作者要感谢亚利桑那大学病理学系的财政支持。该项目得到了WZ的Mark和Jane Gibson捐赠基金的部分支持。

亚利桑那大学获得了机构审查委员会的批准，用于进行这项研究。获得知情的患者同意书。 患者选择注意:获得了亚利桑那大学的机构审查委员会的批准。共招收38名患者。患者年龄32〜86岁，平均55岁，其中绝经后26例，绝经前12例。 <ol><li>获得每名患者的知情同意书。 </li></ol>输卵管细胞收集程序注意:本研究中包括的患者均预计全子宫切除术和双侧输卵管卵巢切除术用于预防性降低风险或恶性肿瘤。进行细胞学研究的病理学家的手术细节是未知的。 <ol><li>手术后立即切除切口，仔细检查包括子宫，双侧卵巢和双侧输卵管在内的新鲜组织标本，以鉴别双侧输卵管。 </li><li> 在夹紧血液供应30分钟内收集输卵管细胞进入防腐液（参见表材料）小瓶;推荐的防腐剂是一种基于甲醇的缓冲防腐剂溶液。 注意:使用温和的触摸刷进行输卵管细胞收集（即将到来的步骤）。除了输卵管从子宫分离出来的罕见病例外，当输卵管连接到子宫时，通常插入刷头以从输卵管fimbria收集细胞。 <ol><li>在小镊子的帮助下，将刷子（参见表格材料 ）插入通过菌毛末端的输卵管内腔。慢慢地顺时针旋转，并将刷子向前移动穿过坠落的凹陷部分opian管，然后是壶腹直到达到峡部。 </li><li>慢慢地逆时针旋转和反转刷子以拉出刷子。确保内腔内刷子的最大速度慢于1厘米/秒。 </li><li>通过在溶液中旋转和旋转刷子10次，在防腐溶液（在小瓶中）冲洗刷子。 </li><li>拧紧小瓶盖。 </li><li>记录患者的信息和样本的变性。 </li><li>将小瓶存放在4°C冰箱（最多6个月）内。 </li></ol></li></ol> 3.管状细胞学幻灯片准备<ol><li>将样品瓶装入自动化处理器进行幻灯片准备。 注意:我们使用自动处理器进行幻灯片准备。将样品瓶放入处理器后，自动执行以下步骤:（i）细胞和碎屑通过样品中测试过滤器的旋转分离和分散小瓶。 （ii）通过温和的真空从过滤器的外表面收集管状细胞。 （iii）将过滤器倒置并压靠在载玻片上，使细胞粘附在载玻片内的圆形区域上。 （iv）将载玻片进一步固定在细胞固定浴中并准备进行染色和细胞学分析。不幸的是，没有其他手动方法，可以产生类似质量的结果。 </li></ol> 4.修改的Papanicolaou染色方案<ol><li>如图1所示，通过运行自动化非gyn染色程序来染色由步骤3.1制成的载玻片。 注意:染色方法代表我们常规用于非妇科标本的Papanicolaou染色技术的修改。我们使用自动组织处理器染色一些幻灯片。我们在本研究中选择了Gyn染色程序的非Gyn染色方法。使用的曙红是EA-65，而不是EA-50妇科标本（Pap涂片标本）。此外，主要用于鳞状细胞染色的Gyn染色程序中的OG-6不包括在染色程序中，因为在本研究中预期不会看到鳞状细胞。 EA65（Eosin Azure）是由3种染料组成的抗污染溶液:曙红Y，快绿FCF和俾斯麦棕Y。程序如表1所示 。 </li><li> 执行手动快速巴氏染色程序如下。 <ol><li>将步骤3.1中制成的载玻片浸入95％乙醇中，10次，每次浸渍约1秒。 </li><li>将载玻片浸入H 2 O中10次，每次浸渍约1秒。 </li><li>在苏木精中出现10-60秒的幻灯片。 </li><li>将载玻片浸入H 2 O中10次，每次浸渍约1秒。 </li><li>将载玻片浸入95％乙醇中，10次，每次浸渍约1秒。 </li><li>在EA65出现幻灯片1 - 3分钟。 </li><li>蘸滑块into H 2 O，10次，每次浸渍约1秒。 </li><li>将载玻片浸入95％乙醇中，10次，每次浸渍约1秒。 </li><li>将载玻片浸在二甲苯中直到载玻片变得清亮。 注意:这是一个修改的Papanicolaou染色程序。对于有限时间或空间的情况，如现场细针抽吸和STAT标本，该方法用作更快速的染色方法。该程序提供与自动化非Gyn染色程序相当的质量染色。程序如表2所示 。 </li></ol></li></ol>旋转细胞到幻灯片上注意:为每个样品制作三个细胞分裂素幻灯片。一个幻灯片是H＆E染色与细胞学幻灯片的形态比较。为未来的IHC染色研究制备了两个未染色的载玻片。本节的详细步骤不是本文的重点;因此，我们不会在扩展d中讨论它们etails。 <ol><li>准备一个细胞离心机与标记的幻灯片，标本漏斗和过滤器每个样品待检查。 </li><li>通过以200×g离心5分钟浓缩细胞。 </li><li>取出约一半的上清液，然后轻轻移液重新悬浮细胞。 </li><li>将每个细胞悬液200μL加入到标本漏斗中。 </li><li>旋转250 xg 5分钟。小心地从细胞离心机中取出载玻片并将其转移到95％乙醇中固定。 </li><li>空气干燥后染色和长期储存。 注意:根据最终幻灯片上的细胞密度，可以跳过或调整步骤5.2和步骤5.3。在这项研究中，我们需要每个高功率视图中存在至少2个细胞簇以获得足够的细胞密度。 </li></ol> 6.分段和广泛审查有条理的结束（SEE-FIM）协议<ol><li>修复整个标本（包括子宫，双侧输卵管和卵巢）在10％的福尔马林之前，通过减少剥落。 </li><li>使用小镊子和剪刀，轻轻小心地将输卵管从峡部的子宫分离。如果存在粘连，请使用小镊子和剪刀从卵巢表面小心地切开融化的末端;这些步骤对于尽量减少卵巢和输卵管之间的肿瘤交叉污染至关重要。 </li><li>从其余的标本截断漏斗和菌毛段（输卵管远端1.0 - 2.0 cm）。 </li><li>以2mm间隔纵向剖开漏斗和菌毛。提交toto的所有部分进行组织学检查。 </li><li>以2 - 3 mm的间隔切开峡部和壶腹，完全提交。 </li></ol>

