作者想承认杰奎琳·罗森博格校对手稿。这项工作得到了美国卫生和人类服务部/国家卫生研究院的支持, 授予雅 DeClerck (grant 5R01 129377) 和 TJ 马爹利基金会。MH Kubala 是洛杉矶儿童医院塞班研究所的研究职业发展奖学金获得者。

使用从健康志愿者获得的人单核细胞的议定书部分遵循儿童医院洛杉矶人类研究伦理委员会的指导方针, 并已得到机构审查委员会在人体材料下的批准协议编号: 08-00208。
1. 用四环素调控的 shRNA 表达肿瘤细胞系的制备
<ol><li>shRNA PAI-1 到 pLKO 普洛向量的克隆8,9<ol>
<li>设计 shRNA 的低聚物, 包含年龄I 和EcoRI 在5的末端, 感官序列, 一个6核苷酸循环, 和反义序列。 注: 典型的寡核苷酸的设计如下: 向前寡聚: 5 ' CCGG-19-21 bp 感觉-CTCGAG-19-21 bp 反义-TTTTT, 反向寡聚: 5 ' AATTAAAAA-19-21 bp 感觉-CTCGAG-19-21 bp 反义 3 "。可在8中找到有关齐聚物设计的详细协议。在这项研究 3 shRNA 序列反对 PAI-1 被测试了为最强的沉寂作用: shRNA 132, shRNA 2, shRNA 333, 并且被搅乱的33包括在表 1。</li>
		<li>退火 shRNA 寡聚物和炒 shRNA 寡聚物。<ol>
<li>重组寡核苷酸到100µM 在水和混合1µL 向前寡核苷酸, 1 µL 反向寡核苷酸, 和8µL H2O。</li>
			<li>要退火的寡核苷酸, 混合如下: 1 µL 寡核苷酸混合物, 5 µL 缓冲10毫米三 (羟甲基) 氨基 (三) pH 7.5, 50 毫米氯化钠 (氯化钠), 10 毫米氯化镁 (氯化镁2), 1 毫米 dithioerythritol (DTE), 和44µL H2i/o.</li>
			<li>在95° c 的5分钟内, 在聚合酶链反应 (PCR) 热循环中孵育混合物, 关掉仪器, 等到室温 (RT) 到达。</li>
		</ol>
</li>
		<li>沉淀退火寡核苷酸加入5µL 3 米醋酸钠 pH 5.2 到50µL 退火寡核苷酸。<ol>
<li>加入100µL 冷100% 乙醇 (乙醇), 孵育30分钟, 在-80 ° c。离心机在台式离心机在最大速度为30分钟在4° c。</li>
			<li>除去上清液, 加入500µL 冷70% 乙醇, 离心30分钟, 除去上清, 并在20µL H2O 中溶解颗粒。</li>
			<li>用分光光度法测定退火寡核苷酸的浓度, 测定260纳米的吸光度。将寡核苷酸稀释到最终浓度为 1 ng/µL。</li>
		</ol>
</li>
		<li>准备 Luria Bertani (lb) 培养基和 lb 琼脂板。<ol>
<li>对于 LB 培养基, 溶解10克胰, 5 克酵母萃取物, 10 克氯化钠在 1 L 水中。使用 1 N 氢氧化钠和高压釜将介质 ph 值调整到 ph 值7.0。</li>
			<li>对于 lb 琼脂板, 添加15克/升的琼脂到 lb 培养基。高压釜和冷却到大约50° c。加入抗生素, 倒入盘子。</li>
		</ol>
</li>
		<li>条纹细菌转与 pLKO-普洛载体 (参见材料目录) 在 LB 琼脂板上补充100µg/毫升氨苄西林和孵化过夜 (O/N) 在37° c。</li>
		<li>接种100毫升 lb 培养基, 辅以100µg/毫升氨苄西林 (lb/氨苄西林) 与1菌落从板和增长 O/N 与震动在37° c。</li>
		<li>IsolateTet-pLKO-普洛从100毫升细菌培养使用质粒隔离试剂盒。</li>
		<li>用 EcoRI 和 AgeI 限制酶制备 pLKO 普洛载体的限制性反应如下: 1 µL EcoRI、1µL AgeI、5µL 10x 缓冲、1µL 质粒 DNA (1 µg)、42µL h2o 在1° c 处孵育 37 h。为了获得足够的裂解载体, 准备 5 x 50 µL 反应。</li>
		<li>使用1% 琼脂糖凝胶纯化从琼脂糖凝胶使用 DNA 提纯试剂盒。为了提高 dna 的产量, 通过使用琼脂糖凝胶梳在大水井中, 并通过手术刀从带上小心地除去大部分琼脂糖, 将琼脂糖在凝胶中的 dna 比降到最小。</li>
		<li>准备结扎反应混合如下: 1 ng 退火寡核苷酸, 50 ng 裂向量, 2 µL 10x 连接缓冲, 1 µL 连接, 水多达20µL. 结扎在16° c 下退火 shRNA 寡核苷酸的载体。<ol>
<li>此外, 还要准备一个无寡核苷酸的负控制反应来解释 uncleaved、部分劈开和 self-ligated 的向量, 这将导致假阳性的菌落。</li>
		</ol>
</li>
		<li>采用标准转化技术, 将结扎的混合物转化为具有化学能力的细菌细胞, 旨在防止含有直接重复的慢载体的同源重组。</li>
		<li>在37° c 时, 在氨苄青霉素 O/N 的板上生长细菌。 注意: 如果卫星的生长发生, 重复的转变和生长细菌在30° c, 以减慢增长, 从而防止卫星殖民地。</li>
		<li>为了验证成功结扎, 选择 10-20 单一的殖民地, 接种2毫升/氨苄西林培养基和一个氨苄西林板作为一个股票板块 (作为一个积极的克隆以后使用)。在37° c 时, 用摇动的 O/N 生长液体培养物。在37° c 下孵育板 O/N。</li>
		<li>在6° c 的时候用膜密封板。</li>
		<li>用质粒分离试剂分离液体培养基中的质粒 DNA。</li>
		<li>采用 XhoI 酶对抗氨苄西林菌落分离的质粒 DNA 进行限制性分析。对于每个克隆, 准备以下反应组合: 0.5 µL XhoI, 2.5 µL 10x 缓冲, 1 µL 质粒 DNA (0.5 µg), 21 µL h2o 在1° c 孵育 37 h。通过在1% 琼脂糖凝胶上运行反应, 分析了限制反应的结果。 注: XhoI 的限制分析将产生3碎片: 8447、1800和 200 bp。1800 bp 的 pLKO-普洛向量的袜序列不存在于含有 shRNA-PAI-1 的阳性克隆中, 而 XhoI 消化会导致大小的 DNA 片段: 8447、190和 130 bp。如图 1所示, 在从氨苄西林抗性菌落中分离出的 DNA 中未发现 2000 bp 片段数: 3、8和11。根据寡核苷酸的设计, 从阳性菌落中获得的 DNA 片段的长度和数量可能会有所不同。</li>
		<li>通过排序8,9, 验证 shRNA 序列是否正确插入到 pLKO-普洛向量中。</li>
		<li>接种100毫升磅/氨苄西林培养与菌落包含正确的克隆和生长 O/N 在37° c。</li>
		<li>用质粒分离试剂分离质粒 DNA。</li>
	</ol></li>
	<li>慢粒子的产生。
	<ol>
<li>用0.01% 聚 l-赖氨酸的无菌水溶液覆盖15厘米的多聚 l-赖氨酸的组织培养皿, 在 RT 中孵育15分钟, 用吸入和风干的方法去除溶液。</li>
		<li>种子 5 x 106人胚肾293细胞 (HEK293) 细胞在聚 l-赖氨酸包覆的 15 cm 盘中, 和文化在 Dulbecco 的修改鹰的培养基 (DMEM) 培养基补充 10% FBS 和1% 青霉素/链霉素混合 (笔/链球菌) 在37° c, 5% CO2 , 直到它们达到 70% 70-100。</li>
		<li>为了产生病毒微粒, 染 HEK293 细胞与25µg pLKO-在 shRNA, 25 µg psPAX, (包装质粒), 和5µg pMD2. 使用转染试剂的质粒 (包封)。 注: psPAX 和 pMD2 质粒是来自德尔 Trono 实验室的礼物 (瑞士洛桑理工联邦)。</li>
		<li>第二天诱导 HEK293 细胞通过改变培养基到 DMEM 补充10毫米丁酸钠和20毫米 HEPES pH 7.2, 并培养8小时。</li>
		<li>用磷酸盐缓冲盐水 (PBS) 冲洗细胞, 加入25毫升的新鲜 DMEM 培养基, 含20毫米 4-(2-羟基乙基)-1-piperazineethanesulfonic 酸 (HEPES) pH 7.2。在37° c 孵育48小时后, 通过移仔细收集含有病毒的培养基;避免溢出。</li>
