作者想感谢 Gráinne 的编辑协助。

sts 细胞与由 sts 患者手术切除的肿瘤肿块分离。该议定书已得到当地道德委员会的批准, 并根据良好的临床实践指导方针和赫尔辛基宣言进行。所有患者都给予书面知情同意, 参加该项研究。手术标本由经验丰富的肉瘤病理学家分析, 并在3小时内进行手术处理。
1. 肿瘤标本的采集与加工:
<ol><li>用100毫升无菌尿液容器中的肉瘤病理学家收集肿瘤标本, 用石蜡膜密封的50毫升 DMEM 低葡萄糖培养基。</li>
	<li>在肿瘤切除术后, 将样品保存在4摄氏度, 最大值为3小时。</li>
	<li>在无菌组织培养罩下执行以下步骤。<ol>
<li>用70% 乙醇消毒罩, 戴上无菌手套。</li>
		<li>用70% 乙醇消毒钢不锈钢镊子 (使用无菌纱布)。</li>
		<li>从尿液容器中取出石蜡膜并丢弃。</li>
		<li>用70% 乙醇清洗尿液容器的外部。</li>
		<li>打开一个方形的培养皿。</li>
		<li>用灭菌的钢不锈钢镊子打开尿液容器, 将肿瘤标本转移到方培养皿中。</li>
	</ol>
</li>
	<li>用 PBS 冲洗肿瘤标本两次。</li>
	<li>打开一把无菌手术手术刀。<ol>
<li>将肿瘤标本细切成 1-2 毫米3片。</li>
		<li>收集一个季度的总样本在2毫升管。</li>
		<li>添加1毫升的 trizol 试剂 (购买) 到管, 并立即冻结在-80 °c (该样本将用于下游分析的 STS 基因表达评估)。</li>
	</ol>
</li>
	<li>收集其余的细粉碎的肿瘤材料在一个15毫升管。<ol>
<li>添加4毫升胶原酶 i 型溶液 (胶原酶 i 型溶于 PBS, 浓度为4µg/毫升)。</li>
		<li>用4毫升的 DMEM 高培养基稀释胶原酶 i 型溶液 (胶原酶的最终浓度为2µg/毫升)。</li>
		<li>关闭15毫升管, 用石蜡膜密封。</li>
		<li>将15毫升管放入一个在37°c 的孵化器中, 在搅拌条件下, 以15分钟活化消化。</li>
		<li>在室温搅拌条件下, 在15分钟内将管放入摇床内。</li>
	</ol>
</li>
	<li>第二天把15毫升的管子放在消毒罩下。</li>
	<li>轻轻地过滤细胞悬浮通过一个100µm 无菌过滤器附着在50毫升管顶部。</li>
	<li>用 DMEM 高培养基冲洗过滤器, 辅以10% 胎牛血清、1% 谷氨酰胺、1% 青霉素/链霉素。</li>
	<li>丢弃100µm 无菌过滤器, 在室温下关闭50毫升管和离心机, 225 x 克5分钟。</li>
	<li>通过倒置管来丢弃上清液。</li>
	<li>重新悬浮细胞与 1-2 毫升 DMEM 高培养基补充10% 胎牛血清, 1% 谷氨酰胺, 1% 青霉素/链霉素。 注意:体积取决于肿瘤样品的处理量。</li>
	<li>用 Neubauer 室数细胞。<ol>
<li>稀释样品1:2 在台盼蓝和使用10µL 稀释计数细胞。</li>
		<li>计数至少两次, 并计算复制的平均值, 以了解所收集的单元格的总平均数。</li>
	</ol>
</li>
	<li>标准单层培养中的平板细胞, 密度为8万细胞/厘米2。 注意:在实验中始终包含控件。为每个条件至少执行3个技术复制。至少执行2生物复制。为临床前药理学、基因表达和蛋白质表达分析, 应在细胞播种 30-45 天内进行少量的培养通道, 以避免癌细胞表型的丧失。</li>
	<li>细胞学样品准备。<ol>
<li>收集10万个细胞在200µL DMEM 高培养基补充10% 胎牛血清, 1% 谷氨酰胺, 1% 青霉素/链霉素。</li>
		<li>将溶液 (200 µL 吸管) 插入一个装有过滤器并安装在玻璃滑梯上的一次性漏斗中。</li>
		<li>打开 cytocentrifuge 并插入示例。</li>
		<li>以 18.6 x g 为20分钟运行 cytocentrifuge。</li>
		<li>丢弃装有过滤器的漏斗。</li>
		<li>固定在丙酮10分钟的幻灯片, 其次是氯仿5分钟。</li>
		<li>将幻灯片存储在摄氏-20 摄氏度。 注意:在染色前解冻并水合样品, 并按照制造商的指示进行染色。至少准备3技术复制。</li>
	</ol>
</li>
</ol>2. STS 细胞培养:
<ol><li>每周更换一次媒体 (DMEM 高培养基培养基补充10% 胎牛血清、1% 谷氨酰胺和1% 青霉素/链霉素)。<ol>
<li>使用 200-1000 µL 吸管丢弃培养基。</li>
		<li>使用 200-1000 µL 吸管添加新的新鲜培养基。</li>
		<li>细胞通道:<ol>
<li>当汇流约为90% 时, 分离细胞: 注意:STS 细胞培养的扩增应根据培养条件每周进行一次。</li>
			<li>丢弃培养基, 用 pbs 洗涤, 在 pbs 中加入2-3 毫升的胰蛋白酶1x。 注意:使用的胰蛋白酶量取决于获得的培养细胞的数量。</li>
			<li>孵育细胞为 3-5 分钟在37°c。</li>
			<li>用 200-1000 µL 吸管从盘子里收集分离出来的细胞。</li>
			<li>将分离的细胞注入15毫升管, 加入4毫升的 DMEM 培养基, 补充10% 胎牛血清、1% 谷氨酰胺和1% 青霉素/链霉素, 以停止酶反应。</li>
			<li>离心机在室温下为 225 x 克, 5 分钟。</li>
			<li>放弃上清。</li>
			<li>添加1毫升的 DMEM 培养基, 补充10% 胎牛血清, 1% 谷氨酰胺, 1% 青霉素/链霉素, 重新悬浮细胞颗粒。</li>
			<li>根据制造商的说明, 在新的培养板或烧瓶中种子细胞。 注意:不要将单元格拆分1:3 以上。</li>
		</ol>
</li>
	</ol>
</li>
</ol>3. 下游分析:
注意:可以使用这个模型进行各种不同的实验 (见下文)。因此, 必须在研究的设计阶段决定进行分析, 以便准备足够数量的样品和复制。
<ol><li>增殖测定, 如 MTT 3-(45-dimethylthiazol-2-基)-25-diphenyltetrazolium 溴化物</li>
	<li>凋亡检测, 如终端 deoxynucleotidyl 转移酶 (TdT) 尼克末端标记 (隧洞) 测定</li>
	<li>免疫组化分析</li>
	<li>基因表达分析</li>
	<li>西方污点分析</li>
	<li>ELISA 检测</li>
	<li>荧光活化细胞分选 (资产管制) 分析</li>
	<li>
体内异种移植模型</li>
</ol>

