作者感谢乳腺癌研究基金会和先进医学研究基金会的支持。

这里描述的所有方法都已被波士顿儿童医院机构生物安全委员会批准。
1. 细胞制剂
<ol><li>
解冻 KHOS 细胞
	<ol>
<li>预热培养基,即, Dulbecco 的改性鹰培养基辅以 10% (卷/卷) 胎小牛血清 (血清) 和 1% (卷/卷) 青霉素/链霉素。</li>
		<li>从液氮罐中取去低温小瓶, 将瓶子的底部浸入37摄氏度水浴中的温水中, 轻轻摇动低温瓶以加速解冻过程。保持低温小瓶的盖子不接触水, 以避免污染。一旦细胞解冻, 通过喷洒70% 乙醇溶液和擦拭瓶子来消毒低温小瓶。</li>
		<li>在层流罩中, 立即从低温小瓶中吸出细胞悬浮液, 并在10毫升加热培养基中轻轻悬浮 (1.1.1 中描述)。离心机细胞悬浮在大约 200 x g 为 5-7 分钟。</li>
		<li>并用重悬细胞颗粒与10毫升加热培养基, 并转移到一个 T75 瓶。用10毫升的吸管轻轻地用吸管多次悬浮细胞。</li>
		<li>将烧瓶放入一个37°c 孵化器与 5% (卷/卷) CO2和湿润气氛。允许单元格附加至少12小时。</li>
		<li>孵育细胞大约 3-7 天直到 80-100% 汇合。每 2-3 天改变培养基。</li>
	</ol>
</li>
	<li>
分裂 KHOS 和 N 细胞
	<ol>
<li>从烧瓶中移除培养基介质。</li>
		<li>用5毫升不含钙和镁的无菌 PBS (1x) 冲洗细胞, 去除不附着的细胞或分数。</li>
		<li>在烧瓶中加入1毫升0.05% 胰蛋白酶-edta 溶液, 并简单地旋转烧瓶, 以确保胰蛋白酶-edta 均匀分散。</li>
		<li>在37摄氏度孵化器或 3-5 分钟的室温下孵育烧瓶 2-3 分钟。检查显微镜下的细胞在大约400x 倍放大, 以确保细胞被四舍五入和分离。</li>
		<li>一旦细胞分离, 添加10毫升培养基, 并轻轻地将培养基上下移几次, 用10毫升的吸管将细胞团聚体分解成单个细胞悬浮。</li>
		<li>使用单元格计数器计数单元格。将细胞悬浮到新的 T75 烧瓶中, 密度为 2 x 106细胞或比例为1:4。</li>
		<li>允许细胞生长到 80-100% 汇合之前, 播种的 QPI 实验。</li>
	</ol>
</li>
	<li>
QPI 种子 KHOS 细胞
	<ol>
<li>种子 KHOS-6 井板中的 A 和 N 细胞密度为 5万-30万细胞/以及5毫升培养基后计数的细胞计数器。 注: 在图像采集过程中, 播种密度依赖于细胞增殖率和成像时间周期, 以 < 100% 汇合。</li>
		<li>孵育细胞至少12小时, 以允许附着。</li>
		<li>在进行 QPI 前, 用新鲜的媒介改变培养基。</li>
	</ol>
</li>
</ol>2. QPI 收购
<ol><li>
盖板滑动设置
	<ol>
<li>用75% 乙醇溶液浸泡15分钟, 清洗好几次, 用运行去离子水冲洗, 并消毒. 把盖子滑入一个不育的层流罩中晾干。</li>
		<li>小心地把盖子滑到一个6井板上, 以避免明显的气泡。确保盖板底部浸入培养基中 (最小5毫升/井)。</li>
