We would like to thank the Analytical Imaging Facility (AIF) in the Albert Einstein College of Medicine for imaging support. This work was supported by grants from the NCI (P30CA013330, CA150344, CA 100324 and CA216248), the SIG 1S10OD019961-01, the Gruss-Lipper Biophotonics Center and its Integrated Imaging Program, and Montefiore&#8217;s Ruth L. Kirschstein T32 Training Grant of Surgeons for the Study of the Tumor Microenvironment (CA200561).

GSK co-wrote the manuscript, performed imaging for figure 1C and 3B, developed fixed tissue analysis protocol, and analyzed and interpreted all data; JMP co-wrote the manuscript, and performed the surgery and intravital imaging for Figure 1B,2C and 3A; LB &#38; AC performed the surgery and intravital imaging for Figure 2B; RJ performed the surgery and intravital imaging for Figure 2A; JSC co-wrote the manuscript and analyzed and interpreted all data; MHO co-wrote the manuscript and analyzed and interpreted all data; and DE performed the surgery and intravital imaging for Figure 2D, co-wrote the manuscript, developed fixed tissue analysis and intravital imaging protocols, and analyzed and interpreted all data.

All experiments using live animals must be conducted in accordance with animal use and care guidelines and regulations. The procedures described in this study were carried out in accordance with the National Institutes of Health regulations concerning the care and use of experimental animals and with the approval of the Albert Einstein College of Medicine Animal Care and Use Committee (IACUC).
1. Evaluation of &#34;bursting permeability&#34;&#160;using live animal imaging
<ol>
	<li>Transplantation of syngeneic breast tumors into mouse hosts with fluorescent macrophages
	<ol>
		<li>Generate pieces of tumor tissue suitable for transplantation.
		<ol>
			<li>Generate fluorescently-labeled tumors in mouse mammary cancer models by crossing the spontaneous, autochthonous, genetically engineered mouse mammary cancer model MMTV-PyMT mice with transgenic mice expressing fluorescent reporters38,39 [e.g., enhanced green fluorescent protein (EGFP), enhanced cyan fluorescent protein (ECFP), or Dendra2].</li>
			<li>Allow the MMTV-PyMT mice with fluorescently labeled tumors to grow to a size of no larger than 2 cm (approximately 10-12 weeks of age).</li>
			<li>Euthanize the tumor bearing MMTV-PyMT mice by placing them into a chamber with 5% isoflurane until 30 seconds after all respirations stop.</li>
			<li>Perform a cervical dislocation.</li>
			<li>Remove hair from the euthanized mouse&#8217;s abdomen using a topical depilatory cream.</li>
			<li>Place a Petri dish with DMEM/F12 cell culture medium on ice.</li>
			<li>Place the mouse and Petri dish on ice into the fume hood.</li>
			<li>Sanitize the mouse&#8217;s abdomen with 70% ethyl alcohol.</li>
			<li>Using sterile gloves and surgical tools (sterilized scissors, forceps, and blade) remove the tumors and place them into the Petri dish.</li>
			<li>Cut up the tumor into small pieces (~2 mm x 2 mm x 2mm in size), discarding any necrotic portions, while they are in the Petri dish.</li>
		</ol>
		</li>
		<li>Transplant the tumor pieces into recipient hosts.
		<ol>
			<li>Raise mice with genetically engineered fluorescently-labeled macrophages, e.g., MacGreen40 or MacBlue 41 mice (Csf1r-GAL4VP16/UAS-ECFP)</li>
			<li>Allow the FVB mice with fluorescently labeled macrophages to grow to an age of ~4-6 weeks).</li>
			<li>Anesthetize the FVB mice with fluorescently labeled macrophages in a chamber using 5% isoflurane with oxygen as a carrier gas.</li>
			<li>Reduce the anesthesia to ~3% isoflurane and apply ophthalmic ointment to the eyes of the mouse to prevent drying.</li>
			<li>Remove hair from over the 4th mammary gland of the mouse.</li>
			<li>Clean the skin with betadine. It is important to maintain sterile conditions throughout the rest of the procedure. This includes using sterilized instruments and reagents.</li>
			<li>Make a small incision ~2-3 mm just inferior to the 4th nipple.</li>
			<li>Dissect until the mammary fat pad is exposed.</li>
			<li>Take a tumor piece from the Petri dish and coat in artificial extracellular matrix.</li>
			<li>Transplant tumors underneath the 4th mammary fat pad.</li>
			<li>Close the incision using cyanoacrylate adhesive.</li>
			<li>Continuously monitor the animal until it fully recovers from anesthesia and is able to maintain sternal recumbency. Also, do not to return the animal to the company of other animals until full recovery.</li>
			<li>To minimize infections, add 1 mL of 100 mg/mL enrofloxacin antibiotic to the animal&#39;s drinking water bottle (8 fl oz).</li>
			<li>Allow tumors to grow until palpable (~5 mm; ~4 weeks).</li>