<ol>
	<li>Siegel, R. L., Miller, K. D., Jemal, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+statistics.">Cancer statistics.</a> CA Cancer J Clin. 66 (1), 7-30 (2016).</li><li>Linch, M., Miah, A. B., Thway, K., Judson, I. R., Benson, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systemic+treatment+of+soft-tissue+sarcoma+gold+standard+and+novel+therapies.">Systemic treatment of soft-tissue sarcoma gold standard and novel therapies.</a> Nat Rev Clin Oncol. 11 (4), 187-202 (2014).</li><li>Fletcher, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+evolving+classification+of+soft+tissue+tumours+-+an+update+based+on+the+new+2013+WHO+classification.">The evolving classification of soft tissue tumours - an update based on the new 2013 WHO classification.</a> Histopathology. 64 (1), 2-11 (2014).</li><li>De Vita, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+classification,+treatment+options,+and+new+perspectives+in+the+management+of+adipocytic+sarcomas.">Current classification, treatment options, and new perspectives in the management of adipocytic sarcomas.</a> Onco Targets Ther. 9 (12), 6233-6246 (2016).</li><li>Recine, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Update+on+the+role+of+trabectedin+in+the+treatment+of+intractable+soft+tissue+sarcomas.">Update on the role of trabectedin in the treatment of intractable soft tissue sarcomas.</a> Onco Targets Ther. 10, 1155-1164 (2017).</li><li>Ducimeti&#232;re, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Incidence+of+sarcoma+histotypes+and+molecular+subtypes+in+a+prospective+epidemiological+study+with+central+pathology+review+and+molecular+testing.">Incidence of sarcoma histotypes and molecular subtypes in a prospective epidemiological study with central pathology review and molecular testing.</a> PLoS One. 6 (8), 20294 (2011).</li><li>Nussbaum, D. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preoperative+or+postoperative+radiotherapy+versus+surgery+alone+for+retroperitoneal+sarcoma:+a+case-control,+propensity+score-matched+analysis+of+a+nationwide+clinical+oncology+database.">Preoperative or postoperative radiotherapy versus surgery alone for retroperitoneal sarcoma: a case-control, propensity score-matched analysis of a nationwide clinical oncology database.</a> Lancet Oncol. 17 (7), 966-975 (2016).</li><li>The ESMO/European Sarcoma Network Working Group. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Soft+tissue+and+visceral+sarcomas:+ESMO+clinical+practice+guidelines+for+diagnosis,+treatment+and+follow-up.">Soft tissue and visceral sarcomas: ESMO clinical practice guidelines for diagnosis, treatment and follow-up.</a> Ann Oncol. 25, 102-112 (2014).</li><li>Paul, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Cancer+Cell+in+Vitro:+A+Review.">The Cancer Cell in Vitro: A Review.</a> Cancer Research. 22, 431-440 (1962).</li><li>Morton, C. L., Houghton, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+of+human+tumor+xenografts+in+immunodeficient+mice.">Establishment of human tumor xenografts in immunodeficient mice.</a> Nat Protoc. 2 (2), 247-250 (2007).</li><li>Centenera, M. M., Raj, G. V., Knudsen, K. E., Tilley, W. D., Butler, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ex+vivo+culture+of+human+prostate+tissue+and+drug+development.">Ex vivo culture of human prostate tissue and drug development.</a> Nat Rev Urol. 10 (8), 483-487 (2013).</li><li>Vannucci, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stroma+as+an+Active+Player+in+the+Development+of+the+Tumor+Microenvironment.">Stroma as an Active Player in the Development of the Tumor Microenvironment.</a> Cancer Microenviron. 8 (3), 159-166 (2015).</li><li>De Vita, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activity+of+Eribulin+in+a+Primary+Culture+of+Well-Differentiated/Dedifferentiated+Adipocytic+Sarcoma.">Activity of Eribulin in a Primary Culture of Well-Differentiated/Dedifferentiated Adipocytic Sarcoma.</a> Molecules. 3 (12), 1662 (2016).</li><li>Failli, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+challenge+of+culturing+human+colorectal+tumor+cells:+establishment+of+a+cell+culture+model+by+the+comparison+of+different+methodological+approaches.">The challenge of culturing human colorectal tumor cells: establishment of a cell culture model by the comparison of different methodological approaches.