포함한 많은 물질이 포함되어 있지만 이러한 물질 중 일부는 질병 부위에서도 유래합니다. 체액에서 이러한 물질의 조사 및 분석은 다양한 질병의 진단에 중추적인 역할을 합니다. 따라서 필요한 물질을 정확하게 분리하는 것이 중요하며,이를 위해 여러 기술이 개발되었습니다.
우리는 CD-PRIME이라는 랩 온 어 디스크 유형의 장치 및 플랫폼을 개발했습니다. 이 장치는 자동화되어 있으며 시료 오염 및 시료 안정성에 대해 우수한 결과를 제공합니다. 또한 우수한 획득 수율, 짧은 작동 시간 및 높은 재현성의 장점이 있습니다. 또한, 장착할 디스크의 종류에 따라, 무세포 DNA를 포함하는 혈장, 순환 종양 세포, 말초 혈액 단핵 세포 또는 버피 코트가 분리될 수 있다. 따라서, 체액에 존재하는 다양한 물질의 획득은 오믹스의 연구를 포함하는 다양한 다운스트림 응용을 위해 수행될 수 있다.

이 기술의 목표는 전혈에서 암 관련 물질을 쉽고 자동으로 분리하는 것입니다. 특히 누구나 모든 적합한 연구 및 분석 분야에서이 기술을 사용할 수 있습니다. 단일 혈액 샘플에서 여러 물질을 동시에 재현 가능하게 분리하는 것은 액체 생검에서 중요합니다. LBx-1 및 LBx-2 디스크는 전혈에서 혈장 및 버피 코트 또는 PBMC를 분리하는 데 사용됩니다. 그림 1 은이 장치의 적용에 ?...

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

이 백서에서는 전혈에서 무세포 DNA 및 순환 종양 세포와 같은 물질을 쉽고 효율적으로 분리하고 수집하기 위한 자동화 장비의 적용에 대해 설명합니다.

