Name: conditional knockdown of gene expression in cancer cell lines to study the recruitment of monocytes/macrophages to the tumor microenvironment
Uploaded: 2017-11-23T14:00:00.000+00:00
Duration: 10 min 59 s
Description: 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

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...

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

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

Universidad San Sebastian

Research

JoVE Journal

Cancer Research

12.3K 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

Real Time Detection of In Vitro Tumor Cell Apoptosis Induced by CD8+ T Cells to Study Immune Suppressive Functions of Tumor-infiltrating Myeloid Cells

Potentiation of the tumor-killing ability of CD8+ T cells in tumors, along with their efficient tumor infiltration, is a key element of successful immunotherapies. Several studies have indicated that tumor infiltrating myeloid cells (e.g., myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMs)) suppress cytotoxicity of CD8+ T cells in the tumor microenvironment, and that targeting these regulatory myeloid cells can improve immunotherapies. Here, we present an in vitro assay system to evaluate immune suppressive effects of monocytic-MDSCs and TAMs on the tumor-killing ability of CD8+ T cells. To this end, we first cultured na&#239;ve splenic CD8+ T cells with anti-CD3/CD28 activating antibodies in the presence or absence of suppressor cells, and then co-cultured the pre-activated T cells with target cancer cells in the presence of a fluorogenic caspase-3 substrate. Fluorescence from the substrate in cancer cells was detected by real-time fluorescence microscopy as an indicator of T-cell induced tumor cell apoptosis. In this assay, we can successfully detect the increase of tumor cell apoptosis by CD8+ T cells and its suppression by pre-culture with TAMs or MDSCs. This functional assay is useful for investigating CD8+ T cell suppression mechanisms by regulatory myeloid cells and identifying druggable targets to overcome it via high throughput screening.

Real Time Detection of In Vitro Tumor Cell Apoptosis Induced by CD8+ T Cells to Study Immune Suppressive Functions of Tumor-infiltrating Myeloid Cells

We describe here a protocol to investigate cytotoxicity of pre-activated CD8+ T cells against cancer cells by detecting apoptotic cancer cells via real-time microscopy. This protocol can investigate mechanisms behind myeloid cell-induced T cell suppression and evaluate compounds aimed at replenishing T cells via blockade of immune suppressive myeloid cells.

cd8+ t 细胞诱导体外肿瘤细胞凋亡的实时检测--研究肿瘤浸润髓内细胞的免疫抑制作用

Potentiation of the tumor-killing ability of CD8+ T cells in tumors, along with their efficient tumor infiltration, is a key element of successful ...

科研

癌症研究

In Vitro Assay to Study Tumor-macrophage Interaction

Tumor associated macrophages (TAMs) account for a large percentage of cells in the tumor mass for different types of cancers. Glioblastoma (GBM), a malignant brain tumor with no cure, has up to a half the tumor mass TAMs. TAMs can be pro-tumoral or anti-tumoral, depending on the activation of specific genes in the cells. Genetic mutations in the tumors, through regulating cytokine expression, can affect recruitment of TAMs to the tumor microenvironment. Here, we describe a quantitative cell-based assay to assess macrophage recruitment by the conditioned medium from the tumor cells. This assay uses the human macrophage cell line MV-4-11 to study macrophage attraction by the conditioned medium from glioblastoma, allowing for high reproducibility and low variability. Data generated with this assay can contribute to a better understanding of the interaction between the tumor and the tumor microenvironment. Similar assay can be used to assess interaction between the tumor cells and other immune cells, including T cells and natural killer (NK) cells.

This article represents a useful in vitro assay to evaluate the capability of conditioned medium from tumor cells to attract macrophages.

体外测定研究肿瘤-巨噬菌体相互作用

Tumor associated macrophages (TAMs) account for a large percentage of cells in the tumor mass for different types of cancers. Glioblastoma (GBM), a ...

Studying the Effects of Tumor-Secreted Paracrine Ligands on Macrophage Activation using Co-Culture with Permeable Membrane Supports

