Bevacizumab delivery mediated by ultrasound-targeted microbubble destruction inhibited tumor growth and angiogenesis in lung cancer xenografts

Bevacizumab delivery mediated by ultrasound-targeted microbubble destruction inhibited tumor growth and angiogenesis in lung cancer xenografts | 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 Bevacizumab delivery mediated by ultrasound-targeted microbubble destruction inhibited tumor growth and angiogenesis in lung cancer xenografts Yu Yao, Ting Xu, Yuxuan Wen, Jing Luo, Yanling Lv, Wenfang Jin, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6286565/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 In this study, we aimed to investigate the influence of ultrasound-targeted microbubble destruction on the anticancer effect of Bevacizumab in a mouse model of lung cancer. A xenograft mouse tumor model was established by subcutaneously injecting Lewis Lung-GFP cells in the flank of nude mice. 15 nude mice bearing subcutaneous tumors were randomly divided into 3 groups. Group A served as a control group without any treatment (Control). Group B received Bevacizumab and administrations of ultrasound contrast agents and sham cavitation treatment (Bevacizumab + sham cavitation). Group C received Bevacizumab combined with ultrasound-targeted microbubble destruction (Bevacizumab +UTMD). Each group was given corresponding treatment once a day for consecutive 3 days. The tumor microvessel density (MVD) was calculated with Contrast-enhanced ultrasound imaging (CEUS) by vascular length and tumor surface area. Tumor weight was measured by scales after resection. Hematoxylin and eosin staining of samples were performed and the expression of CD34 was detected by immunohistochemistry. Results indicated that the tumor size (volume and weight) of group B and group C were smaller than group A (p<0.05) after treatment, and group C was more effective in inhibiting tumor growth than group B (p<0.05). The MVD and CD34 expression of group B and group C were lower than Group A (p<0.05) after treatment, and group C was more significant in the inhibition of angiogenesis than group B(p<0.05). In conclusion, Bevacizumab could inhibit tumor growth and angiogenesis in lung cancer xenografts, and ultrasound-targeted microbubble destruction significantly enhanced the antitumor effect of Bevacizumab. UTMD Bevacizumab lung cancer angiogenesis CD34 Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Ultrasound is emerging as a highly effective, low-cost and truly noninvasive tool for therapy, although it has traditionally been perceived as a tool for diagnostic imaging[1, 2]. One of major physical effects of ultrasound is cavitation, which is the response of gas bubbles in liquid after oscillating to an acoustic field[3]. In the presence of large number of exogenous cavitation nuclei, for instance, lipid microbubbles injected into circulation, inertial cavitation might be induced, which can cause sonoporation or capillary damage[4, 5]. This process leads to hemorrhage, edema, thrombus formation, and wear to the endothelium of capillaries or small vessels in various tissues, which can induce necrosis and apoptosis of cancer cells and inhibit tumor growth[6, 7]. There are several reports indicated that ultrasonic cavitation is an effective application for tumor vasculature destruction or antitumor effects[8-10]. The use of targeted microbubbles may enable concentration of the treatment at the desired location on activation with ultrasound[11, 12], and multiple kinds of targeting ligands have been conjugated to the surface of microbubbles to achieve site-specific accumulation[13]. Lung cancer is the most common malignancy and the leading cause of cancer-related death worldwide[14]. As the main subtype of lung cancer, non-small cell lung cancer (NSCLC) occupies approximately 85% of all lung cancer cases[15]. In the past decades, the advent of various targeted therapies meaningfully improved clinical outcomes of patients with advanced NSCLC[16]. Bevacizumab is a humanized anti-VEGF monoclonal IgG 1 antibody, and it is the first angiogenesis inhibitor approved for the treatment of patients with advanced NSCLC in combination with chemotherapy[17]. In the present study, we investigated an ultrasonic cavitation strategy that targeted tumor angiogenesis with Bevacizumab-loaded microbubble and released its payload on focal ultrasound treatment for anti-tumor efficacy. The efficacy of this strategy was evaluated in a lung cancer model using real-time quantitative CEUS and in vivo fluorescence imaging. Materials and methods Animal care 15 BALB/C male nude mice, aged 4–6 weeks and weighted 20–25 g, were purchased from Beijing Kelihua laboratory animal center (Beijing, P.R. China). All mice were maintained in a HEPA-filtered environment at 25 ℃ and humidity was maintained at 50–60%. All animals were fed with autoclaved laboratory rodent diet. All animal experiments were approved by the Animal Experiment Committee of Nanjing Second Hospital. The animal procedures were performed following the guidelines of the Institutional Animal Care Committee. Establishment of xenograft mouse tumor model Lewis Lung Cancer (LLC) cells transfected with green fluorescence protein gene (Lewis Lung -GFP) were obtained from AntiCancer, Inc., (San Diego, CA). Cells were cultured in RPMI 1640 (GIBCO Life Technologies, New. York, NY) supplemented with 10% fetal bovine serum (FBS, Hyclone, Logan, UT) and penicillin/streptomycin at 37℃ in 5% CO 2 . A mouse model of human lung cancer (LLC -GFP) was used to estimate the efficacy of drug loaded ultrasound contrast agents with ultrasound treatment for inhibition of tumor growth. Stocks of Lewis Lung -GFP tumors were established by subcutaneously injecting 1×10 6 Lewis Lung -GFP cells in the flank of nude mice. Tumor nodules were traced until they became established and reached a mean tumor volume of approximately 100-200 mm 3 . Tumor samples were harvested at the exponential growth phase and resected under aseptic conditions. Preparation of drug loaded ultrasound contrast agent Targestar-SA (MB, Targeson Inc., San Diego, CA; distributed in China by Origin Bioscience) was used as the microbubbles in this study. Targestar-SA is an ultrasound contrast agent composed of a perfluorocarbon gas core encapsulated by a lipid shell. The outer shell is derivatized with streptavidin, which binds biotinylated ligands at a density of 1×10 5 molecules per microbubble. The agents are suspended in aqueous saline at a concentration of approximately 1×10 9 particles/mL, and have a mean diameter of approximately 2.0μm. Bevacizumab (Simcere Pharmaceutical, Nanjing, P.R. China) was used as the therapeutic payload. Microbubbles (MB) were incubated with biotinylated Bevacizumab at room temperature for 20 min at a ratio of 0.7 nmol of biotinylated Bevacizumab per 10 9 microbubbles. The unreacted Bevacizumab was removed from the microbubbles by centrifugal washing, per the manufacturers recommended protocol. The presence of microbubble-bound Bevacizumab was assessed using fluorescence microscopy and flow cytometry. Bevacizumab was conjugated to the MBs and fluorescently labeled using a rabbit anti-Endostatin antibody and a FITC conjugated antirabbit IgG secondary antibody (Abcam). The conjugated microbubble concentration was counted with a hemocytometer. The payload of Bevacizumab was determined by quantitation of microbubble-bound Bevacizumab by BCA Protein Assay (Pierce, Rockford, IL, USA). Naked Targestar-SA microbubbles were used directly from the vial without the addition of Bevacizumab. 