Visualizing murine breast and melanoma tumor microenvironment using intravital multiphoton microscopy.
Ontology highlight
ABSTRACT:
SUBMITTER: Jain R
PROVIDER: S-EPMC8379651 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The abnormal tumor microenvironment fuels tumor progression, metastasis, immune suppression, and treatment resistance. Over last several decades, developments in and applications of intravital microscopy have provided unprecedented insights into the dynamics of the tumor microenvironment. In particular, intravital multiphoton microscopy has revealed the abnormal structure and function of tumor-associated blood and lymphatic vessels, the role of aberrant tumor matrix in drug delivery, invasion and metastasis of tumor cells, the dynamics of immune cell trafficking to and within tumors, and gene expression in tumors. However, traditional multiphoton microscopy suffers from inherently slow imaging rates-only a few frames per second, thus unable to capture more rapid events such as blood flow, lymphatic flow, and cell movement within vessels. Here, we report the development and implementation of a video-rate multiphoton microscope (VR-MPLSM) based on resonant galvanometer mirror scanning that is capable of recording at 30 frames per second and acquiring intravital multispectral images. We show that the design of the system can be readily implemented and is adaptable to various experimental models. As examples, we demonstrate the utility of the system to directly measure flow within tumors, capture metastatic cancer cells moving within the brain vasculature and cells in lymphatic vessels, and image acute responses to changes in a vascular network. VR-MPLSM thus has the potential to further advance intravital imaging and provide new insight into the biology of the tumor microenvironment.
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Project description:BackgroundCancer is a highly complex disease which involves the co-operation of tumor cells with multiple types of host cells and the extracellular matrix. Cancer studies which rely solely on static measurements of individual cell types are insufficient to dissect this complexity. In the last two decades, intravital microscopy has established itself as a powerful technique that can significantly improve our understanding of cancer by revealing the dynamic interactions governing cancer initiation, progression and treatment effects, in living animals. This review focuses on intravital multiphoton microscopy (IV-MPM) applications in mouse models of cancer.Recent findingsIV-MPM studies have already enabled a deeper understanding of the complex events occurring in cancer, at the molecular, cellular and tissue levels. Multiple cells types, present in different tissues, influence cancer cell behavior via activation of distinct signaling pathways. As a result, the boundaries in the field of IV-MPM are continuously being pushed to provide an integrated comprehension of cancer. We propose that optics, informatics and cancer (cell) biology are co-evolving as a new field. We have identified four emerging themes in this new field. First, new microscopy systems and image processing algorithms are enabling the simultaneous identification of multiple interactions between the tumor cells and the components of the tumor microenvironment. Second, techniques from molecular biology are being exploited to visualize subcellular structures and protein activities within individual cells of interest, and relate those to phenotypic decisions, opening the door for "in vivo cell biology". Third, combining IV-MPM with additional imaging modalities, or omics studies, holds promise for linking the cell phenotype to its genotype, metabolic state or tissue location. Finally, the clinical use of IV-MPM for analyzing efficacy of anti-cancer treatments is steadily growing, suggesting a future role of IV-MPM for personalized medicine.ConclusionIV-MPM has revolutionized visualization of tumor-microenvironment interactions in real time. Moving forward, incorporation of novel optics, automated image processing, and omics technologies, in the study of cancer biology, will not only advance our understanding of the underlying complexities but will also leverage the unique aspects of IV-MPM for clinical use.
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Project description:Corneal immune privilege is integral in maintaining the clear avascular window to the foreign world. The presence of distinct populations of corneal leukocytes (CLs) in the normal cornea has been firmly established. However, their precise function and kinetics remain, as of yet, unclear. Through intravital multiphoton microscopy (IV-MPM), allowing the means to accumulate critical spatial and temporal cellular information, we provide details for long-term investigation of CL morphology and kinetics under steady state and following inflammation. Significant alterations in size and morphology of corneal CD11c+ dendritic cells (DCs) were noted following acute sterile inflammation, including cell volume (4364.4 ± 489.6 vs. 1787.6 ± 111.0 μm3, P < 0.001) and sphericity (0.82 ± 0.01 vs. 0.42 ± 0.02, P < 0.001) compared with steady state. Furthermore, IV-MPM analyses revealed alterations in both the CD11c+ DC and major histocompatibility complex class II (MHC)-II+ mature antigen-presenting cell population kinetics during inflammation, including track displacement length (CD11c: 16.57 ± 1.41 vs. 4.64 ± 0.56 μm, P < 0.001; MHC-II: 9.03 ± 0.37 vs. 4.09 ± 0.39, P < 0.001) and velocity (CD11c: 1.91 ± 0.07 μm/min vs. 1.73 ± 0.1302 μm/min; MHC-II: 2.97 ± 0.07 vs. 1.62 ± 0.08, P < 0.001) compared with steady state. Our results reveal in vivo evidence of sessile CL populations exhibiting dendritic morphology under steady state and increased velocity of spherical leukocytes following inflammation. IV-MPM represents a powerful tool to study leukocytes in corneal diseases in context.-Seyed-Razavi, Y., Lopez, M. J., Mantopoulos, D., Zheng, L., Massberg, S., Sendra, V. G., Harris, D. L., Hamrah, P. Kinetics of corneal leukocytes by intravital multiphoton microscopy.