<ol>
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M., 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=Dysplastic+changes+in+prophylactically+removed+Fallopian+tubes+of+women+predisposed+to+developing+ovarian+cancer.">Dysplastic changes in prophylactically removed Fallopian tubes of women predisposed to developing ovarian cancer.</a> J Pathol. 195 (4), 451-456 (2001).</li><li>Crum, C. P., 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+distal+fallopian+tube:+a+new+model+for+pelvic+serous+carcinogenesis.">The distal fallopian tube: a new model for pelvic serous carcinogenesis.</a> Curr Opin Obstet Gynecol. 19 (1), 3-9 (2007).</li><li>Jarboe, E. 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=Tubal+and+ovarian+pathways+to+pelvic+epithelial+cancer:+a+pathological+perspective.">Tubal and ovarian pathways to pelvic epithelial cancer: a pathological perspective.</a> Histopathology. 53 (2), 127-138 (2008).</li><li>Menon, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ovarian+cancer+screening+has+no+effect+on+disease-specific+mortality.">Ovarian cancer screening has no effect on disease-specific mortality.</a> Evid Based Med. 17 (2), 47-48 (2012).</li><li>Buys, S. 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=Effect+of+screening+on+ovarian+cancer+mortality:+the+Prostate,+Lung,+Colorectal+and+Ovarian+(PLCO)+Cancer+Screening+Randomized+Controlled+Trial.">Effect of screening on ovarian cancer mortality: the Prostate, Lung, Colorectal and Ovarian (PLCO) Cancer Screening Randomized Controlled Trial.</a> JAMA. 305 (22), 2295-2303 (2011).</li><li>Otsuka, I., Kameda, S., Hoshi, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+detection+of+ovarian+and+fallopian+tube+cancer+by+examination+of+cytological+samples+from+the+endometrial+cavity.">Early detection of ovarian and fallopian tube cancer by examination of cytological samples from the endometrial cavity.</a> Br J Cancer. 109 (3), 603-609 (2013).</li><li>Dhanani, M., Nassar, A., Dinh, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Classification+of+Fallopian+Tube+Cytology+Sampling+to+Develop+a+Model+for+Future+Peritoneal+and+Ovarian+Cancer+Screening.">Classification of Fallopian Tube Cytology Sampling to Develop a Model for Future Peritoneal and Ovarian Cancer Screening.</a> J Am Soc Cytopathol. 3 (5), S35-S36 (2014).</li><li>Rodriguez, E. 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作者宣称他们没有竞争的经济利益。

已经显示预防性双侧输卵管切除术或输卵管卵巢切除术可降低高危人群发生HGSC的风险，如BRCA基因突变妇女和家族性癌症史。然而，由手术导致的不育和外科绝经是严重后果，造成困难的决定，特别是对于育龄妇女。以前的研究显示输卵管细胞学和组织学之间有很好的相关性17 。我们认为腹腔镜收集的输卵管细胞的输卵管细胞学具有很大的潜力成为早期癌症检测的实用的，微创?...

