Effect of Intrapleural Anti-Vascular Endothelial Growth Factor (VEGF) Associated With NAB Paclitaxel in a Murine Model of Malignant Pleural Effusion

Effect of Intrapleural Anti-Vascular Endothelial Growth Factor (VEGF) Associated With NAB Paclitaxel in a Murine Model of Malignant Pleural Effusion | 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 Effect of Intrapleural Anti-Vascular Endothelial Growth Factor (VEGF) Associated With NAB Paclitaxel in a Murine Model of Malignant Pleural Effusion Carlos Sergio Rocha SIlva, Lisete Ribeiro Teixeira, Karina Rocha Pereira, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5389405/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 02 Aug, 2025 Read the published version in BMC Cancer → Version 1 posted 4 You are reading this latest preprint version Abstract Background: Malignant pleural effusion (MPE) signals a disease with poor prognosis, high morbid-mortality. Studies of MPE in murine models have reproduced its formation and metastases, and have propose therapies for its control. Drugs associated with nanoparticles (nab) can reduce toxicity and increase action on the tumor cell. Our objective was to evaluate nab-Paclitaxel associated or not with intrapleural anti-VEGF in model of MPE. Methods: Intrapleural LLC-cells were injected in 250 mice. After 7 days, mice received weekly intrapleurally Paclitaxel, nab-Paclitaxel, anti-VEGF, nab-Paclitaxel+anti-VEGF or Saline (untreated). Twenty animals from each group weekly were weighed, evaluated for mobility and followed until death. Ten animals from each group were euthanized on 7th, 14th or 21st day for evaluation of pleural fluid volume, cytology, LDH, IL-6, VEGF, TNF-α and histological analysis. Results: Pleural carcinomatosis was lethal in all groups, with longer survival in nab-Paclitaxel+anti-VEGF group. Pleural fluid was greater in untreated and Paclitaxel groups, with the lowest levels in nab-Paclitaxel+anti-VEGF group. Tumor implantation in pleura was observed in all groups with the lowest scores in animals that received nab-Paclitaxel+anti-VEGF. Conclusions: In this experimental model of MPE, intrapleural nab-Paclitaxel associated with anti-VEGF significantly increased survival time, reduced pleural fluid volume and pleural implants. malignant pleural effusion nanoparticles Vascular Endothelial Growth Factor experimental model Figures Figure 1 Figure 2 Figure 3 Figure 4 Background Malignant Pleural Effusion (MPE) is a common complication of advanced Non-Small Cell Lung Cancer (NSCLC) and is associated with low life expectancy 1,2 . Pleural effusion in lung adenocarcinoma occurs by direct invasion of the visceral and parietal membrane or by vascular dissemination of tumor cells to other lung areas and the visceral membrane, with secondary implants in the parietal pleura 2 . For treatment of malignant pleural effusion, several factors are considered, such as histological type, life expectancy, symptomatology, general clinical status, and response to oncological treatments 3,4 . Studies have tried to determine which is the best combination among the chemotherapeutic drugs available to reduce mortality in patients with local or metastatic advanced lung cancer without driver mutations. Still, it has not yet been possible to determine a standard and consensual treatment 5,6 . Paclitaxel has been widely used in treatments for several types of cancers with healing potential, including NSCLC, as a first-line treatment in combination with a platinum compound or as a single agent in patients who are not candidates to target therapy, surgery, and radiotherapy 7,8 . When Paclitaxel is bound to albumin (nab-Paclitaxel), it allows better drug delivery to tumor cells due to more effective absorption and less loss in its dynamics 9-11 . Another line of treatment is monoclonal antibodies that may have direct or indirect action in controlling MPE, such as anti-Vascular Endothelial Growth Factor (VEGF). Studies have shown that angiogenesis is an essential factor in the formation of MPE and that metastatic pleural tumor cells infiltrate lymphatic vessels and block the absorption of pleural fluid, which triggers local inflammation in the pleura. Preclinical and clinical studies have also shown that lowering VEGF levels in patients with NSCLC can significantly control pleural effusion 12-14 . Therefore, VEGF-targeted therapies may be a viable palliative strategy for patients with MPE. Anti-VEGF is a humanized murine monoclonal antibody (MAbs) that binds to the VEGF receptor of neoplastic cells. It can be used in therapeutic approaches to block VEGF