Name: isolation of proximal fluids to investigate the tumor microenvironment of pancreatic adenocarcinoma
Uploaded: 2020-11-05T13:00:00.000+00:00
Duration: 5 min 44 s
Description: Watch this Scientific Journal Video about Isolation of Proximal Fluids to Investigate the Tumor Microenvironment of Pancreatic Adenocarcinoma at JoVE.com

我们感谢罗伯塔·米廖雷的技术援助。导致这些结果的研究已获得意大利人协会（AIRC）在IG2016-ID.18443项目下的资助 - P.I. Marchesi Federica。资助者在研究设计、数据收集和分析、发表决定或手稿准备方面没有任何作用。

对于所有入组的患者，在手术时根据该机构伦理委员会批准的方案收集外周血和胰液。所有患者均在签署知情同意书后纳入研究，包括收集生物标本和临床数据。该研究已获得该机构伦理委员会的批准（协议号ICH-595，批准于2009年5月发布）。涉及小鼠及其护理的程序符合欧盟和机构指南（协议ID 121 / 2016-PR）。
1.胰液分离
注意:胰液的抽取是在胰腺切除术的开放手术（例如，胰十二指肠切除术，全胰腺切除术，胰腺远端切除术）的背景下由胰腺专家外科医生的设备进行的。
<ol><li>患者选择<ol><li>考虑任何计划进行开放性胰腺切除术的患者。</li>
		<li>如果认为主胰管大小足以进行胰液提取，则确认纳入。在增强 CT 成像中，主胰管直径的最小限制为 2 mm。</li>
	</ol></li>
	<li>增强CT成像下胰管术前研究，帮助胰液回收计划<ol><li>在胰颈水平处三维定位腺体内的主胰管:在横截面载玻片上测量主胰管与前缘、上胰缘和下胰腺缘的距离，以及冠状面和矢状面效果图。进入手术室后，使用这些测量值来估算穿刺胰腺以插管Wirsung导管并取回胰液的正确位置。</li>
	</ol></li>
	<li>材料准备<ol><li>无菌材料:打开一根 25 G 针头和一个 3 mL 注射器的无菌信封，并在擦洗护士的合作下将它们放置在无菌区域。</li>
		<li>未消毒材料:在手术室中准备好 3 mL K2EDTA 真空试管，用于储存液体。</li>
	</ol></li>
	<li>患者的准备<ol><li>将患者放在手术室病床上。使用瑞芬太尼、塞沃兰和罗库溴铵诱导混合全身麻醉，然后插管并开始为患者通气。将患者仰卧位，右臂与身体相连，左臂外展 90°，固定在扶板上。</li>
		<li>在切口部位对腹部皮肤进行消毒。在覆盖患者的腹部创建并保持无菌区域。</li>
	</ol></li>
	<li>手术<ol><li>进行肋下切口并进入腹腔。放置罗查德腹部回缩以进行器官暴露。</li>
		<li>通过Kocher操作暴露和调动胰腺，打开胃结肠韧带，沿着胰腺上缘和下缘切开腹膜后组织，在胰颈和位于后方的肠系膜上静脉之间形成解剖平面。</li>
		<li>一旦胰腺被动员并暴露，在胰颈切片之前进行胰液戒断。</li>
	</ol></li>
	<li>胰管的识别和定位<ol><li>使用成像时进行的测量估计胰管的位置，然后触诊胰腺的前表面以确定其精确位置。</li>
	</ol></li>
	<li>收集胰液<ol><li>从下方握住胰头和十二指肠，用左手抬高，用第一个手指标记胰管的位置。</li>
		<li>用装有 25 G 针头的 3 mL 注射器的右手握住。</li>
		<li>用右手将针头插入左手拇指远端的胰腺中。根据术前测量和对穿透导管壁的感知来确定刺入深度和针头倾斜程度。</li>
		<li>用注射器取出果汁。如果无法取回果汁，请将针头重新定位在四个方向，尝试插管胰管。</li>
		<li>取出胰液后，将其移出无菌区并将其转移到 3 mL K2EDTA 真空试管中。保持在4°C，直到样品转移到实验室，并尽早进行进一步处理。 注意:通过此程序可以回收的胰液体积差异很大，根据我们的经验，大约从 0.2 mL 到 3 mL 不等。取汁量高度取决于患者:Wirsung导管的尺寸和胰腺的功能状态（功能与萎缩腺）。根据我们的经验，没有权宜之计可以用来增加回收的胰液量。</li>
	</ol></li>
</ol>2. 胰液的加工
<ol><li>在4°C下以400× g 离心胰液10分钟以除去任何细胞或碎片。 注意:胰液在离心前应透明。手术过程中有时会发生血液污染，使样本看起来更浑浊和更红。考虑从进一步分析中排除此类样本。</li>
	<li>回收上清液，等分试样并储存在-80°C直至进一步分析。</li>
</ol>3.诱导皮下肿瘤
