Name: isolation of primary cancer-associated fibroblasts from a syngeneic murine model of breast cancer for the study of targeted nanoparticles
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Description: Watch this Scientific Journal Video about Isolation of Primary Cancer-Associated Fibroblasts from a Syngeneic Murine Model of Breast Cancer for the Study of Targeted Nanoparticles at JoVE.com

这项研究得到了意大利协会的支持， 根据 Ig 2017 - id. 20172 项目 - P. i. 科西 · 法比奥。SM承认儿科临床研究中心"罗密欧和恩里卡因弗尼齐"，支持她的立场。AB感谢AIRC（ID.20172项目）和米兰大学的研究奖学金。LS 和 MS 博士后和博士奖学金由米兰大学提供支持。

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作者没有什么可透露的。

CAF正在成为重塑细胞外基质、促进转移进展和限制药物进入肿瘤位点34的关键角色。然而，由于它们的异质性，它们的作用仍然是有争议的，一些CAF是肿瘤原性的，而其他一些亚型似乎具有肿瘤抑制作用。在这种情况下，他们的隔离可以是极为感兴趣的，以揭示他们备受争议的作用，在癌症的进展，这将具有重要的临床影响12，31。此外，从人类和临床前小鼠肿瘤模型中成功提取CAF也将促进新的CAF靶向药物的开发。本文报告了一种有效分离和培养原发性CAF与乳腺癌的同步临床前模型的方法。根据经验，三个实验步骤主要影响协议的成功。
第一种是快速工作，以避免与列中凝固和减缓细胞排泄的风险相关的细胞悬浮聚合：事实上，当通过列的细胞聚集在一起时，亚最佳隔离过程的概率增加，最终培养物包含"污染物"细胞克隆。第二种是消耗通过后从不同肿瘤中汇集细胞，以保证在最后的浓缩步骤中通过足够的细胞。最后一个关键问题是将提取的细胞电镀在高细胞密度下，很可能从24井板的一口井开始，以促进细胞生长和菌落扩张长达4+5段。如果细胞以低密度播种，它们就会在自由表面扩张并迅速停止复制。
一些研究已经描述了人类和小鼠起源的CAF提取方法，以研究它们在促进癌症发展和侵入性方面的作用。这在许多类型的肿瘤中都做过，包括乳腺癌、黑色素瘤、胆囊癌和胰腺癌35、36、37、38。然而，由于缺乏特定的CAF标记及其异质性，这些过程的结果仍然不尽如人意。
在这里，分离的细胞被用来验证HFn纳米穴的CAF靶向能力，该纳米穴具有抗FAP靶向护身盆功能。CAFs 初级培养物的使用是一个显著优势，它使纳米策略 前体内 的验证具有比在纳米药物优化的这个初步阶段直接 在体内 进行简单且具有成本效益的结合实验。
从这个意义上说，CAF的前 体内 测试可以在 活体 动物实验中先行，允许筛选和选择最有前途的纳米粒子，以最佳定位CAF。此处介绍的 CAF 模型也可用于初步疗效研究，当将纳米粒子加载到活性药物时。动物以后将只用于评估生物分布、药理动力学和疗效研究。
几个CAF靶向纳米药物正在开发中，如用于乳腺癌光动力治疗的铁氧体纳米卡和基于肽的颗粒，以在前列腺癌模型39，40中提供多索鲁比辛。然而，没有多少研究集中在隔离CAF作为细胞平台的纳米药物优化。
此协议有一些限制。首先，这是一个耗时的程序，需要准备和购买几个试剂和材料。其次，由于4T1乳腺肿瘤中CAF的稀缺性，其产量较低。纳米粒子实验应在达到所需细胞数后立即进行，因为初级CAF会衰老，不能在培养中长期维持。总之，这种CAF分离、培养和定性的方法可以成为加速开发新的靶向纳米药物对抗癌症的有力工具。

