This work was supported by US Department of Defense (BC075554 to B.G. and BC061403 to D.K.), US National Institutes of Health (U54GM064346 to J.v.R.; CA100324 to J.C., J.E.S. and J.W.; CA77522 to J.E.S., U54CA126511 to J.C. and B.G.). We thank D. Entenberg for help with microscopy, M. Rottenkolber for help in fabrication of the imaging box and J. Pollard (Albert Einstein College of Medicine) for the F4/80 antibody.

1. Generation of fluorescent tumors using injection of tumor cells into the mammary fat pad:
<ol>
<li>Grow a cell line expressing Dendra2 protein (as a cytoplasmic marker) to 40 - 80% confluency.</li>
<li>Rinse dishes at least 3 times with PBS w/o Ca2+ or Mg2+.</li>
<li>Add 3 ml of trypsin per 10 cm dish and incubate at 37 &deg;C until most of the cells detach and then hit the dish against a flat surface to shake it.</li>
<li>Rinse all the cells off the dish, and use a scraper (rubber policeman) to collect matrix as well.&nbsp; Add the 5 ml of PBS.&nbsp;Take an aliquot to count during centrifugation.</li>
<li>Centrifuge at 800 g for 5 min.</li>
<li>Aspirate and resuspend in PBS to a concentration of 5- 10x 106/ml.&nbsp; Store on ice until injected (inject within 30 min).</li>
<li>Place a cage containing 4-5 week old female immune deficient mice (e.g. SCID) inside a sterile hood.</li>
<li>Spray the area around the 4th (abdominal) nipple with 70% ethanol.</li>
<li>Inject 0.1 ml into mammary fat pad. If an assistant can be found, one person can hold the mouse in place while the other inserts the needle inside the mammary fat pad. If you are injecting by yourself, put the mouse under light anesthesia using isoflurane.</li>
<li>Once the tumor has grown to 5-7 mm, the Mammary Imaging Window (MIW) should be inserted. Note: Alternatively, for some applications, the MIW can be inserted on top of the healthy mammary fat pad and the cells can be injected afterwards. That approach allows imaging of transiently transfected cells in a physiological environment.</li>
</ol>
2. Manual fabrication of the Mammary Imaging Window (MIW):
<ol>
<li>MIWs (Figure 1A) are made of tissue grade plastic to guarantee biocompatibility. For manual construction, we use 5 or 10 cm dishes.</li>
<li>Put latex gloves on.</li>
<li>Heat up a tissue-culture dish and creat a curved surface by pushing a rounded, hard object against the heated dish (we use a 1-inch dremmel bit for this).</li>
<li>Cut out the center of the dish using a heated razor blade. </li>
<li>Use a small, cone-shaped dremmel bit to make a hole in the center of the dome-shaped plastic base (6-7mm in diameter). </li>
<li>Make the edges of the plastic base completely smooth by sanding it with the dremmel and further filing it. </li>
<li>File the top of the plastic base making a flat surface for the glass coverslip. The diameter of the filed, flat surface should be 9-10mm.</li>
<li>Glue the 8mm circular glass coverslip (number 1) on the flattened surface using the superglue (cyanoacrylate adhesive).</li>
<li>Wait for the glue to dry (15 minutes).</li>
<li>Use a heated 26G needle to make eight suturing holes puncturing from the outside (where the glass is) to the inside of the base. Holes should be evenly distributed around the coverslip, .5-1mm away from the edge of the coverslip. </li>
<li>Widen the holes using a 5-0 suturing needle.</li>
<li>Puncturing holes in the plastic base will make the inner surface uneven. Using sand paper, make this surface completely smooth.</li>
<li>Change the gloves and brush of small plastic particles from the MIW using a small brush.</li>
<li>Wash the MIW with deionized water.</li>
<li>Wash the MIW using 70% ethanol. Use a Q-tip to clean the glass so it is completely transparent. If there are foggy spots present from the glue vapor, carefully use acetone with a Q-tip.</li>
<li>Sterilize the MIW by UV exposure for 3 h at each side.</li>
</ol>
Semi-Manual fabrication of the Mammary Imaging Window (MIW):
In order to fabricate the plastic base, we are currently using silicone rubber casting molds created using hand-made MIWs. The mold is comprised of two parts of silicone rubber that make an exact replica of both the front and back of the original when filled with polyester resin and then immediately joined together.&nbsp;The liquid polyester resin is mixed together 9:10 and results in a hard structure when fully cured in 48h. The plastic is UV protected and archival without breaking down or becoming yellow over time. After the base is cured, steps 2.8-2.16 are done the same way as for manually-made MIWs.
