作者没有确认。

实验过程和善待动物组织批准匹兹堡IACUC大学（协议＃1104674）。 1.制备G6PD染色混合注:确保每个试剂的最终浓度如下: 5mM葡萄糖-6-磷酸（G6P）; 2mM的NADP，5mM的MgCl 2的，0.35毫1-甲氧基-5-甲基吩甲基硫酸（MMPMS）和0.8mM的NBT。1,8,9-对于每个试剂的初始浓度推导对于40 ml的终体积。解决方案新鲜制备，每天使用。 <ol><li>加入2毫升200毫pH 7.4的磷酸盐缓冲液（PB）到35 ml的十月培养基的50ml管中，并剧烈振荡。确认用pH试纸，该溶液是pH为〜7.4。如果不丢弃溶液。 </li><li>加入61毫克G6P溶解在0.94毫升200mM的PB，61毫克NADP溶解在0.94毫升200mM的PB和41毫克的MgCl 2溶于毫升100mM的0.96 PB。再次确认该pH值为7.4〜机智^ h pH试纸。如果没有〜7.4，丢弃的解决方案。 </li><li>添加5毫克MMPMS溶解在0.25毫升200mM的PB（pH 7.4）中的。用力摇动所得的反应混合物以产生一种清晰的暗红色匀浆。如果溶液混浊，使混合物应该被丢弃。被包裹在箔含有反应混合物的管，以尽量减少暴露于光。在室温储存的反应混合物，而最后一个组件，NBT，制备。 </li><li>通过加入0.4 ml的无水二甲甲酰胺（DMF）和0.4 ml的无水乙醇，以164毫克的NBT在2 ml管的O形环盖单独准备5mM的NBT溶液。松散盖上盖子（未密封），并把该溶液到剧烈沸腾的油浴中，直到NBT是在溶液中。8 </li><li>允许溶解的NBT冷却至RT，然后加入所有溶液到OCT反应混合物并剧烈振荡，获得澄清的溶液。观察到的暗红色或purp从初始橙红色的溶液的颜色变化乐颜色随着时间的推移。这是正常的。 </li><li>在37℃的温暖的室内使用前1小时将最终G6PD染色的混合物。 </li></ol> 2.动物处理和组织收集<ol><li>治疗6周龄的C57BL / 6雄性小鼠与DNA损伤剂，例如，10mg / kg的氧化偶氮甲烷（AOM）的体积为200μl的PBS腹腔注射。治疗的对照组小鼠用200微升PBS腹腔注射。 </li><li> 90日，由二氧化碳安乐死小鼠2窒息颈椎脱位批准的IACUC协议。 </li><li>手术从刚肛门到胸骨的基部上方打开腹腔。移动小肠出体腔，但不切除它。切除肠的下部，正下方的盲肠到直肠。10 </li><li>用微型剪刀解剖沿结肠的一侧仔细切片和通过用干净无菌PBS洗涤除去从打开结肠的粪便。瑞士卷消费税ð结肠与腔侧朝外，并置于组织模具10 </li><li>完全沉浸在OCT中，地方模具结肠中含液态氮杜瓦。保持所述模具以使塑料底仅仅与液体N 2接触，直到在OCT介质被冻结固体。 </li><li>存放冷冻组织块在-80至切割冰冻切片。不使用任何固定剂，这将导致在G6PD酶的失活。 </li></ol> 3.准备冰冻切片的<ol><li>切冰冻组织切片在使用低温恒温器在-25℃的厚度为7微米。 4切连续7微米的部分，并把他们两个人在同一张幻灯片（ 图1）。准备2幻灯片，或四个连续的7微米的部分来表示结肠（图1）中的一个级别。 </li><li>对于一个新的水平&gt; 50微米来自PR的最后一节的最小距离重复evious水平。这个距离保证了评估每个级别隐窝不重复彼此。 </li><li>经常擦拭低温恒温器刀片和100％的乙醇以除去OCT的残余物在切割期间积聚。切片被恢复之前，将干叶片擦拭用抗静电干燥器片消散累积静电荷。存储幻灯片冰冻切片在-80℃。 </li></ol> 4.染色冰冻切片组织<ol><li>请在37℃温暖的室内高湿度组织化学反应。 注意:试图在孵化烤箱或幻灯片上温暖的酶促反应导致穷人和不一致的G6PD染色。 </li><li>建井使用两个1.5厘米×0.15厘米钢垫圈（内径×高），该键合在一起使用硅脂（图1）组织染色。切断封口膜，以适应井与中心的底座切出和阱地方ð在幻灯片上的组织切片。 注:在垫圈的底部的封口膜将创建幻灯片和维持对组织切片粘性G6PD染色混合物垫圈之间形成密封。该构建给出了一个很好地与一个0.4 - 0.5 ml的体积，使得在研究中的每个组织部分接收G6PD反应染色混合物相同体积。 </li><li>在温暖的房间10分钟孵育冷冻组织切片在37℃。在37℃，45-50分钟的葡萄糖-6-磷酸脱氢酶染色反应混合物添加到每个组织切片以及（每张幻灯片2段）。从温暖的房间中取出的幻灯片，并小心地将不锈钢油井关闭的幻灯片。 </li><li>在其长边组幻灯片，以使反应介质排出。在100mM的PB（pH 7.4）中为30〜60分钟的地方滑动以除去来自组织的葡萄糖-6-磷酸脱氢酶染色反应混合物，而不会干扰组织染色。淹没在蒸馏水中的幻灯片5-10分钟，以除去铅盐</li><li>密封由加载组织从滴管荷兰国际集团氟 - 凝胶到组织上并等待〜30分钟它干。避免气泡可以混淆突变隐窝的识别。 注意:如果形成气泡，该过程可以去除氟 - 凝胶后重复，通过浸泡在蒸馏水中。不要使用盖玻片，因为他们往往​​陷阱气泡。 </li></ol> 5.测定葡萄糖-6-磷酸脱氢酶突变隐窝和突变频率<ol><li>在光学显微镜下分析幻灯片G6PD突变隐窝。 注意:用于识别一个完全突变体隐窝的标准是:（i）该整个隐窝是缺乏蓝色; （ii）所述隐窝的外结构完整，（ⅲ）的突变体隐窝上观察到相邻的7微米的部分和，（ⅳ）两个观察者不得不标识相同隐窝作为突变体。上相邻的7微米的部分观察到的突变体计为一个突变。 </li><li>采用500万像素的摄像头，图像中的每个葡萄糖-6-磷酸脱氢酶突变隐窝放大40倍和目录的图像图像处理软件。 </li><li>通过选择有代表性的部分，用于每只小鼠从具有最小表面面积和图像它在10倍的放大倍率（图1C）的水平量化隐窝检查在小鼠的总数。 注:A级代表四个连续7微米的部分（ 图1D），这是由于他们的接近常表现突变隐窝上的多个部分。的水平通过&gt;50μm的分离，以可视化从结肠的不同区域隐窝。 </li><li>打开每个图像文件和视觉通过分析文件（ 图 1C中示出为框）依靠一个水平隐窝数。图像文件将在从低至20至&gt; 300隐窝在一个文件中观察到隐窝的数目而变化。 </li><li>通过筛选突变体的G6PD隐窝每个结肠级别数相乘从所选择的水平（图1C）对每只小鼠隐窝的总数。例如，总计数每个选定的级别1250隐窝乘以8级筛选突变隐窝等于10000隐窝的总计数。评价最低的万隐窝每个结肠进行统计分析。 </li><li>组合由隐窝筛选以计算对MF每个治疗的总数在所有动物的观察突变体的数量。未处理小鼠背景干细胞的MF水平是&lt;0.1×10 -4（表1），这是接近先前在C57BL / 6小鼠的报道。11 </li></ol>