		<li>将盘子放入孵化器 (37 °c, 5% CO2和湿润气氛) 到平衡至少15分钟。如果在盖子上有雾, 用无菌棉签擦拭干净。</li>
	</ol>
</li>
	<li>
用显微镜成像细胞
	<ol>
<li>打开软件初始化系统, 包括曝光时间的自标定、模式对比度和全息图噪声。请确保这些值是可接受的 (以绿色或黄色范围显示)。</li>
		<li>把6井板放到 QPI 显微镜的舞台上。</li>
		<li>单击 "实时捕获", 然后在 "软件焦点" 中选择 "手动"。通过调整 "显微镜设置" 中的工作距离, 粗略地聚焦细胞的相位图像, 获得相图中细胞的轮廓。在 "软件焦点" 中更改为 "自动"。</li>
		<li>点击 "新实验" 来创建一个新的实验。首先使用鼠标或数字键盘上的箭头键选择图像采集位置。在每次选择后单击 "记住"。通常 3-5 个位置为每个样品被选择。</li>
		<li>设置 "Timelapse" 部分的成像间隔和时间段。 注意: 要清楚地跟踪癌细胞, 间隔应短于5分钟, 48 小时设置为组合 QPI 分析的时间段。</li>
		<li>通过单击 "捕获" 来启动实验, 它将自动聚焦并在设置时间点获取图像。</li>
	</ol>
</li>
</ol>3. 数据分析
<ol><li>
细胞形态学分析
	<ol>
<li>单击 "识别单元格"。调整 "背景阈值" 的数值设置, 使单元格区域与背景噪音很好地分离。调整 "对象大小" 的设置编号, 以确保每个单元格都有一个核。为需要比较的样本保持一致的参数, 因为这可能会影响最终的面积和体积测量。如果需要, 请使用 "手动更改" 手动修改单元格段。</li>
		<li>使用软件中的 "分析数据" 功能对单元格进行分析。通过单击 "新分析" 开始新的数据分析。将所选图像拖动到 "源框架" 选项卡和单元格形态学参数, 包括单元格区域 (µm2)、光学厚度 (µm) 和每个单元格的卷 (µm3)。选择散点图或直方图模式, 并将数据导出为表或数字。</li>
	</ol>
</li>
	<li>
细胞增殖分析
	<ol>
<li>为不同的时间点选择至少5个图像 (例如、初始、12 h、24 h、36 h 和 48 h)。记录由软件提供的单元格编号。</li>
		<li>为了估计加倍时间, 在与初始数正常化后绘制细胞数, 并拟合指数生长曲线。</li>
	</ol>
</li>
	<li>
细胞运动分析
	<ol>
<li>在软件中使用 "跟踪单元" 功能。单击 "新分析" 开始新的数据分析。在 "源框架" 选项卡中, 将选定时间段 (例如、4小时后的 24 h 孵化) 中的图像拖动一系列. 通过单击 "添加单元格" 选择模式下的单元格随机选择 10-30 单元格。排除边缘的单元格和移出视图的区域。</li>
		<li>仔细检查图像序列中的跟踪。如果系统失去对单元格的跟踪或跟踪错误的单元格, 请通过单击 "确定" 以识别单元格, 或单击 "选择" 模式下的 "修改位置" 来修改单元格位置, 手动调整跟踪单元格。</li>
		<li>选择 "剧情运动" 中的细胞轨迹的玫瑰图, 或在 "剧情特征" (如运动速度 (µm)、运动 (µm)、迁移 (µm) 和迁移直接性等与运动相关的参数上绘制图。将数据导出为表或数字。</li>
	</ol>
</li>
</ol>