在过去的几十年里，很明显，杀死肿瘤细胞通常不足以消除恶性肿瘤，因为肿瘤微环境可能促使肿瘤复发并诱导治疗耐药性1，2。一个新的范式已经出现：瞄准肿瘤频闪剥夺肿瘤的支撑因子，从而，提高化疗的疗效3，4，5。特别是，癌症相关的成纤维细胞（CAFs）是许多固体肿瘤6，7有趣的频闪靶点。CAF是一组非常异质的细胞，通过生长因子、细胞因子和化疗因子的分泌与癌细胞和免疫系统细胞相互作用：建立和改造细胞外矩阵;并启用转移形成8，9，10，11，12。根据肿瘤类型，CAF显示亲肿瘤功能，而其他亚型的CAF似乎有肿瘤抑制功能13，14。为了更好地澄清这种二分法，从原发性肿瘤和转移性肿瘤中彻底描述CAF是很重要的。
在这方面，一个新兴的研究领域侧重于纳米化剂的开发，旨在通过提供活性分子和药物来瞄准和/或摧毁CAF，从而能够重塑肿瘤微环境15、16、17、18。几种类型的纳米粒子被设计成通过细胞毒性药物实现CAF消融，诱导CAF靶向光动力治疗，或通过将CAF恢复到静止状态或诱导TNF相关凋亡诱导配体表达，从而诱导邻近癌细胞16、19的凋亡。此外，许多纳米粒子积极瞄准特定生物标记的潜力，使人们希望选择CAF子集作为目标。虽然它对CAF的绝对特异性仍然受到质疑，但成纤维细胞活化蛋白（FAP）是亲肿瘤频闪最有前途的目标之一，并被利用来引导纳米药物的输送，从而为CAF靶向纳米疗法20、21、22的发展铺平了道路。
本文描述了原发性CAF与乳腺癌的合成模型的分离，并报告了它们在研究纳米粒子的靶向能力时所用，该粒子旨在识别CAF标记，FAP。铁蛋白纳米穴被用作候选纳米系统来建立这种方法，因为其交付特异性可能由目标moieties23，24的表面暴露所塑造。此外，铁蛋白已被成功证明是抗肿瘤应用的优秀生物相容穿梭体，触发肿瘤质量25、26、27的有效载荷的快速积累。迄今为止，CAF靶向纳米系统临床前研究已经涉及对培养中刺激的成纤维细胞系进行体外测试，通过转化生长因子β来诱导细胞活化和CAFs28、29的一些免疫表型特征的表达。这种方法通常应用于不朽的细胞系（如NIH3T3，LX-2），并且非常快速和简单，在几个小时或几天内产生激活的细胞。一个限制是，虽然体外刺激诱导一些基因的表达归因于激活肌纤维细胞，它不能完全回顾所有生物特征的真正的CAF，特别是其异质性在体内。
另一项策略是从人类或小鼠肿瘤样本中提取原发性CAF，30，31。这确保了 CAF 激活发生在生理环境中，并且保持 CAF 子群的异质性。根据研究目标，CAF可能来自不同的来源，从而提供了研究最可靠的条件的可能性。这里报道的协议对于寻求对新型纳米粒子的功能进行初步评估的科学家来说很有价值，这些纳米粒子旨在从乳腺癌模型中瞄准CAF。分离的CAF对于筛选那些有希望进行癌症动物模型体内评估的纳米粒子是有用的。这将在纳米粒子生产的第一步中具有相关性，通过主要考虑配体固定策略，以实现最佳目标特性，推动纳米技术专家向纳米粒子设计的精细化迈进。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>ACK Lysing buffer</td>
			<td>Lonza</td>
			<td>10-548E</td>
			<td>Store at +15&#176; to +30 &#176;C.</td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 goat anti-human antibody</td>
			<td>Immunological Science</td>
			<td>IS-20022</td>
			<td>Protect from light.</td>
		</tr>
		<tr>
			<td>Anti-FAP Fab fragment (3F2)</td>
			<td>Creative Biolabs</td>
			<td>TAB-024WM-F(E)</td>
			<td></td>
		</tr>
		<tr>
			<td>BALB/c mice</td>
			<td>Charles River</td>
			<td>028BALB/C</td>
			<td>7 weeks old female mice</td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Sigma-Aldrich</td>
			<td>A7906</td>
			<td>Store at 2-8 &#176;C.</td>
		</tr>
		<tr>
			<td>CD45 antibody</td>
			<td>Miltenyi Biotec</td>
			<td>130-110-796</td>
			<td>FITC-conjugated fluorescent antibody; clone REA 737; Store protected from light at 2&#8211;8 &#176;C.</td>
		</tr>
		<tr>
			<td>CD90.2 antibody</td>
			<td>Miltenyi Biotec</td>
			<td>130-102-960</td>
			<td>PE-conjugated fluorescent antibody; clone 30-H12; Store protected from light at 2&#8211;8 &#176;C.</td>
		</tr>
		<tr>
			<td>Cell strainers 70 &#181;m</td>
			<td>VWR</td>
			<td>732-2758</td>
			<td></td>
		</tr>
		<tr>
			<td>CytoFLEX</td>
			<td>Beckman Coulter</td>
			<td></td>
			<td>laser configuration B4-R2-V0</td>
		</tr>
		<tr>
			<td>Dead cell Removal Kit</td>
			<td>Miltenyi Biotec</td>
			<td>130-090-101</td>
			<td>contains 10 mL of Dead Cell Removal MicroBeads; 25 mL of 20&#215; Binding Buffer Stock Solution. Store protected from light at 2&#8722;8 &#176;C.</td>
		</tr>
		<tr>
			<td>D-Luciferin</td>
			<td>Caliper</td>
			<td>760504</td>
			<td>reagent for in vivo imaging</td>
		</tr>
		<tr>
			<td>DMEM High Glucose w/o L-Glutamine&#160; w/ Sodium Pyruvate</td>
			<td>Euroclone</td>
			<td>ECB7501L</td>
			<td>Warm at 37 &#176;C in a water bath before use.</td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Sigma-Aldrich</td>
			<td>D2650</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS w/o Calcium, w/o Magnesium</td>
			<td>Euroclone</td>
			<td>ECB4004L</td>
			<td>Keep at room temperature.</td>
		</tr>
		<tr>
			<td>Ethylenediaminetetraacetic acid (EDTA)</td>
			<td>Sigma-Aldrich</td>
			<td>E9884</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Euroclone</td>
			<td>ECS0180L</td>
			<td>Before use, heat at 56 &#176;C for 30 min to kill all the complement proteins.</td>
		</tr>
		<tr>
			<td>Fluorescein Istothiocyanate isomer I (FITC)</td>
			<td>Sigma-Aldrich</td>
			<td>F7250</td>
			<td>Store protected from light at 2&#8211;8 &#176;C.</td>
		</tr>
		<tr>
			<td>GentleMACS C Tubes</td>
			<td>Miltenyi Biotec</td>
			<td>130-096-334</td>
			<td>They are used in combination with the gentleMACS Dissociator.</td>
		</tr>
		<tr>
			<td>GentleMACS dissociator</td>
			<td>Miltenyi Biotec</td>
			<td>130-093-235</td>
			<td>Two samples can be processed in parallel. Special protocols have been developed for various tissues.&#160;</td>
		</tr>
		<tr>
			<td>Goat serum</td>
			<td>Euroclone</td>
			<td>ECS0200D</td>
			<td></td>
		</tr>
		<tr>
			<td>Ham&#39;s F12&#160; w/o L-Glutamine</td>
			<td>Euroclone</td>