注意:小鼠Panc02和DT6606细胞系分别从Lorenzo Piemonti教授（意大利米兰圣拉斐尔糖尿病研究所）和Francesco Novelli教授（意大利都灵实验研究和医学研究中心）获得，如前所述12。
<ol><li>肿瘤细胞的生长<ol><li>罗斯威尔公园纪念研究所（RPMI）1640培养基中的培养Panc02和DT6606细胞含有10%胎牛血清（FBS），2mM L-谷氨酰胺和1%青霉素 - 链霉素抗生素。</li>
		<li>在肿瘤注射前1-2周解冻冷冻细胞，根据细胞系的生长速度。</li>
		<li>在无菌条件下在37°C，5%CO2 和95%湿度下培养细胞。</li>
		<li>用0.025%胰蛋白酶/ EDTA溶液分离细胞在37°C下5分钟，当它们达到80%汇合时，并通过离心消除胰蛋白酶。 注意:DT6606是原代细胞，非永生化细胞，来源于LSL-KrasG12D-Pdx1-Cre小鼠，在 体内 注射前不应传代超过3次，以保持其原始特性。建议在注射前7-10天解冻DT6606细胞。</li>
	</ol></li>
	<li>体内注射肿瘤细胞
	<ol><li>胰蛋白酶消化细胞（见步骤3.1.4）并用磷酸盐缓冲盐水（PBS）洗涤一次。通过离心消除PBS，并在计数前将细胞重悬于新鲜PBS中。</li>
		<li>计数细胞并将其重悬于PBS中，浓度为0.5-1 x 107 个细胞/ mL，以使终浓度为0.5-1 x 106 个细胞/ 100μL以注入每只小鼠。准备多余的细胞。将细胞保持在4°C或冰上，直到程序结束。</li>
		<li>根据不同的细胞系或处理将动物（8周龄雌性C57BL / 6J小鼠）分组在不同的笼子中。</li>
		<li>手动约束动物并使用氯胺酮（80mg / kg）和甲苯噻嗪（10mg / kg）的混合物或根据当地批准的程序麻醉它们。</li>
		<li>用电动剃须刀剃除注射部位，通常是腿上方的侧腹，并用酒精仔细清洁注射部位。</li>
		<li>用 1 mL 注射器上下移液细胞悬液，通过上下移动活塞去除任何气泡。将25G针头连接到注射器上，并向上推动活塞，直到细胞悬液到达针头开口。</li>
		<li>用平头镊子捏住侧腹的皮肤，并小心地将针头插入镊子之间的皮肤褶皱底部，不要刺穿腹膜腔或肌肉组织。要验证针头的正确位置，请轻轻尝试将针尖侧向移动到皮肤下。针头应自由移动。</li>
		<li>缓慢注入100μL细胞悬液（含有0.5-1 x 106 个细胞），轻轻夹住注射部位几秒钟，然后缓慢抽出针头，没有任何侧向运动。</li>
		<li>将鼠标放回笼子并监测麻醉恢复情况。</li>
		<li>使用卡尺检查肿瘤生长3-4周。当肿瘤达到约 0.5-1 cm3 时，使用 CO2 或根据当地批准的程序对动物实施安乐死。</li>
	</ol></li>
</ol>4. 肿瘤间质液（TIF）的分离
<ol><li>皮下肿瘤切除<ol><li>用纸胶带堵住动物的四肢，并用酒精清洁皮肤。切开腹部的皮肤，将其与腹膜分开并一直到四肢。在剪刀，夹子和最终手术刀的帮助下切除在侧腹皮肤下生长的肿瘤。</li>
		<li>称量肿瘤并将其保存在冰上的干净管中，直到分离TIF为止。</li>
	</ol></li>
	<li>通过离心分离TIF<ol><li>将肿瘤切成两半，用PBS快速冲洗两部分，并在滤纸上轻轻吸干以去除多余的PBS。尽快执行这些步骤，以避免肿瘤蒸发。</li>
		<li>立即将肿瘤转移到固定在50mL锥形管上的20μm尼龙细胞过滤器中。</li>
		<li>在4°C下以400× g 离心管10分钟。 注意:这种低速离心可保持细胞完整性，避免细胞内隔室污染TIF。细胞内内容物泄漏可以在下游应用中进行测试，例如通过评估细胞内管家蛋白（例如核糖体蛋白）的存在25。</li>
		<li>从管底部回收TIF，最终将其等分并立即将其冷冻在干冰上并储存在-80°C直至进一步分析。 可选:根据要执行的下游蛋白质组学分析，用蛋白酶抑制剂混合物在PBS中稀释样品，以避免特定分子的降解。 注意:根据肿瘤的组成，在某些情况下，非常小的肿瘤不会产生任何液体。</li>
	</ol></li>
	<li>通过洗脱分离TIF的<ol><li>用剪刀或手术刀将肿瘤切成小块（≈1-3毫米3），并用冷PBS仔细冲洗。 注意:在此步骤中，快速工作并执行最少的操作以避免细胞损坏非常重要。</li>
		<li>将肿瘤碎片转移到 1.5 mL 管中，并加入 500 μL PBS 和蛋白酶抑制剂混合物，以避免分析物降解。在37°C和5%CO2下孵育1小时。</li>
		<li>回收上清液并将其转移到新的 1.5 mL 管中。在4°C下以1，000× g 离心5分钟，以从样品中除去任何细胞。</li>
		<li>将上清液转移到新管中，并在4°C下再次以2，000× g 离心8分钟。</li>
		<li>将上清液转移到新管中，并在4°C下以20，000× g 再次离心30分钟以除去任何碎屑。回收上清液。立即将TIF样品分装并在干冰上冷冻，并储存在-80°C直至进一步分析。</li>
	</ol></li>
</ol>