			<li>Depending on the experiment, allocate the mice into treatment groups, and perform the corresponding treatments, if applicable.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Setup for intravital imaging&#160;&#160;&#160;&#160;&#160;&#160; 
	<ol>
		<li>Turn on microscope&#39;s two-photon laser and detectors.</li>
		<li>Turn on the heating box and pre-heat the microscope&#39;s x-y stage.</li>
		<li>Place the custom-made stage insert42 into the x-y stage.</li>
	</ol>
	</li>
	<li>Preparation of imaging window
	<ol>
		<li>Prior to setting up for imaging, autoclave the custom-made circular imaging window frame42.</li>
		<li>Use a pipette or an insulin syringe to place a thin layer of cyanoacrylate adhesive to the window frame and affix in place an 8 mm circular cover glass. 
		NOTE: 1) It is important to avoid getting residue on the clear aperture of the cover glass. 2) It is important to adhere the cover glass to the window frame at least 1 h before use for imaging.</li>
		<li>Carefully wipe clean any excess cyanoacrylate on the clear aperture of the cover glass using a laboratory wipe wetted with a small amount of acetone.</li>
	</ol>
	</li>
	<li>Preparation of tail vein catheter for administration of fluids and fluorescent dyes during imaging
	<ol>
		<li>Cut a 30 cm piece of polyethylene tubing to construct a tail vein catheter.</li>
		<li>Detach the needle portion of a 30 G needle from its Luer taper by gently bending the needle back and forth until it breaks. 
		NOTE: The needle should be held close to the Luer taper and not the needle tip. This can be performed with pliers or forceps to grasp the needle in order to prevent needle stick injury.</li>
		<li>Insert the blunt end of the needle into one end of the polyethylene tubing.</li>
		<li>Insert the sharp end of another 30 G needle, keeping its Luer taper attached, to the opposite end of the tubing.</li>
		<li>Fill a 1 cc syringe with phosphate buffered saline, attach it to Luer taper of the assembled catheter, and flush the tail vein catheter making sure that there are no air bubbles in the system.</li>
		<li>For vascular labeling, fill a 1 cc syringe with 100 &#956;L of 10 mg/kg 155 kDa dextran-tetramethylrhodamine (TMR) or quantum dots.</li>
	</ol>
	</li>
	<li>Preparation of mouse for imaging
	<ol>
		<li>Anesthetize the mouse in a cage underneath (~1 foot below) a heat lamp using 4%-5% isoflurane mixed with 100% oxygen set to a flow of 1.5-2 L per min.</li>
		<li>Turn on heat lamp over surgical working area. This step is critical for maintaining the core physiologic body temperature of the mouse during the surgery.</li>
		<li>Place the mouse under the heat lamp and lower the anesthesia to 2%-3% for the duration of the surgery. 
		NOTE: Be sure to keep the heat lamp at a safe distance (~1 foot) away from the mouse to avoid overheating.</li>
		<li>Place ophthalmic ointment on the mouse&#8217;s eyes to prevent drying of the eyes and blindness.</li>
		<li>Test that the mouse is anesthetized by performing toe-pinch test. If animal withdraws, increase dosage of isoflurane by 1% and retest in 1-2 min.</li>
		<li>Insert the tail vein catheter into the most distal point on the tail possible.</li>
		<li>Affix the tail vein catheter to the tail with a small piece of lab tape that wraps around the tail and sticks to the needle to insure it does not get dislodged.</li>
		<li>Inject 50 &#956;L per h of PBS through the tail vein catheter to provide adequate hydration. It is critical to avoid injecting too much fluid (no more than 200 &#956;L per h) or any bubbles into the catheter as this can be fatal to the mouse.</li>
		<li>Remove hair on the abdomen over the 4th and 5th mammary glands using depilatory cream.</li>
		<li>Clean the skin with betadine and allow skin to dry.</li>
		<li>Make a longitudinal midline incision starting immediately superior to the genitals and carry the incision up to the level of the superior aspect of the 4th mammary gland.</li>
		<li>Carry the incision transverse to the superior aspect of the 4th mammary gland. It is critical to avoid compromising the blood supply at this point.</li>
		<li>Dissect the mammary fat pad off the peritoneum creating a tissue flap using sterile forceps and scissors. 
		NOTE: Skin flap imaging is susceptible to significant motion artifacts and tissue dehydration. These are avoided, as described previously43,44, by affixing a rigid piece of rubber behind to the skin side of the flap (to stiffen the soft tissue and isolate it from the rest of the body) and then placing the tumor into a shallow imaging window to preserve the hydration. This is critical for stable imaging as the tumor and surrounding tissue in this setting are very compliant.</li>