		<li>通过0.45 µM 注射器过滤器过滤收集的培养基以去除 HEK293 细胞。含有病毒颗粒的培养基可立即使用或储存在-80 ° c 供以后使用。</li>
	</ol></li>
	<li>稳定转染细胞系的生成
	<ol>
<li>种子 HCT116 结肠癌细胞和 MDA-MB-231 乳腺癌细胞的5万细胞/井在 DMEM 培养基补充10% 胎牛血清 (FBS) 和1% 笔/链球菌病在 12-井板。孵育在37° c 在 5% CO2直到细胞是 60-70% 汇合。</li>
		<li>用 PBS 清洗细胞, 并在37° c 5% CO2中加入1毫升的含有病毒的培养基至癌细胞并孵育 O/N。作为一个控制, 添加1毫升的新鲜 DMEM 培养基补充 10% FBS 和1% 笔/链球菌感染2井与癌细胞。</li>
		<li>小心地用吸入剂去除含有病毒的培养基。用 PBS 清洗细胞, 用 10% (v/五) 无四环素 (无 DMEM) FBS 和1% 支笔/链球菌素改变培养基。</li>
		<li>72小时后, 用 PBS 清洗细胞, 将培养基改为 DMEM 10% 无 FBS 1% 笔/链球菌, 1 µg/毫升嘌呤用于选择病毒转染细胞。<ol>
<li>根据所使用的细胞线, 通过测试 non-transduced 细胞中的嘌呤浓度 (0.1、0.5、1、2和5µg/毫升) 的范围, 调整嘌呤浓度以进行最佳选择, 并选择没有控制细胞存活的最低浓度经过3天的文化。</li>
		</ol>
</li>
		<li>通过在嘌呤 3-14 天的存在下培养细胞来选择病毒转细胞。作为控制, 准备两个额外的井与 non-transduced 的细胞, 一个含有中等嘌呤和一个没有嘌呤。观察转细胞与对照井相比, 嘌呤在井中对细胞的部分杀伤作用。 注: 非转细胞不会在嘌呤的情况下生长。</li>
		<li>验证嘌呤抗 HCT116 结肠癌和 MDA-MB-231 乳腺癌细胞中条件 KD 的有效性。 注意: 强力霉素的有效浓度将随使用的细胞线而变化, 必须滴定 (通常从 100 ng/毫升到2µg/毫升)。<ol>
<li>确定对细胞无毒的强力霉素浓度, 但有效的 downregulating 表达的蛋白质的兴趣。在该协议中, 1 µg/毫升成功地用于体外。</li>
			<li>在0.1、1和2µg/毫升强力霉素存在和缺失的情况下培养72小时的细胞。用印迹分析法验证细胞中 downregulated 蛋白 (这里, PAI-1) 的表达水平。比较 HCT116 和 MDA-MB-231 细胞有条件地表达 shRNA 到炒控制单元。</li>
		</ol>
</li>
	</ol></li>
	<li>条件培养基的制备
	<ol>
<li>种子 1 x 106的细胞稳定转染的强力霉素调节 pLKO shRNA 含有慢在10厘米的菜肴。</li>
		<li>生长在罗斯威尔公园纪念研究所 (RPMI) 培养基与10% 无 FBS 和1% 笔/链球菌在缺席和存在1µg/毫升强力霉素 72 h 在37° c 5% CO2, 添加新鲜的强力霉细胞每日。 注: RPMI 培养基与单核细胞培养条件相容。强力霉素是一种光敏化合物。通过铝箔覆盖保护细胞培养物, 避免在直接光照射下工作。</li>
		<li>通过移收集介质, 过滤0.45 µm 过滤器, 在-80 ° c 的等分中冻结。</li>
	</ol></li>
</ol>2. 从人血中分离单核细胞
<ol><li>从人外周血中分离出单核细胞。 注: 人类原发性单核细胞从7毫升的白血球中分离出来, 集中在健康的血小板捐献者身上。根据捐献者的不同, 一个白细胞过滤器产生于 1 x 109和 2 x 109外周血单个核细胞 (PBMCs), 大约10% 为单核。<ol>
<li>在层流柜中准备工作站。</li>
		<li>用紫外光 (UV) 消毒剪刀和层流柜30分钟。</li>
		<li>在层流柜内喷洒70% 乙醇和风干的白细胞过滤器。</li>
		<li>用剪刀把白细胞过滤器的两端都剪掉, 把过滤器的内容清空到50毫升的管子里。</li>
		<li>稀释到90毫升, 加入 pbs 含有 1% (v/v) FBS (pbs/FBS)。</li>
		<li>用15毫升密度梯度溶液制备 3 x 50 ml 管。慢慢地30毫升的 PBS/FBS 稀释血液的密度梯度溶液使用的吸管控制器设置在重力流, 以避免混合的层。</li>
		<li>离心机在一个摆动转子在 400 x g 在 RT 没有刹车25分钟。</li>
		<li>处理顶层, 将含有 PBMCs 的中间层转移到一个新鲜的50毫升管。</li>
		<li>添加 PBS/FBS 高达50毫升和离心机在 120 x g 在 RT 10 分钟, 清除含有血小板的上清液。重复此步骤两次。</li>
		<li>重50毫升的 PBS/FBS 和计数 PBMCs 使用例。离心机在 120 x g 在 RT 和重的颗粒在 pbs/FBS 含有1毫米乙酸酸 (edta) (pbs/FBS/edta) 到最后浓度 5 x 107细胞/毫升。</li>
		<li>使用阴性选择试剂盒通过耗尽 non-monocytic 细胞来丰富单核。 注: 阴性选择试剂盒采用一种抗体鸡尾酒 (CD2、CD3、CD16、CD19、CD20、CD56、CD66b、CD123 和血型 a) 对抗 T 细胞、NK 细胞、中性粒细胞、B 细胞、粒和红细胞。抗体复合物结合到这些 non-monocytic 细胞的表面和对葡聚糖磁性微粒。当管放置在一个磁铁, 珠子坚持管壁, 并从解决方案中删除 non-monocytic 细胞。<ol>
<li>添加50µL 抗体鸡尾酒到1毫升的 5 x 107 PBMCs/毫升, 与吸管混合, 并孵化10分钟在 rt. 将50µL 磁珠加入到 PBMCs 的抗体鸡尾酒中, 与吸管混合, 再在 rt 中孵育10分钟。</li>
			<li>添加 PBS/FBS/EDTA 高达25毫升, 并把 PBMC 混合物的磁铁, 可以容纳50毫升管。在 RT 上孵育10分钟. 磁珠将附着在管壁上, 并将 non-monocytic 细胞从溶液中封存。</li>
		</ol>
</li>
		<li>使用25毫升吸管, 删除含有单核细胞从管在磁铁的解决方案。小心不要用吸管接触管子的两侧。</li>
		<li>在 RT 上离心溶液 250 x g, 除去上清, 并重含有单核细胞的 RPMI 介质中含有10% 无 FBS 和1% 笔/链球菌的颗粒。</li>
	</ol>
</li>
</ol>3. 博伊登室迁移试验
<ol><li>在100毫升的超纯水中溶解1克 bsa, 并通过0.2 µm 孔大小进行过滤, 以制备 1% (w/v) 牛血清白蛋白 (bsa) 溶液。</li>
	<li>在1% 无菌 BSA 溶液中孵育多孔膜插入物, 以提高巨噬细胞对膜的黏附性。对于单核/巨噬细胞, 使用 5-8 µm 孔大小的插入物。<ol>
<li>用 1% BSA 溶液填充无菌组织培养皿, 并将插入物放入盘中。在插入件内应用200µL 1% BSA 溶液。</li>
	</ol>
</li>
	<li>把它们放在一个装满无菌 pbs 的盘子里, 然后在过滤器内应用 pbs, 来清洗两次插入。</li>
	<li>种子 4万 HCT116 或 MDA-MB-231 细胞在0.5 毫升 RPMI 培养基中含有10% 无 FBS 和1% 笔/链球菌感染每井, 在一个24井板, 一式三份。或者, 在井中放置0.5 毫升以前生成的条件介质, 作为趋化因子的来源。</li>
	<li>第二天, 在准备插入的8000单核细胞在0.3 毫升的 RPMI 培养基中含有10% 无 FBS 和1% 笔/链球菌感染, 在一式三份, 并孵化在37° c 5% CO2。</li>
	<li>在48小时后收集刀片。 注意: 实验的长度必须根据特定的条件和所使用的细胞进行优化, 进行时效实验, 在不同的潜伏期收集插入物。<ol>
<li>摆脱多余的介质, 用棉签涂抹内表面, 以去除不迁移的细胞。</li>
		<li>使用赖特-姬姆萨法对插入物的外侧进行着色。</li>
		<li>在高粘度显微镜下仔细安装3插入/玻璃滑油, 确保与迁移的巨噬细胞的过滤器侧面朝上。</li>
	</ol>
</li>
	<li>使用20X 物镜的光学显微镜对幻灯片进行图像处理。拍摄9字段/过滤器的图片。计数迁移的巨噬细胞在9字段/过滤器。</li>
</ol>

