타다 부여 재단 자금이 작품.

1. 셀 확장 특성
<ol><li>SW-13, SW-신, 그리고 남서 DKK3 세포는 표준 습도 인큐베이터 37.0 ° c, 5% CO2 Dulbecco의 수정이 글 중간에 보충 10% 태아 둔감 한 혈 청 및 10000 U/mL 페니실린과 스 ('성장 매체'로 지정)에 유지 합니다.<ol>
<li>Hemocytometer를 사용 하 여 셀을 계산 하 고 별도 6 잘 플레이트에 SW, SW-신, 그리고 남서 DKK3 라인에 대 한 잘 당 5000 셀 접시 하룻밤 불 임 유리 coverslips에 성장 매체에 성장.</li>
	</ol>
</li>
	<li>각 잘 하룻밤 성장 성장 매체 발음, 후 따뜻한 인산 염 버퍼 식 염 수 (PBS)의 1 mL로 세포 세척 하 고 10 분 동안 3.7% 포름알데히드의 1 mL와 함께 셀을 수정.</li>
	<li>30 분 세척 초과 대 한 0.05% 크리스탈 바이올렛의 1 mL와 함께 각 잘에서 aspirate 포름알데히드와 얼룩 셀 멀리 1 mL 이온된 물 3 세척을 사용 하 여 염료. 참고: 염료의 통풍 관의 수준에 따라 몇 가지 추가 세척 배경 셀 시각화 홍보 얼룩을 제거 하려면 필요할 수 있습니다.<ol>
<li>잘 밀고 집게를 사용 하 여 coverslips를 검색 하 고 그들에 게 아래로 얼굴을 배치 분명 장착 시 약의 방울으로 레이블이 지정 된 유리 슬라이드에.</li>
	</ol>
</li>
	<li>가벼운 현미경 임의로 셀 확장 분석 결과 대 한 각 coverslip에서 겹치지 않는 셀의 20 조회를 선택 합니다.<ol>
<li>보기의 각 필드의 400 배 확대에서 photomicrographs를 받아. 직접 각 20 대표 셀의 확장의 각 종류의 수를 계산. 참고: 이와 같이, 20 고립된 세포 시험 되어야 한다 신뢰할 수 있는 결과 보장 하기 위해 각 시험된 조건에 대 한. Filopodia 5 미크론 이하의 기지와 단일 정점 세포 확장에 의해 정의 되었다. Lobopodia는 여러 개의 apices로 길이 포함 된 확장의 5 ~ 20 미크론의 기지와 세포 확장에 의해 정의 되었다. Lamellipodia는 길이 및 또는 없이 apices 20 미크론 보다 큰 셀 확장에 의해 정의 되었다. 테스트 셀 유형에 따라 셀 확장의 크기는 다를 수 있습니다와 조정 매개 변수를 식별 해야 할 수 있습니다.</li>
	</ol>
</li>
	<li>검사 6 우물에 걸쳐 각 셀 형식 (즉, 소프트웨어-13, 남서-네오, 남서 DDK)에서 각 셀 확장의 평균 수를 확인 합니다.</li>
	<li>단방향 분산 분석 (ANOVA) 통계 테스트를 사용 하 여 다른 테스트 세포 유형 사이 세포 막 확장의 평균 비율에 차이 비교 합니다.</li>
</ol>2. 접착 시험
<ol><li>SW-13, SW-신, 그리고 성장 매체에서 37.0 ° C, 5% CO2 표준 습도 인큐베이터에서 남서 DKK3 셀 유지 합니다.<ol>
<li>hemocytometer를 사용 하 여 계산 별도 6 잘 플레이트에 잘 당 SW, SW-신, 그리고 소프트웨어-DKK3의 100000 세포를 플레이트와 하룻밤 성장 매체에 성장.</li>
		<li>아래 테스트 6 시간 포인트의 각 한 격판덮개를 사용 하 여 각 세포 유형에 씨앗 6 잘 플레이트 테스트. 참고: 시간 포인트는 다른 세포 유형에 대 한 달라질 수 있습니다.</li>
	</ol>
</li>
	<li>야간 보육, 따뜻한 PBS 가진 셀을 한 번 씻어 후 각 우물에 비 효소 세포 분리 솔루션 x 1의 0.5 mL를 추가 합니다.<ol>
<li>세포 분리 솔루션 확산 판을 소용돌이 친다. 정의 된 시간 점 (즉, 1, 2, 3, 5, 10 및 15 분)에 지정 된 접시를 발음 하 고 1 mL 따뜻한 PBS 솔루션 세 번 씻어.</li>
		<li>세척, 사이 부드럽게 느슨하게 연결 된 셀을 분리할 접시를 누릅니다.</li>
	</ol>
</li>
	<li>모든 남아 있는 PBS와 3.7% 포름알데히드 10 분의 1 mL와 함께 접시에 남은 셀 첨부 수정 발음.</li>
	<li>30 분에 대 한 0.05% 크리스탈 바이올렛의 1 mL로 세포를 얼룩이 다음 과잉 염료 이온된 물 1 mL로 멀리 세척. 셀 시각화를 촉진 하는 세척 적정 참고: 염료의 통풍 관의 수준에 따라 몇 가지 추가 세척 셀 시각화를 홍보 하기 위해 필요할 수 있습니다.</li>
	<li>100 x 확대 가벼운 현미경을 사용 하 여, 각 잘 각 접시에 나머지 연결 된 셀을 계산 합니다. 참고: 투명 눈금자 또는 좋은 펜 접시의 하단 측면에 가이드 표시의 사용을 동일한 세포의 반복 계산을 피하기 위해 도움이 됩니다.</li>
	<li>각 접시에 잘 당 연결 된 셀의 평균 수를 확인 합니다.</li>
	<li>양방향 ANOVA 통계 테스트를 사용 하 여 주어진된 시간 동안 SW, SW-신, 그리고 남서 DDK3 셀 사이의 셀 분리의 속도 비교 합니다.</li>
</ol>

