The authors would like to thank Dr. Marcus Bosenberg for providing YUMM 1.1 and YUMM 1.7 murine melanoma lines and Dr. Deborah J. Fowell for providing B6.Cg-Tg(CAG-tdKaede)15Utr mice in agreement with RIKEN BRC through the National Bio-Resource of the MEXT, Japan.

All animal protocols have been approved by the Institutional Animal Care and Use Committee at the Oregon Health &#38; Science University.
1. Induction of Inflammation and FITC Painting of Mouse Pinna
<ol>
	<li>In a laminar flow hood, anesthetize a C57Bl/6 mouse using vaporized isofluorane (induce at 3-5% isofluorane and maintain at 1-3% isofluorane; oxygen flow rate at 0.5-1.0 L/min). Ensure proper anesthetization by monitoring the loss of pedal reflex, involuntary movements and reduced resp...

Although the leukocyte egress from peripheral, non-lymphoid tissues is critical for the initiation and resolution of immune responses, the molecular mechanisms that govern egress are poorly understood. This gap in knowledge is largely due to ready availability of tools for the quantification in vivo. Here, we describe the use of photoconvertible mice (Kaede-Tg) to quantify endogenous leukocyte egress from the skin and tumors and provide a direct head-to-head comparison with FITC paint in inflammatory and infecti...

