François Avry

Coralie Mousset

Edward Oujagir

Ayache Bouakaz

Valérie Gouilleux- Gruart

Rose-Anne Thépault

Sylvaine Renault

Sylviane Marouillat

Laurent Machet

Jean-Michel Escoffre
email: jean-michel.escoffre@univ-tours.fr.

PhD J-M Escoffre

Microbubble-assisted ultrasound for imaging and therapy of melanoma skin cancer: A systematic review

Keywords: Melanoma, ultrasound, microbubble, imaging, drug delivery, therapy

Recent technological developments in ultrasound (US) imaging and ultrasound contrast agents (UCAs) have improved the diagnostic confidence in echography. In the clinical management of melanoma, contrast-enhanced ultrasound (CEUS) imaging complements conventional US imaging (i.e., high-resolution US and Doppler imaging) for clinical examination and therapeutic follow-up. These developments have set into motion the combined use of ultrasound and UCAs as a new modality for drug delivery. This modality, called sonoporation, has emerged as a noninvasive, targeted, and safe method for the delivery of therapeutic drugs into melanoma. This review focuses on the results and prospects of using US and UCAs as dual modalities for CEUS imaging and melanoma treatment.

INTRODUCTION

Despite recent improvements in prevention, diagnosis, and treatment, malignant melanoma (MM) is the most aggressive and deadliest skin malignancy, with an increasing incidence in Europe [START_REF] Ferlay | Cancer incidence and mortality patterns in Europe: Estimates for 40 countries and 25 major cancers in 2018[END_REF]. The estimated incidence rates in Europe were 15.8/100 000 men and 14.6/100 000 women in 2018. Although MM is less common than other skin cancers, it is more lethal. In 2018, approximately 27 100 deaths were caused by MM in Europe [START_REF] Ferlay | Cancer incidence and mortality patterns in Europe: Estimates for 40 countries and 25 major cancers in 2018[END_REF]). When MM is diagnosed at an early stage (i.e., localized stage or stages I II), it can be efficiently treated. Indeed, MM is curable with surgery at this stage, with a 5-year specific survival rate of 98% [START_REF] Dowling | Update on Current Treatment Recommendations for Primary Cutaneous Melanoma[END_REF]. Patients with more advanced disease or in metastatic stages have worse outcomes. Conventional treatments, including chemotherapy and radiotherapy, are ineffective, with a 5-year survival of 5% [START_REF] Dowling | Update on Current Treatment Recommendations for Primary Cutaneous Melanoma[END_REF]). Nevertheless, new expensive biological drugs (i.e., immunotherapy with checkpoint inhibitors) or targeted therapy (BRAF and MEK inhibitors) have been designed in recent years, which may prolong survival with a specific 5-year survival of 50% [START_REF] Sarkisian | Current Clinical Trials in the Treatment of Melanomas[END_REF]. Currently, the average cost of diagnosis and treatment of MM is 10 times greater than that of nonmelanoma skin cancer [START_REF] Karimkhani | The global burden of melanoma: results from the Global Burden of Disease Study 2015[END_REF]. The productivity loss due to morbidity and premature mortality also contributes to a socioeconomic burden on society [START_REF] Karimkhani | The global burden of melanoma: results from the Global Burden of Disease Study 2015[END_REF]. In this context, biomedical research is ongoing to develop innovative methods for the early diagnosis and treatment of this aggressive skin cancer to reduce its morbidity and mortality while minimizing its socioeconomic burden.

Detection and staging of skin melanoma are critical with regard to treatment and patient outcomes. Later diagnosis significantly increases the risk of metastatic disease. The gold standard for the diagnosis of MM is based on the histological examination of the total removal of the lesion [START_REF] Ramalingam | Staging Melanoma: What's Old and New[END_REF]. The pathology report must include Breslow thickness (using the ocular micrometer), ulceration, presence of microsatellites, mitotic rate, and margin to perform an accurate staging of melanoma. Breslow thickness, also called the Breslow index, is considered the main prognostic factor for metastatic potential. This index determines the margin sizes of surgical resection of the primary lesion and the probability of microscopic involvement of regional lymph nodes that can be identified using the sentinel lymph node biopsy (SLNB) procedure [START_REF] Bichakjian | Guidelines of care for the management of primary cutaneous melanoma. American Academy of Dermatology[END_REF]. While radionuclide lymphoscintigraphy provides a roadmap for surgeons [START_REF] Tardelli | Sentinel Lymph Node Biopsy in Cutaneous Melanoma: Standard and New Technical Procedures and Clinical Advances. A Systematic Review of the Literature[END_REF], single-photon emission computed tomography/computed tomography (SPECT/CT) imaging provides a more accurate 3D visualization of SLNs than lymphoscintigraphy [START_REF] Chapman | SPECT/CT Improves Detection of Metastatic Sentinel Lymph Nodes in Patients with Head and Neck Melanoma[END_REF].

SPECT/CT helps in planning the surgical procedure by providing valuable anatomical and functional information concerning the tissue and vascular structures surrounding the SLNs. If the presence of metastases in SLNs is confirmed, 1-year adjuvant treatment with immunotherapy or BRAF/MEK inhibitors may be proposed. 18 F-fluorodeoxyglucose positron emission tomography/computer tomography ( 18 F-FDG PET/CT) imaging is currently used to detect suspicious distant melanoma metastases in clinical stage IIc disease [START_REF] Schroer-Gunther | F-18-fluoro-2-deoxyglucose positron emission tomography (PET) and PET/computed tomography imaging in primary staging of patients with malignant melanoma: a systematic review[END_REF]. Both whole-body 18 F-FDG PET/CT and dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) are recommended to assess the location and extent of brain metastases in stage III and higher disease. Finally, elevated blood lactate dehydrogenase levels are associated with poor outcomes and are utilized for stratification at advanced stages in the American Joint Committee on Cancer 8 th edition [START_REF] Gershenwald | Melanoma Staging: American Joint Committee on Cancer (AJCC) 8th Edition and Beyond[END_REF].

More recently, ultrasound (US) imaging has been proposed as a real-time, non-ionizing, and low-cost imaging modality for melanoma diagnosis [START_REF] Cammarota | Current uses of diagnostic high-frequency US in dermatology[END_REF]. In B-mode, the melanoma appears as well-delimited, fusiform, homogeneous, and hypoechoic lesions on highresolution US imaging (7 20 MHz) compared with other lesions [START_REF] Corvino | The Tail and the String Sign: New Sonographic Features of Subcutaneous Melanoma Metastasis[END_REF]. This modality provided thickness measurements that were well correlated with histological Breslow thickness measurements for lesions larger than 1 mm in thickness [START_REF] Serrone | High frequency ultrasound in the preoperative staging of primary melanoma: a statistical analysis[END_REF]. The use of very high-frequency probes, i.e., > 20 MHz, is strongly recommended for the accurate measurement of lesions less than 1 mm in thickness [START_REF] Gambichler | Preoperative ultrasonic assessment of thin melanocytic skin lesions using a 100-MHz ultrasound transducer: a comparative study[END_REF]). In addition, US imaging of draining LNs at low frequency, i.e., 5 10 MHz, is recommended when physical examination presents contradictory findings [START_REF] Catalano | Critical analysis of the ultrasonographic criteria for diagnosing lymph node metastasis in patients with cutaneous melanoma: a systematic review[END_REF]. US examination of regional LNs is advised before SLNB for a broader exploration of LNs in the body regions related to metastatic disease [START_REF] Voit | Ultrasound morphology criteria predict metastatic disease of the sentinel nodes in patients with melanoma[END_REF]. Moreover, an appropriate US imaging-guided preoperative mapping of the tumor lesion and the detection of melanoma infiltration in relevant anatomic structures (e.g., fascia, muscles, and bone cortex) allows improving surgical management, including the best surgical approach and the choice of appropriate surgical margins with low surgical risk, while enhancing the cosmetic prognosis of the patient [START_REF] Chaput | One-step surgical removal of cutaneous melanoma with surgical margins based on preoperative ultrasound measurement of the thickness of the melanoma[END_REF][START_REF] Machet | Preoperative measurement of thickness of cutaneous melanoma using high-resolution 20 MHz ultrasound imaging: A monocenter prospective study and systematic review of the literature[END_REF]). In addition, Doppler imaging has revealed that primary cutaneous melanoma frequently appears hypervascular [START_REF] Lassau | Prognostic value of angiogenesis evaluated with high-frequency and color Doppler sonography for preoperative assessment of melanomas[END_REF], Lassau, et al. 2006). The vascular density was positively correlated with [START_REF] Lassau | Prognostic value of angiogenesis evaluated with high-frequency and color Doppler sonography for preoperative assessment of melanomas[END_REF]. Both vascular density and thickness are significantly correlated with metastatic dissemination (Lassau, et al. 2006).

However, the introduction of ultrasound contrast agents (UCAs) in the late 1990s revolutionized the evaluation of tumor microvasculature by providing more accurate and sensitive information than color/power Doppler imaging. These agents are used to enhance specific body compartments and tissues [START_REF] Frinking | Three Decades of Ultrasound Contrast Agents: A Review of the Past, Present and Future Improvements[END_REF]). Thus, contrast-enhanced ultrasound imaging (CEUS) has become a complementary imaging modality to conventional US imaging for the clinical examination and therapeutic follow-up of patients with melanoma.

In addition, the combined use of US and UCAs as a new modality for drug delivery in the treatment of melanoma has been under investigation for a few years. In this review, we focus on the combined use of US and UCAs as a dual modality for CEUS imaging and melanoma treatment. The limitations of the available literature and future perspectives are discussed.