卵巢高级浆液性癌（HGSC）是最常见和致命的卵巢癌，仍然是对公众健康的主要威胁。在过去十年中，研究人员已经表明，绝大多数的HGSC情况下从该输卵管而不是卵巢本身1，2，3，4，5，6出现。浆液性输卵管上皮内癌（STIC）作为HGSC 1，7，8，9，10，11的前体已被广泛接受。到目前为止，卵巢癌的早期发现仍然很困难。目前的筛选方案与联合癌抗原125（CA-125）和经阴道超声显示对病人的死亡率12，13几乎没有影响。卵巢癌检测的子宫内膜取样显示极低的灵敏度14 。两个先驱研究15,16探索良性输卵管腹腔镜直接采样的使用，并且其特征在于良性输卵管上皮的细胞学特征。我们认为，输卵管的直接取样也可以成为在肿瘤未被成像可视化的早期阶段检测STIC或HGSC的实用且直接的方法。 尽管最终目标是在高风险因素患者中进行微创腹腔镜筛查的实用性，但目前研究的直接目标是:1）建立良性输卵管上皮细胞基线细胞学特征;和2）检测输卵管细胞学检测卵巢癌的敏感性和特异性和癌症前体。因此，我们开发了以下基线输卵管细胞学方法来检测该方案检测HGSC及其前体STIC 17的有效性。在这项研究中，我们利用了大量定期接受的手术标本，除了常规手术程序之外，还对手术切除的输卵管进行了直接取样。 我们最近的研究17显示，恶性肿瘤的恶性或怀疑的细胞学诊断与HGSC的组织学诊断（100％）高度相关。此外，输卵管细胞学评估确定了在本研究中涉及的仅有两个组织学证实的STIC病例的颅内瘤变。我们认为输卵管细胞学在早期检测中具有很大的潜力，将为患者管理提供有价值的信息。

<table>
	<tbody>
		<tr>
			<td>Cytobrush plus GT gentle touch of Medscand sample collection kit</td>
			<td>CooperSurgical</td>
			<td>C0112</td>
			<td></td>
		</tr>
		<tr>
			<td>ThinPrep preservCyt solution</td>
			<td>HOLOGIC</td>
			<td>234005</td>
			<td></td>
		</tr>
		<tr>
			<td>ThinPrep 2000 Processor</td>
			<td>HOLOGIC</td>
			<td>70031-001</td>
			<td></td>
		</tr>
		<tr>
			<td>Papanicolaou Stain, EA 65</td>
			<td>sigma aldrich</td>
			<td>HT40432</td>
			<td></td>
		</tr>
		<tr>
			<td>95% Ethanol</td>
			<td>sigma aldrich</td>
			<td>652261</td>
			<td></td>
		</tr>
		<tr>
			<td>100% Ethanol</td>
			<td>sigma aldrich</td>
			<td>493538</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylene</td>
			<td>sigma aldrich</td>
			<td>214736</td>
			<td></td>
		</tr>
		<tr>
			<td>Hematoxylin</td>
			<td>sigma aldrich</td>
			<td>H9627</td>
			<td></td>
		</tr>
		<tr>
			<td>Sakura Tissue Tek 2000 tissue processor</td>
			<td>SAKURA</td>
			<td>TISSUE-TEK VIP 2000</td>
			<td></td>
		</tr>
		<tr>
			<td>Mayo Dissecting Scissors,5.5 straight</td>
			<td>Pilgrim medical equiment</td>
			<td>FA710-45</td>
			<td></td>
		</tr>
		<tr>
			<td>Chemware Forceps</td>
			<td>United state plastic corp</td>
			<td>76122</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue-Tek Accu-Edge Trimming blades,short</td>
			<td>SAKURA</td>
			<td>4785</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue-Tek Accu-Edge Trimming blades handle,short</td>
			<td>SAKURA</td>
			<td>4786</td>
			<td></td>
		</tr>
	</tbody>
</table>

tubal cytology of the fallopian tube as a promising tool for ovarian cancer early detection

目前，广泛接受的是绝大多数卵巢高级浆液性癌（HGSC）来源于输卵管。然而，由于缺乏用于卵巢癌鉴定的标记物或工具，HGSC的早期检测仍然具有挑战性。当肿瘤不可见时，输卵管的直接取样可增强检测肿瘤细胞的敏感性。我们开发了一种从新鲜手术标本直接收集输卵管细胞的方法，与组织学检查结果具有良好的相关性。这种方法为高风险患者群体中微创腹腔镜筛查的未来应用奠定了基础。

我们以前的研究表明，使用温和的触摸刷直接从切除的输卵管取样可以产生定量和定性的足够的标本用于进一步细胞学评估。此外，自动幻灯片制备和修改的Papanicolaou染色的组合使得病理学家能够更好地可视化细胞学细节以做出准确的诊断。 图1中示出了来自良性输卵管的样品的手动快速pap染色的实例。 如图1所示，刷子操纵导...