3. Insertion of the MIW:
<ol>
<li>The area around the 4th nipple should have a small (5-7 mm diameter) tumor several days/weeks after injection of tumor cells depending on the cell type (Figure 1B). How much time has passed after injection of cells expressing Dendra2 photoswitchable protein or other fluorescent proteins depends on the cell line used. The tumor should not be visibly necrotic, and should have an intact skin with hair on top of the tumor. Mice with necrotic tumors should be euthanized.</li>
<li>Prepare the sterile space for the surgery- lay down a piece of sterile cloth inside a sterile hood. Put a piece of sterile gauze in the middle, and lay a few MIWs and sterile instruments around it: we use small standard scissors, spring scissors, microdissecting tweezers, microdissecting forceps, needle holder. Keep sterile Q-tips, a bottle of 70% ethanol and sterile gloves in the corner of the hood.</li>
<li>The mouse is anesthetized in the sterile hood using IP injection of 2.5% avertin (20 &mu;l/g) in HBSS or, alternatively 10 &mu;g/kg ketamine + 10 &mu;g/g xylazine (contact your Animal Care Institute for approvals and ordering). Note: Avertin solution should be prepared bi-weekly, filter sterilized using a 0.22 &mu;m filter and stored as 500 &mu;l aliquots at 4 &deg;C (in the dark).</li>
<li>Remove hair by shaving the area above the tumor using a small animal shaver.</li>
<li>Remove the rest of the hair using Nair hair removal cream (available in any drugstore). Clean the skin using ethanol-dipped Q-tips.</li>
<li>Transfer the animal onto the sterile gauze and apply ophthalmic ointment to the eyes to keep them from drying and infection.</li>
<li>Sterilize the skin with betadine and clean with 70% ethanol.</li>
<li>Transfer the animal onto a sterile surgical cloth inside the sterile hood. </li>
<li>Pull the skin immediately medial to the nipple using forceps and cut ~2mm incision in the skin.</li>
<li>Separate the underlying mammary fat pad from the skin using dissection scissors and forceps. The skin and hole stretch during this separation step to a size that will accommodate insertion of the MIW.</li>
<li>If accidently a vessel is hit during sugery, use a sterile Q-tips to remove any blood that is produced/to stop the bleeding from becoming excessive. </li>
<li>Insert the MIW such that there is skin on top of the MIW base and suture in place using non-absorbable thread and reverse cutting needle.</li>
<li>Use tissue adhesive or cyanoacrylate to fill in the suturing holes and secure the MIW to the skin.</li>
<li>Add TMP-SMX antibiotic mix: (Sulfamethoxazole 0.6 mg/ml, Trimethoprim 0.12 mg/ml) into the cage water bottle for 3 days before and after the surgery. </li>
<li>The animal stays under anesthesia for 1-4h after avertin IP injection. Since the surgery commonly takes ~45min, it is important to help the animal recover afterwards:</li>
<li>Place a hand heating pad (37 &deg;C) (available in drugstores) on the bottom of the cage and cover it with gauze.</li>
<li>Keep the animal on top of the pad until it turns on its stomach and starts walking.</li>
<li>If the animal is unconscious for more than 3h, inject 0.3 ml of HBSS intraperitoneally for rehydration.</li>
<li>The animal is allowed to recover over the next 3-4 days before the first imaging session takes place.</li>
</ol>
4. Imaging Box Construction
The imaging box assures that the MIW is sitting flat, right above the microscope objective. It also facilitates temperature and anesthesia control through constant airflow of isoflurane. The box is made of plexiglass and glued together using plastic weld. It is fitted for the specific microscope stage and therefore the shape of its bottom varies depending on the microscope used (Figure 2 shows the scheme of the box fitted to Leica SP5 microscope stage). Dimensions of the box in centimeters are l=11.4 cm, w=7.6 cm, h=4.4 cm (Figure 2A). The bottom of the box consists of a hollow plate, 12.7x8.4 cm in size, which fits inside the Leica SP5 microscope stage, and two sliding &ldquo;doors&rdquo;, 5.7 X 5.7 cm_each, which form a circular opening (d=2.22 cm) when closed. The MIW fits into this opening. The front piece contains the inlet hole for the anesthesia delivery while one of the side pieces contains an outlet hole which leads to the vacuum. Note that the condenser and the slide holder need to be removed prior to the imaging box placement.
5. Imaging Box Use