Tumor-derived paracrine signaling is an overlooked component of local immunosuppression and can lead to a permissive environment for continued cancer growth and metastasis. Paracrine signals can involve cell-cell contact between different cell types, such as PD-L1 expressed on the surface of tumors interacting directly with PD-1 on the surface of T cells, or the secretion of ligands by a tumor cell to affect an immune cell. Here we describe a co-culture method to interrogate the effects of tumor-secreted ligands on immune cell (macrophage) activation. This straightforward procedure utilizes commercially available 0.4 &#181;m polycarbonate membrane permeable supports and standard tissue culture plates. In the process described, macrophages are cultured in the upper chamber and tumor cells in the lower chamber. The presence of the 0.4 &#181;m barrier allows for the study of intercellular signaling without the confounding variable of physical contact, because the two cell types share the same medium and exposure to paracrine ligands. This approach can be combined with others, such as genetic alterations of the macrophage (e.g., isolation from genetic knock-out mice) or tumor (e.g., CRISPR-mediated alterations) to study the role of specific secreted factors and receptors. The approach also lends itself to standard molecular biological analyses such as quantitative reverse transcription polymerase chain reaction (qRT-PCR) or Western blot analysis, without the need for flow sorting to separate the two cell populations. Enzyme-linked immunosorbent assays (ELISAs) can similarly be utilized to measure secreted ligands to better understand the dynamic interaction of cell signaling in the multiple cell type context. Duration of co-culture can also be varied for the study of temporally regulated events. This co-culture method is a robust tool that facilitates the study of tumor-secreted signals in the immune context.

Here, we present a method using permeable membrane supports to facilitate the study of non-contact paracrine signaling used by tumor cells to suppress the immune response. The system is amenable to studying the role of tumor-secreted factors in dampening macrophage activation.

研究肿瘤分泌性副体结扎对宏噬细胞活化的影响，使用可渗透膜支架共培养

Tumor-derived paracrine signaling is an overlooked component of local immunosuppression and can lead to a permissive environment for continued cancer ...

Impedance-based Real-time Measurement of Cancer Cell Migration and Invasion

Cancer arises due to uncontrolled proliferation of cells initiated by genetic instability, mutations, and environmental and other stress factors. These acquired abnormalities in complex, multilayered molecular signaling networks induce aberrant cell proliferation and survival, extracellular matrix degradation, and metastasis to distant organs. Approximately 90% of cancer-related deaths are estimated to be caused by the direct or indirect effects of metastatic dissemination. Therefore, it is important to establish a highly reliable, comprehensive system to characterize cancer cell behaviors upon genetic and environmental manipulations. Such a system can give a clear understanding of the molecular regulation of cancer metastasis and the opportunity for successful development of stratified, precise therapeutic strategies. Hence, accurate determination of cancer cell behaviors such as migration and invasion with gain or loss of function of gene(s) allows assessment of the aggressive nature of cancer cells. The real-time measurement system based on cell impedance enables researchers to continually acquire data during a whole experiment and instantly compare and quantify the results under various experimental conditions. Unlike conventional methods, this method does not require fixation, staining, and sample processing to analyze cells that migrate or invade. This method paper emphasizes detailed procedures for real-time determination of migration and invasion of glioblastoma cancer cells.

Cancer is a lethal disease due to its ability to metastasize to different organs. Determining the ability of cancer cells to migrate and invade under various treatment conditions is crucial to assessing therapeutic strategies. This protocol presents a method to assess the real-time metastatic abilities of a glioblastoma cancer cell line.&#160;

基于阻抗的癌细胞迁移和入侵的实时测量

Cancer arises due to uncontrolled proliferation of cells initiated by genetic instability, mutations, and environmental and other stress factors. These ...

Isolation of Human Monocytes by Double Gradient Centrifugation and Their Differentiation to Macrophages in Teflon-coated Cell Culture Bags

Human macrophages are involved in a plethora of pathologic processes ranging from infectious diseases to cancer. Thus they pose a valuable tool to understand the underlying mechanisms of these diseases. We therefore present a straightforward protocol for the isolation of human monocytes from buffy coats, followed by a differentiation procedure which results in high macrophage yields. The technique relies mostly on commonly available lab equipment and thus provides a cost and time effective way to obtain large quantities of human macrophages. Briefly, buffy coats from healthy blood donors are subjected to a double density gradient centrifugation to harvest monocytes from the peripheral blood. These monocytes are then cultured in fluorinated ethylene propylene (FEP) Teflon-coated cell culture bags in the presence of macrophage colony-stimulating factor (M-CSF). The differentiated macrophages can be easily harvested and used for subsequent studies and functional assays. Important methods for quality control and validation of the isolation and differentiation steps will be highlighted within the protocol. In summary, the protocol described here enables scientists to routinely and reproducibly isolate human macrophages without the need for cost intensive tools. Furthermore, disease models can be studied in a syngeneic human system circumventing the use of murine macrophages.

We present a simple and efficient protocol for the generation of human macrophages. Buffy coats are processed by double density gradient centrifugation and isolated monocytes are then differentiated to macrophages in Teflon-coated cell culture bags. This maximizes macrophage yields and facilitates cell harvesting for subsequent experiments.

人类单核双梯度离心法和其分化为巨噬细胞的特富龙涂层的细胞培养袋隔离

Human macrophages are involved in a plethora of pathologic processes ranging from infectious diseases to cancer. Thus they pose a valuable tool to ...