Watch this Scientific Journal Video about Tubal Cytology of the Fallopian Tube as a Promising Tool for Ovarian Cancer Early Detection at JoVE.com

Tubal Cytology of the Fallopian Tube as a Promising Tool for Ovarian Cancer Early Detection

我们探索了输卵管细胞学方法，采用输卵管直接手术切除术作为卵巢癌早期检测的工具。在这里，我们提出一个收集新鲜手术标本的输卵管细胞的方案。

输卵管的管状细胞学作为卵巢癌早期检测的有前景的工具

Currently, it is widely accepted that the vast majority of ovarian high-grade serous carcinoma (HGSC) originate from the fallopian tube. However, due to ...

tubal-cytology-fallopian-tube-as-promising-tool-for-ovarian-cancer

Research

JoVE Journal

Medicine

12.2K 次观看.  University of Arizona College of Medicine. 我们探索了输卵管细胞学方法，采用输卵管直接手术切除术作为卵巢癌早期检测的工具。在这里，我们提出一个收集新鲜手术标本的输卵管细胞的方案。 

视频: Tubal Cytology of the Fallopian Tube as a Promising Tool for Ovarian Cancer Early Detection

In vitro Mesothelial Clearance Assay that Models the Early Steps of Ovarian Cancer Metastasis

Ovarian cancer is the fifth leading cause of cancer related deaths in the United States1. Despite a positive initial response to therapies, 70 to 90 percent of women with ovarian cancer develop new metastases, and the recurrence is often fatal2. It is, therefore, necessary to understand how secondary metastases arise in order to develop better treatments for intermediate and late stage ovarian cancer. Ovarian cancer metastasis occurs when malignant cells detach from the primary tumor site and disseminate throughout the peritoneal cavity. The disseminated cells can form multicellular clusters, or spheroids, that will either remain unattached, or implant onto organs within the peritoneal cavity3 (Figure 1, Movie 1). 
All of the organs within the peritoneal cavity are lined with a single, continuous, layer of mesothelial cells4-6 (Figure 2). However, mesothelial cells are absent from underneath peritoneal tumor masses, as revealed by electron micrograph studies of excised human tumor tissue sections3,5-7 (Figure 2). This suggests that mesothelial cells are excluded from underneath the tumor mass by an unknown process. 
Previous in vitro experiments demonstrated that primary ovarian cancer cells attach more efficiently to extracellular matrix than to mesothelial cells8, and more recent studies showed that primary peritoneal mesothelial cells actually provide a barrier to ovarian cancer cell adhesion and invasion (as compared to adhesion and invasion on substrates that were not covered with mesothelial cells)9,10. This would suggest that mesothelial cells act as a barrier against ovarian cancer metastasis. The cellular and molecular mechanisms by which ovarian cancer cells breach this barrier, and exclude the mesothelium have, until recently, remained unknown. 
Here we describe the methodology for an in vitro assay that models the interaction between ovarian cancer cell spheroids and mesothelial cells in vivo (Figure 3, Movie 2). Our protocol was adapted from previously described methods for analyzing ovarian tumor cell interactions with mesothelial monolayers8-16, and was first described in a report showing that ovarian tumor cells utilize an integrin &#x2013;dependent activation of myosin and traction force to promote the exclusion of the mesothelial cells from under a tumor spheroid17. This model takes advantage of time-lapse fluorescence microscopy to monitor the two cell populations in real time, providing spatial and temporal information on the interaction. The ovarian cancer cells express red fluorescent protein (RFP) while the mesothelial cells express green fluorescent protein (GFP). RFP-expressing ovarian cancer cell spheroids attach to the GFP-expressing mesothelial monolayer. The spheroids spread, invade, and force the mesothelial cells aside creating a hole in the monolayer. This hole is visualized as the negative space (black) in the GFP image. The area of the hole can then be measured to quantitatively analyze differences in clearance activity between control and experimental populations of ovarian cancer and/ or mesothelial cells. This assay requires only a small number of ovarian cancer cells (100 cells per spheroid X 20-30 spheroids per condition), so it is feasible to perform this assay using precious primary tumor cell samples. Furthermore, this assay can be easily adapted for high throughput screening.

In vitro Mesothelial Clearance Assay that Models the Early Steps of Ovarian Cancer Metastasis

The mesothelial clearance assay described here takes advantage of fluorescently labeled cells and time-lapse video microscopy to visualize and quantitatively measure the interactions of ovarian cancer multicellular spheroids and mesothelial cell monolayers. This assay models the early steps of ovarian cancer metastasis.