<ol>
<li>Place the animal under anesthesia with isoflurane and apply ophthalmic ointment to the eyes.</li>
<li>Attach the isoflurane exhaust from the anesthesia machine to the imaging box front, on the outside of the inlet hole (Figure 2B).</li>
<li>Attach a second tube to the outlet hole of the imaging box. This tube should be attached to the gas filter and then to the vacuum. </li>
<li>Open the box lid and open the box doors and place the animal, MIW side facing the bottom, and sliding the doors into the box.</li>
<li>Hold the box and adjust the bottom doors so that the MIW base is held and immobilized between them. The MIW base should be at the same level as the imaging box doors, while the MIW coverslip should be just a few millimeters below this level. Make sure that the positioning of the MIW between the bottom sliding doors ensures that the coverslip (glass portion of the MIW) lies flat, parallel to the bottom doors the box. This will ensure proper focusing. </li>
<li>Lower the microscope objective, to allow space for placement of the box on top of the microscope stage.</li>
<li>Place the box on the microscope stage, and adjust the stage so that the MIW coverslip is above the objective. </li>
<li>Focus the objective and start imaging.</li>
<li>While using isoflurane, the animal should be monitored by visual inspection of the breathing frequency. This can be done visually, or by monitoring the frequency of breathing artifacts appearing during imaging collection (these can interfere with data acquisition). The MouseOx pulse oximetry system (Starr life Sciences Corp) has been used succesfully. That way the data can be gathered continuously without turning the lights on in the room to check on the animal. Note: After each imaging session, animal recovery should be facilitated by wrapping a small hand heating pad in gauze or kimwipes and putting it under the animal in the cage. Note: If an immersion objective is used to image through the MIW, the immersion medium (glycerol, water, oil) should be cleaned off the imaging window. The MIW should be inspected for any cracks or other damage that might have occurred during imaging. This is critical for collecting data during multiple imaging sessions. </li>
</ol>
6. Photoswitching and imaging of Dendra2-labeled cells
The procedure is described for the Leica SP5 confocal microscope; the laser power at the focal plane, available objectives and software options vary when using other confocal microscopes or multiphoton imaging set up, and therefore the protocol described below should be used as a guide to optimize the experiment on your microscope.
<ol>
<li>By observing the tumor through the eyepiece (10x) with the green fluorescence filter, locate major blood vessels which are visibly flowing and laying flat in the same focal plane.</li>
<li>Position one of the vessels in the center of the field and switch to PMT detection.</li>
<li>Set up sequential imaging collection for two channels. One of the channels uses 488nm laser line (10% power), and collects scattering from extracellular matrix (480-495nm) and emission from the green form of Dendra2 (505-540nm). The second channel uses the 543nm laser line (90% power) and collects emission from the red form of Dendra2 (555-600nm). Collect 3D images pre-photoswitching.</li>
<li>Use the ROI scan option to photoswitch a chosen population of cells using the 405nm laser line (30% power, 20-40 scans). Collect emission of the red form of the protein to monitor the switching.</li>
<li>Using the same sequential routine as in 6.3, collect post-switching 3D images.</li>
<li>If photoswitching more than one region in the tumor, it is advised to take pictures through the oculars using a digital camera, in both green and red channels (Figure 3A, B). This will help in the orientation during the subsequent imaging sessions. Note: Additionally, blood vessels can be temporarily labeled using tail-vein injection of fluorescent dextran (Cascade Blue or Alexa Fluor 647, both 10kDa). A few hours after the injection, dextrans will leave the blood vessels and permanently label the macrophages.</li>
</ol>
Representative Results:
Figure 3C shows a rectangular photoswitched region (red) oriented orthogonally relative to the blood vessel (no fluorescence). Non-photoswitched cells are green, while the scattering from the extracellular matrix is purple. Image is a maximum intensity projection of four images along the Z-axis (20-50 &mu;m depth).

