The authors would like to thank S. Burchielli (FTGM, Pisa) for her assistance with animal procedures. This work was supported by ISPRO-Istituto per lo Studio la Prevenzione e la Rete Oncologica institutional funding to LP; MFAG #17095 awarded by AIRC-Associazione Italiana Ricerca sul Cancro to&#160;LP.

All methods described here have been approved by the Italian Ministry of Health (animal protocols #754/2015-PR and #684/2018-PR).
1. Melanoma induction
NOTE: Six-week-old Tyr::CreER+,BrafCA/+,Ptenlox/lox&#160;mice [B6.Cg-Braftm1Mmcm Ptentm1Hwu Tg(Tyr-cre/ERT2)13Bos/BosJ (Braf/Pten)] were used in this study (see the Table of Materials).
<ol>
	<li>Treat the mice with 4-hydroxytamoxifen (4-HT) by applying 3 &#181;L of 5 mM 4-HT on ~1 cm2 of shaved skin of the upper back, as described previously11,16,17, for 3 days in a row. 
	NOTE: This will activate the Cre enzyme and cause a switch from wt Braf to BrafV600E and the loss of Pten. These two hits are enough to induce melanoma formation.</li>
	<li>Observe that&#160;primary tumors develop at the site of the skin painting in 2-3 weeks and reach a volume of 50-100 mm3 in 4 weeks. Also, observe metastases to other skin sites, lymph nodes, and lungs at this time point (t0).</li>
	<li>Use calipers to measure the volume of the primary tumor and of subcutaneous metastases, and ultrasound imaging to measure the volume of the inguinal lymph nodes. Repeat these measurements after one week (t1, 5 weeks after skin painting) and after two weeks (t2, 6 weeks after skin painting).</li>
	<li>At the last time point, euthanize the mice by overdosing with gaseous sevorane.</li>
	<li>Analyze the primary tumor and lymph nodes by visual inspection, then excise them for histological studies, as reported in16.</li>
</ol>
2. Imaging procedure
<ol>
	<li>Place the mouse in an induction chamber for gas anesthesia and supply 3% isoflurane in pure oxygen until the animal is fully anesthetized. Verify the depth of anesthesia by lack of response to paw pinch.</li>
	<li>Transfer the animal to a heated board - a constitutive part of the UHFUS imaging station - holding the animal in a supine position. Use a rectal probe lubricated with petroleum jelly to measure the body temperature. Adjust the board temperature to maintain the mouse&#39;s body temperature in the physiological range (36 &#177; 1&#176;C).</li>
	<li>Moisten the mouse&#39;s eyes with vet ointment to prevent dryness during anesthesia. Supply narcotic gas (1.5% isoflurane in pure oxygen) through a mouse&#39;s nose mask. Adjust the percentage of isoflurane to maintain the correct depth of anesthesia.</li>
	<li>Coat the fore and hind paws with conductive paste and tape them to the ECG plate electrodes embedded in the board. Check that the physiological parameters (heart rate, respiration signal, and core body temperature) are correctly acquired and displayed.</li>
	<li>Remove hair from both inguinal areas by applying a depilatory agent and coat them with an acoustic coupling medium.</li>
	<li>Clamp the UHFUS linear probe (40 MHz center frequency) into a specialized 3D motor embedded in the UHFUS imaging station, allowing automated and stepwise movement of the probe.</li>
	<li>Properly orient and adjust the position of the ultrasound probe to obtain short-axis images of the inguinal lymph node (left/right), and place the region of interest in the focal zone.</li>
	<li>Scan the entire volume of the inguinal lymph node as a sequence of 2D B-mode images, as described previously18. Acquire images at multiple levels of the lymph node by linear movement of the transducer with step sizes on a micrometer scale, to generate 3D data in terms of automatically respiration- and cardiac-gated cine loops.</li>
	<li>Set the image recording with the following parameters: scan distance ranging between 2 and 5 mm (depending on lymph node size); step size 44 &#181;m, with an outcome of 46-114 scan steps/lymph node slices and an acquisition time of 1-3 min per animal. Digitally store the acquired images in raw format (DICOM) for further offline analyses.</li>
	<li>At the end of the imaging session, discontinue the gas anesthesia and allow the animal to recover on the heating board in sternal recumbency. Take care of the animal until it has regained sufficient consciousness to maintain the prone position.</li>
</ol>
3. Post-processing of ultrasound images
<ol>
	<li>Open the dataset of DICOM 3D images of the left/right inguinal lymph node in the software platform for ultrasound image post-processing.</li>
	<li>Segmentation:
	<ol>
		<li>Select Multi-slice Method to visualize both the current frames and thumbnails of all frames corresponding to each image captured during the 3D acquisition.</li>
		<li>Select the thumbnail of the first frame to load it into the contouring view. In the contouring view, left-click on the mouse to drop points along the border of the lymph node. Once the desired number of points has been set (range 10-15), right-click to complete the contour.</li>
		<li>After the first contour is completed, use the thumbnail view to select the next image for contouring. If required, skip over several images (average of 3 frames) between contours to reduce the number of manual traces needed for each 3D volume. 
		NOTE: The software platform for ultrasound image post-processing will automatically generate contours between manual traces, thereby reducing the time of analysis.</li>
		<li>Repeat this process until the entire volume has been outlined. Once complete, click on Finish.</li>
	</ol>
	</li>
	<li>Generation of the 3D wireframe and volume measurement:
	<ol>
		<li>While in the 3D mode window workspace, click on the Volume measurement icon beneath the image display area to activate the surface view. 
		NOTE: The surface view creates a compilation view that maps the user-generated volume to the acquired image. The surface view can be rotated into any desired position.</li>
		<li>Take note of the volume measurement listed in the lower left-hand corner of the cube view. 
		NOTE: Segmentation and 3D volume generation can also be obtained using custom-developed software and/or freely available/commercial software for general image processing. Starting from manual segmentation, the software should provide a mathematical and/or pixel-level description of the lymph node contours. These contours would be combined in a 3D space to render the external surface of lymph nodes. All steps described in the imaging procedure and the post-processing of ultrasound images are summarized in Figure 2.</li>
	</ol>
	</li>
</ol>