<ol>
	<li>Libe, R., Fratticci, A., Bertherat, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adrenocortical+cancer:+pathophysiology+and+clinical+management.">Adrenocortical cancer: pathophysiology and clinical management.</a> Endocrine-related cancer. 14 (1), 13-28 (2007).</li><li>Veeck, J., Dahl, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+the+Wnt+pathway+in+cancer:+the+emerging+role+of+Dickkopf-3.">Targeting the Wnt pathway in cancer: the emerging role of Dickkopf-3.</a> Biochimica et biophysica acta. 1825 (1), 18-28 (2012).</li><li>Friedl, P., Gilmour, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collective+cell+migration+in+morphogenesis,+regeneration+and+cancer.">Collective cell migration in morphogenesis, regeneration and cancer.</a> Nat Rev Mol Cell Biol. 10 (7), 445-457 (2009).</li><li>Jacquemet, 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=L-type+calcium+channels+regulate+filopodia+stability+and+cancer+cell+invasion+downstream+of+integrin+signalling.">L-type calcium channels regulate filopodia stability and cancer cell invasion downstream of integrin signalling.</a> Nat Commun. 7, 13297 (2016).</li><li>Machesky, L. M., Li, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fascin:+Invasive+filopodia+promoting+metastasis.">Fascin: Invasive filopodia promoting metastasis.</a> Commun Integr Biol. 3 (3), 263-270 (2010).</li><li>Petrie, R. J., Harlin, H. M., Korsak, L. I., Yamada, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activating+the+nuclear+piston+mechanism+of+3D+migration+in+tumor+cells.">Activating the nuclear piston mechanism of 3D migration in tumor cells.</a> J Cell Biol. 216 (1), 93-100 (2017).</li><li>Korah, 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=Epigenetic+silencing+of+RASSF1A+deregulates+cytoskeleton+and+promotes+malignant+behavior+of+adrenocortical+carcinoma.">Epigenetic silencing of RASSF1A deregulates cytoskeleton and promotes malignant behavior of adrenocortical carcinoma.</a> Molecular cancer. 12, 87 (2013).</li><li>Cheng, J. 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=A+novel+FOXO1-mediated+dedifferentiation+blocking+role+for+DKK3+in+adrenocortical+carcinogenesis.">A novel FOXO1-mediated dedifferentiation blocking role for DKK3 in adrenocortical carcinogenesis.</a> BMC cancer. 17 (1), 164 (2017).</li><li>Barry, D. J., Durkin, C. H., Abella, J. V., Way, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Open+source+software+for+quantification+of+cell+migration,+protrusions,+and+fluorescence+intensities.">Open source software for quantification of cell migration, protrusions, and fluorescence intensities.</a> J Cell Biol. 209 (1), 163-180 (2015).</li><li>Cliffe, A., 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=Quantitative+3D+analysis+of+complex+single+border+cell+behaviors+in+coordinated+collective+cell+migration.">Quantitative 3D analysis of complex single border cell behaviors in coordinated collective cell migration.</a> Nat Commun. 8, 14905 (2017).</li></ol>

저자는 공개 없다.