15 tumor-bearing mice were randomly divided into 3 groups once the average tumor size had reached 100 mm 3 . A dose of 1×10 8 microbubbles in 70 μl per mouse was administered by retro-orbital injection with a gauge needle. Group A served as an untreated control. Group B received Bevacizumab and administrations of ultrasound contrast agents and sham cavitation treatment (Bevacizumab + sham cavitation). Group C received Bevacizumab combined with ultrasound-targeted microbubble destruction (Bevacizumab + UTMD). Cavitation therapy and contrast-enhanced ultrasound imaging The animals in group C received 3 consecutive daily cavitation therapies, using same acoustic conditions. For ultrasonic cavitation, the transducer from a sonicator equipped with a 1 cm 2 transducer cone tip (Haiying Medical Electronic Instrument Company, Wuxi, China) was placed over the tumor and coupled using acoustic coupling gel. Ultrasonic cavitation was performed at a frequency of 238 kHz, 400 mV, 0.5 MPa 60-s sonication duration, 10 pulses with 10-ms pulse length and 50% duty cycle. The animals in group B received 3 consecutive daily administrations of ultrasound contrast agents and sham cavitation, in which the microbubbles were injected and the transducer was placed over the tumor with acoustic coupling gel but not powered on. Contrast-enhanced ultrasound imaging (CEUS) was used to analyze tumor vasculature in day 1, 5, 8 and 11 after the third cavitation therapy. This technique enables real-time evaluation of active microvascular perfusion in the intact tumor. CEUS was performed on a Mylab90 ultrasound scanner with 4.0–11.0 MHz LA332 linear transducer (Esaote, Genova ITALY). The transducer was coupled to the skin by covering the tumor with acoustic coupling gel. Imaging was performed in CnTI mode at a mechanical index (MI) of 0.04 and transmission frequency of 8 MHz. Imaging gain settings were optimized and held constant during the experiment. Right after injection of microbubbles, ultrasound images were captured to obtain the contrast signal from the tumor tissue as well as from adherent and freely circulating microbubbles. Tumor volume, weight and angiogenesis measurement The maximum diameter length and maximum diameter width perpendicular to the length diameter of the tumor were measured with Image-Pro Plus 6.0 software (MediaCybernetics, Silver Spring, MD, USA). Approximate tumor sizes were calculated as follows: Volume (V) = Length × Width 2 /2. Tumor growth curves were made based on the tumor volume at different time points. The weight of tumor was measured on the scale after injection. The angiogenesis was assessed with tumor microvascular density (MVD) and immunohistochemical staining for CD34. And MVD was calculated by Vascular Length (mm)/ Tumor Surface Area (mm 2 ). Immunohistochemistry At the end of the study, all mice were sacrificed on day 12 after the last cavitation therapy and the tumors were resected. The parts of tumor sample were fixed in 10% buffered formalin and paraffin-embedded. For immunohistochemistry, sections were incubated with primary antibodies against CD34 (BD Biosciences, San Diego, CA) overnight at 4 °C after permeabilization with a solution of 0.1% sodium citrate and 0.1%Triton X-100 and blocked with 10% rabbit serum. After washing in PBS, the slices were incubated with horseradish peroxidase-labeled secondary antibody (1:200, Maixin Bio-Tech Co., Ltd., Fuzhou, China) for 30 min at room temperature. After color development using diaminobenzidine (Maixin Bio-Tech Co., Ltd.), the slices were counterstained in hematoxylin and mounted with a neutral resin medium. The whole slide was first viewed at 100-times magnification in order to identify a hot spot representing the area of the highest vessel density. The field was then switched to 400× magnification for analysis. Statistical analysis Data are expressed as the mean ± standard deviation (mean ± SD). SPSS software, Version 17.0 for Windows (IBM, Armonk, NY, USA), was used for variable analyses. Student’s T-test was used to determine statistical significance between two groups. P<0.05 was considered statistically significant. And data were graphically displayed using GraphPad Prism v.5.0 for Windows (GraphPad Software, Inc., La Jolla, CA, USA). Results Inhibition of tumor growth Mice weight were measured in day1, 5, 8, and 11 after the third cavitation therapy. Compared with control group (26.46±1.14g), the Bevacizumab + sham cavitation group (24.14±0.35g, p<0.01) and Bevacizumab + UTMD group (23.18±0.63g, p<0.001) exhibited significant decrease of mice weight in day11 (Figure1A). Likewise, tumor volume was calculated according to the length and width in day1, 5, 8, and 11. Analogously, the tumor volume of the Bevacizumab + sham cavitation group (1870±269.3mm 3 , p<0.01) and Bevacizumab + UTMD group (1325±167.2mm 3 , p<0.001) was smaller than that of control group (2763±320.8mm 3 ) (Figure1B) in day 11. The tumors were weighed after resection in day11, and results suggested that the tumor weight of the Bevacizumab + sham cavitation group (2.568±0.6078g, p<0.01) and Bevacizumab + UTMD group (1.712±0.2697g, p<0.001) was lighter than that of control group (4.780±1.195g) (Figure1C). Taken together, these results implied that Bevacizumab inhibited tumor growth of lung cancer in vivo, and UTMD facilitated the anti-cancer effect of Bevacizumab. Histologic changes in each group The hematoxylin and eosin staining of samples were performed in each experimental group. Compared with control group, experimental group presented sparser tumor cells and more necrotic areas in tumor tissues. Moreover, the effect of Bevacizumab + UTMD group was especially more pronounced than Bevacizumab + sham cavitation group (Figure2). The results indicated that Bevacizumab with UTMD effectively suppressed tumor growth and promoted tumor necrosis. Inhibition of tumor angiogenesis The subcutaneous tumor nodules were photographed in day1, 5, 8 and 11 after the third cavitation therapy (Figure3A). And the subcutaneous blood vessels of each group were recorded (Figure3B). MVD was calculated according to the max length of blood vessels in microscope and the superficial area of tumor nodules. MVD of the Bevacizumab + sham cavitation group (0.2371±0.05275mm -1 , p<0.01) and Bevacizumab + UTMD group (0.1661±0.01335mm-1, p<0.001) was smaller than that of control group (0.3966±0.05364mm -1 ) (Figure3C) in day 11. Results suggested that Bevacizumab inhibited angiogenesis of lung cancer in vivo, and the inhibition effect was enhanced by UTMD. Expression of CD34 CD34 was identified as the marker of vascular endothelial cell. To further verify the effect of Bevacizumab on angiogenesis, immunohistochemical staining was used to detect the expression of CD34 in each group (Figure4A). And we found that the average gray values for the CD34 protein in the Bevacizumab + sham cavitation group (33.89±7.32, p<0.01) and Bevacizumab + UTMD group (25.67±7.47, p<0.01) were significantly decreased compared with the average gray value in the control group (50.89±15.32) (Figure4B). Consistently, the results indicated that angiogenesis of lung cancer was suppressed by Bevacizumab, and UTMD strengthened the suppression. Discussion Cancer cells require excess nutrients and oxygen to enable tumor growth and progression, therefore growing solid tumors must recruit new blood vessels for provision of nutrients and oxygen and disposal of metabolic waste products[18]. Angiogenesis has been identified as an emerging hallmark of cancer[19] and the activated angiogenesis causing normally quiescent vasculature to continually sprout new vessels that helps to sustain expanding proliferation[20]. Tumor angiogenesis is essentially initiated by secreting angiogenic growth factor molecules, of which the most pivotal factor is vascular endothelial growth factor (VEGF)[21]. VEGF gene encodes ligands that are involved in orchestrating new blood vessel growth and in homeostatic survival of endothelial cells, via receptor tyrosine kinases (VEGFR) [22]. Numerous attempts have been undertaken to explore anti-angiogenic drugs against various types of tumors in the past decades. Bevacizumab, a humanized monoclonal antibody that binds to soluble VEGF, was developed in the 1980s[23]. It has been demonstrated that Bevacizumab induces regression of newly formed vessels and inhibits tumor growth in vivo[24]. In a randomized study including 878 patients with recurrent or advanced non–small-cell lung (NSCLC) cancer, Bevacizumab was proved to have a significant survival benefit[25]. Approval of Bevacizumab in the first-line setting was based on the result and further clinical studies confirmed the benefits of Bevacizumab in overall survival (OS) and progression-free survival (PFS) for NSCLC patients[26-28]. Although the use of Bevacizumab in association with chemotherapy has sensibly improved outcomes of patients with unresectable or metastatic nonsquamous-NSCLC[29], safer and more effective delivery of Bevacizumab is still being explored. Ultrasound (US) consists of pressure waves that can be focused, reflected and refracted through a medium[30]. Hence, US can be precisely controlled and focused on the target site or specific tissue in the body[31]. Since the advantages of convenience, safety and real-time imaging, US has long been an important tool in clinical diagnosis of disease[32]. Increasing evidences are showing the prospect of US in the field of disease treatment, especially