The authors would like to acknowledge NCI R01 CA216248, CA206458, and CA179556 grants for funding this work. We would also like to acknowledge Dr. Kevin Eliceiri and his imaging group for their technical expertise in the early development of our intravital program. We also thank Dr. Ben Cox and other members of the Eliceiri Fabrication Group at the Morgridge Institute for Research for their essential technical design during the early phases of the MIW. Dr. Ellen Dobson assisted with useful conversations about the ImageJ trainable WEKA segmentation tool. In addition, we would like to thank Dr. Melissa Skala and Dr. Alexa Barres-Heaton for the timely use of their microscope. Lastly, we would like to thank Dr. Brigitte Raabe, D.V.M, for all the thoughtful discussions and advice on our mouse handling and care.

The authors have no conflicts of interest to disclose.

4D intravital imaging is a powerful tool to investigate dynamic physiological interactions within the spatial and temporal context of the native tumor microenvironment. This manuscript provides a very basic and adaptable operational framework to compartmentalize dynamic cell interactions within the tumor mass, the adjacent stroma, or within proximity to the vascular network using only endogenous signals from second harmonic generation or NAD(P)H autofluorescence. This protocol provides a comprehensive, step-by-step metho...

The extracellular matrix (ECM) in the tumor microenvironment is known to be dynamically deposited and remodeled by multiple cell types to further facilitate disease progression1,2,3. These matrix alterations provide both mechanical and biological cues that alter cell behavior and often result in a continuing cycle of matrix remodeling4. Investigation into the dynamic, reciprocal interplay between tumor cells and the extracellular matrix is often conducted using three-dimensional (3D) in vitro culture or microfluidic systems. While these bottom-up approaches have demonstrated mechanisms of ECM remodeling5,6,7, increased proliferation8, epithelial to mesenchymal transition9,10,11,12, and tumor cell migration and invasion7, 13,14,15,16, their focus has been primarily on a few cell types (e.g., tumor cells or fibroblasts) within a homogeneous 3D matrix compared to the diversity and heterogeneity of interactions present within a physiological tissue. In addition to in vitro systems, ex vivo tumor histology can also provide some insight into these cell-cell and cell-ECM interactions17. Immunohistochemistry has the advantage of being able to analyze multiple cell types with respect to the spatially heterogeneous composition and architecture of the ECM, but the static endpoints of fixed tissue do not capture the dynamic nature of interactions between cells and the microenvironment. Intravital imaging has opened the door to interrogate diverse and dynamic interactions within the physiological&#160;context of the native tumor microenvironment.
The capabilities of intravital tumor imaging are rapidly advancing. Improvements in the design of imaging windows and surgical techniques to implant the windows have enabled long-term longitudinal tumor imaging at a variety of anatomical locations (i.e., primary tumor, lymph nodes, metastatic sites18,19,20). Moreover, the capacity of optical instrumentation to visualize and collect data in multiple dimensions (i.e., spectral, spatial fluorescence intensity, and lifetime), and at high resolution and speed (video rate) is becoming widely accessible. The improved technology provides an opportunity to explore rapid changes in cell signaling and phenotypic dynamics within a physiological environment. Lastly, the expansion of optogenetic tools and the wide array of genetic fluorescent constructs allow for the tagging of specific cell types to capture cell migration in the tumor microenvironment or cell lineage tracing during development or disease progression21,22. The use of these tools in combination with CRISPR/Cas9 technology provides researchers the opportunity to generate unique animal models in a timely manner.