여기 우리는 생체 외에서 양적 방법 용이성, 몇 구 덩이-폭포, 그리고 다양 한 테스트 조건에 대 한 적용할 수 있는 안정적인 재현성 셀 확장의 특성을 설명 합니다. 또한, 간단 하 고, 정량 접착 및 운동 성 분석 실험 세포 막 확장에 관찰된 변경의 잠재적인 기능적 중요성을 상관 관계를 동시에 수행할 수 있습니다.
그러나, 이러한 방법을 몇 가지 잠재적인 제한이 발생?...

Dysregulated WNT 신호 adrenocortical 악성1에 중요 한 역할을 한다. 이 연구에 사용 하는 방법 조사 여부 DKK3의 입을, WNT 신호의 부정적인 레 귤 레이 터 부 신 피 질에서 dedifferentiation 이벤트 나타내고 셀 확장 레 퍼 토리 변화의 맥락에서 종양의 형성을 촉진 한다. DKK3 38 kDa 분 비는 N 맨끝 신호 펩 티 드와 당단백질 이며 그 적용된 식 세포 주기 검거 귀착되 공격적인 악성 행동을 저해 및 엽 상피 반전 이전 학문은 설명 했다 전환2.
Adrenocortical 암 (ACC) 및 다른 암 악성 동작, 부분에서 주변 표면으로 인터페이스 하는 종양 세포의 기능에 의해 영향을 포함 하 여 기질에 종양 세포 내 습을 촉진 하 고 마이그레이션3. 암 진행에 특정 세포 막 확장의 역할은 filopodia 형성을 통해 주로 다양 한 문맥에서 점점 시연 되 고. 예를 들어 L-타입 칼슘 채널의 overexpression filopodia 형성을 유도 하 여 종양 세포 침공4홍보 발견 되었습니다. 마찬가지로, Fascin, 최소한 단백질 바인딩 하는 걸 정상 조직에서 표현 이기도 overexpressed filopodia 형성5와 협회에서 암 세포에 합니다. 그러나 Lobopodia 대형 엑스트라 세포 매트릭스를 통해 효과적으로 마이그레이션할 비 악성 섬유 아 세포,, 그것은 보였다 fibrosarcoma 셀 셀 마이그레이션을 촉진 하기 위하여 lobopodia 대신 metalloproteinase 활동에 의존 하 고 침략6. 우리는 종양으로 나타났습니다 진압, Ras 연결 도메인 가족 1 isoform (RASSF1A)를 포함 하 여 및 DKK3, 함수 cytoskeletal 요소를 변경 하 고 lamellipodia 형성 증진과 침략 속성7,8방해 구를 두 다 수 있습니다.
이와 같이, 그것은 발암 및 세포 막 확장 변경, 특히 filopodia, lobopodia, 및 lamellipodia 테스트 조건 하에서 형성 평가의 관계에 관련 된 유전자의 영향을 특성에 중요 한. 현재 상태-최첨단 기술을 데이터 수집 및 해석에 대 한 점점 더 정교한 현미경 방법, 형광 라벨, 복잡 한 컴퓨터 알고리즘의 사용을 포함 합니다. 이러한 방법은 새롭고 강력한 분석 도구를 제공 하는 동안 그들의 광범위 한 사용과 세포 생물학 실험에 그들의 복잡도 제한 합니다. 또한, 정확한 정량화 및 셀 확장 형태에 변화의 관찰 아니다 일반적으로 측정된9,10. 반면, 여기 셀 확장 변경 표준 현미경 기술과 쉽게 적응할 수 있는 생체 외에서 메서드를 사용 하 여 정확 하 게 단정 하는 기술을 소개 합니다. 이러한 메서드는 또한 동시에 분석 하는 각 셀에 대 한 각 셀 확장 종류 계량 하 고 세포 막 확장 레 퍼 토리에 전반적인 변화를 결정 합니다. 우리는 또한 어떻게 이러한 변화 세포 접착 속성에 관련 된 수 보여줍니다.
실험 예를 들어, 우리는 소프트웨어-13, SW-DDK3, pCMV6-항목/DKK3 플라스 미드 벡터와 안정적으로 페 되었습니다 하 고 constitutively DKK3, 아드레날린 선에 차별화 요소를 overexpresses 지정의 이전에 만든된 셀 라인을 사용 합니다. 비 페 SW-13 셀과 남서-13 셀 안정적으로 빈 벡터 (pCMV6-항목), 소프트웨어-네오, 지정 된 페 실험 컨트롤 될 것입니다.