这些模型在体外间皮间隙检测卵巢癌转移的早期步骤

Ovarian cancer is the fifth leading cause of cancer related deaths in the United States1. Despite a positive initial response to therapies, 70 to 90 ...

科研

医学

A Human Fallopian Tube Model for Investigation of C. trachomatis Infections

Genital tract infections with Chlamydia trachomatis (C. trachomatis) are the most frequent transmitted sexually disease in women worldwide. Inefficient clearance or persistence of the pathogens may lead to ascending infections of the upper genital tract and are supposed to cause chronic inflammatory damage to infected tissues 1,2. As a consequence, severe clinical sequelae like pelvic inflammatory disease (PID), tubal occlusion and infertility may occur 3,4. 
Most of the research with C. trachomatis has been conducted in epithelial cell lines (e.g. HEp-2 cells and HeLa-229) or in mice. However, as with cell- culture based models, they do neither reflect the physiology of native tissue nor the pathophysiology of C. trachomatis genital tract infections in vivo 5. Further limitations are given by the fact that central signaling cascades (e.g. IFN-&gamma; mediated JAK/STAT signaling pathway) that control intracellular chlamydial growth fundamentally differ between mice and humans 6,7. We and others therefore established a whole organ fallopian tube model to investigate direct interactions between C. trachomatis and human fallopian tube cells ex vivo 8,9.
For this purpose, human fallopian tubes from women undergoing hysterectomy were collected and infected with C. trachomatis serovar D. Within 24 h post infection, specimen where analyzed using scanning electron microscopy (SEM) and transmission electron microscopy (TEM) to detect Chlamydia trachomatis mediated epithelial damage as well as C. trachomatis inclusion formation in the fallopian tissue.

A Human Fallopian Tube Model for Investigation of C. trachomatis Infections

We describe an ex vivo infection model for visualisation of direct interactions from bacterial pathogens with human fallopian tube cells. The whole organ tissue model was established to investigate C. trachomatis induced pathology to the female fallopian tube under "life-like" conditions.

一个人的调查输卵管模型<em&gt; C。沙眼</em&gt;感染

Genital tract infections with Chlamydia trachomatis (C. trachomatis) are the most frequent transmitted sexually disease in women worldwide. Inefficient ...

Heterotypic Three-dimensional In Vitro Modeling of Stromal-Epithelial Interactions During Ovarian Cancer Initiation and Progression

Epithelial ovarian cancers (EOCs) are the leading cause of death from gynecological malignancy in Western societies. Despite advances in surgical treatments and improved platinum-based chemotherapies, there has been little improvement in EOC survival rates for more than four decades 1,2. Whilst stage I tumors have 5-year survival rates &gt;85%, survival rates for stage III/IV disease are &lt;40%. Thus, the high rates of mortality for EOC could be significantly decreased if tumors were detected at earlier, more treatable, stages 3-5. At present, the molecular genetic and biological basis of early stage disease development is poorly understood. More specifically, little is known about the role of the microenvironment during tumor initiation; but known risk factors for EOCs (e.g. age and parity) suggest that the microenvironment plays a key role in the early genesis of EOCs. We therefore developed three-dimensional heterotypic models of both the normal ovary and of early stage ovarian cancers. For the normal ovary, we co-cultured normal ovarian surface epithelial (IOSE) and normal stromal fibroblast (INOF)&nbsp;cells, immortalized by retrovrial transduction of the catalytic subunit of human telomerase holoenzyme (hTERT) to extend the lifespan of these cells in culture. To model the earliest stages of ovarian epithelial cell transformation, overexpression of the CMYC oncogene in IOSE cells, again co-cultured with INOF cells. These heterotypic models were used to investigate the effects of aging and senescence on the transformation and invasion of epithelial cells. Here we describe the methodological steps in development of these three-dimensional model; these methodologies aren't specific to the development of normal ovary and ovarian cancer tissues, and could be used to study other tissue types where stromal and epithelial cell interactions are a fundamental aspect of the tissue maintenance and disease development.

Heterotypic Three-dimensional In Vitro Modeling of Stromal-Epithelial Interactions During Ovarian Cancer Initiation and Progression

We describe methodologies for establishing in vitro heterotypic three-dimensional models comprising ovarian fibroblasts and normal ovarian surface or ovarian cancer epithelial cells. We discuss the use of these models to study stromal-epithelial interactions that occur during ovarian cancer development.

异型三维<em在体外</em&gt;卵巢癌的发生和发展过程中的间质 - 上皮细胞相互作用模型

Epithelial ovarian cancers (EOCs) are the leading cause of death from gynecological malignancy in Western societies. Despite advances in surgical ...