METHODS

PubMed ® and Web of Science TM electronic databases were screened by two authors of this review (FA and JME) using predefined search dates (January 1995 December 2021) and terms for in vivo and clinical studies investigating the contributions of microbubble-assisted ultrasound for the management of melanoma. The PubMed ® database search terms used were:

(melanoma

CONTRAST-ENHANCED ULTRASOUND IMAGING (CEUS)

Principle

Ultrasound contrast agents (UCAs) are used to enhance specific body compartments and tissues [START_REF] Frinking | Three Decades of Ultrasound Contrast Agents: A Review of the Past, Present and Future Improvements[END_REF]. This relative enhancement provides key anatomical, morphological, functional, and molecular information, which is essential for prognosis, diagnosis, lesion characterization, and therapeutic follow-up. UCAs are aqueous solutions of gas microbubbles (MBs) [START_REF] Sennoga | Microbubble-mediated ultrasound drug-delivery and therapeutic monitoring[END_REF]. Perifluorinated gases are preferred for UCAs. These gas MBs are surrounded by a biocompatible shell, which prevents coalescence and reduces gas dissolution in the blood. A large majority of commercially available MBs are stabilized by a phospholipid monolayer that is more acoustically effective because of its thin (i.e., 2 3 nm) and highly elastic shell than polymer MBs. Their size is in the 2 5 m range [START_REF] Frinking | Three Decades of Ultrasound Contrast Agents: A Review of the Past, Present and Future Improvements[END_REF]). Thus, after intravenous (i.v.) injection, these MBs are confined to the entire vasculature, including the smallest capillaries, making them pure intravascular tracers. The half-life of UCAs after i.v.

injection is typically few minutes [START_REF] Sennoga | Microbubble-mediated ultrasound drug-delivery and therapeutic monitoring[END_REF]). This duration is sufficient for imaging purposes and has the added advantage that several consecutive injections or infusions of UCAs can be achieved safely [START_REF] Frinking | Three Decades of Ultrasound Contrast Agents: A Review of the Past, Present and Future Improvements[END_REF]. Because of their compressibility, MBs exposed to US undergo nonlinear volumetric oscillations at a high mechanical index (MI) [START_REF] Frinking | Ultrasound contrast imaging: current and new potential methods[END_REF]. In this nonlinear oscillation mode, the nonlinear signal components of echo signals backscattered by the MBs are specific to MBs and are not present at a significant magnitude in tissue echoes [START_REF] Frinking | Ultrasound contrast imaging: current and new potential methods[END_REF]. Several imaging modes (e.g., second harmonic imaging, pulse inversion, and power modulation) have been developed to preferentially capture these nonlinear components while reducing echoes from nonperfused tissues [START_REF] Averkiou | Imaging Methods for Ultrasound Contrast Agents[END_REF].

These new advances have considerably improved the sensitivity of UCA detection, allowing real-time two-dimensional and three-dimensional (3D) imaging. Recent advances in image and signal processing methods (e.g., deconvolution and skeletonization methods) have allowed for an accurate description and characterization of the tumor microvascular network [START_REF] Gauthier | Assessment of quantitative perfusion parameters by dynamic contrast-enhanced sonography using a deconvolution method: an in vitro and in vivo study[END_REF][START_REF] Molinari | Quantitative assessment of cancer vascular architecture by skeletonization of highresolution 3-D contrast-enhanced ultrasound images: role of liposomes and microbubbles[END_REF]). These methods provide reliable quantitative parameters for assessing tumor perfusion for diagnostic and therapeutic follow-up purposes.

Moreover, ultrasound molecular imaging relies on the i.v. injection of targeted MBs that bind specific overexpressed endothelial biomarkers of angiogenesis (i.e., endoglin, vascular endothelial growth factor receptor-2) and adhesion (i.e., V 3 integrin) processes, which play a major role in tumor progression [START_REF] Klibanov | Ultrasound Molecular Imaging of Cancer: Design and Formulation Strategies of Targeted Contrast Agents[END_REF]. These MBs are functionalized by attaching a targeting agent (i.e., ligand, full, or fragment of antibody or peptide). After their i.v. injection, a fraction of targeted MBs binds the endothelial biomarkers, whereas several of them freely circulate into the blood stream because of the speed of blood flow. The acoustic signal from adherent MBs can be differentiated into circulating MBs using dedicated CEUS modes (Leguerney, et al. 2015). Thus, US imaging assesses the expression levels of target endothelial biomarkers to identify and quantify changes in angiogenesis and adhesion processes involved in melanoma progression.

Diagnosis and characterization of primary melanoma

The performance of CEUS in the diagnosis and characterization of primary melanoma has mainly been assessed in a mouse melanoma model (Figure 2; Table 2). As expected, Schroeder et al., reported that Doppler imaging is not as sensitive as CEUS for detecting melanoma neovascularity in a syngeneic mouse melanoma model [START_REF] Schroeder | Tumor vascularization in experimental melanomas: correlation between unenhanced and contrast enhanced power Doppler imaging and histological grading[END_REF]. The correlation between the percentage of vessel area and the histologic grade of vascularization was significantly improved when CEUS was used. In addition, Gauthier et al., demonstrated that CEUS can provide seven semiquantitative (i.e., peak intensity, slope to wash-in, time to peak intensity, full width at half maximum, area under the curve, area under the wash-out, and area under the wash-in) and three quantitative perfusion parameters (i.e., blood flow, blood volume, and mean transit time) with low intraoperator variability to quantitatively assess the perfusion of melanoma [START_REF] Gauthier | Estimation of intra-operator variability in perfusion parameter measurements using DCE-US[END_REF][START_REF] Gauthier | Assessment of quantitative perfusion parameters by dynamic contrast-enhanced sonography using a deconvolution method: an in vitro and in vivo study[END_REF]. Among these perfusion parameters, Pitre-Champagnat et al., reported that the area under the curve and the peak intensity are significantly correlated to the microvessel density but not to the diameter and maturity of blood vessels [START_REF] Pitre-Champagnat | Dynamic contrast-enhanced ultrasound parametric maps to evaluate intratumoral vascularization[END_REF]. These preclinical investigations demonstrated that CEUS provides reliable perfusion parameters for characterizing the microvasculature of primary melanomas.

Moreover, Forsberg et al., described that CEUS measures of tumor neovascularization provide a noninvasive biomarker of neoangiogenesis, corresponding to the expression of molecular biomarkers of neoangiogenesis (e.g., cyclooxygenase-2 and vascular endothelial growth factor (VEGF)) in both human melanoma xenograft models [START_REF] Forsberg | Comparing contrast-enhanced ultrasound to immunohistochemical markers of angiogenesis in a human melanoma xenograft model: preliminary results[END_REF][START_REF] Forsberg | Monitoring angiogenesis in human melanoma xenograft model using contrast-enhanced ultrasound imaging[END_REF]. In this context, in-vitro and preclinical studies have reported the design and validation of V 3 integrin-, endoglin-, and VEGF receptor 2 (VEGFR2)-targeted UCAs in the US molecular imaging of melanoma neovascularization (Leguerney, et al. 2015). These biomarkers are overexpressed at the surface of the melanoma microvasculature, and their expression is positively correlated with tumor grade. Thus, Leguerney et al., investigated the expression levels of endoglin, V 3 integrin, and VEGFR2 using US molecular imaging in murine melanoma models (Leguerney, et al. 2015). To achieve this goal, the microvasculature and expression levels of these endothelial biomarkers were assessed using UCAs conjugated with dedicated and biotinylated monoclonal antibodies. The results revealed that endoglin is more highly expressed on the surface of melanoma microvasculature than V 3 integrin and VEGFR2, suggesting that US molecular imaging using endoglin-targeted UCAs may be used for the noninvasive longitudinal evaluation of tumor angiogenesis during melanoma growth (Figure 3). In addition, the authors also demonstrated that the efficacy of anti-angiogenic treatments, e.g., sorafenib, on tumor angiogenesis can be detected by assessing the downregulation of endoglin expression at the surface of the tumor microvasculature (Leguerney, et al. 2015).

All of these studies show that the clinical potential of CEUS as well as molecular US imaging are complementary modalities for the detection and characterization of melanoma, as well as for the prediction of the efficacy of antiangiogenic treatment. Unfortunately, these studies were mainly conducted on animal models of melanoma. Further clinical investigations should be performed to confirm these preclinical studies and determine the performance of this imaging modality in a clinical context.

CEUS of advanced stages

In this subsection, we discuss the performance of dynamic contrast-enhanced ultrasound (DCEUS) in the diagnosis of melanoma metastases in the lymph nodes, liver, and gallbladder because their presence in other organs is preferentially detected and characterized using PET-CT imaging and/or MRI (Table 2) [START_REF] Dinnes | CT, MRI, or PET-CT for staging and re-staging of adults with cutaneous melanoma[END_REF].

Lymph nodes

SLNB is the current standard procedure for detecting and identifying metastases in LNs.

However, Bertelsen et al., identified three main limitations of this method [START_REF] Bertelsen | Contrast-Enhanced Ultrasound With Perflubutane for Sentinel Lymph Node Mapping in Cutaneous Melanoma: A Pilot Study[END_REF]): 1) the radiotracer detects secondary LNs, resulting in their dissections, thereby increasing operative time and morbidity; 2) if the primary lesion is located near the draining nodal basins in areas with a dense lymphatic supply, the use of a hand-held gamma probe is more challenging; and 3) lymphoscintigraphy does not offer depth information for anatomic SLN localization. To overcome this issue, the surgeon removes LNs and assesses them ex-vivo using a gamma probe. In this context, different imaging modalities, more specifically CEUS, have been investigated in swine melanoma models and in clinics [START_REF] Ungureanu | The role of lymph node ultrasound evaluation in melanoma -review of the literature[END_REF]).

Goldberg et al., investigated the performance of CEUS (also termed lymphatic ultrasonography or lymphosonography) for the detection of lymphatic channels, SLNs, and metastases after the peritumoral administration of MBs in a swine melanoma model [START_REF] Goldberg | Contrast-enhanced ultrasound imaging of sentinel lymph nodes after peritumoral administration of Sonazoid in a melanoma tumor animal model[END_REF][START_REF] Goldberg | Sentinel lymph nodes in a swine model with melanoma: contrast-enhanced lymphatic US[END_REF]. They reported that CEUS detected half as many false positives as lymphoscintigraphy did. The accuracy of SLN detection was 81.8% and 63.2% for CEUS and lymphoscintigraphy, respectively. The accuracy of CEUS for SLN characterization was 80%. However, when the size of the enhanced SLNs was considered in the characterization of SLNs, the accuracy was 86%. These results suggest that CEUS is statistically more efficient than lymphoscintigraphy in SLN detection in preclinical models. Nevertheless, the efficiency of CEUS for SLN characterization (benign versus malignant) is limited. Recently, Bertelsen et al., confirmed these results and suggested that CEUS may be useful for accurate SLN dissection (Figure 4) [START_REF] Bertelsen | Contrast-Enhanced Ultrasound With Perflubutane for Sentinel Lymph Node Mapping in Cutaneous Melanoma: A Pilot Study[END_REF]. Moreover, Liu et al., reported that secondary LNs can also be detected with CEUS after direct injection of MBs into contrast-enhanced SLNs (Liu, et al. 2014). Secondary LNs detected using CEUS were nearly five times more likely to contain melanoma metastases than those removed using radical LN dissection. Recently, Nam et al., investigated the performance of molecular US imaging with dual-targeted MBs to detect melanoma metastases in SLNs in a swine melanoma model [START_REF] Nam | Sentinel Lymph Node Characterization with a Dual-Targeted Molecular Ultrasound Contrast Agent[END_REF][START_REF] Nam | Performance of Molecular Lymphosonography for Detection and Quantification of Metastatic Involvement in Sentinel Lymph Nodes[END_REF]).