and inhibit tumor neovascularization by depriving oxygen and nutrients of the tumor environment 12,13 . Experimental animal models mimic the human condition, contributing to a better understanding of the mechanisms involved in the formation of malignant pleural fluid and in possible therapeutic strategies 14 . Recent studies using animal model have demonstrated the possibility of obtaining a less permissive microenvironment for the development of MPE using a single drug or a combination of chemotherapeutic agents administered intrapleurally 11,12,15 . Therefore, the aim of this study was to analyze in a murine model of MPE the performance of intrapleural therapy with Paclitaxel, nab-Paclitaxel, anti-VEGF or the association of nab-Paclitaxel plus anti-VEGF MPE. Methods Cell culture Lewis Lung Carcinoma (LLC) cells were purchased from the American Type Culture Collection (Manassas, VA – USA) and were cultured at 37°C in 5% CO2 –95% air using Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum. Animal model Two hundred and fifty male (6-8 weeks old) C57Bl/6 mice were obtained from the University of São Paulo/School of Medicine - Laboratory Animal Center. Animal care, methods and procedures experimental were approved by the University Ethics Committee (CEUA 1216/2019). Animals were anesthetized using 35 mg/kg of ketamine hydrochloride (Cristalia, Brazil) and 5 mg/kg of xylazine hydrochloride (Bayer, Brazil) prior to all procedures. The intrapleural injection of 0.5 x 10 5 LLC cells was performed according to previous described methodology 16 . Four groups of 50 mice each received treatment with anti-VEGF (15mg/kg), nab-Paclitaxel (10mg/kg), anti-VEGF + nab-Paclitaxel, Paclitaxel (30 mg/kg) or saline (untreated) at 7, 14, and 21 days after LLC implantation. These animals were subdivided into two study groups; the first consisted of five sets of 30 animals each, which were euthanized after 7, 14, and 21 days; the second group (five sets of 20 animals) was evaluated for survival expectancy. The mice were euthanized according to the study calendar. The abdominal wall was opened, and the viscera were retracted to visualize the diaphragm. Pleural fluid, when present, was gently aspirated, and the volume was measured and placed in tubes for posterior evaluation 14 . Weight, mobility, and survival analysis After the inoculation procedure, all animals were observed until complete recovery, and they were evaluated for weight (g) and mobility by a subjective score of 0-4 (0 = normal and 4 = stillness) 16 . We monitored mortality daily for all groups to obtain the survival curve. Biochemical assays Lactic dehydrogenase (kinetic UV method) and total protein (Biuret method) were quantified in the pleural fluid using commercial kits (Wienner, Argentina) and analyzed in a semi-automatic device. Cytology Pleural fluid cells were counted in a Neubauer chamber. After centrifugation, cells were prepared, and the slides were stained using Leishman to determine the cell differential. Cytokine analysis For Interleukin-6 (IL-6), Tumor Necrosis Factor-alpha (TNF-α), and VEGF analysis (R&D System, Minneapolis, USA), samples of pleural effusion were collected at the same time points in EDTA tubes, centrifuged and the supernatant was removed and stored for later determination. Cytokine levels were measured by Enzyme-Linked Immunoabsorbent Assay (ELISA) according to the protocol suggested by the manufacturer. Minimum detection levels were 15.6 pg/mL. Histological analysis Tissue samples were stained with Hematoxylin & Eosin (H&E) and evaluated qualitatively for indices such as inflammatory infiltrate in the pulmonary parenchyma, tumors in the visceral pleura, parietal pleura, diaphragm, and heart (cardiac muscle and pericardium). These indices were evaluated by two observers and graded on a scale from 0 to 4, according to their extent and severity 14 . Statistical analysis Data were submitted to descriptive analysis demonstrated through mean and standard deviation for each parameter. For comparative analysis, the one-way ANOVA test was used, followed by a multiple comparison test. In the analysis of survival, comparisons were made between the curves using the Kaplan-Meier statistical test, and the survivors were compared using the log-rank test. All statistical procedures were performed using the SigmaStat 3.1 program (Systat, California/United States), with a statistical significance level set at p < 0.05. Data Availability The datasets used and/or analysed during the current study available from the corresponding author on reasonable request. Results After