作者没有什么可透露的。

在这项研究中，我们描述了术中收集胰液的技术，这是一种在很大程度上未开发的液体活检。我们最近表明，胰液可以用作疾病12代谢标志物的来源。对其他液体活检（如血液5、6、7、尿液8 和唾液9）的代谢组学分析在区分 PDAC 与健康受试者或胰腺炎方面显示出有希望的结果。然而，胰液是由胰腺导管细胞直接分泌的，并且由于其靠近肿瘤部位，即使在疾病的早期阶段，它也可以更可靠地指示周围组织的变化。事实上，虽然胰液的H-NMR波谱数据实现了PDAC患者与其他良性胰腺疾病的区分，但血浆样品却没有。因此，尽管胰液比其他液体活检更难获得，但它是临床生物标志物的宝贵来源，可以帮助识别快速发展的疾病。我们在这里描述了胰液收集的术中技术;然而，它也可以在微创手术期间进行，以确定有价值的疾病早期标志物。不同的收集程序是否会影响胰液的蛋白质组组成仍有待确定。
虽然其他液体活检在PDAC小鼠模型中很容易获得，但如果考虑遗传或原位模型，体内胰液的收集可能非常具有挑战性，并且在皮下模型中根本不可行。因此，在本文中，我们建议使用肿瘤间质液作为一种有价值且广泛适用的替代方案。事实上，与胰液类似，间质液组成直接受到局部环境改变的影响。来自不同肿瘤（如肝细胞45，46，肾细胞41，卵巢22，47，48和乳腺癌42，49）的TIF的蛋白质组学分析在候选生物标志物的研究中显示出令人鼓舞的结果。然而，鉴定的候选蛋白质几乎没有重叠，这表明用于分离TIF的方法以及执行的下游分析和数据收集可能会影响检测到的分析物。最近一项关于鳞状细胞癌的研究比较了这里描述的两种TIF分离方法，离心和洗脱，观察到TIF组成与所用方法无关的强烈一致性，尽管离心产生了更高含量的细胞外蛋白25。
这里描述的用于TIF分离，组织离心和组织洗脱的两种方法都具有技术要求低，快速且只需要基本实验室设备的优点。与其他方法（例如微透析和毛细管超滤）相比，离心和洗脱技术存在仅在 离体可行的内在局限性。在选择合适的方法时，重要的是要考虑几个问题，例如实验的分析目的，回收的体积和细胞破裂。还应考虑肿瘤的组成，因为它会影响恢复的TIF体积。组织离心最初是为细胞贫乏和富含胶原蛋白的组织（如角膜38）开发的;另一方面，肿瘤通常是以高细胞性和丰富的血管化为特征的组织，这两个特征都增加了导水性，因此应有助于通过离心分离TIF。然而，一些肿瘤，包括PDAC，具有丰富的基质或纤维化成分，富含细胞外基质蛋白，如胶原蛋白，其倾向于将大分子保留在组织中21。
另一方面，组织洗脱是基于蛋白质从组织到洗脱液的被动扩散，并且已成功用于多种肿瘤43。洗脱可以回收更大的体积，但蛋白质将被稀释。因此，组织洗脱可能不适用于丰度非常低的分子。此外，洗脱需要更大程度的肿瘤操作，可能导致TIF中的细胞内内容物泄漏。这可能与生物标志物的发现无关，但如果目的是确定蛋白质的局部生产，则可能会引入偏差。应在下游应用中测试细胞破碎量，以便根据肿瘤类型选择最合适的方法。这可以通过多种方式进行，例如通过测试TIF25中管家蛋白（例如核糖体蛋白）的存在。另一种建议的方法是比较TIF和血浆中选定的细胞外物质（如肌酐或Na+）的浓度，两者应相似22。离心方法也应考虑细胞内内容物泄漏;事实上，对于什么应该被认为是"低速"以避免细胞破裂，仍然没有普遍的共识，这可能是由于肿瘤成分的差异。我们选择使用400 x g速度，因为已经表明对于 g 力<42438，细胞内内容物不会发生稀释。
无论采用何种方法，TIF都是对肿瘤微环境进行采样的宝贵且广泛适用的来源。我们已经证明它概括了肿瘤的内在特征，可用于识别疾病或治疗靶点的生物标志物。