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没有宣布利益冲突。

在本研究中, 我们描述了一个体外, 无创, 无标签的方法, 使用 QPI 定量表征人骨肉瘤细胞的血管生成和非血管生成表型。采用综合、高通量的方法对多细胞参数进行了分析, 包括细胞面积、细胞厚度、细胞体积、增殖率、加倍时间、迁移直接、运动速度、迁移和运动。
与常规的细胞形态学和细胞行为评估方法相比, 该法不仅节省了时间和人工, 而且提供了更准确、更详细?...

在固体肿瘤的发展和进展中, 最早的检查点之一是获得血管生成表型, 这是癌症的标志。这一进展涉及各种生物化学和分子过程1,2,3。研究肿瘤进展这一关键步骤的技术挑战是缺乏连续和定量地对活癌细胞的血管生成和非血管生成性表型进行无偏定性和鉴别的工具。传统的检测方法用于研究血管生成和非血管生成细胞的细胞行为, 通常需要昂贵的试剂和仪器, 例如细胞增殖/迁移分析4,5, 6,7,8,9,10,11,12,13,14或互补的在体内评估4,5,6,8,15,16, 以及需要大量的专业知识和密集的时间和劳动消费。
最近, 定量相位成像 (QPI) 已经成为一种新的技术, 它能够对各种细胞形态学和行为参数进行时间推移和无标记的评估17,18,19,20,21,22. 与常规光学显微术不同, QPI 在光通过光学物体后按像素量化相移像素的变化, 并重建具有转换的光学厚度和体积的全息图像, 从而使直接对活细胞的分析和以下特点: (1) 定量成像, (2) 无创和时间推移成像, (3) 无标签成像, (4) 同时多参数成像。这些特性使 QPI 成为评估和理解细胞级病理过程的有力工具。
在最近的一项研究中, 我们利用 QPI 定量地定性和区分人骨肉瘤细胞的血管生成 KHOS 和非血管生成 KHOS-N 表型, 结合细胞形态学分析,扩散和运动23。利用图像分析软件, 对血管生成体与非血管生成性人骨肉瘤细胞的细胞形态和行为参数进行了定量比较, 并在这两个方面确定了五种特征差异。型.这种新的方法提供了一个综合和定量的平台, 以评估各种生物相关的细胞特征。

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	<tbody>
		<tr height="33">
			<td height="33" style="height:34px;width:227px;">T75 flask</td>
			<td style="width:227px;">Corning, NY, USA</td>
			<td style="width:227px;">353136</td>
			<td style="width:227px;"></td>
		</tr>
		<tr height="33">
			<td height="33" style="height:34px;width:227px;">6-well plates&#160;</td>
			<td style="width:227px;">Corning, NY, USA</td>
			<td style="width:227px;">3506</td>
			<td></td>
		</tr>
		<tr height="48">
			<td height="48" style="height:48px;width:227px;">Dulbecco&#8217;s modified Eagle medium (DMEM)</td>
			<td style="width:227px;">Thermo Fisher Scientific, MA, USA</td>
			<td style="width:227px;">11965092</td>
			<td></td>
		</tr>
		<tr height="33">
			<td height="33" style="height:34px;width:227px;">Fetal bovine serum (FBS)&#160;</td>
			<td style="width:227px;">Atlanta Biologicals, GA, USA</td>
			<td style="width:227px;">S11550</td>
			<td></td>
		</tr>
		<tr height="33">
			<td height="33" style="height:34px;width:227px;">Penicillin Streptomycin</td>
			<td style="width:227px;">Thermo Fisher Scientific, MA, USA</td>
			<td style="width:227px;">15140122</td>
			<td></td>
		</tr>
		<tr height="33">
			<td height="33" style="height:34px;width:227px;">Phosphate buffered saline (PBS)</td>
			<td style="width:227px;">Thermo Fisher Scientific, MA, USA</td>
			<td style="width:227px;">10010023</td>
			<td></td>
		</tr>
		<tr height="33">
			<td height="33" style="height:34px;width:227px;">Beckman Z1 Coulter counter</td>
			<td style="width:227px;">Beckman Coulter, IN, USA</td>
			<td style="width:227px;">Z1&#160;</td>
			<td></td>
		</tr>
		<tr height="51">
			<td height="51" style="height:51px;width:227px;">HoloMonitor M4</td>
			<td style="width:227px;">Phase Holographic Imaging Phi AB, Lund, Sweden</td>
			<td style="width:227px;">M4</td>
			<td>Microscope</td>
		</tr>
		<tr height="51">
			<td height="51" style="height:51px;width:227px;">Hololid</td>
			<td style="width:227px;">Phase Holographic Imaging Phi AB, Lund, Sweden</td>
			<td style="width:227px;">PHI 8020</td>
			<td></td>
		</tr>
		<tr height="51">
			<td height="51" style="height:51px;width:227px;">HStudioM4</td>
			<td style="width:227px;">Phase Holographic Imaging Phi AB, Lund, Sweden</td>
			<td style="width:227px;">HStudioM4</td>
			<td>Software</td>
		</tr>
	</tbody>
</table>

a time-lapse, label-free, quantitative phase imaging study of dormant and active human cancer cells