</a> Tumori. 95 (3), 343-347 (2009).</li><li>Steinstraesser, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+of+a+primary+human+sarcoma+model+in+athymic+nude+mice.">Establishment of a primary human sarcoma model in athymic nude mice.</a> Hum. Cell. 23, 50-57 (2010).</li><li>Raouf, A., Sun, Y. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+methods+to+culture+primary+human+breast+epithelial+cells.">In vitro methods to culture primary human breast epithelial cells.</a> Methods Mol. Biol. 946, 363-381 (2013).</li><li>Daigeler, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterogeneous+in+vitro+effects+of+doxorubicin+on+gene+expression+in+primary+human+liposarcoma+cultures.">Heterogeneous in vitro effects of doxorubicin on gene expression in primary human liposarcoma cultures.</a> BMC Cancer. 29, 313 (2008).</li><li>Pan, X., Yoshida, A., Kawai, A., Kondo, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+status+of+publicly+available+sarcoma+cell+lines+for+use+in+proteomic+studies.">Current status of publicly available sarcoma cell lines for use in proteomic studies.</a> Expert Rev Proteomics. 13 (2), 227-240 (2016).</li><li>Liverani, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Innovative+approaches+to+establish+and+characterize+primary+cultures:+an+ex+vivo.+3D+system+and+the+zebrafish+model.">Innovative approaches to establish and characterize primary cultures: an ex vivo. 3D system and the zebrafish model.</a> Biol Open. 15 (2), 309 (2017).</li><li>De Vita, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myxofibrosarcoma+primary+cultures:+molecular+and+pharmacological+profile.">Myxofibrosarcoma primary cultures: molecular and pharmacological profile.</a> Ther Adv Med Oncol. 9 (12), 755-767 (2017).</li><li>Blay, J. Y., Ray-Coquard, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sarcoma+in+2016:+Evolving+biological+understanding+and+treatment+of+sarcomas.">Sarcoma in 2016: Evolving biological understanding and treatment of sarcomas.</a> Nat Rev Clin Oncol. 14 (2), 78-80 (2017).</li><li>Dairkee, S. H., Deng, G., Stampfer, M. R., Waldman, F. M., Smith, H. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+cell+culture+of+primary+breast+carcinoma.">Selective cell culture of primary breast carcinoma.</a> Cancer Res. 55 (12), 2516-2519 (1995).</li><li>Kar, R., Sharma, C., Sen, S., Jain, S. K., Gupta, S. D., Singh, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Response+of+primary+culture+of+human+ovarian+cancer+cells+to+chemotherapy:+In+vitro+individualized+therapy.">Response of primary culture of human ovarian cancer cells to chemotherapy: In vitro individualized therapy.</a> J Cancer Res Ther. 12 (2), 1050-1055 (2016).</li><li>Ravi, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proliferation+and+Tumorigenesis+of+a+Murine+Sarcoma+Cell+Line+in+the+Absence+of+DICER1.">Proliferation and Tumorigenesis of a Murine Sarcoma Cell Line in the Absence of DICER1.</a> Cancer Cell. 21 (6), 848-855 (2012).</li><li>Zhong, X. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+vegf+gene+knockdown+on+growth+of+the+murine+sarcoma+cell+line+MS-K.">Effect of vegf gene knockdown on growth of the murine sarcoma cell line MS-K.</a> Cells. 16 (6), 625-638 (2011).</li><li>Kidani, T., Yasuda, R., Miyawaki, J., Oshima, Y., Miura, H., Masuno, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bisphenol+A+Inhibits+Cell+Proliferation+and+Reduces+the+Motile+Potential+of+Murine+LM8+Osteosarcoma+Cells.">Bisphenol A Inhibits Cell Proliferation and Reduces the Motile Potential of Murine LM8 Osteosarcoma Cells.</a> Anticancer Res. 37 (4), 1711-1722 (2017).</li><li>Sugiyasu, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Radio-sensitization+of+the+murine+osteosarcoma+cell+line+LM8+with+parthenolide,+a+naturalinhibitor+of+NF-&#954;B.">Radio-sensitization of the murine osteosarcoma cell line LM8 with parthenolide, a naturalinhibitor of NF-&#954;B.</a> Oncol Lett. 2 (3), 407-412 (2011).</li><li>Daigeler, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterogeneous+in+vitro+effects+of+doxorubicin+on+gene+expression+in+primary+human+liposarcoma+cultures.">Heterogeneous in vitro effects of doxorubicin on gene expression in primary human liposarcoma cultures.</a> BMC Cancer. 29 (8), 313 (2008).</li></ol>