胰腺导管腺癌（PDAC）是最具侵袭性的肿瘤之一，很快就会成为第二大死因1，2，3。它以其免疫抑制微环境和对免疫治疗方案无反应而闻名4。目前，手术切除仍然是PDAC的唯一治愈选择，但早期复发和术后并发症的频率很高。直到晚期才有特定症状，无法进行早期诊断，从而导致疾病的最后期限。此外，PDAC与其他良性胰腺病变之间的症状重叠会阻碍使用当前的诊断策略实现及时可靠的诊断。识别与特定胰腺病变相关的变量可以促进手术决策过程并改善患者分析。
使用易于获取的体液（例如血液5，6，7，尿液8，唾液9和胰液10，11，12）在生物标志物发现方面取得了有希望的结果。许多研究利用全面的"组学"方法，如基因组学、蛋白质组学和代谢组学技术，来识别可以区分PDAC和其他良性胰腺疾病的候选分子或特征。我们最近证明，胰液是一种相对未开发的体液，可用于识别具有不同临床特征的患者的代谢特征12。胰液是一种富含蛋白质的液体，它积累胰腺导管细胞的分泌组并流向主胰管，然后流向主胆总管。由于它靠近胰腺，它可能受到肿瘤肿块引起的微环境扰动的强烈影响（图1），因此比血液或尿液或基于组织的分析更具信息性。一些研究已经探索了胰液使用各种方法鉴定疾病新型生物标志物的潜力，包括细胞学分析13，质谱分析14，15，评估遗传和表观遗传标志物，如K-ras和p53突变16，17，DNA甲基化改变18和miRNA19.从技术上讲，胰液可以通过术中或微创手术收集，例如内窥镜超声、逆行胰胆管造影或通过内镜收集十二指肠液分泌物20。目前尚不清楚所使用的收集技术在多大程度上影响胰液组成。我们在这里描述了术中收集程序，并表明胰液可以代表PDAC生物标志物的宝贵来源。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61687/61687fig01.jpg"> 图1:胰液收集的示意图。 （A）描述胰液分泌到胰管中的示意图，并在手术过程中收集胰液。插图显示了肿瘤微环境的特写:胰液收集胰管中肿瘤和基质细胞释放的分子。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/61687/61687fig01large.jpg" target="_blank">请点击此处查看此图的大图。</a>
在PDAC的遗传和原位小鼠模型中收集胰液将受到赞赏，以便在临床前机制研究中利用这种生物流体;然而，这种手术在技术上可能非常具有挑战性，对于皮下肿瘤等更简单的模型是不可行的。出于这个原因，我们将肿瘤间质液（TIF）确定为胰液的替代来源，因为它具有作为周围扰动指标的相似特征。间质液（IF）是在血液和淋巴管外发现的细胞外液体，可沐浴组织细胞21。IF组成受器官血液循环和局部分泌的影响;事实上，周围细胞在IF21中积极产生和分泌蛋白质。间质反映了周围组织的微环境变化，因此可以代表在几种病理环境中发现生物标志物的宝贵来源，例如肿瘤。TIF中高浓度的局部分泌蛋白可用于鉴定要作为血浆22，23，24中的预后或诊断生物标志物进行测试的候选分子。一些研究已经证明TIF是高通量蛋白质组学方法的合适样品，例如质谱技术23，24，25，多重ELISA方法26和microRNA分析27。
已经提出了几种用于分离肿瘤中IF的方法，其大致可分为体内（毛细管超滤28，29，30，31和微透析32，33，34，35）和离体方法（组织离心22，36，37，38和组织洗脱39，40，41，42）。这些技术已经进行了广泛的详细审查43，44。适当方法的选择应考虑下游分析和应用以及回收量等问题。我们最近使用这种方法作为原理证明，以证明来自两种小鼠胰腺腺癌细胞系的肿瘤的不同代谢活性12。基于文献24，38，我们选择使用低速离心方法，以避免细胞破裂和细胞内内容物稀释。TIF中葡萄糖和乳酸的含量都反映了两种不同细胞系的不同糖酵解特性。在这里，我们详细描述了两种最常用的TIF分离方法的方案:组织离心和组织洗脱（图2）。
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61687/61687fig02.jpg"> 图2:肿瘤间质液分离方法的示意图。 方案中详细描述的技术的示意图，即组织离心（A）和组织洗脱（B）。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/61687/61687fig02large.jpg" target="_blank">请点击此处查看此图的大图。</a>

<table><tbody><tr><td>1 mL syringe</td><td>BD Biosciences</td><td>309659</td><td></td></tr><tr><td>1.5 mL Eppendorf tube</td><td>Greiner BioOne</td><td>GR616201</td><td></td></tr><tr><td>20 &amp;micro;m nylon cell strainer</td><td>pluriSelect</td><td>43-50020-03</td><td></td></tr><tr><td>25G needle</td><td>BD Biosciences</td><td>305122</td><td></td></tr><tr><td>3 mL K2EDTA vacutainer</td><td>BD Biosciences</td><td>366473</td><td></td></tr><tr><td>3 mL syringe</td><td>BD Biosciences</td><td>309656</td><td></td></tr><tr><td>50 mL Falcon tube</td><td>Corning</td><td>352098</td><td></td></tr><tr><td>Clamps</td><td>Medicon</td><td>06.20.12</td><td></td></tr><tr><td>Disposable scalpel</td><td>Medicom</td><td>9000-10</td><td></td></tr><tr><td>Fetal bovine serum</td><td>Microtech</td><td>MG10432</td><td></td></tr><tr><td>Flat-tipped forceps</td><td>Medicon</td><td>06.00.10</td><td></td></tr><tr><td>Penicillin-Streptomycin</td><td>Lonza</td><td>ECB3001D</td><td></td></tr><tr><td>Phosphate-Buffered Saline (PBS)</td><td>Sigma-Aldrich</td><td>D8537</td><td></td></tr><tr><td>Protease inhibitor cocktail</td><td>Roche</td><td>34044100</td><td></td></tr><tr><td>RPMI medium</td><td>Euroclone</td><td>ECB9006L</td><td></td></tr><tr><td>Scissors</td><td>Medicon</td><td>02.04.09</td><td></td></tr><tr><td>Trypsin/EDTA 1x</td><td>Lonza</td><td>BE17-161F</td><td></td></tr><tr><td>Ultraglutamine 100x</td><td>Lonza</td><td>BE17-605E/U1</td><td></td></tr></tbody></table>

isolation of proximal fluids to investigate the tumor microenvironment of pancreatic adenocarcinoma