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作者没有什么可透露的。

由多种细胞类型组成, 对癌症的发展至关重要。为了确定最佳生长条件, 癌细胞通过分泌趋因子来吸引单核细胞。在这里, 我们提出了一个协议, 以研究的机制, 单核细胞招募的癌细胞体外。为此, 采用诱导基因表达系统, 纯化初级人类单核细胞, 并作为一个例子的迁移试验, 博伊登室测定, 使用。
所提出的协议的第一个关键步骤是将 shRNA 序列的正确克隆确认为 "春节-对" 向量, 该方法应始终按顺序进行评估。进一步建立的功能稳定的春节细胞系需要通过测试 KD 在存在的强力霉素的西部印迹。应用的强力霉素的数量在细胞系之间可能有所不同, 因此最好测试 KD 的有效浓度和对细胞的毒性。四环素诱导系统的一个已知的缺点是它们的泄漏6,7。在存在和无强力霉素的情况下检测到的蛋白质数量必须被测量, 并与被炒的控件进行比较, 以此来解释这种效果。在体外实验中, shRNA2 和 shRNA3 KD 观察到低级别的泄漏, 如图 2所示。此外, 重要的是始终使用无四环素血清在体外, 以避免下调的蛋白质水平在控制条件。本协议中使用的系统比其他的泄漏系统具有优势, 因为它需要一个单一的向量克隆, 允许快速生成多个克隆, 并且呈现非常低的级别。
从血液中获取初级人类单核细胞的一个关键步骤是获得良好的梯度分离, 这依赖于稀释血液在密度梯度上的缓慢分层。该协议的另一个关键步骤是正确估计在阴性选择步骤之前的细胞数量, 因为它将决定加到血液悬浮液中的抗体鸡尾酒和磁性珠子的数量。为了保证实验的重现性, 在进行复制实验时应使用不止一个献血者。这项技术优于现有的单核细胞纯化方法, 包括密度梯度和 adhesion-based 协议34和一个 two-step 过程与单一梯度21, 因为它保证了低淋巴细胞污染水平和高纯度 (> 95%) 获得的单核细胞。由于 non-monocytic 细胞类型从 PBMC 混合物中被阴性选择去除, 单核病毒与抗体鸡尾酒中存在的抗体没有直接接触, 因此通过与抗体的相互作用是最小的。所获得的细胞的替代应用可能包括通过体外和活体实验将单核生物分化为巨噬细胞。该协议的修改通常包括在 PBMCs 洗涤步骤中使用红细胞裂解缓冲液。这种改变的目的是通过施加渗透压, 额外去除红血球。该技术的局限性包括有限数量的巨噬细胞, 可以获得使用指定的数量的试剂。
博伊登室迁移试验是一种快速和直接的方法来分析, 如果一个分泌蛋白或细胞线刺激其他细胞类型的迁移。这种方法的缺陷包括低灵敏度和不核算细胞死亡。解决这些问题的方法将是通过一种不同的化验结果, 如微流控分析法和时效实验, 在短时间内测量迁移。除小种群外, 成熟单核细胞不增殖在体内35,36;但是, 如果使用其他单元格, 则应选择比其分区周期短的时间线, 以避免单元格数增加。另外, 应使用试剂阻断细胞分裂, 以避免改变原来镀的细胞数。
在这里提出的协议可以很容易地适应研究的其他蛋白质分泌的癌细胞和他们的分泌功能。有条件的 kd 癌细胞系的一个可能的应用是研究蛋白质的作用, 它的 kd 是有毒的细胞不仅在体外, 而且还在体内, 在那里的春节调控细胞系移植到小鼠, kd 是诱导在肿瘤的创立以后由增加的强力霉素对饮用水。