			<td>ECB7502L</td>
			<td>Warm at 37 &#176;C in a water bath before use.</td>
		</tr>
		<tr>
			<td>IVIS Lumina II imaging system</td>
			<td>Caliper Life Sciences</td>
			<td></td>
			<td>This imaging system is easy to use for both fluorescent and bioluminescent imaging in vivo. Equipped with a Living Image Software for analysis</td>
		</tr>
		<tr>
			<td>LD columns</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-901</td>
			<td>Composed of ferromagnetic spheres, designed for stringent depletion of unwanted cells. Position the required number of columns on the magnetic Separation Unit and equilibrate with buffer before use</td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>Euroclone</td>
			<td>ECB3000D</td>
			<td>200 mM stock</td>
		</tr>
		<tr>
			<td>Light/fluorescence microscope equipped with camera</td>
			<td>Leica Microsystems</td>
			<td>DM IL LED Fluo/ ICC50 W CameraModule</td>
			<td>inverted microscope for live cells with camera</td>
		</tr>
		<tr>
			<td>MACS MultiStand</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-303</td>
			<td></td>
		</tr>
		<tr>
			<td>MACS Separation Unit</td>
			<td>Miltenyi Biotec</td>
			<td>130-090-976</td>
			<td>Position the magnet on appropriate stand before use.</td>
		</tr>
		<tr>
			<td>MACS Tissue Storage Solution</td>
			<td>Miltenyi Biotec</td>
			<td>130-100-008</td>
			<td></td>
		</tr>
		<tr>
			<td>MS columns</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-201</td>
			<td>Composed of ferromagnetic spheres, designed for positive selection of cells. Position the required number of columns on the magnetic Separation Unit and equilibrate with buffer before use.</td>
		</tr>
		<tr>
			<td>Mycoplasma Removal Agent</td>
			<td>Euroclone</td>
			<td>ECMC210A</td>
			<td>Dilute in culture medium 1:100.</td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>Invitrogen</td>
			<td>A10235</td>
			<td></td>
		</tr>
		<tr>
			<td>Nanodrop spectrophotometer</td>
			<td>Thermo Fisher Scientific</td>
			<td>ND-2000C</td>
			<td></td>
		</tr>
		<tr>
			<td>Non essential aminoacids</td>
			<td>Euroclone</td>
			<td>ECB3054D</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Euroclone</td>
			<td>ECB3001D</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Human apoferritin H-homopolymer (HFn)&#160;</td>
			<td>Molirom</td>
			<td>MLR-P018</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI 1640 w/o L-Glutamine</td>
			<td>Euroclone</td>
			<td>ECB9006L</td>
			<td>Warm at 37 &#176;C in a water bath before use.</td>
		</tr>
		<tr>
			<td>Tumor-Associated Fibroblast Isolation kit</td>
			<td>Miltenyi Biotec</td>
			<td>130-116-474</td>
			<td>Contains 1 mL of Non-Tumor-Associated Fibroblast Depletion Cocktail, mouse; 1 mL of CD90.2 (Tumor-Associated Fibroblast) MicroBeads,mouse. Store protected from light at 2-8 &#176;C.</td>
		</tr>
		<tr>
			<td>Tumor Dissociation Kit</td>
			<td>Miltenyi Biotec</td>
			<td>130-096-730</td>
			<td>Contains lyophilized enzymes (D, R, A) and buffer A; reconstitute enzymes according to the manufacturer&#39;s instructions, aliquot and store at - 20 &#176;C.</td>
		</tr>
		<tr>
			<td>Trypan blue 0.4%</td>
			<td>Lonza</td>
			<td>17-942E</td>
			<td>dilute 1:1 with a sample of cell suspension before counting the cells</td>
		</tr>
		<tr>
			<td>TrypLE select</td>
			<td>Gibco</td>
			<td>12563-029</td>
			<td>use at room temperature</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA</td>
			<td>Lonza</td>
			<td>BE17-161E</td>
			<td>Warm at 37 &#176;C in a water bath before use</td>
		</tr>
		<tr>
			<td>T75 Primo TC flask</td>
			<td>Euroclone</td>
			<td>ET7076</td>
			<td></td>
		</tr>
		<tr>
			<td>Zeba Spin Desalting Columns</td>
			<td>Thermo Fisher Scientific</td>
			<td>89890</td>
			<td>7K MWCO, 2 mL</td>
		</tr>
		<tr>
			<td>1.5 mL tubes</td>
			<td>Biosigma</td>
			<td>CL15.002.0500</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL tubes</td>
			<td>Euroclone</td>
			<td>ET5015B</td>
			<td></td>
		</tr>
		<tr>
			<td>4T1-luc2</td>
			<td>Caliper</td>
			<td></td>
			<td>Mouse mammary gland cancer cell line stably transfected with firefly luciferase gene</td>
		</tr>
		<tr>
			<td>50 mL tubes</td>
			<td>Euroclone</td>
			<td>ET5050B</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation of primary cancer-associated fibroblasts from a syngeneic murine model of breast cancer for the study of targeted nanoparticles