<ol>
	<li>Farina, K. L., Wyckoff, J. B., Rivera, J., Lee, H., Segall, J. E., Condeelis, J. S., Jones, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+motility+of+tumor+cells+visualized+in+living+intact+primary+tumors+using+green+fluorescent+protein.">Cell motility of tumor cells visualized in living intact primary tumors using green fluorescent protein.</a> Cancer Res. 58, 2528-2532 (1998).</li><li>Naumov, G. N., Wilson, S. M., MacDonald, I., Schmidt, E. E., Morris, V. L., Groom, A. C., Hoffman, R. M., Chambers, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+expression+of+green+fluorescent+protein,+coupled+with+high-resolution+in+vivo+videomicroscopy,+to+monitor+steps+in+tumor+metastasis.">Cellular expression of green fluorescent protein, coupled with high-resolution in vivo videomicroscopy, to monitor steps in tumor metastasis.</a> J Cell Sci. 112, 1835-1842 (1999).</li><li>Brown, E. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+measurement+of+gene+expression,+angiogenesis+and+physiological+function+in+tumors+using+multiphoton+laser+scanning+microscopy.">In vivo measurement of gene expression, angiogenesis and physiological function in tumors using multiphoton laser scanning microscopy.</a> Nature Med. 7, 864-868 (2001).</li><li>Wang, W., Wyckoff, J. B., Frohlich, V. C., Oleynikov, Y., Huttelmaier, S., Zavadil, J., Cermak, L., Bottinger, E. P., Singer, R. H., White, J. G., Segall, J. E., Condeelis, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+cell+behavior+in+metastatic+primary+mammary+tumors+correlated+with+gene+expression+patterns+revealed+by+molecular+profiling.">Single cell behavior in metastatic primary mammary tumors correlated with gene expression patterns revealed by molecular profiling.</a> Cancer Res. 62, 6278-6288 (2002).</li><li>Sidani, M., Wyckoff, J., Xue, C., Segall, J. E., Condeelis, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Probing+the+microenvironment+of+mammary+tumors+using+multiphoton+microscopy.">Probing the microenvironment of mammary tumors using multiphoton microscopy.</a> J Mammary Gland Biol Neoplasia. 11, 151-163 (2006).</li><li>Wyckoff, J. B., Wang, Y., Lin, E. Y., Li, J. F., Goswami, S., Stanley, E. R., Segall, J. E., Pollard, J. W., Condeelis, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+visualization+of+macrophage-assisted+tumor+cell+intravasation+in+mammary+tumors.">Direct visualization of macrophage-assisted tumor cell intravasation in mammary tumors.</a> Cancer Res. 67, 2649-2656 (2007).</li><li>Egeblad, M., Ewald, A. J., Askautrud, H. A., Truitt, M. L., Welm, B. E., Bainbridge, E., Peeters, G., Krummel, M. F., Werb, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualizing+stromal+cell+dynamics+in+different+tumor+microenvironments+by+spinning+disk+confocal+microscopy.">Visualizing stromal cell dynamics in different tumor microenvironments by spinning disk confocal microscopy.</a> Dis Model Mech. 1, 155-167 (2008).</li><li>Mempel, T. R., Henrickson, S. E., von Adrian, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T-cell+priming+by+dendritic+cells+in+lymph+nodes+occurs+in+three+distinct+phases.">T-cell priming by dendritic cells in lymph nodes occurs in three distinct phases.</a> Nature. 427, 154-159 (2004).</li><li>Miller, M. J., Wei, S. H., Cahalan, M. D., Parker, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autonomous+T+cell+trafficking+examined+in+vivo+with+intravital+two-photon+microscopy.">Autonomous T cell trafficking examined in vivo with intravital two-photon microscopy.</a> Proc Natl Acad Sci USA. 100, 2604-2609 (2003).</li><li>Kedrin, D., Gligorijevic, B., Wyckoff, J., Verkhusha, V. V., Condeelis, J., Segall, J. E., van Rheenen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravital+imaging+of+metastatic+behavior+through+a+mammary+imaging+window.">Intravital imaging of metastatic behavior through a mammary imaging window.</a> Nat. Methods. 5, 1019-1021 (2008).</li><li>Chudakov, D. M., Lukyanov, S., Lukyanov, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tracking+intracellular+protein+movements+using+photoswitchable+fluorescent+proteins+PS-CFP2+and+Dendra2.">Tracking intracellular protein movements using photoswitchable fluorescent proteins PS-CFP2 and Dendra2.</a> Nat. Protocols. 2, 2024-2032 (2007).</li></ol>