intrapleural injection of LLC cells, all animals developed MPE and presented progressive weight loss (Figure 1). Weight loss was more pronounced after 14 days in the untreated anti-VEGF and Paclitaxel groups when compared to the nab-Paclitaxel and anti-VEGF + nab-Paclitaxel groups. After 21 days, the anti-VEGF + nab-Paclitaxel group showed the lowest weight loss (p<0.05) (Figure 1). There was a progressive reduction in mobility in all groups, with the highest score earlier in the Paclitaxel group and later in the anti-VEGF + nab-Paclitaxel group (p<0.05). All animals developed pleural tumors with diffuse implantation and evident hemorrhagic pleural effusion. In the temporal evaluation, there was a progressive increase in the volume of pleural effusion in all groups. However, the volume was significantly lower in animals receiving anti-VEGF, nab-Paclitaxel, and anti-VEGF + nab-paclitaxel (p<0.05) (Figure 2). A progressive increase in pleural fluid cellularity, LDH, VEGF, and IL-6 was observed with the evolution of the disease (Figure 2). Pleural fluid cytological analysis showed significantly lower cellularity in anti-VEGF (14d = 8388 ± 6453; 21d = 11222 ± 7601), anti-VEGF + nab-Paclitaxel (14d = 7600 ± 4537; 21d = 7900 ± 5201), and nab-Paclitaxel (14d = 7500 ± 4380; 21d = 8652 ± 6308) groups than in untreated control (14d = 9400 ± 5297; 21d = 13565 ± 12576) and Paclitaxel (14d = 11900 ± 3227) groups at all times (14 d - p=0.042 and 21 d - p=0.028). MPE presented a mixed inflammatory infiltrate and macrophages interspersed with malignant cells. LDH levels (U/L) were progressively increased in all groups, especially in the groups Paclitaxel (17000 ± 372) and saline (16220 ± 780) on the 14th day after neoplasm induction. The group treated with anti-VEGF + nab-Paclitaxel (5630 ± 376) presented the lowest levels of LDH compared to other groups (p<0.001). We did not observe statistical differences in total protein levels in the pleural fluid of treated or the control group. Pleural fluid TNF-α levels in treated and control mice were indetectable (<15.6 pg/mL). Nevertheless, the levels of VEGF and IL-6 were significantly higher on the 21st day in the control untreated group (p<0.05) (Figure 2). Tumor implantation in the pleura was observed in all groups from the 14th day, with the lowest scores in animals that received anti-VEGF + nab-Paclitaxel (Figure 3). On the 21st day, the untreated group showed the maximum degree pleural implantation. Tumor implants were observed in the heart (cardiac muscle and pericardium) after the 14th day, mainly in the untreated group. Metastasis to the kidneys, spleen, and liver were not noted. Pleural carcinomatosis was lethal in all groups, with a mortality of 100% after 20 days in the Paclitaxel group, 24 days in the anti-VEGF group, 25 days in the untreated and nab-Paclitaxel groups, and after 30 days in the group that received anti-VEGF + nab-Paclitaxel (p=0.032) (Figure 4). Discussion Lung cancer is a disease with high mortality and represents a challenge for therapeutic approach. Of note, MPE becomes an aggravating factor of the pathology since malignant cells in suspension may fall into the systemic circulation leading to metastasis 17,18 . Our experimental model of neoplasia induced by LLC cells intended to simulate a MPE environment for better understand the pathophysiology of lung cancer implantation in the parietal and visceral pleura, and analyze the evolution of MPE after treatment with intrapleural infusion of paclitaxel, anti-VEGF, nab-paclitaxel, and association of anti-VEGF plus nab-paclitaxel, compared to a control group injected with saline. After intrapleural injection of LLC cells, all animals developed intrapleural tumor implantation with MPE. With the evolution of the disease, we observed progressive weight loss, evidenced from the 14th day after LLC cell implantation. Comparing the groups, the more pronounced weight loss after 14 days was in control, anti-VEGF, and Paclitaxel groups, when compared with nab-Paclitaxel and anti-VEGF + nab-Paclitaxel groups. After 21 days, the anti-VEGF + nab-Paclitaxel group showed the lowest weight loss compared to other groups. These results suggest a less aggressive effect of the tumor in the group treated with the combination of anti-VEGF + nab-Paclitaxel in relation to mobility and metabolic activity with caloric expenditure, promoting a need for food intake with the maintenance of muscle mass, as suggested in previous studies that evaluated weight loss and quality of life in patients with advanced cancer 19 . Similar to weight loss, the activity of animals