Acellular scaffolds for tumor tissue engineering

doi:10.3969/j.issn.2095-4344.0973

Abstract

Abstract:

BACKGROUND: The development of acellular technology provides a new platform and approach for tumor tissue engineering, which can be used to fully simulate an in vivo microenvironment for tumor growth.

OBJECTIVE: To review the application of acellular scaffolds in tumor tissue engineering.

METHODS: PubMed database was retrieved for relevant articles published from 1995 to 2017, with the keywords of “tumor engineering; biomaterials; scaffold; 3D culture; decellularized; tumor microenvironment; ECM”.

RESULTS AND CONCLUSION: Extracellular matrix prepared via decellularization is an ideal platform for cell culture in the terms of physiological and biochemical properties and spatial structure. It contains not only the basic components of the extracellular matrix-proteins and polysaccharides, but also specific growth factors and cytokines. In addition, a complete and specific vascular structure is also retained, which can simulate in vivo nutrient exchange via bioreactors perfusion devices. This platform for research on tumors is very close to the actual environment in the body. In recent years, with the development of tissue engineering, this three-dimensional cultivation method is becoming a hot issue in the field of tumor biology. Cell-cell and cell-extracellular matrix interactions are established in vitro, to simulate the actual tumor microenvironment, and rebuild tumor models in vitro. With the wide application and improvement of acellular technology, it will become an effective tool for the study on tumor biological behavior, drug screening and targeted therapy.

中国组织工程研究杂志出版内容重点：生物材料；骨生物材料; 口腔生物材料; 纳米材料; 缓释材料; 材料相容性；组织工程

Key words: Stents, Tumor Microenvironment, Tissue Engineering

CLC Number:

R318

Ji Zhongjiao, Guo Yibing, Lu Yuhua. Acellular scaffolds for tumor tissue engineering[J]. Chinese Journal of Tissue Engineering Research, 2018, 22(34): 5571-5576.

