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

Summary

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.

Abstract

Tyr::CreER+,Braf^CA/+,Pten^lox/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.

Introduction

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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Protocol

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+,Braf^CA/+,Pten^lox/lox mice [B6.Cg-Braf^tm1Mmcm Pten^tm1Hwu 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-HT) by applying 3 µL of 5 mM 4-HT on ~1 cm² of shaved skin of the upper back, as described previously^11,....

Results

After skin painting of Tyr::CreER+,Braf^CA/+,Pten^lox/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.......

Discussion

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.......

Materials

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