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The authors have nothing to disclose.

常的化合物的基因毒性作用是通过其来修改DNA的能力来确定。这通常是通过分离组织和测量DNA加合物的全球水平上进行。对于AOM，这将涉及量化O 6 -甲基鸟嘌呤加合物在结肠。上使用的特定细胞类型，如在干细胞小生境中的损害此方法的信息，将丢失。此外，DNA加合物的是不一样的突变，因为加合物的只有一小部分被最终转化成固定的突变。12我们提供本文所述细节基于由格里菲?...

结肠癌被认为涉及暴露于环境剂和膳食成分，可以破坏DNA和产生激活体细胞突变在致癌基因（例如，β-catenin的）或失活突变的肿瘤抑制基因（例如，APC）。据推测，这些关键突变发生在结肠的干细胞。由于结肠上皮的独特隐窝结构的，有可能让动物接触与结肠癌的发生​​相关联的化学品之后测量干细胞突变在结肠。几个X连锁酶可作为指标发生在干细胞突变，如突变将存在于一个隐窝内的所有细胞。 以前，一个过程被出版表明化学品诱导结肠肿瘤小鼠也通过在X连接的葡萄糖-6-磷酸脱氢酶（G6PD）基因突变的分析生成体干细胞突变在结肠1-3该方法量化了结肠干细胞随机体细胞突变的发生没有任何选择压力。该过程涉及从处理的小鼠和对照小鼠未固定的冷冻切片结肠癌的生产和隐窝缺乏产生官能G6PD活性细胞的鉴定。这些突变隐窝，呈现白色，表明突变发生在给人们带来的后代也携带突变基因G6PD干细胞。在酶测定中，葡萄糖-6-磷酸脱氢酶缺陷突变隐窝不能氧化葡萄糖-6-磷酸，这是需要的硝基四氮唑蓝（NBT）的还原。当G6PD酶是功能性的，在NBT在染色混合物降低到不溶性甲臜和沉淀物，积累在酶和"染色"细胞蓝色的位置。细胞，葡萄糖-6-磷酸脱氢酶突变体仍然发白而相比之下，蓝染野生型隐窝。这种方法测量的是有一个"空"的表型变异。因为恩P450酶活，如葡萄糖-6-磷酸脱氢酶，可以扩散上，如果节放在水溶液未定影组织部分的细胞出来，有必要稳定酶在组织切片进行分析。4，在酶的稳定化组织不得干扰必需的G6PD酶反应试剂的小分子的扩散。 我们已经取得了一些显着的变化，以原来的程序。用于关键酶染色的介质已从聚乙烯醇到最佳切削温度化合物（OCT），这有利于控制pH值，并在测定中使用的组分的增溶。 0.5 ml的井由钢制成的垫圈，以便每个组织部分接收的葡萄糖-6-磷酸脱氢酶反应混合物相同体积的 - 使用0.4进行染色。的程序被开发来估计在成像的部分，它提供了结肠组织的大采样，而无需隐窝数手动计数&gt; 10 5隐窝每个冒号。相邻的7微米的组织切片的分析，得到的突变体隐窝可视化多个幻灯片，这减少了潜在的染色工件。这些变化使程序更加高效和可重复性。 使用这个修订协议，我们已量化结肠干细胞的突变频率之C57B1 / 6小鼠，以氧化偶氮甲烷，在啮齿类动物中公知的结肠的致癌物质的暴露后5-7

<table><tbody><tr><td>reagents</td><td></td><td></td><td></td></tr><tr><td>nitroblue tetrazolium</td><td></td><td></td><td></td></tr><tr><td>NADP</td><td></td><td></td><td></td></tr><tr><td>glucose-6-phosphate</td><td></td><td></td><td></td></tr><tr><td>1-methoxy-5-methylphenazinium methyl sulfate</td><td></td><td></td><td></td></tr><tr><td>dimethyl formamide</td><td></td><td></td><td></td></tr><tr><td>phosphate buffer (pH 7.4</td><td></td><td></td><td></td></tr><tr><td>Optimal Cutting Temperature Compound (OCT)&amp;nbsp;</td><td></td><td></td><td></td></tr><tr><td>Equipment</td><td></td><td></td><td></td></tr><tr><td>light microscope equipped with 5 megapixel camera</td><td></td><td></td><td></td></tr><tr><td>cryostat</td><td></td><td></td><td></td></tr><tr><td>warm room&amp;nbsp;</td><td></td><td></td><td></td></tr></tbody></table>

