독일 암 센터(DKFZ)의 단일 세포 오픈 랩(scOpenLab)에 유용한 토론을 해 주셔서 감사합니다. 이 연구는 독일 암 원조에 의해 지원 되었다, 맥스 에데르 프로그램 보조금 수 70111964 (S.T.).

신선한 냉동 신경교종 샘플은 독일 하이델베르크에 있는 종양 질병 (NCT) 조직 은행에서 국가 종양 질병 센터 (NCT)에서 얻어졌습니다. 환자 자료의 사용은 하이델베르크의 의학 학부에서 기관 검토 위원회에 의해 승인되었으며, 연구 결과에 포함 된 모든 환자로부터 통보 된 동의를 얻었다.
1. 실험 준비
<ol><li>얼음 또는 4 °C에서 모든 단계를 수행합니다.</li>
	<li>사전 냉각 튜브, 접시, 면도날, 두드리거 및 유봉을 4 °C로 합니다.</li>
	<li>모든 버퍼를 미리 준비합니다. 이러한 버퍼는 실온에서 안정적입니다. 멸균 여과는 특히 자당에 권장됩니다. 주식 준비는 Corces 외19에서수정됩니다. 표 1-7을참조하십시오.</li>
	<li>액체 질소 또는 -80°C 냉동고 저장에서 샘플을 제거하고 2.1 단계까지 드라이 아이스를 유지합니다.</li>
</ol>2. 조직 해부 및 해조
<ol><li>신선한 냉동 조직 샘플 (10-60 mg)을 미리 차가운 페트리 접시로 옮춥니다. 신선한 냉동 조직을 면도날로 얼음 위에 작은 조각으로 잘라냅니다.</li>
	<li>냉각된 핵 용해 버퍼 500μL을 사전 냉각된 1.5mL 튜브에 추가합니다. 핵 리시스 완충제가 함유된 1.5mL 튜브에 조직 조각을 놓고, Douncer(재료의표)로이송한다. 참고: 초기 시료 감소를 위한 "느슨한" 또는 "A" 유봉과 완전한 시료 균질화를 위한 "타이트" 또는 "B" 유봉의 두 가지 두 가지 다이온스 균질화 유봉이 있습니다.</li>
	<li>마찰이 감소 될 때까지 약 20 스트로크에 대한 "느슨한"유봉으로 조직 조각을 두드리고. 파편이 존재하는 경우, 샘플은 100mm 여과로 여과에 의해 미리 지워질 수 있습니다.</li>
	<li>완전한 조직 균질화를 달성하기 위해 20 스트로크에 대한 "꽉"유봉으로 두드리고.</li>
	<li>호모전(약 500 μL)을 미리 냉각된 2mL 튜브로 옮긴다. 차가운 리시스 버퍼 1mL를 추가합니다. 부드럽게 섞어서 얼음에 5분간 배양합니다. 넓은 보어 파이펫 팁과 부드럽게 섞고 인큐베이션 중에 1-2회 반복합니다.</li>
	<li>30 μm 스트레이너 메쉬를 사용하여 전체 호모게네이트를 필터링하고 15mL 팔콘 튜브로 수집하고 새로운 미리 냉각된 2mL 튜브로 다시 이송합니다. 단일 여과기는 일반적으로 전체 동형에 충분합니다.</li>
	<li>경현미경아래에서 큰 파편의 제거와 핵멤브레인의 손상성을 확인하십시오. 핵은 둥글게 될 필요가 있고 핵막은 왜곡되어서는 안됩니다. 파편이 존재하는 경우 핵을 다시 여과할 수 있습니다.</li>
	<li>벤치 상단 원심분리기의 4°C에서 5분 동안 500 x g의 핵원심분리기. 핵을 함유한 펠릿으로 ~50 μL을 남기고 상체를 제거합니다. 다른 1mL의 핵 용해 완충제에서 펠릿을 부드럽게 재중단하고 얼음에서 5 분 동안 배양합니다.</li>
	<li>4°C에서 5분 동안 500 x g의 핵 원심분리기. 펠릿을 방해하지 않고 상체를 제거하고, 1x 균질화 버퍼(HB)의 500mL(600mL)를 추가하고, 재차질 없이 5분 동안 배양한다. 이어서, 1x HB의 또 다른 1.0 mL에서 핵을 다시 중단한다.</li>
	<li>4°C에서 5분 동안 500 x g의 핵 원심분리기. 상체를 제거하고 새로운 2.0 mL 튜브로 1x HB의 200 μL에서 핵을 부드럽게 다시 분리합니다.</li>
</ol>3. 그라데이션 원심분리
<ol><li>50% 이디엑스산올 용액(표5)의200 μL을 추가하여 최종 농도를 25% 이오디산올을 제공합니다. 300mL에 세팅된 파이펫과 10번 잘 섞으세요.</li>
	<li>25% 혼합물 아래에 29% 이오디산올 용액(표6)의300 μL을 추가합니다. 층의 혼합물을 피하기 위해 P1000 미세 팁을 사용합니다.</li>
	<li>29% 혼합물 아래에 35% 이오디산올 용액(표7)의300 μl을 추가합니다. 층의 혼합물을 피하기 위해 P1000 미세 팁을 사용합니다. 주의: 이 단계는 과도한 볼륨 변위를 방지하기 위해 파이펫 팅 중에 파이펫 팁을 점진적으로 제거해야 합니다.</li>
	<li>시료를 스윙 버킷 원심분리기에 놓고 브레이크를 끄고 4°C에서 3,500x g에서 20분 동안 회전합니다.</li>
	<li>흔들리지 않고 샘플을 부드럽게 제거하고 빛 아래에서 관찰하십시오. 95% 순수 핵의 명확한 백색 대역은 두 번째와 제 3 층 사이에 표시되어야한다(도 1).</li>
</ol>4. 핵 분리
<ol><li>29%-35%의 중간 단계에서 백색 핵 밴드까지 상부 층을 흡인한다.</li>
	<li>200mL 부피로 핵 밴드를 수집하고, 20 μm필터(재료 표)로신선한 튜브및 필터로 옮김한다. 참고: 핵은 여과 전에 재중단될 필요가 없습니다.</li>
	<li>경현미경아래에서 큰 파편의 제거와 핵멤브레인의 손상성을 확인하십시오. 핵은 둥글게 될 필요가 있고 핵막은 왜곡되어서는 안됩니다.</li>
	<li>snRNA-seq/snATAC-seq를 위한 혈류계 및 알리쿼트 핵에 Trypan 블루 염색을 사용하여 핵을 계산합니다.</li>
</ol>5. 단일 핵 RNA 및 ATAC 세크
<ol><li>단일 세포 유전자 발현 및 단일 세포 ATAC 시약키트(재료표)를사용하여 핵을 즉시 처리한다. 참고: 10x 유전체학 계통에 대한 핵 샘플 농도는 snRNA-seq에 대한 μL당 1500-3000 핵, 및 snATAC-seq를 위한 m μL당 3500-7000 핵이다. 핵은 1x PBS를 사용하여 희석될 수 있다.</li>
	<li>유전체학 코어 시설에서 생성된 라이브러리를 시퀀스합니다.</li>
	<li>데이터의 품질 관리 분석을 수행합니다. 핵은 독특한 분자 식별자 (UMI) >1000, 유전자의 수 >500 및 미토콘드리아 유전자의 퍼센트 <5%,를 포함하는 경우에 추가 분석을 위해 포함되며, UMIs 및 유전자의 평균 + 3개의 표준 편차 내에 있습니다.</li>
</ol>

