IntraVital最新文献

Cancer cell invasion is an adaptive process based on cell-intrinsic properties to migrate individually or collectively, and their adaptation to encountered tissue structure acting as barrier or providing guidance. Whereas molecular and physical mechanisms of cancer invasion are well-studied in 3D in vitro models, their topographic relevance, classification and validation toward interstitial tissue organization in vivo remain incomplete. Using combined intravital third and second harmonic generation (THG, SHG), and three-channel fluorescence microscopy in live tumors, we here map B16F10 melanoma invasion into the dermis with up to 600 µm penetration depth and reconstruct both invasion mode and tissue tracks to establish invasion routes and outcome. B16F10 cells preferentially develop adaptive invasion patterns along preformed tracks of complex, multi-interface topography, combining single-cell and collective migration modes, without immediate anatomic tissue remodeling or destruction. The data suggest that the dimensionality (1D, 2D, 3D) of tissue interfaces determines the microanatomy exploited by invading tumor cells, emphasizing non-destructive migration along microchannels coupled to contact guidance as key invasion mechanisms. THG imaging further detected the presence and interstitial dynamics of tumor-associated microparticles with submicron resolution, revealing tumor-imposed conditioning of the microenvironment. These topographic findings establish combined THG, SHG and fluorescence microscopy in intravital tumor biology and provide a template for rational in vitro model development and context-dependent molecular classification of invasion modes and routes.

In mammary tumors, intravital imaging techniques have uncovered an essential role for macrophages during tumor cell invasion and metastasis mediated by an epidermal growth factor (EGF) / colony stimulating factor-1 (CSF-1) paracrine loop. It was previously demonstrated that mammary tumors in mice derived from rat carcinoma cells (MTLn3) exhibited high velocity migration on extracellular matrix (ECM) fibers. These cells form paracrine loop-dependent linear assemblies of alternating host macrophages and tumor cells known as "streams." Here, we confirm by intravital imaging that similar streams form in close association with ECM fibers in a highly metastatic patient-derived orthotopic mammary tumor (TN1). To understand the in vivo cell motility behaviors observed in streams, an in vitro model of fibrillar tumor ECM utilizing adhesive 1D micropatterned substrates was developed. MTLn3 cells on 1D fibronectin or type I collagen substrates migrated with higher velocity than on 2D substrates and displayed enhanced lamellipodial protrusion and increased motility upon local interaction and pairing with bone marrow-derived macrophages (BMMs). Inhibitors of EGF or CSF-1 signaling disrupted this interaction and reduced tumor cell velocity and protrusion, validating the requirement for an intact paracrine loop. Both TN1 and MTLn3 cells in the presence of BMMs were capable of co-assembling into linear arrays of alternating tumor cells and BMMs that resembled streams in vivo, suggesting the stream assembly is cell autonomous and can be reconstituted on 1D substrates. Our results validate the use of 1D micropatterned substrates as a simple and defined approach to study fibrillar ECM-dependent cell pairing, migration and relay chemotaxis as a complementary tool to intravital imaging.

The innate immune system is comprised of cellular sentinels that often serve as the first responders to injury and invading pathogens. Our basic understanding of innate immunity is derived from research conducted in peripheral lymphoid tissues. However, it is now recognized that most non-lymphoid tissues throughout the body are equipped with specialized innate immune cells that are uniquely adapted to the niches in which they reside. The central nervous system (CNS) is a particularly interesting compartment because it contains a population of post-mitotic cells (neurons) that are intolerant of robust, cytopathic inflammatory responses observed in many peripheral tissues. Thus, evolutionary adaptations have fitted the CNS with a unique array of innate immune sentinels that facilitate the development of local inflammatory responses but attempt to do so in a manner that preserves the integrity of its post-mitotic residents. Interestingly, studies have even suggested that CNS resident innate immune cells contribute to the homeostasis of this compartment and promote neural activity. In this review we discuss recent advances in our understanding of CNS innate immune sentinels and how novel imaging approaches such as intravital two-photon laser scanning microscopy (TPLSM) have shed light on these cells during states of health and disease.