in cancer treatment[33]. US is usually combined with acoustically responsive microbubbles (MB) or droplets to exert the maximal mechanical effect[34]. Targeted microbubbles are linked to various targeted ligands, including polymer, lipid, and protein anchors, which have been developed and used in experimental and clinical studies[35]. Ultrasound-targeted microbubble destruction (UTMD) is widely used as a novel tool for locally drug delivery or gene transfection in cancer treatment and is considered an effective method of monitoring the status of tumors[36]. It was reported in 2014 that UTMD-mediated HSV-TK effectively transfected the HSV-TK gene into target tissues and exerted a significant inhibitory effect on ovarian cancer in mice[37]. Gemcitabine and nab-paclitaxel is a standard of care chemotherapy combination used in the treatment of patients with advanced pancreatic cancer and Keiran A Logan, et al highlighted the potential of UTMD mediated Gem / PTX as a treatment for pancreatic cancer[38]. In pancreatic and colon cancer, UTMD enhanced delivery of 5-fluorouridine, irinotecan and oxaliplatin, thus to make the treatment more tolerable and to reduce the adverse effects associated with chemotherapy[39]. Moreover, UTMD-targeted microbubbles can significantly enhance the antitumor effect of doxorubicin on a mouse hepatocellular carcinoma (HCC) model[40]. As it has demonstrated that cavitation is the main mechanism of UTMD-enhanced drug delivery[41], sham cavitation was used as control to clarify the influence of UTMD in our study. Consistently with those previous researches, we found that Bevacizumab could inhibit tumor growth and angiogenesis in lung cancer xenografts, and UTMD significantly enhanced the antitumor effect of Bevacizumab compared with sham cavitation. The advantages of UTMD are apparent compared to other methods; it is feasible for drug delivery, does not involve radiation, and can be monitored in real time. However, to ensure both safe and effective treatment is still the major challenge in the future clinical application of UTMD. For example, some MBs are used to enhance vascular permeability of chemotherapy drugs, and these bioeffects raise safety concerns[42]. By now, the safety of UTMD in cancer therapy is mainly evaluated in animal models, and the systemic toxicity of the treatment in humans needs to be further investigated. The validation of the long-term safety and efficacy of UTMD in humans would pave the way for clinical applications. Conclusion In summary, our studies highlighted that Bevacizumab inhibited tumor growth and angiogenesis in lung cancer xenografts, and ultrasound-targeted microbubble destruction (UTMD) could significantly enhance the antitumor effect of Bevacizumab. With the development of drug-loaded microbubbles suitable for clinical use, UTMD‑mediated drug delivery might be a novel and effective therapeutic strategy for the treatment of NSCLC. Declarations Acknowledgement This research was supported by National Natural Science Foundation for Youth of China (No. 81902354). Declaration of interest statement The authors report no conflicts of interest in this work. Author Contribution Siqing Sun and Feng Hua designed and supervised the study. Yu Yao, Ting Xu and Yuxuan Wen performed experiments and wrote the manuscript. Jing Luo, Yanling Lv and Wenfang Jin conducted statistical analysis and edited the manuscript. All authors read and approved the final version of the manuscript. References Datta S, Coussios CC, McAdory LE, Tan J, Porter T, De Courten-Myers G, et al: Correlation of cavitation with ultrasound enhancement of thrombolysis. Ultrasound Med Biol 2006, 32(8):1257-1267. doi:10.1016/j.ultrasmedbio.2006.04.008 Hitchcock KE, Caudell DN, Sutton JT, Klegerman ME, Vela D, Pyne-Geithman GJ, et al: Ultrasound-enhanced delivery of targeted echogenic liposomes in a novel ex vivo mouse aorta model. J Control Release 2010, 144(3):288-295. doi:10.1016/j.jconrel.2010.02.030 Zolochevska O, Figueiredo ML: Advances in sonoporation strategies for cancer. Front Biosci (Schol Ed) 2012, 4:988-1006. doi:10.2741/s313 Marmottant P, Hilgenfeldt S: Controlled vesicle deformation and lysis by single oscillating bubbles. Nature 2003, 423(6936):153-156. doi:10.1038/nature01613 Miller MW, Miller DL, Brayman AA: A review of in vitro bioeffects of inertial ultrasonic cavitation from a mechanistic perspective. Ultrasound Med Biol 1996, 22(9):1131-1154. doi:10.1016/s0301-5629(96)00089-0 Sheikov N, McDannold N, Vykhodtseva N, Jolesz F, Hynynen K: Cellular mechanisms of the blood-brain barrier opening induced by ultrasound in presence of microbubbles. Ultrasound Med Biol 2004, 30(7):979-989. doi:10.1016/j.ultrasmedbio.2004.04.010 Wood AK, Sehgal CM: A review of low-intensity ultrasound for cancer therapy. Ultrasound Med Biol 2015, 41(4):905-928. doi:10.1016/j.ultrasmedbio.2014.11.019 Wang Y, Hu B, Diao X, Zhang J: Antitumor effect of microbubbles enhanced by low frequency ultrasound cavitation on prostate carcinoma xenografts in nude mice. Exp Ther Med 2012, 3(2):187-191. doi:10.3892/etm.2011.377 Huang P, You X, Pan M, Li S, Zhang Y, Zhao Y, et al: A novel therapeutic strategy using ultrasound mediated microbubbles destruction to treat colon cancer in a mouse model. Cancer Lett 2013, 335(1):183-190. doi:10.1016/j.canlet.2013.02.011 Zhang C, Huang P, Zhang Y, Chen J, Shentu W, Sun Y, et al: Anti-tumor efficacy of ultrasonic cavitation is potentiated by concurrent delivery of anti-angiogenic drug in colon cancer. Cancer Lett 2014, 347(1):105-113. doi:10.1016/j.canlet.2014.01.022 Bazan-Peregrino M, Arvanitis CD, Rifai B, Seymour LW, Coussios CC: Ultrasound-induced cavitation enhances the delivery and therapeutic efficacy of an oncolytic virus in an in vitro model. J Control Release 2012, 157(2):235-242. doi:10.1016/j.jconrel.2011.09.086 Arvanitis CD, Bazan-Peregrino M, Rifai B, Seymour LW, Coussios CC: Cavitation-enhanced extravasation for drug delivery. Ultrasound Med Biol 2011, 37(11):1838-1852. doi:10.1016/j.ultrasmedbio.2011.08.004 Willmann JK, Kimura RH, Deshpande N, Lutz AM, Cochran JR, Gambhir SS: Targeted contrast-enhanced ultrasound imaging of tumor angiogenesis with contrast microbubbles conjugated to integrin-binding knottin peptides. J Nucl Med 2010, 51(3):433-440. doi:10.2967/jnumed.109.068007 Siegel RL, Miller KD, Jemal A: Cancer statistics, 2020. CA Cancer J Clin 2020, 70(1):7-30. doi:10.3322/caac.21590 Jonna S, Subramaniam DS: Molecular diagnostics and targeted therapies in non-small cell lung cancer (NSCLC): an update. Discov Med 2019, 27(148):167-170. Sankar K, Gadgeel SM, Qin A: Molecular therapeutic targets in non-small cell lung cancer. Expert Rev Anticancer Ther 2020, 20(8):647-661. doi:10.1080/14737140.2020.1787156 Kazazi-Hyseni F, Beijnen JH, Schellens JH: Bevacizumab. Oncologist 2010, 15(8):819-825. doi:10.1634/theoncologist.2009-0317 Carmeliet P, Jain RK: Angiogenesis in cancer and other diseases. Nature 2000, 407(6801):249-257. doi:10.1038/35025220 Hanahan D, Weinberg RA: Hallmarks of cancer: the next generation. Cell 2011, 144(5):646-674. doi:10.1016/j.cell.2011.02.013 Hanahan D, Folkman J: Patterns and emerging mechanisms of the angiogenic switch during tumorigenesis. Cell 1996, 86(3):353-364. doi:10.1016/s0092-8674(00)80108-7 Siveen KS, Prabhu K, Krishnankutty R, Kuttikrishnan S, Tsakou M, Alali FQ, et al: Vascular Endothelial Growth Factor (VEGF) Signaling in Tumour Vascularization: Potential and Challenges. Curr Vasc Pharmacol 2017, 15(4):339-351. doi:10.2174/1570161115666170105124038 Ferrara N, Gerber HP, LeCouter J: The biology of VEGF and its receptors. Nat Med 2003, 9(6):669-676. doi:10.1038/nm0603-669 Ferrara N, Hillan KJ, Gerber HP, Novotny W: Discovery and development of bevacizumab, an anti-VEGF antibody for treating cancer. Nat Rev Drug Discov 2004, 3(5):391-400. doi:10.1038/nrd1381 Ferrara N, Adamis AP: Ten years of anti-vascular endothelial growth factor therapy. Nat Rev Drug Discov 2016, 15(6):385-403. doi:10.1038/nrd.2015.17 Sandler A, Gray R, Perry MC, Brahmer J, Schiller JH, Dowlati A, et al: Paclitaxel-carboplatin alone or with bevacizumab for non-small-cell lung cancer. N Engl J Med 2006, 355(24):2542-2550. doi:10.1056/NEJMoa061884 Reck M, von Pawel J, Zatloukal P, Ramlau R, Gorbounova V, Hirsh V, et al: Overall survival with cisplatin-gemcitabine and bevacizumab or placebo as first-line therapy for nonsquamous non-small-cell lung cancer: results from a randomised phase III trial (AVAiL). Ann Oncol 2010, 21(9):1804-1809. doi:10.1093/annonc/mdq020 