肿瘤相关巨噬细胞 (tam) 促进肿瘤生长, 转移, 并调节免疫应答2。癌细胞会吸收具有炎症的单核细胞, 浸润肿瘤并分化为 pro-tumorigenic tam3。肿瘤浸润与 tam 相关性不佳的临床结果, 并已与免疫抑制作用的巨噬细胞4,5。然而, 对肿瘤的巨噬细胞的招募机制没有很好的探索和更好地了解有关的途径是至关重要的进一步发展的领域和有前途的治疗方法。研究肿瘤细胞与正常细胞在肿瘤微环境中的相互作用的一个挑战是所涉及的机制和细胞的复杂性, 需要体外方法, 允许对串扰进行解剖。在这里, 我们提出了一个多用途的方法, 可用于研究的分泌效应的癌细胞衍生的, 分泌蛋白的迁移其他细胞类型如巨噬细胞,体外。使用的系统, shRNA 的表达与癌细胞来源的蛋白质参与招募的单核细胞是在控制的四环素诱导启动子, 分泌的作用, 分泌的蛋白质单核定量。在这个协议中, shRNA 序列的克隆在四环素调控的传染媒介然后被提出稳定的癌细胞线的世代。此外, 对原人单核细胞和博伊登室的纯化进行了研究, 以分析癌细胞衍生蛋白对分泌的迁移作用。
蛋白质编码基因的下调通常应用于 siRNA 和 shRNA 技术, 尽管在其使用中并非没有限制。基因的长期击倒 (KD) 可能引起细胞的次级自适应反应, 干扰实验结果。缺乏对基因表达的时间控制, 使得研究蛋白质在时间上的动态作用或蛋白质对细胞生存至关重要的作用具有挑战性。这个问题在体内的设置中尤为重要, 在这种情况下, 蛋白质在肿瘤的发展和进展中的作用可能需要在肿瘤建立后下调表达感兴趣的蛋白。有条件 kd 的优点是防止 kd 对细胞的过早致死作用, 并能够分析蛋白质在肿瘤生长的不同阶段的作用, 而无条件的 kd 可能导致缺乏肿瘤的发展。
为了解决稳定 kd 的局限性, 开发了一些有条件的 kd 系统。有条件基因表达系统包括蜕皮诱导的过氧化系统、细胞色素 P-450 诱导系统1和四环素调控的基因表达系统。四环素调控的基因表达系统允许在添加抗生素四环素 (或其更稳定的模拟-强力霉素) 时控制 shRNA 的表达。在系统中, shRNA 的表达在四环素/强力霉素的存在中诱发了一个基因表达, 而在 shRNA 的系统中, 在四环素的存在下抑制了四环素的表达, 从而导致基因表达。四环素诱导系统的一个缺点是以前报告 shRNA 的表达水平低, 在没有强力霉素-so-called 泄漏6,7。在这里描述的春节系统中, 在四环素的管理下, 组成表达的四环素阻遏 (TetR) 蛋白与 shRNA 感兴趣的 H1 启动子区内的春节反应性元素 (的) 序列的结合是抑制.这导致 shRNA 的表达和对感兴趣的蛋白质的翻译的抑制以四环素依赖方式8,9。
其他可用的系统包括同时基因的 TetO 序列之间的塔塔盒和近端序列元素 (PSE), 并之间的塔塔盒和转录开始网站开发的 Chan等。10这个系统需要少于毒性剂量的四环素来调节 shRNA 表达, 但是, 在 non-induced 状态下, 低浓度的 shRNA 表示。基于 Krüppel 的关联框 (硬壳) 的系统11包括硬壳, 一个锌指蛋白, 它将基因定位在 3 kb 范围内的硬壳结合位点的转录抑制。嵌合蛋白 tTRKRAB 可以结合到 TetO, 由于 DNA 可控容量大, TetO 不需要限制在转录起始点和启动子之间, 对启动子的活动有很低的影响。在 non-induced 状态下, 这种可控 RNA 干扰系统被报告为显示 shRNA11,12的泄漏表达式的较低级别;然而, 它需要一个顺序的, 双矢量的克隆方法。与以前开发的条件 KD 系统, 如蜕皮诱导过度表达系统或细胞色素 P-450 诱导系统相比, 四环素调节系统具有鲁棒性和可逆性的优点, 因此最常规使用的系统13。本协议中使用的系统优于双 TetO 基因系统和硬壳的系统, 因为它需要直接的单矢量克隆, 允许快速生成多个克隆, 并且它在无强力霉素。
对癌症的发展至关重要。为了促进肿瘤生长, 癌细胞通过分泌趋蛋白来吸收炎症单核细胞。招募单核细胞浸润肿瘤, 分化成 pro-tumorigenic tam, 促进肿瘤生长和转移。体外免疫细胞招募的研究利用迁移测定, 用博伊登室分析法广泛使用14、15、16、17。在这个实验中, 细胞趋化蛋白的来源, 例如, 癌细胞, 或纯化蛋白, 被放置在底部的房间。免疫细胞被放置在上部室与多孔膜分离从底部车厢。细胞向趋化因子的渐增梯度迁移, 在膜的下部发现的, 在显微镜下染色和计数。在这里, 我们测试了纤溶酶原激活剂抑制剂 1 (PAI-1) 对单核细胞的趋化作用, 产生稳定的, 诱导的癌细胞线, PAI-1 的表达是由强力霉素加入调控。我们在博伊登室迁移试验中使用人原核细胞, 以评估 PAI-1 在单核移植中的作用。人类原发性单核细胞与广泛使用的细胞 THP1 的差异已经报告, 并包括不同的细胞因子表达模式18;例如, 5-10 倍增加的 TNF-α表达水平的 THP1 细胞相比, 人单核19。THP1 细胞来源于人类白血病细胞细胞, 易于维持, 并增殖, 平均加倍时间为 19-50 h20。相反, 人单核细胞的特点是在没有生长因子的情况下寿命短。由于单核细胞是从献血者的血液中纯化出来的, 在个体中发生了相当数量的变异, 并取决于纯化方法, 可能会发生其他类型的污染。然而, 主要单核细胞是相关的, 它已经被推荐使用或确认的结果, 在生物研究中使用的原单核细胞干细胞线,19。在这里, 我们描述了一个从外周血纯化人类原发性单核细胞的协议。单核细胞纯化的替代方法包括密度梯度和粘附协议和两个步骤的单一梯度聚-Hypaque 后跟一个 Percoll 梯度21。这些方法获得的单核细胞的纯度范围在 70-90% 之间。这里描述的方法使用的密度梯度, 其次是阴性免疫选择22 , 使人单核细胞的纯化与抗体没有直接接触, 从而避免其意外激活, 并导致了 >95% 单核细胞的纯种群。
本文所提出的协议用于建立人类癌细胞系基因表达的功能性的 PAI-1 系统, 以研究肿瘤衍生的分泌蛋白对单核细胞的趋化作用。PAI-1 是表达的各种肿瘤和它的表达似是而非地关联与不良临床结局23,24。PAI-1 的 pro-tumorigenic 角色是其 pro-angiogenic 和亡函数的结果25,26。PAI-1 已被证明有助于炎症, 促进招募巨噬细胞到炎症部位27。PAI-1 显示, 以促进平滑肌 cellmigration28,29和参与 Mac-1 依赖性巨噬细胞迁移30。PAI-1 过度表达也显示, 以显著提高招募原始264.7 巨噬细胞为 B16F10 黑色素瘤肿瘤31。然而, PAI-1 在 TAM 迁移中的作用没有得到详细的研究。我们使用所描述的协议来回答是否 PAI-1 吸引单核细胞到癌细胞的问题。这种方法可以通过抑制癌细胞的分泌蛋白, 分析肿瘤细胞之间的串扰, 并对其成分进行解剖。

