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Cancer Research

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

Published: September 8th, 2021

DOI:

10.3791/62527

1Institute of Clinical Physiology, National Research Council (IFC-CNR), 2Oncogenomics Unit, Core Research Laboratory, ISPRO

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 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 µm). 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 µm 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.

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.

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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 mice [B6.Cg-Braftm1Mmcm Ptentm1Hwu Tg(Tyr-cre/ERT2)13Bos/BosJ (Braf/Pten)] were used in this study (see the Table of Materials).

  1. Treat the mice with 4-hydroxytamoxifen (4.......

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After skin painting of Tyr::CreER+,BrafCA/+,Ptenlox/lox 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.......

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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 t2 with those colle.......

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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 LP.

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

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