血管生成表型的获得是肿瘤休眠逃逸的重要组成部分。虽然一些经典的体外检测 (例如、增殖、迁移和其他) 和在体内模型已经开发, 以调查和表征血管生成和非血管生成细胞表型, 这些方法是时间和劳动密集型, 往往需要昂贵的试剂和仪器, 以及重要的专业知识。在最近的一项研究中, 我们用一种新的定量相成像 (QPI) 技术来进行血管生成和非血管生成性人骨肉瘤 KHOS 细胞的时间推移和无标记的特征。用 QPI 定量测量和分析了细胞参数的一个小组, 包括胞形态学、增殖和运动。这一新颖和定量的方法提供了机会, 以持续和非侵入性研究相关的细胞过程, 行为, 和肿瘤细胞的特点和其他细胞类型的简单和综合的方式。本报告描述了我们的实验性协议, 包括细胞制备、QPI 采集和数据分析。

图 1描述了典型的细胞形态学特征。图像显示为全息图(图1A-b) 和2D 图像 (图1C D)。光学细胞厚度 (从折射率和光学路径长度计算) 通过线剖面或整个细胞测量来量化。绘制了用于整个单元格的 KHOS 和 KHOS 单元的面积和厚度的散射图, 如图 1EG所示, 这...

Watch this Scientific Journal Video about A Time-lapse, Label-free, Quantitative Phase Imaging Study of Dormant and Active Human Cancer Cells at JoVE.com

A Time-lapse, Label-free, Quantitative Phase Imaging Study of Dormant and Active Human Cancer Cells

应用定量相成像技术对休眠和活性癌细胞表型进行了表征。用一种简单的方法对细胞增殖、迁移和形态学测定进行了综合分析。

休眠和活性人肿瘤细胞的时间推移、无标签、定量相成像研究

The acquisition of the angiogenic phenotype is an essential component of the escape from tumor dormancy. Although several classic in vitro assays (e.g., ...

a-time-lapse-label-free-quantitative-phase-imaging-study-dormant

Research

JoVE Journal

Cancer Research

7.3K 次观看.  Boston Children's Hospital. 应用定量相成像技术对休眠和活性癌细胞表型进行了表征。用一种简单的方法对细胞增殖、迁移和形态学测定进行了综合分析。

视频: A Time-lapse, Label-free, Quantitative Phase Imaging Study of Dormant and Active Human Cancer Cells

A Syngeneic Mouse Model of Metastatic Renal Cell Carcinoma for Quantitative and Longitudinal Assessment of Preclinical Therapies

Renal cell carcinoma (RCC) affects &#62; 60,000 people in the United States annually, and ~ 30% of RCC patients have multiple metastases at the time of diagnosis. Metastatic RCC (mRCC) is incurable, with a median survival time of only 18 months. Immune-based interventions (e.g., interferon (IFN) and interleukin (IL)-2) induce durable responses in a fraction of mRCC patients, and multikinase inhibitors (e.g., sunitinib or sorafenib) or anti-VEGF receptor monoclonal antibodies (mAb) are largely palliative, as complete remissions are rare. Such shortcomings in current therapies for mRCC patients provide the rationale for the development of novel treatment protocols. A key component in the preclinical testing of new therapies for mRCC is a suitable animal model. Beneficial features that recapitulate the human condition include a primary renal tumor, renal tumor metastases, and an intact immune system to investigate any therapy-driven immune effector responses and the formation of tumor-induced immunosuppressive factors. This report describes an orthotopic mRCC mouse model that has all of these features. We describe an intrarenal implantation technique using the mouse renal adenocarcinoma cell line Renca, followed by the assessment of tumor growth in the kidney (primary site) and lungs (metastatic site).

Implementation of an orthotopic model of renal cell carcinoma in immunocompetent mice affords the investigator a clinically-relevant system defined by the presence of a primary renal tumor and lung metastases in the same animal. This system can be used to preclinically test a variety of treatments in vivo.

转移性的型号同源小鼠肾细胞癌的临床前疗法的定量和纵向评估

Renal cell carcinoma (RCC) affects &#62; 60,000 people in the United States annually, and ~ 30% of RCC patients have multiple metastases at the time of ...