<table><tbody><tr><td>PBS w/o Ca, Mg</td><td>GIBCO, by Life Technologies</td><td>14190-144</td><td></td></tr><tr><td>Trypsin</td><td>GIBCO, by Life Technologies</td><td>25300</td><td></td></tr><tr><td>HBSS</td><td>GIBCO, by Life Technologies</td><td>14025</td><td></td></tr><tr><td>DMEM media (MDA-MB-231 cells)</td><td>GIBCO, by Life Technologies</td><td>11965</td><td></td></tr><tr><td>Isoflurane(Aerrane)</td><td>Baxter Internationl Inc.</td><td># NDC 10019-773-40</td><td></td></tr><tr><td>SCID mouse</td><td>National Cancer Institute</td><td>N/A</td><td></td></tr><tr><td>Culture dishes</td><td>BD Biosciences</td><td>353003</td><td>Custom Order</td></tr><tr><td>Circular coverslip 8 mm</td><td>Scientific, Inc.</td><td>8CIR-1-FIS</td><td></td></tr></tbody></table>

dendra2 photoswitching through the mammary imaging window

In the last decade, intravital microscopy of breast tumors in mice and rats at single-cell resolution1-4 has resulted in important insights into mechanisms of metastatic behavior such as migration, invasion and intravasation of tumor cells5, 6, angiogenesis3 and immune cells response7-9. We have recently reported a technique to image orthotopic mammary carcinomas over multiple intravital imaging sessions in living mice10. For this, we have developed a Mammary Imaging Window (MIW) and optimized imaging parameters for Dendra211 photoswitching and imaging in vivo. Here, we describe the protocol for the manufacturing of MIW, insertion of the MIW on top of a tumor and imaging of the Dendra2- labeled tumor cells using a custom built imaging box. This protocol can be used to image the metastatic behavior of tumor cells in distinct microenvironments in tumors and allows for long term imaging of blood vessels, tumor cells and host cells.

Watch this Scientific Journal Video about Dendra2 Photoswitching through the Mammary Imaging Window at JoVE.com

Dendra2 Photoswitching through the Mammary Imaging Window

Intravital photoswitching and tracking of Dendra2-labeled tumor cells through the Mammary Imaging Window is a technique which allows us to image the metastatic behavior of tumor cells in chosen tumor microenvironments over a timescale of days.

In the last decade, intravital microscopy of breast tumors in mice and rats at single-cell resolution1-4  has resulted in important insights into ...

dendra2-photoswitching-through-the-mammary-imaging-window

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

11.6K Views.  Albert Einstein College of Medicine - Yeshiva University. Intravital photoswitching and tracking of Dendra2-labeled tumor cells through the Mammary Imaging Window is a technique which allows us to image the metastatic behavior of tumor cells in chosen tumor microenvironments over a timescale of days.

Video: Dendra2 Photoswitching through the Mammary Imaging Window

A Method for 2-Photon Imaging of Blood Flow in the Neocortex through a Cranial Window

The ability to image the cerebral vasculature (from large vessels to capillaries) and record blood flow dynamics in the intact brain of living rodents is a powerful technique. Using in vivo 2-photon microscopy through a cranial window it is possible to image fluorescent dyes injected intravenously. This permits one to image the cortical vasculature and also to obtain measurements of blood flow. This technique was originally developed by David Kleinfeld and Winfried Denk. The method can be used to study blood flow dynamics during or after cerebral ischemia, in neurodegenerative disorders, in brain tumors, or in normal brain physiology. For example, it has been used to study how stroke causes shifts in blood flow direction and changes in red blood cell velocity or flux in and around the infarct. Here we demonstrate how to use 2-photon microscopy to image blood flow dynamics in the neocortex of living mice using fluorescent dyes injected into the tail vein.

Cortical blood flow dynamics can be studied in vivo by imaging fluorescent dextran dyes injected into the tail vein of rodents with 2-photon microscopy. This video shows how to image blood flow dynamics in neocortex of mice through a glass-covered cranial window preparation.