<ol>
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The authors have nothing to disclose.

The data obtained in this study attest the ability of ultrasound imaging to monitor the metastatic involvement of inguinal lymph nodes of the Braf/Pten mouse model of metastatic melanoma. As shown previously16, this technique is especially useful to assess the efficacy of drug treatment. This is because it allows the monitoring of the change in lymph node volume in the same animal over time, by comparing the measurements collected at t1 and&#160;t2 with those collected at t0. This, in turn, contributes to an increase in the robustness of the obtained results, because inter-mouse variability and other factors that might influence basal lymph node size are all accounted for. In addition, ultrasound imaging allows compliance with the 3R principle by reducing the number of animals per experimental group.
In Braf/Pten mice, not only inguinal, but also brachial and axillary lymph nodes are sites of metastatic spreading. However, it is advisable to focus on inguinal lymph nodes because the others are too close to the primary tumor site, which usually alters their localization and morphology during the development of the primary tumor itself. Alternatively, brachial and axillary lymph nodes might become suitable for ultrasound imaging if a different site of tumor induction is chosen, such as ears or paws8. As far as other metastatic sites are concerned, lungs cannot be studied using ultrasound imaging, because of the presence of air in the tissue. Theoretically, only superficial pulmonary metastases reaching the pleural interface could be visualized with this technique. Although micro computed tomography/positron emission tomography (CT/PET) could be used instead, this approach has several drawbacks, including high costs and limited availability. Furthermore, being based on ionizing radiations, micro CT/PET is hardly compatible with longitudinal measurements at multiple time points. Conversely, ultrasound imaging can be easily applied to the study of subcutaneous metastases and allows the measurement of both volume and vascularization16.
If a 2-week time frame is too short to appreciate the effects of the drug under study, a more peripheral induction site (e.g., the tip of the tail9,11) or a less tumor-prone genotype (Tyr::CreER+,BrafCA/+,Ptenlox/+ mice instead of Tyr::CreER+, BrafCA/+, Ptenlox/lox mice) could be selected9. In both cases, the growth of the primary tumor is expected to be much slower, allowing metastasis monitoring for far more than 6 weeks after induction with 4-HT.
From a more technical point of view, it is important to note that 2D segmentation of ultrasound images is the most critical step in this protocol, because it may affect the measurement of 3D volume. Luckily, in the Braf/Pten animal model, the contrast between lymph nodes and surrounding tissues is quite marked, so that the outlining of the lymph node borders by manual segmentation is relatively simple. However, the segmentation process should be facilitated by the high quality of the ultrasound images acquired by the sonographer, who, in turn, should be highly experienced and focused on acquiring the same ultrasound projection of the lymph node, even in scan sessions performed at different time points.
B-mode ultrasound imaging cannot highlight cancer cells directly; instead, it allows the inference of their presence from the increase in the volume of inguinal lymph nodes. In light of this information, it is recommended that ultrasound imaging be coupled with appropriate IHC staining of the lymph nodes, so that the presence of cancer cells can be confirmed at the molecular level. However, a lymph node enlargement observed in an induced Braf/Pten mouse is typically attributable to cancer spreading and not to some other cause , e.g., an ongoing infection. This is likely because experimental mice used for ultrasound imaging are bred in controlled conditions and are routinely subjected to sanitary screening, so that sickness is promptly spotted and treated.