		<li>Stabilize the skin flap by affixing (with cyanoacrylate glue) a small piece of rigid rubber measuring 2 cm x 2 cm to the skin side of the flap. The tumor should be in the center of the area being stabilized by the rubber.</li>
		<li>Keep exposed tissue hydrated with drops of PBS.</li>
		<li>Apply a small film of cyanoacrylate to the outer rim of the custom-made imaging window frame.</li>
		<li>Apply a small droplet of PBS (~10&#8211;20 &#956;L) to the center of the cover glass.</li>
		<li>Dry the surrounding flap tissue with a laboratory wipe. It is critical to make sure that the cyanoacrylate on the window frame does not come into contact with the PBS on the glass, as this can cause the cyanoacrylate to polymerize and set prematurely.</li>
		<li>Affix the small imaging window to the tissue flap with the tumor at the center of the clear aperture.</li>
		<li>Remove the heating box from the stage.</li>
		<li>Transfer the anesthetized mouse and tail vein catheter to the microscope stage. Use extreme caution to ensure the tail vein catheter does not fall out.</li>
		<li>Place mouse on the stage in the prone position.</li>
		<li>Place the nose cone of isoflurane over the snout to ensure maintenance of anesthesia.</li>
		<li>Insert the window into the bore on the custom x-y stage plate.</li>
		<li>Place the heating box back onto the stage to maintain a physiological temperature.</li>
		<li>Monitor the animal&#39;s vital signs by attaching a pulse oximeter probe via clip sensor to the back paw.</li>
		<li>Slowly decrease isoflurane to 0.5%-1% to maintain adequate blood flow and avoid over anesthetizing the mouse.</li>
	</ol>
	</li>
	<li>Intravital imaging 
	NOTE: The imaging we describe in this section was performed on a custom-built two-laser multiphoton microscope that has been previously described5,39,45. Briefly, a femtosecond laser is used to generate 90 femtosecond pulsed laser light centered at 880 nm. Fluorescence light is detected with three of the four simultaneously acquiring detectors (Blue = 447/60, Green = 520/65, and Red 580/60; central wavelength/bandwidth) after separation from the excitation light by a dichroic (Chroma, Z720DCXXR). The microscope stand contains a 25x, 1.05 NA (numerical aperture) long working distance (2 mm) objective lens. It is important to note that, while we have used a custom-built microscope, the protocol described below can be accomplished on any commercially available multiphoton microscope as well.
	<ol>
		<li>Place a drop of distilled water between the 25x, 1.05 NA microscope objective and the window&#8217;s cover glass to make optical contact.</li>
		<li>Use the microscope eyepiece to focus on areas with fluorescent tumor cells near to the surface of the window.</li>
		<li>Find flowing blood vessels and labeled macrophages. It is critical to have flowing blood vessels to assess the dynamics of the vasculature.</li>
		<li>Switch the microscope into multiphoton mode.</li>
		<li>Set the upper and lower limits of a z-series, which measures approximately 50 &#956;m.
		<ol>
			<li>Set the upper limit of the z-series by using the focus adjuster to move the objective to the desired start location, at the most superficial position, and marking this position as zero within the software by clicking on the Z position Top button.</li>
			<li>Set the lower limit of the z-series moving the objective to the deepest layer (typically 50-70 &#956;m from the top for smaller tumors) and clicking the Z position Bottom button.</li>
			<li>Set the z-step size to 5 &#956;m.</li>
		</ol>
		</li>
		<li>Click the Time-Lapse panel button and set the time interval between acquisitions to at least 10 s to provide adequate time to replenish the water above the objective lens. This is done manually with a squeeze pipette on the objective during the long time lapse.</li>
		<li>Remove the syringe with PBS in the tail vein catheter and replace it with another syringe containing the 155 kDa dextran-TMR (tetramethyl rhodamine).</li>
		<li>Inject 100 &#956;L of 155 kDa dextran-TMR via the tail vein catheter.</li>
		<li>After injection, replace the TMR syringe with the PBS syringe.</li>
		<li>Acquire a z-stack time-lapse imaging by clicking on the Z-Stack and Time-Lapse buttons, then clicking on the record button.</li>
		<li>Inject 50 &#181;L of PBS every 30-45 min to maintain adequate hydration of the animal. Avoid injecting more than 200 &#956;L at time as this can cause fluid overload.</li>
	</ol>
	</li>
	<li>Euthanasia
	<ol>
		<li>Increase the isoflurane to 5% and keep the animal under 5% isoflurane with nose cone in place until 30 s after respirations cease.</li>
		<li>Remove the mouse from the stage.</li>
		<li>Perform cervical dislocation.</li>
	</ol>
	</li>