Peripheral tissue immune responses are shaped not only by leukocyte recruitment to the sites of inflammation but also by mechanisms that regulate their subsequent retention. Thus, protective immunity is dictated by cumulative cellular and molecular mechanisms that determine whether a leukocyte enters, stays within, or rather migrates out of peripheral tissue via lymphatic vessels. Importantly, the propensity for leukocytes to exit tissue through lymphatic vessels (termed egress) is linked to their specialized functions. Dendritic cells (DC) acquire migratory behavior in response to maturation signals leading to antigen transport and presentation in draining lymph nodes (dLN), a process that is necessary for adaptive immunity1. Scavenging myeloid cells, such as macrophages and neutrophils, serve to clear apoptotic debris through phagocytosis. During bacterial infection, neutrophils egress tissue and ultimately undergo apoptosis in dLNs2 and in a model of DSS-induced colitis, data supports the hypothesis that macrophage egress is necessary to resolve local inflammation3. Whether neutrophil and macrophage egress occurs in all inflammatory contexts, however, is unknown. Evidence for T lymphocyte egress from steady state4,5,6,7, infected8, and inflamed4,9,10,11,12 peripheral, non-lymphoid tissues indicates that T cells actively recirculate, though the tissue-based signals that drive this exit remain poorly understood. Several studies have identified signals necessary for directional migration towards draining lymphatic capillaries and subsequent egress including chemokine (C-C motif) ligand 21 (CCL21) and its receptor CCR74,11,13, chemokine (C-X-C motif) ligand 12 (CXCL12) and its receptor CXCR42,14, and sphingosine-1-phosphate (S1P)10,15,16. These mechanisms are not active in all contexts, however, and whether they determine egress of all cell types remains an open question. Importantly, further insight into the mechanisms that govern egress and its functional relevance in disease requires quantitative in vivo methods of analysis.
Several methods have been used to quantify egress in multiple animal models in vivo including direct cannulation of lymphatic vessels, adoptive transfer of ex vivo labeled leukocytes, transdermal application of fluorescent tracers, injection of labeled particles, and in vivo photoconversion17,18. Direct cannulation of afferent mouse lymphatic vessels is difficult and limited in small animals by the volumes of fluid that can be collected. Thus, cannulation has largely been performed in large animals (e.g., sheep) where such surgical manipulations are practical. These studies provide direct evidence for the presence of both lymphoid and myeloid cells in lymph10,19,20. Furthermore, ovine models reveal that acute and chronic inflammation increased lymphocyte presence in lymph by nearly 100-fold10,21.
Adoptive transfer of labeled and genetically manipulated lymphocytes has importantly revealed that CCR7 is required for the egress of CD4+ T cells from acutely inflamed skin5,11, while the pretreatment of lymphocytes with the small molecule S1P receptor agonist, FTY720, only partially inhibits their egress10. Interestingly, the egress of transferred lymphocytes from chronically inflamed skin is CCR7-independent10, but may partially require CXCR49. Adoptive transfer experiments, however, deliver non-physiological numbers of ex vivo activated and labeled lymphocytes into tissue through injection, which alters the biomechanical environment of tissues and elevated interstitial fluid pressures that open initial lymphatic capillaries and alter their transport properties22. As an alternative, transdermal application of fluorescein isothiocyanate (FITC) in the presence or absence of dermal irritants (e.g., dibutyl phthalate, DBP) or infection23,24 allows for the tracking of phagocytic cells that accumulate tracer and migrate to dLNs. Similarly, fluorescently labeled tumors provide a means to track phagocytic cells that have engulfed tumor material25. These methods have provided important insight into the mechanisms that govern DC egress13,14,17,26,27 but are unable to track non-phagocytic lymphocytes and, interpretation can be complicated by free lymphatic drainage of soluble FITC thus labeling non-migratory, LN resident DCs.
Alternatively, intravital microscopy is a powerful tool that allows for in vivo tracking of physiologically relevant leukocyte populations in real time28,29. Used in combination with the reporter mice and antibody-based in vivo immunofluorescent labeling, intravital microscopy has revealed the complex spatial and temporal dynamics of immune cell trafficking, including interstitial migration30, transmigration across the lymphatic endothelium, passage within the lymphatic lumen, and migration upon LN entry28,31. Broad adoption of intravital imaging techniques is limited by expense, necessary expertise for set up, and limited throughput for quantifying multiple cell types. Still, coupling quantitative methods that analyze population dynamics tissues with intravital imaging will provide additional and important mechanistic insight with respect to the mechanisms of motility and migration toward and within lymphatic capillaries18,31,32.
Consequently, in vivo photoconversion has emerged as a method that allows for in situ labeling, independent of phagocytic activity, and for the quantification of physiological leukocyte egress (when coupled with flow cytometry) in the absence or presence of challenge. Kaede-Tg mice constitutively express a protein isolated from stony coral that exhibits green fluorescence (Kaede green) until exposed to violet light, after which it irreversibly converts to red fluorescence (Kaede red)33. Photoconverted cells can be tracked as they egress from peripheral tissue sites and accumulate in dLNs. This and other similar photoconvertible mouse models34,35 have revealed important biology including constitutive egress of regulatory T cells from skin36, CXCR4-dependent B cell egress from Peyer&#39;s patches37, mobilization of resident memory T cells upon peptide re-challenge38, and broad leukocyte egress from tumor microenvironments39. Herein, we perform a head-to-head comparison of photoconversion with transdermal FITC application in the context of cutaneous inflammation and infection to allow for direct comparison of existing data with the photoconvertible method. Furthermore, we demonstrate photoconversion in implanted tumors and describe the conversion efficiency and selective egress from tumor microenvironments. As such, we argue that further application of these methods is needed to elucidate the critical biology of leukocyte egress from tumors, which will have significant implications for interpreting intratumoral leukocyte complexity, anti-tumor immunity, and response to therapy.