Melanoma is an aggressive form of skin cancer that often spreads to other skin sites (subcutaneous metastases), as well as to lymph nodes, lungs, liver, brain, and bones1. In the last decade, new drugs have been introduced into clinical practice and have contributed to improving the life expectancy of metastatic melanoma patients. However, limitations remain, including variable time to and degree of response, severe side effects, and the insurgence of acquired resistance1. Therefore, it is crucial to detect metastatic spreading at its early stages, i.e., when it gets to the local lymph nodes.
A biopsy of the local lymph nodes (sentinel lymph nodes) is usually performed to check for the presence of melanoma cells. However, ultrasound imaging is taking hold as a non-invasive method of detecting metastatic involvement, as it outperforms clinical evaluation and can help avoid an unnecessary biopsy2,3,4. Furthermore, ultrasound imaging seems appropriate for lymph node surveillance, especially in the case of advanced age and/or comorbidities5,6. The features that are detected by ultrasound analysis and allow the differentiation between normal and metastatic lymph nodes comprise increased size (volume), change of shape from oval to round, irregular margin, altered echogenic pattern, and altered (increased) vascularization7.
Tyr::CreER+,BrafCA/+,Ptenlox/lox genetically engineered mice (Braf/Pten mice) have recently been made available to the scientific community as a tissue-specific and inducible model for metastatic melanoma8. In this animal model, primary tumors develop very quickly: they become visible within 2-3 weeks after the induction of the switch from wild-type (wt) Braf to BrafV600E and of the loss of Pten, while they reach a volume of 50-100 mm3 within 4 weeks. In the following 2 weeks, the growth of the primary tumor is accompanied by a progressive increase in metastatic burden in other skin sites, lymph nodes, and lungs.
Braf/Pten mice have been extensively used for multiple purposes including the dissection of signaling pathways involved in melanomagenesis9,10, the identification of melanoma cells of origin11,12,13, and the testing of new therapeutic options in terms of both targeted therapy and immunotherapy8,14,15,16. Specifically, we used Braf/Pten mice to demonstrate that attenuated Listeria monocytogenes (Lmat) works as an anti-melanoma vaccine. When systemically administered in the therapeutic setting, Lmat is not associated with overall toxicity as it selectively accumulates at tumor sites. Furthermore, it causes a remarkable decrease in primary melanoma mass and a reduction in metastatic burden in the lymph nodes and lungs. At the molecular level, Lmat causes apoptotic killing of melanoma cells, which is due, at least in part, to non-cell-autonomous activities (recruitment on-site of CD4+ and CD8+ T lymphocytes)16.
When Braf/Pten mice are used for melanoma modeling, the growth of primary tumors and subcutaneous metastases can be monitored by caliper measurements. However, the involvement of lymph nodes and lungs needs to be investigated using an alternative technique, possibly a non-invasive one that allows researchers to follow the same animal over time. This paper describes the use of ultrasound imaging (Figure 1), coupled with a subsequent 3D volumetric analysis of the obtained data, for the longitudinal monitoring of the increase in size (volume) of inguinal lymph nodes.

<table><tbody><tr><td>4-hydroxytamoxifen</td><td>Merck</td><td>H6278</td><td>drug used for tumor induction</td></tr><tr><td>B6.Cg-Braftm1Mmcm Ptentm1Hwu Tg(Tyr-cre/ERT2)13Bos/BosJ (Braf/Pten) mice</td><td>The Jackson Laboratory</td><td>013590</td><td></td></tr><tr><td>Blu gel</td><td>Sooft Ialia</td><td></td><td>ophthalmic solution gel</td></tr><tr><td>BRAFV600E antibody</td><td>Spring Bioscience Corporation</td><td>E19290</td><td></td></tr><tr><td>IsoFlo (isoflorane)</td><td>Zoetis</td><td></td><td>liquid for gaseous anaesthesia</td></tr><tr><td>MLANA antibody</td><td>Thermo Fisher Scientific</td><td>M2-7C10</td><td></td></tr><tr><td>Sigma gel</td><td>Parker</td><td></td><td>electrode gel</td></tr><tr><td>Transonic gel clear</td><td>Telic SAU</td><td></td><td>ultrasound gel</td></tr><tr><td>Veet</td><td>Reckitt Benckiser IT</td><td></td><td>depilatory cream</td></tr><tr><td>Compact Dual Anesthesia System</td><td>Fujifilm, Visualsonics Inc.</td><td></td><td>Isoflurane-based anesthesia system equipped with nose cone and induction chamber</td></tr><tr><td>MX550S</td><td>Fujifilm, Visualsonics Inc.</td><td></td><td>UHFUS linear probe</td></tr><tr><td>Vevo 3100</td><td>Fujifilm, Visualsonics Inc.</td><td></td><td>UHFUS system</td></tr><tr><td>Vevo Imaging Station</td><td>Fujifilm, Visualsonics Inc.</td><td></td><td>UHFUS imaging station and Advancing Physiological Monitoring Unit endowed with heated board</td></tr><tr><td>Vevo Lab</td><td>Fujifilm, Visualsonics Inc.</td><td></td><td>software platform for ultrasound image post-processing</td></tr></tbody></table>

analysis of lymph node volume by ultra-high-frequency ultrasound imaging in the braf/pten genetically engineered mouse model of melanoma