While all these advances make intravital imaging an increasingly powerful method to explore dynamic and physiological cellular interactions, there is still an important need to develop strategies that provide spatial, temporal, and structural context at the tissue level to these biological interactions. Currently, many intravital imaging studies compensate for the lack of visual landmarks such as blood vessels by injecting fluorescent dyes into the vasculature or employing mouse models that exogenously express fluorescent proteins to delineate physical features. Injectable dyes and substrates like fluorescent dextrans are broadly utilized to successfully label the vasculature in intravital collections19, 23, 24. However, this approach is not without limitations. For one, it requires additional mouse manipulations and its utility is limited to short-term experiments. For longitudinal studies, fluorescent dextran can be problematic as we observe the accumulation of dextran in phagocytic cells or diffusion into the surrounding tissue over time25. Exogenous fluorescent protein incorporation into the mouse model has been presented as an alternative to fluorescent dextrans but presents limitations of its own. The availability and diversity of exogenous fluorophores within mouse models are still limited and expensive to create. Additionally, in specific models, such as PDX models, genetic manipulations are not desirable or possible. It has also been shown that the presence of fluorescent or bioluminescent proteins within cells are recognized as foreign within the mouse, and within immunocompetent mouse models, this reduces the amount of metastasis due to the response of the host immune system26,27. Lastly, exogenous fluorescent proteins or fluorescent dyes used for spatial context or to segment subsequent data often occupy prime ranges of the light spectrum that could otherwise be used to investigate the physiological interactions of interest.
The use of the intrinsic signal from the ECM or endogenous fluorescence from cells within the tissue represents a potential universal label-free means to segment intravital data for more in-depth cellular and spatial analysis. Second harmonic generation (SHG) has long been used to visualize the ECM28. With the subsequent development of important tools to aid in the characterization of fiber organization29,30,31, it is possible to characterize cell behavior relative to local ECM structure. In addition, autofluorescence from the endogenous metabolite, NAD(P)H, provides another label-free tool to compartmentalize the tumor microenvironment in vivo. NAD(P)H fluoresces brightly in tumor cells and can be used to discriminate the boundaries of the growing tumor nest from its surrounding stroma21,32. Lastly, the vasculature is an important physiological structure in the tumor microenvironment and the site of key cell-type-specific interactions33,34,35. The excitation of red blood cells (RBC) or blood plasma has been used to visualize the tumor vasculature, and using two- or three-photon excitation (2P; 3P) the measurement of blood flow rates has been shown to be possible36. However, while larger blood vessels are easily identifiable by their endogenous fluorescence signatures, the identification of subtle, variable, and less fluorescent small blood vessels requires more expertise. These inherent difficulties hinder optimal image segmentation. Fortunately, these sources of endogenous fluorescence (i.e., red blood cells and blood plasma) can also be measured by fluorescence lifetime imaging37, which capitalizes on the unique photophysical properties of the vasculature and represents a useful addition to the growing intravital toolbox.
In this protocol, a workflow for the segmentation of four-dimensional (4D) intravital imaging explicitly using intrinsic signals like endogenous fluorescence and SHG is described from acquisition to analysis. This protocol is particularly pertinent for longitudinal studies through a mammary imaging window where exogenous fluorescence may not be practical or possible, as is the case with PDX models. The segmentation principles outlined here, however, are broadly applicable to intravital users investigating tumor biology, tissue development, or even normal tissue physiology. The reported suite of analysis approaches will allow the users to differentiate cellular behavior between regions of aligned or random collagen fiber configurations, compare numbers or behaviors of cells residing in specific regions of the tumor microenvironment, and map the vasculature to the tumor microenvironment using only label-free or intrinsic signal. Together, these methods create an operational framework for maximizing the depth of information gained from 4D intravital imaging of the mammary gland while minimizing the need for additional exogenous labels.