<table>
	<tbody>
		<tr>
			<td>Collagenase D</td>
			<td>Roche</td>
			<td>11088866001</td>
			<td></td>
		</tr>
		<tr>
			<td>DNase</td>
			<td>Roche</td>
			<td>4536282001</td>
			<td></td>
		</tr>
		<tr>
			<td>Silver-LED-405B light source with optical fiber and collimtor</td>
			<td>Prizmatix Ltd</td>
			<td>V8144</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescein isothiocyanate isomer I</td>
			<td>Sigma-Aldrich</td>
			<td>F4274</td>
			<td></td>
		</tr>
		<tr>
			<td>dibutyl phthalate</td>
			<td>Sigma-Aldrich</td>
			<td>524980</td>
			<td></td>
		</tr>
		<tr>
			<td>acetone</td>
			<td>Macron Fine Chemicals</td>
			<td>2440-02</td>
			<td></td>
		</tr>
		<tr>
			<td>29-guage syringes</td>
			<td>Exel International</td>
			<td>26029</td>
			<td></td>
		</tr>
		<tr>
			<td>Evans Blue</td>
			<td>Sigma-Aldrich</td>
			<td>E2129</td>
			<td></td>
		</tr>
		<tr>
			<td>70 um cell strainers</td>
			<td>VWR</td>
			<td>732-2758</td>
			<td></td>
		</tr>
		<tr>
			<td>paraformaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>P6148</td>
			<td></td>
		</tr>
		<tr>
			<td>HBSS</td>
			<td>Caisson</td>
			<td>HBL06</td>
			<td></td>
		</tr>
		<tr>
			<td>LIVE/DEAD Fixable Aqua Dead Cell Stain Kit</td>
			<td>Invitrogen</td>
			<td>L34966</td>
			<td></td>
		</tr>
		<tr>
			<td>Purified Anti-mouse CD16/CD32</td>
			<td>Tonbo Biosciences</td>
			<td>70-0161-M001</td>
			<td></td>
		</tr>
		<tr>
			<td>BV605 CD11c (clone N418)</td>
			<td>Biolegend</td>
			<td>117334</td>
			<td></td>
		</tr>
		<tr>
			<td>PerCP-Cy5.5 MHCII (clone M5/114.15.2)</td>
			<td>BD Pharmingen</td>
			<td>562363</td>
			<td></td>
		</tr>
		<tr>
			<td>BV421 CD3e (clone 145-2C11)</td>
			<td>Biolegend</td>
			<td>100341</td>
			<td></td>
		</tr>
		<tr>
			<td>APC CD8a (clone 53-6.7)</td>
			<td>TonBo Biosciences</td>
			<td>20-0081-u100</td>
			<td></td>
		</tr>
		<tr>
			<td>APC-Cy7 CD45 (clone 30-F11)</td>
			<td>Biolegend</td>
			<td>103116</td>
			<td></td>
		</tr>
		<tr>
			<td>BV650 CD19 (clone 6D5)</td>
			<td>Biolegend</td>
			<td>115541</td>
			<td></td>
		</tr>
		<tr>
			<td>PercCP-Cy5.5 Ly6C (clone HK1.4)</td>
			<td>Biolegend</td>
			<td>128011</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 647 F4/80 (clone BM8)</td>
			<td>Biolegend</td>
			<td>123121</td>
			<td></td>
		</tr>
		<tr>
			<td>APC-Cy7 Ly6G (clone 1A8)</td>
			<td>Biolegend</td>
			<td>127623</td>
			<td></td>
		</tr>
		<tr>
			<td>BV711 CD11b (clone M1/70)</td>
			<td>Biolegend</td>
			<td>101241</td>
			<td></td>
		</tr>
		<tr>
			<td>BV605 CD45 (clone 30-F11)</td>
			<td>Biolegend</td>
			<td>103155</td>
			<td></td>
		</tr>
		<tr>
			<td>BV711 CD4 (clone RM4-5)</td>
			<td>BD Biosciences</td>
			<td>563726</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin (Fraction V)</td>
			<td>Fisher Scientific</td>
			<td>BP1600-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Anit-Rat and Anti-Hamster Igk / Negative Control Compensation Particle Set</td>
			<td>BD Biosciences</td>
			<td>552845</td>
			<td></td>
		</tr>
		<tr>
			<td>Fortessa Flow Cytometer</td>
			<td>BD Biosciences</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>FlowJo v10 Software</td>
			<td>FlowJo</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