The ability to image the cerebral vasculature (from large vessels to capillaries) and record blood flow dynamics in the intact brain of living rodents is ...

Longitudinal Intravital Imaging Through Clear Silicone Windows

Intravital microscopy (IVM) enables visualization of cell movement, division, and death at single-cell resolution. IVM through surgically inserted imaging windows is particularly powerful because it allows longitudinal observation of the same tissue over days to weeks. Typical imaging windows comprise a glass coverslip in a biocompatible metal frame sutured to the mouse&#8217;s skin. These windows can interfere with the free movement of the mice, elicit a strong inflammatory response, and fail due to broken glass or torn sutures, any of which may necessitate euthanasia. To address these issues, windows for long-term abdominal organ and mammary gland imaging&#160;were developed from a thin film of polydimethylsiloxane (PDMS), an optically clear silicone polymer previously used for cranial imaging windows. These windows can be glued directly to the tissues, reducing the time needed for insertion. PDMS is flexible, contributing to its durability in mice over time&#8212;up to 35 days have been tested. Longitudinal imaging is imaging of the same tissue region during separate sessions. A stainless-steel grid was embedded within the windows to localize the same region, allowing the visualization of dynamic processes (like mammary gland involution) at the same locations, days apart. This silicone window also allowed monitoring of single disseminated cancer cells developing into micro-metastases over time. The silicone windows used in this study are simpler to insert than metal-framed glass windows and cause limited inflammation of the imaged tissues. Moreover, embedded grids allow for straightforward tracking of the same tissue region in repeated imaging sessions.

An approach is here presented for long-term intravital imaging using optically clear, silicone windows that can be glued directly to the tissue/organ of interest and the skin.&#160;These windows are cheaper and more versatile than others currently used in the field, and the surgical insertion causes limited inflammation and distress to the animals.

Intravital microscopy (IVM) enables visualization of cell movement, division, and death at single-cell resolution. IVM through surgically inserted imaging ...

Cancer Research

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 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 ...

Murine Pancreatic Imaging Using Implanted Stabilized Window

Stabilized Window for Intravital Imaging of the Murine Pancreas

The physiology and pathophysiology of the pancreas are complex. Diseases of the pancreas, such as pancreatitis and pancreatic adenocarcinoma (PDAC) have high morbidity and mortality. Intravital imaging (IVI) is a powerful technique enabling the high-resolution imaging of tissues in both healthy and diseased states, allowing for real-time observation of cell dynamics. IVI of the murine pancreas presents significant challenges due to the deep visceral and compliant nature of the organ, which make it highly prone to damage and motion artifacts. 
Described here is the process of implantation of the Stabilized Window for Intravital imaging of the murine Pancreas (SWIP). The SWIP allows IVI of the murine pancreas in normal healthy states, during the transformation from the healthy pancreas to acute pancreatitis induced by cerulein, and in malignant states such as pancreatic tumors. In conjunction with genetically labeled cells or the administration of fluorescent dyes, the SWIP enables the measurement of single-cell and subcellular dynamics (including single-cell and collective migration) as well as serial imaging of the same region of interest over multiple days. 
The ability to capture tumor cell migration is of particular importance as the primary cause of cancer-related mortality in PDAC is the overwhelming metastatic burden. Understanding the physiological dynamics of metastasis in PDAC is a critical unmet need and crucial for improving patient prognosis. Overall, the SWIP provides improved imaging stability and expands the application of IVI in the healthy pancreas and malignant pancreas diseases.

Author Spotlight: Revolutionizing Pancreatic Disease Understanding Through Advanced Intravital Imaging 

We present a protocol for the surgical implantation of a stabilized indwelling optical window for subcellular-resolution imaging of the murine pancreas, allowing serial and longitudinal studies of the healthy and diseased pancreas.

The physiology and pathophysiology of the pancreas are complex. Diseases of the pancreas, such as pancreatitis and pancreatic adenocarcinoma (PDAC) have ...