<table width="653">
	<tbody>
		<tr height="63">
			<td height="63" style="width:216px;height:63px;">Dulbecco&#39;s Modified Eagle Medium (DMEM)</td>
			<td style="width:115px;">ThermoFisher</td>
			<td style="width:119px;">11965-092</td>
			<td style="width:203px;">Supplemented with fetal bovine serum and penicllin and streptomycin</td>
		</tr>
		<tr height="42">
			<td height="42" style="width:216px;height:42px;">Deulbeco&#39;s Phosphate Buffered Saline</td>
			<td style="width:115px;">Sigma-Aldrich</td>
			<td style="width:119px;">D8537</td>
			<td>1X solution, used directly</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:216px;height:21px;">3.7% formaldehyde&#160;</td>
			<td style="width:115px;">Sigma-Aldrich</td>
			<td style="width:119px;">F8775</td>
			<td>Dilute stock solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:216px;height:21px;">0.05% crystal violet&#160;</td>
			<td style="width:115px;">Harleco</td>
			<td style="width:119px;">192-12</td>
			<td>Dilute stock solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:216px;height:21px;">De-ionized water</td>
			<td style="width:115px;">NA</td>
			<td style="width:119px;">NA</td>
			<td>NA</td>
		</tr>
		<tr height="42">
			<td height="42" style="width:216px;height:42px;">Microscope with 400X magnification&#160;</td>
			<td style="width:115px;">NA</td>
			<td style="width:119px;">NA</td>
			<td>NA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">6-well plates</td>
			<td>Corning</td>
			<td>353046</td>
			<td>NA</td>
		</tr>
		<tr height="43">
			<td height="43" style="width:216px;height:43px;">Cell dissociation solution non-enzymatic 1X</td>
			<td style="width:115px;">Sigma-Aldrich</td>
			<td style="width:119px;">C5914&#160;</td>
			<td>1X solution, used directly</td>
		</tr>
	</tbody>
</table>

characterization of cell membrane extensions and studying their roles in cancer cell adhesion dynamics

세포 막의 확장 레 퍼 토리 그들의 접착제와 철새 잠재력을 포함 하 여 암 세포의 다양 한 악성 동작을 조절 한다. 정확 하 게 분류 셀 확장을 계량 하 고 세포의 접착 능력에 미치는 영향을 측정 하는 능력은 어떻게 셀 신호 이벤트 영향 암 세포 행동 및 공격 성을 결정 하는 중요 한. 여기, 우리가 시험관에서 설계 및 방법의 사용은 셀 확장 정량화 함께에서 설립 체 외에 모델에 접착 용량 분석 결과와 adrenocortical 암 (ACC)에 대 한 설명. 특히, 우리는 효과 DKK3의 putative 종양 억제기와 프로 차별화 요소, ACC 셀 라인, 소프트웨어-13의 막 확장 표현 형에 테스트합니다. 비교적 간단 하 고 신뢰할 수 있는, 제공 하기 위해 이러한 분석을 제안 하 고 쉽게 해석할 통계를 다양 한 실험 조건에서 이러한 특성을 측정 합니다.

위의 분석 실험을 사용 하 여 셀 확장 형태학 및 세포 접착 속성에 DKK3 overexpression의 효과 설립된 ACC 셀 라인 SW-13, 체 외에서테스트 되었습니다. 셀 DKK3 overexpressing는 안정적으로 남서 13 셀 Myc DDK 태그 pCMV6-항목/DKK3 플라스 미드 벡터 및 소프트웨어 DKK3로 지정 된 transfecting에 의해 생성 되었다. 마찬가지로, 안정적으로 빈 벡터 pCMV6 항목과 transfected 세포 transfection 고 뿌?...