<table><tbody><tr><td>#1.5 12mm round cover glass</td><td>Warner Instruments</td><td># 64-0712</td><td>MIW construction</td></tr><tr><td>1.0 mL syringe for SQ injection</td><td>BD</td><td>309659</td><td>Syringe</td></tr><tr><td>20x objective</td><td>Zeiss</td><td>421452-988</td><td>Water immersion</td></tr><tr><td>27G needle for SQ injection</td><td>Covidien</td><td>1188827012</td><td>Needle</td></tr><tr><td>40x objective</td><td>Nikon</td><td>MRD77410</td><td>Water immersion</td></tr><tr><td>5-0 silk braided suture</td><td>Ethicon</td><td>K870</td><td>Suture for MIW implantation</td></tr><tr><td>Artificial tears gel</td><td>Akorn</td><td>NDC 59399-162-35</td><td>Eye gel</td></tr><tr><td>Betadine solution, 5%</td><td>Fisher Scientific</td><td>NC1558063</td><td>Surgery antiseptic</td></tr><tr><td>cotton-tipped applicator</td><td>Fisher Scientific</td><td>23-400-101</td><td></td></tr><tr><td>Cyanoacrylate adhesive</td><td>Loctite</td><td>1365882</td><td>MIW construction</td></tr><tr><td>fluorescent dextran</td><td>Sigma</td><td>T1287-50mg</td><td>intravenous labelling of vasculature</td></tr><tr><td>forceps</td><td>Mckesson.com</td><td>Miltex&amp;nbsp;#18-782</td><td>stainless, 4 inch, curved</td></tr><tr><td>GaAsP photomultiplier tube</td><td>Hamamatsu&amp;nbsp;</td><td></td><td></td></tr><tr><td>heating blanket</td><td>CARA 72 heating pad</td><td>&amp;nbsp;038056000729</td><td>Temperature selectable</td></tr><tr><td>heating chamber</td><td>home built</td><td></td><td></td></tr><tr><td>Fluorescent lifetime handbook</td><td>Becker and Hickl</td><td></td><td>https://www.becker-hickl.com/literature/handbooks</td></tr><tr><td>inverted microscope base</td><td>Nikon</td><td></td><td></td></tr><tr><td>Isoflurane</td><td>Akorn</td><td>NDC 59399-106-01</td><td>Anesthesia</td></tr><tr><td>Liqui-Nox</td><td>Fisher Scientific</td><td>16-000-125</td><td>MIW cleaning</td></tr><tr><td>Meloxicam</td><td>Norbrook</td><td>NDC 55529-040-10</td><td>Analgesic</td></tr><tr><td>Micro Hose</td><td>Scientific Commodities INC.</td><td>&amp;nbsp;BB31695-PE/1</td><td></td></tr><tr><td>multiphoton scan head</td><td>Bruker Ultima II</td><td></td><td>Multiphoton scanhead and imaging platform</td></tr><tr><td>NADH FLIM filter</td><td>Chroma</td><td>284994</td><td>ET 440/80 m-2P</td></tr><tr><td>Nair</td><td>CVS</td><td>339826</td><td>Depilatory cream</td></tr><tr><td>objective heater</td><td>Tokai Hit</td><td>STRG-WELSX-SET</td><td></td></tr><tr><td>SHG/FAD filter</td><td>Chroma</td><td>320740</td><td>ET450/40m-2P</td></tr><tr><td>Sparkle glass cleaner</td><td>Amazon.com</td><td>B00814ME24</td><td>Glass Cleaner for implanted MIW</td></tr><tr><td>SPC-150 photon counting board</td><td>Becker and Hickl</td><td></td><td></td></tr><tr><td>surgical light</td><td>FAJ</td><td>B06XV1VQVZ</td><td>Magnetic LED gooseneck light</td></tr><tr><td>surgical micro-scissors</td><td>Excelta</td><td>366</td><td>stainless, 3 inch</td></tr><tr><td>Triple antibiotic ointment</td><td>Actavis Pharma</td><td>NDC 0472-0179-34</td><td>Antibiotic</td></tr><tr><td>TV catheter</td><td>Custom</td><td>BD 30G needle: 305106</td><td>Catheter for TV injection</td></tr><tr><td>Two photon filter</td><td>Chroma</td><td>320282</td><td>ET585/65m-2P</td></tr><tr><td>two-photon laser</td><td>Coherent charmeleon</td><td></td><td>Tunable multiphoton laser</td></tr><tr><td>ultrasound gel</td><td>Parker</td><td>PKR-03-02</td><td>Water immersion gel</td></tr><tr><td>Urea crystals</td><td>Sigma</td><td>U5128-5G</td><td>Optional: FLIM IRF</td></tr></tbody></table>

a label-free segmentation approach for intravital imaging of mammary tumor microenvironment