Tyr::CreER+,BrafCA/+,Ptenlox/lox&#160;genetically engineered mice (Braf/Pten mice) are widely used as an in vivo model of metastatic melanoma. Once a primary tumor has been induced by tamoxifen treatment, an increase in metastatic burden is observed within 4-6 weeks after induction. This paper shows how Ultra-High-Frequency UltraSound (UHFUS) imaging can be exploited to monitor the increase in metastatic involvement of the inguinal lymph nodes by measuring the increase in their volume.
The UHFUS system is used to scan anesthetized mice with a UHFUS linear probe (22-55 MHz, axial resolution 40 &#181;m). B-mode images from the inguinal lymph nodes (both left and right sides) are acquired in a short-axis view, positioning the animals in dorsal recumbency. Ultrasound records are acquired using a 44 &#181;m step size on a motorized mechanical arm. Afterward, two-dimensional (2D) B-mode acquisitions are imported into the software platform for ultrasound image post-processing, and inguinal lymph nodes are identified and segmented semi-automatically in the acquired cross-sectional 2D images. Finally, a total reconstruction of the three-dimensional (3D) volume is automatically obtained along with the rendering of the lymph node volume, which is also expressed as an absolute measurement.
This non-invasive in vivo technique is very well tolerated and allows the scheduling of multiple imaging sessions on the same experimental animal over 2 weeks. It is, therefore, ideal to assess the impact of pharmacological treatment on metastatic disease.

After skin painting of Tyr::CreER+,BrafCA/+,Ptenlox/lox&#160;mice with 4-HT, Cre activity is induced, due to which there is a switch at the genomic level from wt Braf to BrafV600E, while Pten is lost (Figure 3A). In 2-3 weeks, mice develop on-site primary tumors with 100% penetrance. After four weeks from 4-HT treatment (t0), primary tumors reach a volume of 50-100 mm3, and their growth can be measured by calipers for an additional 2 weeks ((t1 and t2; Figure 3B, upper panels). Later time points cannot be reached because the tumor becomes so big that the mice require euthanasia.
As far as the metastatic burden is concerned, a gradual increase in pigmentation is observed in the inguinal lymph nodes within 4-6 weeks from 4-HT treatment (Figure 3B, lower panels). Such an increase in pigmentation is due to the presence of melanin deposits, as can be confirmed by hematoxylin and eosin staining performed without removing the melanin. In turn, the melanin deposits are invariably due to the presence of metastasized melanoma cells, as confirmed by immunohistochemical (IHC) staining of the melanoma antigen MLANA and of BRAFV600E (Figure 3C).
The accumulation of pigmented melanoma cells inside inguinal lymph nodes is accompanied by a progressive increase in their volume, as evident by visual inspection (Figure 3B, lower panels). Ultrasound imaging offers the unique opportunity to quantify such an increase longitudinally, in each experimental mouse, as described previously16. Volumetric measurements, segmentation results, and 3D rendering, all referring to a representative case, are shown in Figure 4. The volume of each lymph node is obtained by manual segmentation of the axial sections acquired during a 3D scan.
At the end of the segmentation phase, all the sections show the overlay of the external contour of the lymph node (Figure 4A). These contours are connected frame-by-frame in the rendering phase, and the external surface of the entire lymph node is projected in the 3D space. As a representative example, the 3D rendering of a right inguinal lymph node analyzed at t0, t1, and t2 is shown in Figure 4B, Figure 4C, and Figure 4D, respectively. The graph in Figure 4E quantifies the increase in volume displayed by the left and right inguinal lymph nodes of the same animal over time.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62527/62527fig01.jpg" /> 
Figure 1: The ultrasound imaging system used to monitor the increase in&#160;volume of the inguinal lymph nodes in the Braf/Pten genetically engineered mouse model of melanoma. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62527/62527fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62527/62527fig2v3.jpg" /> 
Figure 2: Step-by-step summary of the imaging procedure and the post-processing of ultrasound images. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62527/62527fig2v3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/62527/62527fig03.jpg" /> 
Figure 3: Visual inspection and histological analyses of inguinal lymph nodes in the tissue-specific and inducible Braf/Pten metastatic melanoma model in the mouse. (A) Cre enzyme causes the switch of wt Braf into BrafV600E and the loss of Pten (excision of exons 4 and 5). This system is melanocyte-specific because the expression of the Cre enzyme is under the control of the promoter of tyrosinase, an enzyme involved in melanin synthesis. Therefore, the two oncogenic hits are restricted to the melanocytic lineage. This system is also inducible because Cre is expressed as a fusion protein with the estrogen receptor and requires skin painting with 4-HT to be translocated into the nucleus, where it can exert its function. (B) The appearance of primary melanoma tumors (upper pictures) and inguinal lymph nodes (lower pictures) after 4, 5, and 6 weeks after 4-HT treatment (t0, t1, and t2, respectively). In lymph nodes, the increase in melanin accumulation and size is detected by visual inspection. Scale bars = 0.5 cm (upper pictures); 0.2 cm (lower pictures). (C) Histological analyses of inguinal lymph nodes, 6 weeks after 4-HT treatment. (upper left)&#160;H&#38;E staining: melanin deposits are removed by incubating slices with 1% KOH and 3% H2O2.&#160;(upper right)&#160;Melanin detection performed by H&#38;E staining without 1% KOH and 3% H2O2 treatment. (bottom left)&#160;MLANA detection by immunoperoxidase staining (DAB chromogen substrate and hematoxylin counterstaining). (bottom right) BRAFV600E detection by immunoperoxidase staining (DAB chromogen substrate and hematoxylin counterstaining). For all panels, original magnification: 40x; scale bars = 20 &#181;m. Abbreviations: wks = weeks;&#160;wt = wild-type; 4-HT = 4-hydroxytamoxifen; H&#38;E = hematoxylin and eosin; DAB = 3,3&#39;-diaminobenzidine. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62527/62527fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/62527/62527fig04.jpg" /> 
Figure 4: Measurements, segmentation, and 3D rendering of the volume of the inguinal lymph nodes in the tissue-specific and inducible Braf/Pten metastatic melanoma model in the mouse. (A) Overlay of the external contours of the right inguinal lymph node in 4 representative scanned sections obtained by manual segmentation. (B-D) Rendering of the 3D volume of the right inguinal lymph node, as measured at 4, 5, and 6 weeks after 4-HT treatment (t0, t1, and t2 time points, respectively). The numerical value of the volume is also reported (in mm3). (E) The volume of the left (black circle) and the right (white circle) inguinal lymph node of the same animal at t0, t1, and t2 time points. Abbreviations: 3D = three-dimensional; 4-HT = 4-hydroxytamoxifen. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62527/62527fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Analysis of Lymph Node Volume by Ultra-High-Frequency Ultrasound Imaging in the Braf/Pten Genetically Engineered Mouse Model of Melanoma at JoVE.com