quantification of  colonic stem cell mutations

测量干细胞突变的能力是一个强有力的工具中的一个关键的细胞群进行量化如果，以及在何种程度，化学可诱导的突变可能导致癌症。使用的酶测定法来量化干细胞突变在X连接的葡萄糖-6-磷酸脱氢酶基因已被报道 。1此方法要求与反应混合物能够产生一个在制备冰冻切片的切片组织和培养如果细胞产生功能葡萄糖-6-磷酸脱氢酶（G6PD）酶的蓝色。如果不是，该细胞出现发白。我们已修改代替聚乙烯醇使用最佳切削温度化合物（OCT）中的反应混合物中。这有利于pH测量，提高了G6PD染色成分的溶解和限制G6PD酶的扩散。为了证明突变发生在干细胞中，整个隐窝必须缺乏G6PD酶促活动。只有当一个干细胞藏着表型G6PD基因突变都会在地下室的后代缺乏G6PD酶的活性。要确定隐窝干细胞突变，连续四个相邻的冰冻切片（A级）切成7微米的厚度。使得相邻削减这种方法提供了构象地穴充分突变，因为相同的突变隐窝将相邻路段进行观察。幻灯片与组织样本均大于50μm相距已准备共&gt; 10 4每只小鼠隐窝来评估。突变频率是所观察到的突变的（白色）由野生型（蓝色）隐窝中一个治​​疗组的数量÷隐窝数。

衡量结肠干细胞基因突变的动物的能力提供了一个独特的方式来关联突变诱发癌症。通常情况下，假定在癌变的关键步骤包括激活突变在癌基因和/或失活的肿瘤抑制基因的突变。我们注射C57BL / 6小鼠用200μl的PBS或10mg / kg的急性中耳炎在200μlPBS中。 AOM是一种已知的致癌物质冒号5-7在90日进行了结肠干细胞基因突变分析。这个时间是必需的，以允许干细胞能够充分填充隐窝，只有当整个隐窝?...

Watch this Scientific Journal Video about Quantification of  Colonic Stem Cell Mutations at JoVE.com

Quantification of  Colonic Stem Cell Mutations

We report significant improvements for the reproducible measurement of somatic colonic stem cell mutations after exposure of mice to potential DNA damaging agents.

结肠干细胞突变定量

The ability to measure stem cell mutations is a powerful tool to quantify in a critical cell population if, and to what extent, a chemical can induce ...

quantification-of-colonic-stem-cell-mutations

Research

JoVE Journal

Developmental Biology

6.6K 次观看.  University of Pittsburgh. We report significant improvements for the reproducible measurement of somatic colonic stem cell mutations after exposure of mice to potential DNA damaging agents.

视频: Quantification of  Colonic Stem Cell Mutations

Simple and Rapid Method to Obtain High-quality Tumor DNA from Clinical-pathological Specimens Using Touch Imprint Cytology

It is critical to determine the mutational status in cancer before administration and treatment of specific molecular targeted drugs for cancer patients. In the clinical setting, formalin-fixed paraffin-embedded (FFPE) tissues are widely used for genetic testing. However, FFPE DNA is generally damaged and fragmented during the fixation process with formalin. Therefore, FFPE DNA is sometimes not adequate for genetic testing because of low quality and quantity of DNA. Here we present a method of touch imprint cytology (TIC) to obtain genomic DNA from cancer cells, which can be observed under a microscope. Cell morphology and cancer cell numbers can be evaluated using TIC specimens. Furthermore, the extraction of genomic DNA from TIC samples can be completed within two days. The total amount and quality of TIC DNA obtained using this method was higher than that of FFPE DNA. This rapid and simple method allows researchers to obtain high-quality DNA for genetic testing (e.g., next generation sequencing analysis, digital PCR, and quantitative real time PCR) and to shorten the turnaround time for reporting results.

Obtaining high-quality genomic DNA from tumor tissues is an essential first step for analyzing genetic alterations using next generation sequencing. In this article, we present a simple and rapid method to enrich tumor cells and obtain intact DNA from touch imprint cytology specimens.

用触印细胞学方法快速获得临床病理标本中高质量肿瘤 DNA

It is critical to determine the mutational status in cancer before administration and treatment of specific molecular targeted drugs for cancer patients. ...

科研

癌症研究

Colorectal Cancer Cell Surface Protein Profiling Using an Antibody Microarray and Fluorescence Multiplexing