The ability to visualize complex and dynamic physiological interactions between numerous cell types and the extracellular matrix (ECM) within a live tumor microenvironment is an important step toward understanding mechanisms that regulate tumor progression. While this can be accomplished through current intravital imaging techniques, it remains challenging due to the heterogeneous nature of tissues and the need for spatial context within the experimental observation. To this end, we have developed an intravital imaging workflow that pairs collagen second harmonic generation imaging, endogenous fluorescence from the metabolic co-factor NAD(P)H, and fluorescence lifetime imaging microscopy (FLIM) as a means to non-invasively compartmentalize the tumor microenvironment into basic domains of the tumor nest, the surrounding stroma or ECM, and the vasculature. This non-invasive protocol details the step-by-step process ranging from the acquisition of time-lapse images of mammary tumor models to post-processing analysis and image segmentation. The primary advantage of this workflow is that it exploits metabolic signatures to contextualize the dynamically changing live tumor microenvironment without the use of exogenous fluorescent labels, making it advantageous for human patient-derived xenograft (PDX) models and future clinical use where extrinsic fluorophores are not readily applicable.

The installation of the MIW and basic experimental planning are the first steps in this process. This particular MIW design and protocol are more amenable to longitudinal studies19 and has been successfully utilized with both upright and inverted microscopes. In this case, an inverted microscope was used as it has resulted in greater image stability of the mammary gland with fewer breathing artifacts. In Figure 1A, we provide the dimensions of the rigid MIW and a grap...

Watch this Scientific Journal Video about A Label-Free Segmentation Approach for Intravital Imaging of Mammary Tumor Microenvironment at JoVE.com

A Label-Free Segmentation Approach for Intravital Imaging of Mammary Tumor Microenvironment

The intravital imaging method described here utilizes collagen second harmonic generation and endogenous fluorescence from the metabolic co-factor NAD(P)H to non-invasively segment an unlabeled tumor microenvironment into tumor, stromal, and vascular compartments for in-depth analysis of 4D intravital images.

The ability to visualize complex and dynamic physiological interactions between numerous cell types and the extracellular matrix (ECM) within a live tumor ...

a-label-free-segmentation-approach-for-intravital-imaging-mammary

Research

JoVE Journal

Cancer Research

2.3K Views.  University of Wisconsin-Madison. Intravitale beeldvorming stelt ons in staat om fysiologische interacties in de micro-omgeving van de tumor te observeren. En door deze methode te gebruiken, kunnen specifieke dynamische gedragingen die worden waargenomen, worden gecompartimenteerd met behulp van lokale weefselkenmerken. Dit protocol gebruikt alleen een alomtegenwoordig labelvrij signaal van collageenvezels of autofluorescente metabolieten om de TME te segmenteren, waardoor de hoeveelheid manipulaties voor de muis wordt gemini...

Video: A Label-Free Segmentation Approach for Intravital Imaging of Mammary Tumor Microenvironment

Assessing Tumor Microenvironment of Metastasis Doorway-Mediated Vascular Permeability Associated with Cancer Cell Dissemination using Intravital Imaging and Fixed Tissue Analysis

The most common cause of cancer related mortality is metastasis, a process that requires dissemination of cancer cells from the primary tumor to secondary sites. Recently, we established that cancer cell dissemination in primary breast cancer and at metastatic sites in the lung occurs only at doorways called Tumor MicroEnvironment of Metastasis (TMEM). TMEM doorway number is prognostic for distant recurrence of metastatic disease in breast cancer patients. TMEM doorways are composed of a cancer cell which over-expresses the actin regulatory protein Mena in direct contact with a perivascular, proangiogenic macrophage which expresses high levels of TIE2 and VEGF, where both of these cells are tightly bound to a blood vessel endothelial cell. Cancer cells can intravasate through TMEM doorways due to transient vascular permeability orchestrated by the joint activity of the TMEM-associated macrophage and the TMEM-associated Mena-expressing cancer cell. In this manuscript, we describe two methods for assessment of TMEM-mediated transient vascular permeability: intravital imaging and fixed tissue immunofluorescence. Although both methods have their advantages and disadvantages, combining the two may provide the most complete analyses of TMEM-mediated vascular permeability as well as microenvironmental prerequisites for TMEM function. Since the metastatic process in breast cancer, and possibly other types of cancer, involves cancer cell dissemination via TMEM doorways, it is essential to employ well established methods for the analysis of the TMEM doorway activity. The two methods described here provide a comprehensive approach to the analysis of TMEM doorway activity, either in na&#239;ve or pharmacologically treated animals, which is of paramount importance for pre-clinical trials of agents that prevent cancer cell dissemination via TMEM.

