We thank Marleen De Meulemeester for demonstrating the assay technique in the video film. B. I. R. is a Postdoctoral Research Fellow of the Research Foundation &#8211; Flanders (FWO &#8211; Vlaanderen). L.M.M. is a recipient of an Emmanuel van der Schueren grant from the Flemish League against Cancer (Vlaamse Liga tegen Kanker).

<ol>
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The authors declare that they have no competing financial interests.

PHFs의 제조 동안, 단편 현탁액에 체류하지 않을 수도 있지만, 혈관 벽에 부착; 이는 배양액의 부피를 증가시킴으로써 극복 될 수있다. PHFs 수가 너무 낮고, 그 크기가 너무 큰 경우, 배양액의 부피를 감소시킨다. 집계 테스트 세포의 실패는 온도 나 미생물 감염에 변동 때문일 수 있습니다. 또한, 집계 무능력 세포의 고유 한 특성 일 수있다. PHF에 골재의 부착 동안에, 접착 불량은 반고체 한천 배지 ?...

침략은 악성 종양의 특징이다. 이 활동은 주변 조직의 파괴에 이르게뿐만 아니라 전이 형성에 연루되어 아닙니다. 암 환자가 침윤 및 전이, 효율적인 안티 침습적 치료법으로 사망 때문에 여전히 종양 세포의 침윤이 개발되었다 모방 부족한 실험실 분석이다. 병아리 심장 분석의 목적은 입체적 종양 침윤을 연구하는 중요한 장기 배양 방법을 제공하는 것이다. 분석은 침습적 및 비 침습적 세포를 구별하고, 종양 침윤에 대한 시험 화합물의 효과를 연구 할 수있다. 분석의 사용 배후의 이론적 근거는 종양은 종양 세포가 지속적으로 기질 (숙주 세포 및 세포 외 기질)과 상호 작용 생태계 있다는 실제적인 개념이며, 이들 분자 상호 침입 통해 그 미세 조정 (1)이다. 그래서, 분석에서 종양 세포 livi 직면종양 세포에 의해 침윤 용 기판으로서, 또한 간질 세포와 행렬 요소의 다른 유형의 소스로서 역할뿐만 NG 병아리 배아 심장 단편 2. 병아리 심장 근육 세포, 섬유 아세포 및 내피 세포를 포함하고, 세포 외 매트릭스는 라미닌, 피브로넥틴 및 콜라겐의 상이한 유형으로 구성된다. 이와 같이, 삼차원 기관 배양 기술은 환자의 종양 침윤에 관련 많은 세포와 분자의 상호 작용을 포함한다. 병아리 심장 분석의 주요 장점은 간질 효과를 구현 한 것이다. 이 측면은 기저막 3 간질 매트릭스 4 분자 구성이 아닌 생활 젤로 종양 세포 침공을 기반으로 체외에서 다른 침략 분석보다 더 완료됩니다. 장기 배양 실험에서 발견 된 바와 같이, 종양 세포 및 정상 생활 숙주 조직 사이의 대결의 개념은 여러 가지로 도입 된독일 7 울프와 슈나이더 프랑스 5, 영국 6 Easty과 Easty 및 Schleich을 포함하여 저자. 위에 인용 된 방법 병아리 심장 침윤 분석의 기술적 장점은 두 조각의 양이 용이하게 표준화 될 수 있도록, 그들은 기관 배양 물 중에 기능적 무결성 모니터링을 허용 수축성 남아 있다는 것이다. 그들은 쉽게 계란의 멸균 콘텐츠 해부 될 수 있기 때문에 또한, 조류 배아 바람직하다. 분석은 종양 세포로 둘러싸 복잡한 기질을 제공하여 병아리 chorioallantois 막 세이 8 컨셉 유사성을 갖는다. 분석은 성공적 9 및 HCT-8 (결장) 10 세포주 패밀리 MCF-7 (유방)과 같은 인간의 종양 침입 및 비 침습성 세포 변형을 구별하기 위해 적용되어왔다. 기술뿐만 아니라 잠재적으로 항 - 침습 화합물을 테스트하는 데 유용 <suP&gt; (11, 12). 상기 한 바와 같이, 그것은 작은 유기 분자의 구조 - 활성 관계의 확립을 위해 사용될 수있다. 분석은, 그러나, 계정으로 암 침공에 면역 세포의 기여를 고려하지 않습니다. 이는 기술이 때문에 조작의 높은 번호, 분석 런 제한된 숫자 (최대 30 배양)과 긴 턴어라운드 시간 (약 1 개월)의, 높은 처리량 분석 시스템으로 간주 될 수 없다는 것을 강조한다.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Ringer&#39;s salt solution</td>
			<td>Braun</td>
			<td>CEO123</td>
			<td></td>
		</tr>
		<tr>
			<td>Bacto-agar</td>
			<td>Becton Dickinson</td>
			<td>214010</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris-(hydroxymethyl)-aminomethaan Analar</td>
			<td>VWR</td>
			<td>103157P</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA : albumin from bovine serum, cohn V fract.</td>