evaluated for mobility was temporally decreased with disease progression. The decrease in mobility was precocious in the group treated with Paclitaxel (after the 7th day) when compared with the group anti-VEGF + nab-Paclitaxel, indicating a beneficial effect with the combination of these two drugs 19 . In the daily follow-up to assess survival after neoplasm induction, we observed that pleural carcinomatosis was lethal in all evaluated groups, regardless of treatment, with a mortality rate of 100%. Animal death occurred earlier in the Paclitaxel group (after 20 days), and with 24 days in the anti-VEGF group, 25 days in the untreated and nab-Paclitaxel groups, and with 30 days in the group that received Anti-VEGF + nab-Paclitaxel (p=0.032). In this experimental study, we observed, an increase in the overall survival of the group treated with anti-VEGF + nab-Paclitaxel, five days higher than the other groups. Our results are consistent with previous results, which indicate that the best treatment for this neoplasm is a combination of the two drugs 20 . A progressive increase in pleural fluid volume was observed in all groups from the 14th day. The Paclitaxel group had a greater pleural fluid volume recovered than all other groups, possibly due to the caustic action of the vehicles castor oil and ethanol used in its formulation. Other chemotherapy drugs are known to be toxic e.g., docetaxel that solubilized in sorbitol (Tween® 80) promotes intense systemic and local adverse effects, and is being routinely administered intravenously and slowly; however, this agent is not recommended for instillation in the pleural space 21 . Our results showed that these agents injected in the pleural space led to an intense inflammatory reaction, promoting the exudation of pleural mesothelial cells and, consequently, an abundant production of pleural effusion. Previous studies used the same method with Paclitaxel as a sclerosing agent to promote an inflammatory response for pleurodesis induction and for local chemotherapy treatment. The results in relation to pleurodesis seemed promising. However, despite using different concentrations from those used in our work, the adverse effects were the same as those observed by us 22,23 . Pleural fluid volume was significantly lower on the 14th day in all animals receiving anti-VEGF, nab-Paclitaxel, and Anti-VEGF + nab-Paclitaxel. The results found in the targeted therapy with anti-VEGF alone were somewhat satisfactory if compared to the group treated with Paclitaxel, and mainly with the untreated group, suggesting its use as an adjuvant in association with a chemotherapy drug 14 . Similar to the group treated with anti-VEGF alone, the group that received treatment on the 7th and 14th days with nab-Paclitaxel had lower levels of pleural fluid volume when compared with the groups treated with Paclitaxel and the untreated group on the 14th and 21st days. Such behavior makes us believe that the action of nanoparticles is responsible for a more effective delivery of the chemotherapy drug to the tumor environment. By providing the tumor cells with albumin carried by the chemotherapy drug Paclitaxel in an encapsulated format, less loss between instillation and absorption by the tumor environment is observed 23 . This effect would promote a more effective chemotherapeutic action in the pleural space, with fewer adverse effects, reducing the tumor mass, the formation of new tumors and, consequently, reducing the number of new vascularization, one of the main causes of the formation of malignant pleural effusion 25,26 . Associated treatments, chemotherapy, and targeted therapy has shown promising action in reducing pleural fluid volume. The association of drugs demonstrated the contribution of each one, nab-Paclitaxel in tumor mass reduction by acting on cell division, through a more effective delivery and a greater concentration of nano-particles, and anti-VEGF in the reduction of neovascularization and permeability by blocking VEGFR 27 . In the group treated only with Paclitaxel, the results found in relation to the levels of pleural fluid suggest an intense inflammatory activity caused by the vehicle used in its formulation 22,23 . Malignant pleural effusion is characterized by a large number of nucleated cells. These cells are recruited after injury suffered by the pleural mesothelium in response to the production of cytokines and by tumor cells in large quantities 28,29 . In our study, we observed this characteristic with an intense inflammatory infiltrate with a high number of neutrophils, lymphocytes, macrophages, and