We describe two methods for assessing transient vascular permeability associated with tumor microenvironment of metastasis (TMEM) doorway function and cancer cell intravasation using intravenous injection of high-molecular weight (155 kDa) dextran in mice. The methods include intravital imaging in live animals and fixed tissue analysis using immunofluorescence.

The most common cause of cancer related mortality is metastasis, a process that requires dissemination of cancer cells from the primary tumor to secondary ...

Live Imaging of Drug Responses in the Tumor Microenvironment in Mouse Models of Breast Cancer

The tumor microenvironment plays a pivotal role in tumor initiation, progression, metastasis, and the response to anti-cancer therapies. Three-dimensional co-culture systems are frequently used to explicate tumor-stroma interactions, including their role in drug responses. However, many of the interactions that occur in vivo in the intact microenvironment cannot be completely replicated in these in vitro settings. Thus, direct visualization of these processes in real-time has become an important tool in understanding tumor responses to therapies and identifying the interactions between cancer cells and the stroma that can influence these responses. Here we provide a method for using spinning disk confocal microscopy of live, anesthetized mice to directly observe drug distribution, cancer cell responses and changes in tumor-stroma interactions following administration of systemic therapy in breast cancer models. We describe procedures for labeling different tumor components, treatment of animals for observing therapeutic responses, and the surgical procedure for exposing tumor tissues for imaging up to 40 hours. The results obtained from this protocol are time-lapse movies, in which such processes as drug infiltration, cancer cell death and stromal cell migration can be evaluated using image analysis software.

We describe a method for imaging response to anti-cancer treatment in vivo and at single cell resolution.

The tumor microenvironment plays a pivotal role in tumor initiation, progression, metastasis, and the response to anti-cancer therapies. Three-dimensional ...

Medicine

In vivo Imaging of Tumor Angiogenesis using Fluorescence Confocal Videomicroscopy

Fibered confocal fluorescence in vivo imaging with a fiber optic bundle uses the same principle as fluorescent confocal microscopy. It can excite fluorescent in situ elements through the optical fibers, and then record some of the emitted photons, via the same optical fibers. The light source is a laser that sends the exciting light through an element within the fiber bundle and as it scans over the sample, recreates an image pixel by pixel. As this scan is very fast, by combining it with dedicated image processing software, images in real time with a frequency of 12 frames/sec can be obtained.
We developed a technique to quantitatively characterize capillary morphology and function, using a confocal fluorescence videomicroscopy device. The first step in our experiment was to record 5 sec movies in the four quadrants of the tumor to visualize the capillary network. All movies were processed using software (ImageCell, Mauna Kea Technology, Paris France) that performs an automated segmentation of vessels around a chosen diameter (10 &mu;m in our case). Thus, we could quantify the 'functional capillary density', which is the ratio between the total vessel area and the total area of the image. This parameter was a surrogate marker for microvascular density, usually measured using pathology tools.
The second step was to record movies of the tumor over 20 min to quantify leakage of the macromolecular contrast agent through the capillary wall into the interstitium. By measuring the ratio of signal intensity in the interstitium over that in the vessels, an 'index leakage' was obtained, acting as a surrogate marker for capillary permeability.

In vivo Imaging of Tumor Angiogenesis using Fluorescence Confocal Videomicroscopy

In this paper, we present a method to analyze tumor microvessels in vivo using dynamic contrast-enhanced fluorescence videomicroscopy. Two quantitative parameters were acquired: functional capillary density reflecting the vascularity of the tumor, and index leakage reflecting the leakiness of the endothelial wall.

Fibered confocal fluorescence in vivo imaging with a fiber optic bundle uses the same principle as fluorescent confocal microscopy. It can excite ...