	<li>Image processing
	<ol>
		<li>Load all images into ImageJ and format them into a 5 dimensional hyperstack (x, y, z, t, and color channel).</li>
		<li>Perform separation of spectral overlap (i.e.: GFP and CFP) and elimination of x-y drift from the hyperstacks using established methods38.</li>
		<li>Use the brightness and contrast adjustment to increase the white level of the blood channel so that the background signal becomes visible.</li>
		<li>For each z slice, carefully inspect each movie for signs of transient vascular leakage (a &#34;burst&#34;). Running the movies at fast frame rates (40 fps) may aid this identification.</li>
		<li>Once a burst has been identified, return the brightness for this channel to a normal level and crop the hyperstack to this region and z-slice.</li>
	</ol>
	</li>
</ol>
2. Evaluation of extravascular dextran using fixed tissue analysis
<ol>
	<li>Tumor and sample preparation 
	NOTE: The 2nd part of this protocol assumes that breast tumors have been harvested from an orthotopic transplantation mouse model of breast carcinoma (i.e. the MMTV-PyMT). This model could be the same as the one described in the 1st part of the protocol, although fluorescently-labeled tumors are not necessary at this point.
	<ol>
		<li>Following the termination of the experimental pipeline (i.e. drug treatments, etc.), perform a tail-vain injection of 100 &#956;L of 10 mg/mL 155 kDa dextran-tetramethyl rhodamine, 1 h before sacrificing the mice.</li>
		<li>Sacrifice the mice and harvest the breast tumors.</li>
		<li>Fix tumors in 10% formalin for 48-72 h and proceed to paraffin-embedding.</li>
		<li>Using a microtome, cut two 5 &#956;m-thick sequential slides from the formalin-fixed paraffin-embedded (FFPE) tissues. One slide is used for staining the dextran, while the other will be used for performing TMEM triple-IHC, for reference. 
		NOTE: The TMEM triple-immunohistochemistry protocol has been described elsewhere 10.</li>
	</ol>
	</li>
	<li>IF staining and scanning for the first of the two sequential sections
	<ol>
		<li>Submit slides to a standard de-paraffinization protocol. This includes two subsequent immersions in xylene (10 min each), followed by dehydration in serially diluted alcohol solutions (100%, 95%, 70%, and 50% EtOH in H2O for 2 min each immersion).</li>
		<li>Perform antigen retrieval by heating (close to boiling point) the sections submerged in citrate (pH 6.0-adjusted) for 20 min.</li>
		<li>Let the samples cool to room temperature for 15-20 min and then wash in PBS 3x for 2 min each wash.</li>
		<li>Block for 60-90 min in blocking buffer (10% FBS; 1% BSA; 0.0025% fish skin gelatin; 0.05% PBST, i.e. PBS with 0.05% Tween-20).</li>
		<li>Incubate samples with a mixture of primary rat and rabbit antibodies which target Endomucin and TMR, respectively, and then wash in PBST 3x, 2 min each.</li>
		<li>Incubate samples with a mixture of secondary donkey antibodies against rat IgG (conjugated to Alexa-647) and rabbit IgG (conjugated to Alexa-488), and then wash in PBST 3x 2 min each.</li>
		<li>Perform a routine DAPI staining (i.e. immersion in DAPI solution for 5-6 min), mount the slides using a glycerol-free &#34;hard&#34;&#160;mounting medium, and store in a dark place until scanning.</li>
		<li>For optimal results, scan the slides on a digital whole slide scanner.</li>
	</ol>
	</li>
	<li>Image Analysis
	<ol>
		<li>Capture 10 High Power Fields (HPFs) per case, using any software suitable for digital pathology.</li>
		<li>Save the Endomucin (Red) and TMR (Green) channels separately as TIFF.</li>
		<li>Using ImageJ, upload the TIFF files and convert them into 8-bit images.</li>
		<li>Threshold the 8-bit images to the level of the negative control, and generate two binarized images, showing the Endomucin and TMR &#34;masks&#34;.</li>
		<li>From the Binary tools, select Fill Holes on the Endomucin mask.</li>
		<li>Generate and save the following five regions of interest: 1) The thresholded dextran image as &#34;Dextran ROI&#34;&#160;(ROI1), 2) the thresholded endomucin image as &#34;Vascular ROI&#34;&#160;(ROI2), 3) the inverted endomucin image as &#34;Extravascular ROI&#34;&#160;(ROI3), 4) the intersected &#34;Extravascular ROI&#34;&#160;and &#34;Dextran ROI&#34;&#160;image (ROI1 &#8745; ROI3) as &#34;Extravascular Dextran ROI&#34;&#160;(ROI4), and 5) the entire image as &#34;Tumor ROI&#34;&#160;(ROI5).</li>
		<li>Divide the ROI4 area by the ROI5 area and multiply by 100 to generate the percent area that the extravascular dextran covers in the entire tumor.</li>
		<li>Repeat the process for 10-20 HPFs per case (depending on tissue availability) and generate an average Extravascular Dextran (% area) for each case.</li>
	</ol>
	</li>
</ol>

