纤维状纤连蛋白网络用作三维组织支架

细胞外基质（ECM）构成了所有多细胞组织的蛋白微环境，并且为细胞体外3D培养提供了模型。ECM结构和组成的变化对生物体的生理功能发挥着一定的驱动作用。正常组织中，纤连蛋白（Fn）是最丰富的ECM蛋白之一，而Fn表达增加与多种癌症密切相关。例如在乳腺癌中，纤维状纤连蛋白（fFn）促进肿瘤发生和转移，部分原因是恶性肿瘤细胞中的上皮向间质转化（EMT）。

Fn是机械敏感性蛋白，它在溶解时（例如在血浆中）以致密状态存在，而位于ECM中时以各种拉伸状态（例如fFn）存在。Fn的结构和构象控制着其结合位点的暴露或螯合，并进一步影响其生物学功能和细胞响应。Fn原纤维的形成涉及到通过结合至细胞表面受体的整联蛋白的机械移位而使蛋白质解折叠。在结构上，解折叠主要发生在机械敏感的III型结构域（Fn-3）处，从而暴露出自缔合位点以形成原纤维。但是，对I，II和III型Fn结构域的每个区域，以及它们如何通过彼此相互作用或与细胞作用形成原纤维的机制仍有待研究。

最近，密歇根大学的Joerg Lahann教授团队发现，在空气、Fn溶液和棋盘状多孔支架之间的三相交界处，通过流体动力学诱导形成的原纤维可产生非常稳定的fFn网络，从而促进细胞侵袭和增殖，能够在体外扩展原发性癌细胞并诱导EMT。通过该方法设计制备的fFn网络可作为包括肿瘤模型在内的体外3D培养系统的工程平台。

本研究中，作者在空气、Fn溶液和经3D喷射的聚丙交酯乙交酯（PLGA）三相接触线处设计形成fFn网络。在短短的15分钟内，流体动力诱导形成的原纤维形成不溶性Fn原纤维的新网络。该网络在整个支架上延伸，长度接近几毫米。通过共聚焦显微镜可看出，该纤维网络与成纤维细胞分泌的胞外基质形态类似。细胞可在fFn网络内外生长，同时fFn网络可为细胞在三维空间中生长提供大量的蛋白基质（Figure 1）。

Figure 1. Hydrodynamically induced fibrillogenesis ofFn. a) Fluid shear is applied at the three-phase interface of a porousscaffold, air, and an Fn solution resulting in the deposition of a network ofinsoluble Fn fibrils (fFn) suspended across the scaffold, i.e., fFn networks.b) Polymer microfiber scaffolds fabricated via 3D jet writing featuringtessellated square pores. Scale bars 500 μm (left) and 25 μm (inset). c) High-resolutionSEM of fFn freely suspended within a pore of the scaffold. Scale bar 1 μm. d)Left) LSCM of fFn (green) suspended within the scaffold (blue) for comparisonto right) Fn deposited by human mammary fibroblasts cultured on glass andsubsequently decellularized. Scale bar 25 μm. e) NIH-3T3 mouse fibroblastscultured three days on either left) an fFn network or right) Fn staticallyadsorbed onto a scaffold. Insets show representative images of the morphologyand distribution of Fn (green) deposited on scaffolds (blue) either byhydrodynamic shearing at the three-phase interface left inset) or staticadsorption right inset). Channels: blue, tessellated scaffold; green, Fn; cyan,cell nucleus; and red, actin. Scale bars 500 μm.

认识到Fn在原发性乳腺癌ECM中的重要性，作者测试了fFn网络对于上皮性乳腺癌小鼠模型中乳腺癌细胞的肿瘤植入增强作用。作者利用在fFn网络上可稳定表达萤火虫荧光素酶的AT-3鼠乳腺癌细胞，将其在支架上培养3天，发现细胞已长满整个支架并仍处于不断增殖状态。研究人员将fFn网络中携带30000个细胞的支架原位植入具有免疫功能的C57BL/6小鼠中，生物发光成像结果显示，最早在支架植入后两天就显示出肿瘤发生的初步迹象，而AT-3细胞的细胞悬液植入体内21天后未能显示出肿瘤植入迹象。不仅如此，移植肿瘤的组织学实验证实癌细胞可从fFn网络自然侵入相邻组织。fFn网络较高的致瘤性与体外培养AT-3细胞的结果一致，在fFn网络上培养的AT-3细胞可增加肿瘤初始阶段（CD29⁺/CD24⁺）和转移性（CD29⁺/CD24⁺/CD90.2⁺）的细胞亚群（Figure 2）。