The current prognosis and classification of CRC relies on staging systems that integrate histopathologic and clinical findings. However, in the majority of CRC cases, cell dysfunction is the result of numerous mutations that modify protein expression and post-translational modification1.
A number of cell surface antigens, including cluster of differentiation (CD) antigens, have been identified as potential prognostic or metastatic biomarkers in CRC. These antigens make ideal biomarkers as their expression often changes with tumour progression or interactions with other cell types, such as tumour-infiltrating lymphocytes (TILs) and tumour-associated macrophages (TAMs).
The use of immunohistochemistry (IHC) for cancer sub-classification and prognostication is well established for some tumour types2,3. However, no single &#x2018;marker&#x2019; has shown prognostic significance greater than clinico-pathological staging or gained wide acceptance for use in routine pathology reporting of all CRC cases. 
A more recent approach to prognostic stratification of disease phenotypes relies on surface protein profiles using multiple 'markers'. While expression profiling of tumours using proteomic techniques such as iTRAQ is a powerful tool for the discovery of biomarkers4, it is not optimal for routine use in diagnostic laboratories and cannot distinguish different cell types in a mixed population. In addition, large amounts of tumour tissue are required for the profiling of purified plasma membrane glycoproteins by these methods. 
In this video we described a simple method for surface proteome profiling of viable cells from disaggregated CRC samples using a DotScan CRC antibody microarray. The 122-antibody microarray consists of a standard 82-antibody region recognizing a range of lineage-specific leukocyte markers, adhesion molecules, receptors and markers of inflammation and immune response5, together with a satellite region for detection of 40 potentially prognostic markers for CRC. Cells are captured only on antibodies for which they express the corresponding antigen. The cell density per dot, determined by optical scanning, reflects the proportion of cells expressing that antigen, the level of expression of the antigen and affinity of the antibody6. 
For CRC tissue or normal intestinal mucosa, optical scans reflect the immunophenotype of mixed populations of cells. Fluorescence multiplexing can then be used to profile selected sub-populations of cells of interest captured on the array. For example, Alexa 647-anti-epithelial cell adhesion molecule (EpCAM; CD326), is a pan-epithelial differentiation antigen that was used to detect CRC cells and also epithelial cells of normal intestinal mucosa, while Phycoerythrin-anti-CD3, was used to detect infiltrating T-cells7. The DotScan CRC microarray should be the prototype for a diagnostic alternative to the anatomically-based CRC staging system.

We described a procedure for the disaggregation of colorectal cancer (CRC) to produce viable single cells, which are then captured on customized antibody microarrays recognizing surface antigens (DotScan CRC microarray). Sub-populations of cells bound to the microarray can be profiled by fluorescence multiplexing using monoclonal antibodies tagged with fluorescent dyes.

大肠癌细胞表面蛋白的分析，使用一种抗体的芯片和荧光多路复用

The current prognosis and classification of CRC relies on staging systems that integrate histopathologic and clinical findings. However, in the majority ...

免疫与感染

Evaluation of Colorectal Cancer Risk and Prevalence by Stool DNA Integrity Detection

Nowadays, stool DNA can be isolated and analyzed by several methods. The long fragments of DNA in stool can be detected by a qPCR assay, which provides a reliable probability of the presence of pre-neoplastic or neoplastic colorectal lesions. This method, called fluorescence long DNA (FL-DNA), is a fast, non-invasive procedure that is an improvement upon the primary prevention system. This method is based on evaluation of fecal DNA integrity by quantitative amplification of specific targets of genomic DNA. In particular, the evaluation of DNA fragments longer than 200 bp allows for detection of patients with colorectal lesions with very high specificity. However, this system and all currently available stool DNA tests present some general issues that need to be addressed (e.g., the frequency at which tests should be carried out and optimal number of stool samples collected at each timepoint for each individual). However, the main advantage of FL-DNA is the possibility to use it in association with a test currently used in the CRC screening program, known as the immunochemical-based fecal occult blood test (iFOBT). Indeed, both tests can be performed on the same sample, reducing costs and achieving a better prediction of the eventual presence of colorectal lesions.

The presented diagnostic FL-DNA kit is a time-saving and user-friendly method to determine the reliable probability of the presence of colorectal cancer lesions.

通过大刀DNA完整性检测评估结肠直肠癌风险和患病率

Nowadays, stool DNA can be isolated and analyzed by several methods. The long fragments of DNA in stool can be detected by a qPCR assay, which provides a ...

Discovery of Driver Genes in Colorectal HT29-derived Cancer Stem-Like Tumorspheres

Cancer stem cells play a vital role against clinical therapies, contributing to tumor relapse. There are many oncogenes involved in tumorigenesis and the initiation of cancer stemness properties. Since gene expression in the formation of colorectal cancer-derived tumorspheres is unclear, it takes time to discover the mechanisms working on one gene at a time. This study demonstrates a method to quickly discover the driver genes involved in the survival of the colorectal cancer stem-like cells in vitro. Colorectal HT29 cancer cells that express the LGR5 when cultured as spheroids and accompany an increase CD133 stemness markers were selected and used in this study. The protocol presented is used to perform RNAseq with available bioinformatics to quickly uncover the overexpressed driver genes in the formation of colorectal HT29-derived stem-like tumorspheres. The methodology can quickly screen and discover potential driver genes in other disease models.