胰腺腺癌（PDAC）是癌症相关死亡的第四大原因，很快就会成为第二大原因。迫切需要与特定胰腺病变相关的变量，以帮助术前鉴别诊断和患者分析。胰液是一种相对未开发的体液，由于其靠近肿瘤部位，它反映了周围组织的变化。在这里，我们详细描述了术中收集程序。不幸的是，将胰液收集转化为PDAC的小鼠模型以进行机理研究在技术上非常具有挑战性。肿瘤间质液（TIF）是血液和血浆外的细胞外液，用于沐浴肿瘤和基质细胞。与胰液类似，由于其收集和浓缩在血浆中稀释的分子的特性，TIF可以用作微环境改变的指标和疾病相关生物标志物的宝贵来源。由于TIF不易获得，因此提出了各种技术来隔离TIF。我们在这里描述了两种简单且技术要求不高的分离方法:组织离心和组织洗脱。

我们按照上述程序从PDAC（n = 31）和其他良性胰腺疾病（非PDAC，n = 9）患者那里获取胰液，包括胰腺炎（n = 2），状壶腹肿瘤（n = 4），神经内分泌肿瘤（n = 2），导管内状粘液瘤变（IPMN; n = 1）12。然后使用核磁共振（1H-NMR）对胰液样品进行代谢组学分析12。通过过滤大分子（例如脂蛋白、脂质等）的广核磁共振信号，我们能够详细了解小分子量代谢物，如 1D Carr-Purcell-Meiboom-Gill （CPMG） 谱图所示（图 3A）。监督OPLS-DA分析表明，胰液中检测到的代谢谱能够区分PDAC和非PDAC患者，通过交叉验证获得的准确率为82.4%（图3B）。有趣的是，对来自相同患者的血浆样本进行的代谢组学分析没有产生相同的鉴别力（通过交叉验证获得的准确度为75.2%）（图3C）。这些结果表明，胰液是一种富含蛋白质的样品，适合进行全面的"组学"分析。此外，与血浆相比，胰液具有更高的辨别能力，这表明将"上游"移动到更靠近肿瘤部位的样本可能是识别生物标志物的成功策略，有助于从其他胰腺疾病中分离出PDAC。
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61687/61687fig03.jpg"> 图3:胰液和血浆中的代谢组学含量。 （A） PDAC（n = 31，蓝色）和非PDAC样品（n = 9，绿色）的 1H-NMR CPMG谱图，显示了胰液中所含小分子量代谢物的详细信息。ppm，百万分之一。（B）对胰汁PDAC样品（蓝色圆圈）和非PDAC样品（紫色三角形）的CPMG光谱进行监督多变量OPLS-DA分析的评分。该分析将两组（每组由蜘蛛图勾勒）分开，通过交叉验证获得的准确率为 82.4%。（C）对血浆PDAC样品（n = 22，蓝色圆圈）和非PDAC样品（n = 7，紫色三角形）的1D-NOESY光谱进行监督多变量OPLS-DA分析的分数。通过交叉验证获得的准确率为 75.2%。该图经许可修改自Cortese等人12 。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/61687/61687fig03large.jpg" target="_blank">请点击此处查看此图的大图。</a>
为了证明TIF含量可靠地反映了肿瘤微环境的变化，我们按照上述方案皮下注射了两种糖酵解速率相反的胰腺癌细胞系Panc02（高度糖酵解）和DT6606（低糖酵解）。然后，我们使用上述低速离心方法从切除的肿瘤中分离TIF（见第4.2段）。从重量在0.25-1g之间的肿瘤中，我们回收了5-15μL的TIF，然后将其用于量化葡萄糖和乳酸的浓度。与低糖酵解肿瘤相比，来自高度糖酵解肿瘤的TIF含有更少的葡萄糖和更多的乳酸（图4）。该数据表明，TIF可以作为肿瘤来源代谢物的来源，并根据肿瘤本身而变化。
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/61687/61687fig04.jpg"> 图4:TIF中的葡萄糖和乳酸含量反映了肿瘤的内在特征。 （A）使用组织离心法从Panc02（高度糖酵解，A 中 n=10，B 中 n=9）和 DT6606（低糖酵解，A 中 n=5，B 中 n=4）皮下植入的肿瘤中分离出组织液中的葡萄糖和 （B） 乳酸浓度。箱形图按箱形给出中位数、下四分位数和上四分位数，按晶须给出最小值和最大值。<a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/61687/61687fig04large.jpg" target="_blank">请点击此处查看此图的大图。</a>

Watch this Scientific Journal Video about Isolation of Proximal Fluids to Investigate the Tumor Microenvironment of Pancreatic Adenocarcinoma at JoVE.com

Isolation of Proximal Fluids to Investigate the Tumor Microenvironment of Pancreatic Adenocarcinoma

