Contrast-enhanced ultrasound with VEGFR2-targeted microbubbles for monitoring combined anti-PD-L1/anti-CTLA-4 immunotherapy effects in a murine melanoma model with immunohistochemical validation

Contrast-enhanced ultrasound with VEGFR2-targeted microbubbles for monitoring combined anti-PD-L1/anti-CTLA-4 immunotherapy effects in a murine melanoma model with immunohistochemical validation | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Contrast-enhanced ultrasound with VEGFR2-targeted microbubbles for monitoring combined anti-PD-L1/anti-CTLA-4 immunotherapy effects in a murine melanoma model with immunohistochemical validation Felix L. Herr, Melissa J. Antons, Larissa V. Blume, Heidrun Hirner-Eppeneder, and 10 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6017170/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Immune checkpoint inhibitors (ICIs) have emerged as a highly effective treatment option for patients with metastatic melanoma. As not all patients respond to ICI immunotherapy imaging biomarkers must be used to accurately monitor early response to therapy. Therefore, the aim of this study was to evaluate contrast-enhanced ultrasound (CEUS) with VEGFR2-targeted microbubbles for monitoring the effects of combined anti-PD-L1/anti-CTLA-4 immunotherapy in a murine melanoma model. Methods Murine melanoma allografts (B16-F10) were implanted subcutaneously in n = 10 therapy and n = 10 control female C57BL/6 mice. CEUS with VEGFR2-targeted microbubbles was performed on day 7 and 12. The therapy group received 3 intraperitoneal injections on days 7, 9, 11 of combined anti-PD-L1/anti-CTLA-4 immunotherapy, the control group received a placebo. CEUS assessed tumour perfusion during an early vascular phase (wash-in area under the curve = WiAUC) and VEGFR2-specific binding during a late molecular phase (signal intensity at 8 minutes (SI 8min ) and 10 minutes (SI 10min )). For pathophysiological validation immunohistochemistry was performed. Results At follow-up, the CEUS perfusion parameter WiAUC demonstrated a significantly higher decrease in the therapy than in the control group (p = 0.021). At follow-up, the signal enhancement in the late phase was significantly lower in the therapy than in the control group (SI 8min p = 0.003; SI 10min p = 0.002). Immunohistochemistry revealed significantly more apoptotic tumour cells (p = 0.001), more tumour infiltrating lymphocytes (p = 0.049), lower tumour cell proliferation (p = 0.001), lower microvascular density (p = 0.003) and lower VEGFR2 expression (p = 0.003) in the therapy than in the control group. Conclusions CEUS with VEGFR2-targeted microbubbles allowed for monitoring early treatment effects of a combined anti-PD-L1/anti-CTLA-4 immunotherapy on melanoma allografts with significantly lower tumour perfusion and significantly lower binding of VEGFR2-targeted microbubbles in the therapy than in the control group. VEGFR2 CEUS immunotherapy melanoma Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Background In recent years, immune checkpoint inhibitors (ICIs) have emerged as a highly efficacious treatment option for patients with metastatic tumours, and have also been approved for use in melanoma (1). ICIs function by blocking the upregulated surface receptors of Cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) or programmed death-ligand 1 (PD-L1) (2). Given that not all patients respond to immunotherapy with ICI and its treatment is associated with adverse effects, imaging biomarkers need to be employed for accurate monitoring of early therapy response (3). Vascular endothelial growth factor (VEGF) is a principal regulator of angiogenesis (4). VEGF and its receptor VEGFR2 are increasingly expressed in solid tumours, such as melanoma, and play an important role in tumours angiogenesis, proliferation and metastasis (5–7). Furthermore, VEGFR2 is associated with the development of resistance to anti-cancer drugs (8). A correlation between tumour immune microenvironment and VEGF(R-2) has already been described: The activation of VEGF(R-2) is associated with the development of an immunosuppressive microenvironment, by inducing the aggregation of immature dendritic cells, bone marrow-derived suppressor cells and regulatory T cells, and inhibiting T lymphocyte migration (9–11). Moreover, VEGF(R-2) has been demonstrated to enhance PD-1 expression (9,12). Studies have shown that mutations of VEGFR2 are linked to better clinical outcomes for tumours under ICI (13). Additionally, VEGFR2 expression level can serve as an important indicator of how well patients will respond to therapies that block PD-1 and PD-L1(14). Further, immunotherapy can reduce the expression of VEGF, which helps counteract its immunosuppressive effects (15,16). Contrast-enhanced ultrasound (CEUS) utilising microbubbles (MB) that bind specifically to VEGFR2 enables the characterisation of functional and molecular parameters (17,18). The MBs are exclusively distributed intravascularly (19). Following intravenous injection, the perfusion of tumours can be quantitatively evaluated in an early phase by assessing various parameters of tissue microcirculation (perfusion). In a subsequent late phase, targeted MBs bound to tumour vascular endothelial cells, which overexpress VEGFR2, permit the non-invasive visualisation of VEGFR2 expression (20,21). Multiparametric CEUS with VEGFR2-targeted MB has demonstrated to be a highly sensitive tool for monitoring therapy effects in a range of tumour entities: In a mouse model of hepatocellular carcinoma, early therapeutic effects of the tyrosine kinase inhibitor sorafenib were analysed (22). Eschbach et al. has already used CEUS to analyse morphology, perfusion and VEGFR2-specific binding in colorectal adenocarcinoma in rats (23). Consequently, VEGFR2-targeted CEUS may provide functional and molecular imaging biomarkers for the assessment of tumour angiogenesis and early therapy response in murine melanoma allografts under immunotherapy. Therefore, we hypothesised that CEUS with VEGFR2-targeted MB enables monitoring of early immunotherapy effects in a murine melanoma allograft tumour model. We aimed to investigate whether the functional and the molecular CEUS parameters have potential as non-invasive in vivo imaging biomarkers of early therapy response with time point-matched multiparametric immunohistochemical validation. Methods Animal model and experimental protocol All animal experiments were conducted in accordance with the guidelines for the use of living animals in scientific studies and the animal study was approved by the Committee for Animal Research of the Government of Upper Bavaria (ROB-55.2-2532.Vet_02-19-32). Female C57BL/6 mice (mean weight ± 20 g, aged 10–12 weeks at the time of inoculation, Charles River, Sulzfeld, Germany) were housed in groups of four per cage in a temperature- and humidity-controlled room with a 12 h light/dark cycle and provided with food and water. In accordance with the approved animal study proposal, animals were excluded from the study if they exhibited any of the following symptoms: a loss of body weight equal to or greater than 19% of the value observed prior to inoculation; a tumour size exceeding 1.5 cm; exulceration; infections or bleeding from the tumour area; bloody diarrhoea; apathy; ascites; or acneiform dermatitis. The murine melanoma cell line B16-F10 (ATCC CRL-6475) was cultured and expanded under standard conditions. After a one-week assimilation period, the melanoma cells (B16-F10; 3 x 10 5 cells) were resuspended in a 1:1 mixture of Matrigel (BD Biosciences, San Jose, CA, USA) and phosphate-buffered saline (PBS, pH 7.4). The tumour cells were injected subcutaneously into the left abdominal flank of C57BL/6 mice ( n = 20) under inhalative isoflurane anaesthesia, with 2.0 vol % isoflurane and 1.5 L/min oxygen. Once tumour diameter reached a maximum of 0.5 cm, animals were randomly assigned to either the therapy ( n = 10) or the control group ( n = 10) and baseline CEUS was conducted. The therapy group received a total of three intraperitoneal injections of anti-PD-L1 and anti-CTLA-4 antibodies (20 µg/kg) on days 7, 9 and 11 after inoculation. The control group received volume equivalent placebo. For imaging, mice were anaesthetised via continuous inhalation of 2.0 vol % isoflurane and 1.5 L/min oxygen and were placed on a heating pad. A multiparametric CEUS protocol was conducted on day 7 (baseline) and on day 12 (follow-up) following inoculation. Tumour allografts were explanted, fixed in formalin and preserved by cryoconservation at baseline and follow-up for multiparametric ex vivo immunohistochemical work-up (Fig. 1 ). MB BR55 (Bracco Suisse SA, Geneva, Switzerland) is an ultrasound molecularly targeted lipid-shelled microbubble that targets VEGFR2. The lipopeptide that binds to VEGFR2 is created by conjugating a heterodimer peptide to the amino group of DSPE-PEG2000-NH2, which is then incorporated into the phospholipid-based MB formulation. The final product, BR55, is provided as a lyophilized powder in a septum-sealed vial, with the gas phase consisting of a mixture of perfluorobutane and nitrogen (24–26). Prior to utilisation, BR55 must be reconstituted by the injection of 2 ml of a 5% glucose solution into the vial via the septum. Once dissolved, the resulting MB suspension is ready for utilisation. The mean diameter of the BR55 MBs is 1.5 µm, with a concentration of approximately 2 x 10⁹ MB per mL. Each MB contains approximately 4 x 10⁵ lipopeptide molecules (24,27). The circulation time of BR55 is approximately 4 minutes (21). The administration of BR55 to each animal was conducted via a standardised manual intravenous injection through a tail vein catheter, with a dose of 200 µL administered. CEUS Imaging CEUS imaging was conducted using a clinical ultrasound system (Philips Epiq 7, Seattle, WA) with a conventional 18L4 linear transducer (transmit frequency 9 MHz, dynamic range 55 dB, depth 25 mm), linear time-gain compensation, and an acoustic focus positioned at the largest tumour cross-section, with the contrast setting adjusted to the image button [35]. The anaesthetised mice were positioned on their sides, and ultrasound coupling gel was applied to the shaved skin. An initial B-mode scan was conducted to visualise the subcutaneous tumour expansion. Video acquisition commenced immediately prior to the administration of BR55, continued for the initial minute, and then resumed