Watch this Scientific Journal Video about Characterization of Cell Membrane Extensions and Studying Their Roles in Cancer Cell Adhesion Dynamics at JoVE.com

Characterization of Cell Membrane Extensions and Studying Their Roles in Cancer Cell Adhesion Dynamics

이 연구는 유틸리티 양적 세포 막 확장 측정 및 셀의 접착 능력의 상관 관계의 용이성을 보여 줍니다. 대표적인 예로 우리 보여 여기 그 Dickkopf 관련 단백질 3 (DKK3) 증가 lobopodia 형성을 촉진 하 고 adrenocortical 암 접착 력 셀 세포 생체 외에서.

세포 막 확장 하 고 공부 하는 암 세포 접착 역동성에 있는 그들의 역할의 특성

The cell membrane&#39;s extension repertoire modulates various malignant behaviors of cancer cells, including their adhesive and migratory potentials. The ...

characterization-cell-membrane-extensions-studying-their-roles-cancer

Research

JoVE Journal

Cancer Research

7.4K 조회수.  Yale University School of Medicine. 이 연구는 유틸리티 양적 세포 막 확장 측정 및 셀의 접착 능력의 상관 관계의 용이성을 보여 줍니다. 대표적인 예로 우리 보여 여기 그 Dickkopf 관련 단백질 3 (DKK3) 증가 lobopodia 형성을 촉진 하 고 adrenocortical 암 접착 력 셀 세포 생체 외에서.

비디오: Characterization of Cell Membrane Extensions and Studying Their Roles in Cancer Cell Adhesion Dynamics

A Protocol for Rapid Post-mortem Cell Culture of Diffuse Intrinsic Pontine Glioma (DIPG)

Diffuse Intrinsic Pontine Glioma (DIPG) is a childhood brainstem tumor that carries a universally fatal prognosis. Because surgical resection is not a viable treatment strategy and biopsy is not routinely performed, the availability of patient samples for research is limited. Consequently, efforts to study this disease have been challenged by a paucity of faithful disease models. To address this need, we describe here a protocol for the rapid processing of post-mortem autopsy tissue samples in order to generate durable patient-derived cell culture models that can be used in in vitro assays or in vivo orthotopic xenograft experiments. These models can be used to screen for potential drug targets and to study fundamental pathobiological processes within DIPG. This protocol can further be extended to analyze and isolate tumor and microenvironmental cells using Fluorescence-activated Cell Sorting (FACS), which enables subsequent analysis of gene expression, protein expression, or epigenetic modifications of DNA at the bulk cell or single cell level. Finally, this protocol can also be adapted to generate patient-derived cultures for other central nervous system tumors.

A Protocol for Rapid Post-mortem Cell Culture of Diffuse Intrinsic Pontine Glioma DIPG

This protocol describes a method for the rapid processing of post-mortem diffuse intrinsic pontine glioma samples for the establishment of patient-derived cell culture models or direct characterization of tumor and microenvironmental cells.

확산 내장 뇌교 신경 교종의 신속한 사후 세포 배양을위한 프로토콜 (DIPG)

Diffuse Intrinsic Pontine Glioma (DIPG) is a childhood brainstem tumor that carries a universally fatal prognosis. Because surgical resection is not a ...

연구

암 연구학

Detection of Mitochondria Membrane Potential to Study CLIC4 Knockdown-induced HN4 Cell Apoptosis In Vitro

Depletion of the mitochondrial membrane potential (MMP, &#916;&#936;m) is considered the earliest event in the apoptotic cascade. It even occurs ahead of nuclear apoptotic characteristics, including chromatin condensation and DNA breakage. Once the MMP collapses, cell apoptosis will initiate irreversibly. A series of lipophilic cationic dyes can pass through the cell membrane and aggregate inside the matrix of mitochondrion, and serve as fluorescence marker to evaluate MMP change. As one of the six members of the Cl- intracellular channel (CLIC) family, CLIC4 participates in the cell apoptotic process mainly through the mitochondrial pathway. Here we describe a detailed protocol to measure MMP via monitoring the fluorescence fluctuation of Rhodamine 123 (Rh123), through which we study apoptosis induced by CLIC4 knockdown. We discuss the advantages and limitations of the application of confocal laser scanning and normal fluorescence microscope in detail, and also compare it with other methods.