癌症相关成纤维细胞 （CAF） 是肿瘤微环境中的关键角色。尽管与肿瘤细胞相比数量减少，但CAF可以调节肿瘤进展，并提供抗肿瘤免疫保护。新兴的抗癌策略旨在通过消融亲肿瘤的CAF或重新编程CAF功能及其激活状态来重塑肿瘤微环境。一种很有前途的方法是开发纳米化输送剂，能够瞄准CAF，从而允许药物和活性分子的特定输送。在这方面，CAF的细胞模型可以为 体外 筛选和这种纳米形态的初步研究提供有用的工具。
本研究描述了原发性CAF与三阴性乳腺癌的合成4T1粘膜模型的分离和文化。磁珠用于两步分离过程，从分离的肿瘤中提取CAF。免疫表型控制在每次通过后使用流细胞测量来验证过程产量。可利用分离的CAF来研究用于处理肿瘤微环境的不同纳米成形物的瞄准能力。荧光标记的H-铁蛋白纳米穴被用作候选纳米粒子来建立这种方法。对纳米粒子进行分析，分析其是否与目标配体结合，以增强其与CAF的结合性。结果表明，乳房CAF的 体外 提取可能是测试和验证纳米粒子的一个有用的系统，用于肿瘤性CAF的特定靶向。

