이 간행물에 보고된 연구는 수여번호 UL1TR003096 (MDE), 국립 심장, 폐 및 혈액 연구소 전 박사 과정 5T32HL134640 (MLD)에 따라 번역 과학 을 발전시키기위한 국립 센터에 의해 지원되었다.

여기에 설명 된 모든 방법은 버밍엄에서 알라바마 대학의 기관 동물 관리 및 사용위원회 (IACUC)에 의해 승인되었습니다.
1. 실험 프로토콜
<ol><li>승인된 IACUC 방법을 사용하여 마우스를 희생합니다. 여기서는 5%의 이소플루란으로 마취된 마우스의 자궁경부 탈구를 사용했습니다. 연구에 적합한 마우스를 사용; 여기에서 는 8주 된 FVB 마우스를 사용합니다.</li>
	<li>수술 가위를 사용하여 하복부 중간에 3.5-5mm 수평 절개를 합니다. 다음으로, 절개로 만든 작은 구멍에 수술 가위를 삽입하고 마우스의 목 바로 아래 중앙 중간선을 수직으로 잘라.</li>
	<li>손가락으로 피부를 다시 당기고 축산 림프절을 검사합니다.</li>
	<li>외과 가위를 사용하여 3.5mm 측면 절개를 하여 복강을 열고 흉부 바닥까지 전방 방향으로 자른다. 간, 비장, 신장 등 복강기관의 장기검사를 한다.</li>
	<li>수술 가위의 평평한 또는 집게를 사용하여 간을 움직여 다이어프램을 노출시합니다. 종양 성장 또는 전이에 대한 다이어프램을 검사합니다. 그런 다음 작업자의 오른쪽에 있는 다이어프램을 부드럽게 잘라서 확장합니다. 흉부 구멍과 폐를 노출시키기 위해 횡격막을 오른쪽에서 왼쪽으로 부드럽게 자릅니다. 폐를 자르지 않도록 주의하십시오.</li>
	<li>왼쪽 늑골 케이지(작업자의 오른쪽)의 측면 극단을 잘라 폐의 왼쪽 엽을 검사합니다.</li>
	<li>폐의 오른쪽 엽을 부드럽게 움직이며 오른쪽 갈비뼈 케이지의 측면 극단을 잘라 내고 흉곽을 제거합니다. 참고: 제거는 나중에 폐 인플레이션을 명확하게 볼 수 있지만, 흉곽의 제거는 선택 사항입니다.<ol>
<li>신선하거나 냉동된 폐 조직이 필요한 경우, 후면 집게를 사용하여 왼쪽 엽의 기관지를 고정하고 관류 전에 외과 가위를 사용하여 왼쪽 폐를 절제하십시오.</li>
	</ol>
</li>
	<li>집게를 사용하여 기관진을 덮는 조직을 들어 올리고 과도한 조직을 잘라냅니다. 그런 다음 기관지 안감의 얇은 조직을 부드럽게 잘라 기도를 노출시합니다.</li>
	<li>외과 가위로 신장 동맥을 잘라냅니다.</li>
	<li>폐를 견딜 수 있도록 22G 바늘이 달린 3mL 주사기를 사용하여 심장의 오른쪽 심실로 10 개의 U /mL 헤파린이있는 1 x PBS를 주입하십시오. PBS/헤파린으로 약 300 μL/s의 폐를 천천히 침투합니다. 폐는 자주 하얗게 변합니다. PBS의 2.5 mL은 일반적으로이 단계에서 사용된다.</li>
	<li>폐 인플레이션의 경우, 22 G 바늘로 3mL 주사기를 사용하시며, 이번에는 기관지와 평행하게 유지됩니다. 바늘을 기관체에 삽입하고 폐가 완전히 팽창 할 때까지 - 200 μL / s보다 큰 흐름의 속도로 10 %의 포르말린을 주입하십시오. 폐가 팽창되면 포르말린이 기관에서 흘러 나올 것입니다. 바늘을 몇 초 더 제자리에 고정한 다음 철회하십시오.<ol>
<li>(선택 사항) 바늘과 폐 인플레이션을 철회하기 전에 봉합사실을 사용하여 기관지를 묶습니다. 이를 위해 작은 한 쌍의 집게로 스레드의 점을 잡고 있는 4인치 의 봉합사 스레드를 사용합니다. 기관지의 등쪽 에 실을 놓고 당겨 바늘 주위에 루프를 만듭니다. 다음으로 바늘 주위에 오버핸드 매듭을 만듭니다. 매듭을 단단히 당기고, 기관지에서 바늘을 제거하고, 매듭을 닫습니다.</li>
	</ol>
</li>
	<li>집게를 사용하여 심장을 들어 올리고, 폐 바로 뒤에 수술 가위를 삽입하고, 폐를 절제하기 위해 심장을 들어 올리는 동안 결합 조직을 잘라냅니다.</li>
	<li>폐에서 제거하는 심장을 잘라.</li>
	<li>폐에 마우스 ID 또는 연구 ID로 표시된 카세트를 배치합니다. 카세트를 10% 버퍼링된 포르말린에 놓고 24-48h를 수정합니다. 폐는 원하는 경우 1 년 이상 고정에 남아있을 수 있습니다.</li>
	<li>폐를 함유한 카세트를 70%에탄올로 옮기고 히스토로지 처리를 진행한다.</li>
</ol>