Figures/Tables 1

2.1 脱细胞支架在乳腺癌研究中的应用细胞外基质被认为是乳腺癌进展过程中的重要调节因子，其对乳腺癌细胞的生长、运动有很强的影响，这与细胞外基质的空间拓扑结构、刚性等物理学性质相关，研究发现通过二维培养的MDA-MB-231乳腺癌细胞表现为单细胞纺锤形形态[12]，而通过三维支架灌注培养的MDA-MB-231乳腺癌细胞则能够形成肿瘤结节[13]。在人脂肪组织衍生的脱细胞支架中培养的乳腺癌MCF-7和BT-474细胞能够形成占据纳米纤维多孔支架的球形细胞聚集体[14]，表明细胞培养在这种高度多孔的三维脱细胞支架上可能有助于形成类似肿瘤组织。除了对细胞形态的影响，细胞外基质中的蛋白可直接调节乳腺癌进展期间的细胞黏附、迁移、侵袭和定植[15]。Xiong等[16]用小鼠肺脱细胞支架种植MDA-MB-231、4T-1和MCF-7乳腺癌细胞，发现转移性MDA-MB-231和4T-1细胞能侵入和定植在肺脱细胞支架中，而非转移性MCF-7细胞在肺脱细胞支架中几乎不能存活，表明肺细胞外基质含有诱导MCF-7细胞凋亡的成分，抑制非转移性乳腺癌细胞的定植，这与体内情况一致。Lü等[17]的研究发现MCF-7在来自A-549人肺腺癌细胞构建的异种肺癌移植模型的脱细胞支架上可形成类似实体瘤的细胞簇。这表明了不同组织来源的细胞外基质其组成以及结构影响着细胞的定植。肿瘤的发生和发展与血管新生密切相关，肿瘤血管生成受到相关血管生成因子的调控，培养在脱细胞支架上的MCF-7细胞的白细胞介素8、碱性成纤维细胞生长因子和血管内皮生长因子的分泌上调[17]，说明细胞中这些因子的表达是受细胞-细胞外基质相互作用以及三维肿瘤细胞培养条件调控的。利用脱细胞产生的支架用作三维培养模型有助于指导乳腺上皮细胞的发育和乳腺癌的发展[18]。上皮-间质转化是一个与乳腺癌的进展和转移有关的发育过程，研究发现敲除Zeb-1基因显着减少了在肺脱细胞支架上培养的乳腺癌细胞的上皮细胞-间充质转化过程[16]。使用脱细胞和蛋白质组学技术，人们可以适当地模拟转移前的生态位，Aguado等[19]利用健康小鼠和接种过乳腺癌细胞小鼠的肺以及肝脏制成脱细胞支架涂层来种植癌细胞，发现相较于健康小鼠的肺和肝脏脱细胞支架涂层，LM2-4或4T-1细胞接种的肺和肝脏的细胞外基质涂层更有利于细胞的定植。此外，由于在三维基质中生长的癌细胞具有不同的黏附，生长，迁移和侵袭表型以及基因和蛋白质谱表达，在二维和三维培养条件下，癌细胞呈递其受体和对抗癌药物作出反应的方式是不同的，乳腺癌细胞在三维培养中表现出比二维培养更高的耐药性，与二维培养的细胞相比，在脱细胞支架中培养的MCF-7和BT-474乳腺癌细胞对多柔比星的耐药性增加，SK-BR-3和BT-474细胞表现出对拉帕替尼更高的更敏感[14]。这些结果表明脱细胞支架可以提供生理或病理相关的微环境来支持乳腺癌细胞侵袭和定植，为研究乳腺癌发生发展的机制提供可靠的体外模型，见表1。 2.2 脱细胞支架在肝癌方面的应用早在1986年Fujita 等[20]通过分析细胞外基质组成，发现其对正常肝细胞以及肝脏肿瘤细胞的生长和分化都具有重要的影响。细胞外基质生物支架为肝细胞植入和功能表达提供了一个更加生理的平台[21-22]。2004年Lin等[25]第一次报道了肝脏组织脱细胞化的方法，为多种类型肝细胞的体外生长提供了更好的模型。Takeda等[23]将肝脏脱细胞后利用酶消化的方法进一步加工以形成水凝胶，可以通过改变细胞外基质的浓度来控制支架孔隙度。此外，2015年Mazza等[24]制作了人肝脏脱细胞，之后该研究组又通过高重力振荡/高应力剪切手段改进了脱细胞方法[25-26]，获得了更为理想的小型脱细胞支架。虽然不同的肝脏脱细胞支架存在结构及组分的差异，但支架都保留了使肝癌细胞黏附、生长所必需的基本特征。转移是恶性肿瘤的基本特征，基质金属蛋白酶9是侵袭性肿瘤的重要标志物，此外过往研究表明局部黏着斑激酶、人封闭蛋白6和E-钙黏蛋白1等黏附蛋白参与了转移过程，相比二维单层培养相比，在三维脱细胞支架培养的HepG-2细胞中局部黏着斑激酶、人封闭蛋白6、E-钙黏蛋白1和基质金属蛋白酶9的表达明显增加[27]，说明三维培养体系增强了肝癌细胞的侵袭和转移。目前，肿瘤细胞的多药耐药性问题是肿瘤化疗的主要障碍之一，人肿瘤细胞的三维培养已经被广泛地用作替代模型来评价不同的抗癌药物的功效，有报道证明肝脱细胞支架内培养的HepG-2细胞比二维培养时对甲氨蝶呤拥有更强的耐药性[37]，这是因为二维培养缺乏脱细胞支架阻止药物扩散的屏障功能，凭借脱细胞技术的应用潜力，未来将有更多的关于不同药物和各种肝癌细胞系相关性的研究。此外，使用门静脉灌注获得了具有完整血管通路的支架，通过其来培养肝癌细胞是成功的，这种循环灌注可以模拟体内营养供应以及物质交换的状态。Hiller等[29]使用三维肝脏模型来研究腺相关病毒的分布和转基因表达，腺相关病毒载体处理的再细胞化肝支架中绿色荧光蛋白强表达，而未处理的支架则没有表达，这说明可以通过药物以及病毒的反应来设计新的抗病毒策略，而且有希望通过三维器官模型来开发新的细胞生长抑制剂来治疗癌症和开展毒理学研究。