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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=Decoupling+genetics,+lineages,+and+microenvironment+in+IDH-mutant+gliomas+by+single-cell+RNA-seq.">Decoupling genetics, lineages, and microenvironment in IDH-mutant gliomas by single-cell RNA-seq.</a> Science. 355 (6332), (2017).</li><li>Tirosh, I., 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=Single-cell+RNA-seq+supports+a+developmental+hierarchy+in+human+oligodendroglioma.">Single-cell RNA-seq supports a developmental hierarchy in human oligodendroglioma.</a> Nature. 539 (7628), 309-313 (2016).</li><li>Weng, Q., 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=Single-Cell+Transcriptomics+Uncovers+Glial+Progenitor+Diversity+and+Cell+Fate+Determinants+during+Development+and+Gliomagenesis.">Single-Cell Transcriptomics Uncovers Glial Progenitor Diversity and Cell Fate Determinants during Development and Gliomagenesis.</a> Cell Stem Cell. 24 (5), 707-723 (2019).</li><li>Neftel, 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=An+Integrative+Model+of+Cellular+States,+Plasticity,+and+Genetics+for+Glioblastoma.">An Integrative Model of Cellular States, Plasticity, and Genetics for Glioblastoma.</a> Cell. 178 (4), 835-849 (2019).</li><li>Al-Ali, 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=Single-nucleus+chromatin+accessibility+reveals+intratumoral+epigenetic+heterogeneity+in+IDH1+mutant+gliomas.">Single-nucleus chromatin accessibility reveals intratumoral epigenetic heterogeneity in IDH1 mutant gliomas.</a> Acta Neuropathologica Communications. 7 (1), 201 (2019).</li></ol>

저자는 공개 할 것이 없습니다.