Quantifying the Brain Metastatic Tumor Micro-Environment using an Organ-On-A Chip 3D Model, Machine Learning, and Confocal Tomography

Brain metastases are the most lethal cancer lesions; 10-30% of all cancers metastasize to the brain, with a median survival of only ~5-20 months, depending on the cancer type. To reduce the brain metastatic tumor burden, gaps in basic and translational knowledge need to be addressed. Major challenges include a paucity of reproducible preclinical models and associated tools. Three-dimensional models of brain metastasis can yield the relevant molecular and phenotypic data used to address these needs when combined with dedicated analysis tools. Moreover, compared to murine models, organ-on-a-chip models of patient tumor cells traversing the blood brain barrier into the brain microenvironment generate results rapidly and are more interpretable with quantitative methods, thus amenable to high throughput testing. Here we describe and demonstrate the use of a novel 3D microfluidic blood brain niche (&#181;mBBN) platform where multiple elements of the niche can be cultured for an extended period (several days), fluorescently imaged by confocal microscopy, and the images reconstructed using an innovative confocal tomography technique; all aimed to understand the development of micro-metastasis and changes to the tumor micro-environment (TME) in a repeatable and quantitative manner. We demonstrate how to fabricate, seed, image, and analyze the cancer cells and TME cellular and humoral components, using this platform. Moreover, we show how artificial intelligence (AI) is used to identify the intrinsic phenotypic differences of cancer cells that are capable of transit through a model &#181;mBBN and to assign them an objective index of brain metastatic potential. The data sets generated by this method can be used to answer basic and translational questions about metastasis, the efficacy of therapeutic strategies, and the role of the TME in both.

Here, we present a protocol for preparing and culturing a blood brain barrier metastatic tumor micro-environment and then quantifying its state using confocal imaging and artificial intelligence (machine learning).

Brain metastases are the most lethal cancer lesions; 10-30% of all cancers metastasize to the brain, with a median survival of only ~5-20 months, ...

Real-time Imaging of Myeloid Cells Dynamics in ApcMin/+ Intestinal Tumors by Spinning Disk Confocal Microscopy

Myeloid cells are the most abundant immune cells within tumors and have been shown to promote tumor progression. Modern intravital imaging techniques enable the observation of live cellular behavior inside the organ but can be challenging in some types of cancer due to organ and tumor accessibility such as intestine. Direct observation of intestinal tumors has not been previously reported. A surgical procedure described here allows direct observation of myeloid cell dynamics within the intestinal tumors in live mice by using transgenic fluorescent reporter mice and injectable tracers or antibodies. For this purpose, a four-color, multi-region, micro-lensed spinning disk confocal microscope that allows long-term continuous imaging with rapid image acquisition has been used. ApcMin/+ mice that develop multiple adenomas in the small intestine are crossed with c-fms-EGFP mice to visualize myeloid cells and with ACTB-ECFP mice to visualize intestinal epithelial cells of the crypts. Procedures for labeling different tumor components, such as blood vessels and neutrophils, and the procedure for positioning the tumor for imaging through the serosal surface are also described. Time-lapse movies compiled from several hours of imaging allow the analysis of myeloid cell behavior in situ in the intestinal microenvironment.

Real-time Imaging of Myeloid Cells Dynamics in ApcMin/+ Intestinal Tumors by Spinning Disk Confocal Microscopy

By using transgenic reporter mice and injectable fluorescent labels, long-term intravital spinning disk confocal microscopy enables direct visualization of myeloid cell behavior into intestinal adenoma in the ApcMin/+ colorectal cancer model.

Myeloid cells are the most abundant immune cells within tumors and have been shown to promote tumor progression. Modern intravital imaging techniques ...