<table border="0" cellpadding="0" cellspacing="0" width="868">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:405px;">Tet-pLKO-puro lentiviral vector</td>
			<td style="width:177px;">Addgene</td>
			<td style="width:119px;">21915</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FBS (Tetracycline-free)</td>
			<td style="width:177px;">Omega Scientific</td>
			<td style="width:119px;">FB-15</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Histopaque</td>
			<td style="width:177px;">Sigma</td>
			<td>10771-500ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EasySep Human Monocyte Enrichment Kit</td>
			<td style="width:177px;">StemCell</td>
			<td>19059</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EasySep Magnet</td>
			<td style="width:177px;">StemCell</td>
			<td>18002</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EcoRI HF</td>
			<td style="width:177px;">NEB</td>
			<td>R3101S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">AgeI HF</td>
			<td style="width:177px;">NEB</td>
			<td>R3552S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cut Smart buffer</td>
			<td style="width:177px;">NEB</td>
			<td>B7200S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EndoFree Plasmid Maxi Kit</td>
			<td style="width:177px;">Qiagen</td>
			<td>12362</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PROMEGA Wizard SV Gel and PCR Cleanup System</td>
			<td style="width:177px;">PROMEGA</td>
			<td>A9281</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">psPAX vector</td>
			<td style="width:177px;">Addgene</td>
			<td style="width:119px;">12260</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">pMD2.G vector</td>
			<td style="width:177px;">Addgene</td>
			<td style="width:119px;">12259</td>
			<td></td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:405px;">One Shot Stbl3 Chemically Competent&#160;E. coli</td>
			<td style="width:177px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">C737303</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hema 3 stain set for Wright-Giemsa stain</td>
			<td style="width:177px;">Protocol</td>
			<td style="width:119px;">123-869</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HEK293 cells</td>
			<td style="width:177px;">ATCC</td>
			<td style="width:119px;">CRL-1573</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PBS</td>
			<td style="width:177px;">Corning</td>
			<td style="width:119px;">21-031-CV</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">XhoI restriction enzyme</td>
			<td style="width:177px;">NEB</td>
			<td>R0146S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ligase</td>
			<td style="width:177px;">NEB</td>
			<td>M0202S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ligase buffer</td>
			<td style="width:177px;">NEB</td>
			<td>M0202S</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">EDTA 0.5 M solution</td>
			<td style="width:177px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">R1021</td>
			<td></td>
		</tr>
		<tr height="21">
			<td>Lipofectamine 2000</td>
			<td style="width:177px;">Invitrogen</td>
			<td>11668019</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:405px;">The Big Easy&#34; EasySep Magnet</td>
			<td style="width:177px;">Stem Cell</td>
			<td style="width:119px;">18001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:405px;">0.1% Poly-L-Lysine solution</td>
			<td style="width:177px;">Sigma-Aldrich</td>
			<td style="width:119px;">P8920</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium Acetate</td>
			<td style="width:177px;">Sigma-Aldrich</td>
			<td style="width:119px;">S7670</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium butyrate</td>
			<td style="width:177px;">Sigma-Aldrich</td>
			<td style="width:119px;">B5887</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.45 &#956;M syringe filters</td>
			<td style="width:177px;">VWR International</td>
			<td>28145-479</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NaOH</td>
			<td style="width:177px;">Sigma-Aldrich</td>
			<td style="width:119px;">S5881-500g</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tris(hydroxymethyl)aminomethane (Tris)</td>
			<td style="width:177px;">Invitrogen</td>
			<td style="width:119px;">15504-020</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium Chloride(NaCl)</td>
			<td style="width:177px;">Sigma-Aldrich</td>
			<td style="width:119px;">S7653-5 kg</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Magnesium Chloride (MgCl2)</td>
			<td style="width:177px;">Sigma-Aldrich</td>
			<td style="width:119px;">M8266-100g</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">dithioerythritol (DTE)</td>
			<td style="width:177px;">Sigma-Aldrich</td>
			<td style="width:119px;">B8255-5g</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ethanol (EtOH)</td>
			<td style="width:177px;">Sigma-Aldrich</td>
			<td style="width:119px;">792780</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">tryptone</td>
			<td style="width:177px;">Amresco</td>
			<td style="width:119px;">J859-500g</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">yeast extract</td>
			<td style="width:177px;">Fluka</td>
			<td style="width:119px;">70161-500g</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bacto agar</td>
			<td style="width:177px;">BD</td>
			<td style="width:119px;">214010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ampicillin</td>
			<td style="width:177px;">Sigma-Aldrich</td>
			<td style="width:119px;">A9393-5g</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dulbecco&#8217;s Modified Eagle&#8217;s Medium (DMEM)</td>
			<td style="width:177px;">Corning</td>
			<td style="width:119px;">10-013-CV</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">phosphate-buffered saline (PBS)</td>
			<td style="width:177px;">Corning</td>
			<td style="width:119px;">21-031-CV</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)</td>
			<td style="width:177px;">Sigma-Aldrich</td>
			<td style="width:119px;">H3375</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">puromycin</td>
			<td style="width:177px;">Sigma-Aldrich</td>
			<td style="width:119px;">P9620</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Doxycycline hyclate</td>
			<td style="width:177px;">Sigma-Aldrich</td>
			<td style="width:119px;">D9891-25g</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Roswell Park Memorial Institute (RPMI)</td>
			<td style="width:177px;">Corning</td>
			<td style="width:119px;">10-040-CV</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:405px;">0.5 M EDTA pH 8.0</td>
			<td style="width:177px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">R1021</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:405px;">Falcon Permeable Support for 24 Well Plate with 8.0&#956;m Transparent PET Membrane</td>
			<td style="width:177px;">Becton Dickinson</td>
			<td style="width:119px;">353097</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:405px;">Bovine Serum Albumin</td>
			<td style="width:177px;">Sigma-Aldrich</td>
			<td style="width:119px;">A7906</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cotton-Tipped Applicator&#160;</td>
			<td style="width:177px;">Medline</td>
			<td style="width:119px;">MDS202000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Protocol Hema 3 STAT pack&#160;</td>
			<td style="width:177px;">Fisher Scientific Company</td>
			<td style="width:119px;">123-869</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Resolve Immersion Oil, High Viscosity</td>
			<td style="width:177px;">Richard Allan Scientific</td>
			<td style="width:119px;">M4004</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:405px;">Penicillin Streptomycin</td>
			<td style="width:177px;">Gibco</td>
			<td style="width:119px;">15140122</td>
			<td></td>
		</tr>
	</tbody>
</table>

conditional knockdown of gene expression in cancer cell lines to study the recruitment of monocytes/macrophages to the tumor microenvironment

siRNA 和 shRNA 介导的敲掉 (KD) 调控基因表达的方法是了解基因和蛋白质功能的宝贵工具。但是, 在有兴趣的蛋白质的 kd 对细胞有致命影响或 kd 的预期效应是时间依赖性的情况下, 无条件 kd 方法是不适当的。条件系统更适合在这些情况下, 并已成为一个很大的兴趣的主题。这些包括蜕皮诱导的过度表达系统, 细胞色素 P-450 诱导系统1, 和四环素调控的基因表现系统。
四环素调控基因表达系统能够通过诱导 shRNA 表达在四环素的存在下对蛋白质表达进行可逆控制。在本协议中, 我们提出了一个实验设计, 利用功能性的, 在人类癌症细胞系的基因表达的条件调节。然后, 我们证明了该系统在肿瘤细胞-单核相互作用的研究中的应用。