			<td>Sigma</td>
			<td>A4503-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>MEM-Rega 3</td>
			<td>Life Technologies&#160;</td>
			<td>19993013</td>
			<td></td>
		</tr>
		<tr>
			<td>Gyrotory shakers</td>
			<td>New Brunswick Scientific</td>
			<td>G10 and G33</td>
			<td></td>
		</tr>
		<tr>
			<td>Erlenmeyer flasks 50 ml</td>
			<td>Novolab</td>
			<td>92717</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass Petri dishes</td>
			<td>Novolab</td>
			<td>68516</td>
			<td></td>
		</tr>
		<tr>
			<td>Iridectomy scissors</td>
			<td>Rumex</td>
			<td>11-0625</td>
			<td></td>
		</tr>
		<tr>
			<td>Macroscope with calibrated ocular grid</td>
			<td>Wild</td>
			<td>157702</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraffin wax</td>
			<td>International Medical products</td>
			<td>8599956</td>
			<td></td>
		</tr>
		<tr>
			<td>Eosin Y</td>
			<td>Sigma</td>
			<td>230251-25g</td>
			<td></td>
		</tr>
		<tr>
			<td>Harris&#39; hematoxylin</td>
			<td>Sigma</td>
			<td>HHS32-1L</td>
			<td></td>
		</tr>
		<tr>
			<td>Coverslipping resin (Tissue-Tek)</td>
			<td>Sakura</td>
			<td>4494</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraffin melting apparatus</td>
			<td>GFL</td>
			<td>1052</td>
			<td></td>
		</tr>
		<tr>
			<td>Microtome for paraffin sectioning</td>
			<td>Reichert</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Stppers for Erlemeyer flasks with gas in- and outlets</td>
			<td>Novolab</td>
			<td>2602421 and 260243</td>
			<td></td>
		</tr>
		<tr>
			<td>Diaminobenzidine</td>
			<td>Sigma</td>
			<td>D8001</td>
			<td></td>
		</tr>
		<tr>
			<td>REAX rocking table</td>
			<td>Heidolph</td>
			<td>54131</td>
			<td></td>
		</tr>
		<tr>
			<td>24-well tissue dishes</td>
			<td>VWR</td>
			<td>NUNC142475</td>
			<td></td>
		</tr>
		<tr>
			<td>Ophtalmological enucleation spoon</td>
			<td>Rumex</td>
			<td>16-060</td>
			<td></td>
		</tr>
		<tr>
			<td>Sharp forceps</td>
			<td>Rumex</td>
			<td>4-111T</td>
			<td></td>
		</tr>
		<tr>
			<td>Blunt forceps</td>
			<td>Nickel-Electro LTD</td>
			<td>7112</td>
			<td></td>
		</tr>
		<tr>
			<td>Erlenmeyer flasks 5 ml</td>
			<td>Novolab</td>
			<td>211901</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope slides washed and degreased</td>
			<td>International Medical products</td>
			<td>8613908</td>
			<td></td>
		</tr>
	</tbody>
</table>

chick heart invasion assay for testing the invasiveness of cancer cells and the activity of potentially anti-invasive compounds