The authors disclose no conflicts of interest.

Here, we outline two protocols that can be applied to visualize and quantify a specific type of vascular permeability which is present at TMEM doorways and is associated with the disruption of vascular tight and adherens junctions. This type of vascular permeability is transient and controlled by the tripartite TMEM cell complex, as explained above5. The ability to identify and quantify TMEM-associated vascular permeability is crucial for the assessment of a pro-metastatic cancer cell microenviron...

Recent advances in our understanding of cancer metastasis have uncovered that epithelial-to-mesenchymal transition (EMT) and the induction of a migratory/invasive cancer cell subpopulation are not, by themselves, sufficient for hematogenous dissemination1. Indeed, it was previously thought that metastasizing cancer cells intravasate through the entirety of cancer-associated endothelium as the tumor neovasculature is often characterized by low pericyte coverage, and as such, is highly permeable and unstable2,3,4. Although highly suggestive of defective functions within the tumor, vascular modifications during carcinogenesis do not provide evidence per se that tumor cells can penetrate blood vessels easily and in an uncontrolled fashion. Insights from intravital imaging (IVI) studies, in which tumor cells are fluorescently-tagged and the vasculature is labeled via the intravenous injection of fluorescent probes (such as dextran or quantum dots), show that, while tumor vessels are uniformly permeable to low molecular weight dextrans (e.g. 70 kD), high molecular weight dextrans (155 kD) and tumor cells can cross the endothelium only at specialized sites of intravasation which are preferentially located at vascular branch point5,6,7. Immunohistochemical (IHC) analyses using animal models and human patient-derived material have shown that these sites are &#34;doorways&#34;&#160;that specialize in regulating vascular permeability, locally and transiently, providing a brief window of opportunity for migratory/invasive cancer cells to enter the circulation. These doorways are called &#34;Tumor Microenvironment of Metastasis&#34;&#160;or &#34;TMEM&#34;, and, quite expectedly, their density correlates with an increased risk of developing metastatic disease in breast cancer patients8,9,10.
Each TMEM doorway consists of three distinct types of cells: a perivascular macrophage, a tumor cell over-expressing the actin-regulatory protein mammalian enabled (Mena), and an endothelial cell, all in direct physical contact with each other1,5,9,10,11,12,13. The key event for the function of TMEM as an intravasation doorway is the localized release of vascular endothelial growth factor (VEGF) onto the underlying vessel by the perivascular macrophage14. VEGF can disrupt homotypic junctions between endothelial cells15,16,17,18,19, a phenomenon that results in transient vascular leakage, also known as &#34;bursting&#34;&#160;permeability as described in IVI studies 5. TMEM macrophages have been shown to express the tyrosine kinase receptor TIE2, which is required for VEGF-mediated TMEM function and homing of these macrophages to the perivascular niche5,20,21,22. In addition to regulating cancer cell dissemination and metastasis, TIE2+ macrophages have been shown to be central regulators of tumor angiogenesis21,22,23,24,25,26,27,28,29,30,31. As such, TIE2+ macrophages represent a critical constituent of the tumor microenvironment and the main regulator of the metastatic cascade.
To better conceptualize TMEM-mediated vascular permeability (i.e. &#34;bursting&#34;), it is very important to distinguish it from other modes of vascular permeability that are not associated with the dissolution of endothelial cell-cell junctions. In an intact endothelium (one whose tight and adherens junctions are not disrupted), there are three main types of vascular permeability: (a) pinocytosis, which may, or may not, be coupled to transcytosis of the ingested material; (b) transportation of material through endothelial fenestrae; and (c) transportation of material through the paracellular pathway, which is regulated by endothelial tight junctions15,16,17,18,19,32,33,34. Although deregulated in many tumors, the aforementioned modes of vascular permeability have been described mostly in the context of normal tissue physiology and homeostasis, the extremes of which are tissues with either limited permeability (e.g., blood-brain barrier, blood-testis barrier), or abundant permeability (e.g., fenestrated capillaries of the kidney glomerular apparatus)34,35,36,37.
Using multiphoton intravital imaging and multiplexed immunofluorescence microscopy, we are able to distinguish between TMEM-mediated vascular permeability (&#34;bursting&#34;) and other modes of vascular permeability in breast tumors. To achieve this, we perform a single intravenous injection of a high-molecular weight, fluorescently-labeled probe in mice. Spontaneous bursting events can then be captured using intravital imaging in live mice; or alternatively, extravasation of the probe can be quantified by co-localization studies with blood vasculature (e.g. CD31+ or Endomucin+) and TMEM doorways using immunofluorescence microscopy. The protocols presented here describe both of these techniques, which could be used either independently or in conjunction with one another.

<table border="0" cellpadding="0" cellspacing="0" style="width:681px;" width="682">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Anti-rabbit IgG (Alexa 488)</td>
			<td style="width:224px;">Life Technologies Corporation</td>
			<td style="width:155px;">A-11034</td>
			<td style="width:64px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-rat IgG (Alexa 647)</td>
			<td>Life Technologies Corporation</td>
			<td>A-21247</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bovine Serum Albumin</td>
			<td>Fisher Scientific</td>
			<td>BP1600-100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Citrate</td>
			<td>Eng Scientific Inc</td>
			<td>9770</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cover Glass Slips</td>
			<td>Electron Microscopy Sciences</td>
			<td>72296-08</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cyanoacrylate Adhesive</td>
			<td>Henkel Adhesive</td>
			<td>1647358</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DAPI</td>
			<td>Perkin Elmer</td>
			<td>FP1490</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dextran-Tetramethyl-Rhodamine</td>
			<td>Sigma Aldrich</td>
			<td>T1287</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMEM/F12</td>
			<td>Gibco</td>
			<td>11320-033</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Endomucin (primary antibody)</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-65495</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Enrofloxacin</td>
			<td>Bayer</td>
			<td>84753076 v-06/2015</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fetal Bovine Serum</td>
			<td>Sigma Aldrich</td>
			<td>F2442</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fish Skin Gelatin</td>
			<td>Fisher Scientific</td>
			<td>G7765</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Insulin Syringe</td>
			<td>Becton Dickinson</td>
			<td>309659</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Isofluorane</td>
			<td>Henry Schein</td>
			<td>NDC 11695-6776-2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Matrigel</td>
			<td>Corning</td>
			<td>CB40234</td>
			<td>Artificial extracellular matrix</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Needle (30 G)</td>
			<td>Becton Dickinson</td>
			<td>305128</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phosphate Buffered Saline</td>
			<td>Life Technologies Corporation</td>
			<td>PBS</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Polyethylene Tubing</td>
			<td>Scientific Commodities Inc</td>
			<td>BB31695-PE/1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pulse Oximeter</td>
			<td>Kent Scientific</td>
			<td>MouseOx</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Puralube Vet Ointment</td>
			<td>Dechra</td>
			<td>NDC 17033-211-38</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Quantum Dots</td>
			<td>Life Technologies Corporation</td>
			<td>Q21561MP</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Rubber</td>
			<td>McMaster Carr</td>
			<td>1310N14</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TMR (primary antibody)</td>
			<td>Invitrogen</td>
			<td>A6397</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tween-20</td>
			<td>MP Biologicals</td>
			<td>TWEEN201</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Xylene</td>
			<td>Fisher Scientific</td>
			<td>184835</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

The experimental procedures described in this protocol article are briefly summarized and illustrated in Figure 1A-C.
To measure TMEM-mediated vascular permeability (&#34;bursting activity&#34;) and to reduce experimental noise from other modes of vascular permeability (i.e. transcellular and paracellular, as explained in the introduction), we performed intravenous (i.v.) injection of high molecular weight probes, such as 155 kDa Dextran, conjugat...