Cerenkov Luminescence Imaging (CLI) for Cancer Therapy Monitoring

In molecular imaging, positron emission tomography (PET) and optical imaging (OI) are two of the most important and thus most widely used modalities1-3. PET is characterized by its excellent sensitivity and quantification ability while OI is notable for non-radiation, relative low cost, short scanning time, high throughput, and wide availability to basic researchers. However, both modalities have their shortcomings as well. PET suffers from poor spatial resolution and high cost, while OI is mostly limited to preclinical applications because of its limited tissue penetration along with prominent scattering optical signals through the thickness of living tissues.
 Recently a bridge between PET and OI has emerged with the discovery of Cerenkov Luminescence Imaging (CLI)4-6. CLI is a new imaging modality that harnesses Cerenkov Radiation (CR) to image radionuclides with OI instruments. Russian Nobel laureate Alekseyevich Cerenkov and his colleagues originally discovered CR in 1934. It is a form of electromagnetic radiation emitted when a charged particle travels at a superluminal speed in a dielectric medium7,8. The charged particle, whether positron or electron, perturbs the electromagnetic field of the medium by displacing the electrons in its atoms. After passing of the disruption photons are emitted as the displaced electrons return to the ground state. For instance, one 18F decay was estimated to produce an average of 3 photons in water5. 
 Since its emergence, CLI has been investigated for its use in a variety of preclinical applications including in vivo tumor imaging, reporter gene imaging, radiotracer development, multimodality imaging, among others4,5,9,10,11. The most important reason why CLI has enjoyed much success so far is that this new technology takes advantage of the low cost and wide availability of OI to image radionuclides, which used to be imaged only by more expensive and less available nuclear imaging modalities such as PET.
 Here, we present the method of using CLI to monitor cancer drug therapy. Our group has recently investigated this new application and validated its feasibility by a proof-of-concept study12. We demonstrated that CLI and PET exhibited excellent correlations across different tumor xenografts and imaging probes. This is consistent with the overarching principle of CR that CLI essentially visualizes the same radionuclides as PET. We selected Bevacizumab (Avastin; Genentech/Roche) as our therapeutic agent because it is a well-known angiogenesis inhibitor13,14. Maturation of this technology in the near future can be envisioned to have a significant impact on preclinical drug development, screening, as well as therapy monitoring of patients receiving treatments.

Cerenkov Luminescence Imaging CLI for Cancer Therapy Monitoring

Use of Cerenkov Luminescence Imaging (CLI) for monitoring preclinical cancer treatment is described here. This method takes advantage of Cerenkov Radiation (CR) and optical imaging (OI) to visualize radiolabeled probes and thus provides an alternative to PET in preclinical therapeutic monitoring and drug screening.

癌症治疗监测的切伦科夫发光成像（CLI）

In  molecular imaging, positron emission tomography (PET) and optical imaging (OI)  are two of the most important and thus most widely used modalities1-3. ...

The In ovo CAM-assay as a Xenograft Model for Sarcoma

Sarcoma is a very rare disease that is heterogeneous in nature, all hampering the development of new therapies. Sarcoma patients are ideal candidates for personalized medicine after stratification, explaining the current interest in developing a reproducible and low-cost xenotransplant model for this disease. The chick chorioallantoic membrane is a natural immunodeficient host capable of sustaining grafted tissues and cells without species-specific restrictions. In addition, it is easily accessed, manipulated and imaged using optical and fluorescence stereomicroscopy. Histology further allows detailed analysis of heterotypic cellular interactions.
This protocol describes in detail the in ovo grafting of the chorioallantoic membrane with fresh sarcoma-derived tumor tissues, their single cell suspensions, and permanent and transient fluorescently labeled established sarcoma cell lines (Saos-2 and SW1353). The chick survival rates are up to 75%. The model is used to study graft- (viability, Ki67 proliferation index, necrosis, infiltration) and host (fibroblast infiltration, vascular ingrowth) behavior. For localized grafting of single cell suspensions, ECM gel provides significant advantages over inert containment materials. The Ki67 proliferation index is related to the distance of the cells from the surface of the CAM and the duration of application on the CAM, the latter determining a time frame for the addition of therapeutic products.

The In ovo CAM-assay as a Xenograft Model for Sarcoma

The in ovo chorioallantoic membrane (CAM) is grafted with fresh sarcoma-derived tumor tissues, their single cell suspensions, and permanent and transient fluorescently labeled established sarcoma cell lines. The model is used to study graft- (viability, Ki67 proliferation index, necrosis, infiltration) and host (fibroblast infiltration, vascular ingrowth) behavior.

该<em&gt;在OVO</em&gt; CAM作为一个肉瘤Xenograftmodel的检测

Sarcoma is a very rare disease that is heterogeneous in nature, all hampering the development of new therapies. Sarcoma patients are ideal candidates for ...