at two-minute intervals for a period of up to ten minutes post-injection (at a frame rate of 16 Hz). This enabled the measurement of MB wash-in and wash-out in the tumour. The binding of the VEGFR2-specific agent was evaluated at 8 and 10 minutes after the injection of BR55, when the circulating MB had largely cleared from the bloodstream, while the tumour tissue remained highlighted, indicating the specific accumulation of targeted MB on the VEGFR2-expressing endothelium. Data post processing The CEUS data were processed using dedicated software on an external workstation with Vuebox® (Version v7.5.0.7051–64-bit version - Bracco Suisse SA, Geneva, Switzerland). A region of interest (ROI) was delineated within a highly perfused area of the tumour's vital outer rim without necrotic areas. The software quantifies contrast enhancement in the ROI, presenting results as relative echo-power values that correspond to MB concentration, measured in arbitrary units (a.u.) (20,22,24) (Fig. 2 ). The key parameters analysed included the wash-in area under the curve (WiAUC) during the early vascular phase, which provides insights into blood flow and volume (28,29), as well as signal intensity measured at 8 minutes (SI 8min ) and 10 minutes (SI 10min ) post-injection, which serve as surrogate indicators of VEGFR2-specific MB binding in the late molecular phase. In addition, the size of the tumour was measured in two dimensions. Immunohistochemistry CD31 First deparaffinization, rehydration and antigen retrieval (SignalStain® EDTA-Unmasking Solution, Cell signaling Technology, Leiden, Netherlands) was performed. For immunohistochemical assessment of tumour microvascular density, non-specific binding sides were blocked in 5% Donkey-Serum in TBS-Tween20. Subsequently tumours slices were incubated with a polyclonal rabbit anti-CD31 primary antibody (1:50; Abcam ab28364, Cambridge, UK) overnight. Tissue samples underwent further processing utilising secondary antibodies conjugated with Alexa Fluor 488 (1:200; ab150073, Abcam Limited, Cambridge, UK), to facilitate fluorescence detection. Counterstaining was conducted using DAPI (Carl Roth, Karlsruhe, Germany) and the slides were covered with Fluoromount-G (ThermoFisher Scientific, Waltham, Massachusetts, USA). The number of tumour microvessels was quantified as the mean of the endothelial cells in 10 random fields at 200x magnification, as previously described (30). CD8 antigen staining The tissue slides were initially deparaffinised and rehydrated, followed by permeabilization with a solution of 1x PBS (Gibco, Carlsbad, CA) and 0.25% Triton-X-100 (Merck KGaA, Darmstadt, Germany). Subsequently, antigen retrieval was conducted by boiling the tissue slides in a 10 mM Tris-EDTA buffer for CD8 (Life Technologies, Carlsbad, CA). Following the retrieval of antigens, thorough washing with 1x PBS was conducted to ensure the removal of residual buffers. Non-specific binding sites were blocked with a solution containing bovine serum albumin (BSA) and primary antibodies against CD8 (1:50; ab217344, Abcam Limited, Cambridge, UK) were applied. Following an overnight incubation period and additional washing steps, secondary antibodies conjugated with Alexa Fluor 488 (1:200; ab150073, Abcam Limited, Cambridge, UK) were applied to facilitate fluorescence detection. Counterstaining with DAPI enabled the visualisation of nuclei. Finally, slides were mounted using Fluoromount-G, ensuring the preservation and optimal visualisation of the stained specimens. Results were quantified as the mean percentage of CD8-positive T-cells in 10 randomly selected fields at 200x magnification. Ki-67 antigen staining A Ki-67-specific monoclonal rabbit anti-human antibody (1:50; SP6, Thermo Fisher MA5-14420, Thermo Fisher) was employed for the purpose of quantifying tumour cell proliferation. The tissue was de-masked in Universal antigen retrival reagent (ab208572, Abcam Limited, Cambridge, UK) using microwave irradiation at 600 W. Following a wash in distilled water and TBS-Tween (0.05%), secondary antibodies conjugated with Alexa Fluor 488 (1:200; ab150073, Abcam Limited, Cambridge, UK) were applied to facilitate fluorescence detection. Subsequently, counterstaining was conducted using DAPI (Carl Roth, Karlsruhe, Germany) and slides were covered with Fluoromount-G (ThermoFisher Scientific, Waltham, Massachusetts, USA). Results were quantified as the mean percentage of proliferating cells in 10 random fields at 200x magnification. Terminal desoxynucleotidyl transferase dUTP nick-end labelling (TUNEL) The initial step involved the deparaffinisation and rehydration of the tissue slides, which were then permeabilised using a solution comprising Triton X-100 (Merck KGaA, Darmstadt, Germany) and sodium citrate (Sigma-Aldrich, Steinheim, Germany). Subsequently, antigen retrival reagent (ab208572, Abcam Limited, Cambridge, UK) was conducted through microwave treatment. The staining was conducted by combining solutions from the provided kit, with meticulous attention to maintaining darkness and temperature control. Following comprehensive washing steps, slides were further stained with DAPI to visualise nuclei. Ultimately, slides were mounted using Fluoromount-G (ThermoFisher Scientific, Waltham, Massachusetts, USA) ensuring the preservation of the stained samples for microscopic examination. Results were quantified as the mean percentage of apoptotic cells in 10 random fields at 200x magnification. VEGFR2 The expression of VEGFR2 was analysed using a VEGFR2-specific monoclonal rabbit anti-human antibody (1:50; Cell Signalling, Cambridge, UK). Following standard procedures, the tissue samples were dewaxed, rehydrated and subsequently demasked in target-retrieval solution (SignalStain EDTA Unmasking Solution, Cell Signalling, Cambridge, UK) using microwave irradiation at 600 W. After an overnight incubation period with the primary antibody, the tissue samples were further processed using secondary antibodies conjugated with Alexa Fluor 488 (1:200; ab150073, Abcam Limited, Cambridge, UK) and counterstaining was conducted using DAPI (Carl Roth, Karlsruhe, Germany). The number of tumour vessels stained positively for VEGFR2 was quantified at a magnification of 200x, with the analysis conducted on 10 random high-power fields. Statistical analysis Continuous variables are presented as means with standard deviations (mean ± SD). For intergroup comparisons between treatment and control group the Mann-Whitney U-test was employed. A Wilcoxon signed-rank test was used for intragroup comparisons of CEUS parameters between baseline (day 7) and follow-up (day 12). P-values < 0.05 were considered statistically significant. All statistical analyses were performed using GraphPad Prism (Graphpad Software, Inc., Boston, MA) and Microsoft Excel (Microsoft Corporation, Redmond, WA). Results The experimental protocol, including imaging with CEUS, was successfully completed in n = 16 animals; in n = 3 animals the ultrasound could not be accomplished due to technical issues, n = 1 animal of the therapy group had to be excluded due to anaesthesia complications. Tumour size Tumour size was assessed at baseline (7 days after inoculation) and follow-up (12 days after inoculation). For calliper measurements in two dimensions there were no significant differences in mean tumour sizes between the therapy and the control group at baseline (29.9 ± 12.7 mm 2 vs. 32.1 ± 17.3 mm 2 ; p = 0.909) and follow-up (104.2 ± 35.3 mm 2 vs. 123.0 ± 66.1 mm 2 ; p = 0.733). There were significant changes in tumour size between baseline and follow-up in the therapy (29.9 ± 12.7 mm 2 vs. 104.2 ± 35.3 mm 2 ; p = 0.006) and in the control group (32.1 ± 17.3 mm 2 vs. 123.0 ± 66.1 mm 2 ; p = 0.002, supplemental table 1 ). For CEUS measurements in two dimensions there were no significant differences in mean tumour sizes between the therapy and the control group at baseline (21.3 ± 13.2 mm 2 vs. 17.4 ± 19.8 mm 2 ; p = 0.173) and follow-up (60.2 ± 28.1 mm 2 vs. 63.2 ± 48.6 mm 2 ; p = 1). There were significant increases in tumour size between baseline and follow-up in the therapy (21.3 ± 13.2 mm 2 vs. 60.2 ± 28.1 mm 2 ; p = 0.004) and in the control group (17.4 ± 19.8 mm 2 vs. 63.2 ± 48.6 mm 2 ; p = 0.002, supplemental table 2 ). CEUS Using BR55-CEUS, functional parameters of tumour perfusion were assessed during an early vascular phase and VEGFR2-specific binding of the MB during a late molecular phase (Fig. 3 ). In the therapy group, a significant decrease of tumour perfusion (WiAUC) was observed following the one-week treatment course with combined anti-PD-L1/anti-CTLA-4 immunotherapy (23767 ± 16937 a.u. at baseline to 8054 ± 10083 a.u. at follow-up; p = 0.008). Also, in the control group significant changes in WiAUC (from 21744 ± 17585 a.u. at baseline to 16402 ± 14813 a.u. at follow-up; p = 0.008) were noted. Moreover, WiAUC was significantly (p = 0.021) lower in the therapy compared to the control group at follow-up (Fig. 4 and supplemental table 3 ). In the late molecular phase, a significant decline in tumour signal intensity between baseline and follow-up was observed in the therapy group (SI 8min : from 517.7 ± 103.7 to 220.9 ± 62.6 a.u., p = 0.008; SI 10min : from 419.7 ± 90.4 to 181.9 ± 48.4 a.u., p = 0.008) and in the control group (SI 8min : from 459.8 ± 51.5 to 350.7 ± 43.3 a.u., p p = 0.008; SI 10min : from 400.3 ± 59 to 281.5 ± 37.3 a.u., p = 0.008). However, significantly lower SI 8min (220.9 ± 62.6 vs. 350.7 ± 43.3 a.u., p = 0.003) and SI 10min (181.9 ± 48.4 vs. 281.5 ± 37.3 a.u., p = 0.002) values were detected in the therapy than in the control group at follow-up (Fig. 5 and supplemental table 4 ). Immunohistochemistry Ex vivo analysis revealed significant anti-tumour effects in the therapy ( n = 8) compared to the control group ( n = 8). Compared to the control group, the tumour samples of the therapy group had a significantly higher percentage of apoptotic cells (TUNEL 52.3 ± 17.2% vs. 17.1 ± 5.6%; p = 0.001) at follow-up. Furthermore, the therapy group showed a significantly higher number of TILs than the control group (CD8 395.4 ± 522.5 vs. 71.9 ± 55.6; p = 0.049) at follow-up. At follow-up, tumour cell proliferation was significantly lower in the therapy group than in the control group (Ki-67 27.2 ± 9.7% vs. 58.7 ± 8.6%; p = 0.001). At follow-up, anti-angiogenic effects of combined anti-PD-L1/anti-CTLA-4 immunotherapy were observed in the investigated