科研

癌症研究

Phosphopeptide Enrichment Coupled with Label-free Quantitative Mass Spectrometry to Investigate the Phosphoproteome in Prostate Cancer

Phosphoproteomics involves the large-scale study of phosphorylated proteins. Protein phosphorylation is a critical step in many signal transduction pathways and is tightly regulated by kinases and phosphatases. Therefore, characterizing the phosphoproteome may provide insights into identifying novel targets and biomarkers for oncologic therapy. Mass spectrometry provides a way to globally detect and quantify thousands of unique phosphorylation events. However, phosphopeptides are much less abundant than non-phosphopeptides, making biochemical analysis more challenging. To overcome this limitation, methods to enrich phosphopeptides prior to the mass spectrometry analysis are required. We describe a procedure to extract and digest proteins from tissue to yield peptides, followed by an enrichment for phosphotyrosine (pY) and phosphoserine/threonine (pST) peptides using an antibody-based and/or titanium dioxide (TiO2)-based enrichment method. After the sample preparation and mass spectrometry, we subsequently identify and quantify phosphopeptides using liquid chromatography-mass spectrometry and analysis software.

This protocol describes a procedure to extract and enrich phosphopeptides from prostate cancer cell lines or tissues for an analysis of the phosphoproteome via mass spectrometry-based proteomics.

磷酸浓缩结合无标签定量质谱法研究前列腺癌的 Phosphoproteome

Phosphoproteomics involves the large-scale study of phosphorylated proteins. Protein phosphorylation is a critical step in many signal transduction ...

Using Microarrays to Interrogate Microenvironmental Impact on Cellular Phenotypes in Cancer

Understanding the impact of the microenvironment on the phenotype of cells is a difficult problem due to the complex mixture of both soluble growth factors and matrix-associated proteins in the microenvironment in vivo. Furthermore, readily available reagents for the modeling of microenvironments in vitro typically utilize complex mixtures of proteins that are incompletely defined and suffer from batch to batch variability. The microenvironment microarray (MEMA) platform allows for the assessment of thousands of simple combinations of microenvironment proteins for their impact on cellular phenotypes in a single assay. The MEMAs are prepared in well plates, which allows the addition of individual ligands to separate wells containing arrayed extracellular matrix (ECM) proteins. The combination of the soluble ligand with each printed ECM forms a unique combination. A typical MEMA assay contains greater than 2,500 unique combinatorial microenvironments that cells are exposed to in a single assay. As a test case, the breast cancer cell line MCF7 was plated on the MEMA platform. Analysis of this assay identified factors that both enhance and inhibit the growth and proliferation of these cells. The MEMA platform is highly flexible and can be extended for use with other biological questions beyond cancer research.

The purpose of the method presented here is to show how microenvironment microarrays (MEMA) can be fabricated and used to interrogate the impact of thousands of simple combinatorial microenvironments on the phenotype of cultured cells.

利用微阵列研究微环境对癌症细胞表型的影响

Understanding the impact of the microenvironment on the phenotype of cells is a difficult problem due to the complex mixture of both soluble growth ...

Yeast As a Chassis for Developing Functional Assays to Study Human P53

The finding that the well-known mammalian P53 protein can act as a transcription factor (TF) in the yeast S. cerevisiae has allowed for the development of different functional assays to study the impacts of 1) binding site [i.e., response element (RE)] sequence variants on P53 transactivation specificity or 2) TP53 mutations, co-expressed cofactors, or small molecules on P53 transactivation activity. Different basic and translational research applications have been developed. Experimentally, these approaches exploit two major advantages of the yeast model. On one hand, the ease of genome editing enables quick construction of qualitative or quantitative reporter systems by exploiting isogenic strains that differ only at the level of a specific P53-RE to investigate sequence-specificity of P53-dependent transactivation. On the other hand, the availability of regulated systems for ectopic P53 expression allows the evaluation of transactivation in a wide range of protein expression. Reviewed in this report are extensively used systems that are based on color reporter genes, luciferase, and the growth of yeast to illustrate their main methodological steps and to critically assess their predictive power. Moreover, the extreme versatility of these approaches can be easily exploited to study different TFs including P63 and P73, which are other members of TP53 gene family.

Presented here are four protocols to construct and exploit yeast Saccharomyces cerevisiae reporter strains to study human P53 transactivation potential, impacts of its various cancer-associated mutations, co-expressed interacting proteins, and the effects of specific small molecules.

酵母作为开发功能分析研究人类P53的机箱

The finding that the well-known mammalian P53 protein can act as a transcription factor (TF) in the yeast S. cerevisiae has allowed for the development of ...