<ol>
	<li>Hsia, C. C., Hyde, D. M., Ochs, M., Weibel, E. R., Structure, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+official+research+policy+statement+of+the+American+Thoracic+Society/European+Respiratory+Society:+standards+for+quantitative+assessment+of+lung+structure.">An official research policy statement of the American Thoracic Society/European Respiratory Society: standards for quantitative assessment of lung structure.</a> American Journal of Respiratory and Critical Care Medicine. 181 (4), 394-418 (2010).</li><li>Vasilescu, D. M., Knudsen, L., Ochs, M., Weibel, E. R., Hoffman, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimized+murine+lung+preparation+for+detailed+structural+evaluation+via+micro-computed+tomography.">Optimized murine lung preparation for detailed structural evaluation via micro-computed tomography.</a> Journal of Applied Physiology. 112 (1), 159-166 (2012).</li><li>Blumler, P., Acosta, R. H., Thomas-Semm, A., Reuss, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lung+fixation+for+the+preservation+of+air+spaces.">Lung fixation for the preservation of air spaces.</a> Experimental Lung Research. 30 (1), 73-82 (2004).</li><li>Oldmixon, E. H., Suzuki, S., Butler, J. P., Hoppin, F. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perfusion+dehydration+fixes+elastin+and+preserves+lung+air-space+dimensions.">Perfusion dehydration fixes elastin and preserves lung air-space dimensions.</a> Journal of Applied Physiology. 58 (1), 105-113 (1985).</li><li>Braber, S., Verheijden, K. A., Henricks, P. A., Kraneveld, A. D., Folkerts, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparison+of+fixation+methods+on+lung+morphology+in+a+murine+model+of+emphysema.">A comparison of fixation methods on lung morphology in a murine model of emphysema.</a> American Journal of Physiology-Lung Cellular and Molecular Physiology. 299 (6), 843-851 (2010).</li><li>Renne, 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=Recommendation+of+optimal+method+for+formalin+fixation+of+rodent+lungs+in+routine+toxicology+studies.">Recommendation of optimal method for formalin fixation of rodent lungs in routine toxicology studies.</a> Toxicologic Pathology. 29 (5), 587-589 (2001).</li><li>Limjunyawong, N., Mock, J., Mitzner, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Instillation+and+Fixation+Methods+Useful+in+Mouse+Lung+Cancer+Research.">Instillation and Fixation Methods Useful in Mouse Lung Cancer Research.</a> Journal of Visualized Experiments. (102), e52964 (2015).</li><li>Lum, H., Mitzner, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+10%+formalin+fixation+on+fixed+lung+volume+and+lung+tissue+shrinkage.+A+comparison+of+eleven+laboratory+species.">Effects of 10% formalin fixation on fixed lung volume and lung tissue shrinkage. A comparison of eleven laboratory species.</a> American Review of Respiratory Disease. 132 (5), 1078-1083 (1985).</li><li>Edmonds, M. D., 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=MicroRNA-31+initiates+lung+tumorigenesis+and+promotes+mutant+KRAS-driven+lung+cancer.">MicroRNA-31 initiates lung tumorigenesis and promotes mutant KRAS-driven lung cancer.</a> Journal of Clinical Investigation. 126 (1), 349-364 (2016).</li><li>Zhao, 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=Wogonin+suppresses+melanoma+cell+B16-F10+invasion+and+migration+by+inhibiting+Ras-medicated+pathways.">Wogonin suppresses melanoma cell B16-F10 invasion and migration by inhibiting Ras-medicated pathways.</a> PLoS One. 9 (9), 106458 (2014).</li></ol>

저자는 공개 할 것이 없습니다.