melanoma allografts with a significantly lower microvascular density in combined anti-PD-L1/anti-CTLA-4-treated than in non-treated animals (CD31 142.1 ± 59.8 vs. 286.3 ± 88.1; p = 0.003), quantified by CD31 staining. VEGFR2 staining showed a significantly lower VEGFR2 expression in combined anti-PD-L1/anti-CTLA-4-treated than in placebo-treated animals (VEGFR2 72.5 ± 18.6 vs. 119 ± 29.6; p = 0.003, Fig. 6 ). Discussion Due to severe adverse events and the fact that not all patients respond to ICI, it is necessary to evaluate non-invasive in vivo imaging biomarkers of early therapy response in order to provide effective therapy guidance (31). Preclinical and clinical studies have investigated the therapy effects, particularly under anti-angiogenic drugs, in melanoma using CEUS (32,33). However, imaging biomarkers of early therapy response under combined immunotherapy in melanoma are still scarce and require further investigation to ascertain the feasibility of clinical use. Tumour size under ICI therapy Tumour size, measured using callipers or CEUS, did not show a significant difference between the therapy and control group, but morphological assessment of tumours does not reveal early physiological changes in tumours (33). This finding is in accordance with Mellinger (32), who observed no difference in tumour size but a decrease in tumour perfusion under immunotherapy in melanoma xenografts. In contrast to us, Mellinger (32) used one type of antibody (anti-PD1) for checkpoint inhibition, and the therapy was administered twice a week within a 18-day period. This supports the observation that immunotherapy does not affect tumour size significantly at early follow-up. Tumour perfusion under ICI therapy A significantly higher reduction of WiAUC at follow-up was observed in the therapy compared to the control group. This is in accordance with Eschbach et al. (23) who demonstrated a significant reduction in WiAUC under regorafenib for colorectal carcinoma xenografts. However, their analysis was conducted following a one-week daily treatment of regorafenib. Regorafenib is an multi-tyrosine kinase inhibitor with a distinct mechanism of action that demonstrated anti-angiogenic and anti-proliferative effects in vivo in different experimental tumour models, including breast cancer, renal cell carcinoma and glioblastoma (34). The significant decrease of the CEUS perfusion parameter WiAUC was consistent with the immunohistochemical CD31 staining, that demonstrated significantly lower microvascular density. This finding is in accordance with Brloznik et al. (35), who were able to correlate a reduction of tumour perfusion in CEUS with tumour histological analyses of CD31 in a murine melanoma model. Differing from our study Brloznik et al. treated the mice with gene electrotransfer and radiotherapy. According to our results, the early vascular CEUS parameter WiAUC indicates to serve as functional perfusion biomarker of early therapy response under combined anti-PD-L1/anti-CTLA-4 immunotherapy in melanoma. ICI therapy effects on VEGFR-2 expression Regarding the evaluation of VEGFR2-targeted microbubbles in the late molecular phase, the treatment group exhibited significantly lower SI 8min and SI 10min values at follow-up. This is in line with Baetke et al. who demonstrated a significant reduction in the amount of VEGFR2-targeted MB in heterotopic squamous cell carcinoma xenografts in mice undergoing anti-VEGF antibody therapy (21). In a murine colorectal carcinoma model anti-angiogenic treatment with sunitinib resulted in decreased tumour perfusion and VEGFR2 expression. In contrast to us, they observed treatment effects already 24 hours post-treatment (36). In accordance with SI 8min and SI 10min parameters, immunohistochemical measures demonstrated a significant decline in VEGFR2. This is in line with Palmowski et al. (37), who demonstrated, complementary to CEUS, a significantly lower stained area fraction for VEGFR2 in immunohistochemical analysis of treated tumours. Differing from our study, Palmowski et al. investigated squamous cell carcinoma xenografts under a matrix metalloproteinase inhibitor. The necrotic tumour areas observed in our study, similar to Eschbach et al. (23), are responsible for the reduction in effective vital tumour mass in rapidly growing tumours, which could explain the transient reduction in vascular proliferation and VEGFR2 expression with decreased BR55 binding (22). It can be suggested that the development of necrosis may be a confounding factor contributing to the decreased differential targeted enhancement observed in treated melanoma allografts. Therefore, we selected ROIs over the vital outer rim of therapy and control tumours to avoid areas of necrosis and elevated interstitial pressure. The significantly lower number of MB on the tumour endothelial surface following combined immunotherapy most likely reflects the significantly higher decrease of tumour perfusion and VEGFR2 expression compared to the control group. In our experimental setup, the observed reduction of specifically bound MB with VEGFR2-targeted CEUS under anti-PD-L1/anti-CTLA-4 immunotherapy of malignant melanoma may be explained by the pathophysiological connection between VEGF and tumour immunology. The ex vivo validation at follow-up demonstrated a significantly elevated number of TILs and apoptotic cells, as well as a significantly lower number of proliferating cells within the therapy group. These findings indicate the presence of an antitumor immune response under combined anti-PD-L1/anti-CTLA-4 therapy. VEGF via its receptor VEGFR2 contributes to the exhaustion of CD8 + T cells, which is characterised by the expression of negative immune checkpoints, such as PD-1 receptors (38). In this context, VEGF promotes the expression of checkpoint molecules(9). Immune checkpoint therapy regulates endothelial cell functions and reverses the immunosuppressive effects of VEGF in metastatic melanoma by decreasing intratumoral VEGF(R2) expression and consecutively activating CD8 + T cells via the IFNγ signalling pathway (15,16,39). The observed combination of pronounced pro-immunogenic, pro-apoptotic, anti-proliferative and anti-VEGFR2 effects in the immunohistochemistry in our study underscores the effectiveness of the combined immunotherapy and the pathophysiological link between VEGF(R2) and tumour immunology. Limitations The results of our study are limited. Firstly, the investigation was conducted on a single tumour-therapy combination within an allograft model of melanoma. Secondly, the observation period was limited to six days with three doses of therapy. Additionally, CEUS imaging was conducted only until day 12 following tumour inoculation, which was constrained by the dynamics of tumour growth. An extension of the observational period and prolongation of the duration of therapy administration may result in not only further inhibition of tumour growth but also a reduction in tumour size, which is not captured in this study. Conclusions This study offers insights into early immunotherapy response in a murine melanoma model, providing time matched correlation of in vivo and ex vivo biomarkers of therapy response. The significant reduction of tumour perfusion and VEGFR2-targeted MB under combined immunotherapy assessed by CEUS were paralleled by significant pro-immunogenic, pro-apoptotic, anti-angiogenic and anti-proliferative effects in immunohistochemistry. Our findings demonstrate that CEUS parameters have potential to serve as non-invasive imaging biomarkers for immunotherapy response assessment in melanoma. Declarations Funding This work was supported by a research grant from Bracco Imaging SA, Geneva, Switzerland, which provided the BR55 free of charge. The authors of this manuscript have no other relationships with these companies, whose products or services may be related to the subject matter of this article. Competing Interests The authors have no relevant financial or non-financial interests to disclose. Author Contributions All authors contributed to the study conception and design. Material preparation, data collection, and analysis were performed by FLH, MJA, LVB, HHE, SKL, AC, JS, TB, DAC, MMH, IT, CCC. The first draft of the manuscript was written by FLH, LVB, HHE, MMH and CCC and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript. All authors read and approved the final manuscript. Availability of data and material The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request. Ethics approval and consent to participate All animal experiments were conducted in accordance with the guidelines for the use of living animals in scientific studies and the animal study was officially approved by the Committee for Animal Research of the Government of Upper Bavaria (ROB-55.2-2532.Vet_02-19-32). The authors have ensured compliance with the ARRIVE guidelines. Clinical trial number: not applicable. Acknowledgements This work was supported by a research grant from Bracco Imaging SA, Geneva, Switzerland, which provided BR55 free of charge. Consent for publication Not applicable. References Shiravand Y, Khodadadi F, Kashani SMA, Hosseini-Fard SR, Hosseini S, Sadeghirad H, u. a. Immune Checkpoint Inhibitors in Cancer Therapy. Curr Oncol Tor Ont. 24. 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Squamous Cell Carcinoma Xenografts: Use of VEGFR2-targeted Microbubbles for Combined Functional and Molecular US to Monitor Antiangiogenic Therapy Effects. Radiology. Februar 2016;278(2):430–40. Baron Toaldo M, Salvatore V, Marinelli S, Palamà C, Milazzo M, Croci L, u. a. Use of VEGFR-2 targeted ultrasound contrast agent for the early evaluation of response to sorafenib in a mouse model of hepatocellular carcinoma. Mol Imaging Biol. Februar 2015;17(1):29–37. Eschbach RS, Clevert DA, Hirner-Eppeneder H, Ingrisch M, Moser M, Schuster J, u. a. Contrast-Enhanced Ultrasound with VEGFR2-Targeted Microbubbles for Monitoring Regorafenib Therapy Effects in Experimental Colorectal Adenocarcinomas in Rats with DCE-MRI and Immunohistochemical Validation. PloS One. 2017;12(1):e0169323. Pochon S, Tardy I, Bussat P, Bettinger T, Brochot J, von Wronski M, u. a. BR55: a lipopeptide-based VEGFR2-targeted ultrasound contrast agent for molecular imaging of angiogenesis. Invest Radiol. Februar 2010;45(2):89–95. Bzyl J, Lederle W, Rix A, Grouls C, Tardy I, Pochon S, u. a. Molecular and functional ultrasound imaging in differently aggressive breast cancer xenografts using two novel ultrasound contrast agents (BR55 and BR38). Eur Radiol. September 2011;21(9):1988–95. Pillai R, Marinelli ER, Fan H, Nanjappan P, Song B, von Wronski MA, u. a. A phospholipid-PEG2000 conjugate of a vascular endothelial growth factor receptor 2 (VEGFR2)-targeting heterodimer peptide for contrast-enhanced ultrasound imaging of angiogenesis. Bioconjug Chem. 17. März 2010;21(3):556–62. Lassau N, Lamuraglia M, Chami L, Leclère J, Bonvalot S, Terrier P, u. a. Gastrointestinal stromal tumors treated with imatinib: monitoring response with contrast-enhanced sonography. AJR Am J Roentgenol. November 2006;187(5):1267–73. Roccarina D, Garcovich M, Ainora ME, Riccardi L, Pompili M, Gasbarrini A, u. a. Usefulness of contrast enhanced ultrasound in monitoring therapeutic response after hepatocellular carcinoma treatment. World J Hepatol. 