Name: lung tumor cell recruitment assay
Uploaded: 2019-02-26T12:30:00
Description: Watch this Scientific Journal Video about Lung Tumor Cell Recruitment Assay at JoVE.com

마우스를 사용 하 여 수행 하는 모든 절차는 동물 연구 위원회의 도쿄 여자의과 대학에 의해 승인 되었다.
1. 루이스 폐 암 (LLC) 세포의 꼬리 정 맥 주사
<ol><li>습도 5%에서 10 %fcs 보충 DMEM LLC 셀 유지 37 ° c.에 CO2 배양 기 세포 형광 단백질 식 시스템 (예를 들어, GFP)를 포함 해야 합니다.</li>
	<li>비 효소 세포 분리 시 약을 사용 하 여 문화 접시에서 셀을 제거 합니?...

<ol>
	<li>Valastyan, S., Weinberg, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+metastasis:+molecular+insights+and+evolving+paradigms.">Tumor metastasis: molecular insights and evolving paradigms.</a> Cell. 147 (2), 275-292 (2011).</li><li>Kaplan, R. N., 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=VEGFR1-positive+haematopoietic+bone+marrow+progenitors+initiate+the+pre-metastatic+niche.">VEGFR1-positive haematopoietic bone marrow progenitors initiate the pre-metastatic niche.</a> Nature. 438 (7069), 820-827 (2005).</li><li>Hiratsuka, S., Watanabe, A., Aburatani, H., Maru, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumour-mediated+upregulation+of+chemoattractants+and+recruitment+of+myeloid+cells+predetermines+lung+metastasis.">Tumour-mediated upregulation of chemoattractants and recruitment of myeloid cells predetermines lung metastasis.</a> Nature Cell Biology. 8 (12), 1369-1375 (2006).</li><li>Hiratsuka, 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=The+S100A8-serum+amyloid+A3-TLR4+paracrine+cascade+establishes+a+pre-metastatic+phase.">The S100A8-serum amyloid A3-TLR4 paracrine cascade establishes a pre-metastatic phase.</a> Nature Cell Biology. 10 (11), 1349-1355 (2008).</li><li>Tomita, T., Sakurai, Y., Ishibashi, S., Maru, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imbalance+of+Clara+cell-mediated+homeostatic+inflammation+is+involved+in+lung+metastasis.">Imbalance of Clara cell-mediated homeostatic inflammation is involved in lung metastasis.</a> Oncogene. 30 (31), 3429-3439 (2011).</li><li>Deguchi, 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=Serum+amyloid+A3+binds+MD-2+to+activate+p38+and+NF-&#954;B+pathways+in+a+MyD88-dependent+manner.">Serum amyloid A3 binds MD-2 to activate p38 and NF-&#954;B pathways in a MyD88-dependent manner.</a> Journal of Immunology. 191 (4), 1856-1864 (2013).</li><li>Erler, J. T., 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=Hypoxia-induced+lysyl+oxidase+is+a+critical+mediator+of+bone+marrow+cell+recruitment+to+form+the+premetastatic+niche.">Hypoxia-induced lysyl oxidase is a critical mediator of bone marrow cell recruitment to form the premetastatic niche.</a> Cancer Cell. 15, 35-44 (2009).</li><li>Huang, 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=Pulmonary+vascular+destabilization+in+the+premetastatic+phase+facilitates+lung+metastasis.">Pulmonary vascular destabilization in the premetastatic phase facilitates lung metastasis.</a> Cancer Research. 69 (19), 7292-7237 (2009).</li><li>Sceneay, J., Smyth, M. J., M&#246;ller, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+pre-metastatic+niche:+finding+common+ground.">The pre-metastatic niche: finding common ground.</a> Cancer Metastasis Reviews. 32 (3-4), 449-464 (2013).</li><li>Castle, J., Shaker, H., Morris, K., Tugwood, J. D., Kirwan, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+significance+of+circulating+tumor+cells+in+breast+cancer:+A+review.">The significance of circulating tumor cells in breast cancer: A review.</a> The Breast. 23, 552-560 (2014).</li><li>Wolf, M. 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=Endothelial+CCR2+signaling+induced+by+colon+carcinoma+cells+enables+extravasation+via+the+JAK2-Stat5+and+p38MAPK+pathway.">Endothelial CCR2 signaling induced by colon carcinoma cells enables extravasation via the JAK2-Stat5 and p38MAPK pathway.</a> Cancer Cell. 22 (1), 91-105 (2012).</li><li>Hiratsuka, 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=Primary+tumours+modulate+innate+immune+signalling+to+create+pre-metastatic+vascular+hyperpermeability+foci.">Primary tumours modulate innate immune signalling to create pre-metastatic vascular hyperpermeability foci.</a> Nature Communications. 4, 1853 (2013).</li><li>Deguchi, 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=Eritoran+inhibits+S100A8-mediated+TLR4/MD-2+activation+and+tumor+growth+by+changing+the+immune+microenvironment.">Eritoran inhibits S100A8-mediated TLR4/MD-2 activation and tumor growth by changing the immune microenvironment.</a> Oncogene. 35 (19), 1445-1456 (2016).</li><li>Ieguchi, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ADAM12-cleaved+ephrin-A1+contributes+to+lung+metastasis.">ADAM12-cleaved ephrin-A1 contributes to lung metastasis.</a> Oncogene. 33 (17), 2179-2190 (2014).</li></ol>