Analysis of Lymph Node Volume by Ultra-High-Frequency Ultrasound Imaging in the Braf/Pten Genetically Engineered Mouse Model of Melanoma

Melanoma is a very aggressive disease that quickly spreads to other organs. This protocol describes the application of ultra-high-frequency ultrasound imaging, coupled with 3D rendering, to monitor the volume of the inguinal lymph nodes in the Braf/Pten mouse model of metastatic melanoma.

Tyr::CreER+,BrafCA/+,Ptenlox/lox&#160;genetically engineered mice (Braf/Pten mice) are widely used as an in vivo model of metastatic melanoma. Once a ...

analysis-lymph-node-volume-ultra-high-frequency-ultrasound-imaging

Nanyang Technological University

Research

JoVE Journal

Cancer Research

2.9K Views.  National Research Council (IFC-CNR). Onze methode maakt het mogelijk om de toename van gemetastaseerde betrokkenheid van inguinale lymfeklieren te monitoren door de toename van hun volume te meten. Deze niet-invasieve methode wordt zeer goed verdragen, daarom kunnen meerdere beeldvormingssessies in de loop van twee weken op hetzelfde proefdier worden gepland. Deze methode is ideaal voor het beoordelen van de impact van farmacologische behandeling op gemetastaseerde ziekte.We hebben deze methode toegepast op het Braf/Pten ...

Video: Analysis of Lymph Node Volume by Ultra-High-Frequency Ultrasound Imaging in the Braf/Pten Genetically Engineered Mouse Model of Melanoma