Intraductal Delivery to the Rabbit Mammary Gland

Localized intraductal treatments for breast cancer offer potential advantages, including efficient delivery to the tumor and reduced systemic toxicity and adverse effects1,2,3,4,5,6,7. However, several challenges remain before these treatments can be applied more widely. The development and validation of intraductal therapeutics in an appropriate animal model facilitate the development of intraductal therapeutic strategies for patients. While the mouse mammary gland has been widely used as a model system of mammary development and tumorigenesis, the anatomy is distinct from the human gland. A larger animal model, such as the rabbit, may serve as a better model for mammary gland structure and intraductal therapeutic development. In contrast to mice, in which ten ductal trees are spatially distributed along the body axis, each terminating in a separate teat, the rabbit mammary gland more closely resembles the human gland, with multiple overlapping ductal systems that exit through separate openings in one teat. Here, we present minimally invasive methods for the delivery of reagents directly into the rabbit mammary duct and for visualization of the delivery itself with high-resolution ultrasound imaging.

Here, we describe a technique for the localized delivery of reagents to the rabbit mammary gland via an intraductal injection. In addition, we describe a protocol for visualization and the confirmation of delivery by high-resolution ultrasound imaging of contrast agents.

Localized intraductal treatments for breast cancer offer potential advantages, including efficient delivery to the tumor and reduced systemic toxicity and ...

Bioengineering

Intraductal Injection for Localized Drug Delivery to the Mouse Mammary Gland

Herein we describe a protocol to deliver various reagents to the mouse mammary gland via intraductal injections. Localized drug delivery and knock-down of genes within the mammary epithelium has been difficult to achieve due to the lack of appropriate targeting molecules that are independent of developmental stages such as pregnancy and lactation. Herein, we describe a technique for localized delivery of reagents to the mammary gland at any stage in adulthood via intraductal injection into the nipples of mice. The injections can be performed on live mice, under anesthesia, and allow for a non-invasive and localized drug delivery to the mammary gland. Furthermore, the injections can be repeated over several months without damaging the nipple. Vital dyes such as Evans Blue are very helpful to learn the technique. Upon intraductal injection of the blue dye, the entire ductal tree becomes visible to the eye. Furthermore, fluorescently labeled reagents also allow for visualization and distribution within the mammary gland. This technique is adaptable for a variety of compounds including siRNA, chemotherapeutic agents, and small molecules.

A protocol for the non-invasive intraductal delivery of aqueous reagents to the mouse mammary gland is described. The method takes advantage of localized injection into the nipples of mammary glands targeting mammary ducts specifically. This technique is adaptable for a variety of compounds including siRNA, chemotherapeutic agents and small molecules.

Herein we describe a protocol to deliver various reagents to the mouse mammary gland via intraductal injections. Localized drug delivery and knock-down of ...

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 ...

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 ...

Intraductal Injection: Delivering Injection Mix into the Ducts of the Mouse Mammary Gland

Source: Xiang, D., et al. <a href="https://jove.unicartagenaproxy.elogim.com/v/59502/modeling-breast-cancer-via-an-intraductal-injection-cre-expressing">Modeling Breast Cancer via an Intraductal Injection of Cre-expressing Adenovirus into the Mouse Mammary Gland.</a> J. Vis. Exp (2019).
This video describes an intraductal injection technique where we deliver injection mix into the ducts of mouse mammary glands.

Source: Xiang, D., et al. Modeling Breast Cancer via an Intraductal Injection of Cre-expressing Adenovirus into the Mouse Mammary Gland. J. Vis. Exp ...

JoVE Encyclopedia of Experiments

Breast Cancer

Procedures and techniques

Cranial Window-based Intravital Imaging in a Mouse Model: An Imaging Technique to Study the Behavior of Pre-injected Cancer Cells in the Brain of a Murine Model

Source: Alieva, M. and Rios, A. C. <a href="https://jove.unicartagenaproxy.elogim.com/v/59278/longitudinal-intravital-imaging-brain-tumor-cell-behavior-response-to">Longitudinal intravital imaging of brain tumor cell behavior in response to an invasive surgical biopsy.</a>&#160;J. Vis. Exp.&#160;(2019)
In this video, we perform intravital imaging of pre-injected cancer cells in a mouse model via an implanted cranial window. This&#160;high-resolution time-lapse imaging technique enables the study of dynamic behavior of cancer cells in the live mouse.

Source: Alieva, M. and Rios, A. C. Longitudinal intravital imaging of brain tumor cell behavior in response to an invasive surgical biopsy.&#160;J. Vis. ...