항 체, 약물, 높은 지방 다이어트 또는 종양 세포 주입 전에 종양 조절 중간 마우스의 전처리 가능 하다. 이 분석 결과 가장 어려운 단계가입니다 꼬리 정 맥 주입. 불완전 주입 결정적 데이터에서 발생합니다. C57BL/6에서 꼬리 정 맥은 실패 한 주사에 따른 식별, 특히 어렵습니다. 생리대 (37 ° C)에 쥐를 배치 도움이 되도록 주입 쉽게 꼬리 정 맥 팽창.
이 분석 결과 환자의 혈액에...

종양 전이 암 관련 질병에 높은 사망률을 차지 한다. 분자 수준에서 조사 관점에서 전이 여러 단계로 분할 될 수 있다: 기본 종양 사이트, 기본 종양 성장 및 주변 조직, intravasation, 혈관을 통해 순환으로 침공에 종양 개시 먼 장기에 종양이 다시 성장을 넘쳐 흐름. 각 단계는 분자/신호1의 다른 세트를 포함 한다.
종양 세포는 또한로 알려져 미리 전이성 단계2,3,,45, 혈액에서 회람 시작 전에 먼 장기에 발생 하는 이벤트와 우려는 최신 연구 6. 우리의 마우스 모델 연구 밝혔다 미리 전이성 단계 폐에 지속적인 저수준 염증 반응을 만들어 폐 전이 촉진 한다. 미리 전이성 단계 themicrovasculature의 hyperpermeability, 파생 하는 골 수 세포 (BMDC)의 신규 모집 그리고 S100A8 및 SAA33,4 를 포함 하 여 cytokine/chemokine 같은 분자의 upregulation 특징 이다 . 그것은 그 lysyl 산화 효소 BMDCs7을 폐에서 세포 외 매트릭스 수정 보고 되었습니다. 또 다른 보고서는 미리 전이성 단계8에서 Angpt2, MMP3, 및 MMP10의 중요성을 밝혔다. 즉, 1 차 종양, 골 수, 및 원격 기관 간의 복잡 한 상호 작용 해야 이해 하 고 제대로 (예를 들어, 사용 하 여 이러한 상호 작용에 관련 된 단백질을 대상으로 마약) 변조 미래 전이9 방지 하기 위해 . 미리 전이성 단계 규제를 그것은 해야 먼저 시각화 되며 진단 또는 치료 개입에 대 한 평가. 여기, 우리는 폐 종양 세포 모집 분석 결과 폐에 미리 전이성 위상의 연구를 가능 하 게 보고 합니다.
마우스의 혈관에 직접 주입 하는 종양 세포는 배양된 세포 사이 차이가 있을 수 있지만 암 환자에서 종양 세포를 순환 하 고 순환 종양 세포에 비슷합니다. 순환 종양 세포 스스로 받 다 몇 가지 특성 변경을 기본 종양에서 순환 들어가면. 그럼에도 불구 하 고, 배양된 세포 형성할 수 있다 종양 immunodeficient 쥐에서 꼬리 정 맥에 주사 하는 경우. 또한, 마우스 모델 시스템에서 종양 세포를 분류 하는 형광 사용할 수 있습니다 본문에이 셀의 추적을 허용 하는. 순환 종양 세포의 행동은 암 환자10에 궁극적인 결과 결정할 때 중요 합니다. 혈액 종양 세포2의 생존에 대 한 좋은 곳이 아니다. 그들은 숙주 면역 체계에 의해 공격에서 탈출 필요 실제 지원의 부족 때문에 apoptosis를 방지 하기 위해 안티-apoptotic 신호를 해야 합니다. 종양 세포에서 전이성 종양 개시의 높은 능력을 가진 더 많은 종양 작은 혹을 생성합니다. 종양 세포가 특정 기관 차 있는 굴곡 운동, 보여주면 그 특정 장기에 전이 관찰 해야. 이 분석 결과에서 전이성 캐스케이드 (기본 종양의 성장 및 내 습)의 초기 단계는 포함 하 고 그래서 초점은 순환 종양 세포 및 stromal 세포 (내 피 세포, 상피 세포와 혈액 세포) 사이 상호 작용에. 기존의 종양 세포 주입 분석 실험에서 연구팀은 종양 혹 전이 검출 하는 확실 한 크기에 성장 될 때까지 기다려야 했습니다. 이 경우에 혈관에 종양 세포의 정확한 수는 측정할 수 없는. 또한,이 과정에는 종양 세포에 있는 다른 요소는 결과 영향을 미칠 지 수를 나타내는 종양 자라나 단계를 포함 됩니다.