abundant neoplastic cells. VEGF levels in all groups showed a progressive increase following the evolution of the disease. On the 21st day, the saline group presented with three times the value of the treated with anti-VEGF + nab-Paclitaxel group. These results suggest that neovascularization of the tumor environment is necessary for neoplastic cell nutrition and tumor establishment. We observed that IL-6 detected in pleural fluid in our experimental model showed the same behavior as VEGF production. IL-6 is secreted by various cell populations within MPE, including cancer cells, macrophages, and pleural mesothelial cells. The levels of this cytokine increased slightly in all groups, except in the group treated with Paclitaxel at 14 days and the control group at 21 days, which could indicate a progression of the local inflammatory reaction in response to the pleural injury by the developing tumors. The group treated with Anti-VEGF + nab-Paclitaxel had the lowest levels of IL-6 at all times compared to other groups, allowing us to consider the beneficial effects of these associated drugs in decreasing the inflammatory response. In all study groups, we found tumor implants in the pleura and the presence of loose tumors in the pleural space. Extensive tumor masses formed bridges between the lung parenchyma and the rib cage and infiltrated neighboring anatomical structures, including the chest wall, mediastinum, and diaphragm. All groups had high implant scores in the visceral and parietal pleura on the 21 st day, corroborating our previous study 16 . Regarding the histological evaluations of tumor implants in different organs, we observed metastases only in the pericardium and in the cardiac muscle. Although Lam et al. described that the incidence of metastatic tumors involving the heart is rarely diagnosed, their occurrence can vary from 1.2 to 19.1%. Furthermore, Desai et al. 30 described that, despite the low probability, the occurrence of cardiac metastases is closely linked to tumor progression and disease evolution 30 . Conclusions In this experimental murine model of malignant pleural effusion with lung adenocarcinoma cells, intrapleural treatment with paclitaxel loaded with lipid nanoparticles associated with anti-VEGF significantly increased survival time, decreased weight loss and mobility, pleural fluid volume, and, in addition, reduced tumor implant scores. Declarations Ethics approval The methods and procedures experimental were approved by the University Ethics Committee (CEUA 1216/2019). Consent for publication Not applicable Availability of data and materials The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. Competing interests All authors have no conflicts of interest to declare Funding The São Paulo Research Foundation (FAPESP) # 2013/13295-9 Authors' contributions CSRS: Execution of experimental techniques; revision of manuscript and references; LRT: Interpretation of the data and writing the manuscript; KRP: Execution of experimental and laboratorial techniques; PFBC: Contributed to the conception and design of the study; VAA: Execution of experimental and biochemical and cytological analysis; RKSB: Execution of experimental and statistical analysis; ACR: Laboratorial techiques revision of the manuscript; EM: Contributed to the conception and design of the study and revision of manuscript; RLM: Pathological analysis and critical revision of the manuscript for important intellectual content; MMPA: conception and design of the study, acquisition and interpretation of the data, statistical analysis, writing and final review of the manuscript. 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Cite Share Download PDF Status: Published Journal Publication published 02 Aug, 2025 Read the published version in BMC Cancer → Version 1 posted Editorial decision: Revision requested 07 Nov, 2024 Editor assigned by journal 07 Nov, 2024 Submission checks completed at journal 05 Nov, 2024 First submitted to journal 04 Nov, 2024 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. 