Immunology and Infection

In Vivo Imaging to Measure Spontaneous Lung Metastasis of Orthotopically-injected Breast Tumor Cells

Metastasis remains the primary cause of cancer-related death. The succession of events that characterize the metastatic cascade presents multiple opportunities for therapeutic intervention, and the ability to accurately model them in mice is critical to evaluate their effects. Here, a step-by-step protocol is presented for the establishment of orthotopic primary breast tumors and the subsequent monitoring of the establishment and growth of metastatic lesions in the lung using in vivo bioluminescence imaging. This methodology allows for the evaluation of treatment or its biological effects along the entire range of metastatic development, from primary tumor escape to outgrowth in the lungs. Breast orthotopic tumors are generated in mice via injection of a luciferase-labeled cell suspension in the 4th mammary gland. Tumors are allowed to grow and disseminate for a specific amount of time and are then surgically resected. Upon resection, spontaneous lung metastasis is detected, and the growth over time is monitored using in vivo bioluminescence imaging. At the desired experimental endpoint, lung tissue can be collected for downstream analysis. The treatment of established, clinically evident metastasis is critical to improve outcomes for stage IV cancer patients, and it can be evaluated through tail vein models of experimental lung metastasis. However, metastatic dissemination occurs early in breast cancer, and many patients have latent, subclinical disseminated disease after surgery. Utilization of spontaneous models such as this one provides the opportunity to study the whole spectrum of the disease, especially the systemic effects driven by treatment of the primary tumor such as pre-metastatic niche priming, and evaluate treatments on dormant and subclinical disease after surgery.

In Vivo Imaging to Measure Spontaneous Lung Metastasis of Orthotopically-injected Breast Tumor Cells

The manuscript describes a methodology for the establishment as well as longitudinal growth monitoring of spontaneous lung metastasis from orthotopically-injected breast tumors, amenable to intervention at all stages of the metastatic cascade.

Metastasis remains the primary cause of cancer-related death. The succession of events that characterize the metastatic cascade presents multiple ...

Extended Time-lapse Intravital Imaging of Real-time Multicellular Dynamics in the Tumor Microenvironment

In the tumor microenvironment, host stromal cells interact with tumor cells to promote tumor progression, angiogenesis, tumor cell dissemination and metastasis. Multicellular interactions in the tumor microenvironment can lead to transient events including directional tumor cell motility and vascular permeability. Quantification of tumor vascular permeability has frequently used end-point experiments to measure extravasation of vascular dyes. However, due to the transient nature of multicellular interactions and vascular permeability, the kinetics of these dynamic events cannot be discerned. By labeling cells and vasculature with injectable dyes or fluorescent proteins, high-resolution time-lapse intravital microscopy has allowed the direct, real-time visualization of transient events in the tumor microenvironment. Here we describe a method for using multiphoton microscopy to perform extended intravital imaging in live mice to directly visualize multicellular dynamics in the tumor microenvironment. This method details cellular labeling strategies, the surgical preparation of a mammary skin flap, the administration of injectable dyes or proteins by tail vein catheter and the acquisition of time-lapse images. The time-lapse sequences obtained from this method facilitate the visualization and quantitation of the kinetics of cellular events of motility and vascular permeability in the tumor microenvironment.

This protocol describes the use of multiphoton microscopy to perform extended time-lapse imaging of multicellular interactions in real time, in vivo at single cell resolution.

In the tumor microenvironment, host stromal cells interact with tumor cells to promote tumor progression, angiogenesis, tumor cell dissemination and ...

Intravital Microscopy of Tumor-associated Vasculature Using Advanced Dorsal Skinfold Window Chambers on Transgenic Fluorescent Mice

Tumor and tumor vessel development, as well as tumor response to therapeutics, are highly dynamic biological processes. Histology provides static information and is often not sufficient for a correct interpretation. Intravital evaluation, in which a process is followed in time, provides extra and often unexpected information. With the creation of transgenic animals expressing cell-specific markers and live cell tracers, improvements to imaging equipment, and the development of several imaging chambers, intravital microscopy has become an important tool to better understand biological processes. This paper describes an experimental design for the investigation of tumor vessel development and of therapeutic effects in a spatial and temporal manner. Using this setup, the stage of vessel development, tip cell and lumen formation, blood flow, extravasation, an established vascular bed, and vascular destruction can be visualized and followed. Furthermore, therapeutic effects, intratumoral fate, and the localization of chemotherapeutic compounds can also be followed.