quantifying leukocyte egress via lymphatic vessels from murine skin and tumors

Leukocyte egress from peripheral tissues to draining lymph nodes is not only critical for immune surveillance and initiation but also contributes to the resolution of peripheral tissue responses. While a variety of methods are used to quantify leukocyte egress from non-lymphoid, peripheral tissues, the cellular and molecular mechanisms that govern context-dependent egress remain poorly understood. Here, we describe the use of in situ photoconversion for quantitative analysis of leukocyte egress from murine skin and tumors. Photoconversion allows for the direct labeling of leukocytes resident within cutaneous tissue. Though skin exposure to violet light induces local inflammatory responses characterized by leukocyte infiltrates and vascular leakiness, in a head-to-head comparison with transdermal application of fluorescent tracers, photoconversion specifically labeled migratory dendritic cell populations and simultaneously enabled the quantification of myeloid and lymphoid egress from cutaneous microenvironments and tumors. The mechanisms of leukocyte egress remain a missing component in our understanding of intratumoral leukocyte complexity, and thus the application of the tools described herein will provide unique insight into the dynamics of tumor immune microenvironments both at steady state and in response to therapy.

We first sought to replicate photoconversion results published in the literature to evaluate the efficiency and determine the associated inflammation in the mouse skin. The ear pinna was exposed to 100 mW violet light (405 nm) for 3 min as previously described33. Single cell suspensions generated from the ear skin or cervical dLNs immediately following the exposure revealed a 78% conversion efficiency of all CD45+ leukocytes in the skin with no converted...

Watch this Scientific Journal Video about Quantifying Leukocyte Egress via Lymphatic Vessels from Murine Skin and Tumors at JoVE.com

Quantifying Leukocyte Egress via Lymphatic Vessels from Murine Skin and Tumors

Here, we demonstrate the methods for in vivo quantification of leukocyte egress from na&#239;ve, inflamed, and malignant murine skin. We perform a head-to-head comparison of two models: transdermal FITC application and in situ photoconversion. Furthermore, we demonstrate the utility of photoconversion for tracking leukocyte egress from cutaneous tumors.

Leukocyte egress from peripheral tissues to draining lymph nodes is not only critical for immune surveillance and initiation but also contributes to the ...