Reck M, von Pawel J, Zatloukal P, Ramlau R, Gorbounova V, Hirsh V, et al: Phase III trial of cisplatin plus gemcitabine with either placebo or bevacizumab as first-line therapy for nonsquamous non-small-cell lung cancer: AVAil. J Clin Oncol 2009, 27(8):1227-1234. doi:10.1200/JCO.2007.14.5466 Patel JD, Socinski MA, Garon EB, Reynolds CH, Spigel DR, Olsen MR, et al: PointBreak: a randomized phase III study of pemetrexed plus carboplatin and bevacizumab followed by maintenance pemetrexed and bevacizumab versus paclitaxel plus carboplatin and bevacizumab followed by maintenance bevacizumab in patients with stage IIIB or IV nonsquamous non-small-cell lung cancer. J Clin Oncol 2013, 31(34):4349-4357. doi:10.1200/JCO.2012.47.9626 Assoun S, Brosseau S, Steinmetz C, Gounant V, Zalcman G: Bevacizumab in advanced lung cancer: state of the art. Future Oncol 2017, 13(28):2515-2535. doi:10.2217/fon-2017-0302 Wu J, Nyborg WL: Ultrasound, cavitation bubbles and their interaction with cells. Adv Drug Deliv Rev 2008, 60(10):1103-1116. doi:10.1016/j.addr.2008.03.009 Sboros V: Response of contrast agents to ultrasound. Adv Drug Deliv Rev 2008, 60(10):1117-1136. doi:10.1016/j.addr.2008.03.011 Neumann D, Kollorz E: Ultrasound . In: Medical Imaging Systems: An Introductory Guide. edn. Edited by Maier A, Steidl S, Christlein V, Hornegger J. Cham (CH); 2018: 237-249. Matthews MJ, Stretanski MF: Ultrasound Therapy . In: StatPearls. edn. Treasure Island (FL); 2021. Liu Z, Gao S, Zhao Y, Li P, Liu J, Li P, et al: Disruption of tumor neovasculature by microbubble enhanced ultrasound: a potential new physical therapy of anti-angiogenesis. Ultrasound Med Biol 2012, 38(2):253-261. doi:10.1016/j.ultrasmedbio.2011.11.007 Fan X, Wang L, Guo Y, Xiong X, Zhu L, Fang K: Inhibition of prostate cancer growth using doxorubicin assisted by ultrasound-targeted nanobubble destruction. Int J Nanomedicine 2016, 11:3585-3596. doi:10.2147/IJN.S111808 Ibsen S, Benchimol M, Simberg D, Esener S: Ultrasound mediated localized drug delivery. Adv Exp Med Biol 2012, 733:145-153. doi:10.1007/978-94-007-2555-3_14 Zhou XL, Shi YL, Li X: Inhibitory effects of the ultrasound-targeted microbubble destruction-mediated herpes simplex virus-thymidine kinase/ganciclovir system on ovarian cancer in mice. Exp Ther Med 2014, 8(4):1159-1163. doi:10.3892/etm.2014.1877 Logan KA, Nesbitt H, Callan B, Gao J, McKaig T, Taylor M, et al: Synthesis of a gemcitabine-modified phospholipid and its subsequent incorporation into a single microbubble formulation loaded with paclitaxel for the treatment of pancreatic cancer using ultrasound-targeted microbubble destruction. Eur J Pharm Biopharm 2021, 165:374-382. doi:10.1016/j.ejpb.2021.05.018 Gao J, Logan KA, Nesbitt H, Callan B, McKaig T, Taylor M, et al: A single microbubble formulation carrying 5-fluorouridine, Irinotecan and oxaliplatin to enable FOLFIRINOX treatment of pancreatic and colon cancer using ultrasound targeted microbubble destruction. J Control Release 2021, 338:358-366. doi:10.1016/j.jconrel.2021.08.050 Wu Y, Sun T, Tang J, Liu Y, Li F: Ultrasound-Targeted Microbubble Destruction Enhances the Antitumor Efficacy of Doxorubicin in a Mouse Hepatocellular Carcinoma Model. Ultrasound Med Biol 2020, 46(3):679-689. doi:10.1016/j.ultrasmedbio.2019.09.017 Paliwal S, Mitragotri S: Ultrasound-induced cavitation: applications in drug and gene delivery. Expert Opin Drug Deliv 2006, 3(6):713-726. doi:10.1517/17425247.3.6.713 ter Haar G: Safety and bio-effects of ultrasound contrast agents. Med Biol Eng Comput 2009, 47(8):893-900. doi:10.1007/s11517-009-0507-3 Additional Declarations No competing interests reported. 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-6286565","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":465356191,"identity":"0a9fa3b9-6eac-4ac4-829b-f05aff3aa55d","order_by":0,"name":"Yu Yao","email":"","orcid":"","institution":"Nanjing Second Hospital","correspondingAuthor":false,"prefix":"","firstName":"Yu","middleName":"","lastName":"Yao","suffix":""},{"id":465356192,"identity":"ae49d7c9-6347-41a6-b52f-f57bc08ffd28","order_by":1,"name":"Ting Xu","email":"","orcid":"","institution":"Nanjing Chest Hospital","correspondingAuthor":false,"prefix":"","firstName":"Ting","middleName":"","lastName":"Xu","suffix":""},{"id":465356193,"identity":"9903d143-4b32-44da-aa61-fedebc4946e5","order_by":2,"name":"Yuxuan Wen","email":"","orcid":"","institution":"Medical School of Nanjing University","correspondingAuthor":false,"prefix":"","firstName":"Yuxuan","middleName":"","lastName":"Wen","suffix":""},{"id":465356194,"identity":"b2cb1a2f-6c56-4206-aad8-806e43e8ced1","order_by":3,"name":"Jing Luo","email":"","orcid":"","institution":"jinling hospital","correspondingAuthor":false,"prefix":"","firstName":"Jing","middleName":"","lastName":"Luo","suffix":""},{"id":465356195,"identity":"7a696d31-1c01-4298-b9de-e4a6da6ed2f9","order_by":4,"name":"Yanling Lv","email":"","orcid":"","institution":"Nanjing Second Hospital","correspondingAuthor":false,"prefix":"","firstName":"Yanling","middleName":"","lastName":"Lv","suffix":""},{"id":465356196,"identity":"6199615f-4ef7-4910-890d-816c1a1723bd","order_by":5,"name":"Wenfang Jin","email":"","orcid":"","institution":"Nanjing Second Hospital","correspondingAuthor":false,"prefix":"","firstName":"Wenfang","middleName":"","lastName":"Jin","suffix":""},{"id":465356197,"identity":"cee5b6fa-8dd9-4693-9b9d-739f2125665f","order_by":6,"name":"Feng Hua","email":"","orcid":"","institution":"The Affiliated Huzhou Hospital, Zhejiang University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Feng","middleName":"","lastName":"Hua","suffix":""},{"id":465356198,"identity":"69dfadfe-8cef-4b73-9135-027caa6ebbd6","order_by":7,"name":"Siqing Sun","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA8klEQVRIiWNgGAWjYDACZgaGA2AGewODAUQogVgtPAeI1QIHEnCVBLTItzNvPFzwqy5xu+TbA8U8NdsY+NlzDBh+7sCtxeAwW8HhmX2HE3fOzksw5jl2m0Gy540BY+8ZPFqYeQwO8/YcSNxwO8fAmLfhNoPBjRwDZsY2PA5rBmupS9xw8wxEiz0hLQyHgVp4fjAnbrjBA7VFgoAWsF94Gw4bbziTY2A459htHokzzwoO9uJzWP/hzZ95/tTJbjh+xszgTc1tOf725I0PfuJzGNAiBqgz2EBRyQNiHcCrAaSF4Q+YwfyAgMpRMApGwSgYoQAATllTiiChmLsAAAAASUVORK5CYII=","orcid":"","institution":"Nanjing Second Hospital","correspondingAuthor":true,"prefix":"","firstName":"Siqing","middleName":"","lastName":"Sun","suffix":""}],"badges":[],"createdAt":"2025-03-23 05:23:10","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6286565/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6286565/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":84187879,"identity":"19b76dc4-cbd5-4736-9384-a7d4a6e2a0d3","added_by":"auto","created_at":"2025-06-09 06:02:02","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":62632,"visible":true,"origin":"","legend":"\u003cp\u003eThe in vivo anti-tumor efficacy. A. The mice weight of each group. B. The tumor volume of each group. C. The tumor weight of each group.\u003c/p\u003e","description":"","filename":"Figure1201.png","url":"https://assets-eu.researchsquare.com/files/rs-6286565/v1/5fbfe06db4013d781af6cd83.png"},{"id":84186190,"identity":"9cdc75f3-d736-4b82-9ca0-f42c2eff802d","added_by":"auto","created_at":"2025-06-09 05:38:02","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":3594570,"visible":true,"origin":"","legend":"\u003cp\u003eThe HE staining of each group was illustrated.\u003c/p\u003e","description":"","filename":"Figure1202.png","url":"https://assets-eu.researchsquare.com/files/rs-6286565/v1/b153836814e72e8c521fc7f4.png"},{"id":84186189,"identity":"faa4a54f-61e6-4c79-a1e6-d0c6ec7d64ee","added_by":"auto","created_at":"2025-06-09 05:38:02","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1034610,"visible":true,"origin":"","legend":"\u003cp\u003eThe effect of Bevacizumab on angiogenesis in lung cancer. A. Whole-body fluorescence imaging of mice. B. The angiogenesis of each group under a fluorescence imaging system. C. MVD of each group was illustrated.\u003c/p\u003e","description":"","filename":"Figure1203.png","url":"https://assets-eu.researchsquare.com/files/rs-6286565/v1/8ff4018e97bfe6b598c5996c.png"},{"id":84186191,"identity":"d9060070-4e06-43f5-92de-8e48199c04c7","added_by":"auto","created_at":"2025-06-09 05:38:02","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":3088205,"visible":true,"origin":"","legend":"\u003cp\u003eImmunohistochemical staining for CD34. A. Immunohistochemical staining for CD34 in each group (400 × magnification) were presented. B. CD34 gray values of the treatment groups were significantly decreased than control group.