Güç Esra, Fankhauser Manuel, Lund Amanda W, Swartz Melody A, Kilarski Witold W
Institute of Bioengineering and Swiss Institute of Experimental Cancer Research (ISREC), École Polytechnique Fédérale de Lausanne.
Institute of Bioengineering and Swiss Institute of Experimental Cancer Research (ISREC), École Polytechnique Fédérale de Lausanne; Department of Cell and Developmental Biology and Knight Cancer Institute, Oregon Health & Science University.
J Vis Exp. 2014 Apr 22(86):51388. doi: 10.3791/51388.
Besides being a physical scaffold to maintain tissue morphology, the extracellular matrix (ECM) is actively involved in regulating cell and tissue function during development and organ homeostasis. It does so by acting via biochemical, biomechanical, and biophysical signaling pathways, such as through the release of bioactive ECM protein fragments, regulating tissue tension, and providing pathways for cell migration. The extracellular matrix of the tumor microenvironment undergoes substantial remodeling, characterized by the degradation, deposition and organization of fibrillar and non-fibrillar matrix proteins. Stromal stiffening of the tumor microenvironment can promote tumor growth and invasion, and cause remodeling of blood and lymphatic vessels. Live imaging of matrix proteins, however, to this point is limited to fibrillar collagens that can be detected by second harmonic generation using multi-photon microscopy, leaving the majority of matrix components largely invisible. Here we describe procedures for tumor inoculation in the thin dorsal ear skin, immunolabeling of extracellular matrix proteins and intravital imaging of the exposed tissue in live mice using epifluorescence and two-photon microscopy. Our intravital imaging method allows for the direct detection of both fibrillar and non-fibrillar matrix proteins in the context of a growing dermal tumor. We show examples of vessel remodeling caused by local matrix contraction. We also found that fibrillar matrix of the tumor detected with the second harmonic generation is spatially distinct from newly deposited matrix components such as tenascin C. We also showed long-term (12 hours) imaging of T-cell interaction with tumor cells and tumor cells migration along the collagen IV of basement membrane. Taken together, this method uniquely allows for the simultaneous detection of tumor cells, their physical microenvironment and the endogenous tissue immune response over time, which may provide important insights into the mechanisms underlying tumor progression and ultimate success or resistance to therapy.
细胞外基质（ECM）除了作为维持组织形态的物理支架外，在发育和器官稳态过程中还积极参与调节细胞和组织功能。它通过生化、生物力学和生物物理信号通路发挥作用，例如通过释放生物活性ECM蛋白片段、调节组织张力以及为细胞迁移提供途径。肿瘤微环境的细胞外基质会经历大量重塑，其特征是纤维状和非纤维状基质蛋白的降解、沉积和组织化。肿瘤微环境的基质硬化可促进肿瘤生长和侵袭，并导致血管和淋巴管重塑。然而，到目前为止，基质蛋白的活体成像仅限于使用多光子显微镜通过二次谐波产生检测到的纤维状胶原蛋白，这使得大多数基质成分在很大程度上不可见。在这里，我们描述了在小鼠薄背耳皮肤中接种肿瘤、对细胞外基质蛋白进行免疫标记以及使用落射荧光和双光子显微镜对活体小鼠中暴露组织进行活体成像的方法。我们的活体成像方法能够在生长的皮肤肿瘤背景下直接检测纤维状和非纤维状基质蛋白。我们展示了由局部基质收缩引起的血管重塑实例。我们还发现，通过二次谐波产生检测到的肿瘤纤维状基质在空间上与新沉积的基质成分（如腱生蛋白C）不同。我们还展示了T细胞与肿瘤细胞相互作用以及肿瘤细胞沿基底膜IV型胶原迁移的长期（12小时）成像。综上所述，这种方法独特地允许同时检测肿瘤细胞、其物理微环境和内源性组织免疫反应随时间的变化，这可能为肿瘤进展以及最终对治疗的成功或抵抗的潜在机制提供重要见解。
