Long-term Intravital Immunofluorescence Imaging of Tissue Matrix Components with Epifluorescence and Two-photon Microscopy

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.

Intravital Immunofluorescence for Visualizing the Microcirculatory and Immune Microenvironments in the Mouse Ear Dermis

Witold Waldemar Kilarski, Amanda Waite Lund, Scott Oliver, Melody Swartz, Jeremy Teo

Visualizing the dynamic behaviors of immune cells in living tissue has dramatically increased our understanding of how cells interact with their surroundings, contributing important insights into mechanisms of leukocyte trafficking, tumor cell invasion, and T cell education by dendritic cells, among others. Despite substantial advances with various intravital imaging techniques including two-photon microscopy and the generation of multitudes of reporter mice, there is a growing need to assess cell interactions in the context of specific extracellular matrix composition and microvascular functions, and as well, simpler and more widely accessible methods are needed to image cell behaviors in the context of living tissue physiology. Here we present an antibody-based method for intravital imaging of cell interactions with the blood, lymphatic, and the extracellular matrix compartments of the living dermis while simultaneously assessing capillary permeability and lymphatic drainage function. Using the exposed dorsal ear of the anesthetized mouse and a fluorescence stereomicroscope, such events can be imaged in the context of specific extracellular matrix proteins, or matrix-bound chemokine stores. We developed and optimized the method to minimize tissue damage to the ear, rapidly immunostain for multiple extracellular or cell surface receptors of interest, minimize immunotoxicity with pre-blocking Fc gamma receptors and phototoxicity with extracellular antioxidants, and highlight the major dermal tissue structures with basement membrane markers. We demonstrate differential migration behaviors of bone marrow-derived dendritic cells, blood-circulating leukocytes, and dermal dendritic cells, with the latter entering sparse CCL21-positive areas of pre-collecting lymphatic vessels. This new method allows simultaneous imaging of cells and tissue structures, microvascular function, and extracellular microenvironment in multiple skin locations for 12 hours or more, with the flexibility of immunolabeling in addition to genetic-based fluorescent reporters.

Public Library of Science2013

Migration dynamics of breast cancer cells in a tunable 3D interstitial flow chamber

Ulrike Haessler, Philippe Renaud, Melody Swartz, Jeremy Teo

The migration of cells such as leukocytes, tumor cells, and fibroblasts through 3D matrices is critical for regulating homeostasis and immunity and for driving pathogenesis. Interstitial flow through the extracellular matrix, which can substantially increase during inflammation and in the tumor microenvironment, can influence cell migration in multiple ways. Leukocytes and tumor cells are heterogeneous in their migration responses to flow, yet most 3D migration studies use endpoint measurements representing average characteristics. Here we present a robust new microfluidic device for 3D culture with live imaging under well-controlled flow conditions, along with a comparison of analytical methods for describing the migration behavior of heterogeneous cell populations. We then use the model to provide new insight on how interstitial flow affects MDA-MB-231 breast cancer cell invasion, phenomena that are not seen from averaged or endpoint measurements. Specifically, we find that interstitial flow increases the percentage of cells that become migratory, and increases migrational speed in about 20% of the cells. It also increases the migrational persistence of a subpopulation (5-10% of cells) in the positive or negative flow direction. Cells that migrated upstream moved faster but with less directedness, whereas cells that migrated in the direction of flow moved at slower speeds but with higher directedness. These findings demonstrate how fluid flow in the tumor microenvironment can enhance tumor cell invasion by directing a subpopulation of tumor cells in the flow direction; i.e., towards the draining lymphatic vessels, a major route of metastasis.

2012

Engineering Lymphangiogenesis

Manuel Andreas Fankhauser

Melanoma causes the vast majority of skin cancer deaths, and there is currently no conventional treatment available that is able to cure metastatic melanoma patients. However, a novel treatment modality has recently emerged: cancer immunotherapy. Cancer immunotherapy aims at raising potent, tumor-specific T cell responses that are able to recognize and eliminate cancerous cells. Despite the fact that for the first time in history some metastatic melanoma patients can be cured, it is currently unclear why a large fraction of patients does not respond to immunotherapy. Thus, the overarching goal of this thesis was to gain a better understanding of the molecular mechanisms dictating the outcome to immunotherapy. Pre-existing inflammation within the tumor microenvironment is associated with good clinical prognosis following immunotherapy. Because our lab has previously shown that tumor associated lymphatic vessels (LVs) can modulate tumor inflammation, we hypothesized that LVs might be involved in modulating the outcome of cancer immunotherapy. To explore this hypothesis, we characterized two mouse models that recapitulate primary human lymphangiogenic melanoma. Using these models, we found that tumor-associated lymphatics secrete CCL21 to actively attract naïve T cells into the tumor microenvironment. Interestingly, we found that antigen-specific immunotherapy induced activation of these naïve T cell infiltrates, which not only resulted in eradication of the primary melanoma tumors, but also conferred the mice with long-term protection against tumor re-challenge. We thus demonstrate that LVs increase the quantity and quality of tumor inflammation, thereby establishing a microenvironment that is able to potentiate antigen-specific immunotherapy. We then show that controlled release of VEGFC from injectable hydrogels leads to lymphangiogenesis in the mouse dermis. Similarly to what we found in lymphangiogenic tumors, engineered lymphangiogenic skin sites displayed increased expression of CCL21, and increased infiltrations of CD4+ and CD8+ T cells. Delivery of the model antigen ovalbumin into lymphangiogenic sites led to enhanced release of the effector cytokine interferon-¿ upon antigen-restimulation, suggesting that engineered lymphangiogenic sites could be exploited for therapeutic immunomodulation. Finally, to better understand mechanisms underlying successful cancer immunotherapy, we established a tool for the observation of anti-tumor immune responses in the context of the native tumor microenvironment. By combining existing methods in a novel way, we developed an intravital microscopy method based on immunofluorescence that allows simultaneous, high resolution and dynamic visualization of the tumor microenvironment including immune cells and extracellular matrix proteins. In conclusion, this thesis demonstrates that lymphatic endothelial cells (LECs) orchestrate immune cell recruitment into peripheral tissues by secretion of CCL21 chemokine, both in steady-state and disease. Our findings add more evidence to the hypothesis that LECs might play an underappreciated role in driving the formation of peripheral sites of immunomodulation. Our findings have translational potential, as immune cell infiltrates attracted by local lymphangiogenesis can be exploited for therapeutic immune regulation, such as to improving the efficacy of cancer immunotherapy.