종양 내 이질성 분야는 흥미로운 단계에 있으며, 기존의 지식에 도전하고 확장하기 위해 새로운 에세이와 플랫폼이 개발되고 있습니다. 종양 내 이질성은진모(28)의현재 치료 양식에 대한 질병 진행 및 저항에 기여하는 중요한 요소입니다. 뇌종양에대한 최근 연구는 동일한 종양29,30,31, 32내의 세포 이질성을 ?...

성인에서 가장 흔한 1 차 뇌종양인 확산 저학년 진모(LGG)는 종종 대뇌 반구에서 발생하는 침을 일으키는 신생물입니다. LGGs는 종양 집단뿐만 아니라 종양 발달 및 진행1,2,3,4,5에복잡하게 관여하는 비악성 세포에 의해 구동되는 종양 내 이질성 및 종양 내 이질성을 모두 나타낸다.
지난 10 년 동안, gliomas의 필드에 수집 된 게놈 데이터의 눈사태가 있었다. 이들 데이터는 주로 벌크 종양 시퀀싱 연구에서 비롯되며 분자 특성화, 그리고 뇌종양의 현재분류5,6,7,8,9,10,11에 대단히기여하였다. 그러나, 비록 이러한 연구는 gliomas와 관련 된 광범위 한 분자 풍경을 공개, 치료 개입에 관한 진행의 실망 스러운 부족 여전히 있다. 뇌종양의 치료 내성에 대한 장애물 중 하나는 종양 내 이질성입니다. 이 문제를 해결하기 위해, 다양한 연구는 단일 세포 수준12,13,14,15,16,17에서종양 내에 존재하는 게놈, 전사, 프로테오믹및후생유전학 이질성에 초점을 맞추고 있다.
지난 몇 년 동안 단일 세포 분야에서 놀라운 기술 발전이 있었지만, 주요 제한 요인 중 하나는 세포를 분리하고 이러한 실험을 수행하는 데 필요한 신선한 표본의 가용성입니다. 이러한 한계를 극복하기 위해, 세포18,19가아닌 핵을 사용하여 냉동 조직에서 snRNA-seq 및 snATAC-seq와 같은 분석을 수행하는 데 몇 가지 성공적인 시도가 있었다. 이러한 방법의 대부분은 형광 활성화 세포 정렬 (FACS) 또는 여과 전략에 의존. 단일 세포와 단일 핵 접근법 모두 강점과 단점이 있습니다. 단세포 접근은 미토콘드리아 성적증명서를 유지하며, 이는 유익할지라도 높은 풍부성으로 인한 전사적 적용범위를 감소시킬 수 있다. 단일 핵 절연 접근법은 미토콘드리아 분획의 높은 비율을 제거하여 핵전사체(20)의보다 심층적인 커버리지를 허용한다.
RNA-seq 및 ATAC-seq를 포함하여 단세포 유전체학 데이터를 분석하기 위하여 최근에 이용된 각종 상업적으로 이용가능한 플랫폼이 있습니다. 가장 눈에 띄는 플랫폼 중 하나는 단일 세포 유전자 발현 및 단일 세포 ATAC 프로파일링을 위한 10x 유전체학 크롬 플랫폼입니다. 플랫폼이 미세 유체 챔버의 도움으로 작동함에 따라 모든 파편이나 골재는 데이터, 시약 및 귀중한 임상 샘플의 손실로 이어지는 시스템을 막을 수 있습니다. 따라서 단일 세포 연구의 성공은 주로 단일 세포/핵의 정확한 격리에 달려 있습니다.
본 명세서에서 입증할 프로토콜은 DroNc-seq 및 Omni-ATAC-seq 프로토콜의 약간 변형된 조합이며, 인간 뇌18,19,21,22,23,24에서신경 장애 및 신경 세포 유형을 이해하기 위해 snRNA-seq를 활용하는 최근 연구에 유사한접근법을활용한다. 이 프로토콜은 동결된 시료의 효소/기계적 해리의 조합을 사용하여 여과 및 그라데이션 원심분리를 거쳐 신선한 냉동 신경교종 조직에서 단일 핵을 빠르고 정확하게 분리할 수 있게 합니다. 우리는 성공적으로 뇌종양 견본에서 동일 핵 준비에서 snRNA-seq 및 snATAC-seq 데이터를 생성하기 위하여 이 프로토콜을 이용했습니다.