Time-Lapse 2D Imaging of Phagocytic Activity in M1 Macrophage-4T1 Mouse Mammary Carcinoma Cells in Co-cultures

Tumor-associated macrophages (TAMs) have been identified as an important component for tumor growth, invasion, metastasis, and resistance to cancer therapies. However, tumor-associated macrophages can be harmful to the tumor depending on the tumor microenvironment and can reversibly alter their phenotypic characteristics by either antagonizing the cytotoxic activity of immune cells or enhancing anti-tumor response. The molecular actions of macrophages and their interactions with tumor cells (e.g., phagocytosis) have not been extensively studied. Therefore, the interaction between immune cells (M1/M2-subtype TAM) and cancer cells in the tumor microenvironment is now a focus of cancer immunotherapy research. In the present study, a live cell coculture model of induced M1 macrophages and mouse mammary 4T1 carcinoma cells was developed to assess the phagocytic activity of macrophages using a time-lapse video feature using phase-contrast, fluorescent, and differential interference contrast (DIC) microscopy. The present method can observe and document multipoint live-cell imaging of phagocytosis. Phagocytosis of 4T1 cells by M1 macrophages can be observed using fluorescent microscopy before staining 4T1 cells with carboxyfluorescein succinimidyl ester (CFSE). The current publication describes how to coculture macrophages and tumor cells in a single imaging dish, polarize M1 macrophages, and record multipoint events of macrophages engulfing 4T1 cells during 13 h of coculture.

Macrophage phagocytic activity against cancer cells, specifically 4T1 mouse mammary carcinoma cells, was imaged in this study. The live cell coculture model was established and observed using a combination of fluorescent and differential interference contrast microscopy. This assessment was imaged using imaging software to develop multipoint time-lapse video.

M1巨噬细胞-4T1小鼠乳腺癌细胞在共培养中噬菌体活性的时移2D成像

Tumor-associated macrophages (TAMs) have been identified as an important component for tumor growth, invasion, metastasis, and resistance to cancer ...

Studying Triple Negative Breast Cancer Using Orthotopic Breast Cancer Model

Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype with limited therapeutic options. When compared to patients with less aggressive breast tumors, the 5-year survival rate of TNBC patients is 77% due to their characteristic drug-resistant phenotype and metastatic burden. Toward this end, murine models have been established aimed at identifying novel therapeutic strategies limiting TNBC tumor growth and metastatic spread. This work describes a practical guide for the TNBC orthotopic model where MDA-MB-231 breast cancer cells suspended in a basement membrane matrix are implanted in the fourth mammary fat pad, which closely mimics the cancer cell behavior in humans. Measurement of tumors by caliper, lung metastasis assessment via in vivo and ex vivo imaging, and molecular detection are discussed. This model provides an excellent platform to study therapeutic efficacy and is especially suitable for the study of the interaction between the primary tumor and distal metastatic sites.

This work presets an advanced protocol to accurately assess tumor loading by detection of green fluorescent protein and bioluminescence signals as well as the integration of quantitative molecular detection technique.

研究使用正交乳腺癌模型的三阴性乳腺癌

Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype with limited therapeutic options. When compared to patients with less ...

Combined Conditional Knockdown and Adapted Sphere Formation Assay to Study a Stemness-Associated Gene of Patient-derived Gastric Cancer Stem Cells

Cancer stem cells (CSCs) are implicated in tumor initiation, development and recurrence after treatment, and have become the center of attention of many studies in the last decades. Therefore, it is important to develop methods to investigate the role of key genes involved in cancer cell stemness. Gastric cancer (GC) is one of the most common and mortal types of cancers. Gastric cancer stem cells (GCSCs) are thought to be the root of gastric cancer relapse, metastasis and drug resistance. Understanding GCSCs&#160;biology is needed to advance the development of targeted therapies and eventually to reduce mortality among patients. In this protocol, we present an experimental design using a conditional knockdown system and an adapted sphere formation assay to study the effect of clusterin on the stemness of patient-derived GCSCs. The protocol can be easily adapted to study both in vitro and in vivo function of stemness-associated genes in different types of CSCs.

In this protocol, we present an experimental design using a conditional knockdown system and an adapted sphere formation assay to study the effect of clusterin on the stemness of patient-derived GCSCs. The protocol can be easily adapted to study both in vitro and in vivo function of stemness-associated genes in different types of CSCs.