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Paulo","correspondingAuthor":false,"prefix":"","firstName":"Amanda","middleName":"Cabral","lastName":"Roque","suffix":""},{"id":377651576,"identity":"8d54161d-07df-4531-ba70-e709e6b5be65","order_by":7,"name":"Evaldo Marchi","email":"","orcid":"","institution":"Medical College of Jundiai","correspondingAuthor":false,"prefix":"","firstName":"Evaldo","middleName":"","lastName":"Marchi","suffix":""},{"id":377651577,"identity":"3f521f98-4d1f-4535-9c3c-7fcdccaa3783","order_by":8,"name":"Ronei Luciano Mamoni","email":"","orcid":"","institution":"Medical College of Jundiai","correspondingAuthor":false,"prefix":"","firstName":"Ronei","middleName":"Luciano","lastName":"Mamoni","suffix":""},{"id":377651578,"identity":"a0bd7b15-a760-43c5-86d6-799c42433338","order_by":9,"name":"Milena Marques Pagliarelli Acencio","email":"","orcid":"","institution":"Pulmonary Division, Instituto do Coracao (InCor), Faculdade de Medicina, Universidade de Sao Paulo","correspondingAuthor":false,"prefix":"","firstName":"Milena","middleName":"Marques Pagliarelli","lastName":"Acencio","suffix":""}],"badges":[],"createdAt":"2024-11-04 15:38:41","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5389405/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5389405/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12885-025-14622-x","type":"published","date":"2025-08-02T16:05:22+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":69375916,"identity":"c8428674-72c8-466e-a595-7c809e99edde","added_by":"auto","created_at":"2024-11-19 17:08:53","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":56373,"visible":true,"origin":"","legend":"\u003cp\u003eWeight and mobility/activity of mice injected with LLC cells treated with anti-VEGF and/or nab Paclitaxel, Paclitaxel and untreated. *p\u0026lt;0.05 when compared by temporal evaluation and # p\u0026lt;0.05 when compared the groups.\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5389405/v1/55147cff0d7e5da7f1b91748.jpg"},{"id":69375915,"identity":"eac9e90b-543b-45ae-ab42-8dedc90070b7","added_by":"auto","created_at":"2024-11-19 17:08:53","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":94543,"visible":true,"origin":"","legend":"\u003cp\u003eVolume, VEGF and IL-6 of malignant pleural effusion of mice injected with LLC cells treated with anti-VEGF and/or nab Paclitaxel, Paclitaxel and untreated. *p\u0026lt;0.05 when compared by temporal evaluation and # p\u0026lt;0.05 when compared the groups.\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5389405/v1/e3cd8210d9328d60b05fdd69.jpg"},{"id":69375918,"identity":"e70025a6-e851-42c1-b2ac-bb50abd733d1","added_by":"auto","created_at":"2024-11-19 17:08:53","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":188006,"visible":true,"origin":"","legend":"\u003cp\u003eMicroscopic appearance of pleural fragments after 14 days of mice injected with LLC cells in differents groups. A: Visceral pleura with tumor implants in untreated group; B = Parietal pleura with tumor implants in untreated group; C = Parietal pleura with tumor implants in Paclitaxel group, D = Parietal pleura with tumor implants in Anti-VEGF; E = Visceral pleura with tumor implants in nab Paclitaxel group; F = Parietal pleura with tumor implants in anti-VEGF + nab Paclitaxel group. 100x\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5389405/v1/d02de9ee59c7bfb77bfdec97.jpg"},{"id":69376288,"identity":"80c47f01-19a0-4c62-b0bc-a95a1d3595f3","added_by":"auto","created_at":"2024-11-19 17:16:53","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":62721,"visible":true,"origin":"","legend":"\u003cp\u003eSurvival time evaluation of mice injected with LLC cells treated with anti-VEGF and/or nab Paclitaxel, Paclitaxel and untreated. p=0.032\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5389405/v1/864cb70dd2a83cf653435544.jpg"},{"id":88268204,"identity":"f7d2d7c6-7cf6-40ae-b2c1-4952a9bb482c","added_by":"auto","created_at":"2025-08-04 16:50:01","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":986689,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5389405/v1/b3418565-8d51-4b42-bdb2-223fea1b7513.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eEffect of Intrapleural Anti-Vascular Endothelial Growth Factor (VEGF) Associated With NAB Paclitaxel in a Murine Model of Malignant Pleural Effusion\u003c/p\u003e","fulltext":[{"header":"Background","content":"\u003cp\u003eMalignant Pleural Effusion (MPE) is a common complication of advanced Non-Small Cell Lung Cancer (NSCLC) and is associated with low life expectancy\u003csup\u003e1,2\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003ePleural effusion in lung adenocarcinoma occurs by direct invasion of the visceral and parietal membrane or by vascular dissemination of tumor cells to other lung areas and the visceral \u0026nbsp;membrane, with secondary implants in the parietal pleura\u003csup\u003e2\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eFor treatment of malignant pleural effusion, several factors are considered, such as histological type, life expectancy, symptomatology, general clinical status, and response