\u003c/p\u003e","description":"","filename":"Figure1204.png","url":"https://assets-eu.researchsquare.com/files/rs-6286565/v1/a71dd16ed22b307488c9b004.png"},{"id":92391975,"identity":"e210d2a0-3e25-41e4-86f3-84acb73308ca","added_by":"auto","created_at":"2025-09-29 08:47:43","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":8302070,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6286565/v1/16278087-2bdd-477d-b685-f928fe6b5a97.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Bevacizumab delivery mediated by ultrasound-targeted microbubble destruction inhibited tumor growth and angiogenesis in lung cancer xenografts","fulltext":[{"header":"Introduction","content":"\u003cp\u003eUltrasound is emerging as a highly effective, low-cost and truly noninvasive tool for therapy, although it has traditionally been perceived as a tool for diagnostic imaging[1, 2]. One of major physical effects of ultrasound is cavitation, which is the response of gas bubbles in liquid after oscillating to an acoustic field[3]. In the presence of large number of exogenous cavitation nuclei, for instance, lipid microbubbles injected into circulation, inertial cavitation might be induced, which can cause sonoporation or capillary damage[4, 5]. This process leads to hemorrhage, edema, thrombus formation, and wear to the endothelium of capillaries or small vessels in various tissues, which can induce necrosis and apoptosis of cancer cells and inhibit tumor growth[6, 7]. There are several reports indicated that ultrasonic cavitation is an effective application for tumor vasculature destruction or antitumor effects[8-10]. The use of targeted microbubbles may enable concentration of the treatment at the desired location on activation with ultrasound[11, 12], and multiple kinds of targeting ligands have been conjugated to the surface of microbubbles to achieve site-specific accumulation[13].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eLung cancer is the most common malignancy and the leading cause of cancer-related death worldwide[14]. As the main subtype of lung cancer, non-small cell lung cancer (NSCLC) occupies approximately 85% of all lung cancer cases[15]. In the past decades, the advent of various targeted therapies meaningfully improved clinical outcomes of patients with advanced NSCLC[16]. Bevacizumab is a humanized anti-VEGF monoclonal IgG\u003csub\u003e1\u003c/sub\u003e antibody, and it is the first angiogenesis inhibitor approved for the treatment of patients with advanced NSCLC in combination with chemotherapy[17]. In the present study, we investigated an ultrasonic cavitation strategy that targeted tumor angiogenesis with Bevacizumab-loaded microbubble and released its payload on focal ultrasound treatment for anti-tumor efficacy. The efficacy of this strategy was evaluated in a lung cancer model using real-time quantitative CEUS and in vivo fluorescence imaging.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003e\u003cstrong\u003eAnimal care\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e15 BALB/C male nude mice, aged 4\u0026ndash;6 weeks and weighted 20\u0026ndash;25 g, were purchased from Beijing Kelihua laboratory animal center (Beijing, P.R. China). All mice were maintained in a HEPA-filtered environment at 25 ℃ and humidity was maintained at 50\u0026ndash;60%. All animals were fed with autoclaved laboratory rodent diet. All animal experiments were approved by the Animal Experiment Committee of Nanjing Second Hospital. The animal procedures were performed following the guidelines of the Institutional Animal Care Committee.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEstablishment of xenograft mouse tumor model\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLewis Lung Cancer (LLC) cells transfected with green fluorescence protein gene (Lewis Lung -GFP) were obtained from AntiCancer, Inc., (San Diego, CA). Cells were cultured in RPMI 1640 (GIBCO Life Technologies, New. York, NY) supplemented with 10% fetal bovine serum (FBS, Hyclone, Logan, UT) and penicillin/streptomycin at 37℃ in 5% CO\u003csub\u003e2\u003c/sub\u003e. A mouse model of human lung cancer (LLC -GFP) was used to estimate the efficacy of drug loaded ultrasound contrast agents with ultrasound treatment for inhibition of tumor growth. Stocks of Lewis Lung -GFP tumors were established by subcutaneously injecting 1\u0026times;10\u003csup\u003e6\u003c/sup\u003e Lewis Lung -GFP cells in the flank of nude mice. Tumor nodules were traced until they became established and reached a mean tumor volume of approximately 100-200 mm\u003csup\u003e3\u003c/sup\u003e.\u003csup\u003e\u0026nbsp;\u003c/sup\u003eTumor samples were harvested at the exponential growth phase and resected under aseptic conditions.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePreparation of drug loaded ultrasound contrast agent\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTargestar-SA (MB, Targeson Inc., San Diego, CA; distributed in China by Origin Bioscience) was used as the microbubbles in this study. Targestar-SA is an ultrasound contrast agent composed of a perfluorocarbon gas core encapsulated by a lipid shell. The outer shell is derivatized with streptavidin, which binds biotinylated ligands at a density of 1\u0026times;10\u003csup\u003e5\u003c/sup\u003e molecules per microbubble. The agents are suspended in aqueous saline at a concentration of approximately 1\u0026times;10\u003csup\u003e\u0026nbsp;9\u0026nbsp;\u003c/sup\u003eparticles/mL, and have a mean diameter of approximately 2.0\u0026mu;m. Bevacizumab (Simcere Pharmaceutical, Nanjing, P.R. China) was used as the therapeutic payload. Microbubbles (MB) were incubated with biotinylated Bevacizumab at room temperature for 20 min at a ratio of 0.7 nmol of biotinylated Bevacizumab per 10\u003csup\u003e9\u003c/sup\u003e microbubbles. The unreacted Bevacizumab was removed from the microbubbles by centrifugal washing, per the manufacturers recommended protocol. The presence of microbubble-bound Bevacizumab was assessed using fluorescence microscopy and flow cytometry. Bevacizumab was conjugated to the MBs and fluorescently labeled using a rabbit anti-Endostatin antibody and a FITC conjugated antirabbit IgG secondary antibody (Abcam). The conjugated microbubble concentration was counted with a hemocytometer. The payload of Bevacizumab was determined by quantitation of microbubble-bound Bevacizumab by BCA Protein Assay (Pierce, Rockford, IL, USA). Naked Targestar-SA microbubbles were used directly from the vial without the addition of Bevacizumab. 15 tumor-bearing mice were randomly divided into 3 groups once the average tumor size had reached 100 mm\u003csup\u003e3\u003c/sup\u003e. A dose of 1\u0026times;10\u003csup\u003e8\u0026nbsp;\u003c/sup\u003emicrobubbles in 70 \u0026mu;l per mouse was administered by retro-orbital injection with a gauge needle. Group A served as an untreated control. Group B received Bevacizumab and administrations of ultrasound contrast agents and sham cavitation treatment (Bevacizumab + sham cavitation). Group C received Bevacizumab combined with ultrasound-targeted microbubble destruction (Bevacizumab + UTMD).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCavitation therapy and contrast-enhanced ultrasound imaging\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe animals in group C received 3 consecutive daily cavitation therapies, using same acoustic conditions. For ultrasonic cavitation, the transducer from a sonicator equipped with a 1 cm\u003csup\u003e2\u003c/sup\u003e transducer cone tip (Haiying Medical Electronic Instrument Company, Wuxi, China) was placed over the tumor and coupled using acoustic coupling gel. Ultrasonic cavitation was performed at a frequency of 238 kHz, 400 mV, 0.5 MPa 60-s sonication duration, 10 pulses with 10-ms pulse length and 50% duty cycle. The animals in group B received 3 consecutive daily administrations of ultrasound contrast agents and sham cavitation, in which the microbubbles were injected and the transducer was placed over the tumor with acoustic coupling gel but not powered on.\u003c/p\u003e\n\u003cp\u003eContrast-enhanced ultrasound imaging (CEUS) was used to analyze tumor vasculature in day 1, 5, 8 and 11 after the third cavitation therapy. This technique enables real-time evaluation of active microvascular perfusion in the intact tumor. CEUS was performed on a Mylab90 ultrasound scanner with 4.0\u0026ndash;11.0 MHz LA332 linear transducer (Esaote, Genova ITALY). The transducer was coupled to the skin by covering the tumor with acoustic coupling gel. Imaging was performed in CnTI mode at a mechanical index (MI) of 0.04 and transmission frequency of 8 MHz. Imaging gain settings were optimized and held constant during the experiment. Right after injection of microbubbles, ultrasound images were captured to obtain the contrast signal from the tumor tissue as well as from adherent and freely circulating microbubbles.