Photoconversion provides a trackable in vivo method for the quantitative analysis of leukocyte egress for a mechanistic insight into leukocyte residence and exit dynamics from multiple tissue sites in diseased states. Well, here we demonstrate the utility of leukocyte tracking from cutaneous tumors. The application of this assay to other tissues and diseases is limited only by the ability to administer light.The main advantage of this technique is that to allows the quantification of endogenous, rather than transferred immune cell populations as a egress from inflamed tissues in vivo. To induce inflammation, after confirming a lack of response to toe pinch, use a micropipette to administer 20 microliters of dibutyl phthalate or DBP, to the ventral side of the ear pinna of an anesthetized Kaede transgenic mouse. After allowing the DBP to dry, pull the ear through a slit in a piece of aluminum foil and use double-sided tape to secure the ear flatly to the foil with the dorsal side facing upward.Then, position that you're directly under a 405 nanometer light source and photoconvert for three minutes at 100 milliwatts of power. For vaccinia infection, use a micropipette to administer 5 times 10 to the 6th plaque forming units of vaccinia virus in 10 microliters of PBS on to the ear pinna of an anesthetized Kaede transgenic mouse and poke the pinna 25 times. 24 hours later, photoconvert the pinna as just demonstrated.At the appropriate experimental time point, harvest the pinnae and the cervical and inguinal lymph nodes and use two pairs of tweezers to peel apart the ventral and dorsal side of each pinna. Then place the lymph nodes and the pinna pieces, with the inside of the ear facing down, into individual wells of a 24-well plate containing 1 milligram per milliliter of Collagenase D and 80 units per milliliter of Dnase diluted in HBSS containing calcium and magnesium. Use two 29 gauge needles to tease open each lymph node capsule and place the plate at 37 degrees Celsius for 30 minutes.At the end of the incubation, press the digested pinna and lymph node tissues through separate 70 Micron nylon cell strainers to obtain single cell suspensions of each tissue. When the tumors have grown to the appropriate experimental size, shave any newly regrown fur around the tumor site and pull the tumor through a circular hole cut in a piece of aluminum foil. Position the tumor directly below the 405 nanometer light source and photoconvert for 5 minutes at 200 milliwatts of power.24 hours after the photoconversion, harvest the tumors and brachial and inguinal lymph nodes from each animal and use scissors to mince the tumors within individual wells of a 24-well plate containing Collagenase D and DNase in HBSS. Place the lymph nodes into separate wells and tease open the capsules as just demonstrated. After incubating the tumors for one hour and the lymph nodes for a half hour at 37 degrees Celsius, press the digested tissues through separate 70 Micron strainers to obtain single cell suspensions of each tissue.To analyze the single cell suspensions by flow cytometry, first collect the spleen or blood from a Kaede transgenic mouse and lyse the red blood cells with ammonium chloride potassium buffer. Split the cells into one unconverted Kaede FITC and one converted Kaede PE population and resuspend the single color Kaede PE cells in one milliliter of PBS in one well of a 24-well plate, then photoconvert the cells for 5 minutes under a 405 nanometer light source at 100 milliwatts of power. Next use the photoconverted Kaede PE cells and the unconverted Kaede FITC cells to set the photomultiplier voltages to 70 to 80%of the total range of each fluorescent channel.Once the voltages for the Kaede proteins have been set, compensate for all of the other primary antibody stains using single fluorophore labeled compensation beads, according to the manufacturer's instructions. Then, run the samples on the flow cytometer according to standard flow cytometric analysis protocols. Single cell suspensions generated from the ear skin or cervical draining lymph nodes immediately following photoconversion exposure reveal a 78%conversion efficiency of all CD45 positive leukocytes in the skin, with no converted cells observed in the draining lymph nodes.Quantification of the numbers of infiltrating CD45 positive leukocytes in photoconverted ears zero and 24 hours post-conversion, reveals that violet light exposure causes a statistically significant increase in leukocyte infiltration. This increase in CD45 positive leukocyte infiltration however, is small relative to the infiltration induced by a potent inflammatory stimulus such as vaccinia virus. Photoconversion also increases vascular permeability in the ear pinnae as measured by Miles assay, together indicating that effects of violet light on tissue inflammation should be considered when optimizing dose, exposure time and data interpretation.Flow cytometric analysis of dissociated tumor cells from photoconverted animals reveals a significant conversion of intratumoral CD45 positive cells from Kaede FITC positive to Kaede PE positive, while draining lymph node cells remain unconverted. The photoconversion efficiency of CD45 positive leukocytes varied significantly between tumor types and sizes, with CD45 positive leukocytes within small melanoma tumors demonstrating the most efficient photoconversion. As the tumor volumes increase, the conversion efficiency decreases to around 50%Notably, melanin has a striking impact on photoconversion efficiency with a maximum photoconversion of CD45 positive cells observed within melanin-producing tumors of only about 30%in smaller tumors, that drops to 10%as the tumor volumes approach 400 cubic millimeters.The examination of tumor draining lymph nodes 24 hours after photoconversion reveals the presence of Kaede red positive immune cells of various phenotypes, thus demonstrating leukocyte emigration from the photoconverted tumor. After its development, this technique paved the way for researchers in the field of Immunology to uncover the kinetics of steady state and inflamed leukocyte turnover in peripheral tissue. While attempting this procedure, it is important to ensure that only the tissue of interest is photoconverted, as inadvertent photoconversion of the surrounding tissue will compromise your results.Individuals new to this method should optimize the exposure time for conversion of their tissue, as well as duration of the times before the tissue collection occurs based on the dynamics of leukocyte of interest.

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8.4K visualizações.  Oregon Health and Science University. A fotoconversão fornece um método in vivo rastreável para a análise quantitativa da saída de leucócitos para uma visão mecanicista sobre a residência leucócito e a dinâmica de saída de múltiplos sítios teciduais em estados doentes. Bem, aqui demonstramos a utilidade do rastreamento de leucócitos de tumores cutâneos. A aplicação deste ensaio a outros tecidos e doenças é limitada apenas pela capacidade de administrar a luz.A principal vantagem dessa técnica é que per...