Presented here is a protocol to discover the overexpressed driver genes maintaining the established cancer stem-like cells derived from colorectal HT29 cells. RNAseq with available bioinformatics was performed to investigate and screen gene expression networks for elucidating a potential mechanism involved in the survival of targeted tumor cells.

大肠癌HT29衍生癌症干细胞类肿瘤球中的驱动基因发现

Cancer stem cells play a vital role against clinical therapies, contributing to tumor relapse. There are many oncogenes involved in tumorigenesis and the ...

Identifying DNA Mutations in Purified Hematopoietic Stem/Progenitor Cells

In recent years, it has become apparent that genomic instability is tightly related to many developmental disorders, cancers, and aging. Given that stem cells are responsible for ensuring tissue homeostasis and repair throughout life, it is reasonable to hypothesize that the stem cell population is critical for preserving genomic integrity of tissues. Therefore, significant interest has arisen in assessing the impact of endogenous and environmental factors on genomic integrity in stem cells and their progeny, aiming to understand the etiology of stem-cell based diseases.
LacI transgenic mice carry a recoverable &#955;&#160;phage vector encoding the LacI reporter system, in which the LacI gene serves as the mutation reporter. The result of a mutated LacI gene is the production of &#946;-galactosidase that cleaves a chromogenic substrate, turning it blue. The LacI reporter system is carried in all cells, including stem/progenitor cells and can easily be recovered and used to subsequently infect E. coli. After incubating infected E. coli on agarose that contains the correct substrate, plaques can be scored; blue plaques indicate a mutant LacI gene, while clear plaques harbor wild-type. The frequency of blue (among clear) plaques indicates the mutant frequency in the original cell population the DNA was extracted from. Sequencing the mutant LacI gene will show the location of the mutations in the gene and the type of mutation.
The LacI transgenic mouse model is well-established as an in vivo mutagenesis assay. Moreover, the mice and the reagents for the assay are commercially available. Here we describe in detail how this model can be adapted to measure the frequency of spontaneously occurring DNA mutants in stem cell-enriched Lin-IL7R-Sca-1+cKit++(LSK) cells and other subpopulations of the hematopoietic system.

Here we describe an in vivo mutagenesis assay for small numbers of purified hematopoietic cells using the LacI transgenic mouse model.&#160;The LacI gene can be isolated to determine the frequency, location, and type of DNA mutants&#160;spontaneously arisen or after exposure to genotoxins.

在纯化造血干/祖细胞识别DNA突变

In recent years, it has become apparent that genomic instability is tightly related to many developmental disorders, cancers, and aging. Given that stem ...

A Three-dimensional Model of Spheroids to Study Colon Cancer Stem Cells

Colorectal cancers are characterized by heterogeneity and a hierarchical organization comprising a population of cancer stem cells (CSCs) responsible for tumor development, maintenance, and resistance to drugs. A better understanding of CSC properties for their specific targeting is, therefore, a pre-requisite for effective therapy. However, there is a paucity of suitable preclinical models for in-depth investigations. Although in vitro two-dimensional (2D) cancer cell lines provide valuable insights into tumor biology, they do not replicate the phenotypic and genetic tumor heterogeneity. In contrast, three-dimensional (3D) models address and reproduce near-physiological cancer complexity and cell heterogeneity. The aim of this work was to design a robust and reproducible 3D culture system to study CSC biology. The present methodology describes the development and optimization of conditions to generate 3D spheroids, which are homogenous in size, from Caco2 colon adenocarcinoma cells, a model that can be used for long-term culture. Importantly, within the spheroids, the cells which were organized around lumen-like structures, were characterized by differential cell proliferation patterns and by the presence of CSCs expressing a panel of markers. These results provide the first proof-of-concept for the appropriateness of this 3D approach to study cell heterogeneity and CSC biology, including the response to chemotherapy.

This protocol presents a novel, robust, and reproducible culture system to generate and grow three-dimensional spheroids from Caco2 colon adenocarcinoma cells. The results provide the first proof-of-concept for the appropriateness of this approach to study cancer stem cell biology, including the response to chemotherapy.