胰液是人类胰腺癌生物标志物的宝贵来源。我们在这里描述一种术中收集程序的方法。为了克服在小鼠模型中采用该程序的挑战，我们建议使用替代样品，肿瘤组织液，并在此处描述其分离的两种方案。

分离近端液研究胰腺腺癌肿瘤微环境

Pancreatic adenocarcinoma (PDAC) is the fourth leading cause of cancer-related death, and soon to become the second. There is an urgent need of variables ...

isolation-proximal-fluids-to-investigate-tumor-microenvironment

Universidad San Sebastian

Research

JoVE Journal

Cancer Research

3.3K 次观看.  Humanitas Clinical and Research Center-IRCCS. 胰液是人类胰腺癌生物标志物的宝贵来源。我们在这里描述一种术中收集程序的方法。为了克服在小鼠模型中采用该程序的挑战，我们建议使用替代样品，肿瘤组织液，并在此处描述其分离的两种方案。

视频: Isolation of Proximal Fluids to Investigate the Tumor Microenvironment of Pancreatic Adenocarcinoma

Immunophenotyping of Orthotopic Homograft (Syngeneic) of Murine Primary KPC Pancreatic Ductal Adenocarcinoma by Flow Cytometry

Homograft (syngeneic) tumors are the workhorse of today&#39;s immuno-oncology (I/O) preclinical research. The tumor microenvironment (TME), particularly its immune-components, is vital to the prognosis and prediction of treatment outcomes, especially those of immunotherapy. TME immune-components are composed of different subsets of tumor-infiltrating immune cells assessable by multi-color FACS. Pancreatic ductal adenocarcinoma (PDAC) is among the deadliest malignances lacking good treatment options, thus an urgent and unmet medical need. One important reason for its non-responsiveness to various therapies (chemo-, targeted, I/O) has been its abundant TME, consisting of fibroblasts and leukocytes that protect tumor cells from these therapies. Orthotopically implanted PDAC is believed to more accurately recapture the TME of human pancreatic cancers than conventional subcutaneous (SC) models.
Homograft tumors (KPC) are transplants of mouse spontaneous PDAC originating from genetically engineered KPC-mice (KrasG12D/+/P53-/-/Pdx1-Cre) (KPC-GEMM). The primary tumor tissue is cut into small fragments (~2 mm3) and transplanted subcutaneously (SC) to the syngeneic recipients (C57BL/6, 7&#8211;9 weeks old). The homografts were then surgically orthotopically transplanted onto the pancreas of new C57BL/6 mice, along with SC-implantation, which reached tumor volumes of 300&#8211;1,000 mm3 by 17 days. Only tumors of 400&#8211;600 mm3 were harvested per approved autopsy procedure and cleaned to remove the adjacent non-tumor tissues. They were dissociated per protocol using a tissue dissociator into single-cell suspensions, followed by staining with designated panels of fluorescently-labeled antibodies for various markers of different immune cells (lymphoid, myeloid and NK, DCs). The stained samples were analyzed using multi-color FACS to determine numbers of immune cells of different lineages, as well as their relative percentage within tumors. The immune profiles of orthotopic tumors were then compared to those of SC tumors. The preliminary data demonstrated significantly elevated infiltrating TILs/TAMs in tumors over the pancreas, and higher B-cell infiltration into orthotopic rather than SC tumors.

Immunophenotyping of Orthotopic Homograft Syngeneic of Murine Primary KPC Pancreatic Ductal Adenocarcinoma by Flow Cytometry

The experimental procedure on the immunophenotyping of murine orthotopic PDAC homografts aims at profiling the tumor immuno-microenvironment. Tumors are orthotopically implanted via surgery. Tumors of 200&#8211;600 mm3 in size were harvested and dissociated to prepare single-cell suspensions, followed by multi-immune marker FACS analysis using different fluorescently-labeled antibodies.

流式细胞术对小鼠原发性胰腺导管腺癌原位同种异体 (自体) 免疫分型的研究

Homograft (syngeneic) tumors are the workhorse of today&#39;s immuno-oncology (I/O) preclinical research. The tumor microenvironment (TME), particularly ...

科研

癌症研究

Isolating and Analyzing Cells of the Pancreas Mesenchyme by Flow Cytometry

The pancreas is comprised of epithelial cells that are required for food digestion and blood glucose regulation. Cells of the pancreas microenvironment, including endothelial, neuronal, and mesenchymal cells were shown to regulate cell differentiation and proliferation in the embryonic pancreas. In the adult, the function and mass of insulin-producing cells were shown to depend on cells in their microenvironment, including pericyte, immune, endothelial, and neuronal cells. Lastly, changes in the pancreas microenvironment were shown to regulate pancreas tumorigenesis. However, the cues underlying these processes are not fully defined. Therefore, characterizing the different cell types that comprise the pancreas microenvironment and profiling their gene expression are crucial to delineate the tissue development and function under normal and diseased states. Here, we describe a method that allows for the isolation of mesenchymal cells from the pancreas of embryonic, neonatal, and adult mice. This method utilizes the enzymatic digestion of mouse pancreatic tissue and the subsequent fluorescence-activated cell sorting (FACS) or flow-cytometric analysis of labeled cells. Cells can be labeled by either immunostaining for surface markers or by the expression of fluorescent proteins. Cell isolation can facilitate the characterization of genes and proteins expressed in cells of the pancreas mesenchyme. This protocol was successful in isolating and culturing highly enriched mesenchymal cell populations from the embryonic, neonatal, and adult mouse pancreas.