Non-invasive Optical Imaging of the Lymphatic Vasculature of a Mouse

The lymphatic vascular system is an important component of the circulatory system that maintains fluid homeostasis, provides immune surveillance, and mediates fat absorption in the gut. Yet despite its critical function, there is comparatively little understanding of how the lymphatic system adapts to serve these functions in health and disease1. Recently, we have demonstrated the ability to dynamically image lymphatic architecture and lymph "pumping" action in normal human subjects as well as in persons suffering lymphatic dysfunction using trace administration of a near-infrared fluorescent (NIRF) dye and a custom, Gen III-intensified imaging system2-4. NIRF imaging showed dramatic changes in lymphatic architecture and function with human disease. It remains unclear how these changes occur and new animal models are being developed to elucidate their genetic and molecular basis. In this protocol, we present NIRF lymphatic, small animal imaging5,6 using indocyanine green (ICG), a dye that has been used for 50 years in humans7, and a NIRF dye-labeled cyclic albumin binding domain (cABD-IRDye800) peptide that preferentially binds mouse and human albumin8. Approximately 5.5 times brighter than ICG, cABD-IRDye800 has a similar lymphatic clearance profile and can be injected in smaller doses than ICG to achieve sufficient NIRF signals for imaging8. Because both cABD-IRDye800 and ICG bind to albumin in the interstitial space8, they both may depict active protein transport into and within the lymphatics. Intradermal (ID) injections (5-50 &mu;l) of ICG (645 &mu;M) or cABD-IRDye800 (200 &mu;M) in saline are administered to the dorsal aspect of each hind paw and/or the left and right side of the base of the tail of an isoflurane-anesthetized mouse. The resulting dye concentration in the animal is 83-1,250 &mu;g/kg for ICG or 113-1,700 &mu;g/kg for cABD-IRDye800. Immediately following injections, functional lymphatic imaging is conducted for up to 1 hr using a customized, small animal NIRF imaging system. Whole animal spatial resolution can depict fluorescent lymphatic vessels of 100 microns or less, and images of structures up to 3 cm in depth can be acquired9. Images are acquired using V++ software and analyzed using ImageJ or MATLAB software. During analysis, consecutive regions of interest (ROIs) encompassing the entire vessel diameter are drawn along a given lymph vessel. The dimensions for each ROI are kept constant for a given vessel and NIRF intensity is measured for each ROI to quantitatively assess "packets" of lymph moving through vessels.

Recently developed imaging techniques using near-infrared fluorescence (NIRF) may help elucidate the role the lymphatic system plays in cancer metastasis, immune response, wound repair, and other lymphatic-associated diseases.

The lymphatic  vascular system is an important component of the circulatory system that  maintains fluid homeostasis, provides immune surveillance, and ...

Immunology and Infection

Studying Triple Negative Breast Cancer Using Orthotopic Breast Cancer Model

Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype with limited therapeutic options. When compared to patients with less aggressive breast tumors, the 5-year survival rate of TNBC patients is 77% due to their characteristic drug-resistant phenotype and metastatic burden. Toward this end, murine models have been established aimed at identifying novel therapeutic strategies limiting TNBC tumor growth and metastatic spread. This work describes a practical guide for the TNBC orthotopic model where MDA-MB-231 breast cancer cells suspended in a basement membrane matrix are implanted in the fourth mammary fat pad, which closely mimics the cancer cell behavior in humans. Measurement of tumors by caliper, lung metastasis assessment via in vivo and ex vivo imaging, and molecular detection are discussed. This model provides an excellent platform to study therapeutic efficacy and is especially suitable for the study of the interaction between the primary tumor and distal metastatic sites.

This work presets an advanced protocol to accurately assess tumor loading by detection of green fluorescent protein and bioluminescence signals as well as the integration of quantitative molecular detection technique.

Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype with limited therapeutic options. When compared to patients with less ...

Detection of Lung Tumor Progression in Mice by Ultrasound Imaging

With ~1.6 million victims per year, lung cancer contributes tremendously to the worldwide burden of cancer. Lung cancer is partly driven by genetic alterations in oncogenes such as the KRAS oncogene, which constitutes ~25% of lung cancer cases. The difficulty in therapeutically targeting KRAS-driven lung cancer partly stems from having poor models that can mimic the progression of the disease in the lab. We describe a method that permits the relative quantification of primary KRAS lung tumors in a Cre-inducible LSL-KRAS G12D mouse model via ultrasound imaging. This method relies on brightness (B)-mode acquisition of the lung parenchyma. Tumors that are initially formed in this model are visualized as B-lines and can be quantified by counting the number of B-lines present in the acquired images. These would represent the relative tumor number formed on the surface of the mouse lung. As the formed tumors develop with time, they are perceived as deep clefts within the lung parenchyma. Since the circumference of the formed tumor is well-defined, calculating the relative tumor volume is achieved by measuring the length and width of the tumor and applying them in the formula used for tumor caliper measurements. Ultrasound imaging is a non-invasive, fast and user-friendly technique that is often used for tumor quantifications in mice. Although artifacts may appear when obtaining ultrasound images, it has been shown that this imaging technique is more advantageous for tumor quantifications in mice compared to other imaging techniques such as computed tomography (CT) imaging and bioluminescence imaging (BLI). Researchers can investigate novel therapeutic targets using this technique by comparing lung tumor initiation and progression between different groups of mice.

This protocol describes the steps taken to induce KRAS lung tumors in mice as well as the quantification of formed tumors by ultrasound imaging. Small tumors are visualized in early timepoints as B-lines. At later timepoints, relative tumor volume measurements are achieved by the measurement tool in the ultrasound software.

With ~1.6 million victims per year, lung cancer contributes tremendously to the worldwide burden of cancer. Lung cancer is partly driven by genetic ...