Figure 2. Engineered fFn networks enhance tumorengraftment efficiency in a mouse breast cancer model. a) AT-3 mouse breastcancer cells formed 3D cell volumes approximately 70 μm thick in vitro afterthree days on fFn networks. Scale bar 25 μm. b) Large-scale view shows that AT-3cells in (a) proliferated and filled the 3D space within the fFn network. Scalebar 500 μm. a,b) Channels: cyan, cell nucleus; red, actin. c) Bioluminescenceimage of tumor formation in immune-competent mice 21 d after AT-3 cells wereorthotopically implanted (image exposure time 10 s). Mice on the left afterimplantation of fFn networks carrying about 30 000 AT-3 cells into the mammaryfat pads indicated by arrows (group 1). The contralateral mammary fat padreceived an injection of approximately the same number of cells suspended in anFn solution as indicated by circles (group 2). The mouse on the right is a positivecontrol having received the group 3 fFn network in the left mammary fat pad(arrow), and the group 4 injection in the right, each delivering 200000 AT-3cells (the minimum required for tumor formation by cell injection). d) Mason’sTrichrome staining of a group 1 tumor graft that formed after 21 d showing AT-3cells invading the surrounding tissues. Scale bar: 25 μm. e,f ) Quantificationof the CD29+/CD24+ population in AT-3 cells capable ofself-renewal (P < 0.05) (e) and the CD29+/CD24+/CD90.2+ tumor initiating population in AT-3 cells (f). AT-3 cells were cultured threedays on TCPS, TCPS with Fn conformally adsorbed (Fn on TCPS), or fFn networks.A single star indicates that the fFn networks are statistically different fromTCPS and Fn on TCPS; a double star indicates that TCPS and Fn on TCPS arestatistically similar.

在人乳腺癌三阴性乳腺癌细胞中，作者进一步评估了fFn网络造成的致瘤性。结果发现，MDA-MB-468细胞在fFn网络上培养4天后，在约10 mm²的扩展区域中形成了密集的3D癌症微环境。不仅如此，fFn网络上形成的CD44⁺/CD24⁻细胞亚群明显多于对照组和Fn处理组，此外，每个时间点上检测的fFn网络中的CD44⁺/CD24⁻/ALDH⁺细胞亚群均高于对照组。上述结果与前面观察到的在fFn网络上扩展后MDA-MB-468细胞中EMT的水平升高现象一致，表明fFn网络具备优异的致瘤性（Figure 3），可用于肿瘤学扩展研究。

Figure 3. Engineered fFn networks increase thetumor-initiating population in MDA-MB-468 human breast cancer cells. a)MDA-MB-468 breast cancer cells cultured four days on fFn networks formcell–cell and cell–ECM contacts. Scale bar 25 μm. b) MDA-MB-468 cells formlarge interconnected volumes throughout fFn networks after four days. Scale bar500 μm. a,b) Channels: green, Fn; orange, laminin; cyan, cell nucleus; and red,actin. c,d) Population of MDA-MB-468s on fFn networks (black solid line andsquare marker), TCPS (black dotted line and crisscross marker), or Fn adsorbedconformally onto TCPS (gray line and triangular marker) that are CD44⁺/CD24⁻ (c) and CD44⁺/CD24⁻/ALDH⁺ (d) measured atdifferent culture time points. Marker expression for cells on TCPS or Fn onTCPS is significantly lower than that of the fFn network at values nearing zeroin d). The starred time point is statistically different from the other threetime points within the fFn network data set.

本研究由密歇根大学的Joerg Lahann教授团队完成，于2019年9月30日在线发表于Advanced Materials。

论文信息：

Stacy Jordahl, Luis Solorio, Dylan B. Neale, Sean McDermott, Jacob H. Jordahl, Alexandra Fox, Christopher Dunlay, Annie Xiao, Martha Brown, Max Wicha, Gary D. Luker, and Joerg Lahann.* Engineered Fibrillar Fibronectin Networks as Three-Dimensional Tissue Scaffolds. Adv. Mater. 2019, 46：1904580

供稿：李家颖

审校：陈嵩

编辑：韩峰