병아리 심장 분석의 목적은 입체적 종양 침윤을 연구하는 중요한 장기 배양 방법을 제공하는 것이다. 분석은 침습적 및 비 침습적 세포를 구별하고, 종양 침윤에 대한 시험 화합물의 효과에 대한 연구를 가능하게 할 수있다. 암 세포 - 중 집계 또는 단일 세포로 - 배아 병아리 마음의 조각에 직면하고 있습니다. 며칠 또는 몇 주 동안 정지에서 기관 배양 후 직면 문화는 고정 및 조직 학적 분석을 위해 파라핀에 포함. 암세포 및 정상 조직 사이의 입체적인 상호 작용은 다음 헤 마톡 실린 - 에오신으로 또는 심장 조직의 에피토프 또는 대향하는 암 세포에 대한 면역 조직 화학 염색을 한 후 염색 직렬 섹션에서 재구성된다. 분석은 암세포 침윤이 암세포 및 그들의 이웃 기질 호스트 소자 (근육 섬유 모세포, endoth 분자 사이의 상호 작용의 결과 인 것으로 최근의 개념과 일치elial 세포, 세포 외 매트릭스 성분 등). 여기서,이 기질은 환경 생체 조직 절편 같은 암세포에 제공된다. 분석의 관련성에 대한 지원은 여러 양태이다. 프로그레시브 직업 및 교체 시간과 숙주 조직의 공간 및 침입과 대향 세포의 생체 내에서 비 - 침입 일반적 분석의 결과와 상관 관계 : 분석에서 내습 암 침윤의 기준에 따른다. 또한, 생체 내에서 세포의 침입 패턴은 병리학 자에 의해 정의 된 바와 같이, 분석에서 조직 학적 이미지에 반영된다. 많은 잠재적으로 항 - 침습 유기 동족체 화합물과 얻어진 결과의 정량 구조 - 활성 관계 (QSAR) 분석은 임상에서 사용되는 플라보노이드 및 칼콘, 공지 된 항 - 전이성 약물 구조 - 활성 관계 (예를 들어, 미세 소관 억제제의 연구를 허용 )뿐만 아니라 분석에서 침입을 억제한다. HoweveR, 분석은 암 침공 계정 면역 학적 기여를 고려하지 않습니다.

도 5에 제시된 조직 학적 단면은 성공적인 분석의 숫자의 최종 결과를 나타낸다. 문화의 소리 조직학가 가능한 세포를 표시하고 종양 세포와 정상 조직 사이의 상호 작용을 해석 할 수 있습니다. 또한, 정상 조직에서 어떠한 면역 반응은 면역 거부 시스템이 개발되기 전에, 예를 들어 사용 된 병아리 배아의 정확한 기간을 확인하는 관찰 할 수 없다. 배양 기간 동안 (총) 오염의 부재를 나타?...

Watch this Scientific Journal Video about Chick Heart Invasion Assay for Testing the Invasiveness of Cancer Cells and the Activity of Potentially Anti-invasive Compounds at JoVE.com

Chick Heart Invasion Assay for Testing the Invasiveness of Cancer Cells and the Activity of Potentially Anti-invasive Compounds

여기서는 입체적 정상 조직 절편에 살아있는 종양 세포의 침윤을 연구 프로토콜을 제시한다. 이 기관 배양 기술은 주로 시험 관내에서 잠재적으로 항 - 침습 약물을 시험에 적용된다.

테스트를위한 병아리 심장 침략 분석 암 세포의 침윤과 잠재적 안티 침략 화합물의 활동

The goal of the chick heart assay is to offer a relevant organ culture method to study tumor invasion in three dimensions. The assay can distinguish ...

chick-heart-invasion-assay-for-testing-invasiveness-cancer-cells

Research

JoVE Journal

Medicine

8.9K 조회수.  University of Ghent.  여기서는 입체적 정상 조직 절편에 살아있는 종양 세포의 침윤을 연구 프로토콜을 제시한다. 이 기관 배양 기술은 주로 시험 관내에서 잠재적으로 항 - 침습 약물을 시험에 적용된다. 