마우스 폐의 관류, 인플레이션 및 고정을 위해 위에서 설명한 절차는 폐 종양 조직학 및 병리학 분석을 위한 마우스 폐의 빠르고 효율적인 준비에 이상적입니다. 절차는 특별한 장비를 필요로하지 않으며 마우스 당 6 분 이내에 수행 할 수 있습니다. 이 절차는 인플레이션또는 일정한 유체 압력에 대한 고정 된 볼륨을 필요로하지 않습니다. 이 절차는 표준화되지 않기 때문에 폐의 입체적 또는 형?...

폐 종양 발생 및 암 전이의 다양한 마우스 모델은 복잡한 GEMM에서 발암 물질 유발 모델, 합성 및 xenograft 모델에 이르기까지 존재하며, 암세포는 심장 내, 내트라토라시, 꼬리 정맥 또는 폐 내종양을 확립하는 다른 방법을 통해 주입된다. 이 모든 모형은 폐 조직학 및 병리학의 조직학 평가를 위한 일반적인 필요를 공유합니다. 따라서, 폐를 흡수하면서 쥐의 부검을 수행하는 견고하면서도 신속한 방법이 필요하며, 폐를 팽창시키고 고정하여 폐 아키텍처를 명확하게 시각화할 필요가 있다. 속도는 단일 시점에서 수십 마리의 마우스로부터 폐를 수집해야 할 수도 있기 때문에 이 절차의 중요한 구성 요소입니다. 이 절차는 마우스 당 6 분 이내에 수행 할 수 있습니다.
이 절차는 종양 조직학을 평가하기에 충분하지만 폐의 입체학 또는 형태 측정을 수행하고자하는 사람들에게는 권장되지 않습니다. 이러한 측정은 폐의 절대 표면적, 절대 부피 및 폐포 크기 및 번호1의계산과 마찬가지로 폐 인플레이션을 표준화할 필요가 있다. 이 방법은 또한 몇몇 화상 진찰 접근에 대 한 최적이 아니다. 예를 들어, 전 생체 형태 분석을 위해 μCT를 통한 폐의 이미징은 폐가 공기2로채워져 있어야 합니다. 공기 공간과 치수의 보존이 주요 관심사인 경우, 관류 탈수 기술3,4에의해 폐를 고치는 것이 좋습니다. 이 모델의 가장 큰 관심사 중 하나는 폐포 벽의 파열에 대 한 잠재력, 폐기종의 연구에 그것의 사용을 감소; 그러나, 폐기종의 연구를 위한 폐의 고정을 위한 권장절차는 10% 포르말린(여기에 기술된 프로토콜과 유사)의 장내 주입에 의해 또는 정시 고정5에의해 폐를 고치는 것이 좋습니다.
여기서 설명된 절차의 장점은 일정한 유체 압력을 필요로 하지 않고, 완전히 팽창할 때까지 폐를 팽창시켜 시술에 필요한 시간을 감소시킨다는 것입니다. 여기서 설명된 절차는 독성학 연구를 위한 폐 고정의 가장 좋은 방법을 추천하기 위하여 소위원회가 형성된 독성병리학의 사회의 무기학에 의해 추천된 방법과 유사합니다. 이 소위원회 내의 과학자의 대다수는 폐가 고정6에남아 있던 시간에 변화한 권고가 있더라도, 주사기를 가진 장 내 주입에 의하여 폐를 고정하는 것을 추천했습니다. 따라서, 다양한 폐 인플레이션 및 고정 방법이 존재하지만, 본 원에 기재된 방법은 다운스트림 종양 조직학적 평가를 위해 폐를 신속하게 팽창시키고 고치는 최적의 방법이 될 것을 제안한다.

