CN103108547B - 用于分离具有干细胞性质的化学治疗剂抗性癌细胞的方法 - Google Patents

用于分离具有干细胞性质的化学治疗剂抗性癌细胞的方法 Download PDF

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CN103108547B
CN103108547B CN201180031067.6A CN201180031067A CN103108547B CN 103108547 B CN103108547 B CN 103108547B CN 201180031067 A CN201180031067 A CN 201180031067A CN 103108547 B CN103108547 B CN 103108547B
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L.加斯达
B.史密斯
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Abstract

本发明涉及癌细胞在琼脂糖包被的、含琼脂糖珠中的封装的应用，用于分离具有至少一种干细胞特性例如OCT4的表达的化学治疗剂抗性细胞。因此分离的细胞，如同用于筛选潜在的疗法的方法一样，也是本发明的特征。

Description

用于分离具有干细胞性质的化学治疗剂抗性癌细胞的方法
相关申请
本申请要求临时专利申请61/458,390和61/458,391的优先权，两者都于2010年11月23日提交，且整体引入作为参考。
发明领域
本发明涉及用于分离癌症干细胞的方法，和因此分离的癌症干细胞。它还涉及用于筛选目的化合物以测定它们是否具有作为抗肿瘤剂的潜在功效的方法。
背景和现有技术
在化学治疗和缓解后来自癌症的大多数死亡起因于一种或多种原始治疗肿瘤的复发。这看起来是略微违反直觉的，因为已知的癌症治疗有时但不一定消除肿瘤至其中可以宣布患者“治愈”的点。
已进展为解释肿瘤复发以及肿瘤生长本身的理论是“癌症干细胞”或“CSC”理论。简言之，这个理论断定具有干细胞的一些特征的癌细胞罕见群体经历不对称分裂，其又导致替换干细胞，和肿瘤扩增细胞的更加谱系限制群体。这些“新”细胞快速增殖，并且构成大多数肿瘤，与缓慢循环、休眠且对靶向快速分裂细胞的疗法有抗性的干细胞形成对比。其结果是虽然肿瘤中的大多数细胞对这些靶向治疗中的一种或多种敏感，但干细胞样、化学抗性癌细胞的小群体未被破坏，并且在上文讨论的循环重复其自身。
明确的是，存在鉴定这些干细胞样癌细胞的需要，以及定量其在特定受试者或患者的肿瘤中的存在的需要。此外，虽然肿瘤学领域认识到用于癌症的许多化学疗法“一种方法不能解决所有问题（one size does not fit all）”，并且仍存在用于开发靶向治疗、以及用于鉴定潜在有用的抗肿瘤药物的更一般方法的需要。
全都整体引入作为参考的美国专利号5,888,497；6,303,151；6,808,705；6,818,230；7,041,504；和7,297,331，描述用于将癌细胞封装到琼脂糖珠中的方法，所述琼脂糖珠依次又由琼脂糖包被。按照例如也整体引入作为参考的公开的申请2007/0071732，琼脂糖类型可以改变，如其他细胞类型（胰岛）的背景中所示。
关于这些癌细胞封装的工作已导致细胞群体发展的观察，所述细胞群体可能具有并行干细胞的性质。因此有利的是测定这些参考文献中所述的材料是否可以用于分离化学疗法抗性的癌细胞，其还具有干细胞样性质。在如此进行中，还学习到这些封装可以用于筛选目的化合物，以测定化合物是否针对癌症有效。
本发明的这些和其他方面如何达到将在下述公开内容中可见。
优选实施方案的详述
实施例1
琼脂糖包被的、含RENCA细胞的琼脂糖珠依照Smith等人，Cane Res.，71（3）：716-724（2011）；和Smith等人，Cane. Res. 71（3）：725-735（2011）进行制备，所述参考文献的公开内容引入作为参考。所得到的珠在培养基中培养12周。
随后使珠的样品暴露于以三个可变浓度之一的单一的已知抗癌药物。暴露涉及基于药物已知的半衰期，使珠在药物的存在下温育一段时间。
在温育后，将珠洗涤两次，并且转移至首次用于实验的培养基。还制备包括未处理的珠的对照，以及暴露于用于稳定药物的无论何种载体的珠。
珠的组织学检查在暴露于药物后经历一周。在一些情况下，不存在变化，而在其他情况下，药物导致细胞生存的完全丧失，并且在其他情况下，并且存在中间效应。结果如下：
实施例2
用多西他赛和紫杉醇处理不破坏封装的肿瘤集落内的所有细胞，并且因此选择已用这些药物处理的珠用于进一步研究。用以低浓度的卡铂（Caboplatin）或以中等浓度的长春瑞滨处理的珠可以同样使用。
紫杉醇和多西他赛处理的珠在标准条件下培养，如已暴露于DMSO的对照珠一样，所述DMSO是用于递送两种药物的载体。培养期是18周。
在DMSO处理的珠中封装的细胞显示出正常形态，其是椭圆形肿瘤集落，由围绕内部碎片的1-2个细胞厚的细胞边缘组成。相比之下，已用3.5 μg/ml紫杉醇处理的珠显示共6周的细胞丧失，到第18周回复处理前的细胞数目。在处理后6周时，用多西他赛（5 μg/ml）处理的珠显示仅1-2个细胞/集落的一致模式。在第18周时，约10%的珠发展1或2个大集落。
为了定量细胞丧失，使用标准方法，将来自DMSO对照、紫杉醇和多西他赛组的代表珠切开且染色，以便允许细胞核的计数。结果针对DMSO处理的珠标准化。