Watch this Scientific Journal Video about Assessing Tumor Microenvironment of Metastasis Doorway-Mediated Vascular Permeability Associated with Cancer Cell Dissemination using Intravital Imaging and F

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

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 ...

assessing-tumor-microenvironment-metastasis-doorway-mediated-vascular

Research

JoVE Journal

Cancer Research

8.7K Views.  Albert Einstein College of Medicine. 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.

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

The Drosophila Imaginal Disc Tumor Model: Visualization and Quantification of Gene Expression and Tumor Invasiveness Using Genetic Mosaics

Drosophila melanogaster has emerged as a powerful experimental system for functional and mechanistic studies of tumor development and progression in the context of a whole organism. Sophisticated techniques to generate genetic mosaics facilitate induction of visually marked, genetically defined clones surrounded by normal tissue. The clones can be analyzed through diverse molecular, cellular and omics approaches. This study describes how to generate fluorescently labeled clonal tumors of varying malignancy in the eye/antennal imaginal discs (EAD) of Drosophila larvae using the Mosaic Analysis with a Repressible Cell Marker (MARCM) technique. It describes procedures how to recover the mosaic EAD and brain from the larvae and how to process them for simultaneous imaging of fluorescent transgenic reporters and antibody staining. To facilitate molecular characterization of the mosaic tissue, we describe a protocol for isolation of total RNA from the EAD. The dissection procedure is suitable to recover EAD and brains from any larval stage. The fixation and staining protocol for imaginal discs works with a number of transgenic reporters and antibodies that recognize Drosophila proteins. The protocol for RNA isolation can be applied to various larval organs, whole larvae, and adult flies. Total RNA can be used for profiling of gene expression changes using candidate or genome-wide approaches. Finally, we detail a method for quantifying invasiveness of the clonal tumors. Although this method has limited use, its underlying concept is broadly applicable to other quantitative studies where cognitive bias must be avoided.

The Drosophila Imaginal Disc Tumor Model: Visualization and Quantification of Gene Expression and Tumor Invasiveness Using Genetic Mosaics

This protocol demonstrates how to generate fluorescently marked, genetically defined clonal tumors in the Drosophila eye/antennal imaginal discs (EAD). It describes how to dissect the EAD and brain from the third instar larvae and how to process them to visualize and quantify gene expression changes and tumor invasiveness.

Drosophila melanogaster has emerged as a powerful experimental system for functional and mechanistic studies of tumor development and progression in the ...

Micromanipulation of Circulating Tumor Cells for Downstream Molecular Analysis and Metastatic Potential Assessment

Blood-borne metastasis accounts for&#160;most cancer-related deaths and involves circulating tumor cells (CTCs) that are successful in establishing new tumors at distant sites. CTCs are found in the bloodstream of patients as single cells (single CTCs) or as multicellular aggregates (CTC clusters and CTC-white blood cell clusters), with the latter displaying a higher metastatic ability. Beyond enumeration, phenotypic and molecular analysis is extraordinarily important to dissect CTC biology and to identify actionable vulnerabilities. Here, we provide a detailed description of a workflow that includes CTC immunostaining and micromanipulation, ex vivo culture to assess&#160;proliferative and survival capabilities of individual cells, and in vivo metastasis-formation assays. Additionally, we provide a protocol to achieve the dissociation of CTC clusters into individual cells and the investigation of intra-cluster heterogeneity. With these approaches, for instance, we precisely quantify survival and proliferative potential of single CTCs and individual cells within CTC clusters, leading us to the observation that cells within clusters display better survival and proliferation in ex vivo cultures compared to single CTCs. Overall, our workflow offers a platform to dissect the characteristics of CTCs at the single cell level, aiming towards the identification of metastasis-relevant pathways and a better understanding of CTC biology.

Here, we present an integrated workflow to identify phenotypic and molecular features that characterize circulating tumor cells (CTCs). We combine live immunostaining and robotic micromanipulation of single and clustered CTCs with single cell-based techniques for downstream analysis and assessment of metastasis-seeding ability.

Blood-borne metastasis accounts for&#160;most cancer-related deaths and involves circulating tumor cells (CTCs) that are successful in establishing new ...

Analysis of Side Population in Solid Tumor Cell Lines

Cancer stem cells (CSCs) are an important cause of tumor growth, metastasis, and recurrence. Isolation and identification of CSCs are of great significance for tumor research. Currently, several techniques are used for the identification and purification of CSCs from tumor tissues and tumor cell lines. Separation and analysis of side population (SP) cells are two of the commonly used methods. The methods rely on the ability of CSCs to rapidly expel fluorescent dyes, such as Hoechst 33342. The efflux of the dye is associated with the ATP-binding cassette (ABC) transporters and can be inhibited by ABC transporter inhibitors. Methods for staining cultured tumor cells with Hoechst 33342 and analyzing the proportion of their SP cells by flow cytometry are described. This assay is convenient, fast, and cost-effective. Data generated in this assay can contribute to a better understanding of the effect of genes or other extracellular and intracellular signals on the stemness properties of tumor cells.

A convenient, fast, and cost-effective method to measure the proportion of side population cells in solid tumor cell lines is presented.