Osteopathic Manipulative Treatment as a Useful Adjunctive Tool for Pneumonia

Pneumonia, the inflammatory state of lung tissue primarily due to microbial infection, claimed 52,306 lives in the United States in 20071 and resulted in the hospitalization of 1.1 million patients2. With an average length of in-patient hospital stay of five days2, pneumonia and influenza comprise significant financial burden costing the United States $40.2 billion in 20053. Under the current Infectious Disease Society of America/American Thoracic Society guidelines, standard-of-care recommendations include the rapid administration of an appropriate antibiotic regiment, fluid replacement, and ventilation (if necessary). Non-standard therapies include the use of corticosteroids and statins; however, these therapies lack conclusive supporting evidence4. (Figure 1)

Osteopathic Manipulative Treatment (OMT) is a cost-effective adjunctive treatment of pneumonia that has been shown to reduce patients&#8217; length of hospital stay, duration of intravenous antibiotics, and incidence of respiratory failure or death when compared to subjects who received conventional care alone5. The use of manual manipulation techniques for pneumonia was first recorded as early as the Spanish influenza pandemic of 1918, when patients treated with standard medical care had an estimated mortality rate of 33%, compared to a 10% mortality rate in patients treated by osteopathic physicians6. When applied to the management of pneumonia, manual manipulation techniques bolster lymphatic flow, respiratory function, and immunological defense by targeting anatomical structures involved in the these systems7,8, 9, 10.

The objective of this review video-article is three-fold: a) summarize the findings of randomized controlled studies on the efficacy of OMT in adult patients with diagnosed pneumonia, b) demonstrate established protocols utilized by osteopathic physicians treating pneumonia, c) elucidate the physiological mechanisms behind manual manipulation of the respiratory and lymphatic systems. Specifically, we will discuss and demonstrate four routine techniques that address autonomics, lymph drainage, and rib cage mobility: 1) Rib Raising, 2) Thoracic Pump, 3) Doming of the Thoracic Diaphragm, and 4) Muscle Energy for Rib 1.5,11

Pneumonia is one of the top ten leading causes of death in the U.S. Osteopathic manipulative techniques (OMT) may be utilized as an adjunctive therapy in the treatment of pneumonia to enhance biomechanical and immune function; and ultimately, to reduce hospital stay, duration of antibiotics, incidence of respiratory failure, and mortality. In this video-article, we will review randomized controlled studies on the use of OMT in pneumonia patients, as well as demonstrate specific hands-on techniques that are routinely practiced by osteopathic physicians when treating pulmonary infections.

整骨手法治疗为肺炎是非常好的辅助工具

Pneumonia, the inflammatory state of lung tissue primarily due to microbial infection, claimed 52,306 lives in the United States in 20071 and resulted in ...

Method for Obtaining Primary Ovarian Cancer Cells From Solid Specimens

Reliable tools for investigating ovarian cancer initiation and progression are urgently needed. While the use of ovarian cancer cell lines remains a valuable tool for understanding ovarian cancer, their use has many limitations. These include the lack of heterogeneity and the plethora of genetic alterations associated with extended in vitro passaging.&#160;Here we describe a method that allows for rapid establishment of primary ovarian cancer cells form solid clinical specimens collected at the time of surgery. The method consists of subjecting clinical specimens to enzymatic digestion for 30 min. The isolated cell suspension is allowed to grow and can be used for downstream application including drug screening. The advantage of primary ovarian cancer cell lines over established ovarian cancer cell lines is that they are representative of the original specific clinical specimens they are derived from and can be derived from different sites whether primary or metastatic ovarian cancer.

This study describes a detailed method for isolation and characterization of primary ovarian cancer cells from solid clinical specimens. Ovarian cancer clinical specimens are subjected to enzymatic digestion to obtain viable, fibroblast-free epithelial ovarian cancer (EOC) cells highly suitable for downstream applications.

为获取原发性卵巢癌细胞固体样品的方法

Reliable tools for investigating ovarian cancer initiation and progression are urgently needed. While the use of ovarian cancer cell lines remains a ...

Evaluation of the Efficacy of the H. pylori Protein HP-NAP as a Therapeutic Tool for Treatment of Bladder Cancer in an Orthotopic Murine Model

Bladder cancer is one of the most common malignancies of the urogenital tract. Intravesical injection of Bacillus Calmette-Gu&#233;rin (BCG) is the gold standard treatment for the high-grade non-muscle invasive bladder cancer (NMIBC). However, since the treatment-related side effects are relevant, newer biological response modifiers with a better benefit/side effects ratio are needed.
The tumour microenvironment can influence both tumour development and therapy efficacy. In order to obtain a good model, it is desirable to implant tumour cells in the organ from which the cancer originates.
In this protocol, we describe a method for establishing a tumour in the bladder cavity of female mice and subsequent delivery of therapeutic agents; the latter are exemplified by our use of Helicobacter pylori neutrophil activating protein (HP-NAP). A preliminary chemical burn of the mucosa, followed by the injection of mouse urothelial carcinoma cell line MB49 via urethral catheterization, enables the cells to attach to the bladder mucosa. After a period, required to allow an initial proliferation of the cells, mice are treated with HP-NAP, administrated again via catheterization. The anti-tumour activity of HP-NAP is evaluated comparing the tumour volume, the extent of necrosis and the degree of vascularization between vehicle- and HP-NAP-treated animals.