在用紫杉醇处理的情况下，存在经过第1-3周约25%的细胞/集落的最初丧失。大多数集落的确含有活细胞。细胞数目在18周后已增加至等价于对照DMSO珠。在多西他赛处理的珠的情况下，它们非常快速地丧失活细胞，从而使得在处理后6周仅1-2个活细胞存在于集落中。在第18周时，约10%的珠已发展1或2个大细胞集落，从而指示罕见的、多西他赛抗性RENCA细胞可以在珠内形成新集落。
实施例3
近期证据暗示OCT4（“八聚体结合转录因子4”），关于胚胎干细胞的标记，可以与其他转录因子结合使用，以诱导成年细胞至多能干细胞样。参见例如，Park等人，Nature 451（7475）：141-146（2008）。干细胞特有的另外标记可以在例如引入本文作为参考的International Stem Cell Initiative，等人，Nat. Biotechnol 25（7）:803-16（2007）中找到。
执行实验，以测定在处理后六周在多西他赛处理的细胞中保留的细胞是否显示出这种标记。
将细胞用DAPI染色，以鉴定活的细胞，同时标准免疫化学程序用于染色OCT4，使用针对OCT4的兔多克隆抗体和用Fluor 488缀合物标记的山羊抗IgG抗体。
结果指出活的细胞表达OCT4。与对于多西他赛的抗性一起获得的表达暗示剩余细胞是癌症干细胞。
实施例4
癌症干细胞的必要条件是形成癌细胞集落的能力。为了测定上文描述的细胞是否可以做到这点，从珠中取出集落，并且在去除后5-6周经由机械破碎收集存活细胞，并且将集落用镊子在RPMI 1640加上10%新生小牛血清中切碎。随后将悬液通过40 μm细胞滤网，以便使任何琼脂糖碎片降到最低，并且经由离心形成团块。将细胞团块重悬浮于首次用于实验的培养基中，并且在体外培养（200细胞/ml，在补充有10%新生小牛血清的RPMI 1640中），或在体内培养。为了精炼，将200个细胞与来自接受者小鼠的血滴混合，从而形成凝块，随后将所述凝块植入从其中获得血液的小鼠的肾囊下。随后就肿瘤的生长观察小鼠。在纯化的干细胞的体内移植后肿瘤的发展视为领域公认的用于鉴定癌症干细胞的“黄金标准”。
在体外生长的RENCA细胞比以单层生长的正常RENCA细胞大得多，并且未观察到它们经历细胞生长共约16周。在培养后16-17周，细胞形成斑块且在每周传代后开始增殖，并且在培养中作为单层生长。但在单层生长起始后两周，这些细胞具有与正常RENCA细胞相当的生长率，并且作为正常RENCA单层细胞出现，具有等价大小和形态。
在接受植入物的十只小鼠中，一只发展在囊下的肿瘤，并且在诱导后98天死亡。它也已发展肺转移。
结果指出这些细胞因此可以视为癌症干细胞。
前述实施例阐述本发明的多个方面，其包括用于分离癌细胞的方法，所述癌细胞对于化学治疗剂例如多西他赛是有抗性的，并且具有干细胞的一种或多种性质例如OCT4的表达。干细胞的其他性质是本领域众所周知的并且在此处不重复。该方法涉及将癌细胞的样品封装在含有琼脂糖的珠中，所述珠随后用琼脂糖包被，培养所得到的珠以生长其中含有的癌细胞，使珠与化学治疗剂接触，并且测定所述剩余细胞中的哪个原位或通过由其去除而表达OCT4。
这种方法可以用于例如开发用于患有癌症的受试者的预后，因为如上所述，本文描述类型的细胞负责受试者中的癌症复发。基本上，高百分比的所述细胞指示对于患者比在封装的样品中显示出少数此类细胞或无此类细胞的患者的情况更差的预后。
任何化学治疗剂都可以用于本发明的方法中，如任何类型的癌症一样。技术人员将知道用于癌症治疗的许多其他已知的治疗剂。类似地，由例如上文引用的参考文献认识到，除本文描述的那些外，还可以使用多种癌症和琼脂糖。此外，本发明的珠可以包括材料例如胶原，或与琼脂糖相容的其他材料。癌细胞优选是哺乳动物细胞，并且最优选地，人细胞。
然而，本文报道的分离的细胞对已知化学治疗剂有抗性的事实并不意味着它们“本身”是化学抗性的。如所示的，本文测试的试剂是在快速增殖细胞的破坏中一般有用的药物。存在可用于由琼脂糖包被的、琼脂糖珠中剩余的细胞代表类型的细胞的其他治疗，其将是技术人员已知的。虽然在本文中未报道，但此类药物可以针对这些干细胞样、化学治疗剂抗性的OCT4表达细胞进行测试，并且可以开发治疗方案，其中例如受试者接受标准抗癌剂作为第一个治疗过程，随后为针对本文分离且描述的细胞类型的试剂。此类细胞是分离的癌细胞，其对化学治疗剂例如多西他赛是有抗性的，并且表达OCT4。在本发明的这个方面，目的物质可以针对在与第一种试剂接触后剩余的干细胞样癌细胞进行测试，或可以封装干细胞样癌细胞的分开样品且以相同方式进行。
技术人员还将注意到本发明涉及用于测定目的化合物是否具有作为抗癌剂的功效的方法。如可见的，该方法涉及使目的化合物与琼脂糖包被的琼脂糖珠以所选时间段和所选浓度接触，所述琼脂糖珠含有癌细胞的样品，并且测定所述化合物是否破坏比对照更大百分比的细胞。在此类情况下，化合物可以视为治疗上有用的，以及在非治疗背景中有用的，例如在破坏混合细胞群体中的癌细胞中，或在消除来自细胞混合物的非干细胞癌细胞中使用。还考虑的是超过一种潜在有用的治疗剂的“混合物（cocktails）”，或已知治疗剂与测试化合物的组合，以测定组合治疗或在非治疗背景中的应用是否是合适的。
本发明的其他特征对于技术人员将是明确的并且无需在此处重复。
已采用的术语和表述用作描述性而不是限制性的术语，并且在此类术语和表述的使用中不预期排除所示且描述的特征或其部分的任何等价物，认识到多种修饰在本发明的范围内是可能的。