Here, we describe a method for the isolation of cells in the pancreas microenvironment from embryonic, neonatal and adult mouse tissue, focusing on the isolation of mesenchymal cells. This method allows profiling of cell gene expression and protein secretion in order to elucidate the extrinsic signals that regulate pancreas development, function, and tumorigenesis.

隔离并通过流式细胞仪分析胰腺间充质细胞

The pancreas is comprised of epithelial cells that are required for food digestion and blood glucose regulation. Cells of the pancreas microenvironment, ...

发育生物学

Isolation and Characterization of Patient-derived Pancreatic Ductal Adenocarcinoma Organoid Models

Pancreatic ductal adenocarcinoma (PDAC) is amongst the most lethal malignancies. Recently, next-generation organoid culture methods enabling the 3-dimensional (3D) modeling of this disease have been described. Patient-derived organoid (PDO) models can be isolated from both surgical specimens as well as small biopsies and form rapidly in culture. Importantly, organoid models preserve the pathogenic genetic alterations detected in the patient&#39;s tumor and are predictive of the patient&#39;s treatment response, thus enabling translational studies. Here, we provide comprehensive protocols for adapting tissue culture workflow to study 3D, matrix embedded, organoid models. We detail methods and considerations for isolating and propagating primary PDAC organoids. Furthermore, we describe how bespoke organoid media is prepared and quality controlled in the laboratory. Finally, we describe assays for downstream characterization of the organoid models such as isolation of nucleic acids (DNA and RNA), and drug testing. Importantly we provide critical considerations for implementing organoid methodology in a research laboratory.

Patient-derived organoid cultures of pancreatic ductal adenocarcinoma are a rapidly established 3-dimensional model that represent epithelial tumor cell compartments with high fidelity, enabling translational research into this lethal malignancy. Here, we provide detailed methods to establish and propagate organoids as well as to perform relevant biological assays using these models.

患者衍生胰腺腺腺腺腺癌有机体模型的分离和特征

Pancreatic ductal adenocarcinoma (PDAC) is amongst the most lethal malignancies. Recently, next-generation organoid culture methods enabling the ...

Generation of Orthotopic Pancreatic Tumors and Ex vivo Characterization of Tumor-Infiltrating T Cell Cytotoxicity

In vivo models of pancreatic cancer provide invaluable tools for studying disease dynamics, immune infiltration and new therapeutic strategies. The orthotopic murine model can be performed on large cohorts of immunocompetent mice simultaneously, is relatively inexpensive and preserves the cognate tissue microenvironment. The quantification of T cell infiltration and cytotoxic activity within orthotopic tumors provides a useful indicator of an antitumoral response.
This protocol describes the methodology for surgical generation of orthotopic pancreatic tumors by injection of a low number of syngeneic tumor cells resuspended in 5 &#181;L basement membrane directly into the pancreas. Mice bearing orthotopic tumors take approximately 30 days to reach endpoint, at which point tumors can be harvested and processed for characterization of tumor-infiltrating T cell activity. Rapid enzymatic digestion using collagenase and DNase allows a single-cell suspension to be extracted from tumors. The viability and cell surface markers of immune cells extracted from the tumor are preserved; therefore, it is appropriate for multiple downstream applications, including flow-assisted cell sorting of immune cells for culture or RNA extraction, flow cytometry analysis of immune cell populations. Here, we describe the ex vivo stimulation of T cell populations for intracellular cytokine quantification (IFN&#947; and TNF&#945;) and degranulation activity (CD107a) as a measure of overall cytotoxicity. Whole-tumor digests were stimulated with phorbol myristate acetate and ionomycin for 5 h, in the presence of anti-CD107a antibody in order to upregulate cytokine production and degranulation. The addition of brefeldin A and monensin for the final 4 h was performed to block extracellular transport and maximize cytokine detection. Extra- and intra-cellular staining of cells was then performed for flow cytometry analysis, where the proportion of IFN&#947;+, TNF&#945;+ and CD107a+ CD4+ and CD8+ T cells was quantified.
This method provides a starting base to perform comprehensive analysis of the tumor microenvironment.

Generation of Orthotopic Pancreatic Tumors and Ex vivo Characterization of Tumor-Infiltrating T Cell Cytotoxicity

This protocol describes the surgical generation of orthotopic pancreatic tumors and the rapid digestion of freshly isolated murine pancreatic tumors. Following digestion, viable immune cell populations can be used for further downstream analysis, including ex vivo stimulation of T cells for intracellular cytokine detection by flow cytometry.

正交胰腺肿瘤的产生与肿瘤渗透T细胞细胞毒性的外体特征

In vivo models of pancreatic cancer provide invaluable tools for studying disease dynamics, immune infiltration and new therapeutic strategies. The ...