三 shRNA 序列被测试为最有效的 KD PAI-1。为此, 根据上述协议, 将 shRNA 序列与 PAI-1 和被打乱的 (表 1) 复制到 pLKO 普洛表达式向量中。HT-1080 纤维细胞系稳定地转染了生成的结构, 细胞被强力霉素治疗3天。用印迹(图 2A) 对 PAI-1 的表达进行了验证, 并通过密度测量分析了与 PAI-1 和蛋白相对应的波段强度。使用最有效的 KD 序列 (shRNA 2), 我们还产生了 MDA-MB-231 上皮性乳腺腺癌细胞 (图 2B) 和 HCT116 大肠癌细胞 (图 2C) 稳定转染四环素调控pLKO-shRNA2-PAI-1 表达式向量 (和 shRNA 作为控件被打乱)。在 MDA-MB-231 细胞系 (图 2B) 中, 在 HCT116 细胞系 (1 µg/毫升)(图 2C) 的存在中, 我们实现了74% 的 PAI-1 表达减少, 这是由西方印迹和密度分析乐队。用博伊登室法测定单核细胞向癌细胞迁移的数量。我们推测, 癌细胞衍生的 PAI-1 促进单核细胞向肿瘤细胞的迁移, 因此, 当 PAI-1 downregulated 通过添加强力霉素时, 会减少迁移。肿瘤细胞治疗3天的强力霉素, 并在4万细胞/井的 24-井板的底部播种。纯化的人单核细胞被应用于该试验的顶室, 24 小时后, 附着在膜底的单核细胞在显微镜下染色和计数。我们证明, 在没有强力霉素, 因此存在的 PAI-1, 在 MDA-MB-231 和 HCT116 细胞 (既 PAI-1 shRNA 和炒控制), 大大增加了人类单核移植 (图 3)。当在 co-cultures 中添加强力霉素时, 单核细胞的迁移被抑制33% 和 74%, 而 PAI-1 的产生则被下调 (图 3A, C)。转与 shRNA 控制的肿瘤细胞无单核移植的抑制作用, 同时表明强力霉素对单核移植无抑制作用。当化 (图 3B, D) 被放置在井底的强力霉素治疗的癌细胞的条件培养基上时, 也会得到类似的结果。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56333/56333fig1.jpg"> 图 1.从16细菌菌落中提取的 DNA XhoI 限制性分析.从16细菌菌落中提取 DNA, 并与 XhoI 酶进行限制性反应。用琼脂糖凝胶电泳法对其产生的 DNA 片段进行了分析。在 XhoI 限制摘要后缺少 2 kb 片段的正克隆 (泳道3、8和 11) 用箭头标记。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56333/56333fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56333/56333fig2.jpg"> 图 2.肿瘤细胞系中 PAI-1 表达的条件 KD.pLKO-shRNA-PAI-1 (shRNA 1-3) 和3天强力霉素治疗后, 在细胞系中稳定转染四环素的 PAI-1 表达的印迹分析。(A) HT1080。(B) MDA-MB-231。(C) HCT116。在蛋白带强度的 PAI-1 强度的比值下, 条纹的密度分析显示在西印迹的通道下面。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56333/56333fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56333/56333fig3.jpg"> 图 3.PAI-1 促进巨噬细胞迁移.人类单核细胞向 (A) MDA-MB-231 和 (C) HCT116 的迁移, 用 shRNA 室法测定, 以有条件表达 shRNA 2 对 PAI-1 和炒博伊登进行稳定转染。用强力霉素治疗癌细胞3天, 并在一份24块的平板上播种。人原发性单核细胞被应用在插入物的顶端。24小时后, 过滤器下部的单核细胞被染色并在显微镜下计数。人单核细胞向条件培养基的迁移生成超过 72 h (B) MDA-MB-231 和 (D) HCT116 在存在和无强力霉素的情况下有条件地表达 PAI-1。数据表示技术 triplicates 的平均值 [+/-标准偏差 (SD)]。对于每一个复制, 迁移单核细胞计数在9场20X 客观放大。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56333/56333fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>寡聚的名称</td>
			<td></td>
			<td colspan="4">序列</td>
		</tr>
<tr>
<td>shRNA 1 (反 PAI-1 序列 1- 8 )</td>
			<td>提出:</td>
			<td colspan="4">shPAI-1A5 "CCGGCTGACTTCACGAGTCTTTCCTCGAGGAAAGACTCGTGAAGTCAGTTTTT 3"</td>
		</tr>
<tr>
<td></td>
			<td>反向:</td>
			<td colspan="4">shPAI-1B 5 "AATTAAAAACTGACTTCACGAGTCTTTCCTCGAGGAAAGACTCGTGAAGTCAG 3"</td>
		</tr>
<tr>
<td>shRNA 2: (反 PAI-1 序列 2)</td>
			<td>提出:</td>
			<td colspan="4">shPAI-1C 5 "-CCGGCAGACAGTTTCAGGCTGACTTCTCGAGAAGTCAGCCTGAAACTGTCTGTTTTT-3"</td>
		</tr>
<tr>
<td></td>
			<td>反向:</td>
			<td colspan="4">shPAI-1D 5 "-AATTAAAAACAGACAGTTTCAGGCTGACTTCTCGAGAAGTCAGCCTGAAACTGTCTG-3"</td>
		</tr>
<tr>
<td>shRNA 3 (反 PAI-1 序列-3 9)</td>
			<td>提出:</td>
			<td colspan="4">shPAI-1E 5 "-CCGGAAGGATGAGATCAGCACCACACTCGAGTGTGGTGCTGATCTCATCCTTTTTTT-3"</td>
		</tr>
<tr>
<td></td>
			<td>反向:</td>
			<td colspan="4">shPAI-1F 5 "-AATTAAAAAAAGGATGAGATCAGCACCACACTCGAGTGTGGTGCTGATCTCATCCTT-3"</td>
		</tr>
<tr>
<td>混乱 (打乱序列-4 9)</td>
			<td>提出:</td>
			<td colspan="4">争夺战 1 5 "-CCGGAATTCTCCGAACGTGTCACGTCTCGAGACGTGACACGTTCGGAGAATTTTTTT-3"</td>
		</tr>
<tr>
<td></td>
			<td>反向:</td>
			<td colspan="4">争夺战 2 5 "-AATTAAAAAAATTCTCCGAACGTGTCACGTCTCGAGACGTGACACGTTCGGAGAATT-3"</td>
		</tr>
</tbody></table>表 1. shRNA 序列.这里 3 shRNA 序列反对 PAI-1 被测试了为最强的沉默作用: shRNA 1, shRNA 2, shRNA 3, 和被炒

Watch this Scientific Journal Video about Conditional Knockdown of Gene Expression in Cancer Cell Lines to Study the Recruitment of Monocytes/Macrophages to the Tumor Microenvironment at JoVE.com

Conditional Knockdown of Gene Expression in Cancer Cell Lines to Study the Recruitment of Monocytes/Macrophages to the Tumor Microenvironment

本议定书作为一个计划, 建立一个功能性的癌症细胞系的系统, 并随后使用, 特别是研究肿瘤细胞源性蛋白质在招募单核/巨噬细胞到肿瘤微环境的作用。

肿瘤细胞系基因表达的条件性击倒研究单核/巨噬细胞在微环境中的招募

siRNA and shRNA-mediated knock down (KD) methods of regulating gene expression are invaluable tools for understanding gene and protein function. However, ...