계산 간단한 과정은 종양 세포에서 형광 stereomicroscope 표시. stereomicroscope 전체 폐 스캔 수 있도록 넓은 시야를 제공 합니다. Cytometry 즉시 조직에서 종양 세포의 정확한 수를 제공 하는 유용한 도구 이기도 합니다. Confocal 현미경 조직에 형광 셀 계산도 가능 하 고 전이성 종양 세포의 가장 정확한 사진을 표시 하지만 시간이 많이 걸리는 이며 선택된 영역을 계산 하는 경우에 치우치는 결과 줄 수 있습니다. 이 분석 결과의 궁극적인 목표는 종양 세포는 폐에 축적 하는 방법을 쉽게 관찰 하는. 보고 이전3,4, 미리 전이성 단계 기본 종양에서 염증 신호 때문에 폐가 하는 경우, 주입 된 종양 세포 폐 전이의 더 높은 기회를 암시 하는 따라서 폐에 축적 하는 경향이 있다. 다른 조건 (류 마티스 관절염, 천식, 등), 및 (안티-TNFα) 같은 항 염증 제 대리인 그들의 치료는 폐 전이 감소 수 있습니다. 따라서, 우리는이 분석 결과 폐 전이의 가능성을 평가 하는 강력한 도구입니다 결론.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Collagenase</td>
			<td>Sigma</td>
			<td>C6885</td>
			<td></td>
		</tr>
		<tr>
			<td>Dispase</td>
			<td>Gibco</td>
			<td>17105-041</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>Sigma</td>
			<td>A7030</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS</td>
			<td>Gibco</td>
			<td>14190-144</td>
			<td>no calcium, no magnesium</td>
		</tr>
		<tr>
			<td>Deoxyribonuclease I</td>
			<td>Sigma</td>
			<td>DN-25</td>
			<td>trace amount</td>
		</tr>
		<tr>
			<td>RBC lysis buffer</td>
			<td>Sigma</td>
			<td>R7757</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell strainer</td>
			<td>BD</td>
			<td>352340</td>
			<td>40 micrometer mesh</td>
		</tr>
		<tr>
			<td>Fluorescence labeling kit</td>
			<td>Sigma</td>
			<td>MIN26-KIT</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Gibco</td>
			<td>11965-092</td>
			<td></td>
		</tr>
		<tr>
			<td>0.22 &#956;m syringe filter</td>
			<td>sartorius</td>
			<td>17597K</td>
			<td></td>
		</tr>
		<tr>
			<td>O.C.T. compound</td>
			<td>Sakura Finetek</td>
			<td>4583</td>
			<td></td>
		</tr>
		<tr>
			<td>4% Paraformaldehyde Phosphate Buffer Solution</td>
			<td>Wako</td>
			<td>163-20145</td>
			<td></td>
		</tr>
	</tbody>
</table>