These MBs were functionalized with V 3 integrin and P-selectin antibodies and injected intravenously. They reported that molecular US imaging had higher sensitivity (91% versus 80%) and specificity (67% versus 54%) than B-mode imaging for the diagnosis of metastatic LNs [START_REF] Nam | Performance of Molecular Lymphosonography for Detection and Quantification of Metastatic Involvement in Sentinel Lymph Nodes[END_REF]. The mean contrast intensity from the dual-targeted MBs for metastatic SLNs was 6-fold higher than that of benign LNs, whereas control MBs (i.e., IgG-labeled MBs)

showed no significant difference in contrast intensity between metastatic and benign LNs [START_REF] Nam | Sentinel Lymph Node Characterization with a Dual-Targeted Molecular Ultrasound Contrast Agent[END_REF].

Despite the high sensitivity of CEUS for SLN detection in swine melanoma models, the first clinical study showed contradictory results. [START_REF] Schmid-Wendtner | Improved differentiation of benign and malignant lymphadenopathy in patients with cutaneous melanoma by contrast-enhanced color Doppler sonography[END_REF]. CEUS revealed perfusion primarily in peripheral areas of LN metastases or in a mixed pattern with high perfusion in one region and lack of perfusion in another.

However, Nielsen et al., reported that SLNs were visualized only in 1 patient out of 10 with melanoma, thus suggesting that CEUS might not be used for SLN detection in clinics [START_REF] Nielsen | Sentinel node detection in melanomas using contrast-enhanced ultrasound[END_REF]. They explained the following: i) sows and humans have different LN structures and afferent lymphatic vessels, and ii) the echogenicity of the hilum is more hyperechoic in humans than in sows. Consequently, it is difficult to determine whether this hyperechogenicity is physiologically normal or induced by UCAs; iii) Sonazoid ® [START_REF] Goldberg | Contrast-enhanced ultrasound imaging of sentinel lymph nodes after peritumoral administration of Sonazoid in a melanoma tumor animal model[END_REF][START_REF] Goldberg | Sentinel lymph nodes in a swine model with melanoma: contrast-enhanced lymphatic US[END_REF]) and Levovist ® (Schmid-Wendtner, et al. 2002) fill the LNs more efficiently than SonoVue ® thus providing higher contrast to the surrounding tissues [START_REF] Nielsen | Sentinel node detection in melanomas using contrast-enhanced ultrasound[END_REF]. In this analysis, the administration ways of UCAs are not discussed. Indeed, UCAs were injected peritumorally in swine melanoma models [START_REF] Goldberg | Contrast-enhanced ultrasound imaging of sentinel lymph nodes after peritumoral administration of Sonazoid in a melanoma tumor animal model[END_REF][START_REF] Goldberg | Sentinel lymph nodes in a swine model with melanoma: contrast-enhanced lymphatic US[END_REF]) while they are administered either subcutaneously [START_REF] Nielsen | Sentinel node detection in melanomas using contrast-enhanced ultrasound[END_REF] or intravenously [START_REF] Schmid-Wendtner | Improved differentiation of benign and malignant lymphadenopathy in patients with cutaneous melanoma by contrast-enhanced color Doppler sonography[END_REF]) in clinical trials. The contradictory results observed between preclinical and clinical studies could be also explained by the different administration ways of UCAs used in both type of studies. Other clinical investigations have been conducted to assess the performance of CEUS for SLN detection in melanoma patients after an i.v. injection of UCAs.

De Giorgi et al., found that CEUS showed a negative predictive value of 100% in a sample of 15 patients with melanoma [START_REF] De Giorgi | Contrast-enhanced ultrasound: a filter role in AJCC stage I/II melanoma patients[END_REF]. These data indicate that all negative results were confirmed by negative SLN histopathological analysis; thus, the SLNs were nonmetastatic. Nevertheless, CEUS has a high false positive rate for SLN detection. This modality identified eight out of 13 histologically confirmed negative SLNs, with a specificity of 61.5%. On CEUS, these five false-positive SLNs were categorized as suspicious (4/5) or positive (1/5). This investigation is the first to report a sensitivity of 100% and an accuracy of 73.7% for CEUS in diagnosing metastases in the SLNs of melanoma patients [START_REF] De Giorgi | Contrast-enhanced ultrasound: a filter role in AJCC stage I/II melanoma patients[END_REF]). In addition, Rubaltelli et al., performed CEUS examinations in a series of 436 melanoma patients after standard US imaging to assess the significance of focal thickening of the superficial LNs in patients with cutaneous melanoma [START_REF] Rubaltelli | Contrast-enhanced ultrasound for characterizing lymph nodes with focal cortical thickening in patients with cutaneous melanoma[END_REF]. The CEUS findings were compared with the results of fine-needle aspiration cytology (FNAC). CEUS correctly categorized 42 of 44 LNs showing focal cortical thickening with a sensitivity of 100%; more importantly, the specificity was 99.5 %. This sensitivity was higher than that of standard US imaging (93%). In 29 nodes, the area of focal thickening displayed a contrast enhancement comparable to that of the remaining cortex. In 15 nodes, this area was less perfused than the adjacent parenchyma during the arterial phase. Some areas showed perfusion defects during the parenchymal phase. Their results showed 13 true positives, 31 false positives, and no false negatives. FNAC should only be performed in 15 of 44 patients and avoided in 29 patients with negative results on both CEUS and histopathological examinations. Two years later, Rubaltelli et al., investigated the performance of CEUS in the differential diagnosis of benign and malignant LNs based on the enhancement in the blood stream pattern on CEUS [START_REF] Rubaltelli | Potential use of contrast-enhanced ultrasound (CEUS) in the detection of metastatic superficial lymph nodes in melanoma patients[END_REF]. In 47 of 540 patients, US examination of LNs revealed an absence of echogenic hilus, round shape, and peripheral capsular vascularity. CEUS revealed altered blood perfusion in the LNs. US-FNAC confirmed the presence of LN metastasis in these patients. In 442 of 540 patients, the size, morphology, and vascularity of the LNs were characteristic of benign LNs.

In 51 of 540 patients, standard US imaging showed LNs with focal cortical lobulation. On CEUS imaging, no perfusion defect was observed in 26 of the 51 patients, and US-FNAC revealed the absence of metastases in the LNs. In 25 of the 51 patients, CEUS showed defects in the blood perfusion of the LNs. Using US-FNAC, the presence of metastases in the LNs was confirmed in 19 of 25 patients and was excluded in 6 of 25 patients. Considering the morphological parameters (i.e., echogenic hilus, round shape, and peripheral capsular vascularity), standard US imaging had a sensitivity, specificity, and accuracy of 71%, 100%, and 97%, respectively. This imaging modality showed 19 false negatives and no false positives.

If the presence of focal cortical thickening was considered as a malignancy criterion, the sensitivity, specificity, and accuracy were then 100%, 93%, and 94%, respectively. This modality yielded 32 false positives and no false negatives. However, when CEUS was combined with standard US imaging to examine LNs with focal cortical thickening, the sensitivity and the specificity were 98% and 99%; more importantly, the accuracy was 99%.

The combination of both imaging modalities accurately categorized 534 of 540 melanoma patients, but revealed six false positives and no false negatives. The likelihood ratios established the good performance of the imaging modalities exploited in this clinical trial and the superiority of CEUS in comparison with the recommended values of above 10 and below 0.1 for positive and negative likelihood ratios, respectively.

Altogether, these preclinical and clinical data suggest that CEUS (with bare or targeted MBs) is a promising imaging modality for specifically detecting metastatic SLNs and secondary LNs and quantifying the degree of metastatic involvement.

Liver

The liver is the main solid organ in the abdomen where melanoma metastases have been discovered. Contrast-enhanced CT is the most common imaging modality used to examine liver metastases [START_REF] Barat | MRI and PET/CT features of abdominal manifestations of cutaneous melanoma: a review of current concepts in the era of tumor-specific therapies[END_REF]). However, MRI has been described to be more sensitive than CT for the detection of liver metastases [START_REF] Sofue | MR imaging of hepatic metastasis in patients with malignant melanoma: evaluation of suspected lesions screened at contrast-enhanced CT[END_REF]. Although US and PET imaging may also help to examine liver metastases, both modalities are reported to be less sensitive than CT and MRI because of operator dependence and the low spatial resolution of US [START_REF] Chang | Metastatic melanoma in the chest and abdomen: the great radiologic imitator[END_REF]) and PET imaging [START_REF] Barat | MRI and PET/CT features of abdominal manifestations of cutaneous melanoma: a review of current concepts in the era of tumor-specific therapies[END_REF], respectively. Liver metastases are mostly welldefined and hypoechoic lesions observed on US imaging. However, diffuse hepatic infiltrations of melanoma metastases, as well as very large metastatic lesions, can induce a heterogeneous echotexture with diffusely increased echogenicity, suggesting hepatocellular carcinoma or cirrhosis [START_REF] Velazquez | Diffuse Hepatic Infiltration by Metastatic Melanoma[END_REF]. As previously described [START_REF] Lu | Value of Perfusion Parameters for Differentiating Hepatocellular Carcinoma and Liver Metastasis With Hypervascularity and a Normal Hepatic Background on Contrast-Enhanced Ultrasound Imaging[END_REF], CEUS is an effective modality for the detection and characterization of liver metastases, which are smaller in size and more frequent than B-mode imaging. CEUS displayed a transient and homogenous arterial contrast enhancement in melanoma liver metastases, followed by rapid and complete wash-out initiated within the conventional arterial phase [START_REF] Murphy-Lavallee | Are metastases really hypovascular in the arterial phase? The perspective based on contrast-enhanced ultrasonography[END_REF]). This enhancement pattern was similar to that obtained using MRI and CT imaging. In addition, malignant liver lesions are hypoechoic in the late contrast phase on CEUS compared to healthy liver tissue, whereas benign lesions appear isoechoic or hyperechoic [START_REF] Quaia | Characterization of focal liver lesions with contrast-specific US modes and a sulfur hexafluoride-filled microbubble contrast agent: diagnostic performance and confidence[END_REF][START_REF] Wildner | Differentiation of malignant liver tumors by software-based perfusion quantification with dynamic contrast-enhanced ultrasound (DCEUS)[END_REF]. Corvino et al., reported that CEUS is a promising imaging modality for diagnosing metastatic cystic liver lesions, especially melanoma [START_REF] Corvino | Rectal melanoma presenting as a solitary complex cystic liver lesion: role of contrast-specific low-MI real-time ultrasound imaging[END_REF]. Such lesions are rare, and their differential diagnosis is not easy compared to other liver metastases.

They described the case of a 41-year-old patient with a rectal melanoma with solitary complex cystic liver metastasis. CEUS revealed some distinctive features of healthy liver parenchyma.

This diagnosis was confirmed using ultrasound-guided FNAC. CEUS made it possible to establish a correct and early diagnosis and to provide appropriate management for the patient.