作者没有披露的利益冲突。

为了阐明肿瘤的分子背景, 预测预后不佳, 为癌症患者制定新的治疗策略, 需要明确的临床前模型。这对于像 sts 这样的罕见肿瘤尤其重要, 因为它的形态学、侵袭性潜能和临床行为等方面的高异质性挑战了我们对 sts 生物学和患者管理的理解21。此外, 少数商业肉瘤细胞系可限制对这组间质肿瘤的临床前调查18。因此,前体模型代表了 STS 的宝贵研究资源。我?...

软组织肉瘤 (STS) 包括一组骨髓间质来源的异质性病变, 占所有实体肿瘤的 1%,1,2, 新的世界卫生组织分类确认存在超过50不同的子类型3。其中, 最常见的 histotypes 是脂肪肉瘤或肉瘤和平滑肌肉瘤, 占所有成人 STS 的15% 和 11%, 分别为4,5。虽然 STS 可以发展在身体的不同部分, 四肢和腹腔是最常见的网站, 发生在60% 和20% 的情况下, 分别为6。
治疗局部性疾病的基石是广泛的外科手术切除, 而佐剂和新辅助化疗的好处仍然不清楚, 仅在选定的病例中考虑7。在转移设置中, 化疗是治疗的标准, 但结果显示有限的效果8。更好地了解 STS 的分子生物学将有助于改善目前的鉴别诊断, 优化现有的治疗方法, 并确定新的治疗目标。
在这种背景下, 使用永生化细胞系9进行了多年的癌症研究。这些实验模型通常在标准单层支持或植入 immunodeficient 啮齿动物中培养, 以建立体内异种移植模型10。永生化细胞系代表一种易于管理和容易储存的材料, 可以进行大量的研究实验。事实上, 它们是临床前研究的最便宜和最广泛使用的工具11。
然而, 基于细胞线的癌症模型也有一些局限性,例如在单层系统中长时间的培养以及越来越多的通道诱导表型和遗传漂移, 使细胞不太可能重述关键肿瘤特征。此外, 细胞系的培养不能重现细胞间的相互作用和信号分子串扰, 模仿肿瘤的生物学行为和特征的肿瘤微环境。这些问题构成临床前和临床结果之间存在的差距的基础12。
鉴于上述情况, 对患者派生的初级文化的兴趣增加了13。事实上, 切除恶性和正常组织降低了失去癌细胞表型和异质性的风险, 从而提供了更具代表性的肿瘤微环境的起始材料。此外, 使用从手术切除病人身上采集的新鲜肿瘤标本, 我们可以调查单个肿瘤, 并比较患者身体同一部位的不同病变14。基于上述原因, 组织衍生细胞培养物为研究肿瘤病理生理学和药理学15,16,17, 特别是在 STS 中提供了一种有价值的材料, 可用于商业上可用的肉瘤细胞系。受限18。因此, 我们通过手术切除肿瘤组织13,19,20, 优化了建立病人派生的 STS 原细胞培养的方法。我们的方法包括将肿瘤标本分列到小组织片段, 然后用隔夜酶组织离解来获得单个细胞悬浮。第二天, 通过添加新鲜培养基停止酶消化, 并过滤得到的悬浮物以去除组织碎片、细胞团聚体和过量基质物质或碎片。最后, 将一小部分分离的肿瘤细胞 cytospinned 到玻璃滑梯上, 固定存放, 用于下游分析, 目的是通过项细胞形态、免疫组化和分子细胞遗传学评价确定 STS 原代培养的建立。(例如MDM2 放大的鱼分析代表区分良好的肉瘤和去分化肉瘤肉瘤的标准鉴别诊断)4. 其余的细胞被播种成一个标准的单层培养基因表达谱和药理学研究。所有的实验都是使用低通道初级培养, 以避免选择特定的癌症 subclones, 并分析了经验丰富的肉瘤病理学家。因此, 我们创建了一个完全人类 STS前体模型13,19,20。这种高通用性、易于处理的模型可以代表不同类型的临床前研究的有用工具,例如确定新的诊断和预后分子标记, 或加深对其活动和机制的了解。标准和新药在 STS 管理中的作用。

<table border="0" cellpadding="0" cellspacing="0" style="width:599px;" width="597">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="53">
			<td height="53" style="height:53px;width:163px;">DMEM High Glucose</td>
			<td style="width:180px;">EuroClone</td>
			<td style="width:111px;">ECB7501L</td>
			<td style="width:145px;"></td>
		</tr>
		<tr height="53">
			<td height="53" style="height:53px;width:163px;">Fetal bovine serum</td>
			<td style="width:180px;">EuroClone</td>
			<td style="width:111px;">ECS0180D</td>
			<td style="width:145px;"></td>
		</tr>
		<tr height="51">
			<td height="51" style="height:51px;width:163px;">Glutamine</td>
			<td style="width:180px;">Gibco</td>
			<td style="width:111px;">25030-024</td>
			<td style="width:145px;"></td>
		</tr>
		<tr height="112">
			<td height="112" style="height:112px;width:163px;">Paraformaldehyde 4% aqueous solution, EM grade</td>
			<td style="width:180px;">Electron Microscopy Sciences</td>
			<td style="width:111px;">157-4-100</td>
			<td style="width:145px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:163px;">Penicillin streptomycin</td>
			<td style="width:180px;">Gibco</td>
			<td style="width:111px;">15140122</td>
			<td style="width:145px;"></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:163px;">Triazol reagent</td>
			<td style="width:180px;">Ambion Life-Technologies</td>
			<td style="width:111px;">15596018</td>
			<td></td>
		</tr>
		<tr height="62">
			<td height="62" style="height:62px;">Trypsin</td>
			<td>EuroClone</td>
			<td style="width:111px;">ECB3052D</td>
			<td></td>
		</tr>
	</tbody>
</table>