免疫与感染

A Robust Discovery Platform for the Identification of Novel Mediators of Melanoma Metastasis

Metastasis is a complex process, requiring cells to overcome barriers that are only incompletely modeled by in vitro assays. A systematic workflow was established using robust, reproducible in vivo models and standardized methods to identify novel players in melanoma metastasis. This approach allows for data inference at specific experimental stages to precisely characterize a gene's role in metastasis. Models are established by introducing genetically modified melanoma cells via intracardiac, intradermal, or subcutaneous injections into mice, followed by monitoring with serial in vivo imaging. Once preestablished endpoints are reached, primary tumors and/or metastases-bearing organs are harvested and processed for various analyses. Tumor cells can be sorted and subjected to any of several 'omics' platforms, including single-cell RNA sequencing. Organs undergo imaging and immunohistopathological analyses to quantify the overall burden of metastases and map their specific anatomic location. This optimized pipeline, including standardized protocols for engraftment, monitoring, tissue harvesting, processing, and analysis, can be adopted for patient-derived, short-term cultures and established human and murine cell lines of various solid cancer types.

This article describes a workflow of techniques employed for testing novel candidate mediators of melanoma metastasis and their mechanism(s) of action.

用于鉴定黑色素瘤转移新介质的强大发现平台

Metastasis is a complex process, requiring cells to overcome barriers that are only incompletely modeled by in vitro assays. A systematic workflow was ...

An Ectopic Chemokine Expression Model for Testing Macrophage Recruitment In Vivo

Zebrafish are widely used in basic and biomedical research. Many zebrafish transgenic lines are currently available to label various types of cells. Owing to the transparent embryonic body of zebrafish, it is convenient for us to study the effect of one chemokine on the behavior of a certain type of cells in vivo. Here we provided a workflow to investigate the function of a chemokine on macrophage migration in vivo. We constructed a tissue-specific overexpression plasmid to overexpress IL-34 and injected the plasmid into one-cell stage transgenic fish embryos whose macrophages were specifically labeled by a fluorescent protein. We then used whole mount fluorescent in situ hybridization and immunostaining to detect the pattern of the chemokine expression and the number or location of macrophages. The injected WT embryos were raised to generate a stable transgenic line. Finally, we used confocal live imaging to directly observe macrophage behavior in the stable transgenic fish to study the function of IL-34 on macrophages in vivo.

To test the effect of a chemokine on macrophage recruitment in vivo, the whole mount in situ hybridization was used to detect the ectopic expression of the chemokine, and immunostaining was used to label macrophages. Live imaging was used for real-time observation of macrophage migration.

用于测试 Vivo 中巨噬菌体招募的异位化学表达模型

Zebrafish are widely used in basic and biomedical research. Many zebrafish transgenic lines are currently available to label various types of cells. Owing ...

发育生物学

<meta charset="utf8"></head><body>检查 3D 胶原基质设置中的巨噬细胞-肿瘤球状体相互作用

A Macrophage-Tumor Spheroid Co-Invasion Assay

The interaction of immune and cancer cells and their respective impact on metastasis represents an important aspect of cancer research. So far, only a few protocols are available that allow an in vitro approximation of the in vivo situation. Here, we present a novel approach to observing the impact of human macrophages on the invasiveness of cancer cells, using tumor spheroids of H1299 non-small cell lung carcinoma cells embedded in a three-dimensional (3D) collagen I matrix. With this co-cultivation setup, we tested the impact of small interfering RNA (siRNA)-based depletion of regulatory factors in macrophages on the 3D invasion of cancer cells from the tumor spheroid compared to controls. This method allows the determination of different parameters, such as spheroid area or the number of invading cancer cells, and thus, to detect differences in cancer cell invasion. In this article, we present the respective setup, discuss the subsequent analysis, as well as the advantages and potential pitfalls of this method.

<meta charset="utf8"></head><body>巨噬细胞-肿瘤球状体共侵袭测定

Here, we present a protocol to investigate the interaction of primary human monocyte-derived macrophages with a tumor spheroid in a three-dimensional (3D) collagen I matrix, with the possibility to compare the impact of soluble and physical properties of the microenvironment on cell invasion.

巨噬细胞-肿瘤球状体共侵袭测定

The interaction of immune and cancer cells and their respective impact on metastasis represents an important aspect of cancer research. So far, only a few ...

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

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用于鉴定黑色素瘤转移新介质的强大发现平台

用于测试 Vivo 中巨噬菌体招募的异位化学表达模型

巨噬细胞-肿瘤球状体共侵袭测定

巨噬细胞-肿瘤球状体共侵袭测定

巨噬细胞-肿瘤球状体共侵袭测定