Claims (11)

1.一种用于分离具有干细胞性质的化学治疗剂抗性癌细胞的方法，其包括：
（i）将癌细胞的样品封装在琼脂糖包被的、含有琼脂糖的珠中；
（ii）使所述珠与至少一种抗癌化学治疗剂接触足以杀死所述癌细胞的至少一部分的时间；
（iii）从所述珠中取出任何存活细胞；
（iv）就至少一种干细胞性质测定所述存活细胞，和
（v）使显示出所述干细胞性质的任何细胞与并非如此的任何细胞分离。
2.权利要求1的方法，其中所述干细胞性质是OCT4的表达。
3.权利要求1的方法，其中所述化学治疗剂是多西他赛。
4.权利要求1的方法，其中所述癌细胞是哺乳动物细胞。
5.权利要求4的方法，其中所述哺乳动物细胞是人细胞。
6.一种用于测定目的物质是否具有抗癌功效的方法，其包括（i）使所述物质与在含有琼脂糖的、琼脂糖包被的珠中封装的癌细胞样品以预定浓度和预定时间段接触，（ii）测定由所述物质杀死的所述癌细胞的百分比，和（iii）比较所述百分比与由对照试剂杀死的在含有琼脂糖的、琼脂糖包被的珠中封装的所述癌细胞的百分比，其中由所述物质杀死的更高百分比指示对于所述物质的抗癌功效。
7.权利要求6的方法，其中所述癌细胞是哺乳动物癌细胞。
8.权利要求7的方法，其中所述哺乳动物癌细胞是人癌细胞。
9.一种用于测定目的物质是否具有抗癌干细胞活性的方法，其包括使在含有琼脂糖的、琼脂糖包被的珠中封装的癌症干细胞与所述物质接触，并且比较剩余细胞的数目与未接触所述物质的含有癌症干细胞的珠，其中在两者之间的差异指示所述物质具有抗癌干细胞活性。
10.含有琼脂糖的、琼脂糖包被的珠在制备用于测定患有癌症的受试者的预后的药物中的用途，所述测定包括：
(i) 将从所述受试者取得的癌细胞的样品封装在含有琼脂糖的、琼脂糖包被的珠中；
(ii) 使所述含有琼脂糖的、琼脂糖包被的珠与已知针对所述癌细胞有效的抗癌剂接触，所述抗癌剂与所述珠在已知有效的浓度下接触已知有效的时间段；
(iii) 在(ii)所述的时间段之后，测定在所述珠中剩余的癌细胞的数目；和
(iv) 其中与预后好的患者相比高数目的所述癌细胞指示对于所述受试者的差的预后。
11.权利要求10的用途，其中在(ii)所述的时间段之后在所述珠中没有细胞剩余指示对于所述受试者积极的预后。
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