Detection of Mitochondria Membrane Potential to Study CLIC4 Knockdown-induced HN4 Cell Apoptosis In Vitro

Here we present a detailed protocol for the application of rhodamine 123 to identify the mitochondrial membrane potential (MMP) and study CLIC4 knockdown-induced HN4 cell apoptosis in vitro. Under common fluorescence microscope and confocal laser scanning fluorescence microscope, the real-time change of the MMP was recorded.

CLIC4 최저 유도 HN4 세포 Apoptosis에서 체 외에 미토 콘 드리 아 막 잠재력의 발견

Depletion of the mitochondrial membrane potential (MMP, &#916;&#936;m) is considered the earliest event in the apoptotic cascade. It even occurs ahead of ...

Target Cell Pre-enrichment and Whole Genome Amplification for Single Cell Downstream Characterization

Rare target cells can be isolated from a high background of non-target cells using antibodies specific for surface proteins of target cells. A recently developed method uses a medical wire functionalized with anti-epithelial cell adhesion molecule (EpCAM) antibodies for in vivo isolation of circulating tumor cells (CTCs)1. A patient-matched cohort in non-metastatic prostate cancer showed that the in vivo isolation technique resulted in a higher percentage of patients positive for CTCs as well as higher CTC counts as compared to the current gold standard in CTC enumeration. As cells cannot be recovered from current medical devices, a new functionalized wire (referred to as Device) was manufactured allowing capture and subsequent detachment of cells by enzymatic treatment. Cells are allowed to attach to the Device, visualized on a microscope and detached using enzymatic treatment. Recovered cells are cytocentrifuged onto membrane-coated slides and harvested individually by means of laser microdissection or micromanipulation. Single-cell samples are then subjected to single-cell whole genome amplification allowing multiple downstream analysis including screening and target-specific approaches. The procedure of isolation and recovery yields high quality DNA from single cells and does not impair subsequent whole genome amplification (WGA). A single cell&#39;s amplified DNA can be forwarded to screening and/or targeted analysis such as array comparative genome hybridization (array-CGH) or sequencing. The device allows ex vivo isolation from artificial rare cell samples (i.e. 500 target cells spiked into 5 mL of peripheral blood). Whereas detachment rates of cells are acceptable (50 - 90%), the recovery rate of detached cells onto slides spans a wide range dependent on the cell line used (&#60;10 - &#62;50%) and needs some further attention. This device is not cleared for the use in patients.

This protocol is to recover and prepare rare target cells from a mixture with non-target background cells for molecular genetic characterization at the single-cell level. DNA quality is equal to non-treated single cells and allows for single-cell application (both screening based and targeted analysis).

단일 셀 다운스트림 특성화에 대 한 대상 셀 사전 농축 및 전체 게놈 증폭

Rare target cells can be isolated from a high background of non-target cells using antibodies specific for surface proteins of target cells. A recently ...

3-D Cell Culture System for Studying Invasion and Evaluating Therapeutics in Bladder Cancer

Bladder cancer is a significant health problem. It is estimated that more than 16,000 people will die this year in the United States from bladder cancer. While 75% of bladder cancers are non-invasive and unlikely to metastasize, about 25% progress to an invasive growth pattern. Up to half of the patients with invasive cancers will develop lethal metastatic relapse. Thus, understanding the mechanism of invasive progression in bladder cancer is crucial to predict patient outcomes and prevent lethal metastases. In this article, we present a three-dimensional cancer invasion model which allows incorporation of tumor cells and stromal components to mimic in vivo conditions occurring in the bladder tumor microenvironment. This model provides the opportunity to observe the invasive process in real time using time-lapse imaging, interrogate the molecular pathways involved using confocal immunofluorescent imaging and screen compounds with the potential to block invasion. While this protocol focuses on bladder cancer, it is likely that similar methods could be used to examine invasion and motility in other tumor types as well.

The processes governing bladder cancer invasion represent opportunities for biomarker and therapeutic development. Here we present a bladder cancer invasion model which incorporates 3-D culture of tumor spheroids, time-lapse imaging and confocal microscopy. This technique is useful for defining the features of the invasive process and for screening therapeutic agents.

침공을 공부 하 고 방광 암에서 치료제를 평가 하기 위한 3 차원 셀 문화 시스템

Bladder cancer is a significant health problem. It is estimated that more than 16,000 people will die this year in the United States from bladder cancer. ...