<table>
	<tbody>
		<tr>
			<td>2-Mercaptoethanol</td>
			<td>Sigma</td>
			<td>M6250</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Sigma</td>
			<td>21115-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Dounce Homogenizer</td>
			<td>Active motif</td>
			<td>40401</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA (0.5 M)</td>
			<td>Thermo Scientific</td>
			<td>R1021</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon 5 mL Round Bottom Polystyrene Test Tube</td>
			<td>Corning</td>
			<td>352235</td>
			<td></td>
		</tr>
		<tr>
			<td>Iodixanol (aka Optiprep)</td>
			<td>Stem cell technologies</td>
			<td>07820</td>
			<td></td>
		</tr>
		<tr>
			<td>MACs Smart Strainers (30 &#181;m)</td>
			<td>Miltenyi Biotec</td>
			<td>130-098-458</td>
			<td></td>
		</tr>
		<tr>
			<td>MACS SmartStrainers (100 &#181;m)</td>
			<td>Miltenyi Biotec</td>
			<td>130-098-463</td>
			<td></td>
		</tr>
		<tr>
			<td>Mg(Ac)2</td>
			<td>Sigma</td>
			<td>63052-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>NP-40</td>
			<td>Abcam</td>
			<td>ab142227</td>
			<td></td>
		</tr>
		<tr>
			<td>Nuclei Isolation Kit: Nuclei EZ Prep</td>
			<td>Sigma</td>
			<td>NUC101-1KT</td>
			<td></td>
		</tr>
		<tr>
			<td>Phenylmethanesulfonyl fluoride (PMSF)</td>
			<td>Sigma</td>
			<td>P7626</td>
			<td></td>
		</tr>
		<tr>
			<td>Pre-Separation Filters (20 &#181;m)</td>
			<td>Miltenyi Biotec</td>
			<td>130-101-812</td>
			<td></td>
		</tr>
		<tr>
			<td>Safe lock tubes 1.5 mL</td>
			<td>Eppendorf</td>
			<td>0030120086</td>
			<td></td>
		</tr>
		<tr>
			<td>Safe lock tubes 2.0 mL</td>
			<td>Eppendorf</td>
			<td>0030120094</td>
			<td></td>
		</tr>
		<tr>
			<td>Single Cell ATAC</td>
			<td>10x Genomics</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Single Cell Gene Expression</td>
			<td>10x Genomics</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma</td>
			<td>S0389</td>
			<td></td>
		</tr>
		<tr>
			<td>Wide Bore pipette tips (1000 &#181;L)</td>
			<td>Themo Fisher Scientific</td>
			<td>2079GPK</td>
			<td></td>
		</tr>
		<tr>
			<td>Wide Bore pipette tips (200 &#181;L)</td>
			<td>Themo Fisher Scientific</td>
			<td>2069GPK</td>
			<td></td>
		</tr>
	</tbody>
</table>

nuclei isolation from fresh frozen brain tumors for single-nucleus rna-seq and atac-seq

성인용 확산 글리오마는 종양 내 이질성을 나타낸다. 최근까지, 대규모 분자 프로파일링 노력의 대부분은 뇌종양의 분자 분류로 이어진 대량 접근에 초점을 맞추고있다. 지난 5 년 동안, 단세포 염기서열 분석 접근은 gliomas의 몇몇 중요한 특징을 강조했습니다. 이러한 연구의 대부분은 유동 세포측정또는 항체 기반 분리 방법을 사용하여 단일 세포를 격리하기 위해 신선한 뇌종양 표본을 활용하였다. 앞으로 바이오뱅크의 신선하게 냉동 된 조직 샘플을 사용하면 단일 세포 응용 분야에 더 큰 유연성을 제공 할 것입니다. 더욱이, 단세포 필드가 발전함에 따라, 다음 과제는 종양 복잡성을 더 잘 해명하기 위해 단일 세포 또는 동일한 샘플 제제로부터 다중 omics 데이터를 생성하는 것입니다. 따라서, 고처리량 시퀀싱(snATAC-seq)을 가진 트랜스포사제-접근 크롬틴을 위한 단일 핵 RNA 시퀀싱(snRNA-seq) 및 단일 핵 분석과 같은 다양한 방법에 대한 데이터 생성을 허용하는 간단하고 유연한 프로토콜이 이 분야에 중요할 것이다.
10x 유전체학 플랫폼과 같은 접근 가능한 미세 유체 계측기와 결합된 단세포 분야의 최근 발전은 실험실에서 단일 세포 응용을 용이하게 했다. 뇌종양 이질성을 연구하기 위해, 우리는 신선한 냉동 진모에서 단 하나 핵의 격리를 위한 향상된 프로토콜을 개발했습니다. 이 프로토콜은 기존 단일 셀 프로토콜을 병합하고 여과 및 버퍼 매개 그라데이션 원심분리가 뒤따르는 균질화 단계를 결합합니다. 결과 샘플은 동일한 핵 제제로부터 단일 핵 유전자 발현 및 크로마티닌 접근성 데이터를 생성하는 데 사용할 수 있는 순수한 단일 핵 현탁액이다.