A Mouse Model to Investigate the Role of Cancer-Associated Fibroblasts in Tumor Growth

Cancer-associated fibroblasts (CAFs) can play an important role in tumor growth by creating a tumor-promoting microenvironment. Models to study the role of CAFs in the tumor microenvironment can be helpful for understanding the functional importance of fibroblasts, fibroblasts from different tissues, and specific genetic factors in fibroblasts. Mouse models are essential for understanding the contributors to tumor growth and progression in an in vivo context. Here, a protocol in which cancer cells are mixed with fibroblasts and introduced into mice to develop tumors is provided. Tumor sizes over time and final tumor weights are determined and compared among groups. The protocol described can provide more insight into the functional role of CAFs in tumor growth and progression.

A protocol to co-inject cancer cells and fibroblasts and monitor tumor growth over time is provided. This protocol can be used to understand the molecular basis for the role of fibroblasts as regulators of tumor growth.

Cancer-associated fibroblasts (CAFs) can play an important role in tumor growth by creating a tumor-promoting microenvironment. Models to study the role ...

High-Resolution Ultrasonography for the Analysis of Orthotopic ATC Tumors in a Genetically Engineered Mouse Model

Anaplastic thyroid carcinoma (ATC) is associated with a poor prognosis and short median survival time, but no effective treatment improves the outcomes significantly. Genetically engineered murine models that mimic ATC&#39;s progression may help researchers to study treatments for this disease. Crossing three different genotypes of mice, a TPO-cre/ERT2; BrafCA/wt; Trp53&#916;ex2-10/&#916;ex2-10&#160;transgenic ATC model was developed. The ATC murine model was induced by an intraperitoneal injection of tamoxifen with overexpression of BrafV600E and deletion of Trp53, and the tumors were generated within about 1 month. High-resolution ultrasound was applied to investigate the tumor initiation and progression, and the dynamic growth curve was obtained by measuring the tumor sizes. Compared to magnetic resonance imaging (MRI) and computed tomography scanning, ultrasound has advantages in observing the ATC murine model, such as being noninvasive, portable, in real-time, and without radiation exposure. High-resolution ultrasound is suitable for dynamic and multiple measurements. However, ultrasonographic examination of the thyroid in mice requires relevant anatomical knowledge and experience. This article provides a detailed procedure for utilizing high-resolution ultrasound to scan tumors in the transgenic ATC model. Meanwhile, ultrasonic parameter adjustment, ultrasound scanning skills, anesthesia and recovery of the animals, and other elements that need attention during the process are listed.

The present protocol describes high-frequency ultrasonography for visualizing the entire mouse thyroid gland and monitoring the growth of anaplastic thyroid carcinoma.

Anaplastic thyroid carcinoma (ATC) is associated with a poor prognosis and short median survival time, but no effective treatment improves the outcomes ...

Implantation and Evaluation of Melanoma in the Murine Choroid via Optical Coherence Tomography

Establishing experimental choroidal melanoma models is challenging in terms of the ability to induce tumors at the correct localization. In addition, difficulties in observing posterior choroidal melanoma in vivo limit tumor location and growth evaluation in real-time. The approach described here optimizes techniques for establishing choroidal melanoma in mice via a multi-step sub-choroidal B16LS9 cell injection procedure. To enable precision in injecting into the small dimensions of the mouse uvea, the complete procedure is performed under a microscope. First, a conjunctival peritomy is formed in the dorsal-temporal area of the eye. Then, a tract into the sub-choroidal space is created by inserting a needle through the exposed sclera. This is followed by the insertion of a blunt needle into the tract and the injection of melanoma cells into the choroid. Immediately after injection, noninvasive optical coherence tomography (OCT) imaging is utilized to determine tumor location and progress. Retinal detachment is evaluated as a predictor of tumor site and size. The presented method enables the reproducible induction of choroid-localized melanoma in mice and the live imaging of tumor growth evaluation. As such, it provides a valuable tool for studying intraocular tumors.

Implantation and Evaluation of Melanoma in the Murine Choroid via Optical Coherence Tomography

The present protocol describes the implantation and evaluation of melanoma in the murine choroid utilizing optical coherence tomography.

Establishing experimental choroidal melanoma models is challenging in terms of the ability to induce tumors at the correct localization. In addition, ...