lung tumor cell recruitment assay

종양 전이 경 세 하는 분자 메커니즘을 조사, 다양 한 분석 실험 모델 동물로 마우스를 사용 하 여 제안 되었습니다. 여기, 종양 세포 넘쳐 흐름 또는 micrometastasis를 평가 하기 위해 간단한 분석 결과 보여 줍니다. 이 분석 결과에서 종양 세포 꼬리 정 맥을 통해 주입 했다 고 짧은 기간 후 폐 해 부 되었고 누적된 레이블이 지정 된 종양 세포를 소화. 이 분석 결과 혈관으로 기본 종양의 초기 단계를 건너뛰고 먼 기관 종양 전이 발생 하는 위치에 있는 이벤트의 연구를 촉진 한다. 제한 된 수의 전이 관찰 하는 혈관에 주입 하는 셀의 수를 최적화할 수 있습니다. 그것은 알려졌다 stromal 셀 먼 장기에 전이에 기여. 따라서,이 분석 결과 잠재적인 치료 약물 이나 종양 전이의 방지를 위한 장치를 탐구 하는 유용한 도구 일 수 있었다.

폐 주사 (그림 1A) 후 많은 GFP LLC 셀 2 시간을 제공합니다. 그것은 또한 빨간 필터에 검출 형광 명소 셀 번호 개수에서 제외 되어야 주목 한다. LLC는 세포의 대부분 주사 (그림 1C) 후 폐 24 h에서에서 사라집니다.
폐에 GFP LLC의 번호를 확인 하는 돌출부의 흐름 cytometric 분석 (<strong class="xf...

Watch this Scientific Journal Video about Lung Tumor Cell Recruitment Assay at JoVE.com

Lung Tumor Cell Recruitment Assay

폐 종양 세포 모집 분석 결과

이 원고 종양 전이의 동물 모델에서 폐에 종양 세포 축적을 계량 하는 방법을 설명 합니다.

To investigate the molecular mechanisms governing tumor metastasis, various assays using the mouse as a model animal have been proposed. Here, we ...