단일 핵 유전체학은 제한된 데이터와 프로토콜을 가진 진화하는 분야입니다. 단일 핵 작용의 결과에 영향을 미치는 중요한 요소는 순수하고 그대로 핵의 분리입니다. 우리는 두 개의 출판 된 프로토콜을 결합 (DroNc-seq 및 Omni-ATAC-seq 프로토콜) 상대적으로 짧은 시간에 신선한 냉동 신경 교종 조직 블록에서 고품질및 순수 핵을 분리하여 성적 증명서의 안정성을 유지(도 1).
<...

Watch this Scientific Journal Video about Nuclei Isolation from Fresh Frozen Brain Tumors for Single-Nucleus RNA-seq and ATAC-seq at JoVE.com

Nuclei Isolation from Fresh Frozen Brain Tumors for Single-Nucleus RNA-seq and ATAC-seq

종양 내 이질성은 진모를 포함한 종양의 내재된 특징입니다. 우리는 단일 핵 RNA 및 ATAC 염기서열 분석 연구를 위한 신선한 동결 신경교종 조직에서 단 하나 핵을 격리하기 위하여 완충과 그라데이션 원심분리의 조합을 이용하는 간단하고 효율적인 프로토콜을 개발했습니다.

단일 핵 RNA-seq 및 ATAC-seq를 위한 신선한 냉동 뇌 종양에서 핵 절연

Adult diffuse gliomas exhibit inter- and intra-tumor heterogeneity. Until recently, the majority of large-scale molecular profiling efforts have focused ...

nuclei-isolation-from-fresh-frozen-brain-tumors-for-single-nucleus

Research

JoVE Journal

Cancer Research

11.9K 조회수.  University Hospital Heidelberg. 종양 내 이질성은 진모를 포함한 종양의 내재된 특징입니다. 우리는 단일 핵 RNA 및 ATAC 염기서열 분석 연구를 위한 신선한 동결 신경교종 조직에서 단 하나 핵을 격리하기 위하여 완충과 그라데이션 원심분리의 조합을 이용하는 간단하고 효율적인 프로토콜을 개발했습니다.

비디오: Nuclei Isolation from Fresh Frozen Brain Tumors for Single-Nucleus RNA-seq and ATAC-seq

Next Generation Sequencing for the Detection of Actionable Mutations in Solid and Liquid Tumors

As our understanding of the driver mutations necessary for initiation and progression of cancers improves, we gain critical information on how specific molecular profiles of a tumor may predict responsiveness to therapeutic agents or provide knowledge about prognosis. At our institution a tumor genotyping program was established as part of routine clinical care, screening both hematologic and solid tumors for a wide spectrum of mutations using two next-generation sequencing (NGS) panels: a custom, 33 gene hematological malignancies panel for use with peripheral blood and bone marrow, and a commercially produced solid tumor panel for use with formalin-fixed paraffin-embedded tissue that targets 47 genes commonly mutated in cancer. Our workflow includes a pathologist review of the biopsy to ensure there is adequate amount of tumor for the assay followed by customized DNA extraction is performed on the specimen. Quality control of the specimen includes steps for quantity, quality and integrity and only after the extracted DNA passes these metrics an amplicon library is generated and sequenced. The resulting data is analyzed through an in-house bioinformatics pipeline and the variants are reviewed and interpreted for pathogenicity. Here we provide a snapshot of the utility of each panel using two clinical cases to provide insight into how a well-designed NGS workflow can contribute to optimizing clinical outcomes.

This manuscript describes clinical protocols for two next-generation sequencing panels. One panel interrogates hematologic malignancies while the other panel targets genes commonly mutated in solid tumors. Molecular classification of driver mutations in human malignancies offers valuable prognostic and predictive information.

고체 및 액체 종양에서 실행 가능한 돌연변이의 검출을위한 차세대 시퀀싱

As our understanding of the driver mutations necessary for initiation and progression of cancers improves, we gain critical information on how specific ...