<table>
	<tbody>
		<tr>
			<td>10% buffered formalin</td>
			<td>Fisher</td>
			<td>23-245685</td>
			<td></td>
		</tr>
		<tr>
			<td>22 G Needle</td>
			<td>BD</td>
			<td>305155</td>
			<td></td>
		</tr>
		<tr>
			<td>3 mL syringe</td>
			<td>BD</td>
			<td>309656</td>
			<td></td>
		</tr>
		<tr>
			<td>70% Ethanol</td>
			<td>Decon</td>
			<td>2405</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Harvard Apparatus</td>
			<td>72-8595</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>Fisher</td>
			<td>H19</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline (PBS)</td>
			<td>Corning</td>
			<td>21-030-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical scissors</td>
			<td>Harvard Apparatus</td>
			<td>72-8428</td>
			<td></td>
		</tr>
	</tbody>
</table>

perfusion and inflation of the mouse lung for tumor histology

폐 장학적 연구를 평가하는 능력은 폐암 연구 및 암 전이 분야에 매우 중요합니다. 조달된 조직의 품질을 희생하지 않고 연구에서 신속하고 효율적으로 부검을 수행하는 것이 똑같이 중요합니다. 이 프로토콜의 목표는 다운스트림 조직 분석을 위해 마우스 폐를 신속하게 퍼퓨즈, 팽창 및 수정하는 방법을 제시하는 것입니다. 이 방법은 폐 인플레이션을 표준화하지 않습니다; 따라서 특별한 절차 나 장비가 필요하지 않으며 대신 단순히 심장을 통해 관류 다음 기관을 통해 직접 고정을 주입합니다. 이것은 종양 크기, 작사학 및 점수의 충분한 추정을 허용합니다. 이것은 또한 폐 조직 고정의 앞에 동결된 조직의 집합을 허용합니다. 이 방법은 폐의 나중에 형태화 를 허용하지 않는다는 점에서 제한됩니다. 그러나, 유전자 조작된 마우스 모델(GEMM), 신제모델, 이종이이식 종양 및 전이 연구에서 폐종양 분석을 하기에 충분하다.

위의 프로토콜은 마우스 폐의 빠른 관류, 인플레이션 및 고정을 허용합니다. 아래 그림은 각 단계의 중요성을 나타냅니다. 그림 1은 관류 단계가 건너뛰거나 폐가 올바르게 침투하지 못한 PBS 및 폐와 함께 퍼진 H&E 염색 폐를 묘사합니다. 표시된 바와 같이, 제대로 퍼진 폐에 있는 과잉 혈액은 이상적인 히스토로지 보다는 적게 생성하고 완전히 폐 건축을 관찰하는 것을 어렵?...

Watch this Scientific Journal Video about Perfusion and Inflation of the Mouse Lung for Tumor Histology at JoVE.com

Perfusion and Inflation of the Mouse Lung for Tumor Histology

이 방법의 목적은 폐 종양 병리학의 검사 및 폐에 전이의 평가를 위한 마우스 폐의 관류, 인플레이션 및 고정을 위한 간단하고 효율적인 방법을 제시하는 것입니다.

종양 연고학을 위한 마우스 폐의 관류 및 인플레이션

The ability to evaluate lung histology is critical for the fields of lung cancer research and cancer metastasis. It is equally important to perform ...

perfusion-and-inflation-of-the-mouse-lung-for-tumor-histology

Research

JoVE Journal

Cancer Research

17.6K 조회수.  University of Alabama at Birmingham. 이 방법의 목적은 폐 종양 병리학의 검사 및 폐에 전이의 평가를 위한 마우스 폐의 관류, 인플레이션 및 고정을 위한 간단하고 효율적인 방법을 제시하는 것입니다.

비디오: Perfusion and Inflation of the Mouse Lung for Tumor Histology

Lung Tumor Cell Recruitment Assay

To investigate the molecular mechanisms governing tumor metastasis, various assays using the mouse as a model animal have been proposed. Here, we demonstrate a simple assay to evaluate tumor cell extravasation or micrometastasis. In this assay, tumor cells were injected through the tail vein, and after a short period, the lungs were dissected and digested to count the accumulated labeled tumor cells. This assay skips the initial step of primary tumor invasion into the blood vessel and facilitates the study of events in the distant organ where tumor metastasis occurs. The number of cells injected into the blood vessel can be optimized to observe a limited number of metastases. It has been reported that stromal cells in the distant organ contribute to metastasis. Thus, this assay could be a useful tool to explore potential therapeutic drugs or devices for prevention of tumor metastasis.