Gallbladder

With 50% 65% of all metastatic lesions involving the gallbladder, melanoma is the most common cause of gallbladder metastases [START_REF] Backman | Metastases of malignant melanoma in the gastrointestinal tract[END_REF]. Because such metastases are rare and frequently asymptomatic or rarely associated with obstructive symptoms or acute cholecystitis, their clinical diagnosis can be challenging [START_REF] Chang | Metastatic melanoma in the chest and abdomen: the great radiologic imitator[END_REF]. Clinicians or radiologists can confuse tumor nodules with gallbladder sludge or pay little attention to them [START_REF] Barretta | Gallbladder metastasis: spectrum of imaging findings[END_REF]. Although most patients with gallbladder metastases have widespread cancer and poor survival, the gallbladder may represent the first metastatic site in a few patients [START_REF] Barretta | Gallbladder metastasis: spectrum of imaging findings[END_REF]). These rare cases can be treated surgically if they are diagnosed correctly at an early stage [START_REF] Katz | Surgical management of melanoma of the gallbladder: a report of 13 cases and review of the literature[END_REF]). As previously reported, US imaging is an effective modality for detecting and characterizing gallbladder metastases [START_REF] Holloway | Ultrasound diagnosis of metastatic melanoma of the gallbladder[END_REF]. These metastases appear as single or multiple moderate/hyperechoic lesions (> 1 cm in diameter) attached to the gallbladder wall. These nodules display minimal to absent posterior acoustic shadowing. In addition, gallbladder wall thickening is diffuse in cases of tumor infiltration or associated cholecystitis. Gallbladder metastases are known to be perfused. Indeed, Barretta et al., reported that CEUS showed early, intense, and diffuse contrast enhancement of metastatic lesions in eight patients with primary melanoma gallbladder metastases [START_REF] Barretta | Gallbladder metastasis: spectrum of imaging findings[END_REF].

This enhancement gradually decreased over several minutes after i.v. injection of UCAs.

Andreano et al., described a rapid, intense, but heterogeneous contrast enhancement during the arterial phase and a very rapid wash-out at 60 s after i.v. contrast administration to be indicative of a malignant lesion (Figure 5) [START_REF] Andreano | Asymptomatic metastatic melanoma of the gallbladder diagnosed with contrast-enhanced ultrasonography[END_REF]). In addition, Barretta et al., demonstrated that color Doppler imaging failed to detect any blood perfusion in the gallbladder metastases of 5/11 patients with primary melanoma gallbladder metastases [START_REF] Barretta | Gallbladder metastasis: spectrum of imaging findings[END_REF]. In three of these patients, CEUS showed a significant contrast enhancement of the metastatic lesions, suggesting that these lesions were indeed perfused. The detection of flow signals in color Doppler after i.v. contrast injection in two of these three patients confirmed that CEUS is more sensitive than color Doppler. Overall, these clinical results demonstrate that CEUS is a complementary US imaging modality for precisely identifying metastatic gallbladder metastases.

CEUS-guided interventions

As previously described, US-guided biopsy (e.g., core needle biopsy and SLNB) is an accurate and safe method for obtaining tissue samples for the histopathological diagnosis of melanoma ( [START_REF] Lekht | Versatile utilization of real-time intraoperative contrast-enhanced ultrasound in cranial neurosurgery: technical note and retrospective case series[END_REF]. This lesion showed a realtime dynamic tumor perfusion pattern that was inconsistent with the previously described patterns of brain tumors. After resection of the lesion, CEUS detected a residual tumor in an unsafe brain location for additional resection. In this clinical study, intraoperative CEUS assisted maximal tumor resection with no intraoperative complications. In agreement with previous studies [START_REF] Prada | Intraoperative contrast-enhanced ultrasound for brain tumor surgery[END_REF]), this study confirmed that intraoperative CEUS is a safe, real-time, and dynamic contrast-based imaging technique to guide surgery and biopsy in neurooncology. Further prospective investigations should determine the place of this modality in the diagnostic arsenal and therapeutic management of melanoma.

CEUS monitoring of therapeutic follow-up

Since the development of new targeted therapies (i.e., therapeutic antibody, tyrosine kinase inhibitors, etc.) and US therapies (i.e., high-intensity focused ultrasound, antivascular ultrasound therapy, etc.), it has been clearly established that the assessment of morphological tumor criteria (e.g., tumor dimensions and volume) alone cannot determine the early therapeutic efficacy of these treatments [START_REF] Lassau | Dynamic contrast-enhanced ultrasonography (DCE-US): a new tool for the early evaluation of antiangiogenic treatment[END_REF]. Indeed, morphological evaluation of tumors does not reveal early physiological changes in tumors. Some preclinical and clinical studies have reported that melanoma is susceptible to treatment with antiangiogenic drugs (e.g., sorafenib, bevacizumab, and imatinib) [START_REF] Flaherty | Phase I/II Trial of Imatinib and Bevacizumab in Patients With Advanced Melanoma and Other Advanced Cancers[END_REF], Forsberg, et al. 2014, Leguerney, et al. 2012[START_REF] Seiler | Dose-response relationship of ultrasound contrast agent in an in vivo murine melanoma model[END_REF] or with therapeutic US [START_REF] Rouffiac | Combination of HIFU therapy with contrast-enhanced sonography for quantitative assessment of therapeutic efficiency on tumor grafted mice[END_REF][START_REF] Wood | Acute increases in murine tumor echogenicity after antivascular ultrasound therapy: a pilot preclinical study[END_REF], which leads to a decrease in tumor angiogenesis, followed by tumor hypoxia and cell death. As previously reported [START_REF] Ferrara | Evaluation of tumor angiogenesis with US: imaging, Doppler, and contrast agents[END_REF], Doppler imaging is not very sensitive and accurate for assessing the effects of these treatments on slow blood flow in tumor vessels. However, CEUS allows the visualization and quantification of tissue vascularity with high sensitivity [START_REF] Escudier | Phase I trial of sorafenib in combination with IFN alpha-2a in patients with unresectable and/or metastatic renal cell carcinoma or malignant melanoma[END_REF][START_REF] Lavisse | Early quantitative evaluation of a tumor vasculature disruptive agent AVE8062 using dynamic contrast-enhanced ultrasonography[END_REF].

The contribution of CEUS imaging to targeted treatment assessment has been extensively described and discussed by Lassau et al., highlighting CEUS as a powerful and relevant functional modality for monitoring targeted treatments (Table 2) [START_REF] Escudier | Phase I trial of sorafenib in combination with IFN alpha-2a in patients with unresectable and/or metastatic renal cell carcinoma or malignant melanoma[END_REF][START_REF] Lavisse | Early quantitative evaluation of a tumor vasculature disruptive agent AVE8062 using dynamic contrast-enhanced ultrasonography[END_REF], Leguerney, et al. 2012). Indeed, CEUS enables the detection of tumor microvessels and the quantitative evaluation of tumor perfusion using raw linear data. This modality allows the calculation of perfusion parameters, including blood flow and blood volume, which correspond to the slope of wash-in and the area under the time intensity curve (AUC) on the time intensity curve, respectively. Thus, Lavisse et al., investigated the early tumor vasculature disrupting effects of a pharmacological agent, AVE8062, using CEUS imaging in a murine B16F10 melanoma model [START_REF] Lavisse | Early quantitative evaluation of a tumor vasculature disruptive agent AVE8062 using dynamic contrast-enhanced ultrasonography[END_REF]. They reported that DCEUS allowed for an effective and quantitative assessment of the anti-angiogenic effects of AVE8062. Variations in tumor perfusion parameters were positively correlated with changes in microvascular density. Similarly, Forsberg et al., described that CEUS is a sensitive modality for investigating the functional effects of VEGF traps on tumor neovascularity in a DB-1 human melanoma xenograft model (Forsberg, et al. 2014). A strong correlation between DCEUS and the histological measures of angiogenesis has also been reported. These preclinical data are supported by clinical trials. Lassau et al., demonstrated that a decrease in tumor angiogenesis can be discerned and measured in responders after one or two treatment weeks (Lassau, et al. 2007[START_REF] Lassau | Dynamic contrast-enhanced ultrasonography (DCE-US): a new tool for the early evaluation of antiangiogenic treatment[END_REF]). This decrease is generally correlated with progression-free and overall survival in renal cell and hepatocellular carcinomas. [START_REF] Lassau | Validation of dynamic contrast-enhanced ultrasound in predicting outcomes of antiangiogenic therapy for solid tumors: the French multicenter support for innovative and expensive techniques study[END_REF]. In this clinical study, they performed 1968 CEUS examinations in 539 patients, including 55 patients with melanoma.

They demonstrated that CEUS provides a predictive biomarker of tumor progression related to blood volume, termed as the change in AUC from baseline to day 30. Indeed, patients with a greater than 40% decrease in AUC on day 30 showed better freedom from progression and overall survival. These data suggest that this biomarker should be considered for the early evaluation of anti-angiogenic treatment of melanoma.

In conclusion, preclinical studies have clearly demonstrated that CEUS is a relevant diagnostic tool for assessing the early effects of antiangiogenic treatments in melanoma.

However, further investigations are required to demonstrate its clinical relevance.

DRUG DELIVERY USING MICROBUBBLE-ASSISTED ULTRASOUND

Thus, MB-assisted US (i.e., sonoporation or sonopermeabilization) is a noninvasive and local drug delivery method that offers great promise for improving the therapeutic efficacy of a wide range of therapeutic molecules (e.g., chemotherapeutics, antibodies, recombinant peptides/proteins, and nucleic acids) by enhancing their bioavailability in tumor tissues and reducing their systemic side effects on healthy tissues [START_REF] Kooiman | Ultrasound-Responsive Cavitation Nuclei for Therapy and Drug Delivery[END_REF]. For their in-vivo delivery, these molecules are either co-administered with MBs or loaded on or into MBs [START_REF] Kooiman | Ultrasound-Responsive Cavitation Nuclei for Therapy and Drug Delivery[END_REF]. After in in-vivo administration and sufficient accumulation of therapeutic molecules and MBs in the tumor microvasculature, the tumor tissue is exposed to US waves [START_REF] Presset | Endothelial Cells, First Target of Drug Delivery Using Microbubble-Assisted Ultrasound[END_REF]. Under the action of US, volumetric oscillations of MBs induce several local acoustic phenomena close to the endothelial wall of tumor vessels, which results

in their transient permeabilization [START_REF] Presset | Endothelial Cells, First Target of Drug Delivery Using Microbubble-Assisted Ultrasound[END_REF]. This vascular permeabilization significantly enhances extravasation, penetration, and retention of therapeutic molecules in tumor tissues [START_REF] Kooiman | Ultrasound-Responsive Cavitation Nuclei for Therapy and Drug Delivery[END_REF][START_REF] Presset | Endothelial Cells, First Target of Drug Delivery Using Microbubble-Assisted Ultrasound[END_REF]). Thus, MB-assisted US is an easy-touse and cost-effective delivery method for therapeutic molecules, including chemotherapeutic drugs [START_REF] Baghirov | Ultrasound-mediated delivery and distribution of polymeric nanoparticles in the normal brain parenchyma of a metastatic brain tumour model[END_REF][START_REF] Prasad | Ultrasound-Triggered Spatiotemporal Delivery of Topotecan and Curcumin as Combination Therapy for Cancer[END_REF][START_REF] Prasad | Curcumin Encapsulated Lecithin Nanoemulsions: An Oral Platform for Ultrasound Mediated Spatiotemporal Delivery of Curcumin to the Tumor[END_REF], photothermal agents [START_REF] Hu | Opto-acoustic synergistic irradiation for vaporization of natural melanin-cored nanodroplets at safe energy levels and efficient sono-chemo-photothermal cancer therapy[END_REF], Liu, et al. 2019[START_REF] Wang | Synergistic delivery of gold nanorods using multifunctional microbubbles for enhanced plasmonic photothermal therapy[END_REF], melanoma-associated antigens (Ags), and immunomodulatory molecules [START_REF] Dewitte | The potential of antigen and TriMix sonoporation using mRNA-loaded microbubbles for ultrasound-triggered cancer immunotherapy[END_REF][START_REF] Gao | Gasfilled ultrasound microbubbles enhance the immunoactivity of the HSP70MAGEA1 fusion protein against MAGEA1expressing tumours[END_REF][START_REF] Oda | Prophylactic immunization with Bubble liposomes and ultrasound-treated dendritic cells provided a four-fold decrease in the frequency of melanoma lung metastasis[END_REF]) into melanoma. In addition, this method may be used to treat primary cutaneous melanoma as well as metastases located in deep tissues under the guidance of US imaging [START_REF] Sennoga | Microbubble-mediated ultrasound drug-delivery and therapeutic monitoring[END_REF].