비디오: Chick Heart Invasion Assay for Testing the Invasiveness of Cancer Cells and the Activity of Potentially Anti-invasive Compounds

Modeling and Imaging 3-Dimensional Collective Cell Invasion

A defining characteristic of cancer malignancy is invasion and metastasis 1. In some cancers (e.g. glioma 2), local invasion into surrounding healthy tissue is the root cause of disease and death. For other cancers (e.g. breast, lung, etc.), it is the process of metastasis, in which tumor cells move from a primary tumor mass, colonize distal sites and ultimately contribute to organ failure, that eventually leads to morbidity and mortality 3. It has been estimated that invasion and metastasis are responsible for 90&#37; of cancer deaths 4. As a result, there has been intense interest in identifying the molecular processes and critical protein mediators of invasion and metastasis for the purposes of improving diagnosis and treatment 5.
A challenge for cancer scientists is to develop invasion assays that sufficiently resemble the in vivo situation to enable accurate disease modeling 6. Two-dimensional cell motility assays are only informative about one aspect of invasion and do not take into account extracellular matrix (ECM) protein remodeling which is also a critical element. Recently, research has refined our understanding of tumor cell invasion and revealed that individual cells may move by elongated or rounded modes 7. In addition, there has been greater appreciation of the contribution of collective invasion, in which cells invade in strands, sheets and clusters, particularly in highly differentiated tumors that maintain epithelial characteristics, to the spread of cancer 8.
We present a refined method 9 for examining the contributions of candidate proteins to collective invasion 10. In particular, by engineering separate pools of cells to express different fluorescent proteins, it is possible to molecularly dissect the activities and proteins required in leading cells versus those required in following cells. The use of RNAi provides the molecular tool to experimentally disassemble the processes involved in individual cell invasion as well as in different positions of collective invasion. In this procedure, mixtures of fluorescently-labeled cells are plated on the bottom of a Transwell insert previously filled with Matrigel ECM protein, then allowed to invade "upwards" through the filter and into the Matrigel. Reconstruction of z-series image stacks, obtained by confocal imaging, into three-dimensional representations allows for visualization of collectively invading strands and analysis of the representation of fluorescently-labeled cells in leading versus following positions.

Models of tumor cell invasion into three-dimensional extracellular matrix better reflect the in vivo situation than two-dimensional motility assays. Using matrix invasion assays combined with confocal imaging of fluorescently-labeled cells, detailed information on invasion modes and the distinct contributions of leading versus following cells can be obtained.

모델링 및 3 차원 이미징 단체 셀 침략

A defining characteristic of cancer malignancy is invasion and metastasis 1. In some cancers (e.g. glioma 2), local invasion into surrounding healthy ...

연구

의학

Minimal Erythema Dose (MED) Testing

Ultraviolet radiation (UV) therapy is sometimes used as a treatment for various common skin conditions, including psoriasis, acne, and eczema. The dosage of UV light is prescribed according to an individual's skin sensitivity. Thus, to establish the proper dosage of UV light to administer to a patient, the patient is sometimes screened to determine a minimal erythema dose (MED), which is the amount of UV radiation that will produce minimal erythema (sunburn or redness caused by engorgement of capillaries) of an individual's skin within a few hours following exposure. This article describes how to conduct minimal erythema dose (MED) testing. There is currently no easy way to determine an appropriate UV dose for clinical or research purposes without conducting formal MED testing, requiring observation hours after testing, or informal trial and error testing with the risks of under- or over-dosing. However, some alternative methods are discussed.

Minimal Erythema Dose MED Testing

This article describes how to conduct minimal erythema dose (MED) testing in order to determine the lowest dose of ultraviolet radiation that will cause erythema (burning) when administered to an individual.

최소 홍반 량 (MED) 시험

Ultraviolet radiation (UV) therapy is  sometimes used as a treatment for various common skin conditions, including  psoriasis, acne, and eczema. The ...

Quantification of Breast Cancer Cell Invasiveness Using a Three-dimensional (3D) Model

It is now well known that the cellular and tissue microenvironment are critical regulators influencing tumor initiation and progression. Moreover, the extracellular matrix (ECM) has been demonstrated to be a critical regulator of cell behavior in culture and homeostasis in vivo. The current approach of culturing cells on two-dimensional (2D), plastic surfaces results in the disturbance and loss of complex interactions between cells and their microenvironment. Through the use of three-dimensional (3D) culture assays, the conditions for cell-microenvironment interaction are established resembling the in vivo microenvironment. This article provides a detailed methodology to grow breast cancer cells in a 3D basement membrane protein matrix, exemplifying the potential of 3D culture in the assessment of cell invasion into the surrounding environment. In addition, we discuss how these 3D assays have the potential to examine the loss of signaling molecules that regulate epithelial morphology by immunostaining procedures. These studies aid to identify important mechanistic details into the processes regulating invasion, required for the spread of breast cancer.