18. Juli 2015;7(14):1866–74. Lassau N, Chebil M, Chami L, Bidault S, Girard E, Roche A. Dynamic contrast-enhanced ultrasonography (DCE-US): a new tool for the early evaluation of antiangiogenic treatment. Target Oncol. März 2010;5(1):53–8. Bruns CJ, Harbison MT, Davis DW, Portera CA, Tsan R, McConkey DJ, u. a. Epidermal growth factor receptor blockade with C225 plus gemcitabine results in regression of human pancreatic carcinoma growing orthotopically in nude mice by antiangiogenic mechanisms. Clin Cancer Res Off J Am Assoc Cancer Res. Mai 2000;6(5):1936–48. Nisar S, Bhat AA, Hashem S, Yadav SK, Rizwan A, Singh M, u. a. Non-invasive biomarkers for monitoring the immunotherapeutic response to cancer. J Transl Med. 9. Dezember 2020;18(1):471. Mellinger A, Hersant J, Bourreau C, Lecoq S, Deveze E, Clere N, u. a. Caliper, contrast enhanced-ultrasound or laser speckle contrast imaging: Techniques to follow mice melanoma growth. J Biophotonics. März 2024;17(3):e202300439. Avry F, Mousset C, Oujagir E, Bouakaz A, Gouilleux-Gruart V, Thépault RA, u. a. Microbubble-Assisted Ultrasound for Imaging and Therapy of Melanoma Skin Cancer: A Systematic Review. Ultrasound Med Biol. November 2022;48(11):2174–98. Eschbach RS, Fendler WP, Kazmierczak PM, Hacker M, Rominger A, Carlsen J, u. a. Correlation of perfusion MRI and 18F-FDG PET imaging biomarkers for monitoring regorafenib therapy in experimental colon carcinomas with immunohistochemical validation. PloS One. 2015;10(2):e0115543. Brloznik M, Boc N, Cemazar M, Bosnjak M, Savarin M, Kejzar N, u. a. Contrast-enhanced ultrasound for evaluation of tumor perfusion and outcome following treatment in a murine melanoma model. Bioelectrochemistry Amst Neth. Dezember 2021;142:107932. Payen T, Dizeux A, Baldini C, Le Guillou-Buffello D, Lamuraglia M, Comperat E, u. a. VEGFR2-Targeted Contrast-Enhanced Ultrasound to Distinguish between Two Anti-Angiogenic Treatments. Ultrasound Med Biol. August 2015;41(8):2202–11. Palmowski M, Huppert J, Ladewig G, Hauff P, Reinhardt M, Mueller MM, u. a. Molecular profiling of angiogenesis with targeted ultrasound imaging: early assessment of antiangiogenic therapy effects. Mol Cancer Ther. Januar 2008;7(1):101–9. Kim CG, Jang M, Kim Y, Leem G, Kim KH, Lee H, u. a. VEGF-A drives TOX-dependent T cell exhaustion in anti-PD-1-resistant microsatellite stable colorectal cancers. Sci Immunol. 8. November 2019;4(41):eaay0555. Amersfoort J, Eelen G, Carmeliet P. Immunomodulation by endothelial cells - partnering up with the immune system? Nat Rev Immunol. September 2022;22(9):576–88. Supplementary Files CD31control.tif CD31therapy.tif CD8control.tif CD8therapy.tif KI67control.tif KI67therapy.tif TUNELcontrol.tif TUNELtherapy.tif VEGFR2control.tif VEGFR2therapy.tif SupplementaryInformation11022025.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6017170","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":421726828,"identity":"e18ccc19-1b66-4c6d-a2dd-90cc47b8b848","order_by":0,"name":"Felix L. 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Blume","email":"","orcid":"","institution":"University Hospital Munich: LMU Klinikum","correspondingAuthor":false,"prefix":"","firstName":"Larissa","middleName":"V.","lastName":"Blume","suffix":""},{"id":421726831,"identity":"3b68fa2f-5f5f-41d0-8241-acf6aba94ed2","order_by":3,"name":"Heidrun Hirner-Eppeneder","email":"","orcid":"","institution":"University Hospital Munich: LMU Klinikum","correspondingAuthor":false,"prefix":"","firstName":"Heidrun","middleName":"","lastName":"Hirner-Eppeneder","suffix":""},{"id":421726832,"identity":"c2de68d4-ea32-46fc-b7ed-26dea40069b6","order_by":4,"name":"Sandra Kloiber-Langhorst","email":"","orcid":"","institution":"University Hospital Munich: LMU Klinikum","correspondingAuthor":false,"prefix":"","firstName":"Sandra","middleName":"","lastName":"Kloiber-Langhorst","suffix":""},{"id":421726833,"identity":"6cde2250-ff9e-4202-8201-6910098c7ce2","order_by":5,"name":"Amra Cimic","email":"","orcid":"","institution":"University Hospital Munich: LMU Klinikum","correspondingAuthor":false,"prefix":"","firstName":"Amra","middleName":"","lastName":"Cimic","suffix":""},{"id":421726834,"identity":"0603fd4e-487c-4972-9343-481bbbd7a49e","order_by":6,"name":"Jennifer Stueckl","email":"","orcid":"","institution":"University Hospital Munich: LMU Klinikum","correspondingAuthor":false,"prefix":"","firstName":"Jennifer","middleName":"","lastName":"Stueckl","suffix":""},{"id":421726835,"identity":"8c789758-202d-4855-b622-2098623fdebd","order_by":7,"name":"Isabelle Tardy","email":"","orcid":"","institution":"Bracco Suisse SA","correspondingAuthor":false,"prefix":"","firstName":"Isabelle","middleName":"","lastName":"Tardy","suffix":""},{"id":421726836,"identity":"68c95a45-e0ed-4d01-a140-ccd3b72f8f08","order_by":8,"name":"Tanja Burkard","email":"","orcid":"","institution":"University Hospital Munich: LMU Klinikum","correspondingAuthor":false,"prefix":"","firstName":"Tanja","middleName":"","lastName":"Burkard","suffix":""},{"id":421726837,"identity":"12396d52-82f8-42a0-9a55-22b107c58178","order_by":9,"name":"Jens Ricke","email":"","orcid":"","institution":"University Hospital Munich: LMU Klinikum","correspondingAuthor":false,"prefix":"","firstName":"Jens","middleName":"","lastName":"Ricke","suffix":""},{"id":421726838,"identity":"10c7070b-d2a0-4299-b7c9-bae65cf179ba","order_by":10,"name":"Wolfgang G. Kunz","email":"","orcid":"","institution":"University Hospital Munich: LMU Klinikum","correspondingAuthor":false,"prefix":"","firstName":"Wolfgang","middleName":"G.","lastName":"Kunz","suffix":""},{"id":421726839,"identity":"14a70d9a-a388-42f6-a87a-36c854be34db","order_by":11,"name":"Dirk-Andre Clevert","email":"","orcid":"","institution":"University Hospital Munich: LMU Klinikum","correspondingAuthor":false,"prefix":"","firstName":"Dirk-Andre","middleName":"","lastName":"Clevert","suffix":""},{"id":421726840,"identity":"2f3c7a5e-712b-47c0-ad09-04cac2ac6daa","order_by":12,"name":"Maurice M. Heimer","email":"","orcid":"","institution":"University Hospital Munich: LMU Klinikum","correspondingAuthor":false,"prefix":"","firstName":"Maurice","middleName":"M.","lastName":"Heimer","suffix":""},{"id":421726841,"identity":"d9335c97-b65b-4396-aa30-6c4f7d6ab6c9","order_by":13,"name":"Clemens C. Cyran","email":"","orcid":"","institution":"University Hospital Munich: LMU Klinikum","correspondingAuthor":false,"prefix":"","firstName":"Clemens","middleName":"C.","lastName":"Cyran","suffix":""}],"badges":[],"createdAt":"2025-02-12 17:31:00","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6017170/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6017170/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":77801663,"identity":"0ed98d7c-3817-4ee7-9dbe-93fb90428dfe","added_by":"auto","created_at":"2025-03-05 16:40:24","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":51815,"visible":true,"origin":"","legend":"\u003cp\u003eThe control group received a placebo, while the therapy group received combined anti-PD-L1/anti-CTLA-4 immunotherapy on days 7, 9 and 11 after inoculation. CEUS imaging and immunohistochemistry were conducted on days 7 and 12 after inoculation.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-6017170/v1/0995b5793f5d84719b981c9b.png"},{"id":77802051,"identity":"b40b3f7b-5e79-477f-aca8-3658e8b08e56","added_by":"auto","created_at":"2025-03-05 16:48:24","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":115862,"visible":true,"origin":"","legend":"\u003cp\u003eRepresentative CEUS images acquired seven days after tumour inoculation (baseline) showing tumour (green) and a blood volume parameter map with a ROI in a hypervascular vital tumour site of the outer rim (yellow) are shown. The corresponding signal-intensity-versus-time curves are displayed below.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-6017170/v1/7c8867093fd29b365631aaa4.png"},{"id":77802054,"identity":"b949426a-64fd-4a02-a6bb-f9700fc2255c","added_by":"auto","created_at":"2025-03-05 16:48:25","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":195689,"visible":true,"origin":"","legend":"\u003cp\u003eRepresentative CEUS images with BR55 of mice with a subcutaneous tumour allograft (arrows) under treatment. Therapy (blue) and control group (orange) at baseline and follow-up. Arrows indicating tumour allografts. Left side: early vascular phase as a functional imaging biomarker; right side: late phase demonstrating VEGFR2-specific binding as a molecular imaging biomarker, 10 minutes post-contrast injection. It is notable that the number of circulating MB observed in the early vascular phase was significantly lower in the therapy group compared to both the baseline and the control group. Similarly, signal enhancement in the late phase at follow-up was significantly lower in the therapy group compared to both the baseline and the control group.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-6017170/v1/4eb769d6d1a8f830fa52351f.png"},{"id":77801665,"identity":"793931e0-05e3-4821-8d2d-0dc3e839069d","added_by":"auto","created_at":"2025-03-05 16:40:24","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":57564,"visible":true,"origin":"","legend":"\u003cp\u003eColumn charts of WiAUC in therapy and control group at baseline and follow-up. WiAUC in arbitrary units of control (\u003cem\u003en\u003c/em\u003e = 8) and therapy group (\u003cem\u003en\u003c/em\u003e = 8) at baseline and follow-up. Note the significant difference (** p \u0026lt; 0.02) between the mean values of WiAUC between the therapy (blue) and the control group (red) at follow-up. There is also a significant (** p \u0026lt; 0.02) decline of WiAUC in therapy group between baseline and follow-up. Significantly (* p \u0026lt; 0.05) lower WiAUC values in the therapy compared to the control group at follow-up.