In this section, we provide only a survey of preclinical studies exploring the therapeutic efficacy and safety of delivery of therapeutic molecules using MB-assisted US for the treatment of melanoma because no clinical investigation has been conducted to the best of our knowledge.

Chemotherapy

Conventional anti-cancer chemotherapy is ineffective in treating advanced melanoma. Since 2006, some investigations have been performed to design therapeutic protocols for acoustically mediated delivery of free or encapsulated forms of chemotherapeutics in mouse models of primary tumors and metastases of melanoma [START_REF] Abdalkader | Evaluation of the potential of doxorubicin loaded microbubbles as a theranostic modality using a murine tumor model[END_REF][START_REF] Prasad | Ultrasound-Triggered Spatiotemporal Delivery of Topotecan and Curcumin as Combination Therapy for Cancer[END_REF][START_REF] Tartis | Therapeutic effects of paclitaxel-containing ultrasound contrast agents[END_REF]) (Table 3). Among these anti-cancer drugs, doxorubicin (DOX) is one of the most potent and is administered alone or in combination with other drugs for the treatment of solid tumors [START_REF] Carvalho | Doxorubicin: the good, the bad and the ugly effect[END_REF]). However, its clinical use is severely limited owing to its systemic side effects and, specifically, its cardiotoxicity. To overcome this issue, DOX was encapsulated inside PEGylated liposomes (Doxil ® or Caelyx ® ) [START_REF] Barenholz | Doxil(R)--the first FDA-approved nano-drug: lessons learned[END_REF]).

These DOX liposomal formulations passively accumulate inside the tumor through enhanced permeability and retention effects [START_REF] Barenholz | Doxil(R)--the first FDA-approved nano-drug: lessons learned[END_REF] [START_REF] Abdalkader | Evaluation of the potential of doxorubicin loaded microbubbles as a theranostic modality using a murine tumor model[END_REF]. Nine days after tumor inoculation, the tumors were treated three times over 2 d intervals. Mice received an i.v. injection of DOX-loaded MBs (70 g DOX/330 g lipid in 200 L of 5% glucose solution), and the tumors were immediately exposed to US for 60 s. The results showed that acoustically mediated delivery using DOX-loaded MBs, as compared to DOX-loaded MB treatment alone (i.e., without US exposure; 0.100 g/g tumor tissue), significantly enhanced the i.t. DOX dose (0.536 g/g tumor tissue) (Figure 6A).

Because cardiotoxicity is a major side effect of DOX, the authors assessed the DOX dose in the heart tissue. Surprisingly, there was no significant change in DOX doses in the heart and liver tissues between the experimental groups (Figure 6B-C). Unfortunately, they did not assess the levels of creatine kinase, alanine, and aspartate aminotransferases to evaluate the effects of this treatment on heart and liver functions. In addition, acoustically mediated DOX delivery using DOX-loaded MBs significantly induced a reduction in tumor volume (2453.9 175 mm 3 ) by day 27 after tumor inoculation compared with no treatment (7156.2 1384 mm 3 ) and DOXloaded MB treatment alone (i.e., without US exposure; 4663.2 454 mm 3 ) (Figure 6D).

Furthermore, treatment with DOX-loaded MBs in combination with US exposure did not induce any significant loss in body weight relative to no treatment or DOX-loaded MB treatment alone, suggesting that this treatment was well tolerated (Figure 6E). Altogether, these results demonstrate that the combination of DOX-loaded MBs with US is an efficient and safe strategy for delivering DOX to primary melanoma tumors.

Moreover, Prasad et al., developed different formulations to deliver or co-deliver curcumin (Cur) using MB-assisted US into melanoma (Prasad andBanerjee 2019, Prasad, et al. 2020).

Cur is a plant-derived anticancer agent with negligible side effects. Preclinical studies have reported that the high therapeutic efficacy of Cur requires higher doses because of its low solubility and bioavailability [START_REF] Heger | The molecular basis for the pharmacokinetics and pharmacodynamics of curcumin and its metabolites in relation to cancer[END_REF]). To overcome these issues, Cur has been survival rate of 93.7% and 26.2%, respectively. Surprisingly, the authors did not report the effects of Tpt/Cur codelivery using Tpt/Cur-loaded MBs in combination with US on mouse survival. However, this preclinical study is the first to demonstrate the potential of MB-assisted US to co-deliver more than one anticancer drug for the treatment of melanoma.

In conclusion, preclinical studies have successfully reported the therapeutic benefits of MBassisted US in the delivery of one or more chemotherapeutics in s.c. melanoma murine models.

These findings should be confirmed in small-and large-animal models of primary tumors and metastases of melanoma. In addition, the contribution of host immunity to the tumor response to chemotherapy after acoustically mediated drug delivery is still lacking and needs to be investigated in complementary studies using syngeneic animal models of melanoma.

Photothermal therapy

Photothermal therapy relies on the use of a femtosecond pulse laser to cause irreversible thermal damage to tumor tissues [START_REF] Li | Clinical development and potential of photothermal and photodynamic therapies for cancer[END_REF]. This therapeutic modality is a promising and noninvasive strategy for the treatment of isolated cutaneous melanoma (Liu, et al. 2019).

However, conventional laser irradiation cannot treat deep tumor tissues and generally induces thermal damage to the healthy tissues surrounding the tumor lesion [START_REF] Li | Clinical development and potential of photothermal and photodynamic therapies for cancer[END_REF]). To solve this problem, photothermal agents (e.g., indocyanine green, IR780 dye, gold nanorods, melanin) are loaded in acoustically responsive carriers (e.g., MBs, nanodroplets) to enhance the delivery of these agents into melanoma after US exposure (Table 4). The accumulation of such carriers can be significantly increased in the melanoma microvasculature by functionalizing these carriers with targeting agents (e.g., cyclic arginine-glycine-asparagine, antiVEGFR2 antibody), which bind overexpressed endothelial biomarkers (e.g., integrin V 3, VEGFR2).

This enhanced i.t. bioavailability of photothermal agents improves the thermal efficacy of the pulse laser, thus irreversibly ablating superficial melanoma [START_REF] Hu | Opto-acoustic synergistic irradiation for vaporization of natural melanin-cored nanodroplets at safe energy levels and efficient sono-chemo-photothermal cancer therapy[END_REF][START_REF] Wang | Synergistic delivery of gold nanorods using multifunctional microbubbles for enhanced plasmonic photothermal therapy[END_REF]).

Wang et al., loaded photoabsorbing agents, namely gold nanorods, in VEGFR2-targeted

MBs to effectively deliver these agents into melanoma cells using MB-assisted US, thus improving the efficacy of plasmonic photothermal therapy [START_REF] Wang | Synergistic delivery of gold nanorods using multifunctional microbubbles for enhanced plasmonic photothermal therapy[END_REF] [START_REF] Wang | Synergistic delivery of gold nanorods using multifunctional microbubbles for enhanced plasmonic photothermal therapy[END_REF]. Moreover, Hu et al., designed melanin-cored (i.e., photoabsorbing agent) and DOX-loaded (i.e., chemotherapeutics) perfluoropentane nanodroplets for the photothermal therapy of melanoma in a mouse model (A375) [START_REF] Hu | Opto-acoustic synergistic irradiation for vaporization of natural melanin-cored nanodroplets at safe energy levels and efficient sono-chemo-photothermal cancer therapy[END_REF]). After their i.v. or i.t. injection (200 L containing 10 mg/mL melanin and 110 g/mL DOX), the tumor tissue and, therefore, circulating nanodroplets were exposed to an optoacoustic synergistic irradiation (i.e., laser at 1 W.cm -2 ; US at 1.1 MHz, 1 Hz PRF, 5 cycle/pulse, 0.82 MPa PNP, for 2 min). This method vaporized these nanodroplets into MBs, which cavitated to increase the permeability of the tumor microvasculature, thus enhancing the i.t. delivery of melanin and DOX. These molecules mediated a secondary chemo-photothermal therapy under subsequent laser irradiation (1 W.cm -2 for 5 min; 24 or 5 h after i.v. or i.t.

injection of nanodroplets, respectively), which efficiently eradicated the melanoma. These investigations show that photothermal therapy in combination with or without chemotherapy could have great potential for the treatment of melanoma. Nevertheless, additional studies must be performed in small and large animals to confirm the therapeutic benefits of this therapy in melanoma treatment.