conditional-knockdown-gene-expression-cancer-cell-lines-to-study

Nanyang Technological University

Research

JoVE Journal

Cancer Research

12.2K 次观看.  University of Southern California. 本议定书作为一个计划, 建立一个功能性的癌症细胞系的系统, 并随后使用, 特别是研究肿瘤细胞源性蛋白质在招募单核/巨噬细胞到肿瘤微环境的作用。

视频: Conditional Knockdown of Gene Expression in Cancer Cell Lines to Study the Recruitment of Monocytes/Macrophages to the Tumor Microenvironment

Lung Tumor Cell Recruitment Assay

To investigate the molecular mechanisms governing tumor metastasis, various assays using the mouse as a model animal have been proposed. Here, we demonstrate a simple assay to evaluate tumor cell extravasation or micrometastasis. In this assay, tumor cells were injected through the tail vein, and after a short period, the lungs were dissected and digested to count the accumulated labeled tumor cells. This assay skips the initial step of primary tumor invasion into the blood vessel and facilitates the study of events in the distant organ where tumor metastasis occurs. The number of cells injected into the blood vessel can be optimized to observe a limited number of metastases. It has been reported that stromal cells in the distant organ contribute to metastasis. Thus, this assay could be a useful tool to explore potential therapeutic drugs or devices for prevention of tumor metastasis.

This manuscript describes a method to quantify tumor cell accumulation in the lungs in an animal model of tumor metastasis.

肺肿瘤细胞招募分析

To investigate the molecular mechanisms governing tumor metastasis, various assays using the mouse as a model animal have been proposed. Here, we ...

科研

癌症研究

The Drosophila Imaginal Disc Tumor Model: Visualization and Quantification of Gene Expression and Tumor Invasiveness Using Genetic Mosaics

Drosophila melanogaster has emerged as a powerful experimental system for functional and mechanistic studies of tumor development and progression in the context of a whole organism. Sophisticated techniques to generate genetic mosaics facilitate induction of visually marked, genetically defined clones surrounded by normal tissue. The clones can be analyzed through diverse molecular, cellular and omics approaches. This study describes how to generate fluorescently labeled clonal tumors of varying malignancy in the eye/antennal imaginal discs (EAD) of Drosophila larvae using the Mosaic Analysis with a Repressible Cell Marker (MARCM) technique. It describes procedures how to recover the mosaic EAD and brain from the larvae and how to process them for simultaneous imaging of fluorescent transgenic reporters and antibody staining. To facilitate molecular characterization of the mosaic tissue, we describe a protocol for isolation of total RNA from the EAD. The dissection procedure is suitable to recover EAD and brains from any larval stage. The fixation and staining protocol for imaginal discs works with a number of transgenic reporters and antibodies that recognize Drosophila proteins. The protocol for RNA isolation can be applied to various larval organs, whole larvae, and adult flies. Total RNA can be used for profiling of gene expression changes using candidate or genome-wide approaches. Finally, we detail a method for quantifying invasiveness of the clonal tumors. Although this method has limited use, its underlying concept is broadly applicable to other quantitative studies where cognitive bias must be avoided.

The Drosophila Imaginal Disc Tumor Model: Visualization and Quantification of Gene Expression and Tumor Invasiveness Using Genetic Mosaics

This protocol demonstrates how to generate fluorescently marked, genetically defined clonal tumors in the Drosophila eye/antennal imaginal discs (EAD). It describes how to dissect the EAD and brain from the third instar larvae and how to process them to visualize and quantify gene expression changes and tumor invasiveness.

该<em&gt;果蝇</em&gt;成虫盘瘤模型:基因的表达与肿瘤侵袭性的可视化和定量遗传马赛克

Drosophila melanogaster has emerged as a powerful experimental system for functional and mechanistic studies of tumor development and progression in the ...

Detection of Mitochondria Membrane Potential to Study CLIC4 Knockdown-induced HN4 Cell Apoptosis In Vitro

Depletion of the mitochondrial membrane potential (MMP, &#916;&#936;m) is considered the earliest event in the apoptotic cascade. It even occurs ahead of nuclear apoptotic characteristics, including chromatin condensation and DNA breakage. Once the MMP collapses, cell apoptosis will initiate irreversibly. A series of lipophilic cationic dyes can pass through the cell membrane and aggregate inside the matrix of mitochondrion, and serve as fluorescence marker to evaluate MMP change. As one of the six members of the Cl- intracellular channel (CLIC) family, CLIC4 participates in the cell apoptotic process mainly through the mitochondrial pathway. Here we describe a detailed protocol to measure MMP via monitoring the fluorescence fluctuation of Rhodamine 123 (Rh123), through which we study apoptosis induced by CLIC4 knockdown. We discuss the advantages and limitations of the application of confocal laser scanning and normal fluorescence microscope in detail, and also compare it with other methods.

Detection of Mitochondria Membrane Potential to Study CLIC4 Knockdown-induced HN4 Cell Apoptosis In Vitro

Here we present a detailed protocol for the application of rhodamine 123 to identify the mitochondrial membrane potential (MMP) and study CLIC4 knockdown-induced HN4 cell apoptosis in vitro. Under common fluorescence microscope and confocal laser scanning fluorescence microscope, the real-time change of the MMP was recorded.

线粒体膜电位检测 CLIC4 击倒诱导 HN4 细胞凋亡的实验研究

Depletion of the mitochondrial membrane potential (MMP, &#916;&#936;m) is considered the earliest event in the apoptotic cascade. It even occurs ahead of ...

In Vivo EPR Assessment of pH, pO2, Redox Status, and Concentrations of Phosphate and Glutathione in the Tumor Microenvironment

This protocol demonstrates the capability of low-field electron paramagnetic resonance (EPR)-based techniques in combination with functional paramagnetic probes to provide quantitative information on the chemical tumor microenvironment (TME), including pO2, pH, redox status, concentrations of interstitial inorganic phosphate (Pi), and intracellular glutathione (GSH). In particular, an application of a recently developed soluble multifunctional trityl probe provides unsurpassed opportunity for in vivo concurrent measurements of pH, pO2 and Pi in Extracellular space (HOPE probe). The measurements of three parameters using a single probe allow for their correlation analyses independent of probe distribution and time of the measurements.

In Vivo EPR Assessment of pH, pO2, Redox Status, and Concentrations of Phosphate and Glutathione in the Tumor Microenvironment

Low-field (L-band, 1.2 GHz) electron paramagnetic resonance using soluble nitroxyl and trityl probes is demonstrated for assessment of physiologically important parameters in the tumor microenvironment in mouse models of breast cancer.

体内在肿瘤微环境中 pH 值、 pO2、氧化还原状态、磷酸盐和谷胱甘肽浓度的 EPR 评价

This protocol demonstrates the capability of low-field electron paramagnetic resonance (EPR)-based techniques in combination with functional paramagnetic ...