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-6017170/v1/1e264e3a4bc34dc5b1ce0c4d.png"},{"id":77802052,"identity":"bea6d5f8-8cde-4375-bc4c-5c55846ec6f3","added_by":"auto","created_at":"2025-03-05 16:48:24","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":100921,"visible":true,"origin":"","legend":"\u003cp\u003eColumn charts of SI\u003csub\u003e8min \u003c/sub\u003eand SI\u003csub\u003e10min\u003c/sub\u003e in the therapy and control group at follow-up. Signal intensity 8 (left side) and 10 minutes post injection (right side) in arbitrary units of therapy (\u003cem\u003en\u003c/em\u003e = 8) and control (\u003cem\u003en\u003c/em\u003e = 8) group at baseline and follow-up. Note the significant difference (** p \u0026lt; 0.02) between the mean values of SI\u003csub\u003e8min \u003c/sub\u003eand SI\u003csub\u003e10min\u003c/sub\u003e between the therapy (blue) and the control group (red) at follow-up.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-6017170/v1/f7c5aa0e17e524c2e60fddbd.png"},{"id":77802860,"identity":"d4a234c0-419a-4229-81f7-f49561c543b1","added_by":"auto","created_at":"2025-03-05 16:56:24","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":268960,"visible":true,"origin":"","legend":"\u003cp\u003eCD8 (TILs) (A, B), CD31 (microvascular density) (C, D), Ki-67 (cell proliferation) (E, F), TUNEL (apoptosis) (G, H) and VEGFR2 (I, J) expression in control group (left column) and therapy group (right column). Note that the therapy group exhibited a significantly higher immune response and apoptosis rate, as well as a significantly lower microvascular density, tumour cell proliferation and VEGFR2 expression.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-6017170/v1/bb0a5980888a0a8f81f86bb2.png"},{"id":79149099,"identity":"f263bca1-886c-4179-9005-53b60422520a","added_by":"auto","created_at":"2025-03-25 03:53:35","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1588517,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6017170/v1/c376bbfd-8306-4083-ac78-bf8244bb1a68.pdf"},{"id":77802065,"identity":"53f3954a-2dee-425a-8ed1-540b10900de2","added_by":"auto","created_at":"2025-03-05 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16:40:25","extension":"tif","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":14745766,"visible":true,"origin":"","legend":"","description":"","filename":"KI67control.tif","url":"https://assets-eu.researchsquare.com/files/rs-6017170/v1/0f5ed15adf6e391ca5be5c5f.tif"},{"id":77801695,"identity":"d6865dcb-70bf-4737-81bf-c1150ec579fc","added_by":"auto","created_at":"2025-03-05 16:40:25","extension":"tif","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":14745766,"visible":true,"origin":"","legend":"","description":"","filename":"KI67therapy.tif","url":"https://assets-eu.researchsquare.com/files/rs-6017170/v1/796b0ef863e880c6b9179143.tif"},{"id":77801697,"identity":"87f1614c-560c-4d11-87ac-0023ca2205a9","added_by":"auto","created_at":"2025-03-05 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16:48:24","extension":"docx","order_by":11,"title":"","display":"","copyAsset":false,"role":"supplement","size":31238,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryInformation11022025.docx","url":"https://assets-eu.researchsquare.com/files/rs-6017170/v1/fb68c425cf29bb8806c78821.docx"}],"financialInterests":"","formattedTitle":"Contrast-enhanced ultrasound with VEGFR2-targeted microbubbles for monitoring combined anti-PD-L1/anti-CTLA-4 immunotherapy effects in a murine melanoma model with immunohistochemical validation","fulltext":[{"header":"Background","content":"\u003cp\u003eIn recent years, immune checkpoint inhibitors (ICIs) have emerged as a highly efficacious treatment option for patients with metastatic tumours, and have also been approved for use in melanoma (1). ICIs function by blocking the upregulated surface receptors of Cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) or programmed death-ligand 1 (PD-L1) (2). Given that not all patients respond to immunotherapy with ICI and its treatment is associated with adverse effects, imaging biomarkers need to be employed for accurate monitoring of early therapy response (3).\u003c/p\u003e \u003cp\u003eVascular endothelial growth factor (VEGF) is a principal regulator of angiogenesis (4). VEGF and its receptor VEGFR2 are increasingly expressed in solid tumours, such as melanoma, and play an important role in tumours angiogenesis, proliferation and metastasis (5\u0026ndash;7). Furthermore, VEGFR2 is associated with the development of resistance to anti-cancer drugs (8). A correlation between tumour immune microenvironment and VEGF(R-2) has already been described: The activation of VEGF(R-2) is associated with the development of an immunosuppressive microenvironment, by inducing the aggregation of immature dendritic cells, bone marrow-derived suppressor cells and regulatory T cells, and inhibiting T lymphocyte migration (9\u0026ndash;11). Moreover, VEGF(R-2) has been demonstrated to enhance PD-1 expression (9,12). Studies have shown that mutations of VEGFR2 are linked to better clinical outcomes for tumours under ICI (13). Additionally, VEGFR2 expression level can serve as an important indicator of how well patients will respond to therapies that block PD-1 and PD-L1(14). Further, immunotherapy can reduce the expression of VEGF, which helps counteract its immunosuppressive effects (15,16).\u003c/p\u003e \u003cp\u003eContrast-enhanced ultrasound (CEUS) utilising microbubbles (MB) that bind specifically to VEGFR2 enables the characterisation of functional and molecular parameters (17,18). The MBs are exclusively distributed intravascularly (19). Following intravenous injection, the perfusion of tumours can be quantitatively evaluated in an early phase by assessing various parameters of tissue microcirculation (perfusion). In a subsequent late phase, targeted MBs bound to tumour vascular endothelial cells, which overexpress VEGFR2, permit the non-invasive visualisation of VEGFR2 expression (20,21). Multiparametric CEUS with VEGFR2-targeted MB has demonstrated to be a highly sensitive tool for monitoring therapy effects in a range of tumour entities: In a mouse model of hepatocellular carcinoma, early therapeutic effects of the tyrosine kinase inhibitor sorafenib were analysed (22). Eschbach et al. has already used CEUS to analyse morphology, perfusion and VEGFR2-specific binding in colorectal adenocarcinoma in rats (23). Consequently, VEGFR2-targeted CEUS may provide functional and molecular imaging biomarkers for the assessment of tumour angiogenesis and early therapy response in murine melanoma allografts under immunotherapy.\u003c/p\u003e \u003cp\u003eTherefore, we hypothesised that CEUS with VEGFR2-targeted MB enables monitoring of early immunotherapy effects in a murine melanoma allograft tumour model. We aimed to investigate whether the functional and the molecular CEUS parameters have potential as non-invasive in vivo imaging biomarkers of early therapy response with time point-matched multiparametric immunohistochemical validation.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eAnimal model and experimental protocol\u003c/h2\u003e \u003cp\u003eAll animal experiments were conducted in accordance with the guidelines for the use of living animals in scientific studies and the animal study was approved by the Committee for Animal Research of the Government of Upper Bavaria (ROB-55.2-2532.Vet_02-19-32). Female C57BL/6 mice (mean weight\u0026thinsp;\u0026plusmn;\u0026thinsp;20 g, aged 10\u0026ndash;12 weeks at the time of inoculation, Charles River, Sulzfeld, Germany) were housed in groups of four per cage in a temperature- and humidity-controlled room with a 12 h light/dark cycle and provided with food and water. In accordance with the approved animal study proposal, animals were excluded from the study if they exhibited any of the following symptoms: a loss of body weight equal to or greater than 19% of the value observed prior to inoculation; a tumour size exceeding 1.5 cm; exulceration; infections or bleeding from the tumour area; bloody diarrhoea; apathy; ascites; or acneiform dermatitis.\u003c/p\u003e \u003cp\u003eThe murine melanoma cell line B16-F10 (ATCC CRL-6475) was cultured and expanded under standard conditions. After a one-week assimilation period, the melanoma cells (B16-F10; 3 x 10\u003csup\u003e5\u003c/sup\u003e cells) were resuspended in a 1:1 mixture of Matrigel (BD Biosciences, San Jose, CA, USA) and phosphate-buffered saline (PBS, pH 7.4). The tumour cells were injected subcutaneously into the left abdominal flank of C57BL/6 mice (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;20) under inhalative isoflurane anaesthesia, with 2.0 vol % isoflurane and 1.5 L/min oxygen. Once tumour diameter reached a maximum of 0.5 cm, animals were randomly assigned to either the therapy (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;10) or the control group (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;10) and baseline CEUS was conducted. The therapy group received a total of three intraperitoneal injections of anti-PD-L1 and anti-CTLA-4 antibodies (20 \u0026micro;g/kg) on days 7, 9 and 11 after inoculation. The control group received volume equivalent placebo. For imaging, mice were anaesthetised via continuous inhalation of 2.0 vol % isoflurane and 1.5 L/min oxygen and were placed on a heating pad. A multiparametric CEUS protocol was conducted on day 7 (baseline) and on day 12 (follow-up) following inoculation. Tumour allografts were explanted, fixed in formalin and preserved by cryoconservation at baseline and follow-up for multiparametric ex vivo immunohistochemical work-up (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eMB\u003c/h3\u003e\n\u003cp\u003eBR55 (Bracco Suisse SA, Geneva, Switzerland) is an ultrasound molecularly targeted lipid-shelled microbubble that targets VEGFR2. The lipopeptide that binds to VEGFR2 is created by conjugating a heterodimer peptide to the amino group of DSPE-PEG2000-NH2, which is then incorporated into the phospholipid-based MB formulation. The final product, BR55, is provided as a lyophilized powder in a septum-sealed vial, with the gas phase consisting of a mixture of perfluorobutane and nitrogen (24\u0026ndash;26). Prior to utilisation, BR55 must be reconstituted by the injection of 2 ml of a 5% glucose solution into the vial via the septum. Once dissolved, the resulting MB suspension is ready for utilisation. The mean diameter of the BR55 MBs is 1.5 \u0026micro;m, with a concentration of approximately 2 x 10⁹ MB per mL. Each MB contains approximately 4 x 10⁵ lipopeptide molecules (24,27). The circulation time of BR55 is approximately 4 minutes (21). The administration of BR55 to each animal was conducted via a standardised manual intravenous injection through a tail vein catheter, with a dose of 200 \u0026micro;L administered.