연구

암 연구학

Transplantation of Zebrafish Pediatric Brain Tumors into Immune-competent Hosts for Long-term Study of Tumor Cell Behavior and Drug Response

Tumor cell transplantation is an important technique to define the mechanisms controlling cancer cell growth, migration, and host response, as well as to assess potential patient response to therapy. Current methods largely depend on using syngeneic or immune-compromised animals to avoid rejection of the tumor graft. Such methods require the use of specific genetic strains that often prevent the analysis of immune-tumor cell interactions and/or are limited to specific genetic backgrounds. An alternative method in zebrafish takes advantage of an incompletely developed immune system in the embryonic brain before 3 days, where tumor cells are transplanted for use in short-term assays (i.e., 3 to 10 days). However, these methods cause host lethality, which prevents the long-term study of tumor cell behavior and drug response. This protocol describes a simple and efficient method for the long-term orthotopic transplantation of zebrafish brain tumor tissue into the fourth ventricle of a 2-day-old immune-competent zebrafish. This method allows: 1) long-term study of tumor cell behaviors, such as invasion and dissemination; 2) durable tumor response to drugs; and 3) re-transplantation of tumors for the study of tumor evolution and/or the impact of different host genetic backgrounds. In summary, this technique allows cancer researchers to assess engraftment, invasion, and growth at distant sites, as well as to perform chemical screens and cell competition assays over many months. This protocol can be extended to studies of other tumor types and can be used to elucidate mechanisms of chemoresistance and metastasis.

The transplantation of cancer cells is an important tool for the identification of cancer mechanisms and therapeutic responses. Current techniques depend on immune-incompetent animals. Here, we describe a method to transplant zebrafish tumor cells into immune-competent embryos for the long-term analysis of tumor cell behavior and in vivo drug responses.

종양 세포의 행동과 약물 반응에 대한 장기 연구를 위해 면역 능력이있는 숙주 내로 Zebrafish Pediatric Brain 종양을 이식

Tumor cell transplantation is an important technique to define the mechanisms controlling cancer cell growth, migration, and host response, as well as to ...

Development of a Human Preclinical Model of Osteoclastogenesis from Peripheral Blood Monocytes Co-cultured with Breast Cancer Cell Lines

The crosstalk between tumor cells and bone cells in the bone microenvironment is crucial to understanding the mechanism of bone metastasis formation. We developed an in vitro fully human preclinical model of a co-culture of breast cancer cells and monocytes undergoing differentiation towards osteoclasts. We optimized a model of osteoclastogenesis starting from a sample of peripheral blood collected from healthy donors. Peripheral blood mononuclear cells (PBMCs) were first separated by density gradient centrifugation, seeded at a high density and induced to differentiate by adding two growth factors (GFs): receptor activator of nuclear factor-&#954;B ligand (RANKL) and macrophage colony-stimulating factor (MCSF). The cells were left in culture for 14 days and then fixed and analyzed by downstream analysis. In osteolytic bone metastases, one of the effects of cancer cell arrival in bone is the induction of osteoclastogenesis. We thus challenged our model with co-cultures of breast cancer cells to study the differentiation power of cancer cells with respect to GFs. A straightforward way of studying cancer cell-osteoclast interaction is to perform indirect co-cultures based on the use of conditioned medium collected from breast cancer cell cultures and mixed with fresh medium. This mixture is then used to induce osteoclast differentiation. We also optimized a method of direct co-culture in which cancer cells and monocytes undergoing differentiation share the medium and exchange secreted factors. This is a significant improvement over the original indirect co-culture method as researchers can observe the reciprocal interactions of the two cell types and perform downstream analyses for both cancer cells and osteoclasts. This method enables us to study the effect of drugs on the metastatic bone microenvironment and to seed cell lines other than those derived from breast cancer. The model can also be used to study other diseases such as osteoporosis or other bone conditions.

This protocol describes development of an in vitro human preclinical model of osteoclastogenesis from peripheral blood monocytes cultured with breast cancer cell lines to mimic the cancer cell-osteoclast interaction. The model could be used to further our understanding of bone metastasis formation and improve therapeutic options.

말 초 혈액 Monocytes 유 방 암 세포 라인 공동 경작에서 Osteoclastogenesis의 인간의 전 임상 모델의 개발

The crosstalk between tumor cells and bone cells in the bone microenvironment is crucial to understanding the mechanism of bone metastasis formation. We ...

Detection of a CDH1 Rare Transcript Variant in Fresh-frozen Gastric Cancer Tissues by Chip-based Digital PCR

CDH1a, a non-canonical transcript of the CDH1 gene, has been found to be expressed in some gastric cancer (GC) cell lines, whereas it is absent in normal gastric mucosa. Recently, we detected CDH1a transcript variant in fresh-frozen tumor tissues obtained from patients with GC. The expression of this variant in tissue samples was investigated by the chip-based digital PCR (dPCR) approach presented here. dPCR offers the potential for an accurate, robust, and highly sensitive measurement of nucleic acids and is increasingly utilized for many applications in different fields. dPCR is capable of detecting rare targets; in addition, dPCR offers the possibility for absolute and precise quantification of nucleic acids without the need for calibrators and standard curves. In fact, the reaction partitioning enriches the target from the background, which improves amplification efficiency and tolerance to inhibitors. Such characteristics make dPCR an optimal tool for the detection of the CDH1a rare transcript.