用于 最佳 CAF 隔离的体内模型设置 将10个5 4T1-luc细胞注射到雌性BALB/c小鼠的乳腺脂肪垫中，可在植入后5天导致可检测肿瘤质量的生长。通过通过卡钳测量肿瘤体积和 BLI 测量肿瘤细胞的生存能力，在植入后监测肿瘤生长一个月。为了找到一个牺牲窗口，足以为CAFs隔离，寻求最佳妥协之间的肿瘤大小和BLI一方面，另一方面新兴的肿瘤溃疡和坏死（图1）。由于坏死核在植入后20天出现，并在25天和30天（ 图1C中的BLI图像记录）放大，因此将第20天设定为隔离过程后优化细胞恢复的时间点。即使在肿瘤处理 前体内的第一步仔细切除所有可见的坏死区域后，在分离成单细胞的末端也发现了高比例的死细胞（表1）。由于这个百分比可能相关，特别是随着肿瘤大小的增加，在处理4T1模型时，死细胞切除总是必要的。
优化 CAF 隔离程序、培养和特征 还需要两段话来分离CAF（CD90.2+ CD45-）的种群，从收集的可行细胞面板中分离出：非肿瘤相关成纤维细胞的耗竭和肿瘤相关成纤维细胞的富集（图2）。耗竭珠鸡尾酒可有效去除CD45+细胞（分别占消耗前和衰竭后细胞总数的67.35%和0.69%），并且始终用于处理每列单个肿瘤。如表1所示，在耗竭步骤后，被精化的细胞数量从平均3×106下降到1×105。由于细胞总数大幅减少，在进行浓缩步骤之前，将收集的细胞从至少 2 个集中到单个管中最多 4 个肿瘤是很方便的。通过这样做，获得足够数量的细胞与肿瘤相关的成纤维细胞珠孵育，并通过一个单一的分离列获得CD90.2+ CD45-细胞的93%的最终平均值（表2）。
一旦播种到组织培养板上，这些恢复的细胞附着在塑料上，并揭示了一个大型主轴形状的形态典型的成纤维细胞（图3A，B），不同于4T1肿瘤细胞（图3D）。耗竭后不汇集肿瘤会导致与肿瘤相关的成纤维细胞微珠的细胞产量过低，无法建立培养。在其他情况下，当微珠孵化的持续时间和温度没有仔细维护时，可能会发生一些非特定的结合。在这种情况下，浓缩步骤效率较低，与 CD90.2+ CD45+和 CD90.2- CD45-细胞的百分比较低，以及 CD90.2= CAF（分别为 5.97 ± 1.5 和 16.75 ± 1.1）（图 4和表 3）。这些污染物细胞可能是不同形态的小克隆（图4B，黑色箭头）在培养中出现的原因，这些克隆的生长速度比CAF快，并战胜了主要的CAF培养物（图4C）。这些次优结果证实了始终仔细检查 CD90.2 和 CD45 表达式以及浓缩过程结束时和培养细胞生长过程中的细胞形态的重要性。
使用分离细胞来评估工程纳米药物的CAF靶向潜力 新分离的CAF可用于从基础研究到药理学研究等多个应用。这个小组的目标是开发能够专门针对CAF的HFn纳米穴。HFn 的功能与特定的抗 FAP 抗体片段 （HFn-FAP） 在两种不同的蛋白质： 抗体比 （较低的 1：1 和更高的 1：5）， 并与 CAF 的结合进行了测试。由于FAP被用作亲肿瘤CAF的表面生物标志物，因此在分离的CAF（图3C）上检查FAP表达是至关重要的。遵循了5段以上的文化表达，以确认主要的CAF文化保持了其原始特征。
发现与裸HFn相比，HFn的FAP功能化有助于向CAF靶向的重大转变，而抗体（1：1）的降低足以观察这种效应（图5A）。然而，这并没有观察到肿瘤4T1细胞用于建立体内肿瘤模型，其中裸HFn显示比功能化HFn（图5B）更高的结合性。这很可能是由于4T1细胞中没有FAP过度表达，以及HFn与TFR1的优先相互作用，TfR1调节细胞中的HFn吸收，正如该组27，33广泛报道的那样。这些结果证实了使用乳房CAF的初级培养物来初步筛选纳米粒子的靶向能力，旨在解决肿瘤微环境的有用性。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62504/62504fig01.jpg"> 图1：4T1肿瘤模型的建立。细胞（105个细胞/小鼠）被注射到乳腺脂肪垫中。植入后（A）通过用卡钳测量肿瘤体积，通过生物发光成像测量肿瘤体积（B），在植入后第5天、第10天、第15天、第20天、第25天和第30天跟踪肿瘤生长。肿瘤体积和 BLI 分别表示为 mm3和计数。结果报告为平均± SEM （n=6）。（C） BLI代表性图像获得5，10，15，20，25和30天后，细胞植入确认肿瘤生长，直到第25天，当它似乎达到一个高原。在最后一次分析点（30 天）中，BLI 不会比第 25 天增加。从第20天开始，坏死和溃疡区域开始在肿瘤的中心部分变得可见。颜色刻度： 最小 = 1，194， 最大 = 20，462.缩写：BLI = 生物发光成像;SEM = 平均值的标准错误。<a href="https://www.jove.com/files/ftp_upload/62504/62504fig01large.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/62504/62504fig02.jpg"> 图2：孤立CAF的肿瘤样本制备和流细胞学特征。肿瘤被切除，并减少到（A） 小块约 1+2 毫米的帮助下，手术刀：（B） 切除肿瘤组织消化和机械分离后单细胞悬浮：（C） 红血球裂解后获得的细胞颗粒：（D） 红血球和死细胞切除后获得的细胞中的CD45和CD90.2表达的流动细胞学分析，（E） 非癌症相关成纤维细胞耗尽后，以及（F） 富集与癌症相关的成纤维细胞后，其中大多数细胞为CD90.2× CD45- （蓝色矩形）。缩写：CAF® 癌症相关成纤维细胞;CD = 差异化集群;PE-A = 植物红素区域;FITC-A = 荧光素异氰酸酯区域。<a href="https://www.jove.com/files/ftp_upload/62504/62504fig02large.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/62504/62504fig03.jpg"> 图3：通过光学显微镜（第2段和第5段的不同汇合水平），与（D）4T1肿瘤细胞相比，通过光学显微镜检查了CAF和FAP表达的形态分析。（A、B）CAF形态在培养的所有段落中进行了检查：比例杆 = 10μm.（C）纤维细胞活化蛋白 （FAP） 表达在收集的 CD90.2= CD45 的隔离过程结束时通过流细胞测量进行评估，以确认其分子特征。