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTumor volume, weight and angiogenesis measurement\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe maximum diameter length and maximum diameter width perpendicular to the length diameter of the tumor were measured with Image-Pro Plus 6.0 software (MediaCybernetics, Silver Spring, MD, USA). Approximate tumor sizes were calculated as follows: Volume (V) = Length \u0026times; Width\u003csup\u003e2\u003c/sup\u003e/2. Tumor growth curves were made based on the tumor volume at different time points. The weight of tumor was measured on the scale after injection. The angiogenesis was assessed with tumor microvascular density (MVD) and immunohistochemical staining for CD34. And MVD was calculated by Vascular Length (mm)/ Tumor Surface Area (mm\u003csup\u003e2\u003c/sup\u003e).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImmunohistochemistry\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAt the end of the study, all mice were sacrificed on day 12 after the last cavitation therapy and the tumors were resected. The parts of tumor sample were fixed in 10% buffered formalin and paraffin-embedded. For immunohistochemistry, sections were incubated with primary antibodies against CD34 (BD Biosciences, San Diego, CA) overnight at 4 \u0026deg;C after permeabilization with a solution of 0.1% sodium citrate and 0.1%Triton X-100 and blocked with 10% rabbit serum. After washing in PBS, the slices were incubated with horseradish peroxidase-labeled secondary antibody (1:200, Maixin Bio-Tech Co., Ltd., Fuzhou, China) for 30 min at room temperature. After color development using diaminobenzidine (Maixin Bio-Tech Co., Ltd.), the slices were counterstained in hematoxylin and mounted with a neutral resin medium. The whole slide was first viewed at 100-times magnification in order to identify a hot spot representing the area of the highest vessel density. The field was then switched to 400\u0026times; magnification for analysis.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData are expressed as the mean \u0026plusmn; standard deviation (mean \u0026plusmn; SD). SPSS software, Version 17.0 for Windows (IBM, Armonk, NY, USA), was used for variable analyses. Student\u0026rsquo;s T-test was used to determine statistical significance between two groups. P\u0026lt;0.05 was considered statistically significant. And data were graphically displayed using GraphPad Prism v.5.0 for Windows (GraphPad Software, Inc., La Jolla, CA, USA).\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eInhibition of tumor growth\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMice weight were measured in day1, 5, 8, and 11 after the third cavitation therapy. Compared with control group (26.46\u0026plusmn;1.14g), the Bevacizumab + sham cavitation group (24.14\u0026plusmn;0.35g, p\u0026lt;0.01) and Bevacizumab + UTMD group (23.18\u0026plusmn;0.63g, p\u0026lt;0.001) exhibited significant decrease of mice weight in day11 (Figure1A). Likewise, tumor volume was calculated according to the length and width in day1, 5, 8, and 11. Analogously, the tumor volume of the Bevacizumab + sham cavitation group (1870\u0026plusmn;269.3mm\u003csup\u003e3\u003c/sup\u003e, p\u0026lt;0.01) and Bevacizumab + UTMD group (1325\u0026plusmn;167.2mm\u003csup\u003e3\u003c/sup\u003e, p\u0026lt;0.001) was smaller than that of control group (2763\u0026plusmn;320.8mm\u003csup\u003e3\u003c/sup\u003e) (Figure1B) in day 11. The tumors were weighed after resection in day11, and results suggested that the tumor weight of the Bevacizumab + sham cavitation group (2.568\u0026plusmn;0.6078g, p\u0026lt;0.01) and Bevacizumab + UTMD group (1.712\u0026plusmn;0.2697g, p\u0026lt;0.001) was lighter than that of control group (4.780\u0026plusmn;1.195g) (Figure1C). Taken together, these results implied that Bevacizumab inhibited tumor growth of lung cancer in vivo, and UTMD facilitated the anti-cancer effect of Bevacizumab.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHistologic changes in each group\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe hematoxylin and eosin staining of samples were performed in each experimental group. Compared with control group, experimental group presented sparser tumor cells and more necrotic areas in tumor tissues. Moreover, the effect of Bevacizumab + UTMD group was especially more pronounced than Bevacizumab + sham cavitation group (Figure2). The results indicated that Bevacizumab with UTMD effectively suppressed tumor growth and promoted tumor necrosis.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInhibition of tumor angiogenesis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe subcutaneous tumor nodules were photographed in day1, 5, 8 and 11 after the third cavitation therapy (Figure3A). And the subcutaneous blood vessels of each group were recorded (Figure3B). MVD was calculated according to the max length of blood vessels in microscope and the superficial area of tumor nodules. MVD of the Bevacizumab + sham cavitation group (0.2371\u0026plusmn;0.05275mm\u003csup\u003e-1\u003c/sup\u003e, p\u0026lt;0.01) and Bevacizumab + UTMD group (0.1661\u0026plusmn;0.01335mm-1, p\u0026lt;0.001) was smaller than that of control group (0.3966\u0026plusmn;0.05364mm\u003csup\u003e-1\u003c/sup\u003e) (Figure3C) in day 11. Results suggested that Bevacizumab inhibited angiogenesis of lung cancer in vivo, and the inhibition effect was enhanced by UTMD.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExpression of CD34\u0026nbsp;\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCD34 was identified as the marker of vascular endothelial cell. To further verify the effect of Bevacizumab on angiogenesis, immunohistochemical staining was used to detect the expression of CD34 in each group (Figure4A). And we found that the average gray values for the CD34 protein in the Bevacizumab + sham cavitation group (33.89\u0026plusmn;7.32, p\u0026lt;0.01) and Bevacizumab + UTMD group (25.67\u0026plusmn;7.47, p\u0026lt;0.01) were significantly decreased compared with the average gray value in the control group (50.89\u0026plusmn;15.32) (Figure4B). Consistently, the results indicated that angiogenesis of lung cancer was suppressed by Bevacizumab, and UTMD strengthened the suppression.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eCancer cells require excess nutrients and oxygen to enable tumor growth and progression, therefore growing solid tumors must recruit new blood vessels for provision of nutrients and oxygen and disposal of metabolic waste products[18]. Angiogenesis has been identified as an emerging hallmark of cancer[19] and the activated angiogenesis causing normally quiescent vasculature to continually sprout new vessels that helps to sustain expanding proliferation[20]. Tumor angiogenesis is essentially initiated by secreting angiogenic growth factor molecules, of which the most pivotal factor is vascular endothelial growth factor (VEGF)[21]. VEGF gene encodes ligands that are involved in orchestrating new blood vessel growth and in homeostatic survival of endothelial cells, via receptor tyrosine kinases (VEGFR) [22]. Numerous attempts have been undertaken to explore anti-angiogenic drugs against various types of tumors in the past decades. Bevacizumab, a humanized monoclonal antibody that binds to soluble VEGF, was developed in the 1980s[23]. It has been demonstrated that Bevacizumab induces regression of newly formed vessels and inhibits tumor growth in vivo[24]. In a randomized study including 878 patients with recurrent or advanced non\u0026ndash;small-cell lung (NSCLC) cancer, Bevacizumab was proved to have a significant survival benefit[25]. Approval of Bevacizumab in the first-line setting was based on the result and further clinical studies confirmed the benefits of Bevacizumab in overall survival (OS) and progression-free survival (PFS) for NSCLC patients[26-28]. \u0026nbsp;Although the use of Bevacizumab in association with chemotherapy has sensibly improved outcomes of patients with unresectable or metastatic nonsquamous-NSCLC[29], safer and more effective delivery of Bevacizumab is still being explored.