\u003c/p\u003e\n\u003ch3\u003eCEUS Imaging\u003c/h3\u003e\n\u003cp\u003eCEUS imaging was conducted using a clinical ultrasound system (Philips Epiq 7, Seattle, WA) with a conventional 18L4 linear transducer (transmit frequency 9 MHz, dynamic range 55 dB, depth 25 mm), linear time-gain compensation, and an acoustic focus positioned at the largest tumour cross-section, with the contrast setting adjusted to the image button [35]. The anaesthetised mice were positioned on their sides, and ultrasound coupling gel was applied to the shaved skin. An initial B-mode scan was conducted to visualise the subcutaneous tumour expansion. Video acquisition commenced immediately prior to the administration of BR55, continued for the initial minute, and then resumed at two-minute intervals for a period of up to ten minutes post-injection (at a frame rate of 16 Hz). This enabled the measurement of MB wash-in and wash-out in the tumour. The binding of the VEGFR2-specific agent was evaluated at 8 and 10 minutes after the injection of BR55, when the circulating MB had largely cleared from the bloodstream, while the tumour tissue remained highlighted, indicating the specific accumulation of targeted MB on the VEGFR2-expressing endothelium.\u003c/p\u003e\n\u003ch3\u003eData post processing\u003c/h3\u003e\n\u003cp\u003eThe CEUS data were processed using dedicated software on an external workstation with Vuebox\u0026reg; (Version v7.5.0.7051\u0026ndash;64-bit version - Bracco Suisse SA, Geneva, Switzerland). A region of interest (ROI) was delineated within a highly perfused area of the tumour's vital outer rim without necrotic areas. The software quantifies contrast enhancement in the ROI, presenting results as relative echo-power values that correspond to MB concentration, measured in arbitrary units (a.u.) (20,22,24) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The key parameters analysed included the wash-in area under the curve (WiAUC) during the early vascular phase, which provides insights into blood flow and volume (28,29), as well as signal intensity measured at 8 minutes (SI\u003csub\u003e8min\u003c/sub\u003e) and 10 minutes (SI\u003csub\u003e10min\u003c/sub\u003e) post-injection, which serve as surrogate indicators of VEGFR2-specific MB binding in the late molecular phase. In addition, the size of the tumour was measured in two dimensions.\u003c/p\u003e \n\u003ch3\u003eImmunohistochemistry\u003c/h3\u003e\n\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eCD31\u003c/h2\u003e \u003cp\u003eFirst deparaffinization, rehydration and antigen retrieval (SignalStain\u0026reg; EDTA-Unmasking Solution, Cell signaling Technology, Leiden, Netherlands) was performed. For immunohistochemical assessment of tumour microvascular density, non-specific binding sides were blocked in 5% Donkey-Serum in TBS-Tween20. Subsequently tumours slices were incubated with a polyclonal rabbit anti-CD31 primary antibody (1:50; Abcam ab28364, Cambridge, UK) overnight. Tissue samples underwent further processing utilising secondary antibodies conjugated with Alexa Fluor 488 (1:200; ab150073, Abcam Limited, Cambridge, UK), to facilitate fluorescence detection. Counterstaining was conducted using DAPI (Carl Roth, Karlsruhe, Germany) and the slides were covered with Fluoromount-G (ThermoFisher Scientific, Waltham, Massachusetts, USA). The number of tumour microvessels was quantified as the mean of the endothelial cells in 10 random fields at 200x magnification, as previously described (30).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eCD8 antigen staining\u003c/h3\u003e\n\u003cp\u003eThe tissue slides were initially deparaffinised and rehydrated, followed by permeabilization with a solution of 1x PBS (Gibco, Carlsbad, CA) and 0.25% Triton-X-100 (Merck KGaA, Darmstadt, Germany). Subsequently, antigen retrieval was conducted by boiling the tissue slides in a 10 mM Tris-EDTA buffer for CD8 (Life Technologies, Carlsbad, CA). Following the retrieval of antigens, thorough washing with 1x PBS was conducted to ensure the removal of residual buffers. Non-specific binding sites were blocked with a solution containing bovine serum albumin (BSA) and primary antibodies against CD8 (1:50; ab217344, Abcam Limited, Cambridge, UK) were applied. Following an overnight incubation period and additional washing steps, secondary antibodies conjugated with Alexa Fluor 488 (1:200; ab150073, Abcam Limited, Cambridge, UK) were applied to facilitate fluorescence detection. Counterstaining with DAPI enabled the visualisation of nuclei. Finally, slides were mounted using Fluoromount-G, ensuring the preservation and optimal visualisation of the stained specimens. Results were quantified as the mean percentage of CD8-positive T-cells in 10 randomly selected fields at 200x magnification.\u003c/p\u003e\n\u003ch3\u003eKi-67 antigen staining\u003c/h3\u003e\n\u003cp\u003eA Ki-67-specific monoclonal rabbit anti-human antibody (1:50; SP6, Thermo Fisher MA5-14420, Thermo Fisher) was employed for the purpose of quantifying tumour cell proliferation. The tissue was de-masked in Universal antigen retrival reagent (ab208572, Abcam Limited, Cambridge, UK) using microwave irradiation at 600 W. Following a wash in distilled water and TBS-Tween (0.05%), secondary antibodies conjugated with Alexa Fluor 488 (1:200; ab150073, Abcam Limited, Cambridge, UK) were applied to facilitate fluorescence detection. Subsequently, counterstaining was conducted using DAPI (Carl Roth, Karlsruhe, Germany) and slides were covered with Fluoromount-G (ThermoFisher Scientific, Waltham, Massachusetts, USA). Results were quantified as the mean percentage of proliferating cells in 10 random fields at 200x magnification.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eTerminal desoxynucleotidyl transferase dUTP nick-end labelling (TUNEL)\u003c/h2\u003e \u003cp\u003eThe initial step involved the deparaffinisation and rehydration of the tissue slides, which were then permeabilised using a solution comprising Triton X-100 (Merck KGaA, Darmstadt, Germany) and sodium citrate (Sigma-Aldrich, Steinheim, Germany). Subsequently, antigen retrival reagent (ab208572, Abcam Limited, Cambridge, UK) was conducted through microwave treatment. The staining was conducted by combining solutions from the provided kit, with meticulous attention to maintaining darkness and temperature control. Following comprehensive washing steps, slides were further stained with DAPI to visualise nuclei. Ultimately, slides were mounted using Fluoromount-G (ThermoFisher Scientific, Waltham, Massachusetts, USA) ensuring the preservation of the stained samples for microscopic examination. Results were quantified as the mean percentage of apoptotic cells in 10 random fields at 200x magnification.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eVEGFR2\u003c/h2\u003e \u003cp\u003eThe expression of VEGFR2 was analysed using a VEGFR2-specific monoclonal rabbit anti-human antibody (1:50; Cell Signalling, Cambridge, UK). Following standard procedures, the tissue samples were dewaxed, rehydrated and subsequently demasked in target-retrieval solution (SignalStain EDTA Unmasking Solution, Cell Signalling, Cambridge, UK) using microwave irradiation at 600 W. After an overnight incubation period with the primary antibody, the tissue samples were further processed using secondary antibodies conjugated with Alexa Fluor 488 (1:200; ab150073, Abcam Limited, Cambridge, UK) and counterstaining was conducted using DAPI (Carl Roth, Karlsruhe, Germany). The number of tumour vessels stained positively for VEGFR2 was quantified at a magnification of 200x, with the analysis conducted on 10 random high-power fields.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eContinuous variables are presented as means with standard deviations (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD). For intergroup comparisons between treatment and control group the Mann-Whitney U-test was employed. A Wilcoxon signed-rank test was used for intragroup comparisons of CEUS parameters between baseline (day 7) and follow-up (day 12). P-values\u0026thinsp;\u0026lt;\u0026thinsp;0.05 were considered statistically significant. All statistical analyses were performed using GraphPad Prism (Graphpad Software, Inc., Boston, MA) and Microsoft Excel (Microsoft Corporation, Redmond, WA).\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003eThe experimental protocol, including imaging with CEUS, was successfully completed in \u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;16 animals; in \u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;3 animals the ultrasound could not be accomplished due to technical issues, \u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;1 animal of the therapy group had to be excluded due to anaesthesia complications.