This manuscript describes a method to quantify tumor cell accumulation in the lungs in an animal model of tumor metastasis.

폐 종양 세포 모집 분석 결과

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

연구

암 연구학

The Colon-26 Carcinoma Tumor-bearing Mouse as a Model for the Study of Cancer Cachexia

Cancer cachexia is the progressive loss of skeletal muscle mass and adipose tissue, negative nitrogen balance, anorexia, fatigue, inflammation, and activation of lipolysis and proteolysis systems. Cancer patients with cachexia benefit less from anti-neoplastic therapies and show increased mortality1. Several animal models have been established in order to investigate the molecular causes responsible for body and muscle wasting as a result of tumor growth. Here, we describe methodologies pertaining to a well-characterized model of cancer cachexia: mice bearing the C26 carcinoma2-4. Although this model is heavily used in cachexia research, different approaches make reproducibility a potential issue. The growth of the C26 tumor causes a marked and progressive loss of body and skeletal muscle mass, accompanied by reduced muscle cross-sectional area and muscle strength3-5. Adipose tissue is also lost. Wasting is coincident with elevated circulating levels of pro-inflammatory cytokines, particularly Interleukin-6 (IL-6)3, which is directly, although not entirely, responsible for C26 cachexia. It is well-accepted that a primary mechanism by which the C26 tumor induces muscle tissue depletion is the activation of skeletal muscle proteolytic systems. Thus, expression of muscle-specific ubiquitin ligases, such as atrogin-1/MAFbx and MuRF-1, represent an accepted method for the evaluation of the ongoing muscle catabolism2. Here, we present how to execute this model in a reproducible manner and how to excise several tissues and organs (the liver, spleen, and heart), as well as fat and skeletal muscles (the gastrocnemius, tibialis anterior, and quadriceps). We also provide useful protocols that describe how to perform muscle freezing, sectioning, and fiber size quantification.

Mice bearing the Colon-26 (C26) carcinoma represent a classical model of cancer cachexia. Progressive muscle wasting occurs in association with tumor growth, over-expression of muscle-specific ubiquitin ligases, and reductions in muscle cross-sectional area. Fat loss is also observed. Cachexia is studied in a time-dependent manner with increasing severity of wasting.

콜론-26 암 종양 베어링 암 악액질의 연구를위한 모델로 마우스를

Cancer cachexia is the progressive loss of skeletal muscle mass and adipose tissue, negative nitrogen balance, anorexia, fatigue, inflammation, and ...

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 ...

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 ...

Utilizing 18F-FDG PET/CT Imaging and Quantitative Histology to Measure Dynamic Changes in the Glucose Metabolism in Mouse Models of Lung Cancer

A hallmark of advanced tumors is a switch to aerobic glycolysis that is readily measured by [18F]-2-fluoro-2-deoxy-D-glucose positron emission tomography (18F-FDG PET) imaging. Co-mutations in the KRAS proto-oncogene and the LKB1 tumor suppressor gene are frequent events in lung cancer that drive hypermetabolic, glycolytic tumor growth. A critical pathway regulating the growth and metabolism of these tumors is the mechanistic target of the rapamycin (mTOR) pathway, which can be effectively targeted using selective catalytic mTOR kinase inhibitors. The mTOR inhibitor MLN0128 suppresses glycolysis in mice bearing tumors with Kras and Lkb1 co-mutations, referred to as KL mice. The therapy response in KL mice is first measured by 18F-FDG PET and computed tomography (CT) imaging before and after the delivery of MLN0128. By utilizing 18F-FDG PET/CT, researchers are able to measure dynamic changes in the glucose metabolism in genetically engineered mouse models (GEMMs) of lung cancer following a therapeutic intervention with targeted therapies. This is followed by ex vivo autoradiography and a quantitative immunohistochemical (qIHC) analysis using morphometric software. The use of qIHC enables the detection and quantification of distinct changes in the biomarker profiles following treatment as well as the characterization of distinct tumor pathologies. The coupling of PET imaging to quantitative histology is an effective strategy to identify metabolic and therapeutic responses in vivo in mouse models of disease.