Quantification of Breast Cancer Cell Invasiveness Using a Three-dimensional 3D Model

This article provides detailed methodologies for the use of three-dimensional (3D) assays to quantify breast cancer cell invasion. Specifically, we discuss the procedures required to set up such assays, quantification, and data analysis, as well as methods to examine the loss of membrane integrity that occurs when cells invade.

3 차원 (3D) 모델을 사용하여 유방암 세포 침윤의 정량화

It is now well known that the cellular and tissue microenvironment are critical regulators influencing tumor initiation and progression. Moreover, the ...

A Modified In vitro Invasion Assay to Determine the Potential Role of Hormones, Cytokines and/or Growth Factors in Mediating Cancer Cell Invasion

Blood serum serves as a chemoattractant towards which cancer cells migrate and invade, facilitating their intravasation into microvessels. However, the actual molecules towards which the cells migrate remain elusive. This modified invasion assay has been developed to identify targets which drive cell migration and invasion. This technique compares the invasion index under three conditions to determine whether a specific hormone, growth factor, or cytokine plays a role in mediating the invasive potential of a cancer cell. These conditions include i) normal fetal bovine serum (FBS), ii) charcoal-stripped FBS (CS-FBS), which removes hormones, growth factors, and cytokines and iii) CS-FBS + molecule (denoted &#8220;X&#8221;). A significant change in cell invasion with CS-FBS as compared to FBS, indicates the involvement of hormones, cytokines or growth factors in mediating the change. Individual molecules can then be added back to CS-FBS to assay their ability to reverse or rescue the invasion phenotype. Furthermore, two or more factors can be combined to evaluate the additive or synergistic effects of multiple molecules in driving or inhibiting invasion. Overall, this method enables the investigator to determine whether hormones, cytokines, and/or growth factors play a role in cell invasion by serving as chemoattractants or inhibitors of invasion for a particular type of cancer cell or a specific mutant. By identifying specific chemoattractants and inhibitors, this modified invasion assay may help to elucidate signaling pathways that direct cancer cell invasion.

A Modified In vitro Invasion Assay to Determine the Potential Role of Hormones, Cytokines and/or Growth Factors in Mediating Cancer Cell Invasion

This video article describes a modified in vitro method to examine the role of hormones, cytokines and/or growth factors in driving cancer cell invasion.

수정<em&gt; 시험관</em&gt; 침공 분석은 호르몬, 중재 암 세포 침략의 사이토 카인 및 / 또는 성장 인자의 잠재적 인 역할을 확인하는

Blood serum serves as a chemoattractant towards which cancer cells migrate and invade, facilitating their intravasation into microvessels. However, the ...

Assessment of Ovarian Cancer Spheroid Attachment and Invasion of Mesothelial Cells in Real Time

Ovarian cancers metastasize by shedding into the peritoneal fluid and dispersing to distal sites within the peritoneum. Monolayer cultures do not accurately model the behaviors of cancer cells within a nonadherent environment, as cancer cells inherently aggregate into multicellular structures which contribute to the metastatic process by attaching to and invading the peritoneal lining to form secondary tumors. To model this important stage of ovarian cancer metastasis, multicellular aggregates, or spheroids, can be generated from established ovarian cancer cell lines maintained under nonadherent conditions. To mimic the peritoneal microenvironment encountered by tumor cells in vivo, a spheroid-mesothelial co-culture model was established in which preformed spheroids are plated on top of a human mesothelial cell monolayer, formed over an extracellular matrix barrier. Methods were then developed using a real-time cell analyzer to conduct quantitative real time measurements of the invasive capacity of different ovarian cancer cell lines grown as spheroids. This approach allows for the continuous measurement of invasion over long periods of time, which has several advantages over traditional endpoint assays and more laborious real time microscopy image analyses. In short, this method enables a rapid, determination of factors which regulate the interactions between ovarian cancer spheroid cells invading through mesothelial and matrix barriers over time.