Cancer stem cells (CSCs) are an important cause of tumor growth, metastasis, and recurrence. Isolation and identification of CSCs are of great ...

Pathological Analysis of Lung Metastasis Following Lateral Tail-Vein Injection of Tumor Cells

Metastasis, the primary cause of morbidity and mortality for most cancer patients, can be challenging to model preclinically in mice. Few spontaneous metastasis models are available. Thus, the experimental metastasis model involving tail-vein injection of suitable cell lines is a mainstay of metastasis research. When cancer cells are injected into the lateral tail-vein, the lung is their preferred site of colonization. A potential limitation of this technique is the accurate quantification of the metastatic lung tumor burden. While some investigators count macrometastases of a pre-defined size and/or include micrometastases following sectioning of tissue, others determine the area of metastatic lesions relative to normal tissue area. Both of these quantification methods can be exceedingly difficult when the metastatic burden is high. Herein, we demonstrate an intravenous injection model of lung metastasis followed by an advanced method for quantifying metastatic tumor burden using image analysis software. This process allows for investigation of multiple end-point parameters, including average metastasis size, total number of metastases, and total metastasis area, to provide a comprehensive analysis. Furthermore, this method has been reviewed by a veterinary pathologist board-certified by the American College of Veterinary Pathologists (SEK) to ensure accuracy.

Intravenous injection of cancer cells is often used in metastasis research, but the metastatic tumor burden can be difficult to analyze. Herein, we demonstrate a tail-vein injection model of metastasis and include a novel approach to analyze the resulting metastatic lung tumor burden.

Metastasis, the primary cause of morbidity and mortality for most cancer patients, can be challenging to model preclinically in mice. Few spontaneous ...

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 ...

Expanding the Comprehension of the Tumor Microenvironment using Mass Spectrometry Imaging of Formalin-Fixed and Paraffin-Embedded Tissue Samples

Advances in immune-based therapies have revolutionized cancer treatment and research. This has triggered growing demand for the characterization of the tumor immune landscape. Although standard immunohistochemistry is suitable for studying tissue architecture, it is limited to the analysis of a small number of markers. Conversely, techniques such as flow cytometry can evaluate multiple markers simultaneously, although information about tissue morphology is lost. In recent years, multiplexed strategies that integrate phenotypic and spatial analysis have emerged as comprehensive approaches to the characterization of the tumor immune landscape. Herein, we discuss an innovative technology combining metal-labeled antibodies and secondary ion mass spectrometry focusing on the technical steps in assay development and optimization, tissue preparation, and image acquisition and processing. Before staining, a metal-labeled antibody panel must be developed and optimized. This hi-plex image system supports up to 40 metal-tagged antibodies in a single tissue section. Of note, the risk of signal interference increases with the number of markers included in the panel. After panel design, particular attention should be given to the metal isotope assignment to the antibody to minimize this interference. Preliminary panel testing is performed using a small subset of antibodies and subsequent testing of the entire panel in control tissues. Formalin-fixed, paraffin-embedded tissue sections are obtained and mounted on gold-coated slides and further stained. The staining takes 2 days and closely resembles standard immunohistochemical staining. Once samples are stained, they are placed in the image acquisition instrument. Fields of view are selected, and images are acquired, uploaded, and stored. The final stage is image preparation for the filtering and removal of interference using the system&#39;s image processing software. A disadvantage of this platform is the lack of analytical software. However, the images generated are supported by different computational pathology software.

In the era of cancer immunotherapy, interest in elucidating tumor microenvironment dynamics has increased strikingly. This protocol details a mass spectrometry imaging technique with respect to its staining and imaging steps, which allow for highly multiplexed spatial analysis.

Advances in immune-based therapies have revolutionized cancer treatment and research. This has triggered growing demand for the characterization of the ...

Zebrafish Larvae as a Model to Evaluate Potential Radiosensitizers or Protectors

Zebrafish are extensively used in several kinds of research because they are one of the easily maintained vertebrate models and exhibit several features of a unique and convenient model system. As highly proliferative cells are more susceptible to radiation-induced DNA damage, zebrafish embryos are a front-line in vivo model in radiation research. In addition, this model projects the effect of radiation and different drugs within a short time, along with major biological events and associated responses. Several cancer studies have used zebrafish, and this protocol is based on the use of radiation modifiers in the context of radiotherapy and cancer. This method can be readily used to validate the effects of different drugs on irradiated and control (non-irradiated) embryos, thus identifying drugs as radio sensitizing or protective drugs. Although this methodology is used in most drug screening experiments, the details of the experiment and the toxicity assessment with the background of X-ray radiation exposure are limited or only briefly addressed, making it difficult to perform. This protocol addresses this issue and discusses the procedure and toxicity evaluation with a detailed illustration. The procedure describes a simple approach for using zebrafish embryos for radiation studies and radiation-based drug screening with much reliability and reproducibility.

The zebrafish has recently been exploited as a model to validate potential radiation modifiers. The present protocol describes the detailed steps to use zebrafish embryos for radiation-based screening experiments and some observational approaches to evaluate the effect of different treatments and radiation.

Zebrafish are extensively used in several kinds of research because they are one of the easily maintained vertebrate models and exhibit several features ...