在未染色的控制细胞（红色图）上设置了荧光阈值，以量化平均荧光强度和抗体染色细胞中阳性细胞 （FAP+）的百分比（蓝色图）。缩写：CAF® 癌症相关成纤维细胞;FAP = 成纤维细胞激活蛋白;CD = 差异化集群;MFI = 平均荧光强度;Ab = 抗体;FITC-A = 氟辛异氰酸酯区域;FAP® = FAP 阳性细胞。<a href="https://www.jove.com/files/ftp_upload/62504/62504fig03large.jpg" target="_blank">请单击此处查看此图的较大版本。</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/62504/62504fig04.jpg"> 图4：CFS隔离过程的次优示例。（A ） 在最后隔离步骤后进行流细胞学评估，发现存在污染物CD90.2- CD45+和CD90.2- CD45-细胞。（B） 这些细胞在播种时可被视为具有小圆形态的克隆（黑色箭头和镶板左下角的嵌入），（C）在培养的第三段之后战胜了CAF。秤杆 = 10μm。缩写： CAF = 癌症相关成纤维细胞;CD = 差异化集群;PE-A = 植物红素区域;FITC-A = 荧光素异氰酸酯区域。<a href="https://www.jove.com/files/ftp_upload/62504/62504fig04large.jpg" target="_blank">请单击此处查看此图的较大版本。</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/62504/62504fig05.jpg"> 图5：HFn纳米卡在CAF和4T1上的绑定。HFn 纳米穴在荧光标记上 FITC，在两种不同的蛋白质抗体比率（1：1 和 1：5）下使用抗 FAP 抗体片段 （HFn-FAP） 功能，并在 2 小时的 4 °C 下与（A）目标 CAF 和（B） 4T1 细胞一起孵育。绑定通过流细胞测量进行评估。（A） 与裸HFn相比，HFn-FAP与CAF的结合显著增加三倍，相比之下，在4T1细胞中观察到裸HFn的结合性显著提高，而FAP识别不会增强其结合性。结果报告为三个独立实验的平均±标准偏差。p = 0.0003;•p = 0.0021 ;•+p = 0.0008。缩写：CAF® 癌症相关成纤维细胞;FAP = 成纤维细胞激活蛋白;HFn = 人类铁氧体重链的重组变种，用作裸纳米粒子或结合;HFN-FAP – HFn 纳米粒子功能与抗 FAP 抗体的可变部分，在两个 HFn：Fab@FAP 摩尔比，1：1 和 1：5。<a href="https://www.jove.com/files/ftp_upload/62504/62504fig05large.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></td>
			<td>细胞数（平均±SD）</td>
			<td>提取产量总计（每段）</td>
		</tr>
<tr>
<td>切除肿瘤（红细胞切除后）</td>
			<td>1.27 x 108 ± 9.81 x 107
</td>
			<td>100%</td>
		</tr>
<tr>
<td>切除肿瘤（死细胞切除后）</td>
			<td>3.01 x 106 ± 9.61 x 105
</td>
			<td>2.38%</td>
		</tr>
<tr>
<td>消耗后鸡尾酒</td>
			<td>1.15 x 105 ± 4.95 x 104
</td>
			<td>0.11% (3.82%)</td>
		</tr>
<tr>
<td>浓缩后</td>
			<td>3.00 x 104 ± 1.2 x 104
</td>
			<td>0.027% (26.09%)</td>
		</tr>
</tbody></table>表1：癌症相关成纤维细胞分离的每一步后细胞总数。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>细胞分布 （%）</td>
			<td>CD90.2= CD45-
</td>
			<td>CD90.2= CD45|
</td>
			<td>CD90.2- CD45|
</td>
			<td>CD90.2- CD45-
</td>
		</tr>
<tr>
<td>切除肿瘤（死细胞切除后）</td>
			<td>1.81 ± 0.98</td>
			<td>10.78 ± 4.51</td>
			<td>56.57 ± 14.05</td>
			<td>28.20 ± 17.57</td>
		</tr>
<tr>
<td>消耗后鸡尾酒</td>
			<td>69.33 ± 16.75</td>
			<td>0.14 ± 0.13</td>
			<td>0.55 ± 0.63</td>
			<td>39.89 ± 30.31</td>
		</tr>
<tr>
<td>浓缩后</td>
			<td>93.14 ± 3.3</td>
			<td>0.09 ± 0.1</td>
			<td>0.09 ± 0.08</td>
			<td>6.69 ± 3.4</td>
		</tr>
</tbody></table>表2：每次通过癌症相关成纤维细胞分离后，根据CD90.2和CD45的表达方式进行细胞分布。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>细胞分布 （%）</td>
			<td>CD90.2= CD45-
</td>
			<td>CD90.2= CD45|
</td>
			<td>CD90.2- CD45|
</td>
			<td>CD90.2- CD45-
</td>
		</tr>
<tr>
<td>浓缩后（无池）</td>
			<td>75.80 ± 2.9</td>
			<td>1.49 ± 0.4</td>
			<td>5.97 ± 1.5</td>
			<td>16.75 ± 1.1</td>
		</tr>
</tbody></table>表3：CD90.2和CD45表达细胞在亚最佳癌症相关成纤维细胞分离实验后收集。