establishment of a primary culture of patient-derived soft tissue sarcoma

软组织肉瘤 (STS) 是一种非均质恶性肿瘤, 具有诊断、分类和管理的难度。迄今为止, 已经发现了这些罕见的实体肿瘤的50多种组织学亚型。因此, 由于其独特的多样性和低发病率, 我们对这些肿瘤生物学的理解仍然有限。患者源性培养是研究 STS 病理生理学和药理学的理想平台。因此, 我们开发了一个人类临床前模型的 STS 开始从肿瘤标本采集的病人进行手术切除。采用胶原酶消化和过滤分离的方法, 从手术标本中获得患者源性 STS 细胞培养。细胞计数, 种子, 并在标准单层培养14天左右, 然后由下游分析处理。在进行分子或药物分析之前, 通过对 cytomorphologic 特征的评估, 并在可用时免疫组化标记, 确定 STS 初级文化的建立。这个方法代表了一个有用的工具 1) 来研究这些探索性的恶性恶性肿瘤和2的自然历史, 以测试不同药物的影响, 以努力了解更多有关其行动机制。

我们设计了一种简单的方法, 以获得建立一个病人衍生软组织肉瘤的原代培养, 并在此报告一个例子, 取得的结果在一个特定的 STS histotype13。该议定书用于建立不同 STS histotypes 的初级文化, 包括分化良好的肉瘤、去分化肉瘤肉瘤、粘液肉瘤、多形肉瘤、myxofibrosarcoma、未分化多形肉瘤, 要点和硬纤维纤维瘤病。
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Watch this Scientific Journal Video about Establishment of a Primary Culture of Patient-derived Soft Tissue Sarcoma at JoVE.com

Establishment of a Primary Culture of Patient-derived Soft Tissue Sarcoma

以下协议的重点是建立一个初级文化的病人衍生软组织肉瘤 (STS)。该模型可以帮助我们更好地了解这些罕见的恶性肿瘤的分子背景和药理学特征, 并可作为进一步研究的出发点, 以改善 STS 管理。

病人衍生软组织肉瘤原代培养的建立

Soft tissue sarcomas (STS) represent a spectrum of heterogeneous malignancies with a difficult diagnosis, classification, and management. To date, more ...

establishment-primary-culture-patient-derived-soft-tissue

Research

JoVE Journal

Cancer Research

13.9K 次观看.  Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST) IRCCS. 以下协议的重点是建立一个初级文化的病人衍生软组织肉瘤 (STS)。该模型可以帮助我们更好地了解这些罕见的恶性肿瘤的分子背景和药理学特征, 并可作为进一步研究的出发点, 以改善 STS 管理。

视频: Establishment of a Primary Culture of Patient-derived Soft Tissue Sarcoma

Labeling of Breast Cancer Patient-derived Xenografts with Traceable Reporters for Tumor Growth and Metastasis Studies

The use of preclinical models to study tumor biology and response to treatment is central to cancer research. Long-established human cell lines, and many transgenic mouse models, often fail to recapitulate the key aspects of human malignancies. Thus, alternative models that better represent the heterogeneity of patients' tumors and their metastases are being developed. Patient-derived xenograft (PDX) models in which surgically resected tumor samples are engrafted into immunocompromised mice have become an attractive alternative as they can be transplanted through multiple generations,and more efficiently reflect tumor heterogeneity than xenografts derived from human cancer cell lines. A limitation to the use of PDXs is that they are difficult to transfect or transduce to introduce traceable reporters or to manipulate gene expression. The current protocol describes methods to transduce dissociated tumor cells from PDXs with high transduction efficiency, and the use of labeled PDXs for experimental models of breast cancer metastases. The protocol also demonstrates the use of labeled PDXs in experimental metastasis models to study the organ-colonization process of the metastatic cascade. Metastases to different organs can be easily visualized and quantified using bioluminescent imaging in live animals, or GFP expression during dissection and in excised organs. These methods provide a powerful tool to extend the use of multiple types of PDXs to metastasis research.

We describe a method for stable labeling of patient-derived xenografts (PDXs) with lentiviral particles expressing green-fluorescent protein and luciferase reporters. This method allows for tracking the growth of PDXs at the primary site, as well as detecting spontaneous and experimental metastases using in vivo imaging systems.

可追踪记者乳腺癌患者来源的移植瘤贴标肿瘤生长和转移的研究

The use of preclinical models to study tumor biology and response to treatment is central to cancer research. Long-established human cell lines, and many ...