High-frequency Ultrasound Imaging of Mouse Cervical Lymph Nodes

High-frequency ultrasound (HFUS) is widely employed as a non-invasive method for imaging internal anatomic structures in experimental small animal systems. HFUS has the ability to detect structures as small as 30 &#181;m, a property that has been utilized for visualizing superficial lymph nodes in rodents in brightness (B)-mode. Combining power Doppler with B-mode imaging allows for measuring circulatory blood flow within lymph nodes and other organs. While HFUS has been utilized for lymph node imaging in a number of mouse&#160; model systems, a detailed protocol describing HFUS imaging and characterization of the cervical lymph nodes in mice has not been reported. Here, we show that HFUS can be adapted to detect and characterize cervical lymph nodes in mice. Combined B-mode and power Doppler imaging can be used to detect increases in blood flow in immunologically-enlarged cervical nodes. We also describe the use of B-mode imaging to conduct fine needle biopsies of cervical lymph nodes to retrieve lymph tissue for histological&#160; analysis. Finally, software-aided steps are described to calculate changes in lymph node volume and to visualize changes in lymph node morphology following image reconstruction. The ability to visually monitor changes in cervical lymph node biology over time provides a simple and powerful technique for the non-invasive monitoring of cervical lymph node alterations in preclinical mouse models of oral cavity disease.

This protocol describes the application of high-frequency ultrasound (HFUS) for imaging mouse cervical lymph nodes. This technique optimizes visualization and quantification of cervical lymph node morphology, volume and blood flow. Image-guided biopsy of cervical lymph nodes and processing of lymph tissue for histological evaluation is also demonstrated.

High-frequency ultrasound (HFUS) is widely employed as a non-invasive method for imaging internal anatomic structures in experimental small animal ...

Medicine

Evaluating Antigen-Specific CD8+ T Cells During Lymph Node Metastasis

Draining Lymph Node Metastasis Model for Assessing the Dynamics of Antigen-Specific CD8+ T Cells During Tumorigenesis

Tumor antigen-specific CD8+ T cells from draining lymph nodes gain an accumulating importance in mounting anti-tumor immune response during tumorigenesis. However, in many cases, cancer cells form metastatic loci in lymph nodes before further metastasizing to distant organs. To what extent the local and systematic CD8+ T cell responses were influenced by LN metastasis remains obscure. To this end, we set up a murine LN metastasis model combined with a B16F10-GP melanoma cell line expressing the surrogate neoantigen derived from lymphocytic choriomeningitis virus (LCMV), glycoprotein (GP), and P14 transgenic mice harboring T cell receptors (TCRs) specific to GP-derived peptide GP33-41 presented by the class I major histocompatibility complex (MHC) molecule H-2Db. This protocol enables the study of antigen-specific CD8+ T cell responses during LN metastasis. In this protocol, C57BL/6J mice were subcutaneously implanted with B16F10-GP cells, followed by adoptive transfer with naive P14 cells. When the subcutaneous tumor grew to approximately 5 mm in diameter, the primary tumor was excised, and B16F10-GP cells were directly injected into the tumor draining lymph node (TdLN). Then, the dynamics of CD8+ T cells were monitored during the process of LN metastasis. Collectively, this model has provided an approach to precisely investigate the antigen-specific CD8+ T cell immune responses during LN metastasis.

Author Spotlight: A Model to Study the Systemic and Local Dynamics of CD8+ T Cells During LN Metastasis

The experimental design presented here provides a useful reproductive model for the studies of antigen-specific CD8+ T cells during lymph node (LN) metastasis, which excludes the perturbation of bystander CD8+ T cells.

Tumor antigen-specific CD8+ T cells from draining lymph nodes gain an accumulating importance in mounting anti-tumor immune response during tumorigenesis. ...

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.

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 ...

Biology

Monitoring Pulmonary Nodule Progression Using Micro-Computed Tomography and Blood Sampling in a Mouse Model

Micro-computed tomography (micro-CT) is a real-time, intuitive, sensitive, and minimally invasive technique for monitoring changes from pulmonary nodules (PN) to lung cancer (LC). The integration of submandibular vein blood sampling enables rapid, stable, and straightforward detection of imaging and key target alterations during the progression of PN to LC. In this study, we administered a dosage of 100 mg/kg of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone in A/J mice to develop a lung adenocarcinoma model. Disease progression in the experimental animals was then monitored through submandibular vein blood sampling and micro-CT assay. Experimental results showed the presence of nodular foci in the lungs of some animals by the 10th week, with the development of lung adenocarcinoma images becoming evident by the 21st week. In conclusion, micro-CT can effectively observe pathological changes in the lungs of mice and, when combined with submandibular vein blood sampling, can dynamically monitor changes in blood, protein, and targets. This method provides a highly specific, simple, and sensitive approach for drug screening, pharmacokinetic testing, toxicological experiments, and safety studies.

This protocol describes an efficient, simple, and minimally invasive method for studying pulmonary nodules. Submaxillary vein blood collection and micro-CT imaging are used as investigative techniques.

Micro-computed tomography (micro-CT) is a real-time, intuitive, sensitive, and minimally invasive technique for monitoring changes from pulmonary nodules ...

Analysis of Lymph Node Volume by Ultra-High-Frequency Ultrasound Imaging in the Braf/Pten Genetically Engineered Mouse Model of Melanoma

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