Mesenchymal Stem Cell Isolation from Pulp Tissue and Co-Culture with Cancer Cells to Study Their Interactions

Cancer as a multistep process and complicated disease is not only regulated by individual cell proliferation and growth but also controlled by tumor environment and cell-cell interactions. Identification of cancer and stem cell interactions, including changes in extracellular environment, physical interactions, and secreted factors, might enable the discovery of new therapy options. We combine known co-culture techniques to create a model system for mesenchymal stem cells (MSCs) and cancer cell interactions. In the current study, dental pulp stem cells (DPSCs) and PC-3 prostate cancer cell interactions were examined by direct and indirect co-culture techniques. Condition medium (CM) obtained from DPSCs and 0.4 &#181;m pore sized trans-well membranes were used to study paracrine activity. Co-culture of different cell types together was performed to study direct cell-cell interaction. The results revealed that CM increased cell proliferation and decreased apoptosis in prostate cancer cell cultures. Both CM and trans-well system increased cell migration capacity of PC-3 cells. Cells stained with different membrane dyes were seeded into the same culture vessels, and DPSCs participated in a self-organized structure with PC-3 cells under this direct co-culture condition. Overall, the results indicated that co-culture techniques could be useful for cancer and MSC interactions as a model system.

We provide protocols for evaluation of mesenchymal stem cells isolated from dental pulp and prostate cancer cell interactions based on direct and indirect co-culture methods. Condition medium and trans-well membranes are suitable to analyze indirect paracrine activity. Seeding differentially stained cells together is an appropriate model for direct cell-cell interaction.

펄프 조직 문화와 공동 암 세포와 그들의 상호 작용을 공부 하에서 중간 엽 줄기 세포 분리

Cancer as a multistep process and complicated disease is not only regulated by individual cell proliferation and growth but also controlled by tumor ...

Establishment of Gastric Cancer Patient-derived Xenograft Models and Primary Cell Lines

The use of preclinical models to advance our understanding of tumor biology and investigate the efficacy of therapeutic agents is key to cancer research. Although there are many established gastric cancer cell lines and many conventional transgenic mouse models for preclinical research, the disadvantages of these in vitro and in vivo models limit their applications. Because the characteristics of these models have changed in culture, they no longer model tumor heterogeneity, and their responses have not been able to predict responses in humans. Thus, alternative models that better represent tumor heterogeneity are being developed. Patient-derived xenograft (PDX) models preserve the histologic appearance of cancer cells, retain intratumoral heterogeneity, and better reflect the relevant human components of the tumor microenvironment. However, it usually takes 4-8 months to develop a PDX model, which is longer than the expected survival of many gastric patients. For this reason, establishing primary cancer cell lines may be an effective complementary method for drug response studies. The current protocol describes methods to establish PDX models and primary cancer cell lines from surgical gastric cancer samples. These methods provide a useful tool for drug development and cancer biology research.

The current protocol describes methods to establish patient-derived xenograft (PDX) models and primary cancer cell lines from surgical gastric cancer samples. The methods provide a useful tool for drug development and cancer biology research.

위암 환자 유래 이종이식 모델 및 1차 세포주 구축

The use of preclinical models to advance our understanding of tumor biology and investigate the efficacy of therapeutic agents is key to cancer research. ...

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 ...

Radiosensitivity of Cancer Stem Cells in Lung Cancer Cell Lines

The presence of cancer stem cells (CSCs) has been associated with relapse or poor outcomes after radiotherapy. Studying radioresistant CSCs may provide clues to overcoming radioresistance. Voltage-gated calcium channel &#945;2&#948;1 subunit isoform 5 has been reported as a marker for radioresistant CSCs in non-small cell lung cancer (NSCLC) cell lines. Using calcium channel &#945;2&#948;1 subunit as an example of a CSC marker, methods to study the radiosensitivity of CSCs in NSCLC cell lines are presented. CSCs are sorted with putative markers by flow cytometry, and the self-renewal capacity of sorted cells is evaluated by sphere formation assay. Colony formation assay, which determines how many cells lose the ability to generate descendants forming the colony after a certain dose of radiation, is then performed to assess the radiosensitivity of sorted cells. This manuscript provides initial steps for studying the radiosensitivity of CSCs, which establishes the basis for further understanding of the underlying mechanisms.

The presence of cancer stem cells have been associated with relapse or poor outcomes after radiotherapy. This manuscript describes the methods to study the radiosensitivity of cancer stem cells in lung cancer cell lines.