임상 샘플에서 암 관련 물질의 자동 분리 및 수집

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

우리의 프로토콜은 사용자 친화적입니다. 이 기술을 사용하면 누구나 혈액에서 성분을 쉽게 분리 할 수 있습니다. 오염을 최소화하고 재료를 정확하게 분리할 수 있습니다.자동화 및 이 프로토콜의 확립으로 연구자들은 고순도의 암 관련 물질을 쉽게 얻을 수 있어 다양한 다운스트림 연구에 도움이 됩니다. 특히 최종 사용자 연구에 중요한 이점을 제공 할 것으로 기대됩니다. 그들의 절차를 시연하는 것은 암 유전체학 연구소의 선임 연구원이자 보조 관리자 인 Jay Jeong이 될 것입니다.시작하려면 기기의 터치스크린 패널에서 카트리지 유형을 선택하여 LBx 1 카트리지를 로드합니다. 화살표 버튼을 눌러 샘플 수를 변경합니다. 그런 다음 기기 도어를 열고 사용할 모든 카트리지를 카트리지 홀더의 번호 순서대로 삽입합니다.총 4개의 카트리지의 경우 카트리지 홀더의 빈 공간에 더미 카트리지를 놓습니다. 지지 휠을 사용하여 카트리지를 장착하고 잠금 너트를 조여 고정합니다. 도어를 닫고 터치패드 화면의 실행 버튼을 누릅니다.기기는 카트리지의 밸브를 닫으며 약 30 초가 걸립니다. 메시지에 따라 문을 엽니다. 지지 휠을 제거하고 카트리지 홀더에서 밸브 폐쇄 카트리지를 제거합니다.혈청 학적 피펫을 사용하여 최대 10 밀리리터의 전혈 샘플을 피펫팅합니다. 피펫 팁을 카트리지의 샘플 입구 깊숙이 삽입하고 전혈 샘플을 천천히 주입합니다. 카트리지를 기기에 삽입한 후 기기의 도어를 닫고 OK 버튼을 누릅니다.혈장과 버피 코트는 전혈에서 자동으로 분리되며 약 30 분이 소요됩니다. 플라즈마와 버피 코트가 분리되면 알람 소리와 함께 화면에 메시지가 나타납니다. 도어를 열거나 정지 버튼을 눌러 알람을 중지하십시오.문 여세요. 카트리지를 제거하고 테이블 위에 놓습니다. 1 밀리리터 피펫 팁을 사용하여 플라즈마 배출구에서 3 밀리리터의 혈장을 회수합니다.그런 다음 1밀리리터 피펫 팁을 사용하여 버피 코트 배출구에서 버피 코트 3밀리리터를 회수합니다. 그런 다음 기기의 터치스크린 패널에서 카트리지 이름을 선택하여 LBx 2 카트리지를 로드합니다. 화살표 버튼을 눌러 샘플 수를 변경하고 LBx 1 카트리지에 표시된 것과 동일한 절차에 따라 카트리지 홀더에 카트리지를 삽입합니다.카트리지를 삽입하고 도어를 닫은 후 터치패드 화면의 실행 버튼을 누릅니다. 앞에 표시된 절차에 따라 카트리지 홀더에서 밸브가 닫힌 카트리지를 제거하고 테이블 위에 놓습니다. 혈청학적 피펫을 사용하여 밀도 구배 용액을 피펫팅한다.피펫 팁을 카트리지 입구 깊숙이 삽입하고 용액을 천천히 주입하십시오. 그런 다음 전혈 샘플을 피펫팅하고 샘플 입구에 천천히 주입합니다. 카트리지를 삽입하고 고정한 후 도어를 닫고 OK 버튼을 누릅니다.혈장과 PBMC가 전혈에서 자동으로 분리되면 경보음과 함께 메시지가 나타납니다. 도어를 열고 카트리지를 제거하십시오. 1 밀리리터 피펫 팁을 사용하여 혈장 배출구로부터 3 밀리리터의 혈장을 회수한 다음, PBMC 배출구로부터 3 밀리리터의 PBMC를 회수한다.마찬가지로 FAST-auto 카트리지의 경우 카트리지 홀더에서 밸브 폐쇄 카트리지를 제거한 후 PBS 용액 6ml를 카트리지의 PBS 입구에 주입합니다. 이어서, LBx 1로부터 수득된 전혈 또는 버피코트 3 밀리리터, 또는 LBx 2로부터 수득된 PBMC 샘플을 피펫팅하고, 샘플을 카트리지의 샘플 입구에 천천히 주입한다. 카트리지를 삽입하고 고정한 후 도어를 닫고 OK 버튼을 누릅니다.CTC의 자동 보강이 완료되면 경보를 중지합니다. 카트리지를 제거하고 백 플레이트 리무버 또는 BPR을 카트리지 전면의 구멍 4 개에 삽입합니다. 양손의 엄지 손가락으로 진한 파란색 날개를 누른 다음 딸깍 소리가 날 때까지 하늘색 몸체를 누릅니다.카트리지 본체를 들어 올려 조심스럽게 제거하십시오. 핀셋을 사용하여 가장자리로 필터 멤브레인을 매우 부드럽게 들어 올리고 핵산 준비를 위해 멤브레인을 1.5 밀리리터 튜브에 넣거나 염색을 위해 슬라이드 유리에 놓습니다. 필요한 경우 여과 된 혈액을 1 밀리리터 피펫 팁을 사용하여 혈액 배출구로 회수하십시오.추출된 cfDNA는 바이오분석기를 사용하여 평가하고, cfDNA 스크린 테이프를 전자 사다리와 함께 사용하였다. 녹색 선은 사다리와 샘플을 정렬하는 데 사용되었습니다. 삼각형 표시는 DNA 밴드를 나타냅니다.대부분의 cfDNA는 길이가 166 염기쌍이고 일부는 정수의 배수로 존재합니다. 일반적으로 cfDNA의 농도는 최대 50-700 염기쌍의 크기를 측정합니다. 밀리리터당 8.47나노그램 및 밀리리터당 5.2나노그램과 같이 각 개별 샘플에서 얻은 양에는 차이가 있지만 cfDNA 추출 결과는 모든 경우에 농도와 크기 분포 모두에 양호했습니다.이러한 결과는 LBx 1 방법이 cfDNA를 포함하는 혈장을 정확하게 분리한다는 것을 나타냅니다. FAST-auto에서는 막을 제거하고 형광 항체로 염색하여 CTC와 백혈구를 검출했습니다. 유리 슬라이드 위에 놓인 염색 된 멤브레인을 형광 현미경으로 관찰 하였다.막 염색 후 세포는 DAPI 또는 핵 양성 신호를 파란색으로 표시하고 EpCAM 및 CK 또는 CTC 양성은 녹색으로 강조 표시되고 CD45 또는 WBC 양성은 빨간색으로 강조 표시됩니다. 멤브레인을 제거하는 것은 어렵지 않습니다. 그러나 CTC의 손실 및 오염에주의해야합니다.전혈, PBMC 및 버피 코트를 모두 사용할 수 있습니다. 대신 전체 볼륨을 설정하기만 하면 됩니다. 테스트가 필요하지만 다른 액체도 사용할 수 있습니다.이 기술은 병원에서 직접 수술 할 수 있으며 물질을 신속하게 얻을 수 있습니다.

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.

유방암 관련 림프 부종의 초음파 평가

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

연구

암 연구학

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.

터치 인쇄물 세포학을 사용 하 여 병 적인 임상 표본에서 높은-품질 종양 DNA를 간단 하 고 빠른 방법

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.

암 관련 피로에 미토 콘 드리 아 기능의 역할 평가

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.

22C 3를 사용 하 여 반대로 PD L1 항 체 생 검 및 비 작은 세포 폐 암 환자에서 세포학 샘플에 집중

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.

종양 세포종 필드 치료 치료의 임상 적용

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.

면역형광에 의한 비소세포 폐암 환자의 순환 종양 세포의 반자동 PD-L1 특성화 및 열거

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.

부인과 및 유방암 종양에서 암 줄기 세포 구체 획득

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.

핸드헬드 동력계를 사용하여 암 관련 피로의 모터 측면 측정

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.

세포 및 조직 샘플에서 인간 유방암 줄기 세포의 분리 및 기능 평가

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.

간단하고 빠른 롤링 서클 증폭 기반 조잡한 생물학적 시료에서 토포이소머라제 1 활성 검출

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