Immunotherapy and Vaccination

Immunotherapy and vaccination are promising therapeutic modalities for producing potent and long-lasting immunizations against melanoma [START_REF] Escoffre | Bubble-Assisted Ultrasound: Application in Immunotherapy and Vaccination[END_REF]. Immunotherapy stimulates the immune response in melanoma patients by administering immunomodulating molecules (i.e., interferons, interleukins, antiPD1/PDL1, and antiCTLA4 antibodies) (Yamaguchi, et al. 2011). Moreover, prophylactic and therapeutic vaccines have been designed

and evaluated for melanoma treatment. Prophylactic immunization relies on the delivery of melanoma-associated antigens (MAA) in healthy patients to induce a rapid and efficient immune response during disease occurrence [START_REF] Un | Suppression of melanoma growth and metastasis by DNA vaccination using an ultrasound-responsive and mannose-modified gene carrier[END_REF]). In addition, as an immunotherapy, the therapeutic vaccine aims to stimulate the immune response in diseased patients by delivering MAA [START_REF] Gao | Gasfilled ultrasound microbubbles enhance the immunoactivity of the HSP70MAGEA1 fusion protein against MAGEA1expressing tumours[END_REF][START_REF] Oda | Prophylactic immunization with Bubble liposomes and ultrasound-treated dendritic cells provided a four-fold decrease in the frequency of melanoma lung metastasis[END_REF]. Immunomodulating molecules and MAA can be administered either as recombinant peptides/proteins or as encoded by plasmid DNA (pDNA) or messenger RNA (mRNA). Nevertheless, the pharmacological, immunological, and physicochemical properties of these molecules limit their access to target tissues (e.g., immune cells, LN, skin, skeletal muscle, and melanoma), thus reducing their therapeutic efficacy [START_REF] Escoffre | Bubble-Assisted Ultrasound: Application in Immunotherapy and Vaccination[END_REF]. As previously described for chemotherapy, the clinical development of anti-cancer immunotherapy and vaccination requires the design of a safe and efficient method for the delivery of these molecules. In the remainder of this section, we demonstrate the potential of MB-assisted US for the delivery of these molecules for the treatment of melanoma (Table 5).

Cytokine pDNA-based immunotherapy

Cytokine pDNA-based immunotherapy (e.g., interferon-, interleukin-2, -12 and -27, etc.) is a promising strategy for inducing long-lasting antitumor immunity against primary and metastatic cancer diseases [START_REF] Escoffre | Bubble-Assisted Ultrasound: Application in Immunotherapy and Vaccination[END_REF]. The delivery of cytokine pDNA can result in complete tumor regression, inhibition of metastasis, and increase in survival. This strategy is well tolerated in comparison to immunotherapy based on the use of recombinant cytokines.

Indeed, Yamaguchi et al., reported the therapeutic effects of the delivery of interferon-pDNA using MB-assisted US (1 MHz, 0.5 Hz PRF, 50% DC, 0.22 W.cm -2 , for 3 min; i.t. injection of 100 g pDNA/100 L Sonazoid MBs) in a melanoma mouse model (C32) (Yamaguchi, et al. 2011). Two weeks after tumor inoculation, the tumors were treated once a week for four weeks.

This strategy led to a 2-fold decrease in tumor growth compared to unsonicated tumors.

Histopathological examination of tumor tissues (i.e., terminal deoxynucleotidyl transferase (TDT)-mediated dUT nick end labeling assay and immunohistochemistry against interferonand caspase-3) confirmed the therapeutic efficacy of this immunotherapy. Although these results may demonstrate that cytokine pDNA-based immunotherapy has great potential for the treatment of melanoma, regrettably, the delivery of cytokine pDNA using MB-assisted US has been poorly investigated for the treatment of melanoma, whereas it has been described for the treatment of other types of cancers (Suzuki, et al. 2010[START_REF] Zolochevska | Sonoporation delivery of interleukin-27 gene therapy efficiently reduces prostate tumor cell growth in vivo[END_REF].

Recombinant vaccines

The development of antitumor vaccines has become a major research topic in the field of cancer immunotherapy. Tumor-specific antigens are the main components of tumor vaccines. They induce specific Th-1 and Th-2 immune responses to inhibit tumor occurrence and development.

Melanoma-associated antigen A1 (MAGEA1), gp100, melanoma antigen recognized by Th-1 cells (MART-1), and tyrosinase-related protein (TRP) are the main MAA exploited in recombinant vaccines [START_REF] Gao | Gasfilled ultrasound microbubbles enhance the immunoactivity of the HSP70MAGEA1 fusion protein against MAGEA1expressing tumours[END_REF][START_REF] Un | Suppression of melanoma growth and metastasis by DNA vaccination using an ultrasound-responsive and mannose-modified gene carrier[END_REF]. MAGEA1 is processed and presented by major histocompatibility complex-1 (MHC-1) molecules on cancer cells and induces a Th-1 immune response against MAGEA1-expressing cancer cells. However, MAGEA1-based tumor vaccines do not show significant clinical effects on cancer [START_REF] Slingluff | Helper T-cell responses and clinical activity of a melanoma vaccine with multiple peptides from MAGE and melanocytic differentiation antigens[END_REF]). To overcome this issue, MAGEA1 is fused with heat shock protein 70 (HSP70) to improve the processing and presentation of MAGEA1, thereby significantly enhancing Th-1 and Th-2 immune responses against melanoma. Gao et al., proposed the delivery of this HSP70/MAGEA1 fusion protein using MB-assisted US (MI 0.75; s.c. injection of 10 g/100 L/mouse of HSP70/MAGEA1-loaded MBs) in a mouse melanoma model (B16-MAGEA1) [START_REF] Gao | Gasfilled ultrasound microbubbles enhance the immunoactivity of the HSP70MAGEA1 fusion protein against MAGEA1expressing tumours[END_REF]). To achieve this goal, HSP70/MAGEA1-loaded MBs were developed. One Based on these immunological properties, DC-based vaccines have been developed for melanoma treatment [START_REF] Abbas | Basic immunology: functions and disorders of the immune system[END_REF]. Briefly, the DC precursors were purified from the patient after leukapheresis. Incubation of these precursors with different growth factors and cytokines (e.g., granulocyte-macrophage colony-stimulating factor, and interleukin 4) generates immature DCs. Subsequently, DC maturation occurs when these cells are incubated with immunostimulatory cytokines (e.g., Tumor necrosis factor , interleukin 6, and FMS-like Tyrosine kinase 3 Ligand) and toll-like agonists (e.g., lipopolysaccharides, CpG, and polyI:C).

Mature DCs were loaded with MAA to generate activated DCs. These fully mature and activated DCs were administered back to the patient. Among the different methods for generating melanoma-specific DCs, bubble-assisted US can be used to deliver MAA and immunomodulating molecules as recombinant proteins/peptides or as encoded by pDNA or mRNA into DCs [START_REF] Escoffre | Bubble-Assisted Ultrasound: Application in Immunotherapy and Vaccination[END_REF]. Oda et al., designed a promising method to deliver directly MAA (50 g) into DCs using US (2 MHz, 10% DC, burst rate 2 Hz, 2 W.cm -2 for 3 10 s, with time interval of 10 s) and bubble liposomes (120 g). This method directly delivered the MAA into the cytosol of DCs with an efficiency of approximately 74%, resulting in the presentation of MHC-I restricted Ags on DCs. To assess the efficacy of in-vivo prophylactic vaccination, mice were immunized twice with mature and activated DCs (1 10 6 cells/100 L) at a one-week interval. B16/BL6 melanoma cells were then injected intravenously to generate lung metastases. DC-based vaccination induced a 4-fold decrease in the frequency of melanoma lung metastases (Figure 7). On the other hand, Dewitte et al., preferred to deliver both Ag (ovalbumin, used as Ag model) and immunomodulating mRNAs (TriMix: caTRL4, CD40-L, CD70) into DCs using MB-assisted US (1 MHz, 2 W.cm -2 , 20% DC, for 30 s; defined volume of MBs containing 10 g ovalbumin (OVA) mRNA/40 g TriMix) [START_REF] Dewitte | The potential of antigen and TriMix sonoporation using mRNA-loaded microbubbles for ultrasound-triggered cancer immunotherapy[END_REF]. They then evaluated the efficacy of this DC-based vaccination in a mouse melanoma model (MO4) that overexpresses OVA. Ten days after s.c. inoculation of melanoma cells, mice were immunized twice by subcutaneously injecting mature and activated DCs (50 L at 2 10 6 cells.mL -1 ) at a one-week interval. These DCs induced a potent Ag-specific T cell response, leading the specific lysis of melanoma cells. As a result, this DC-based vaccination resulted in a significant decrease in tumor growth, which naturally translated into an 82% prolongation of median survival for immunized mice in comparison with naive mice. Importantly, tumor eradication was observed in one-third of immunized mice that showed potent and long-lasting antigen-specific immunological memory. These preclinical studies show that bubble-assisted US is a promising method for delivering MAA and immunomodulating molecules into DCs.

DC-based vaccines are mainly generated ex-vivo from autologous DCs, which must be tailormade for each patient. This process is complex and expensive, which prevents the generalization of immunotherapy. The in-vivo delivery of MAA and immunomodulatory molecules in APCs, including CDs and macrophages, bypasses these procedures [START_REF] Un | Development of an ultrasound-responsive and mannose-modified gene carrier for DNA vaccine therapy[END_REF]. Accordingly, Un et al., developed US-responsive and mannose receptor-targeted bubble lipoplexes that were selective to APC and carried pDNA encoding ubiquitylated MAA (i.e., glycoprotein gp10025-33 and tyrosinase-related protein 2181-188 peptide epitopes) [START_REF] Un | Suppression of melanoma growth and metastasis by DNA vaccination using an ultrasound-responsive and mannose-modified gene carrier[END_REF]. They investigated the efficacy of this strategy to induce a prophylactic immunization (1.045 MHz, 50% DC, 10 Hz burst rate, 1 W.cm -2 , for 2 min; i.v. injection of 400 L bubble lipoplexes/50 g pDNA) against primary and metastatic melanoma in a mouse model (B16BL6). To achieve this objective, the mice were immunized three times with a two-week interval between each immunization. Afterward, the mice were inoculated with melanoma cells either subcutaneously to induce a subcutaneous tumor or intravenously to induce lung metastasis. The secretion of Th1 cytokines and CTL activities were significantly enhanced 10and 15-fold in immunized mice compared to untreated mice at two weeks after the last immunization, respectively. In addition, the authors succeeded in obtaining potent and longlasting (100 d post-tumor inoculation) immunization against primary cutaneous melanoma and melanoma lung metastases. Following investigation of this immunization against primary cutaneous melanoma, a complete tumor rejection was observed in 70% of immunized mice.

These mice showed 80% survival rate at 100 d post-tumor inoculation, whereas no nonimmunized mice survived longer than 45 days post-tumor transplantation. In addition, this immunization significantly suppressed the formation of melanoma lung metastases in mice and induced a survival prolongation of immunized mice (75% survival at 100 d post-tumor inoculation) in comparison with naïve mice (no survival after 35 days post-tumor inoculation).