to oncological treatments\u003csup\u003e3,4\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eStudies have tried to determine which is the best combination among the chemotherapeutic drugs available to reduce mortality in patients with local or metastatic advanced lung cancer without driver mutations. Still, it has not yet been possible to determine a standard and consensual treatment\u003csup\u003e5,6\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003ePaclitaxel has been widely used in treatments for several types of cancers with healing potential, including NSCLC, as a first-line treatment in combination with a platinum compound or as a single agent in patients who are not candidates to target therapy, surgery, and radiotherapy\u003csup\u003e7,8\u003c/sup\u003e. When Paclitaxel is bound to albumin (nab-Paclitaxel), it allows better drug delivery to tumor cells due to more effective absorption and less loss in its dynamics\u003csup\u003e9-11\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eAnother line of treatment is monoclonal antibodies that may have direct or indirect action in controlling MPE, such as anti-Vascular Endothelial Growth Factor (VEGF). Studies have shown that angiogenesis is an essential factor in the formation of MPE and that metastatic pleural tumor cells infiltrate lymphatic vessels and block the absorption of pleural fluid, which triggers local inflammation in the pleura. Preclinical and clinical studies have also shown that lowering VEGF levels in patients with NSCLC can significantly control pleural effusion\u003csup\u003e12-14\u003c/sup\u003e. Therefore, VEGF-targeted therapies may be a viable palliative strategy for patients with MPE.\u003c/p\u003e\n\u003cp\u003eAnti-VEGF is a humanized murine monoclonal antibody (MAbs) that binds to the VEGF receptor of neoplastic cells. It can be used in therapeutic approaches to block VEGF and inhibit tumor neovascularization by depriving oxygen and nutrients of the tumor environment\u003csup\u003e12,13\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eExperimental animal models mimic the human condition, contributing to a better understanding of the mechanisms involved in the formation of malignant pleural fluid and in possible therapeutic strategies\u003csup\u003e14\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eRecent studies using animal model have demonstrated the possibility of obtaining a less permissive microenvironment for the development of MPE using a single drug or a combination of chemotherapeutic agents administered intrapleurally\u003csup\u003e11,12,15\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eTherefore, the aim of this study was to analyze in a murine model of MPE the performance of intrapleural therapy with Paclitaxel, nab-Paclitaxel, anti-VEGF or the association of nab-Paclitaxel plus anti-VEGF MPE.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003eCell culture\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLewis Lung Carcinoma (LLC) cells were purchased from the American Type Culture Collection (Manassas, VA \u0026ndash; USA) and were cultured at 37\u0026deg;C in 5% CO2 \u0026ndash;95% air using Dulbecco\u0026rsquo;s modified Eagle\u0026rsquo;s medium (DMEM) with 10% fetal bovine serum.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnimal model\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTwo hundred and fifty male (6-8 weeks old) C57Bl/6 mice were obtained\u0026nbsp;from the University of S\u0026atilde;o Paulo/School of Medicine - Laboratory Animal Center. Animal care, methods and procedures experimental were approved by the University Ethics Committee (CEUA 1216/2019).\u003c/p\u003e\n\u003cp\u003eAnimals were anesthetized using 35 mg/kg of ketamine hydrochloride (Cristalia, Brazil) and 5 mg/kg of xylazine hydrochloride (Bayer, Brazil) prior to all procedures. The intrapleural injection of 0.5 x 10\u003csup\u003e5\u003c/sup\u003e LLC cells was performed according to previous described methodology\u003csup\u003e16\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eFour groups of 50 mice each received treatment with anti-VEGF (15mg/kg), nab-Paclitaxel (10mg/kg), anti-VEGF + nab-Paclitaxel, Paclitaxel (30 mg/kg) or saline (untreated) at 7, 14, and 21 days after LLC implantation. These animals were subdivided into two study groups; the first consisted of five sets of 30 animals each, which were euthanized after 7, 14, and 21 days; the second group (five sets of 20 animals) was evaluated for survival expectancy.