\u003c/p\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eTumour size\u003c/h2\u003e \u003cp\u003eTumour size was assessed at baseline (7 days after inoculation) and follow-up (12 days after inoculation). For calliper measurements in two dimensions there were no significant differences in mean tumour sizes between the therapy and the control group at baseline (29.9\u0026thinsp;\u0026plusmn;\u0026thinsp;12.7 mm\u003csup\u003e2\u003c/sup\u003e vs. 32.1\u0026thinsp;\u0026plusmn;\u0026thinsp;17.3 mm\u003csup\u003e2\u003c/sup\u003e; p\u0026thinsp;=\u0026thinsp;0.909) and follow-up (104.2\u0026thinsp;\u0026plusmn;\u0026thinsp;35.3 mm\u003csup\u003e2\u003c/sup\u003e vs. 123.0\u0026thinsp;\u0026plusmn;\u0026thinsp;66.1 mm\u003csup\u003e2\u003c/sup\u003e; p\u0026thinsp;=\u0026thinsp;0.733). There were significant changes in tumour size between baseline and follow-up in the therapy (29.9\u0026thinsp;\u0026plusmn;\u0026thinsp;12.7 mm\u003csup\u003e2\u003c/sup\u003e vs. 104.2\u0026thinsp;\u0026plusmn;\u0026thinsp;35.3 mm\u003csup\u003e2\u003c/sup\u003e; p\u0026thinsp;=\u0026thinsp;0.006) and in the control group (32.1\u0026thinsp;\u0026plusmn;\u0026thinsp;17.3 mm\u003csup\u003e2\u003c/sup\u003e vs. 123.0\u0026thinsp;\u0026plusmn;\u0026thinsp;66.1 mm\u003csup\u003e2\u003c/sup\u003e; p\u0026thinsp;=\u0026thinsp;0.002, supplemental table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). For CEUS measurements in two dimensions there were no significant differences in mean tumour sizes between the therapy and the control group at baseline (21.3\u0026thinsp;\u0026plusmn;\u0026thinsp;13.2 mm\u003csup\u003e2\u003c/sup\u003e vs. 17.4\u0026thinsp;\u0026plusmn;\u0026thinsp;19.8 mm\u003csup\u003e2\u003c/sup\u003e; p\u0026thinsp;=\u0026thinsp;0.173) and follow-up (60.2\u0026thinsp;\u0026plusmn;\u0026thinsp;28.1 mm\u003csup\u003e2\u003c/sup\u003e vs. 63.2\u0026thinsp;\u0026plusmn;\u0026thinsp;48.6 mm\u003csup\u003e2\u003c/sup\u003e; p\u0026thinsp;=\u0026thinsp;1). There were significant increases in tumour size between baseline and follow-up in the therapy (21.3\u0026thinsp;\u0026plusmn;\u0026thinsp;13.2 mm\u003csup\u003e2\u003c/sup\u003e vs. 60.2\u0026thinsp;\u0026plusmn;\u0026thinsp;28.1 mm\u003csup\u003e2\u003c/sup\u003e; p\u0026thinsp;=\u0026thinsp;0.004) and in the control group (17.4\u0026thinsp;\u0026plusmn;\u0026thinsp;19.8 mm\u003csup\u003e2\u003c/sup\u003e vs. 63.2\u0026thinsp;\u0026plusmn;\u0026thinsp;48.6 mm\u003csup\u003e2\u003c/sup\u003e; p\u0026thinsp;=\u0026thinsp;0.002, supplemental table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eCEUS\u003c/h2\u003e \u003cp\u003eUsing BR55-CEUS, functional parameters of tumour perfusion were assessed during an early vascular phase and VEGFR2-specific binding of the MB during a late molecular phase (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn the therapy group, a significant decrease of tumour perfusion (WiAUC) was observed following the one-week treatment course with combined anti-PD-L1/anti-CTLA-4 immunotherapy (23767\u0026thinsp;\u0026plusmn;\u0026thinsp;16937 a.u. at baseline to 8054\u0026thinsp;\u0026plusmn;\u0026thinsp;10083 a.u. at follow-up; p\u0026thinsp;=\u0026thinsp;0.008). Also, in the control group significant changes in WiAUC (from 21744\u0026thinsp;\u0026plusmn;\u0026thinsp;17585 a.u. at baseline to 16402\u0026thinsp;\u0026plusmn;\u0026thinsp;14813 a.u. at follow-up; p\u0026thinsp;=\u0026thinsp;0.008) were noted. Moreover, WiAUC was significantly (p\u0026thinsp;=\u0026thinsp;0.021) lower in the therapy compared to the control group at follow-up (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e and supplemental table \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn the late molecular phase, a significant decline in tumour signal intensity between baseline and follow-up was observed in the therapy group (SI\u003csub\u003e8min\u003c/sub\u003e: from 517.7\u0026thinsp;\u0026plusmn;\u0026thinsp;103.7 to 220.9\u0026thinsp;\u0026plusmn;\u0026thinsp;62.6 a.u., p\u0026thinsp;=\u0026thinsp;0.008; SI\u003csub\u003e10min\u003c/sub\u003e: from 419.7\u0026thinsp;\u0026plusmn;\u0026thinsp;90.4 to 181.9\u0026thinsp;\u0026plusmn;\u0026thinsp;48.4 a.u., p\u0026thinsp;=\u0026thinsp;0.008) and in the control group (SI\u003csub\u003e8min\u003c/sub\u003e: from 459.8\u0026thinsp;\u0026plusmn;\u0026thinsp;51.5 to 350.7\u0026thinsp;\u0026plusmn;\u0026thinsp;43.3 a.u., p p\u0026thinsp;=\u0026thinsp;0.008; SI\u003csub\u003e10min\u003c/sub\u003e: from 400.3\u0026thinsp;\u0026plusmn;\u0026thinsp;59 to 281.5\u0026thinsp;\u0026plusmn;\u0026thinsp;37.3 a.u., p\u0026thinsp;=\u0026thinsp;0.008). However, significantly lower SI\u003csub\u003e8min\u003c/sub\u003e (220.9\u0026thinsp;\u0026plusmn;\u0026thinsp;62.6 vs. 350.7\u0026thinsp;\u0026plusmn;\u0026thinsp;43.3 a.u., p\u0026thinsp;=\u0026thinsp;0.003) and SI\u003csub\u003e10min\u003c/sub\u003e (181.9\u0026thinsp;\u0026plusmn;\u0026thinsp;48.4 vs. 281.5\u0026thinsp;\u0026plusmn;\u0026thinsp;37.3 a.u., p\u0026thinsp;=\u0026thinsp;0.002) values were detected in the therapy than in the control group at follow-up (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e and supplemental table \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eImmunohistochemistry\u003c/h2\u003e \u003cp\u003eEx vivo analysis revealed significant anti-tumour effects in the therapy (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8) compared to the control group (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8). Compared to the control group, the tumour samples of the therapy group had a significantly higher percentage of apoptotic cells (TUNEL 52.3\u0026thinsp;\u0026plusmn;\u0026thinsp;17.2% vs. 17.1\u0026thinsp;\u0026plusmn;\u0026thinsp;5.6%; p\u0026thinsp;=\u0026thinsp;0.001) at follow-up. Furthermore, the therapy group showed a significantly higher number of TILs than the control group (CD8 395.4\u0026thinsp;\u0026plusmn;\u0026thinsp;522.5 vs. 71.9\u0026thinsp;\u0026plusmn;\u0026thinsp;55.6; p\u0026thinsp;=\u0026thinsp;0.049) at follow-up. At follow-up, tumour cell proliferation was significantly lower in the therapy group than in the control group (Ki-67 27.2\u0026thinsp;\u0026plusmn;\u0026thinsp;9.7% vs. 58.7\u0026thinsp;\u0026plusmn;\u0026thinsp;8.6%; p\u0026thinsp;=\u0026thinsp;0.001). At follow-up, anti-angiogenic effects of combined anti-PD-L1/anti-CTLA-4 immunotherapy were observed in the investigated melanoma allografts with a significantly lower microvascular density in combined anti-PD-L1/anti-CTLA-4-treated than in non-treated animals (CD31 142.1\u0026thinsp;\u0026plusmn;\u0026thinsp;59.8 vs. 286.3\u0026thinsp;\u0026plusmn;\u0026thinsp;88.1; p\u0026thinsp;=\u0026thinsp;0.003), quantified by CD31 staining. VEGFR2 staining showed a significantly lower VEGFR2 expression in combined anti-PD-L1/anti-CTLA-4-treated than in placebo-treated animals (VEGFR2 72.5\u0026thinsp;\u0026plusmn;\u0026thinsp;18.6 vs. 119\u0026thinsp;\u0026plusmn;\u0026thinsp;29.6; p\u0026thinsp;=\u0026thinsp;0.003, Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eDue to severe adverse events and the fact that not all patients respond to ICI, it is necessary to evaluate non-invasive in vivo imaging biomarkers of early therapy response in order to provide effective therapy guidance (31). Preclinical and clinical studies have investigated the therapy effects, particularly under anti-angiogenic drugs, in melanoma using CEUS (32,33). However, imaging biomarkers of early therapy response under combined immunotherapy in melanoma are still scarce and require further investigation to ascertain the feasibility of clinical use.\u003c/p\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eTumour size under ICI therapy\u003c/h2\u003e \u003cp\u003eTumour size, measured using callipers or CEUS, did not show a significant difference between the therapy and control group, but morphological assessment of tumours does not reveal early physiological changes in tumours (33). This finding is in accordance with Mellinger (32), who observed no difference in tumour size but a decrease in tumour perfusion under immunotherapy in melanoma xenografts. In contrast to us, Mellinger (32) used one type of antibody (anti-PD1) for checkpoint inhibition, and the therapy was administered twice a week within a 18-day period. This supports the observation that immunotherapy does not affect tumour size significantly at early follow-up.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eTumour perfusion under ICI therapy\u003c/h2\u003e \u003cp\u003eA significantly higher reduction of WiAUC at follow-up was observed in the therapy compared to the control group. This is in accordance with Eschbach et al. (23) who demonstrated a significant reduction in WiAUC under regorafenib for colorectal carcinoma xenografts. However, their analysis was conducted following a one-week daily treatment of regorafenib. Regorafenib is an multi-tyrosine kinase inhibitor with a distinct mechanism of action that demonstrated anti-angiogenic and anti-proliferative effects in vivo in different experimental tumour models, including breast cancer, renal cell carcinoma and glioblastoma (34). The significant decrease of the CEUS perfusion parameter WiAUC was consistent with the immunohistochemical CD31 staining, that demonstrated significantly lower microvascular density. This finding is in accordance with Brloznik et al. (35), who were able to correlate a reduction of tumour perfusion in CEUS with tumour histological analyses of CD31 in a murine melanoma model. Differing from our study Brloznik et al. treated the mice with gene electrotransfer and radiotherapy. According to our results, the early vascular CEUS parameter WiAUC indicates to serve as functional perfusion biomarker of early therapy response under combined anti-PD-L1/anti-CTLA-4 immunotherapy in melanoma.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eICI therapy effects on VEGFR-2 expression\u003c/h2\u003e \u003cp\u003eRegarding the evaluation of VEGFR2-targeted microbubbles in the late molecular phase, the treatment group exhibited significantly lower SI\u003csub\u003e8min\u003c/sub\u003e and SI\u003csub\u003e10min\u003c/sub\u003e values at follow-up. This is in line with Baetke et al. who demonstrated a significant reduction in the amount of VEGFR2-targeted MB in heterotopic squamous cell carcinoma xenografts in mice undergoing anti-VEGF antibody therapy (21). In a murine colorectal carcinoma model anti-angiogenic treatment with sunitinib resulted in decreased tumour perfusion and VEGFR2 expression. In contrast to us, they observed treatment effects already 24 hours post-treatment (36). In accordance with SI\u003csub\u003e8min\u003c/sub\u003e and SI\u003csub\u003e10min\u003c/sub\u003e parameters, immunohistochemical measures demonstrated a significant decline in VEGFR2. This is in line with Palmowski et al. (37), who demonstrated, complementary to CEUS, a significantly lower stained area fraction for VEGFR2 in immunohistochemical analysis of treated tumours. Differing from our study, Palmowski et al. investigated squamous cell carcinoma xenografts under a matrix metalloproteinase inhibitor. The necrotic tumour areas observed in our study, similar to Eschbach et al. (23), are responsible for the reduction in effective vital tumour mass in rapidly growing tumours, which could explain the transient reduction in vascular proliferation and VEGFR2 expression with decreased BR55 binding (22). It can be suggested that the development of necrosis may be a confounding factor contributing to the decreased differential targeted enhancement observed in treated melanoma allografts. Therefore, we selected ROIs over the vital outer rim of therapy and control tumours to avoid areas of necrosis and elevated interstitial pressure. The significantly lower number of MB on the tumour endothelial surface following combined immunotherapy most likely reflects the significantly higher decrease of tumour perfusion and VEGFR2 expression compared to the control group.