이 방법은 전이의 분자 기계장치에 관하여 종양 생물학 필드에 있는 중요한 질문에 대답하는 것을 도울 수 있습니다. 이 기술의 주요 장점은 원격 기관 내의 종양 세포의 모집이 수량일 수 있다는 것입니다. 표준 프로토콜에 따라 배양 용기에서 형광 단백질 발현 루이스 폐 암종 또는 LLC세포를 제거하기 위해 비 효소 세포 해리 시약을 사용하여 시작합니다.결과 세포 용액을 원심분리 전에 15밀리리터 원원형 튜브로 옮기고, 원심분리기는 세척당 PBS의 5밀리리터에서 펠릿을 두 번 세척합니다. 두 번째 세척 후, 40 마이크로미터 모공 세포 스트레이너를 통해 세포를 필터링하고, 현탁액을 밀리리터 농도당 6세포로 10배로 희석한다. 그런 다음 29 게이지 바늘이 장착 된 1 밀리리터 주사기에 세포를로드하고 8- 10 주 된 C57-Black-6, 수컷 마우스의 꼬리 정맥에 100 마이크로 리터의 셀을 주입하십시오.적절한 실험 적 끝점에서 가위를 사용하여 각 마우스의 복부 표면에서 피부를 제거하고 갈비뼈와 횡격막을 잘라 흉부 구멍을 노출시하십시오. 25 게이지 날개 주입 바늘과 튜브를 사용하여 오른쪽 심실을 통해 PBS의 15 밀리리터를 플러시하고 심장과 흉통을 제거하십시오. 집게로 기관을 잡고, 당겨, 기관 주위의 결합 조직을 해부, 폐를 수확.그런 다음 PBS에서 폐를 세척하고 형광 현미경으로 시상에서 형광 세포의 시각화를 위해 하나의 엽을 분리합니다. 폐 조직을 소화하기 위해, 먼저 가위로 caudal 엽을 주사위, 10 밀리리터 주사기에 폐 조각을 배치. 플런저로 주사기를 닫고 소화용액 5밀리리터를 주사기에 흡인시키고, 플런저를 주사기 샤프트 안팎으로 이동하여 폐 조직이 용액 전체에 보급될 때까지 주사기 샤프트 안팎으로 이동합니다.폐 슬러리를 50 밀리리터 튜브에 분배하고, 섭씨 37도와 150rpm에서 15분 동안 상호 셰이커에 티슈를 소화합니다. 인큐베이션의 끝에서, 폐 세포가 완전히 분산 될 때까지 나머지 조직을 삼각하고, 추가 30 분 동안 셰이커에 튜브를 반환합니다. 그런 다음 40 마이크로미터 모공 스트레이너를 통해 세포를 필터링하고 원심 분리에 의해 세포를 수집합니다.혈중 세포측정에 의해 고립된 폐 세포를 분석하기 위해, 실온에서 1분 동안 적혈구 용해 완충제 의 2밀리리터에서 펠릿을 재보중단하고 원심분리에 의해 세포를 수집한다. 그런 다음 신선한 PBS의 5 밀리리터에서 펠릿을 두 번 원심 분리하여 세척당 1 %소 세럼 알부민으로 보충합니다. 두 번째 원심 분리 후, 신선한 PBS 플러스 BSA의 1 밀리리터에서 펠릿을 재연하고, 혈동 세포에 의해 종양 세포의 정량화를 위한 새로운 40 마이크로미터 모공 스트레이너를 통해 세포를 필터링한다.폐는 주입 후에 2 시간 많은 GFP-LLC 세포를 제출합니다, 세포의 대다수는 24 시간 안에 폐에서 사라지고 있습니다. 빨간색 필터 내에서 감지되는 형광 반점은 셀 수에서 제외되어야 합니다. 폐내GFP-LLC의 수를 확인하기 위해, 로브 중 하나는 입증된 바와 같이 유동 세포 측정 분석에 사용될 수 있다.이 절차를 시도하는 동안, 냉동 단면 절차는 종양 세포의 정확한 위치를 관찰하기위한 보다 정확한 방법입니다 기억하는 것이 중요합니다.

Research

JoVE Journal

Cancer Research

Conditional Knockdown of Gene Expression in Cancer Cell Lines to Study the Recruitment of Monocytes/Macrophages to the Tumor Microenvironment

siRNA and shRNA-mediated knock down (KD) methods of regulating gene expression are invaluable tools for understanding gene and protein function. However, in the case that the KD of the protein of interest has a lethal effect on cells or the anticipated effect of the KD is time-dependent, unconditional KD methods are not appropriate. Conditional systems are more suitable in these cases and have been the subject of much interest. These include Ecdysone-inducible overexpression systems, Cytochrome P-450 induction system1, and the tetracycline regulated gene expression systems. 
The tetracycline regulated gene expression system enables reversible control over protein expression by induction of shRNA expression in the presence of tetracycline. In this protocol, we present an experimental design using functional Tet-ON system in human cancer cell lines for conditional regulation of gene expression. We then demonstrate the use of this system in the study of tumor cell-monocyte interaction.

This protocol serves as a scheme for setting up a functional Tet-ON system in cancer cell lines and its subsequent use, in particular for studying the role of tumor cell-derived proteins in recruitment of monocytes/macrophages to the tumor microenvironment.

암 세포 선 종양 Microenvironment에 Monocytes/대 식 세포의 모집 연구에서 유전자 발현의 조건부 최저

siRNA and shRNA-mediated knock down (KD) methods of regulating gene expression are invaluable tools for understanding gene and protein function. However, ...