Watch this Scientific Journal Video about Isolation of Primary Cancer-Associated Fibroblasts from a Syngeneic Murine Model of Breast Cancer for the Study of Targeted Nanoparticles at JoVE.com

Isolation of Primary Cancer-Associated Fibroblasts from a Syngeneic Murine Model of Breast Cancer for the Study of Targeted Nanoparticles

本文旨在为三阴性乳腺癌的同步粘膜模型中原发性癌症相关成纤维细胞的分离与培养提供协议，并应用于旨在针对肿瘤微环境的新型纳米粒子的临床前研究。

将原发性癌症相关纤维细胞与乳腺癌的合成素 Murine 模型分离，用于靶向纳米粒子的研究

Cancer-associated fibroblasts (CAFs) are key actors in the context of the tumor microenvironment. Despite being reduced in number as compared to tumor ...

该协议使成功分离和培养与纤维细胞相关的原发性癌症与穆林乳腺癌，筛选出新的纳米粒子，旨在瞄准肿瘤微环境。初级CAF是肿瘤频闪体体外研究最可靠、最生理的模型。在此协议中，我们描述了如何实现最佳收益。首先，为肿瘤分离准备消化组合，在紧紧关闭管子后将三到五毫米的肿瘤片段浸泡在溶液中。将管子倒置，检查所有肿瘤的碎片是否在管子底部朝向盖子。然后将管子连接到适当的外壳中的机械分离器上，并运行专为硬肿瘤设计的分离程序。运行后，分离管子，并将其倒置，在37摄氏度下孵育样品40分钟，轻轻摇晃。然后将管子重新连接到适当的外壳分离器上，并运行硬肿瘤的分离程序两次，确保手术结束时不会留下大组织块。在 50 毫升管中通过 40 微米细胞过滤器过滤样品。然后用 RPMI 1640 中离心机的 10 毫升洗涤器。离心后，将颗粒重新沉入PBE缓冲器中，由PBS、0.5%BSA和两毫摩尔EDTA组成，并用Trypan Blue计算细胞。通过用无菌双蒸馏水稀释 20 倍绑定缓冲器库存溶液来修复死细胞去除结合缓冲器。用5毫升1倍结合缓冲器和离心机清洗细胞。离心后，用0.1毫升的死细胞去除微珠将细胞颗粒重新用于10至7个细胞，并在室温下孵育15分钟。在孵化过程中，在引擎盖下准备一个磁立柱，并将铁磁分离柱悬挂到它，提示指向下。用 0.5 毫升的冷结合缓冲器使柱子等价，并等待解决方案流过。孵化后，在珠子和细胞悬浮液中加入400微升冷结合缓冲器。将整个体积加载到柱子上，然后用 15 毫升的管子收集污水。用 0.5 毫升的冷结合缓冲器清洗柱子四次，并在同一管中收集总污水。对于非癌症相关成纤维细胞的耗竭，使用用 PBS 滋润的 70 微米细胞过滤细胞悬浮物，并离心细胞。取出超高颗粒，在80微升冷PBE缓冲器中重新使用颗粒。加入20微升非肿瘤相关成纤维细胞耗竭鸡尾酒。混合好，在4摄氏度下孵育样品，在黑暗中15分钟。在与珠子的孵化过程中，在磁架上准备铁磁耗竭柱。用两毫升冷 PBE 缓冲器使柱子等价，并等待解决方案流过。孵化后，在珠子和细胞悬浮液中加入400微升缓冲液。将整个体积加载到柱子上，然后用 15 毫升的管子收集污水。用两毫升的 PBE 清洗柱子两次。将总污水收集到同一管中，并离心出水。对于癌症相关成纤维细胞的阳性选择，将颗粒重新用于 80 微升冷 PBE 缓冲器中。然后在20微升肿瘤相关的成纤维细胞微珠，并在4摄氏度的黑暗中孵育样品15分钟。在孵化过程中，在磁架上准备铁磁分离柱，并用0.5毫升的冷PBE缓冲器将其平衡。孵化后，在珠子和细胞悬浮中加入400微升PBE。将整个体积加载到柱子上，让未标记的细胞流进 15 毫升管中。用 0.5 毫升 PBE 缓冲器清洗柱子三次，并在同一管中收集总污水。从磁铁中取出柱子，放入 1.5 毫升管中。在柱子上加入一毫升的PBE，并立即将柱塞推入柱子以冲洗细胞。离心后，将细胞颗粒重新消耗在适当的DMEM体积中，并将细胞播种在组织培养板中。在显微镜下检查细胞密度，将板置于37摄氏度和5%的二氧化碳，让细胞粘附和生长。将4T1荧光细胞注射到雌性小鼠的乳腺脂肪垫中，导致植入5天后可检测到的肿瘤质量的生长。为了找到一个足以进行CAF隔离的牺牲窗口，我们一方面在肿瘤体型和生物发光成像之间寻求最佳的妥协，另一方面在肿瘤溃疡和坏死之间寻求最佳的妥协。第20天被设定为在隔离过程之后优化细胞恢复的时间点。还需要两段话来分离CAF的种群，从收集的活细胞面板中分离，非肿瘤相关成纤维细胞的耗竭，以及肿瘤相关成纤维细胞的富集。CAF细胞揭示了大主轴形状的形态，典型的成纤维细胞，与4T1肿瘤细胞不同。FAP的表达经过五段，证实主要的CAF文化保持了其原始特征。当微珠孵化的持续时间和温度得不到维持时，在孤立的培养物中发现了具有不同形态的克隆。这些污染物细胞生长得更快，并战胜了主要的CAF培养。与裸HFN相比，与人类费里汀Hfn-FAP功能纳米药物结合的CAF以1比1和1比5的抗体片段浓度增加了三倍。相反，对于肿瘤4T1细胞，裸HFN表现出比功能化HFN更高的结合。为了增加分离的CAF的产量杂质，保持缓冲器冷，尊重珠子的孵化时间，并在最后的浓缩步骤之前每列拉多达4个肿瘤。分离的CAF可用于筛选几个候选纳米药物的结合，吸收和药理疗效，然后再进行临床前体内实验。CAF隔离使我们能够测试纳米粒子是否可用于瞄准肿瘤微环境。从而为与化疗协同作用的新靶向疗法铺平了道路。