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Project description:Conventional microscopy has limitations in viewing the cochlear microstructures due to three-dimensional spiral structure and the overlying bone. But these issues can be overcome by imaging the cochlea in vitro with intravital multiphoton microscopy (MPM). By using near-infrared lasers for multiphoton excitation, intravital MPM can detect endogenous fluorescence and second harmonic generation of tissues. In this study, we used intravital MPM to visualize various cochlear microstructures without any staining and non-invasively analyze the volume changes of the scala media (SM) without removing the overlying cochlear bone. The intravital MPM images revealed various tissue types, ranging from thin membranes to dense bone, as well as the spiral ganglion beneath the cochlear bone. The two-dimensional, cross-sectional, and serial z-stack intravital MPM images also revealed the spatial dilation of the SM in the temporal bone of pendrin-deficient mice. These findings suggest that intravital MPM might serve as a new method for obtaining microanatomical information regarding the cochlea, similar to standard histopathological analyses in the animal study for the cochlea. Given the capability of intravital MPM for detecting an increase in the volume of the SM in pendrin-deficient mice, it might be a promising new tool for assessing the pathophysiology of hearing loss in the future.
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Project description:The podocyte is a highly specialized kidney glomerular epithelial cell that plays an essential role in glomerular filtration and is believed to be the target of numerous glomerular diseases leading to proteinuria. Despite the leaps in our understanding of podocyte biology, new methodologies are needed to facilitate research into the cell. Multiphoton microscopy (MPM) was used to image the nephrin knockout/green fluorescent protein (GFP) knock-in heterozygote (Nphs1(tm1Rkl)/J) mouse. The nephrin promoter restricts GFP expression to the podocytes that fluoresce green under excitation. From the exterior of an intact kidney, MPM can peer into the renal parenchyma and visualize the podocytes that outline the globular shape of the glomeruli. Details as fine as the podocyte's secondary processes can be resolved. In contrast, podocytes exhibit no fluorescence in the wildtype mouse and are invisible to MPM. Phenotypically, there are no significant differences between wildtype and Nphs1(tm1Rkl)/J mice in body weight, urinary albumin excretion, creatinine clearance, or glomerular depth. Interestingly, the glomeruli are closer to the kidney capsule in female mice, making the gender the preferred choice for MPM. For the first time, green fluorescent podocytes in a mouse model free of confounding phenotypes can be visualized unequivocally and in the "positive" by MPM, facilitating intravital studies of the podocyte.
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Project description:Oncolytic virus (OV) therapy is an emerging cancer treatment that uses replicating viruses to infect and kill tumor cells and incite anticancer immunity. While the approach shows promise, it currently fails most patients, indicating strategies to improve OV activity are needed. Developing these will require greater understanding of OV biology, particularly in the context of OV delivery and clearance, the infection process within a complex tumor microenvironment, and the modulation of anticancer immunity. To help achieve this, we have established a technique for high-resolution 4D imaging of OV-host interactions within intact tissues of live mice using intravital microscopy (IVM). We show that oncolytic vesicular stomatitis virus (VSV) directly labeled with Alexa Fluor dyes is easily visualized by single- or multiphoton microscopy while retaining bioactivity in vivo. The addition of fluorophore-tagged antibodies and genetically encoded reporter proteins to image target cells and the virus infection enables real-time imaging of dynamic interactions between VSV and host cells in blood, tumor, and visceral organs of live mice. The method has sufficient in vivo resolution to observe leukocytes in blood binding to and transporting VSV particles, foci of VSV infection spreading through a tumor, and antigen-presenting cells in the spleen interacting with and being infected by VSV. Visualizing OV-host interactions by IVM represents a powerful new tool for studying OV therapy.
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