Multiplex Immunofluorescence and Spatial Image Analysis of the Tumor Microenvironment

Multiplex Immunofluorescence Combined with Spatial Image Analysis for the Clinical and Biological Assessment of the Tumor Microenvironment

The tumor microenvironment (TME) is composed of a plethora of different cell types, such as cytotoxic immune cells and immunomodulatory cells. Depending on its composition and the interactions between cancer cells and peri-tumoral cells, the TME may affect cancer progression. The characterization of tumors and their complex microenvironment could improve the understanding of cancer diseases and may help scientists and clinicians to discover new biomarkers.
We recently developed several multiplex immunofluorescence (mIF) panels based on tyramide signal amplification (TSA) for the characterization of the TME in colorectal cancer, head and neck squamous cell carcinoma, melanoma, and lung cancer. Once the staining and scanning of the corresponding panels are completed, the samples are analyzed on an image analysis software. The spatial position and the staining of each cell are then exported from this quantification software into R. We developed R scripts that allow us not only to analyze the density of each cell type in several tumor compartments (e.g. the center of the tumor, the margin of the tumor, and the stroma) but also to perform distance-based analyses between different cell types.
This particular workflow adds a spatial dimension to the classical density analysis already routinely performed for several markers. mIF analysis could allow scientists to have a better understanding of the complex interaction between cancer cells and the TME and to discover new predictive biomarkers of response to treatments, such as immune checkpoint inhibitors, and targeted therapies.

Author Spotlight: Multiplex Immunofluorescence Combined with Spatial Image Analysis for the Clinical and Biological Assessment of the Tumor Microenvironment  

In this article, a protocol for manual tyramide signal amplification (TSA) multiplex immunofluorescence (mIF) combined with image analysis and spatial analysis is described. This protocol can be used with formalin-fixed paraffin-embedded (FFPE) sections for the staining of two to six antigens per slide depending on the slide scanner available in the laboratory.

The tumor microenvironment (TME) is composed of a plethora of different cell types, such as cytotoxic immune cells and immunomodulatory cells. Depending ...

Examining Tumor Cell Dissemination from Lung Metastases

Tracking Tumor Cell Dissemination from Lung Metastases Using Photoconversion

Metastasis - the systemic spread of cancer - is the leading cause of cancer-related deaths. Although metastasis is commonly thought of as a unidirectional process wherein cells from the primary tumor disseminate and seed metastases, tumor cells in existing metastases can also redisseminate and give rise to new lesions in tertiary sites in a process known as &#34;metastasis-from-metastases&#34; or &#34;metastasis-to-metastasis seeding.&#34; Metastasis-to-metastasis seeding may increase the metastatic burden and decrease the patient&#39;s quality of life and survival. Therefore, understanding the processes behind this phenomenon is crucial to refining treatment strategies for patients with metastatic cancer.
Little is known about metastasis-to-metastasis seeding, due in part to logistical and technological limitations. Studies on metastasis-to-metastasis seeding rely primarily on sequencing methods, which may not be practical for researchers studying the exact timing of metastasis-to-metastasis seeding events or what promotes or prevents them. This highlights the lack of methodologies that facilitate the study of metastasis-to-metastasis seeding. To address this, we have developed - and describe herein - a murine surgical protocol for the selective photoconversion of lung metastases, allowing specific marking and fate tracking of tumor cells redisseminating from the lung to tertiary sites. To our knowledge, this is the only method for studying tumor cell redissemination and metastasis-to-metastasis seeding from the lungs that does not require genomic analysis.

Author Spotlight: Decoding Metastasis-to-Metastasis Seeding Using a New In Vivo Technique for Tracking Breast Cancer Spread 

We present a method for studying tumor cell redissemination from lung metastases involving a surgical protocol for the selective photoconversion of lung metastases, followed by the identification of redisseminated tumor cells in tertiary organs.

Metastasis - the systemic spread of cancer - is the leading cause of cancer-related deaths. Although metastasis is commonly thought of as a unidirectional ...

Mapping the Tumor Immune Landscape: An In-Depth Multiplex IHC Analysis Protocol

Multiplex Immunohistochemical Analysis of the Spatial Immune Cell Landscape of the Tumor Microenvironment

The immune cell landscape of the tumor microenvironment potentially contains information for the discovery of prognostic and predictive biomarkers. Multiplex immunohistochemistry is a valuable tool to visualize and identify different types of immune cells in tumor tissues while retaining its spatial information. Here we provide detailed protocols to analyze lymphocyte, myeloid, and dendritic cell populations in tissue sections. Starting from cutting formalin-fixed paraffin-embedded sections, automatic multiplex staining procedures on an automated platform, scanning of the slides on a multispectral imaging microscope, to the analysis of images using an in-house-developed machine learning algorithm ImmuNet. These protocols can be applied to a variety of tumor specimens by simply switching tumor markers to analyze immune cells in different compartments of the sample (tumor versus invasive margin) and apply nearest-neighbor analysis. This analysis is not limited to tumor samples but can also be applied to other (non-)pathogenic tissues. Improvements to the equipment and workflow over the past few years have significantly shortened throughput times, which facilitates the future application of this procedure in the diagnostic setting.

Author Spotlight: Unlocking Insights into the Immune Cell Landscape of Tumors

This protocol describes in detail how immune cell characterization of the tumor microenvironment using multiplex immunohistochemistry is carried out.

The immune cell landscape of the tumor microenvironment potentially contains information for the discovery of prognostic and predictive biomarkers. ...