\u003c/p\u003e\n\u003cp\u003eUltrasound (US) consists of pressure waves that \u0026nbsp;can be focused, reflected and refracted through a medium[30]. Hence, US can be precisely controlled and focused on the target site or specific tissue in the body[31]. Since the advantages of convenience, safety and real-time imaging, US has long been an important tool in clinical diagnosis of disease[32]. Increasing evidences are showing the prospect of US in the field of disease treatment, especially in cancer treatment[33]. US is usually combined with acoustically responsive microbubbles (MB) or droplets to exert the maximal mechanical effect[34]. Targeted microbubbles are linked to various targeted ligands, including polymer, lipid, and protein anchors, which have been developed and used in experimental and clinical studies[35].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eUltrasound-targeted microbubble destruction (UTMD) is widely used as a novel tool for locally drug delivery or gene transfection in cancer treatment and is considered an effective method of monitoring the status of tumors[36]. It was reported in 2014 that UTMD-mediated HSV-TK effectively transfected the HSV-TK gene into target tissues and exerted a significant inhibitory effect on ovarian cancer in mice[37]. Gemcitabine and nab-paclitaxel is a standard of care chemotherapy combination used in the treatment of patients with advanced pancreatic cancer and Keiran A Logan, et al highlighted the potential of UTMD mediated Gem / PTX as a treatment for pancreatic cancer[38]. In pancreatic and colon cancer, UTMD enhanced delivery of 5-fluorouridine, irinotecan and oxaliplatin, thus to make the treatment more tolerable and to reduce the adverse effects associated with chemotherapy[39]. Moreover, UTMD-targeted microbubbles can significantly enhance the antitumor effect of doxorubicin on a mouse hepatocellular carcinoma (HCC) model[40]. As it has demonstrated that cavitation is the main mechanism of UTMD-enhanced drug delivery[41], sham cavitation was used as control to clarify the influence of UTMD in our study. Consistently with those previous researches, we found that Bevacizumab could inhibit tumor growth and angiogenesis in lung cancer xenografts, and UTMD significantly enhanced the antitumor effect of Bevacizumab compared with sham cavitation.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe advantages of UTMD are apparent compared to other methods; it is feasible for drug delivery, does not involve radiation, and can be monitored in real time. However, to ensure both safe and effective treatment is still the major challenge in the future clinical application of UTMD. For example, some MBs are used to enhance vascular permeability of chemotherapy drugs, and these bioeffects raise safety concerns[42]. By now, the safety of UTMD in cancer therapy is mainly evaluated in animal models, and the systemic toxicity of the treatment in humans needs to be further investigated. The validation of the long-term safety and efficacy of UTMD in humans would pave the way for clinical applications.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn summary, our studies highlighted that Bevacizumab inhibited tumor growth and angiogenesis in lung cancer xenografts, and ultrasound-targeted microbubble destruction (UTMD) could significantly enhance the antitumor effect of Bevacizumab. With the development of drug-loaded microbubbles suitable for clinical use, UTMD‑mediated drug delivery might be a novel and effective therapeutic strategy for the treatment of NSCLC.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was supported by National Natural Science Foundation for Youth of China (No. 81902354).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of interest statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors report no conflicts of interest in this work.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contribution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSiqing Sun and Feng Hua designed and supervised the study. Yu Yao, Ting Xu and Yuxuan Wen performed experiments and wrote the manuscript. Jing Luo, Yanling Lv and Wenfang Jin conducted statistical analysis and edited the manuscript. All authors read and approved the final version of the manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eDatta S, Coussios CC, McAdory LE, Tan J, Porter T, De Courten-Myers G, et al: Correlation of cavitation with ultrasound enhancement of thrombolysis. Ultrasound Med Biol 2006, 32(8):1257-1267. doi:10.1016/j.ultrasmedbio.2006.04.008\u003c/li\u003e\n \u003cli\u003eHitchcock KE, Caudell DN, Sutton JT, Klegerman ME, Vela D, Pyne-Geithman GJ, et al: Ultrasound-enhanced delivery of targeted echogenic liposomes in a novel ex vivo mouse aorta model. J Control Release 2010, 144(3):288-295. doi:10.1016/j.jconrel.2010.02.030\u003c/li\u003e\n \u003cli\u003eZolochevska O, Figueiredo ML: Advances in sonoporation strategies for cancer. Front Biosci (Schol Ed) 2012, 4:988-1006. doi:10.2741/s313\u003c/li\u003e\n \u003cli\u003eMarmottant P, Hilgenfeldt S: Controlled vesicle deformation and lysis by single oscillating bubbles. Nature 2003, 423(6936):153-156. doi:10.1038/nature01613\u003c/li\u003e\n \u003cli\u003eMiller MW, Miller DL, Brayman AA: A review of in vitro bioeffects of inertial ultrasonic cavitation from a mechanistic perspective. Ultrasound Med Biol 1996, 22(9):1131-1154. doi:10.1016/s0301-5629(96)00089-0\u003c/li\u003e\n \u003cli\u003eSheikov N, McDannold N, Vykhodtseva N, Jolesz F, Hynynen K: Cellular mechanisms of the blood-brain barrier opening induced by ultrasound in presence of microbubbles. Ultrasound Med Biol 2004, 30(7):979-989. doi:10.1016/j.ultrasmedbio.2004.04.010\u003c/li\u003e\n \u003cli\u003eWood AK, Sehgal CM: A review of low-intensity ultrasound for cancer therapy. Ultrasound Med Biol 2015, 41(4):905-928. doi:10.1016/j.ultrasmedbio.2014.11.019\u003c/li\u003e\n \u003cli\u003eWang Y, Hu B, Diao X, Zhang J: Antitumor effect of microbubbles enhanced by low frequency ultrasound cavitation on prostate carcinoma xenografts in nude mice. Exp Ther Med 2012, 3(2):187-191. doi:10.3892/etm.2011.377\u003c/li\u003e\n \u003cli\u003eHuang P, You X, Pan M, Li S, Zhang Y, Zhao Y, et al: A novel therapeutic strategy using ultrasound mediated microbubbles destruction to treat colon cancer in a mouse model. Cancer Lett 2013, 335(1):183-190. doi:10.1016/j.canlet.2013.02.011\u003c/li\u003e\n \u003cli\u003eZhang C, Huang P, Zhang Y, Chen J, Shentu W, Sun Y, et al: Anti-tumor efficacy of ultrasonic cavitation is potentiated by concurrent delivery of anti-angiogenic drug in colon cancer. Cancer Lett 2014, 347(1):105-113. doi:10.1016/j.canlet.2014.01.022\u003c/li\u003e\n \u003cli\u003eBazan-Peregrino M, Arvanitis CD, Rifai B, Seymour LW, Coussios CC: Ultrasound-induced cavitation enhances the delivery and therapeutic efficacy of an oncolytic virus in an in vitro model. J Control Release 2012, 157(2):235-242. doi:10.1016/j.jconrel.2011.09.086\u003c/li\u003e\n \u003cli\u003eArvanitis CD, Bazan-Peregrino M, Rifai B, Seymour LW, Coussios CC: Cavitation-enhanced extravasation for drug delivery. Ultrasound Med Biol 2011, 37(11):1838-1852. doi:10.1016/j.ultrasmedbio.2011.08.004\u003c/li\u003e\n \u003cli\u003eWillmann JK, Kimura RH, Deshpande N, Lutz AM, Cochran JR, Gambhir SS: Targeted contrast-enhanced ultrasound imaging of tumor angiogenesis with contrast microbubbles conjugated to integrin-binding knottin peptides. J Nucl Med 2010, 51(3):433-440. doi:10.2967/jnumed.109.068007\u003c/li\u003e\n \u003cli\u003eSiegel RL, Miller KD, Jemal A: Cancer statistics, 2020. CA Cancer J Clin 2020, 70(1):7-30. doi:10.3322/caac.21590\u003c/li\u003e\n \u003cli\u003eJonna S, Subramaniam DS: Molecular diagnostics and targeted therapies in non-small cell lung cancer (NSCLC): an update. Discov Med 2019, 27(148):167-170.