科研

癌症研究

Isolation and Characterization of Tumor-initiating Cells from Sarcoma Patient-derived Xenografts

The existence and importance of tumor-initiating cells (TICs) have been supported by increasing evidence during the past decade. These TICs have been shown to be responsible for tumor initiation, metastasis, and drug resistance. Therefore, it is important to develop specific TIC-targeting therapy in addition to current chemotherapy strategies, which mostly focus on the bulk of non-TICs. In order to further understand the mechanism behind the malignancy of TICs, we describe a method to isolate and to characterize TICs in human sarcomas. Herein, we show a detailed protocol to generate patient-derived xenografts (PDXs) of human sarcomas and to isolate TICs by fluorescence-activated cell sorting (FACS) using human leukocyte antigen class I (HLA-1) as a negative marker. Also, we describe how to functionally characterize these TICs, including a sphere formation assay and a tumor formation assay, and to induce differentiation along mesenchymal pathways. The isolation and characterization of PDX TICs provide clues for the discovery of potential targeting therapy reagents. Moreover, increasing evidence suggests that this protocol may be further extended to isolate and characterize TICs from other types of human cancers.

We describe a detailed protocol for the isolation of tumor-initiating cells from human sarcoma patient-derived xenografts by fluorescence-activated cell sorting, using human leukocyte antigen-1 (HLA-1) as a negative marker, and for the further validation and characterization of these HLA-1-negative tumor-initiating cells.

来自沙柯玛患者衍生异种移植物的肿瘤启动细胞的分离和表征

The existence and importance of tumor-initiating cells (TICs) have been supported by increasing evidence during the past decade. These TICs have been ...

Utilization of Ultrasound Guided Tissue-directed Cellular Implantation for the Establishment of Biologically Relevant Metastatic Tumor Xenografts

Preclinical testing of anticancer therapies relies on relevant xenograft models that mimic the innate tendencies of cancer. Advantages of standard subcutaneous flank models include procedural ease and the ability to monitor tumor progression and response without invasive imaging. Such models are often inconsistent in translational clinical trials and have limited biologically relevant characteristics with low proclivity to produce metastasis, as there is a lack of a native microenvironment. In comparison, orthotopic xenograft models at native tumor sites have been shown to mimic the tumor microenvironment and replicate important disease characteristics such as distant metastatic spread. These models often require tedious surgical procedures with prolonged anesthetic time and recovery periods. To address this, cancer researchers have recently utilized ultrasound-guided injection techniques to establish cancer xenograft models for preclinical experiments, which allows for rapid and reliable establishment of tissue-directed murine models. Ultrasound visualization also provides a noninvasive method for longitudinal assessment of tumor engraftment and growth. Here, we describe the method for ultrasound-guided injection of cancer cells, utilizing the adrenal gland for NB and renal sub capsule for ES. This minimally invasive approach overcomes tedious open surgery implantation of cancer cells in tissue-specific locations for growth and metastasis, and abates morbid recovery periods. We describe the utilization of both established cell lines and patient derived cell lines for orthotopic injection. Pre-made commercial kits are available for tumor dissociation and luciferase tagging of cells. Injection of cell suspension using image-guidance provides a minimally invasive and reproducible platform for the creation of preclinical models. This method is utilized to create reliable preclinical models for other cancers such as bladder, liver and pancreas exemplifying its untapped potential for numerous cancer models.

Here, we present a protocol to utilize ultrasound-guided injection of neuroblastoma (NB) and Ewing's sarcoma (ES) cells (established cell lines and patient-derived tumor cells) at biologically relevant sites to create reliable preclinical models for cancer research.

超声引导组织定向细胞植入术在生物学相关转移性肿瘤移植中的应用

Preclinical testing of anticancer therapies relies on relevant xenograft models that mimic the innate tendencies of cancer. Advantages of standard ...

A Melanoma Patient-Derived Xenograft Model

Accumulating evidence suggests that molecular and biological properties differ in melanoma cells grown in traditional two-dimensional tissue culture vessels versus in vivo in human patients. This is due to the bottleneck selection of clonal populations of melanoma cells that can robustly grow in vitro in the absence of physiological conditions. Further, responses to therapy in two-dimensional tissue cultures overall do not faithfully reflect responses to therapy in melanoma patients, with the majority of clinical trials failing to show the efficacy of therapeutic combinations shown to be effective in vitro. Although xenografting of melanoma cells into mice provides the physiological in vivo context absent from two-dimensional tissue culture assays, the melanoma cells used for engraftment have already undergone bottleneck selection for cells that could grow under two-dimensional conditions when the cell line was established. The irreversible alterations that occur as a consequence of the bottleneck include changes in growth and invasion properties, as well as the loss of specific subpopulations. Therefore, models that better recapitulate the human condition in vivo may better predict therapeutic strategies that effectively increase the overall survival of patients with metastatic melanoma. The patient-derived xenograft (PDX) technique involves the direct implantation of tumor cells from the human patient to a mouse recipient. In this manner, tumor cells are consistently grown under physiological stresses in vivo and never undergo the two-dimensional bottleneck, which preserves the molecular and biological properties present when the tumor was in the human patient. Notable, PDX models derived from organ sites of metastases (i.e., brain) display similar metastatic capacity, while PDX models derived from therapy naive patients and patients with acquired resistance to therapy (i.e., BRAF/MEK inhibitor therapy) display similar sensitivity to therapy.

Patient-derived xenograft (PDX) models more robustly recapitulate melanoma molecular and biological features and are more predictive of therapy response compared to traditional plastic tissue culture-based assays. Here we describe our standard operating protocol for the establishment of new PDX models and the characterization/experimentation of existing PDX models.