Detection of a CDH1 Rare Transcript Variant in Fresh-frozen Gastric Cancer Tissues by Chip-based Digital PCR

The manuscript describes a chip-based digital PCR assay to detect a rare CDH1 transcript variant (CDH1a) in fresh-frozen normal and tumor tissues obtained from patients with gastric cancer.

칩 기반 디지털 PCR에 의해 신선한 냉동 위 암 조직에서 CDH1 드문 사본 이체의 탐지

CDH1a, a non-canonical transcript of the CDH1 gene, has been found to be expressed in some gastric cancer (GC) cell lines, whereas it is absent in normal ...

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 ...

Generation of Orthotopic Pancreatic Tumors and Ex vivo Characterization of Tumor-Infiltrating T Cell Cytotoxicity

In vivo models of pancreatic cancer provide invaluable tools for studying disease dynamics, immune infiltration and new therapeutic strategies. The orthotopic murine model can be performed on large cohorts of immunocompetent mice simultaneously, is relatively inexpensive and preserves the cognate tissue microenvironment. The quantification of T cell infiltration and cytotoxic activity within orthotopic tumors provides a useful indicator of an antitumoral response.
This protocol describes the methodology for surgical generation of orthotopic pancreatic tumors by injection of a low number of syngeneic tumor cells resuspended in 5 &#181;L basement membrane directly into the pancreas. Mice bearing orthotopic tumors take approximately 30 days to reach endpoint, at which point tumors can be harvested and processed for characterization of tumor-infiltrating T cell activity. Rapid enzymatic digestion using collagenase and DNase allows a single-cell suspension to be extracted from tumors. The viability and cell surface markers of immune cells extracted from the tumor are preserved; therefore, it is appropriate for multiple downstream applications, including flow-assisted cell sorting of immune cells for culture or RNA extraction, flow cytometry analysis of immune cell populations. Here, we describe the ex vivo stimulation of T cell populations for intracellular cytokine quantification (IFN&#947; and TNF&#945;) and degranulation activity (CD107a) as a measure of overall cytotoxicity. Whole-tumor digests were stimulated with phorbol myristate acetate and ionomycin for 5 h, in the presence of anti-CD107a antibody in order to upregulate cytokine production and degranulation. The addition of brefeldin A and monensin for the final 4 h was performed to block extracellular transport and maximize cytokine detection. Extra- and intra-cellular staining of cells was then performed for flow cytometry analysis, where the proportion of IFN&#947;+, TNF&#945;+ and CD107a+ CD4+ and CD8+ T cells was quantified.
This method provides a starting base to perform comprehensive analysis of the tumor microenvironment.

Generation of Orthotopic Pancreatic Tumors and Ex vivo Characterization of Tumor-Infiltrating T Cell Cytotoxicity

This protocol describes the surgical generation of orthotopic pancreatic tumors and the rapid digestion of freshly isolated murine pancreatic tumors. Following digestion, viable immune cell populations can be used for further downstream analysis, including ex vivo stimulation of T cells for intracellular cytokine detection by flow cytometry.

종양 침투 T 세포 독성의 정형 외 췌장 종양 및 Ex 생체 특성의 생성

In vivo models of pancreatic cancer provide invaluable tools for studying disease dynamics, immune infiltration and new therapeutic strategies. The ...

Orthotopic Transplantation of Breast Tumors as Preclinical Models for Breast Cancer

Preclinical models that faithfully recapitulate tumor heterogeneity and therapeutic response are critical for translational breast cancer research. Immortalized cell lines are easy to grow and genetically modify to study molecular mechanisms, yet the selective pressure from cell culture often leads to genetic and epigenetic alterations over time. Patient-derived xenograft (PDX) models faithfully recapitulate the heterogeneity and drug response of human breast tumors. PDX models exhibit a relatively short latency after orthotopic transplantation that facilitates the investigation of breast tumor biology and drug response. The transplantable genetically engineered mouse models allow the study of breast tumor immunity. The current protocol describes the method to orthotopically transplant breast tumor fragments into the mammary fat pad followed by drug treatments. These preclinical models provide valuable approaches to investigate breast tumor biology, drug response, biomarker discovery and mechanisms of drug resistance.

Patient-derived xenograft (PDX) models and transplantable genetically engineered mouse models faithfully recapitulate human disease and are preferred models for basic and translational breast cancer research. Here, a method is described to orthotopically transplant breast tumor fragments into the mammary fat pad to study tumor biology and evaluate drug response.

유방암을 위한 전임상 모형으로 유방 종양의 정형소 이식

Preclinical models that faithfully recapitulate tumor heterogeneity and therapeutic response are critical for translational breast cancer research. ...