\u003c/li\u003e\n \u003cli\u003eSankar K, Gadgeel SM, Qin A: Molecular therapeutic targets in non-small cell lung cancer. Expert Rev Anticancer Ther 2020, 20(8):647-661. doi:10.1080/14737140.2020.1787156\u003c/li\u003e\n \u003cli\u003eKazazi-Hyseni F, Beijnen JH, Schellens JH: Bevacizumab. Oncologist 2010, 15(8):819-825. doi:10.1634/theoncologist.2009-0317\u003c/li\u003e\n \u003cli\u003eCarmeliet P, Jain RK: Angiogenesis in cancer and other diseases. Nature 2000, 407(6801):249-257. doi:10.1038/35025220\u003c/li\u003e\n \u003cli\u003eHanahan D, Weinberg RA: Hallmarks of cancer: the next generation. Cell 2011, 144(5):646-674. doi:10.1016/j.cell.2011.02.013\u003c/li\u003e\n \u003cli\u003eHanahan D, Folkman J: Patterns and emerging mechanisms of the angiogenic switch during tumorigenesis. Cell 1996, 86(3):353-364. doi:10.1016/s0092-8674(00)80108-7\u003c/li\u003e\n \u003cli\u003eSiveen KS, Prabhu K, Krishnankutty R, Kuttikrishnan S, Tsakou M, Alali FQ, et al: Vascular Endothelial Growth Factor (VEGF) Signaling in Tumour Vascularization: Potential and Challenges. Curr Vasc Pharmacol 2017, 15(4):339-351. doi:10.2174/1570161115666170105124038\u003c/li\u003e\n \u003cli\u003eFerrara N, Gerber HP, LeCouter J: The biology of VEGF and its receptors. Nat Med 2003, 9(6):669-676. doi:10.1038/nm0603-669\u003c/li\u003e\n \u003cli\u003eFerrara N, Hillan KJ, Gerber HP, Novotny W: Discovery and development of bevacizumab, an anti-VEGF antibody for treating cancer. Nat Rev Drug Discov 2004, 3(5):391-400. doi:10.1038/nrd1381\u003c/li\u003e\n \u003cli\u003eFerrara N, Adamis AP: Ten years of anti-vascular endothelial growth factor therapy. Nat Rev Drug Discov 2016, 15(6):385-403. doi:10.1038/nrd.2015.17\u003c/li\u003e\n \u003cli\u003eSandler A, Gray R, Perry MC, Brahmer J, Schiller JH, Dowlati A, et al: Paclitaxel-carboplatin alone or with bevacizumab for non-small-cell lung cancer. N Engl J Med 2006, 355(24):2542-2550. doi:10.1056/NEJMoa061884\u003c/li\u003e\n \u003cli\u003eReck M, von Pawel J, Zatloukal P, Ramlau R, Gorbounova V, Hirsh V, et al: Overall survival with cisplatin-gemcitabine and bevacizumab or placebo as first-line therapy for nonsquamous non-small-cell lung cancer: results from a randomised phase III trial (AVAiL). Ann Oncol 2010, 21(9):1804-1809. doi:10.1093/annonc/mdq020\u003c/li\u003e\n \u003cli\u003eReck M, von Pawel J, Zatloukal P, Ramlau R, Gorbounova V, Hirsh V, et al: Phase III trial of cisplatin plus gemcitabine with either placebo or bevacizumab as first-line therapy for nonsquamous non-small-cell lung cancer: AVAil. J Clin Oncol 2009, 27(8):1227-1234. doi:10.1200/JCO.2007.14.5466\u003c/li\u003e\n \u003cli\u003ePatel JD, Socinski MA, Garon EB, Reynolds CH, Spigel DR, Olsen MR, et al: PointBreak: a randomized phase III study of pemetrexed plus carboplatin and bevacizumab followed by maintenance pemetrexed and bevacizumab versus paclitaxel plus carboplatin and bevacizumab followed by maintenance bevacizumab in patients with stage IIIB or IV nonsquamous non-small-cell lung cancer. J Clin Oncol 2013, 31(34):4349-4357. doi:10.1200/JCO.2012.47.9626\u003c/li\u003e\n \u003cli\u003eAssoun S, Brosseau S, Steinmetz C, Gounant V, Zalcman G: Bevacizumab in advanced lung cancer: state of the art. Future Oncol 2017, 13(28):2515-2535. doi:10.2217/fon-2017-0302\u003c/li\u003e\n \u003cli\u003eWu J, Nyborg WL: Ultrasound, cavitation bubbles and their interaction with cells. Adv Drug Deliv Rev 2008, 60(10):1103-1116. doi:10.1016/j.addr.2008.03.009\u003c/li\u003e\n \u003cli\u003eSboros V: Response of contrast agents to ultrasound. Adv Drug Deliv Rev 2008, 60(10):1117-1136. doi:10.1016/j.addr.2008.03.011\u003c/li\u003e\n \u003cli\u003eNeumann D, Kollorz E: \u003cstrong\u003eUltrasound\u003c/strong\u003e. In: \u003cem\u003eMedical Imaging Systems: An Introductory Guide.\u003c/em\u003e edn. Edited by Maier A, Steidl S, Christlein V, Hornegger J. Cham (CH); 2018: 237-249.\u003c/li\u003e\n \u003cli\u003eMatthews MJ, Stretanski MF: \u003cstrong\u003eUltrasound Therapy\u003c/strong\u003e. In: \u003cem\u003eStatPearls.\u003c/em\u003e edn. Treasure Island (FL); 2021.\u003c/li\u003e\n \u003cli\u003eLiu Z, Gao S, Zhao Y, Li P, Liu J, Li P, et al: Disruption of tumor neovasculature by microbubble enhanced ultrasound: a potential new physical therapy of anti-angiogenesis. Ultrasound Med Biol 2012, 38(2):253-261. doi:10.1016/j.ultrasmedbio.2011.11.007\u003c/li\u003e\n \u003cli\u003eFan X, Wang L, Guo Y, Xiong X, Zhu L, Fang K: Inhibition of prostate cancer growth using doxorubicin assisted by ultrasound-targeted nanobubble destruction. Int J Nanomedicine 2016, 11:3585-3596. doi:10.2147/IJN.S111808\u003c/li\u003e\n \u003cli\u003eIbsen S, Benchimol M, Simberg D, Esener S: Ultrasound mediated localized drug delivery. Adv Exp Med Biol 2012, 733:145-153. doi:10.1007/978-94-007-2555-3_14\u003c/li\u003e\n \u003cli\u003eZhou XL, Shi YL, Li X: Inhibitory effects of the ultrasound-targeted microbubble destruction-mediated herpes simplex virus-thymidine kinase/ganciclovir system on ovarian cancer in mice. Exp Ther Med 2014, 8(4):1159-1163. doi:10.3892/etm.2014.1877\u003c/li\u003e\n \u003cli\u003eLogan KA, Nesbitt H, Callan B, Gao J, McKaig T, Taylor M, et al: Synthesis of a gemcitabine-modified phospholipid and its subsequent incorporation into a single microbubble formulation loaded with paclitaxel for the treatment of pancreatic cancer using ultrasound-targeted microbubble destruction. Eur J Pharm Biopharm 2021, 165:374-382. doi:10.1016/j.ejpb.2021.05.018\u003c/li\u003e\n \u003cli\u003eGao J, Logan KA, Nesbitt H, Callan B, McKaig T, Taylor M, et al: A single microbubble formulation carrying 5-fluorouridine, Irinotecan and oxaliplatin to enable FOLFIRINOX treatment of pancreatic and colon cancer using ultrasound targeted microbubble destruction. J Control Release 2021, 338:358-366. doi:10.1016/j.jconrel.2021.08.050\u003c/li\u003e\n \u003cli\u003eWu Y, Sun T, Tang J, Liu Y, Li F: Ultrasound-Targeted Microbubble Destruction Enhances the Antitumor Efficacy of Doxorubicin in a Mouse Hepatocellular Carcinoma Model. Ultrasound Med Biol 2020, 46(3):679-689. doi:10.1016/j.ultrasmedbio.2019.09.017\u003c/li\u003e\n \u003cli\u003ePaliwal S, Mitragotri S: Ultrasound-induced cavitation: applications in drug and gene delivery. Expert Opin Drug Deliv 2006, 3(6):713-726. doi:10.1517/17425247.3.6.713\u003c/li\u003e\n \u003cli\u003eter Haar G: Safety and bio-effects of ultrasound contrast agents. Med Biol Eng Comput 2009, 47(8):893-900. doi:10.1007/s11517-009-0507-3\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":"UTMD, Bevacizumab, lung cancer, angiogenesis, CD34","lastPublishedDoi":"10.21203/rs.3.rs-6286565/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6286565/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"In this study, we aimed to investigate the influence of ultrasound-targeted microbubble destruction on the anticancer effect of Bevacizumab in a mouse model of lung cancer. A xenograft mouse tumor model was established by subcutaneously injecting Lewis Lung-GFP cells in the flank of nude mice. 15 nude mice bearing subcutaneous tumors were randomly divided into 3 groups. Group A served as a control group without any treatment (Control). Group B received Bevacizumab and administrations of ultrasound contrast agents and sham cavitation treatment (Bevacizumab + sham cavitation). Group C received Bevacizumab combined with ultrasound-targeted microbubble destruction (Bevacizumab +UTMD). Each group was given corresponding treatment once a day for consecutive 3 days. The tumor microvessel density (MVD) was calculated with Contrast-enhanced ultrasound imaging (CEUS) by vascular length and tumor surface area. Tumor weight was measured by scales after resection. Hematoxylin and eosin staining of samples were performed and the expression of CD34 was detected by immunohistochemistry. Results indicated that the tumor size (volume and weight) of group B and group C were smaller than group A (p\u003c0.05) after treatment, and group C was more effective in inhibiting tumor growth than group B (p\u003c0.05). The MVD and CD34 expression of group B and group C were lower than Group A (p\u003c0.05) after treatment, and group C was more significant in the inhibition of angiogenesis than group B(p\u003c0.05). In conclusion, Bevacizumab could inhibit tumor growth and angiogenesis in lung cancer xenografts, and ultrasound-targeted microbubble destruction significantly enhanced the antitumor effect of Bevacizumab.","manuscriptTitle":"Bevacizumab delivery mediated by ultrasound-targeted microbubble destruction inhibited tumor growth and angiogenesis in lung cancer xenografts","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-09 05:37:57","doi":"10.21203/rs.3.rs-6286565/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":"c19908e8-945e-4c76-b9fa-347e9fd9cc97","owner":[],"postedDate":"June 9th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-09-27T05:23:47+00:00","versionOfRecord":[],"versionCreatedAt":"2025-06-09 05:37:57","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6286565","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6286565","identity":"rs-6286565","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}