\u003c/p\u003e \u003cp\u003eIn our experimental setup, the observed reduction of specifically bound MB with VEGFR2-targeted CEUS under anti-PD-L1/anti-CTLA-4 immunotherapy of malignant melanoma may be explained by the pathophysiological connection between VEGF and tumour immunology. The ex vivo validation at follow-up demonstrated a significantly elevated number of TILs and apoptotic cells, as well as a significantly lower number of proliferating cells within the therapy group. These findings indicate the presence of an antitumor immune response under combined anti-PD-L1/anti-CTLA-4 therapy. VEGF via its receptor VEGFR2 contributes to the exhaustion of CD8\u0026thinsp;+\u0026thinsp;T cells, which is characterised by the expression of negative immune checkpoints, such as PD-1 receptors (38). In this context, VEGF promotes the expression of checkpoint molecules(9). Immune checkpoint therapy regulates endothelial cell functions and reverses the immunosuppressive effects of VEGF in metastatic melanoma by decreasing intratumoral VEGF(R2) expression and consecutively activating CD8\u0026thinsp;+\u0026thinsp;T cells via the IFNγ signalling pathway (15,16,39). The observed combination of pronounced pro-immunogenic, pro-apoptotic, anti-proliferative and anti-VEGFR2 effects in the immunohistochemistry in our study underscores the effectiveness of the combined immunotherapy and the pathophysiological link between VEGF(R2) and tumour immunology.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eLimitations\u003c/h2\u003e \u003cp\u003eThe results of our study are limited. Firstly, the investigation was conducted on a single tumour-therapy combination within an allograft model of melanoma. Secondly, the observation period was limited to six days with three doses of therapy. Additionally, CEUS imaging was conducted only until day 12 following tumour inoculation, which was constrained by the dynamics of tumour growth. An extension of the observational period and prolongation of the duration of therapy administration may result in not only further inhibition of tumour growth but also a reduction in tumour size, which is not captured in this study.\u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThis study offers insights into early immunotherapy response in a murine melanoma model, providing time matched correlation of in vivo and ex vivo biomarkers of therapy response. The significant reduction of tumour perfusion and VEGFR2-targeted MB under combined immunotherapy assessed by CEUS were paralleled by significant pro-immunogenic, pro-apoptotic, anti-angiogenic and anti-proliferative effects in immunohistochemistry. Our findings demonstrate that CEUS parameters have potential to serve as non-invasive imaging biomarkers for immunotherapy response assessment in melanoma.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by a research grant from Bracco Imaging SA, Geneva, Switzerland, which provided the BR55 free of charge. The authors of this manuscript have no other relationships with these companies, whose products or services may be related to the subject matter of this article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors contributed to the study conception and design. Material preparation, data collection, and analysis were performed by FLH, MJA, LVB, HHE, SKL, AC, JS, TB, DAC, MMH, IT, CCC. The first draft of the manuscript was written by FLH, LVB, HHE, MMH and CCC and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript. \u0026nbsp;All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and material\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll animal experiments were conducted in accordance with the guidelines for the use of living animals in scientific studies and the animal study was officially approved by the Committee for Animal Research of the Government of Upper Bavaria (ROB-55.2-2532.Vet_02-19-32). The authors have ensured compliance with the ARRIVE guidelines. Clinical trial number: not applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements \u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by a research grant from Bracco Imaging SA, Geneva, Switzerland, which provided BR55 free of charge.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eShiravand Y, Khodadadi F, Kashani SMA, Hosseini-Fard SR, Hosseini S, Sadeghirad H, u. a. Immune Checkpoint Inhibitors in Cancer Therapy. Curr Oncol Tor Ont. 24. April 2022;29(5):3044\u0026ndash;60.\u003c/li\u003e\n\u003cli\u003eLjunggren HG, Jonsson R, H\u0026ouml;glund P. Seminal immunologic discoveries with direct clinical implications: The 2018 Nobel Prize in Physiology or Medicine honours discoveries in cancer immunotherapy. Scand J Immunol. 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Correlation of perfusion MRI and 18F-FDG PET imaging biomarkers for monitoring regorafenib therapy in experimental colon carcinomas with immunohistochemical validation. PloS One. 2015;10(2):e0115543.\u003c/li\u003e\n\u003cli\u003eBrloznik M, Boc N, Cemazar M, Bosnjak M, Savarin M, Kejzar N, u. a. Contrast-enhanced ultrasound for evaluation of tumor perfusion and outcome following treatment in a murine melanoma model. Bioelectrochemistry Amst Neth. Dezember 2021;142:107932.\u003c/li\u003e\n\u003cli\u003ePayen T, Dizeux A, Baldini C, Le Guillou-Buffello D, Lamuraglia M, Comperat E, u. a. VEGFR2-Targeted Contrast-Enhanced Ultrasound to Distinguish between Two Anti-Angiogenic Treatments. Ultrasound Med Biol. August 2015;41(8):2202\u0026ndash;11.\u003c/li\u003e\n\u003cli\u003ePalmowski M, Huppert J, Ladewig G, Hauff P, Reinhardt M, Mueller MM, u. a. Molecular profiling of angiogenesis with targeted ultrasound imaging: early assessment of antiangiogenic therapy effects. Mol Cancer Ther. Januar 2008;7(1):101\u0026ndash;9.\u003c/li\u003e\n\u003cli\u003eKim CG, Jang M, Kim Y, Leem G, Kim KH, Lee H, u. a. VEGF-A drives TOX-dependent T cell exhaustion in anti-PD-1-resistant microsatellite stable colorectal cancers. Sci Immunol. 8. November 2019;4(41):eaay0555.\u003c/li\u003e\n\u003cli\u003eAmersfoort J, Eelen G, Carmeliet P. Immunomodulation by endothelial cells - partnering up with the immune system? Nat Rev Immunol. September 2022;22(9):576\u0026ndash;88.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"VEGFR2, CEUS, immunotherapy, melanoma","lastPublishedDoi":"10.21203/rs.3.rs-6017170/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6017170/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eImmune checkpoint inhibitors (ICIs) have emerged as a highly effective treatment option for patients with metastatic melanoma. As not all patients respond to ICI immunotherapy imaging biomarkers must be used to accurately monitor early response to therapy. Therefore, the aim of this study was to evaluate contrast-enhanced ultrasound (CEUS) with VEGFR2-targeted microbubbles for monitoring the effects of combined anti-PD-L1/anti-CTLA-4 immunotherapy in a murine melanoma model.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eMurine melanoma allografts (B16-F10) were implanted subcutaneously in \u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;10 therapy and \u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;10 control female C57BL/6 mice. CEUS with VEGFR2-targeted microbubbles was performed on day 7 and 12. The therapy group received 3 intraperitoneal injections on days 7, 9, 11 of combined anti-PD-L1/anti-CTLA-4 immunotherapy, the control group received a placebo. CEUS assessed tumour perfusion during an early vascular phase (wash-in area under the curve\u0026thinsp;=\u0026thinsp;WiAUC) and VEGFR2-specific binding during a late molecular phase (signal intensity at 8 minutes (SI\u003csub\u003e8min\u003c/sub\u003e) and 10 minutes (SI\u003csub\u003e10min\u003c/sub\u003e)). For pathophysiological validation immunohistochemistry was performed.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eAt follow-up, the CEUS perfusion parameter WiAUC demonstrated a significantly higher decrease in the therapy than in the control group (p\u0026thinsp;=\u0026thinsp;0.021). At follow-up, the signal enhancement in the late phase was significantly lower in the therapy than in the control group (SI\u003csub\u003e8min\u003c/sub\u003e p\u0026thinsp;=\u0026thinsp;0.003; SI\u003csub\u003e10min\u003c/sub\u003e p\u0026thinsp;=\u0026thinsp;0.002). Immunohistochemistry revealed significantly more apoptotic tumour cells (p\u0026thinsp;=\u0026thinsp;0.001), more tumour infiltrating lymphocytes (p\u0026thinsp;=\u0026thinsp;0.049), lower tumour cell proliferation (p\u0026thinsp;=\u0026thinsp;0.001), lower microvascular density (p\u0026thinsp;=\u0026thinsp;0.003) and lower VEGFR2 expression (p\u0026thinsp;=\u0026thinsp;0.003) in the therapy than in the control group.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eCEUS with VEGFR2-targeted microbubbles allowed for monitoring early treatment effects of a combined anti-PD-L1/anti-CTLA-4 immunotherapy on melanoma allografts with significantly lower tumour perfusion and significantly lower binding of VEGFR2-targeted microbubbles in the therapy than in the control group.\u003c/p\u003e","manuscriptTitle":"Contrast-enhanced ultrasound with VEGFR2-targeted microbubbles for monitoring combined anti-PD-L1/anti-CTLA-4 immunotherapy effects in a murine melanoma model with immunohistochemical validation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-03-05 16:40:19","doi":"10.21203/rs.3.rs-6017170/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"7278b8f8-ab0d-4a47-b081-bbb866be3f75","owner":[],"postedDate":"March 5th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-03-25T03:45:28+00:00","versionOfRecord":[],"versionCreatedAt":"2025-03-05 16:40:19","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6017170","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6017170","identity":"rs-6017170","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}