연구

암 연구학

The Establishment of a Lung Colonization Assay for Circulating Tumor Cell Visualization in Lung Tissues

Metastasis is the major cause of cancer death. The role of circulating tumor cells (CTCs) in promoting cancer metastasis, in which lung colonization by CTCs critically contributes to early lung metastatic processes, has been vigorously investigated. As such, animal models are the only approach that captures the full systemic process of metastasis. Given that problems occur in previous experimental designs for examining the contributions of CTCs to blood vessel extravasation, we established an in vivo lung colonization assay in which a long-term-fluorescence cell-tracer, carboxyfluorescein succinimidyl ester (CFSE), was used to label suspended tumor cells and lung perfusion was performed to clear non-specifically trapped CTCs prior to lung removal, confocal imaging, and quantification. Polymeric fibronectin (polyFN) assembled on CTC surfaces has been found to mediate lung colonization in the final establishment of metastatic tumor tissues. Here, to specifically test the requirement of polyFN assembly on CTCs for lung colonization and extravasation, we performed short term lung colonization assays in which suspended Lewis lung carcinoma cells (LLCs) stably expressing FN-shRNA (shFN) or scramble-shRNA (shScr) and pre-labeled with 20 &#956;M of CFSE were intravenously inoculated into C57BL/6 mice. We successfully demonstrated that the abilities of shFN LLC cells to colonize the mouse lungs were significantly diminished in comparison to shScr LLC cells. Therefore, this short-term methodology may be widely applied to specifically demonstrate the ability of CTCs within the circulation to colonize the lungs.

An animal model is needed to decipher the role of circulating tumor cells (CTCs) in promoting lung colonization during cancer metastasis. Here, we established and successfully performed an in vivo assay to specifically test the requirement of polymeric fibronectin (polyFN) assembly on CTCs for lung colonization.

순환 종양 세포 시각화 폐 조직에 대 한 폐 식민 분석 결과의 설립

Metastasis is the major cause of cancer death. The role of circulating tumor cells (CTCs) in promoting cancer metastasis, in which lung colonization by ...

Acellular and Cellular Lung Model to Study Tumor Metastasis

It is difficult to isolate tumor cells at different points of tumor progression. We created an ex vivo lung model that can show the interaction of tumor cells with a natural matrix and continual flow of nutrients, as well as a model that shows the interaction of tumor cells with normal cellular components and a natural matrix. The acellular ex vivo lung model is created by isolating a rat heart-lung block and removing all the cells using the decellularization process. The right main bronchus is tied off and tumor cells are placed in the trachea by a syringe. The cells move and populate the left lung. The lung is then placed in a bioreactor where the pulmonary artery receives a continual flow of media in a closed circuit. The tumor grown on the left lung is the primary tumor. The tumor cells that are isolated in the circulating media are circulating tumor cells and the tumor cells in the right lung are metastatic lesions. The cellular ex vivo lung model is created by skipping the decellularization process. Each model can be used to answer different research questions.

Here, we present a protocol for an ex vivo lung cancer model that mimics the steps of tumor progression and helps to isolate a primary tumor, circulating tumor cells, and metastatic lesions.

Acellular과 세포 폐 종양 전이 연구 모델

It is difficult to isolate tumor cells at different points of tumor progression. We created an ex vivo lung model that can show the interaction of tumor ...

Live-Cell Imaging Assays to Study Glioblastoma Brain Tumor Stem Cell Migration and Invasion

Glioblastoma (GBM) is an aggressive brain tumor that is poorly controlled with the currently available treatment options. Key features of GBMs include rapid proliferation and pervasive invasion into the normal brain. Recurrence is thought to result from the presence of radio- and chemo-resistant brain tumor stem cells (BTSCs) that invade away from the initial cancerous mass and, thus, evade surgical resection. Hence, therapies that target BTSCs and their invasive abilities may improve the otherwise poor prognosis of this disease. Our group and others have successfully established and characterized BTSC cultures from GBM patient samples. These BTSC cultures demonstrate fundamental cancer stem cell properties such as clonogenic self-renewal, multi-lineage differentiation, and tumor initiation in immune-deficient mice. In order to improve on the current therapeutic approaches for GBM, a better understanding of the mechanisms of BTSC migration and invasion is necessary. In GBM, the study of migration and invasion is restricted, in part, due to the limitations of existing techniques which do not fully account for the in vitro growth characteristics of BTSCs grown as neurospheres. Here, we describe rapid and quantitative live-cell imaging assays to study both the migration and invasion properties of BTSCs. The first method described is the BTSC migration assay which measures the migration toward a chemoattractant gradient. The second method described is the BTSC invasion assay which images and quantifies a cellular invasion from neurospheres into a matrix. The assays described here are used for the quantification of BTSC migration and invasion over time and under different treatment conditions.

