Name: analysis of lymph node volume by ultra-high-frequency ultrasound imaging in the braf/pten genetically engineered mouse model of melanoma
Uploaded: 2021-09-08T14:00:00
Description: 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

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...

<ol>
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E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=&#946;-catenin+signaling+controls+metastasis+in+Braf-activated+Pten-deficient+melanomas.">&#946;-catenin signaling controls metastasis in Braf-activated Pten-deficient melanomas.</a> Cancer Cell. 20 (6), 741-754 (2011).</li><li>Xie, X., Koh, J. Y., Price, S., White, E., Mehnert, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Atg7+overcomes+senescence+and+promotes+growth+of+BrafV600E-driven+melanoma.">Atg7 overcomes senescence and promotes growth of BrafV600E-driven melanoma.</a> Cancer Discovery. 5 (4), 410-423 (2015).</li><li>Kohler, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+cutaneous+melanoma+induced+by+mutant+BRaf+arises+from+expansion+and+dedifferentiation+of+mature+pigmented+melanocytes.">Mouse cutaneous melanoma induced by mutant BRaf arises from expansion and dedifferentiation of mature pigmented melanocytes.</a> Cell Stem Cell. 21 (5), 679-693 (2017).</li><li>Yuan, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phenformin+enhances+the+therapeutic+benefit+of+BRAF(V600E)+inhibition+in+melanoma.">Phenformin enhances the therapeutic benefit of BRAF(V600E) inhibition in melanoma.</a> Proceedings of the National Academy of Sciences of the United States of America. 110 (45), 18226-18231 (2013).</li><li>Marsh Durban, V., Deuker, M. 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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 colle...

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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 additiona...

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 ...

Our method enables monitoring the increase in metastatic involvement of inguinal lymph nodes by measuring the increase in their volume. This uninvasive method is very well-tolerated, therefore, multiple imaging sessions can be scheduled on the same experimental animal over the course of two weeks. This method is ideal for assessing the impact of pharmacological treatment on metastatic disease.We applied this method to the Braf/Pten melanoma model, but in principle, it can be adapted to assess the efficacy of various drugs in a variety of preclinical mouse models of metastatic cancer. Demonstrating the procedure will be Dr.Marianna Vitiello, a post-doc from my laboratory, and Dr.Claudia Kusmic, fellow researcher at IFC-CNR. For melanoma induction, apply three microliters of five-millimolar 4-hydroxytamoxifen on approximately one square centimeter of shaved skin of the upper back for three days in a row.Six weeks after skin painting, use calipers to measure the volume of the primary tumor, and eventually, of subcutaneous metastases. Then, use ultrasound imaging to measure the volume of the inguinal lymph nodes. After analyzing the primary tumor and lymph nodes by visual inspection, excise them for histological studies.After confirming anesthesia, transfer the animal to a heated board of the ultra-high-frequency ultrasound imaging station, holding the animal in a supine position. Adjust the board temperature using a rectal probe lubricated with petroleum jelly to maintain the mouse's body temperature in the physiological range. 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 are correctly acquired and displayed. Remove hair from both inguinal areas by applying a depilatory agent and coat them with an acoustic coupling medium. Then, clamp the ultra-high-frequency ultrasound linear probe into a specialized 3D motor embedded in the imaging station, allowing automated and stepwise movement of the probe.Properly orient and adjust the position of the ultrasound probe to obtain short-access images of the inguinal lymph node, and place the region-of-interest the focal zone. Set the Scan Distance between two and five millimeters, Step Size to 44 micrometers with an outcome of 46 to 114 scan steps per lymph node slice, and an acquisition time of one to three minutes per animal. Scan the entire volume of the inguinal lymph node as a sequence of 2D B-mode images, acquiring images at multiple levels by linear movement of the transducer with step sizes on a micrometer scale to generate 3D data in terms of respiration and cardiac-gated cine loops.After imaging, digitally store the acquired images in raw format for further offline analyses. For post-processing of ultrasound images, open the dataset of the 3D inguinal lymph node images, select multi-slice method and press the Start button to visualize both the current frames and thumbnails of all frames corresponding to each image captured during the 3D acquisition. Select the thumbnail of the first frame where the lymph node is evident 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, right lick to complete the contour. After the first contour is completed, use the thumbnail view to select the next image for contouring.If required, skip over several images between contours to reduce the number of manual traces needed for each 3D volume. Repeat this process until the entire volume has been outlined, then, click on Finish. While in the 3D mode window workspace, click on the volume measurement icon beneath the image display area to visualize the numerical value of the lymph node volume, and activate the surface view with the 3D rendering of the lymph node volume.Treating mice with 4-hydroxytamoxifen induces CreER enzyme activity, causing a switch at the genomic level from wild-type Braf to mutated Braf V600E, while Pten is lost. Four weeks after 4-hydroxytamoxifen treatment, primary tumors reach a volume of 50 to 100 cubic millimeters and their growth can be measured by calipers for an additional two weeks, thereby showing a gradual increase in pigmentation observed in the inguinal lymph nodes. The melanin deposits are invariably due to the presence of metastasized melanoma cells, as confirmed by immunohistochemical staining of the melanoma antigen MelanA and Braf V600E.At the end of the segmentation phase, all sections showed the overlay of the external contour of the lymph node. The 3D renderings of a right inguinal lymph node, analyzed at four, five, and six weeks after 4-HT treatment are shown here. The increase in volume of the left and right inguinal lymph nodes of the same animal over time was also quantified.It's recommended to couple ultrasound imaging with appropriate immunohistochemical staining, so that the presence of cancer cells is confirmed at the molecular level. Besides lymph nodes, the volume of subcutaneous metastasis can also be monitored over time using ultrasound imaging. If the two-weeks timeframe is too short to appreciate the effects of the drug under study, select a more peripheral induction site or a less tumor-prone genotype.

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