黑色素瘤患者衍生异种移植模型

Accumulating evidence suggests that molecular and biological properties differ in melanoma cells grown in traditional two-dimensional tissue culture ...

Establishment of Gastric Cancer Patient-derived Xenograft Models and Primary Cell Lines

The use of preclinical models to advance our understanding of tumor biology and investigate the efficacy of therapeutic agents is key to cancer research. Although there are many established gastric cancer cell lines and many conventional transgenic mouse models for preclinical research, the disadvantages of these in vitro and in vivo models limit their applications. Because the characteristics of these models have changed in culture, they no longer model tumor heterogeneity, and their responses have not been able to predict responses in humans. Thus, alternative models that better represent tumor heterogeneity are being developed. Patient-derived xenograft (PDX) models preserve the histologic appearance of cancer cells, retain intratumoral heterogeneity, and better reflect the relevant human components of the tumor microenvironment. However, it usually takes 4-8 months to develop a PDX model, which is longer than the expected survival of many gastric patients. For this reason, establishing primary cancer cell lines may be an effective complementary method for drug response studies. The current protocol describes methods to establish PDX models and primary cancer cell lines from surgical gastric cancer samples. These methods provide a useful tool for drug development and cancer biology research.

The current protocol describes methods to establish patient-derived xenograft (PDX) models and primary cancer cell lines from surgical gastric cancer samples. The methods provide a useful tool for drug development and cancer biology research.

建立胃癌患者衍生异种移植模型和原细胞系

The use of preclinical models to advance our understanding of tumor biology and investigate the efficacy of therapeutic agents is key to cancer research. ...

Drug Screening of Primary Patient Derived Tumor Xenografts in Zebrafish

Patient derived xenograft models are critical in defining how different cancers respond to drug treatment in an in vivo system. Mouse models are the standard in the field, but zebrafish have emerged as an alternative model with several advantages, including the ability for high-throughput and low-cost drug screening. Zebrafish also allow for in vivo drug screening with large replicate numbers that were previously only obtainable with in vitro systems. The ability to rapidly perform large scale drug screens may open up the possibility for personalized medicine with rapid translation of results back to clinic. Zebrafish xenograft models could also be used to rapidly screen for actionable mutations based on tumor response to targeted therapies or to identify new anti-cancer compounds from large libraries. The current major limitation in the field has been quantifying and automating the process so that drug screens can be done on a larger scale and be less labor-intensive. We have developed a workflow for xenografting primary patient samples into zebrafish larvae and performing large scale drug screens using a fluorescence microscope equipped imaging unit and automated sampler unit. This method allows for standardization and quantification of engrafted tumor area and response to drug treatment across large numbers of zebrafish larvae. Overall, this method is advantageous over traditional cell culture drug screening as it allows for growth of tumor cells in an in vivo environment throughout drug treatment, and is more practical and cost-effective than mice for large scale in vivo drug screens.

Zebrafish xenograft models allow for high-throughput drug screening and fluorescent imaging of human cancer cells in an in vivo microenvironment. We developed a workflow for large scale, automated drug screening on patient-derived leukemia samples in zebrafish using an automated fluorescence microscope equipped imaging unit.

斑马鱼原发性患者衍生肿瘤异种移植物的药物筛选

Patient derived xenograft models are critical in defining how different cancers respond to drug treatment in an in vivo system. Mouse models are the ...

The Establishment and Utilization of Patient Derived Xenograft Models of Central Nervous System Metastasis

The development of novel therapies for central nervous system (CNS) metastasis has been hindered by the lack of preclinical models that accurately represent the disease. Patient derived xenograft (PDX) models of CNS metastasis have been shown to better represent the phenotypic and molecular characteristics of the human disease, as well as better reflect the heterogeneity and clonal dynamics of human patient tumors compared to historic cell line models. There are multiple sites that can be used to implant patient-derived tissue when setting up preclinical trials, each with their own advantages and disadvantages, and each suited for studying different aspects of the metastatic cascade. Here, the protocol describes how to establish PDX models and present three different approaches for utilizing CNS metastasis PDX models in pre-clinical studies, discussing each of their applications and limitations. These include flank implantation, orthotopic injection in the brain, and intracardiac injection. Subcutaneous flank implantation is the easiest to monitor and, therefore, most convenient for pre-clinical studies. In addition, metastases to brain and other tissues from flank implantation were observed, indicating that the tumor has undergone multiple steps of metastasis, including intravasation, extravasation, and colonization. Orthotopic injection in the brain is the best option for recapitulating the brain tumor microenvironment and is useful for determining the efficacy of biologics to cross the blood-brain barrier (BBB) but bypasses most steps of the metastatic cascade. Intracardiac injection facilitates metastasis to the brain and is also useful for studying organ tropism. While this method forgoes earlier steps of the metastatic cascade, these cells will still have to survive circulation, extravasate, and colonize. The utility of a PDX model, is therefore, impacted by the route of tumor inoculation and the choice of which one to utilize should be dictated by the scientific question and overall goals of the experiment.

Central nervous system metastasis PDX models represent the phenotypic and molecular characteristics of human metastasis, making them excellent models for preclinical studies. Described here is how to establish PDX models and the inoculation routes that are best used for preclinical studies.