Research

JoVE Journal

Cancer Research

Anticancer Efficacy of Photodynamic Therapy with Lung Cancer-Targeted Nanoparticles

光动力疗法的抗癌功效肺癌靶向纳米粒子

Photodynamic therapy (PDT) is a non-invasive and non-surgical method representing an attractive alternative choice for lung cancer treatment. ...

科研

癌症研究

A Portal Vein Injection Model to Study Liver Metastasis of Breast Cancer

门静脉注射模型来研究肝转移乳腺癌

Breast cancer is the leading cause of cancer-related mortality in women worldwide. Liver metastasis is involved in upwards of 30% of cases with breast ...

A Syngeneic Mouse Model of Metastatic Renal Cell Carcinoma for Quantitative and Longitudinal Assessment of Preclinical Therapies

转移性的型号同源小鼠肾细胞癌的临床前疗法的定量和纵向评估

Renal cell carcinoma (RCC) affects &#62; 60,000 people in the United States annually, and ~ 30% of RCC patients have multiple metastases at the time of ...

A Mouse Model of Fatigue Induced by Peripheral Irradiation

疲劳的小鼠模型的外围照射引起

Cancer-related fatigue (CRF) is a distressing and costly condition that often affects patients receiving cancer treatments, including radiation therapy. ...

A Syngeneic Pancreatic Cancer Mouse Model to Study the Effects of Irreversible Electroporation

自体胰腺癌小鼠模型研究不可逆电穿孔的影响

Pancreatic cancer (PC), a disease which kills approximately 40,000 patients each year in the US, has successfully evaded several therapeutic approaches ...

A Bioluminescent and Fluorescent Orthotopic Syngeneic Murine Model of Androgen-dependent and Castration-resistant Prostate Cancer

雄激素依赖性和去势抗性前列腺癌的荧光自体小鼠模型

Orthotopic tumor modeling is a valuable tool for pre-clinical prostate cancer research, as it has multiple advantages over both subcutaneous and ...

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

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

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 ...

Orthotopic Transplantation of Syngeneic Lung Adenocarcinoma Cells to Study PD-L1 Expression

合成肺腺癌细胞原位移植研究 pd-l1 表达

The use of mouse models is indispensable for studying the pathophysiology of various diseases. With respect to lung cancer, several models are available, ...

A 3D Spheroid Model as a More Physiological System for Cancer-Associated Fibroblasts Differentiation and Invasion In Vitro Studies

3D球形模型作为癌症相关纤维细胞分化和入侵体内研究的更多生理系统

Defining the ideal model for an in vitro study is essential, mainly if studying physiological processes such as differentiation of cells. In the tumor ...