研究结肠癌干细胞的球体三维模型

Colorectal cancers are characterized by heterogeneity and a hierarchical organization comprising a population of cancer stem cells (CSCs) responsible for ...

Comparative Lesions Analysis Through a Targeted Sequencing Approach

Assessing intra-tumoral heterogeneity (ITH) is of paramount importance to anticipate failure of targeted therapies and design accordingly effective anti-tumor strategies. Although concerns are frequently raised due to differences in sample processing and depth of coverage, next-generation sequencing of solid tumors have unraveled a highly variable degree of ITH across tumor types. Capturing the genetic relatedness between primary and metastatic lesions through the identification of clonal and subclonal populations is critical to the design of therapies for advance-stage diseases. Here, we report a method for comparative lesions analysis that allows for the identification of clonal and subclonal populations among different specimens from the same patient. The experimental approach described here integrates three well-established approaches: histological analysis, high-coverage multi-lesion sequencing, and immunophenotypic analyses. In order to minimize the effects on the detection of subclonal events by inappropriate sample processing, we subjected tissues to careful pathological examination and neoplastic cell enrichment. Quality controlled DNA from neoplastic lesions and normal tissues was then subjected to high coverage sequencing, targeting the coding regions of 409 relevant cancer genes. While only looking at a limited genomic space, our approach enables evaluating the extent of heterogeneity among somatic alterations (single-nucleotide mutations and copy-number variations) in distinct lesions from a given patient. Through comparative analysis of sequencing data, we were able to distinguish clonal vs. subclonal alterations. The majority of ITH is often ascribed to passenger mutations; therefore, we also used immunohistochemistry to predict functional consequences of mutations. While this protocol has been applied to a specific tumor type, we anticipate that the methodology described here is broadly applicable to other solid tumor types.

This article describes a method to identify clonal and subclonal alterations among different specimens from a given patient. Although the experiments described here focus on a specific tumor type, the approach is broadly applicable to other solid tumors.

通过有针对性的测序方法进行比较病变分析

Assessing intra-tumoral heterogeneity (ITH) is of paramount importance to anticipate failure of targeted therapies and design accordingly effective ...

行为学

Molecular Profiling of the Invasive Tumor Microenvironment in a 3-Dimensional Model of Colorectal Cancer Cells and Ex vivo Fibroblasts

Invading colorectal cancer (CRC) cells have acquired the capacity to break free from their sister cells, infiltrate the stroma, and remodel the extracellular matrix (ECM). Characterizing the biology of this phenotypically distinct group of cells could substantially improve our understanding of early events during the metastatic cascade.

Tumor invasion is a dynamic process facilitated by bidirectional interactions between malignant epithelium and the cancer associated stroma. In order to examine cell-specific responses at the tumor stroma-interface we have combined organotypic co-culture and laser micro-dissection techniques.
Organotypic models, in which key stromal constituents such as fibroblasts are 3-dimentioanally co-cultured with cancer epithelial cells, are highly manipulatable experimental tools which enable invasion and cancer-stroma interactions to be studied in near-physiological conditions.

Laser microdissection (LMD) is a technique which entails the surgical dissection and extraction of the various strata within tumor tissue, with micron level precision.
By combining these techniques with genomic, transcriptomic and epigenetic profiling we aim to develop a deeper understanding of the molecular characteristics of invading tumor cells and surrounding stromal tissue, and in doing so potentially reveal novel biomarkers and opportunities for drug development in CRC.&#160;&#160;&#160;

Molecular Profiling of the Invasive Tumor Microenvironment in a 3-Dimensional Model of Colorectal Cancer Cells and Ex vivo Fibroblasts

Molecular profiling of laser microdissected cells and stroma from synthetic, tissue-engineered colorectal cancer models represents a novel and manipulatable approach to characterize and dissect the distinctive biology at the interface between tumor cells at the invasive front and cancer associated stromal cells. &#160;

在结直肠癌细胞的三维模型的入侵肿瘤微环境的分子剖析和<em&gt;体外</em&gt;成纤维细胞

Invading colorectal cancer (CRC) cells have acquired the capacity to break free from their sister cells, infiltrate the stroma, and remodel the ...