This protocol describes the intravital imaging of transgenic mice expressing cell-specific fluorescent markers. Intravital imaging provides a non-invasive method for high-resolution observations in living animals using implantable windows through which the microscopic visualization of processes is possible. This method is especially useful to study longitudinal processes.

Tumor and tumor vessel development, as well as tumor response to therapeutics, are highly dynamic biological processes. Histology provides static ...

Longitudinal Intravital Microscopy Using a Mammary Imaging Window with Replaceable Lid

The branched structure of the mammary gland is highly dynamic and undergoes several phases of growth and remodeling after birth. Intravital microscopy in combination with skin flap surgery or implantation of imaging windows has been used to study the dynamics of the healthy mammary gland at different developmental stages. Most mammary imaging technologies are limited to a time frame of hours to days, whereas the majority of mammary gland remodeling processes occur in time frames of days to weeks. To study mammary gland remodeling, methods that allow optical access to the tissue of interest for extended time frames are required. Here, an improved version of the titanium mammary imaging window with a replaceable lid (R.MIW) is described that allows high-resolution imaging of the mammary gland with a cellular resolution for up to several weeks. Importantly, the R.MIW provides tissue access over the entire duration of the intravital imaging experiment and could therefore be used for local tissue manipulation, labeling, drug administration, or image-guided microdissection. Taken together, the R.MIW enables high-resolution characterization of the cellular dynamics during mammary gland development, homeostasis, and disease.

This protocol describes a novel mammary imaging window with a replaceable lid (R.MIW). Intravital microscopy after implantation of the R.MIW allows for longitudinal and multi-day imaging of the healthy and diseased mammary gland with a cellular resolution during the different developmental stages.

The branched structure of the mammary gland is highly dynamic and undergoes several phases of growth and remodeling after birth. Intravital microscopy in ...

Biology

Intravital Subcellular Microscopy of the Mammary Gland

The mammary gland constitutes a model par excellence for investigating epithelial functions, including tissue remodeling, cell polarity, and secretory mechanisms. During pregnancy, the gland expands from a primitive ductal tree embedded in a fat pad to a highly branched alveolar network primed for the formation and secretion of colostrum and milk. Post-partum, the gland supplies all the nutrients required for neonatal survival, including membrane-coated lipid droplets (LDs), proteins, carbohydrates, ions, and water. Various milk components, including lactose, casein micelles, and skim-milk proteins, are synthesized within the alveolar cells and secreted from vesicles by exocytosis at the apical surface. LDs are transported from sites of synthesis in the rough endoplasmic reticulum to the cell apex, coated with cellular membranes, and secreted by a unique apocrine mechanism. Other preformed constituents, including antibodies and hormones, are transported from the serosal side of the epithelium into milk by transcytosis. These processes are amenable to intravital microscopy because the mammary gland is a skin gland and, therefore, directly accessible to experimental manipulation. In this paper, a facile procedure is described to investigate the kinetics of LD secretion in situ, in real-time, in live anesthetized mice. Boron-dipyrromethene (BODIPY)665/676&#160;or monodansylpentane are used to label the neutral lipid fraction of transgenic mice, which either express soluble EGFP (enhanced green fluorescent protein) in the cytoplasm, or a membrane-targeted peptide fused to either EGFP or tdTomato. The membrane-tagged fusion proteins serve as markers of cell surfaces, and the lipid dyes resolve LDs &#8805; 0.7 &#181;m. Time-lapse images can be recorded by standard laser scanning confocal microscopy down to a depth of 15-25 &#181;m or by multiphoton microscopy for imaging deeper in the tissue. The mammary gland may be bathed with pharmacological agents or fluorescent dyes throughout the surgery, providing a platform for acute experimental manipulations as required.

The present protocol describes a facile technique for the intravital imaging of the lactating mouse mammary gland by laser scanning confocal and multiphoton microscopy.

The mammary gland constitutes a model par excellence for investigating epithelial functions, including tissue remodeling, cell polarity, and secretory ...

A Label-Free Segmentation Approach for Intravital Imaging of Mammary Tumor Microenvironment

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