If this method induces an effective prophylactic immunization, its therapeutic efficacy for established melanomas is insufficient because the tumor growth rate is too fast for therapeutic immunization to be successful. The same research group proposed enhancing the in-vivo transfection efficacy of APCs to increase the efficacy of this therapeutic immunization. They reported that transfection efficacy was significantly increased by transcriptional activation using DOX -loaded liposomes [START_REF] Un | Enhancement of gene expression by transcriptional activation using doxorubicin-loaded liposome/pDNA complexes[END_REF]. Indeed, DOX is known to activate transcription factors, such as nuclear factor kappa-light-chain-enhancer of activated B cells (NF-), thus increasing the transcription of transfected pDNA. Accordingly, they designed an immunization method associating the delivery of DOX-loaded liposomes (200 L at 4 mg/kg b.w.) with APC transfection using mannose-targeted bubble lipoplexes (i.v. injection of 400 L bubble lipoplexes/50 g pDNA encoding gp10025-33 and tyrosinase-related protein 2181-188 peptide epitopes) in combination with US (1.045 MHz, 50% DC, 10 Hz burst rate, 1 W.cm -2 , for 1 min) [START_REF] Un | Suppression of melanoma growth and metastasis by DNA vaccination using an ultrasound-responsive and mannose-modified gene carrier[END_REF]. DOX-loaded liposomes activated transcriptional factors (e.g., NF-B and activated Protein-1) in the spleen, and subsequently, pDNA expression was significantly enhanced in splenic cells. Moreover, this method resulted in an effective CTL response against tumor cells. To investigate the efficacy of this method in primary cutaneous melanoma, DOXloaded liposomes were administered once a week after the s.c. inoculation of melanoma cells (B16BL6) in mice. Therapeutic immunization was performed twice, at 24 h and 2 weeks after the delivery of DOX-loaded liposomes. Only one immunization induced a 7-fold decrease in tumor volume compared to that in the nontreatment group. In addition, the survival of mice immunized twice was significantly prolonged (25% survival at 100 d post-tumor inoculation)

in comparison with that of naïve mice (no survival after 40 d post-tumor inoculation). Using the previously described immunization schedule, mice immunized twice showed a lower number of lung metastases than the naïve mice. Taken together, these results demonstrate that the therapeutic effects of this immunization on established melanoma are significantly enhanced by the delivery of DOX-loaded liposomes.

In conclusion, bubble-assisted US is an effective method for the ex-vivo and in-vivo design of DC-based vaccines against melanoma. Current preclinical studies, mainly conducted in mouse melanoma models, should be confirmed in larger animals in the near future before clinical investigation.

FUTURE PERSPECTIVES AND CONCLUSIONS

Using bare or targeted UCAs enables CEUS to be utilized as a noninvasive and complementary imaging modality to US imaging for the detection and the characterization of primary cutaneous melanoma as well as metastatic melanoma (e.g., LNs, liver and gallbladder). Indeed, highresolution US imaging provides precise morphological (e.g., dimensions and volume) and acoustic (e.g., surface sound reflectance, intralesional sound reflection, and total US attenuation) characteristics, whereas CEUS provides more accurate and sensitive information on melanoma microvasculature than color/power Doppler imaging. Unfortunately, the studies investigating the performance of CEUS for the detection and characterization of primary melanoma are mainly conducted on small-(mouse, rat) and large-(swine) animal models.

Further clinical investigations should be performed to corroborate these preclinical studies and determine the performance of CEUS in a clinical context. Moreover, preclinical and clinical studies have revealed that CEUS is a promising imaging modality to specifically detect metastatic SLNs and secondary LNs and to measure the degree of metastatic involvement. MB-assisted US is also an easy-to-use and cost-effective method for the noninvasive and local delivery of chemotherapeutic drugs, photothermal agents, melanoma-associated agents, and immunomodulating molecules into melanoma, which is achieved by increasing their extravasation and i.t. bioavailability and reducing their systemic side effects on healthy tissues. Metastases showed a moderate to intense contrast enhancement in the arterial phase a followed by a more or less rapid wash-out in the venous phase [START_REF] Corvino | Rectal melanoma presenting as a solitary complex cystic liver lesion: role of contrast-specific low-MI real-time ultrasound imaging[END_REF] Detection of cystic hepatic metastases 1 with rectal melanoma SonoVue UCAs i.v. injection 2.4 mL followed by a bolus injection of saline solution (10 mL) Metastasis revealed a hyper-enhancement of the cystic wall and intracystic septation in the arterial phase followed by rapid wash-out in the portal and late phases [START_REF] Wildner | Differentiation of malignant liver tumors by software-based perfusion quantification with dynamic contrast-enhanced ultrasound (DCEUS)[END_REF] Detection of liver metastases 148 but only 7 with liver metastasis from melanoma SonoVue UCAs i.v. injection 1.5 mL followed by a bolus injection of saline solution (10 mL) Metastasis showed a rapid contrast enhancement a followed by a very rapid wash-out, which are characteristic of a malignant lesion [START_REF] Lekht | Versatile utilization of real-time intraoperative contrast-enhanced ultrasound in cranial neurosurgery: technical note and retrospective case series[END_REF]

  encapsulated inside polymer-or phospholipid-based colloidal particles.Prasad et al., designed a Cur-encapsulated lecithin nanoemulsions (Cur-NE) for the oral administration of Cur[START_REF] Prasad | Curcumin Encapsulated Lecithin Nanoemulsions: An Oral Platform for Ultrasound Mediated Spatiotemporal Delivery of Curcumin to the Tumor[END_REF]. They then investigated MB-assisted US as a physical method to improve the spatiotemporal delivery of Cur into the tumor in a B16F10 melanoma mouse model. Cur-NE was administered orally at 40 mg/kg b.w. to mice with subcutaneous (s.c.) melanoma. Two hours later, MBs (250 g) were intravenously injected. The tumors were immediately exposed to US (2 W/cm 2 , 50% DC) for 60 s. Mice were treated five times: first three treatments on alternate days and the last two treatments after a duration of two days. Fifteen days after treatment initiation, this protocol significantly reduced the relative growth rate of the tumor 8fold and 10-fold compared to Cur-NE treatment alone or free Cur treatment alone, respectively.The Kaplan Meier survival study revealed the survival percentage was 90.9% in the mice group treated with Cur-NE and MB-assisted US, whereas this percentage was 75.6% in the mice group receiving free Cur treatment alone. Unfortunately, the authors did not describe the survival percentage in mice treated with Cur-NE alone. Nevertheless, these results clearly established that MB-assisted US significantly improved the therapeutic efficacy of Cur in a mouse model of melanoma. In addition,Prasad et al., developed a new MB formulation to codeliver both anticancer drugs, i.e., Cur and topotecan (Tpt)[START_REF] Prasad | Ultrasound-Triggered Spatiotemporal Delivery of Topotecan and Curcumin as Combination Therapy for Cancer[END_REF]. Topotecan is a hydrophilic analog of camptothecin, which prevents DNA replication by inhibiting topoisomerase I activity. They designed polymeric nanocapsules with a hydrophobic core and hydrophilic polymeric shell, thus enabling the loading of Cur and Tpt, respectively[START_REF] Prasad | Ultrasound-Triggered Spatiotemporal Delivery of Topotecan and Curcumin as Combination Therapy for Cancer[END_REF]. These nanocapsules were then loaded onto the surface of the MBs (Tpt/Curloaded MBs). Such nanocapsule/MB complexes are more stable than liposome/MB complexes and are more suitable for the coencapsulation of hydrophobic and hydrophilic drugs. The authors investigated the therapeutic efficacy of the codelivery of Tpt and Cur using Tpt/Curloaded MBs in combination with US (2 W/cm 2 , 50% DC) in the B16F10 melanoma tumor model. Fifteen days after tumor inoculation, Tpt/Cur-loaded MBs (5 mg/kg and 2.5 mg/kg for Cur and Tpt, respectively) were intravenously injected, and the tumors were immediately exposed to US for 60 s. Mice were treated five times as described above. The codelivery of Tpt and Cur using Tpt/Cur-loaded MBs in combination with US induced a 15-fold and 4-fold decrease in the relative growth of tumors compared to free Tpt/Cur treatment alone and Tpt/Cur-loaded MBs treatment alone, respectively. Further, the mice group receiving Tpt/Curloaded MBs treatment (without US exposure) or free Tpt/Cur treatment on its own showed a

  week post-tumor inoculation, MBs were subcutaneously administered around the LNs under US imaging guidance. The HSP70/MAGEA1 fusion proteins were then released from the MBs after US exposure. This immunization protocol was repeated once a week for three weeks. This strategy enhanced Th-1 and Th-2 immune responses more effectively than the HSP70/MAGEA1 fusion protein alone. Used as a therapeutic recombinant vaccine, this immunotherapy induced 8-fold and 2-fold decreases in tumor volume compared to mice treated with MBs or HSP70/MAGEA1 fusion protein alone, respectively. This result translated into a significant increase in survival for mice immunized with this vaccine (50% survival at 60 d post-tumor inoculation) in comparison with mice treated with HSP70/MAGEA1 fusion protein (10% survival at 52 d post-tumor inoculation) and MBs only (no living mice 28 d post-tumor inoculation). In addition, this strategy provides an efficient prophylactic vaccine against melanoma. Indeed, this vaccine resulted in a 4-fold and 2-fold decrease in tumor volume compared to mice treated with MBs or HSP70/MAGEA1 fusion protein alone, respectively. Subsequently, immunized mice showed prolonged survival (35% survival at 70 d post-tumor inoculation) in comparison with those immunized with the HSP70-MAGEA1 fusion protein (20% survival at 58 d post-tumor inoculation) or MBs only (no living mice 25 d post-tumor inoculation). These results demonstrate that MB-assisted US is a promising method for delivering recombinant vaccines, thus increasing their prophylactic and therapeutic efficacy. Dendritic cell-based vaccines Dendritic cells (DCs) are the most potent antigen-presenting cells (APCs) that prime and activate cytotoxic T-lymphocytes (CTL) and T-helper lymphocytes (i.e., Th cells) (Abbas and Lichtman 2006). CTLs play a major role in the elimination of tumor cells, whereas Th cells support long-lasting and potent T and B lymphocyte responses. DCs also play a major role in the production of antibodies through their interactions with B lymphocytes. As sentinel cells of our organism, DCs recognize, engulf, and process tumor antigens. DCs then migrate to draining LNs and mature into APCs. Mature DCs present epitope peptides derived from tumor antigens via MHC-I and MHC-II molecules, thus initiating the immune response. In addition, tumor antigens may directly access draining LNs, where resident DCs recognize them. Associated with danger signals, these agents trigger a suitable and effective adaptive immune response.

Figure 1 .

 1 Figure 1. Flowchart of publication selection.

Figure 2 .

 2 Figure 2. CEUS of murine melanoma. To get this original data, B16F10 cells (1 10 6 cells

Figure 3 .

 3 Figure 3. Expression levels of endoglin, V 3 integrin and VEGFR2 using US molecular

Figure 4 .

 4 Figure 4. CEUS of cutaneous melanoma and its SLNs in swine melanoma model. (A)

Figure 5 .

 5 Figure 5. CEUS of metastatic melanoma of the gallbladder in patient. (A) Grayscale

Figure 6 .