结合条件敲击和适应球形成测定研究患者衍生胃癌干细胞的干细胞相关基因

Cancer stem cells (CSCs) are implicated in tumor initiation, development and recurrence after treatment, and have become the center of attention of many ...

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.

肿瘤细胞外侧尾静脉注射后肺转移的病理分析

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

Immunofluorescence Imaging of DNA Damage and Repair Foci in Human Colon Cancer Cells

The purpose of the manuscript is to provide a step-by-step protocol for performing immunofluorescence microscopy to study the radiation-induced DNA damage response induced by neutron-gamma mixed-beam used in boron neutron capture therapy (BNCT). Specifically, the proposed methodology is applied for the detection of repair proteins activation which can be visualized as foci using antibodies specific to DNA double-strand breaks (DNA-DSBs). DNA repair foci were assessed by immunofluorescence in colon cancer cells (HCT-116) after irradiation with the neutron-mixed beam. DNA-DSBs are the most genotoxic lesions and are repaired in mammalian cells by two major pathways: non-homologous end-joining pathway (NHEJ) and homologous recombination repair (HRR). The frequencies of foci, immunochemically stained, for commonly used markers in radiobiology like &#947;-H2AX, 53BP1 are associated with DNA-DSB number and are considered as efficient and sensitive markers for monitoring the induction and repair of DNA-DSBs. It was established that &#947;-H2AX foci attract repair proteins, leading to a higher concentration of repair factors near a DSB. To monitor DNA damage at the cellular level, immunofluorescence analysis for the presence of DNA-PKcs representative repair protein foci from the NHEJ pathway and Rad52 from the HRR pathway was planned. We have developed and introduced a reliable immunofluorescence staining protocol for the detection of radiation-induced DNA damage response with antibodies specific for repair factors from NHEJ and HRR pathways and observed radiation-induced foci (RIF). The proposed methodology can be used for investigating repair protein that is highly activated in the case of neutron-mixed beam radiation, thereby indicating the dominance of the repair pathway.

Radiation-induced DNA damage response activated by neutron mixed-beam used in boron neutron capture therapy (BNCT) has not been fully established. This protocol provides a step-by-step procedure to detect radiation-induced foci (RIF) of repair proteins by immunofluorescence staining in human colon cancer cell lines after irradiation with the neutron-mixed beam.

人类结肠癌细胞DNA损伤与修复福西的免疫荧光成像

The purpose of the manuscript is to provide a step-by-step protocol for performing immunofluorescence microscopy to study the radiation-induced DNA damage ...

A Label-Free Segmentation Approach for Intravital Imaging of Mammary Tumor Microenvironment

The ability to visualize complex and dynamic physiological interactions between numerous cell types and the extracellular matrix (ECM) within a live tumor microenvironment is an important step toward understanding mechanisms that regulate tumor progression. While this can be accomplished through current intravital imaging techniques, it remains challenging due to the heterogeneous nature of tissues and the need for spatial context within the experimental observation. To this end, we have developed an intravital imaging workflow that pairs collagen second harmonic generation imaging, endogenous fluorescence from the metabolic co-factor NAD(P)H, and fluorescence lifetime imaging microscopy (FLIM) as a means to non-invasively compartmentalize the tumor microenvironment into basic domains of the tumor nest, the surrounding stroma or ECM, and the vasculature. This non-invasive protocol details the step-by-step process ranging from the acquisition of time-lapse images of mammary tumor models to post-processing analysis and image segmentation. The primary advantage of this workflow is that it exploits metabolic signatures to contextualize the dynamically changing live tumor microenvironment without the use of exogenous fluorescent labels, making it advantageous for human patient-derived xenograft (PDX) models and future clinical use where extrinsic fluorophores are not readily applicable.

The intravital imaging method described here utilizes collagen second harmonic generation and endogenous fluorescence from the metabolic co-factor NAD(P)H to non-invasively segment an unlabeled tumor microenvironment into tumor, stromal, and vascular compartments for in-depth analysis of 4D intravital images.

一种用于乳腺肿瘤微环境活体内成像的无标记分割方法

The ability to visualize complex and dynamic physiological interactions between numerous cell types and the extracellular matrix (ECM) within a live tumor ...