Virus Delivery of CRISPR Guides to the Murine Prostate for Gene Alteration

With an increasing incidence of prostate cancer, identification of new tumor drivers or modulators is crucial. Genetically engineered mouse models (GEMM) for prostate cancer are hampered by tumor heterogeneity and its complex microevolution dynamics. Traditional prostate cancer mouse models include, amongst others, germline and conditional knockouts, transgenic expression of oncogenes, and xenograft models. Generation of de novo mutations in these models is complex, time-consuming, and costly. In addition, most of traditional models target the majority of the prostate epithelium, whereas human prostate cancer is well known to evolve as an isolated event in only a small subset of cells. Valuable models need to simulate not only prostate cancer initiation, but also progression to advanced disease.
Here we describe a method to target a few cells in the prostate epithelium by transducing cells by viral particles. The delivery of an engineered virus to the murine prostate allows alteration of gene expression in the prostate epithelia. Virus type and quantity will hereby define the number of targeted cells for gene alteration by transducing a few cells for cancer initiation and many cells for gene therapy. Through surgery-based injection in the anterior lobe, distal from the urinary track, the tumor in this model can expand without impairing the urinary function of the animal. Furthermore, by targeting only a subset of prostate epithelial cells the technique enables clonal expansion of the tumor, and therefore mimics human tumor initiation, progression, as well as invasion through the basal membrane.
This novel technique provides a powerful prostate cancer model with improved physiological relevance. Animal suffering is limited, and since no additional breeding is required, overall animal count is reduced. At the same time, analysis of new candidate genes and pathways is accelerated, which in turn is more cost efficient.

This protocol describes a newly established method for virus delivery to the murine prostate. Using either CRISPR/Cas9 technology, gene overexpression, or Cre recombinase delivery, the technique allows orthotopic alteration of gene expression and implements a novel mouse model for prostate cancer.

CRISPR 指南对小鼠前列腺病毒传递的基因改变

With an increasing incidence of prostate cancer, identification of new tumor drivers or modulators is crucial. Genetically engineered mouse models (GEMM) ...

Live-Cell Imaging Assays to Study Glioblastoma Brain Tumor Stem Cell Migration and Invasion

Glioblastoma (GBM) is an aggressive brain tumor that is poorly controlled with the currently available treatment options. Key features of GBMs include rapid proliferation and pervasive invasion into the normal brain. Recurrence is thought to result from the presence of radio- and chemo-resistant brain tumor stem cells (BTSCs) that invade away from the initial cancerous mass and, thus, evade surgical resection. Hence, therapies that target BTSCs and their invasive abilities may improve the otherwise poor prognosis of this disease. Our group and others have successfully established and characterized BTSC cultures from GBM patient samples. These BTSC cultures demonstrate fundamental cancer stem cell properties such as clonogenic self-renewal, multi-lineage differentiation, and tumor initiation in immune-deficient mice. In order to improve on the current therapeutic approaches for GBM, a better understanding of the mechanisms of BTSC migration and invasion is necessary. In GBM, the study of migration and invasion is restricted, in part, due to the limitations of existing techniques which do not fully account for the in vitro growth characteristics of BTSCs grown as neurospheres. Here, we describe rapid and quantitative live-cell imaging assays to study both the migration and invasion properties of BTSCs. The first method described is the BTSC migration assay which measures the migration toward a chemoattractant gradient. The second method described is the BTSC invasion assay which images and quantifies a cellular invasion from neurospheres into a matrix. The assays described here are used for the quantification of BTSC migration and invasion over time and under different treatment conditions.

Here, we describe live-cell imaging techniques to quantitatively measure the migration and invasion of glioblastoma brain tumor stem cells over time and under multiple treatment conditions.

活体细胞成像技术研究胶质瘤脑干细胞的迁移和侵袭

Glioblastoma (GBM) is an aggressive brain tumor that is poorly controlled with the currently available treatment options. Key features of GBMs include ...

Establishing Cell Lines Overexpressing DR3 to Assess the Apoptotic Response to Anti-mitotic Therapeutics

Studying the function of a gene of interest can be achieved by manipulating its level of expression, such as decreasing its expression with knockdown cell lines or increasing its expression with overexpression cell lines. Transient and stable transfection are two methods that are often used for exogenous gene expression. Transient transfection is only useful for short-term expression, whereas stable transfection allows exogenous genes to be integrated into the host cell genome where it will be continuously expressed. As a result, stable transfection is usually employed for research into long-term genetic regulation. Here we describe a simple protocol to generate a stable cell line overexpressing tagged death receptor 3 (DR3) to explore DR3 function. We picked single clones after a retroviral infection in order to maintain the homogeneity and purity of the stable cell lines. The stable cell lines generated using this protocol render DR3-deficient HT29 cells sensitive to antimitotic drugs, thus reconstituting the apoptotic response in HT29 cells. Moreover, the FLAG tag on DR3 compensates for the unavailability of good DR3 antibody and facilitates the biochemical study of the molecular mechanism by which antimitotic agents induce apoptosis.

Establishing a stable cell line overexpressing a gene of interest to study gene function can be done by stable transfection-picking single clones after transfecting them via retroviral infection. Here we show that HT29-DR3 cell lines generated in this way elucidate the mechanisms by which death receptor 3 (DR3) contributes to antimitotics-induced apoptosis.

建立过度表达 dr3 的细胞系, 评估抗有丝分裂治疗的凋亡反应

Studying the function of a gene of interest can be achieved by manipulating its level of expression, such as decreasing its expression with knockdown cell ...

Analyzing Tumor Gene Expression Factors with the CorExplorer Web Portal

Differential gene expression analysis is an important technique for understanding disease states. The machine learning algorithm CorEx has shown utility in analyzing differential expression of groups of genes in tumor RNA-seq in a way that may be helpful for advancing precision oncology. However, CorEx produces many factors that can be challenging to analyze and connect to existing understanding. To facilitate such connections, we have built a website, CorExplorer, that allows users to interactively explore the data and answer common questions related to its analysis. We trained CorEx on RNA-seq gene expression data for four tumor types: ovarian, lung, melanoma, and colorectal. We then incorporated corresponding survival, protein-protein interactions, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichments, and heatmaps into the website for association with the factor graph visualization. Here we employ example protocols to illustrate use of the database for comprehending the significance of the learned tumor factors in the context of this external data.

We introduce the CorExplorer web portal, an resource for exploration of tumor RNA sequencing factors found by the machine learning algorithm CorEx (Correlation Explanation), and show how factors can be analyzed relative to survival, database annotations, protein-protein interactions, and one another to gain insight into tumor biology and therapeutic interventions.

分析肿瘤基因表达因子与CorExplorer门户网站

Differential gene expression analysis is an important technique for understanding disease states. The machine learning algorithm CorEx has shown utility ...

Analysis of Side Population in Solid Tumor Cell Lines

Cancer stem cells (CSCs) are an important cause of tumor growth, metastasis, and recurrence. Isolation and identification of CSCs are of great significance for tumor research. Currently, several techniques are used for the identification and purification of CSCs from tumor tissues and tumor cell lines. Separation and analysis of side population (SP) cells are two of the commonly used methods. The methods rely on the ability of CSCs to rapidly expel fluorescent dyes, such as Hoechst 33342. The efflux of the dye is associated with the ATP-binding cassette (ABC) transporters and can be inhibited by ABC transporter inhibitors. Methods for staining cultured tumor cells with Hoechst 33342 and analyzing the proportion of their SP cells by flow cytometry are described. This assay is convenient, fast, and cost-effective. Data generated in this assay can contribute to a better understanding of the effect of genes or other extracellular and intracellular signals on the stemness properties of tumor cells.

A convenient, fast, and cost-effective method to measure the proportion of side population cells in solid tumor cell lines is presented.

固体肿瘤细胞系侧面人口分析

Cancer stem cells (CSCs) are an important cause of tumor growth, metastasis, and recurrence. Isolation and identification of CSCs are of great ...

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 ...