Ovarian cancer cell invasion into the mesothelial lining of the peritoneum is a dynamic process over time. Utilizing a real time analyzer, the invasive capacity of ovarian cancer cells in a spheroid-mesothelial cell co-culture model can be quantified over prolonged time periods, providing insights into factors regulating the metastatic process.

실시간으로 중피 세포의 난소 암 회전 타원체의 부착과 침략의 평가

Ovarian cancers metastasize by shedding into the peritoneal fluid and dispersing to distal sites within the peritoneum. Monolayer cultures do not ...

Three-Dimensional (3D) Tumor Spheroid Invasion Assay

Invasion of surrounding normal tissues is generally considered to be a key hallmark of malignant (as opposed to benign) tumors. For some cancers in particular (e.g., brain tumors such as glioblastoma multiforme and squamous cell carcinoma of the head and neck &#8211; SCCHN) it is a cause of severe morbidity and can be life-threatening even in the absence of distant metastases. In addition, cancers which have relapsed following treatment unfortunately often present with a more aggressive phenotype. Therefore, there is an opportunity to target the process of invasion to provide novel therapies that could be complementary to standard anti-proliferative agents. Until now, this strategy has been hampered by the lack of robust, reproducible assays suitable for a detailed analysis of invasion and for drug screening. Here we provide a simple micro-plate method (based on uniform, self-assembling 3D tumor spheroids) which has great potential for such studies. We exemplify the assay platform using a human glioblastoma cell line and also an SCCHN model where the development of resistance against targeted epidermal growth factor receptor (EGFR) inhibitors is associated with enhanced matrix-invasive potential. We also provide two alternative methods of semi-automated quantification: one using an imaging cytometer and a second which simply requires standard microscopy and image capture with digital image analysis.

Three-Dimensional 3D Tumor Spheroid Invasion Assay

Invasion of surrounding normal tissues is a defining characteristic of malignant tumors. We provide here a simple, semi-automated micro-plate assay of invasion into a natural 3D biomatrix that has been exemplified with a number of models of advanced human cancers.

3 차원 (3D) 종양 회전 타원체 침공 분석

Invasion of surrounding normal tissues is generally considered to be a key hallmark of malignant (as opposed to benign) tumors. For some cancers in ...

Utilization of the Soft Agar Colony Formation Assay to Identify Inhibitors of Tumorigenicity in Breast Cancer Cells

Given the inherent difficulties in investigating the mechanisms of tumor progression in vivo, cell-based assays such as the soft agar colony formation assay (hereafter called soft agar assay), which measures the ability of cells to proliferate in semi-solid matrices, remain a hallmark of cancer research. A key advantage of this technique over conventional 2D monolayer or 3D spheroid cell culture assays is the close mimicry of the 3D cellular environment to that seen in vivo. Importantly, the soft agar assay also provides an ideal tool to rigorously test the effects of novel compounds or treatment conditions on cell proliferation and migration. Additionally, this assay enables the quantitative assessment of cell transformation potential within the context of genetic perturbations. We recently identified peptidylarginine deiminase 2 (PADI2) as a potential breast cancer biomarker and therapeutic target. Here we highlight the utility of the soft agar assay for preclinical anti-cancer studies by testing the effects of the PADI inhibitor, BB-Cl-amidine (BB-CLA), on the tumorigenicity of human ductal carcinoma in situ (MCF10DCIS) cells.

Here, we document the use of the soft agar colony formation assay to test the effects of a peptidylarginine deiminase (PADI) enzyme inhibitor, BB-Cl-amidine, on breast cancer tumorigenicity in vitro.