Utilizing 18F-FDG PET/CT Imaging and Quantitative Histology to Measure Dynamic Changes in the Glucose Metabolism in Mouse Models of Lung Cancer

In this protocol, we describe how to utilize [18F]-2-fluoro-2-deoxy-D-glucose positron emission tomography and computed tomography (18F-FDG PET/CT) imaging to measure the tumor metabolic response to the targeted therapy MLN0128 in a Kras/Lkb1 mutant mouse model of lung cancer and coupled imaging with high resolution ex vivo autoradiography and quantitative histology.

18F FDG PET/CT 영상 및 정량 조직학 폐암의 마우스 모델에서 포도 당 물질 대사에 있는 동적인 변화를 측정 하

A hallmark of advanced tumors is a switch to aerobic glycolysis that is readily measured by [18F]-2-fluoro-2-deoxy-D-glucose positron emission tomography ...

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 ...

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 ...

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 ...

Pathological Analysis of Lung Metastasis Following Lateral Tail-Vein Injection of Tumor Cells

Metastasis, the primary cause of morbidity and mortality for most cancer patients, can be challenging to model preclinically in mice. Few spontaneous metastasis models are available. Thus, the experimental metastasis model involving tail-vein injection of suitable cell lines is a mainstay of metastasis research. When cancer cells are injected into the lateral tail-vein, the lung is their preferred site of colonization. A potential limitation of this technique is the accurate quantification of the metastatic lung tumor burden. While some investigators count macrometastases of a pre-defined size and/or include micrometastases following sectioning of tissue, others determine the area of metastatic lesions relative to normal tissue area. Both of these quantification methods can be exceedingly difficult when the metastatic burden is high. Herein, we demonstrate an intravenous injection model of lung metastasis followed by an advanced method for quantifying metastatic tumor burden using image analysis software. This process allows for investigation of multiple end-point parameters, including average metastasis size, total number of metastases, and total metastasis area, to provide a comprehensive analysis. Furthermore, this method has been reviewed by a veterinary pathologist board-certified by the American College of Veterinary Pathologists (SEK) to ensure accuracy.

Intravenous injection of cancer cells is often used in metastasis research, but the metastatic tumor burden can be difficult to analyze. Herein, we demonstrate a tail-vein injection model of metastasis and include a novel approach to analyze the resulting metastatic lung tumor burden.

종양 세포의 측면 꼬리 정맥 주입 다음 폐 전이의 병리학 적 분석

Metastasis, the primary cause of morbidity and mortality for most cancer patients, can be challenging to model preclinically in mice. Few spontaneous ...

Generation of Zebrafish Larval Xenografts and Tumor Behavior Analysis

Zebrafish larval xenografts are being widely used for cancer research to perform in vivo and real-time studies of human cancer. The possibility of rapidly visualizing the response to anti-cancer therapies (chemo, radiotherapy, and biologicals), angiogenesis and metastasis with single cell resolution, places the zebrafish xenograft model as a top choice to develop preclinical studies.

The zebrafish larval xenograft assay presents several experimental advantages compared to other models, but probably the most striking is the reduction of size scale and consequently time. This reduction of scale allows single cell imaging, the use of a relatively low number of human cells (compatible with biopsies), medium-high-throughput drug screenings, but most importantly enables a significant reduction of the time of the assay. All these advantages make the zebrafish xenograft assay extremely attractive for future personalized medicine applications.

Many zebrafish xenograft protocols have been developed with a wide diversity of human tumors; however, a general and standardized protocol to efficiently generate zebrafish larval xenografts is still lacking. Here we provide a step-by-step protocol, with tips to generate xenografts and guidelines for tumor behavior analysis, whole-mount immunofluorescence, and confocal imaging quantification.

Here, we provide a step-by-step protocol, with tips to generate xenografts and guidelines for tumor behavior analysis, whole-mount immunofluorescence, and confocal imaging quantification.

제브라피시 애벌레 제노이식 및 종양 행동 분석의 생성

Zebrafish larval xenografts are being widely used for cancer research to perform in vivo and real-time studies of human cancer. The possibility of rapidly ...