Here, we describe live-cell imaging techniques to quantitatively measure the migration and invasion of glioblastoma brain tumor stem cells over time and under multiple treatment conditions.

라이브 셀 이미징 분석 실험 연구 세포종 뇌 종양 줄기 세포 이동과 내 습을

Glioblastoma (GBM) is an aggressive brain tumor that is poorly controlled with the currently available treatment options. Key features of GBMs include ...

Digital Polymerase Chain Reaction Assay for the Genetic Variation in a Sporadic Familial Adenomatous Polyposis Patient Using the Chip-in-a-tube Format

The quantitative analysis of human genetic variation is crucial for understanding the molecular characteristics of serious medical conditions, such as tumors. Because digital polymerase chain reactions (PCR) enable the precise quantification of DNA copy number variants, they are becoming an essential tool for detecting rare genetic variations, such as drug-resistant mutations. It is expected that molecular diagnoses using digital PCR (dPCR) will be available in clinical practice in the near future; thus, how to efficiently conduct dPCR with human genetic material is a hot topic. Here, we introduce a method to detect Adenomatous polyposis coli (APC) somatic mosaicism using dPCR with the chip-in-a-tube format, which allows eight dPCR reactions to be simultaneously conducted. Care should be taken when filling and sealing the reaction mixture on the chips. This article demonstrates how to avoid the over- and underestimation of positive partitions. Furthermore, we present a simple procedure for collecting the dPCR product from the partitions on the chips, which can then be used to confirm the specific amplification. We hope that this methods report will help promote the dPCR with the chip-in-a-tube method in genetic research.

Digital polymerase chain reaction (PCR) is a useful tool for the high-sensitivity detection of single nucleotide variants and DNA copy number variants. Here, we demonstrate key considerations for measuring rare variants in the human genome using digital PCR with the chip-in-a-tube format.

튜브에 칩 형식을 사용 하 여 산발적 인 가족 용 대 환자에 있는 유전 변이 대 한 디지털 중 합 효소 연쇄 반응 분석 결과

The quantitative analysis of human genetic variation is crucial for understanding the molecular characteristics of serious medical conditions, such as ...

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 ...

Real Time Detection of In Vitro Tumor Cell Apoptosis Induced by CD8+ T Cells to Study Immune Suppressive Functions of Tumor-infiltrating Myeloid Cells

Potentiation of the tumor-killing ability of CD8+ T cells in tumors, along with their efficient tumor infiltration, is a key element of successful immunotherapies. Several studies have indicated that tumor infiltrating myeloid cells (e.g., myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMs)) suppress cytotoxicity of CD8+ T cells in the tumor microenvironment, and that targeting these regulatory myeloid cells can improve immunotherapies. Here, we present an in vitro assay system to evaluate immune suppressive effects of monocytic-MDSCs and TAMs on the tumor-killing ability of CD8+ T cells. To this end, we first cultured na&#239;ve splenic CD8+ T cells with anti-CD3/CD28 activating antibodies in the presence or absence of suppressor cells, and then co-cultured the pre-activated T cells with target cancer cells in the presence of a fluorogenic caspase-3 substrate. Fluorescence from the substrate in cancer cells was detected by real-time fluorescence microscopy as an indicator of T-cell induced tumor cell apoptosis. In this assay, we can successfully detect the increase of tumor cell apoptosis by CD8+ T cells and its suppression by pre-culture with TAMs or MDSCs. This functional assay is useful for investigating CD8+ T cell suppression mechanisms by regulatory myeloid cells and identifying druggable targets to overcome it via high throughput screening.