Evaluation of the Efficacy of the H. pylori Protein HP-NAP as a Therapeutic Tool for Treatment of Bladder Cancer in an Orthotopic Murine Model

Here the method to establish a syngeneic mouse model of orthotopic bladder tumour to evaluate the anti-tumour efficacy of the bacterial protein HP-NAP is described.

评价疗效<em&gt;小时。幽门螺杆菌</em&gt;蛋白HP-NAP为膀胱癌的治疗在原位小鼠模型的治疗工具

Bladder cancer is one of the most common malignancies of the urogenital tract. Intravesical injection of Bacillus Calmette-Gu&#233;rin (BCG) is the gold ...

Quantitation of Intra-peritoneal Ovarian Cancer Metastasis

Epithelial ovarian cancer (EOC) is the leading cause of death from gynecologic malignancy in the United States. Mortality is due to diagnosis of 75% of women with late stage disease, when metastasis is already present. EOC is characterized by diffuse and widely disseminated intra-peritoneal metastasis. Cells shed from the primary tumor anchor in the mesothelium that lines the peritoneal cavity as well as in the omentum, resulting in multi-focal metastasis, often in the presence of peritoneal ascites. Efforts in our laboratory are directed at a more detailed understanding of factors that regulate EOC metastatic success. However, quantifying metastatic tumor burden represents a significant technical challenge due to the large number, small size and broad distribution of lesions throughout the peritoneum. Herein we describe a method for analysis of EOC metastasis using cells labeled with red fluorescent protein (RFP) coupled with in vivo multispectral imaging. Following intra-peritoneal injection of RFP-labelled tumor cells, mice are imaged weekly until time of sacrifice. At this time, the peritoneal cavity is surgically exposed and organs are imaged in situ. Dissected organs are then placed on a labeled transparent template and imaged ex vivo. Removal of tissue auto-fluorescence during image processing using multispectral unmixing enables accurate quantitation of relative tumor burden. This method has utility in a variety of applications including therapeutic studies to evaluate compounds that may inhibit metastasis and thereby improve overall survival.

Ovarian cancer metastasis is characterized by numerous diffuse intra-peritoneal lesions, such that accurate visual quantitation of tumor burden is challenging. Herein we describe a method for in situ and ex vivo quantitation of metastatic tumor burden using red fluorescent protein (RFP)-labeled tumor cells and optical imaging.

腹膜内卵巢癌转移的定量

Epithelial ovarian cancer (EOC) is the leading cause of death from gynecologic malignancy in the United States.  Mortality is due to diagnosis of 75% of ...

Modeling the Early Steps of Ovarian Cancer Dissemination in an Organotypic Culture of the Human Peritoneal Cavity

The pattern of ovarian cancer metastasis is markedly different from that of most other epithelial tumors, because it rarely spreads hematogenously. Instead, ovarian cancer cells exfoliated from the primary tumor are carried by peritoneal fluid to metastatic sites within the peritoneal cavity. These sites, most notably the abdominal peritoneum and omentum, are organs covered by a mesothelium-lined surface. To investigate the processes of ovarian cancer dissemination, we assembled a complex three-dimensional culture system that reconstructs the lining of the peritoneal cavity in vitro. Primary human fibroblasts and mesothelial cells were isolated from human omentum. The fibroblasts were then mixed with extracellular matrix and covered with a layer of the primary human mesothelial cells to mimic the peritoneal and omental surfaces encountered by metastasizing ovarian cancer cells. The resulting organotypic model is, as shown, used to examine the early steps of ovarian cancer dissemination, including cancer cell adhesion, invasion, and proliferation. This model has been used in a number of studies to investigate the role of the microenvironment (cellular and acellular) in early ovarian cancer dissemination. It has also been successfully adapted to high throughput screening and used to identify and test inhibitors of ovarian cancer metastasis.

Here, we present a protocol to construct a three-dimensional in vitro model of the lining of the peritoneal cavity, composed of primary human mesothelial cells and fibroblasts layered with extracellular matrix, as a tool to investigate ovarian cancer cell adhesion, invasion, and proliferation.

造型卵巢癌传播在人类腹腔的器官文化的早期步骤

The pattern of ovarian cancer metastasis is markedly different from that of most other epithelial tumors, because it rarely spreads hematogenously. ...