Cell-Matrix Interactions and Migration

细胞外基质分子、整联蛋白受体、细胞骨架马达蛋白、蛋白酶和这些分子系统的调节剂通过复杂的整合机制促进细胞迁移、信号传导和癌细胞侵袭。我们正在处理以下具体问题： 1.整合素、细胞外基质和细胞骨架的功能如何整合，它们如何协调以产生有效的细胞迁移或侵袭？ 2.什么样的生物物理特性和信号传导机制对于有效的细胞迁移和侵袭是重要的？ 我们正在使用各种细胞和分子生物学方法来解决这些问题，包括活细胞宽视野，共聚焦和双光子延时显微镜与荧光蛋白嵌合体，生物化学和信号转导分析，以及评估局部基质变形的方法，以应对迁移或入侵哺乳动物细胞的力量。我们使用各种荧光分子嵌合体和突变体的细胞骨架蛋白和各种配置的细胞外基质分子作为一个长期计划的一部分，以分析其功能的整合素介导的过程。 癌细胞通过基底膜的侵袭是肿瘤扩散和转移的起始步骤，而穿过这一组织屏障是侵袭的关键病理标志。然而，关于人类癌细胞如何突破并穿过基底膜进入周围的细胞外基质和组织的知识相对较少。我们使用了一个三维体外侵袭模型，该模型由基底膜包裹的人癌细胞球体组成，并嵌入3D I型胶原凝胶中，以使用显微镜观察癌症侵袭的早期事件。人类乳腺癌细胞产生了大量大小不一的基底膜穿孔或洞，这些穿孔或洞在细胞侵袭过程中随着时间的推移而扩大。各种基质金属蛋白酶（MMP）抑制剂和破坏肌动蛋白聚合的治疗强烈抑制穿孔大小。尽管肌球蛋白II相关的肌动球蛋白收缩性先前已经涉及扩大基底膜穿孔以允许破裂的发育模型，但在这种癌症侵袭模型中抑制收缩性对孔扩张的影响出乎意料地最小。我们的结论是，人类癌细胞可以主要使用MMP-型的蛋白水解和肌动蛋白聚合来破坏基底膜，并扩大孔的大小，可以允许过境的细胞核（最大的细胞器）和癌细胞体的其余部分的跨基底膜的入侵，但肌动球蛋白收缩性的作用有限。这种有效的基底膜破坏过程可以单独使用癌细胞来证明，而无需辅助细胞如癌症相关的成纤维细胞或巨噬细胞的帮助。我们正在扩展这项研究，以评估癌细胞可以通过这些基底膜穿孔挤压随后在3D胶原基质中迁移入侵的机制。我们还完成了一个离体模型系统的开发，用于分析细胞在生理3D环境中的相互作用和迁移，以补充我们在体外对细胞-基质相互作用的研究。后一种方法使用小鼠筋膜作为天然细胞外基质的来源。 这种结合的方法涉及细胞迁移和细胞与各种类型的细胞外基质相互作用的调节机制的表征，应该提供新的方法来理解和潜在地改善异常胚胎发育和癌症侵袭期间细胞所使用的迁移或侵入过程。深入了解细胞与其细胞外基质环境相互作用的确切方式也有助于未来再生医学的组织工程研究。

Extracellular matrix molecules, integrin receptors, cytoskeletal motor proteins, proteases, and regulators of these molecular systems contribute to cell migration, signaling, and cancer cell invasion by complex, integrated mechanisms. We are addressing the following specific questions: 1. How are the functions of integrins, the extracellular matrix, and the cytoskeleton integrated, and how are they coordinated to produce effective cell migration or invasion? 2. What biophysical properties and signaling mechanisms are important for efficient cell migration and invasion? We are using a variety of cell and molecular biology approaches to address these questions, including live-cell wide-field, confocal, and two-photon time-lapse microscopy with fluorescent protein chimeras, biochemical and signal transduction analyses, as well as methods for evaluating local matrix deformations in response to forces from migrating or invading mammalian cells. We use a variety of fluorescent molecular chimeras and mutants of cytoskeletal proteins and various configurations of extracellular matrix molecules as part of a long-term program to analyze their functions in integrin-mediated processes. Cancer invasion through basement membranes is the initial step of tumor dissemination and metastasis, and crossing of this tissue barrier is a key pathological sign of invasion. However, relatively little is known about how human cancer cells breach and traverse basement membranes into surrounding extracellular matrix and tissues. We used a three-dimensional in vitro invasion model consisting of human cancer cell spheroids encapsulated by a basement membrane and embedded in 3D type I collagen gels to visualize early events of cancer invasion using microscopy. Human breast cancer cells generated large numbers of basement membrane perforations, or holes, of varying sizes that expanded over time during the process of cell invasion. Treatment with various matrix metalloproteinase (MMP) inhibitors and disrupting actin polymerization strongly suppressed perforation size. Although myosin II-associated actomyosin contractility had been implicated previously in developmental models for enlarging basement membrane perforations to permit breaching, inhibiting contractility in this cancer invasion model had unexpectedly minimal effects on hole expansion. We conclude that human cancer cells can mainly use MMP-type proteolysis and actin polymerization to perforate the basement membrane and expand holes to a size that can permit transit of the nucleus (the largest cell organelle) and the rest of the cancer cell body for invasion across the basement membrane, but with limited roles for actomyosin contractility. This efficient basement membrane breaching process could be demonstrated using the cancer cells alone without the help of accessory cells such as cancer-associated fibroblasts or macrophages. We are extending this study to evaluate the mechanisms by which cancer cells can squeeze through these basement membrane perforations for subsequent migratory invasion in a 3D collagen matrix. We are also completing development of an ex vivo model system for analyzing cell interactions and migration in a physiological 3D environment to complement our studies on cell-matrix interactions in vitro. This latter approach uses mouse fascia as a source of native extracellular matrix. This combined approach involving mechanistic characterization of the regulation of cell migration and cell interactions with various types of extracellular matrix should provide novel approaches to understanding and potentially ameliorating migratory or invasive processes used by cells during abnormal embryonic development and cancer invasion. An in-depth understanding of the precise ways in which cells interact with their extracellular matrix environment should also facilitate tissue engineering studies for future regenerative medicine.