A Rapid Screening Workflow to Identify Potential Combination Therapy for GBM using Patient-Derived Glioma Stem Cells

The glioma stem cells (GSCs) are a small fraction of cancer cells which play essential roles in tumor initiation, angiogenesis, and drug resistance in glioblastoma (GBM), the most prevalent and devastating primary brain tumor. The presence of GSCs makes the GBM very refractory to most of individual targeted agents, so high-throughput screening methods are required to identify potential effective combination therapeutics. The protocol describes a simple workflow to enable rapid screening for potential combination therapy with synergistic interaction. The general steps of this workflow consist of establishing luciferase-tagged GSCs, preparing matrigel coated plates, combination drug screening, analyzing, and validating the results.

환자 유래 신경종 줄기 세포를 사용하여 GBM에 대한 잠재적인 조합 요법을 식별하는 신속한 선별 워크플로우

The glioma stem cells (GSCs) are a small fraction of cancer cells which play essential roles in tumor initiation, angiogenesis, and drug resistance in ...

ATAC-Seq Optimization for Cancer Epigenetics Research

The assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) probes deoxyribonucleic acid (DNA) accessibility using the hyperactive Tn5 transposase. Tn5 cuts and ligates adapters for high-throughput sequencing within accessible chromatin regions. In eukaryotic cells, genomic DNA is packaged into chromatin, a complex of DNA, histones, and other proteins, which acts as a physical barrier to the transcriptional machinery. In response to extrinsic signals, transcription factors recruit chromatin remodeling complexes to enable access to the transcriptional machinery for gene activation. Therefore, identifying open chromatin regions is useful when monitoring enhancer and gene promoter activities during biological events such as cancer progression. Since this protocol is easy to use and has a low cell input requirement, ATAC-seq has been widely adopted to define open chromatin regions in various cell types, including cancer cells. For successful data acquisition, several parameters need to be considered when preparing ATAC-seq libraries. Among them, the choice of cell lysis buffer, the titration of the Tn5 enzyme, and the starting volume of cells are crucial for ATAC-seq library preparation in cancer cells. Optimization is essential for generating high-quality data. Here, we provide a detailed description of the ATAC-seq optimization methods for epithelial cell types.

ATAC-seq is a DNA sequencing method that uses the hyperactive mutant transposase, Tn5, to map changes in chromatin accessibility mediated by transcription factors. ATAC-seq enables the discovery of the molecular mechanisms underlying phenotypic alterations in cancer cells. This protocol outlines optimization procedures for ATAC-seq in epithelial cell types, including cancer cells.

암 후성유전학 연구를 위한 ATAC-Seq 최적화

The assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) probes deoxyribonucleic acid (DNA) accessibility using the ...

췌장암 유래 종양 오가노이드 및 신선한 조직의 섬유아세포

Establishment of Pancreatic Cancer-Derived Tumor Organoids and Fibroblasts From Fresh Tissue

Tumor organoids are three-dimensional (3D) ex vivo tumor models that recapitulate the biological key features of the original primary tumor tissues. Patient-derived tumor organoids have been used in translational cancer research and can be applied to assess treatment sensitivity and resistance, cell-cell interactions, and tumor cell interactions with the tumor microenvironment. Tumor organoids are complex culture systems that require advanced cell culture techniques and culture media with specific growth factor cocktails and a biological basement membrane that mimics the extracellular environment. The ability to establish primary tumor cultures highly depends on the tissue of origin, the cellularity, and the clinical features of the tumor, such as the tumor grade. Furthermore, tissue sample collection, material quality and quantity, as well as correct biobanking and storage are crucial elements of this procedure. The technical capabilities of the laboratory are also crucial factors to consider. Here, we report a validated SOP/protocol that is technically and economically feasible for the culture of ex vivo tumor organoids from fresh tissue samples of pancreatic adenocarcinoma origin, either from fresh primary resected patient donor tissue or patient-derived xenografts (PDX). The technique described herein can be performed in laboratories with basic tissue culture and mouse facilities and is tailored for wide application in the translational oncology field.

저자 스포트라이트: Establishment of Pancreatic Cancer-Derived Tumor Organoids and Fibroblasts from fresh tissue  

Tumor organoids have revolutionized cancer research and the approach to personalized medicine. They represent a clinically relevant tumor model that allows researchers to stay one step ahead of the tumor in the clinic. This protocol establishes tumor organoids from fresh pancreatic tumor tissue samples and patient-derived xenografts of pancreatic adenocarcinoma origin.

췌장암 유래 종양 오가노이드 및 섬유아세포를 신선한 조직에서 확립

Tumor organoids are three-dimensional (3D) ex vivo tumor models that recapitulate the biological key features of the original primary tumor tissues. ...