Real Time Detection of In Vitro Tumor Cell Apoptosis Induced by CD8+ T Cells to Study Immune Suppressive Functions of Tumor-infiltrating Myeloid Cells

We describe here a protocol to investigate cytotoxicity of pre-activated CD8+ T cells against cancer cells by detecting apoptotic cancer cells via real-time microscopy. This protocol can investigate mechanisms behind myeloid cell-induced T cell suppression and evaluate compounds aimed at replenishing T cells via blockade of immune suppressive myeloid cells.

실시간 탐지의 체 외 종양 세포 Apoptosis에 CD8에 의해 유도 된+ T 세포 종양 침투 골수성 세포의 면역 진압 기능 연구

Potentiation of the tumor-killing ability of CD8+ T cells in tumors, along with their efficient tumor infiltration, is a key element of successful ...

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 ...

In Vitro Assay to Study Tumor-macrophage Interaction

Tumor associated macrophages (TAMs) account for a large percentage of cells in the tumor mass for different types of cancers. Glioblastoma (GBM), a malignant brain tumor with no cure, has up to a half the tumor mass TAMs. TAMs can be pro-tumoral or anti-tumoral, depending on the activation of specific genes in the cells. Genetic mutations in the tumors, through regulating cytokine expression, can affect recruitment of TAMs to the tumor microenvironment. Here, we describe a quantitative cell-based assay to assess macrophage recruitment by the conditioned medium from the tumor cells. This assay uses the human macrophage cell line MV-4-11 to study macrophage attraction by the conditioned medium from glioblastoma, allowing for high reproducibility and low variability. Data generated with this assay can contribute to a better understanding of the interaction between the tumor and the tumor microenvironment. Similar assay can be used to assess interaction between the tumor cells and other immune cells, including T cells and natural killer (NK) cells.

This article represents a useful in vitro assay to evaluate the capability of conditioned medium from tumor cells to attract macrophages.

종양 대식 세포 상호 작용을 연구 하는 시험관 내 분석

Tumor associated macrophages (TAMs) account for a large percentage of cells in the tumor mass for different types of cancers. Glioblastoma (GBM), a ...

Detection of Lung Tumor Progression in Mice by Ultrasound Imaging

With ~1.6 million victims per year, lung cancer contributes tremendously to the worldwide burden of cancer. Lung cancer is partly driven by genetic alterations in oncogenes such as the KRAS oncogene, which constitutes ~25% of lung cancer cases. The difficulty in therapeutically targeting KRAS-driven lung cancer partly stems from having poor models that can mimic the progression of the disease in the lab. We describe a method that permits the relative quantification of primary KRAS lung tumors in a Cre-inducible LSL-KRAS G12D mouse model via ultrasound imaging. This method relies on brightness (B)-mode acquisition of the lung parenchyma. Tumors that are initially formed in this model are visualized as B-lines and can be quantified by counting the number of B-lines present in the acquired images. These would represent the relative tumor number formed on the surface of the mouse lung. As the formed tumors develop with time, they are perceived as deep clefts within the lung parenchyma. Since the circumference of the formed tumor is well-defined, calculating the relative tumor volume is achieved by measuring the length and width of the tumor and applying them in the formula used for tumor caliper measurements. Ultrasound imaging is a non-invasive, fast and user-friendly technique that is often used for tumor quantifications in mice. Although artifacts may appear when obtaining ultrasound images, it has been shown that this imaging technique is more advantageous for tumor quantifications in mice compared to other imaging techniques such as computed tomography (CT) imaging and bioluminescence imaging (BLI). Researchers can investigate novel therapeutic targets using this technique by comparing lung tumor initiation and progression between different groups of mice.

This protocol describes the steps taken to induce KRAS lung tumors in mice as well as the quantification of formed tumors by ultrasound imaging. Small tumors are visualized in early timepoints as B-lines. At later timepoints, relative tumor volume measurements are achieved by the measurement tool in the ultrasound software.

초음파 화상 진찰에 의하여 마우스에 있는 폐 종양 진행의 탐지

With ~1.6 million victims per year, lung cancer contributes tremendously to the worldwide burden of cancer. Lung cancer is partly driven by genetic ...