 6 Figure 6. Evaluation of DOX-loaded MBs (DLMBs) for an acoustically mediated drug

Table 2 )

 2 . When using CEUS, the tumor should be biopsied in the most significant areas (e.g., areas of angiogenesis) to provide an accurate histological diagnosis of the tumor. CEUS-guided core needle biopsy has provided promising results in the diagnosis of soft tissue tumors[START_REF] Coran | Core needle biopsy of soft tissue tumors, CEUS vs US guided: a pilot study[END_REF]. Indeed, De Marchi et al., reported that 94.8% of tumor samples were adequate for studies are needed to assess the performance of CEUS-guided biopsy in the detection and characterization of melanoma.Conventional intraoperative US imaging is an inexpensive and safe method that allows visualization of real-time changes in tumor lesions during surgical intervention. Unlike CEUS, this modality does not provide functional or perfusion information, which may be useful for discriminating normal tissues from tumor tissues. Therefore, CEUS is a promising tool for use in intraoperative imaging.Lekht et al., reported the use of real-time intraoperative CEUS in cranial neurosurgery for metastatic melanoma

diagnosis with 97.1% sensitivity and 92.5% specificity

[START_REF] Marchi | Accuracy of core-needle biopsy after contrast-enhanced ultrasound in soft-tissue tumours[END_REF]

. To the best of our knowledge, no study has been conducted on the diagnosis of melanoma. Consequently, further clinical

  Flaherty et al., hypothesized that significant changes in tumor vasculature assessed by CEUS (and DCE-MRI) would correlate with the clinical efficacy of imatinib and bevacizumab in patients with advanced melanoma and other advanced cancers[START_REF] Flaherty | Phase I/II Trial of Imatinib and Bevacizumab in Patients With Advanced Melanoma and Other Advanced Cancers[END_REF]. However, only a few patients showed evidence of disease control. Indeed, of the five patients imaged with CEUS before and after the therapeutic regimen, two patients showed a significant decrease in tumor perfusion, one patient showed a minor decrease in tumor perfusion, and two patients exhibited no change in tumor perfusion. Consequently, no correlation between the clinical efficacy of the treatment and changes in tumor vasculature could be established. Nevertheless,Lassau et al., reported a multicenter investigation of the performance of CEUS to assess tumor response in different solid tumor types, including melanoma, treated with several anti-angiogenic drugs (e.g.,

sorafenib, bevacizumab, sunitinib, and imatinib)

  , thus significantly reducing the cardiotoxicity of DOX. However, several investigations have reported other adverse effects

(e.g., mucositis and hand and-foot syndrome) and a limited intratumoral (i.t.) DOX concentration

[START_REF] O'brien | Reduced cardiotoxicity and comparable efficacy in a phase III trial of pegylated liposomal doxorubicin HCl (CAELYX/Doxil) versus conventional doxorubicin for first-line treatment of metastatic breast cancer[END_REF]

. One of the strategies to overcome both problems consists of loading DOX inside or onto MBs and then intravenously administering these DOX-loaded MBs before exposing the tumor tissues to US. Accordingly, Abdalkader et al., designed a novel phospholipid-based MB formulation containing DOX for an acoustically mediated drug delivery (2 MHz, 2 W/cm 2 , 50% DC, 10 Hz PRF) in B16BL6 melanoma mouse model

  Exposure of the tumor to US (1 MHz, 100 Hz PRF, 20 cycles, 2.98 MPa for 5 min) induced MB destruction, which permeabilized its microvasculature and released gold nanorods into the tumor tissue. Despite showing that MB-assisted US significantly increased the i.t.

	bioavailability of gold nanorods, the authors did not demonstrate that this process is associated
	with an improvement in the effectiveness of photothermal therapy. Indeed, in a mouse breast
	adenocarcinoma model, the authors reported a significant laser-mediated temperature
	increment (> 45 °C) to eradicate the tumor after the acoustically enhanced i.t. delivery of gold
	nanorods

. Once administered intravenously (200 L at 6.8 10 8 /mL containing 10 nM gold nanorods), these MBs bound VEGFR2 overexpressed in the melanoma microvasculature in mice (B16F10).

  Despite the low number of clinical studies, CEUS has also revealed several distinctive features in terms of tumor microvasculature between healthy liver and gallbladder tissues and metastatic foci present in these tissues. Because CEUS enables the early diagnosis of these metastases and provides the most adequate care for the patient, these results should be confirmed in large cohort

	clinical trials.
	Anecdotally, few clinical studies have reported that CEUS can guide clinical interventions,
	including biopsy and surgery. Thus, CEUS-guided biopsy can provide an accurate histological
	diagnosis of melanoma by biopsying relevant tumor areas, such as those of tumor
	neoangiogenesis. In addition to conventional intraoperative US imaging, CEUS could provide
	perfusion information for helping surgeons to better discriminate healthy tissues from
	melanoma. Further investigations are needed to define the performance of CEUS in the
	supervision of diagnostic and therapeutic interventions. Furthermore, we have preclinically
	established evidence that CEUS is a sensitive and accurate US modality for assessing the early
	vascular changes induced by anti-angiogenic treatments of melanoma. These results have been
	partially confirmed in the clinical setting.

Table 1 . Inclusion and exclusion criteria used to select studies Inclusion criteria Exclusion criteria

 1

	Involving microbubble-assisted ultrasound	Ultrasound	without
		microbubbles
	Concerning melanoma skin cancer	Review	papers,
		comments, Letters
	Studies investigating the use of microbubble-mediated	Other languages
	ultrasound for the imaging of melanoma
	Studies investigating the use of microbubble-mediated
	ultrasound for the treatment of melanoma
	In-vitro, animals and patients
	English

Table 2 . CEUS of melanoma in clinics

 2

	References Application	Number of		Microbubbles	(type,	Main outcomes
			patients		concentration/volume,
					administration way)
	(Schmid-	Detection of	19		Levovist UCAs		Help to differentiate
	Wendtner,	metastases in			i.v. injection		LN metastases from
	et al. 2002)	LNs			8 mL at 312.5 mg/mL	reactive	LNs,
							hematomas	or
							seromas
	(Nielsen, et	Detection of	10		SonoVue UCAs		Detection	of	2
	al. 2009)	metastases in			s.c. injection		contrast-enhanced
		LNs			1 mL at 8 or 16 L/mL	inguinal LNs in one
							patient only
	(De Giorgi,	Detection of	15		SonoVue UCAs		Sensitivity: 100%
	et al. 2010)	metastases in			i.v. injection		Specificity: 61.5%
		LNs			2.4 mL followed by a	PPV: 54.5%
					bolus injection of saline	NPV: 100%
					solution (10 mL)
	(Rubaltelli,	Detection of	436		SonoVue UCAs		Sensitivity: 100%
	et al. 2011)	metastases in			i.v. injection		Specificity: 99.5%
		LNs			4.8 mL followed by a	PPV: 86.7%
					bolus injection of saline	NPV: 100%
					solution (10 mL)
	(Rubaltelli,	Detection of	540		SonoVue UCAs		Sensitivity: 98%
	et al. 2014)	metastases in			i.v. injection		Specificity: 99%
		LNs			4.8 mL followed by a	Accuracy: 99%
					bolus injection of saline
					solution (20 mL)
	(Punjabi, et	Detection of	1		SonoVue UCAs		Metastases are very
	al. 2006)	metastases in			i.v. injection		well perfused
		gall bladder			2.4 mL
	(Murphy-	Detection of	50 but only 3	Definity UCAs		Metastases	are
	Lavallee, et	liver	with	liver	i.v. injection		hypervascular.	A
	al. 2007)	metastases	metastases from	0.1 to 0.3 mL (standard	rapid and transient
			melanoma		dose, 0.2 mL) followed	arterial	contrast
					by a bolus injection of	enhancement
					saline solution (5 mL)	followed by a very
							rapid wash-out are
							observed
	(Andreano,	Detection of	1		SonoVue UCAs		Metastasis showed a
	et al. 2010)	metastases in			i.v. injection		rapid	contrast
		gall bladder			2.4 mL followed by a	enhancement	a
					bolus injection of saline	followed by a very
					solution (10 mL)		rapid	wash-out,
							which	are
							characteristic of a
							malignant lesion
	(Barretta,	Detection of	13 but 11	SonoVue UCAs
	et al. 2011)	metastases in	with		i.v. injection
		gall bladder	cutaneous	2.4 mL followed by a
			melanoma	bolus injection of saline
			8/11		solution (10 mL)
			investigated
			with CEUS

  LNs, lymph nodes; i.v., intravenous; s.c., subcutaneous; NPV, negative prediction value; PPV, positive prediction value; DCEUS, dynamic contrast-enhanced ultrasound; AUC, area under the curve.

		CEUS-guided	1 patient with	Definity UCAs	Metastases revealed
	)	surgery	brain metastases	i.v. injection	an intense arterial
			from melanoma	0.3 mL followed by a	contrast enhancement
				bolus injection of saline	followed by delayed
				solution (10 mL)	wash-out
					The	real-time
					DCEUS evaluation
					helped to a maximal
					tumor	resection
					without
					intraoperative
					complications
	(Escudier,	Monitoring of	13 patients but	SonoVue UCAs	Overall best response
	et al. 2007)	therapeutic	only 1 with	i.v. injection	of stable disease was
		follow-up	melanoma	4.8 mL at 8 L/mL	observed	and
		(Sorafenib/IFN			confirmed	for
		-2a			melanoma patient
		combination)
	(Lassau, et	Monitoring of	52 patients with	SonoVue UCAs	DCEUS provide a
	al. 2014)	therapeutic	metastatic	i.v. injection	predictive biomarker
		follow-up	melanoma	4.8 mL followed by a	of tumor progression,
		(anti-		bolus injection of saline	termed as the change
		angiogenic		solution (5 mL)	in AUC (a perfusion
		treatment			parameter linked to
		including			blood volume) from
		Sorafenib,			baseline to day 30
		Bevacizumab,			Patients	with	a
		Sunitinib,			greater than 40%
		Imatinib, etc.)			decrease in AUC on
					day 30 revealed
					freedom	from
					progression	and
					overall survival

Table 3 .

 3 Chemotherapy

This method provides the possibility of treating primary cutaneous and metastatic melanoma under the guidance of US imaging. However, we must keep in mind that current efficacy studies of acoustically mediated delivery have been reported in small animal models of primary and metastatic melanoma. Indeed, no proof of concept has been validated in preclinical studies on large animal models of melanoma or in clinical studies. Moreover, despite promising results, we can candidly question whether the acoustically mediated delivery of chemotherapeutic drugs and photothermal agents is relevant for the treatment of primary cutaneous melanoma. Indeed, recent skin removal of primary melanoma is minimally invasive, efficient, and safe.

However, the activation and/or stimulation of the anti-tumor immune response by in-vivo acoustically mediated delivery of MAAs and immunomodulatory molecules showed promising results for the treatment of metastatic melanomas. The current data should be confirmed in small-and large-animal models of metastatic melanoma in the near future before clinical investigation.

FUNDING/SUPPORT

The IRIS project (2019-00131873) was supported by the Région Centre-Val de Loire (France) and was approved by the S2E2 cluster.

Conflict of Interest disclosure