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.
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.
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).
2. Imaging procedure
3. Post-processing of ultrasound images
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 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.
Figure 1: The ultrasound imaging system used to monitor the increase in volume of the inguinal lymph nodes in the Braf/Pten genetically engineered mouse model of melanoma. Please click here to view a larger version of this figure.
Figure 2: Step-by-step summary of the imaging procedure and the post-processing of ultrasound images. Please click here to view a larger version of this figure.
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) H&E staining: melanin deposits are removed by incubating slices with 1% KOH and 3% H2O2. (upper right) Melanin detection performed by H&E staining without 1% KOH and 3% H2O2 treatment. (bottom left) 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 µm. Abbreviations: wks = weeks; wt = wild-type; 4-HT = 4-hydroxytamoxifen; H&E = hematoxylin and eosin; DAB = 3,3'-diaminobenzidine. Please click here to view a larger version of this figure.
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. Please click here to view a larger version of this figure.
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 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.
The authors have nothing to disclose.
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.
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 |