医学

Deficient Pms2, ERCC1, Ku86, CcOI in Field Defects During Progression to Colon Cancer

In carcinogenesis, the "field defect" is recognized clinically because of the high propensity of survivors of certain cancers to develop other malignancies of the same tissue type, often in a nearby location. Such field defects have been indicated in colon cancer. The molecular abnormalities that are responsible for a field defect in the colon should be detectable at high frequency in the histologically normal tissue surrounding a colonic adenocarcinoma or surrounding an adenoma with advanced neoplasia (well on the way to a colon cancer), but at low frequency in the colonic mucosa from patients without colonic neoplasia.
Using immunohistochemistry, entire crypts within 10 cm on each side of colonic adenocarcinomas or advanced colonic neoplasias were found to be frequently reduced or absent in expression for two DNA repair proteins, Pms2 and/or ERCC1. Pms2 is a dual role protein, active in DNA mismatch repair as well as needed in apoptosis of cells with excess DNA damage. ERCC1 is active in DNA nucleotide excision repair. The reduced or absent expression of both ERCC1 and Pms2 would create cells with both increased ability to survive (apoptosis resistance) and increased level of mutability. The reduced or absent expression of both ERCC1 and Pms2 is likely an early step in progression to colon cancer.
DNA repair gene Ku86 (active in DNA non-homologous end joining) and Cytochrome c Oxidase Subunit I (involved in apoptosis) had each been reported to be decreased in expression in mucosal areas close to colon cancers. However, immunohistochemical evaluation of their levels of expression showed only low to modest frequencies of crypts to be deficient in their expression in a field defect surrounding colon cancer or surrounding advanced colonic neoplasia.
We show, here, our method of evaluation of crypts for expression of ERCC1, Pms2, Ku86 and CcOI. We show that frequency of entire crypts deficient for Pms2 and ERCC1 is often as great as 70% to 95% in 20 cm long areas surrounding a colonic neoplasia, while frequency of crypts deficient in Ku86 has a median value of 2% and frequency of crypts deficient in CcOI has a median value of 16% in these areas. The entire colon is 150 cm long (about 5 feet) and has about 10 million crypts in its mucosal layer. The defect in Pms2 and ERCC1 surrounding a colon cancer thus may include 1 million crypts. It is from a defective crypt that colon cancer arises.

Reduced/absent expression of Pms2 and/or ERCC1 in entire crypts is a frequent event within 10 cm on each side of colonic adenocarcinomas, likely the basis of a field defect with high mutability and progression to cancer. Deficiency in Ku86 or CcOI is much less frequent in these field defects.

缺乏PMS2，ERCC1，Ku86，视野缺损CcOI在结肠癌进展

In carcinogenesis, the "field defect" is recognized clinically because of the high propensity of survivors of certain cancers to develop other ...

生物学

MLH1 Gene Expression in Peripheral Blood in Colon Cancer Patients and Their Association with Colon Cancer

MutL homolog 1 (MLH1) is a component of the heterodimeric complex MutL&#945; that detects and fixes base-base mismatches and insertion/deletion loops caused by nucleotide misincorporation. In the absence of MLH1 protein, the frequency of non-repaired mismatches increases, resulting in organ cancer. The current study sought to quantify MLH1 gene expression and its relationship with tumor invasion (T) and lymph node invasion (N) in blood samples from patients with colorectal cancer (CRC). Blood samples were obtained from 36 CRC patients. RNA was extracted, and cDNA was synthesized using a kit. The primers were built using the exon-exon junction approach, and MLH1 and &#946;-actin genes were tested 3x using real-time polymerase chain reaction (Real-Time PCR). Gene expression analysis software was used to analyze the data, and a t-test was used to examine the expression of MLH1 and its connection with T and N variables. In this study, 36 patients with colorectal cancer, including 15 (41.6%) women and 21 (58.4%) men, with a mean age of 57.35 &plusmn; 4.22 years and in the age range of 26-87 years, were included. The results showed that the ratio of MLH1 gene expression in patients decreased compared to that in healthy individuals, and the decrease in gene expression at different stages of the disease was significant. The results of this study showed that the reduction of MLH1 gene expression has an effective role in the development of CRC.

This study examines the association between MLH1 gene expression in peripheral blood and colon cancer, utilizing a case-control approach to compare expression levels in patients and matched healthy controls.

MutL homolog 1 (MLH1) is a component of the heterodimeric complex MutL&#945; that detects and fixes base-base mismatches and insertion/deletion loops ...