综上所述，使用脱细胞支架作为体外三维癌症模型为肿瘤研究提供了优越的三维细胞培养环境，将为研究人员评估和预测新型抗癌药物的疗效提供生物平台，不仅如此，多样的制作方式也可以寻找出最符合体外肿瘤细胞培养的肿瘤模型，见表1。 2.3 脱细胞支架在肺癌方面的应用肺癌是世界常见的恶性肿瘤之一。肺脱细胞支架的发展对于肺组织工程器官的构建具有重要意义，目前脱细胞支架在肺癌研究中具有巨大的潜力，用支架作三维培养为细胞提供比常规二维培养体系更复杂的环境，脱细胞支架被开发并用于检测肺癌细胞的生物学行为[30-32]，在肺脱细胞支架内生长的非小细胞肺癌细胞倾向于生长为肉眼可见的结节[31，33]，而这在二维培养中并不产生，通常处于实体瘤中心的细胞接受的营养和氧气少于周边[34]，在脱细胞支架上培养的肺癌细胞可能在营养丰富的地方形成结节，因此靠近血管的营养丰富区域中的细胞增殖更加明显，但三维培养的增殖率总体不如二维培养，表明生长在脱细胞模型中的细胞更加类似于体内增殖，这符合体内实体瘤的表现。基质金属蛋白酶通过降解细胞外基质和基膜，在肿瘤细胞的侵袭、迁移中发挥关键作用[35]，在三维肺模型中生长的A-549细胞产生基质金属蛋白酶9，而在二维培养中未发现。Mayorca-Guiliani等[36]利用高分辨率成像和定量检测细胞外基质蛋白等方法检测脱细胞支架，结构表明癌症驱动的细胞外基质重塑是器官特异性的，并且伴随着细胞外基质组成和拓扑结构的全面改变。癌细胞对基底层的渗透是肿瘤侵袭性的一个标志，细胞获取间充质细胞形态和纤维连接蛋白、E-钙黏蛋白、β-连环蛋白和黏蛋白1的表达下降，也在脱细胞模型中得到证实[37]。此外，评价脱细胞支架内癌症的进展，一般依赖于组织学评估[31]，或者在确定的培养期后分析基因表达谱[38]，但这种分析涉及组织破坏，如果开发在培养期间可重复评估三维灌注组织工程构建体中细胞活力的方法，这将扩大脱细胞支架在癌症研究中的应用。Tapias等[39]运用刃天青试剂灌注保存血管网络的脱细胞肺支架，通过测定它的代谢指数，用以在灌注培养条件下监测肺非小细胞癌细胞的增殖和细胞毒性，此后，Ren等[40]运用刃天青还原法来检测细胞在支架内的生存力和增殖状态，该方法可在长期体外培养过程中非侵入的、重复的和快速估计活细胞数。在肺等的多种组织中，成纤维细胞合成分泌细胞外基质，Scherzer等[41]用人成纤维细胞衍生的细胞外基质作为肺腺癌细胞生长的三维底物模型，描述了成纤维细胞衍生的基质对各种肺源性上皮细胞系(包括癌和非转化细胞)性质的影响，表明了细胞与细胞之间相互作用的重要性及其纳入体外实验的要求。在肺癌研究中，脱细胞基质与天然肿瘤具有相似的微环境，三维脱细胞模型中生长的人肺癌细胞能模拟患者中肺癌生长和转移的特征，可作为研究肿瘤细胞体内行为的理想平台，见表1。 2.4 脱细胞支架在其他癌症方面的应用脱细胞支架在乳腺癌、肝癌以及肺癌研究中已被广泛应用，而在其他肿瘤中的应用报道较少。脱细胞支架可以最大限度保留细胞外基质组织和结构，模拟细胞生存的微环境，Zhao等[42]将舌鳞状细胞癌细胞种植在制备的舌脱细胞支架上，其培养后呈现类似于临床舌鳞状细胞癌组织病理学的特征。有研究表明健康、癌旁、肿瘤组织衍生的细胞外基质在支持细胞增殖的能力上依次增强[43]，这些不同组织来源的细胞外基质的组份及结构上的差异是由于在肿瘤进展过程中细胞外基质重塑造成的，因此，可以运用脱细胞支架研究肿瘤进展。Pinto等[44]将结直肠肿瘤患者的癌和癌旁组织进行脱细胞处理，发现癌组织的脱细胞基质结构具有更高的刚度，同时能够诱导巨噬细胞向M2型抗炎表型转化，促进结肠直肠癌细胞的侵袭迁移。此外，恶性肿瘤的化疗效果随肿瘤恶性程度的增加而降低[45]。Hoshiba等[46]利用不同分期的HT-29细胞衍生的基质中Akt的激活和ATP结合盒亚家族B成员1、ATP结合盒亚家族C成员1表达具有差异，这表明在肿瘤进展过程中，细胞外基质重塑对肿瘤组织获得化学耐药性起重要作用。上皮细胞-间充质转化是癌症进展中的重要步骤，上皮细胞迁移和侵袭的主要状态是上皮细胞-间充质转化，其增加细胞对细胞凋亡的抗性，并降低对化疗和放射治疗的敏感性，通过脱细胞技术肿瘤上皮细胞-间充质转化被证实。Nietzer等[47]在猪空肠的脱细胞培养SW-480结肠癌细胞，发现了波形蛋白在减少，且与成纤维细胞共培养的SW-480细胞形成被成纤维细胞包围的肿瘤样聚集体，证明了肿瘤进展过程中间质向上皮转化过程的存在。此外，在个性化医疗方面，也迫切需要建立与临床表现相关的创新肿瘤模型，有研究者通过猪空肠脱细胞支架来模拟恶性周围神经鞘瘤的体内生长，运用流式生物反应系统调节支架中的氧气和营养供应，结果发现更多的肿瘤样组织形成[48]，这预示可以通过原代细胞与细胞外基质的整合来根据患者的个人情况确定最佳的治疗方法。为了研究前列腺癌骨转移的机制，Reichert等[49]将LNCaP和PC-3细胞种植在原代人成骨细胞分泌的细胞外基质上，发现其促进了细胞的增殖和黏附，以及上皮向间质转化。脱细胞支架作为肿瘤培养平台，在不同癌症上的应用扩大了此技术对于模拟肿瘤微环境具有重要意义，见表1。 "

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