소프트 한천 콜로니 형성 분석의 활용은 유방암 세포에서 종양 유발의 억제를 확인하는

Given the inherent difficulties in investigating the mechanisms of tumor progression in vivo, cell-based assays such as the soft agar colony formation ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor initiation, metastasis and resistance to radio- and chemotherapy. Here we describe a specific methodology for culturing primary human pancreatic CSCs as tumor spheres in anchorage-independent conditions. Cells are grown in serum-free, non-adherent conditions in order to enrich for CSCs while their more differentiated progenies do not survive and proliferate during the initial phase following seeding of single cells. This assay can be used to estimate the percentage of CSCs present in a population of tumor cells. Both size (which can range from 35 to 250 micrometers) and number of tumor spheres formed represents CSC activity harbored in either bulk populations of cultured cancer cells or freshly harvested and digested tumors 1,2. Using this assay, we recently found that metformin selectively ablates pancreatic CSCs; a finding that was subsequently further corroborated by demonstrating diminished expression of pluripotency-associated genes/surface markers and reduced in vivo tumorigenicity of metformin-treated cells. As the final step for preclinical development we treated mice bearing established tumors with metformin and found significantly prolonged survival. Clinical studies testing the use of metformin in patients with PDAC are currently underway (e.g., NCT01210911, NCT01167738, and NCT01488552). Mechanistically, we found that metformin induces a fatal energy crisis in CSCs by enhancing reactive oxygen species (ROS) production and reducing mitochondrial transmembrane potential. In contrast, non-CSCs were not eliminated by metformin treatment, but rather underwent reversible cell cycle arrest. Therefore, our study serves as a successful example for the potential of in vitro sphere formation as a screening tool to identify compounds that potentially target CSCs, but this technique will require further in vitro and in vivo validation to eliminate false discoveries.

Pancreatic cancer stem cells (CSCs) can be expanded in vitro using the anchorage-independent sphere culture technique, which represents a powerful tool to study CSC biology and can serve as the first step to develop novel CSC-targeting therapies. Here the methodology for expanding, analyzing and targeting of pancreatic CSCs is provided.

췌장암 새로운 치료 전략을 개발하기위한 전지 특성 줄기 공부

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor ...

Isolation and Propagation of Circulating Tumor Cells from a Mouse Cancer Model

Cancer metastasis is the foremost cause of cancer-associated deaths. Recent studies have shown that circulating tumor cells (CTCs) are important in cancer metastasis. Indeed, the number of CTCs correlates with tumor size. Here, a detailed description is provided of a methodology for isolation and propagation of CTCs from a syngeneic mouse model of hepatocellular carcinoma (HCC) which allows for downstream analysis of potentially important molecular mechanisms of solid organ tumor metastasis. This method is efficient and reproducible. It is a non-invasive technique and, therefore, has potential to replace the invasive biopsy of tissues from humans which may be associated with complications. Therefore, the method discussed here allows for the isolation and propagation of CTCs from whole blood samples such that they can be examined and characterized. This has potential for future adaptation for clinical applications such as diagnosis, and personalized targeted therapy.

Circulating tumor cells (CTCs) have been shown to play an important role in tumor metastasis. Here, a method for the isolation and propagation of CTCs from the whole blood of a syngeneic mouse tumor model of hepatocellular carcinoma (HCC) metastasis is described.

마우스 종양 모델에서 종양 세포 순환의 분리 및 전파

Cancer metastasis is the foremost cause of cancer-associated deaths. Recent studies have shown that circulating tumor cells (CTCs) are important in cancer ...

Three-Dimensional Culture Assay to Explore Cancer Cell Invasiveness and Satellite Tumor Formation

Mammalian cell culture in monolayers is widely used to study various physiological and molecular processes. However, this approach to study growing cells often generates unwanted artifacts. Therefore, cell culture in a three-dimensional (3D) environment, often using extracellular matrix components, emerged as an interesting alternative due to its close similarity to the native in vivo tissue or organ. We developed a 3D cell culture system using two compartments, namely (i) a central compartment containing cancer cells embedded in a collagen gel acting as a pseudo-primary macrospherical tumor and (ii) a peripheral cell-free compartment made of a fibrin gel, i.e. an extracellular matrix component different from that used in the center, in which cancer cells can migrate (invasion front) and/or form microspherical tumors representing secondary or satellite tumors. The formation of satellite tumors in the peripheral compartment is remarkably correlated to the known aggressiveness or metastatic origin of the native tumor cells, which makes this 3D culture system unique. This cell culture approach might be considered to assess cancer cell invasiveness and motility, cell-extracellular matrix interactions and as a method to evaluate anti-cancer drug properties.

Cancer cells are embedded in a collagen gel and then sandwiched in an acellular fibrin gel to generate a 3D culture system in which the invasiveness and formation of satellite tumors may be monitored.

암 세포 침윤 및 위성 종양 형성을 탐색하는 문화 분석 세 가지 차원

Mammalian cell culture in monolayers is widely used to study various physiological and molecular processes. However, this approach to study growing cells ...