폐암 세포줄에 있는 암 줄기 세포의 방과성

The presence of cancer stem cells (CSCs) has been associated with relapse or poor outcomes after radiotherapy. Studying radioresistant CSCs may provide ...

Comparing Metastatic Clear Cell Renal Cell Carcinoma Model Established in Mouse Kidney and on Chicken Chorioallantoic Membrane

Metastatic clear cell renal cell carcinoma (ccRCC) is the most common subtype of kidney cancer. Localized ccRCC has a favorable surgical outcome. However, one third of ccRCC patients will develop metastases to the lung, which is related to a very poor outcome for patients. Unfortunately, no therapy is available for this deadly stage, because the molecular mechanism of metastasis remains unknown. It has been known for 25 years that the loss of function of the von Hippel-Lindau (VHL) tumor suppressor gene is pathognomonic of ccRCC. However, no clinically relevant transgenic mouse model of ccRCC has been generated. The purpose of this protocol is to introduce and compare two newly established animal models for metastatic ccRCC. The first is renal implantation in the mouse model. In our laboratory, the CRISPR gene editing system was utilized to knock out the VHL gene in several RCC cell lines. Orthotopic implantation of heterogeneous ccRCC populations to the renal capsule created novel ccRCC models that develop robust lung metastases in immunocompetent mice. The second model is the chicken chorioallantoic membrane (CAM) system. In comparison to the mouse model, this model is more time, labor, and cost-efficient. This model also supported robust tumor formation and intravasation. Due to the short 10 day period of tumor growth in CAM, no overt metastasis was observed by immunohistochemistry (IHC) in the collected embryo tissues. However, when tumor growth was extended by two weeks in the hatched chicken, micrometastatic ccRCC lesions were observed by IHC in the lungs. These two novel preclinical models will be useful to further study the molecular mechanism behind metastasis, as well as to establish new, patient-derived xenografts (PDXs) toward the development of novel treatments for metastatic ccRCC.

Metastatic clear cell renal cell carcinoma is a disease without a comprehensive animal model for thorough preclinical investigation. This protocol illustrates two novel animal models for the disease: the orthotopically implanted mouse model and the chicken chorioallantoic membrane model, both of which demonstrate lung metastasis resembling clinical cases.

마우스 신장과 닭 융모알란투암 막에 설립된 전이성 맑은 세포 신장 세포 암종 모델 비교

Metastatic clear cell renal cell carcinoma (ccRCC) is the most common subtype of kidney cancer. Localized ccRCC has a favorable surgical outcome. However, ...

Quantification of Tumor Cell Adhesion in Lymph Node Cryosections

Tumor-draining lymph nodes (LNs) are not merely filters of tumor-produced waste. They are one of the most common regional sites of provisional residence of disseminated tumor cells in patients with different types of cancer. The detection of these LN-residing tumor cells is an important biomarker associated with poor prognosis and adjuvant therapy decisions. Recent mouse models have indicated that LN-residing tumor cells could be a substantial source of malignant cells for distant metastases. The ability to quantify the adhesivity of tumor cells to LN parenchyma is a critical gauge in experimental research that focuses on the identification of genes or signaling pathways relevant for lymphatic/metastatic dissemination. Because LNs are complex 3D structures with a variety of appearances and compositions in tissue sections depending on the plane of section, their matrices are difficult to replicate experimentally in vitro in a fully controlled way. Here, we describe a simple and inexpensive method that allows the quantification of adhesive tumor cells to LN cryosections. Using serial sections of the same LN, we adapt the classic method developed by Brodt to use nonradioactive labels and directly count the number of adhering tumor cells per LN surface area. LN-adherent tumor cells are readily identified by light microscopy and confirmed by a fluorescence-based method, giving an adhesion index that reveals the cell-binding affinity to LN parenchyma, which is suggestive evidence of molecular alterations in the affinity binding of integrins to their correlate LN-ligands.

Here, we describe a simple and inexpensive method that allows the quantification of adhesive tumor cells to lymph node (LN) cryosections. LN-adherent tumor cells are readily identified by light microscopy and confirmed by a fluorescence-based method, giving an adhesion index that reveals the tumor cell-binding affinity to LN parenchyma.

림프절 저온 절에서 종양 세포 접착의 정량화

Tumor-draining lymph nodes (LNs) are not merely filters of tumor-produced waste. They are one of the most common regional sites of provisional residence ...