患者衍生的中枢神经系统转移异种移植模型的建立与利用

The development of novel therapies for central nervous system (CNS) metastasis has been hindered by the lack of preclinical models that accurately ...

Generation and Culturing of High-Grade Serous Ovarian Cancer Patient-Derived Organoids

Organoids are 3D dynamic tumor models that can be grown successfully from patient-derived ovarian tumor tissue, ascites, or pleural fluid and aid in the discovery of novel therapeutics and predictive biomarkers for ovarian cancer. These models recapitulate clonal heterogeneity, the tumor microenvironment, and cell-cell and cell-matrix interactions. Additionally, they have been shown to match the primary tumor morphologically, cytologically, immunohistochemically, and genetically. Thus, organoids facilitate research on tumor cells and the tumor microenvironment and are superior to cell lines. The present protocol describes distinct methods to generate patient-derived ovarian cancer organoids from patient tumors, ascites, and pleural fluid samples with a higher than 97% success rate. The patient samples are separated into cellular suspensions by both mechanical and enzymatic digestion. The cells are then plated utilizing a basement membrane extract (BME) and are supported with optimized growth media containing supplements specific to the culturing of high-grade serous ovarian cancer (HGSOC). After forming initial organoids, the PDOs can sustain long-term culture, including passaging for expansion for subsequent experiments.

Patient-derived organoids (PDO) are a three-dimensional (3D) culture that can mimic the tumor environment in vitro. In high-grade serous ovarian cancer, PDOs represent a model to study novel biomarkers and therapeutics.

高级别浆液性卵巢癌患者来源类器官的生成和培养

Organoids are 3D dynamic tumor models that can be grown successfully from patient-derived ovarian tumor tissue, ascites, or pleural fluid and aid in the ...

Establishment and Culture of Patient-Derived Breast Organoids

Breast cancer is a complex disease that has been classified into several different histological and molecular subtypes. Patient-derived breast tumor organoids developed in our laboratory consist of a mix of multiple tumor-derived cell populations, and thus represent a better approximation of tumor cell diversity and milieu than the established 2D cancer cell lines. Organoids serve as an ideal in vitro model, allowing for cell-extracellular matrix interactions, known to play an important role in cell-cell interactions and cancer progression. Patient-derived organoids also have advantages over mouse models as they are of human origin. Furthermore, they have been shown to recapitulate the genomic, transcriptomic as well as metabolic heterogeneity of patient tumors; thus, they are capable of representing tumor complexity as well as patient diversity. As a result, they are poised to provide more accurate insights into target discovery and validation and drug sensitivity assays. In this protocol, we provide a detailed demonstration of how patient-derived breast organoids are established from resected breast tumors (cancer organoids) or reductive mammoplasty-derived breast tissue (normal organoids). This is followed by a comprehensive account of 3D organoid culture, expansion, passaging, freezing, as well as thawing of patient-derived breast organoid cultures.

A detailed protocol is provided here for establishing human breast organoids from patient-derived breast tumor resections or normal breast tissue. The protocol provides comprehensive step-by-step instructions for culturing, freezing, and thawing human patient-derived breast organoids.

患者来源乳腺类器官的建立和培养

Breast cancer is a complex disease that has been classified into several different histological and molecular subtypes. Patient-derived breast tumor ...

Establishment of Zebrafish Patient-Derived Xenografts from Pancreatic Cancer for Chemosensitivity Testing

Cancer is one of the main causes of death worldwide, and the incidence of many types of cancer continues to increase. Much progress has been made in terms of screening, prevention, and treatment; however, preclinical models that predict the chemosensitivity profile of cancer patients are still lacking. To fill this gap, an in vivo patient-derived xenograft model was developed and validated. The model was based on zebrafish (Danio rerio) embryos at 2 days post fertilization, which were used as recipients of xenograft fragments of tumor tissue taken from a patient's surgical specimen. 
It is also worth noting that bioptic samples were not digested or disaggregated, in order to maintain the tumor microenvironment, which is crucial in terms of analyzing tumor behavior and the response to therapy. The protocol details a method for establishing zebrafish-based patient-derived xenografts (zPDXs) from primary solid tumor surgical resection. After screening by an anatomopathologist, the specimen is dissected using a scalpel blade. Necrotic tissue, vessels, or fatty tissue are removed and then chopped into 0.3 mm x 0.3 mm x 0.3 mm pieces. 
The pieces are then fluorescently labeled and xenotransplanted into the perivitelline space of zebrafish embryos. A large number of embryos can be processed at a low cost, enabling high-throughput in vivo analyses of the chemosensitivity of zPDXs to multiple anticancer drugs. Confocal images are routinely acquired to detect and quantify the apoptotic levels induced by chemotherapy treatment compared to the control group. The xenograft procedure has a significant time advantage, since it can be completed in a single day, providing a reasonable time window to carry out a therapeutic screening for co-clinical trials.

Preclinical models aim to advance the knowledge of cancer biology and predict treatment efficacy. This paper describes the generation of zebrafish-based patient-derived xenografts (zPDXs) with tumor tissue fragments. The zPDXs were treated with chemotherapy, the therapeutic effect of which was assessed in terms of cell apoptosis of the transplanted tissue.

建立胰腺癌斑马鱼患者来源的异种移植物进行化学敏感性测试

Cancer is one of the main causes of death worldwide, and the incidence of many types of cancer continues to increase. Much progress has been made in terms ...