Pancreatic Tissue Dissection to Isolate Viable Single Cells

The pancreas includes two major systems: the endocrine system, which produces and secretes hormones, and the exocrine system, which accounts for approximately 90% of the pancreas and includes cells that produce and secrete digestive enzymes. The digestive enzymes are produced in the pancreatic acinar cells, stored in vesicles called zymogens, and are then released into the duodenum via the pancreatic duct to initiate metabolic processes. The enzymes produced by the acinar cells can kill cells or degrade cell-free RNA. In addition, acinar cells are fragile, and common dissociation protocols result in a large number of dead cells and cell-free proteases and RNases. Therefore, one of the biggest challenges in pancreatic tissue digestion is recovering intact and viable cells, especially acinar cells. The protocol presented in this article shows a two-step method that we developed to meet this need. The protocol can be used to digest normal pancreata, pancreata that include pre-malignant lesions, or pancreatic tumors that include a large number of stromal and immune cells.

Pancreatic metaplastic cells are precursors of malignant cells that give rise to pancreatic tumors. However, isolating intact viable pancreatic cells is challenging. Here, we present an efficient method for pancreatic tissue dissociation. The cells can then be used for single-cell RNA sequencing (scRNA-seq) or for two- or three-dimensional co-culturing.

胰腺组织解剖以分离活的单细胞

The pancreas includes two major systems: the endocrine system, which produces and secretes hormones, and the exocrine system, which accounts for ...

来自新鲜组织的胰腺癌来源的肿瘤类器官和成纤维细胞

Establishment of Pancreatic Cancer-Derived Tumor Organoids and Fibroblasts From Fresh Tissue

Tumor organoids are three-dimensional (3D) ex vivo tumor models that recapitulate the biological key features of the original primary tumor tissues. Patient-derived tumor organoids have been used in translational cancer research and can be applied to assess treatment sensitivity and resistance, cell-cell interactions, and tumor cell interactions with the tumor microenvironment. Tumor organoids are complex culture systems that require advanced cell culture techniques and culture media with specific growth factor cocktails and a biological basement membrane that mimics the extracellular environment. The ability to establish primary tumor cultures highly depends on the tissue of origin, the cellularity, and the clinical features of the tumor, such as the tumor grade. Furthermore, tissue sample collection, material quality and quantity, as well as correct biobanking and storage are crucial elements of this procedure. The technical capabilities of the laboratory are also crucial factors to consider. Here, we report a validated SOP/protocol that is technically and economically feasible for the culture of ex vivo tumor organoids from fresh tissue samples of pancreatic adenocarcinoma origin, either from fresh primary resected patient donor tissue or patient-derived xenografts (PDX). The technique described herein can be performed in laboratories with basic tissue culture and mouse facilities and is tailored for wide application in the translational oncology field.

作者聚焦:从新鲜组织中建立胰腺癌来源的肿瘤类器官和成纤维细胞  

Tumor organoids have revolutionized cancer research and the approach to personalized medicine. They represent a clinically relevant tumor model that allows researchers to stay one step ahead of the tumor in the clinic. This protocol establishes tumor organoids from fresh pancreatic tumor tissue samples and patient-derived xenografts of pancreatic adenocarcinoma origin.

从新鲜组织中建立胰腺癌来源的肿瘤类器官和成纤维细胞

Tumor organoids are three-dimensional (3D) ex vivo tumor models that recapitulate the biological key features of the original primary tumor tissues. ...

分离细胞核用于实体瘤标本中的多组测序

Optimized Nuclei Isolation from Fresh and Frozen Solid Tumor Specimens for Multiome Sequencing

Multiome sequencing, which provides same-cell/paired single-cell RNA- and the assay for transposase-accessible chromatin with sequencing (ATAC-sequencing) data, represents a breakthrough in our ability to discern tumor cell heterogeneity-a primary focus of translational cancer research at this time. However, the quality of sequencing data acquired using this advanced modality is highly dependent on the quality of the input material. 
Digestion conditions need to be optimized to maximize cell yield without sacrificing quality. This is particularly challenging in the context of solid tumors with dense desmoplastic matrices that must be gently broken down for cell release. Freshly isolated cells from solid tumor tissue are more fragile than those isolated from cell lines. Additionally, as the cell types isolated are heterogeneous, conditions should be selected to support the total cell population. 
Finally, nuclear isolation conditions must be optimized based on these qualities in terms of lysis times and reagent types/ratios. In this article, we describe our experience with nuclear isolation for the 10x Genomics multiome sequencing platform from solid tumor specimens. We provide recommendations for tissue digestion, storage of single-cell suspensions (if desired), and nuclear isolation and assessment.

作者聚焦：在具有挑战性的肿瘤微环境中增强用于多组测序的细胞核分离

The protocol provides a reliable and optimized approach to the isolation of nuclei from solid tumor specimens for multiome sequencing using the 10x Genomics platform, including recommendations for tissue dissociation conditions, cryopreservation of single-cell suspensions, and assessment of isolated nuclei.

从新鲜和冷冻实体瘤标本中优化细胞核分离，用于多组学测序

Multiome sequencing, which provides same-cell/paired single-cell RNA- and the assay for transposase-accessible chromatin with sequencing (ATAC-sequencing) ...

分离近端液研究胰腺腺癌肿瘤微环境

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从新鲜和冷冻实体瘤标本中优化细胞核分离，用于多组学测序

从新鲜和冷冻实体瘤标本中优化细胞核分离，用于多组学测序