\u003c/p\u003e\n\u003cp\u003eThe mice were euthanized according to the study calendar. The abdominal wall was opened, and the viscera were retracted to visualize the diaphragm. Pleural fluid, when present, was gently aspirated, and the volume was measured and placed in tubes for posterior evaluation\u003csup\u003e14\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWeight, mobility, and survival analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAfter the inoculation procedure, all animals were observed until complete recovery, and they were evaluated for weight (g) and mobility by a subjective score of 0-4 (0 = normal and 4 = stillness)\u003csup\u003e16\u003c/sup\u003e. We monitored mortality daily for all groups to obtain the survival curve.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBiochemical assays\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLactic dehydrogenase (kinetic UV method) and total protein (Biuret method) were quantified in the pleural fluid using commercial kits (Wienner, Argentina) and analyzed in a semi-automatic device.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCytology\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePleural fluid cells were counted in a Neubauer chamber. After centrifugation, cells were prepared, and the slides were stained using Leishman to determine the cell differential.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCytokine analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor Interleukin-6 (IL-6), Tumor Necrosis Factor-alpha (TNF-\u0026alpha;), and VEGF analysis (R\u0026amp;D System, Minneapolis, USA), samples of pleural effusion were collected at the same time points in EDTA tubes, centrifuged and the supernatant was removed and stored for later determination. Cytokine levels were measured by Enzyme-Linked Immunoabsorbent Assay (ELISA) according to the protocol suggested by the manufacturer. Minimum detection levels were 15.6 pg/mL.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHistological analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTissue samples were stained with Hematoxylin \u0026amp; Eosin (H\u0026amp;E) and evaluated qualitatively for indices such as inflammatory infiltrate in the pulmonary parenchyma, tumors in the visceral pleura, parietal pleura, diaphragm, and heart (cardiac muscle and pericardium). These indices were evaluated by two observers and graded on a scale from 0 to 4, according to their extent and severity\u003csup\u003e14\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData were submitted to descriptive analysis demonstrated through mean and standard deviation for each parameter. For comparative analysis, the one-way ANOVA test was used, followed by a multiple comparison test.\u003c/p\u003e\n\u003cp\u003eIn the analysis of survival, comparisons were made between the curves using the Kaplan-Meier statistical test, and the survivors were compared using the log-rank test.\u003c/p\u003e\n\u003cp\u003eAll statistical procedures were performed using the SigmaStat 3.1 program (Systat, California/United States), with a statistical significance level set at p \u0026lt; 0.05.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analysed during the current study available from the corresponding author on reasonable request.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eAfter intrapleural injection of LLC cells, all animals developed MPE and presented progressive weight loss (Figure 1).\u003c/p\u003e\n\u003cp\u003eWeight loss was more pronounced after 14 days in the untreated anti-VEGF and Paclitaxel groups when compared to the nab-Paclitaxel and anti-VEGF + nab-Paclitaxel groups. After 21 days, the anti-VEGF + nab-Paclitaxel group showed the lowest weight loss (p\u0026lt;0.05) (Figure 1).\u003c/p\u003e\n\u003cp\u003eThere was a progressive reduction in mobility in all groups, with the highest score earlier in the Paclitaxel group and later in the anti-VEGF + nab-Paclitaxel group (p\u0026lt;0.05).\u003c/p\u003e\n\u003cp\u003eAll animals developed pleural tumors with diffuse implantation and evident hemorrhagic pleural effusion. In the temporal evaluation, there was a progressive increase in the volume of pleural effusion in all groups. However, the volume was significantly lower in animals receiving anti-VEGF, nab-Paclitaxel, and anti-VEGF + nab-paclitaxel (p\u0026lt;0.05) (Figure 2).\u003c/p\u003e\n\u003cp\u003eA progressive increase in pleural fluid cellularity, LDH, VEGF, and IL-6 was observed with the evolution of the disease (Figure 2).\u003c/p\u003e\n\u003cp\u003ePleural fluid cytological analysis showed significantly lower cellularity in anti-VEGF (14d = 8388 \u0026plusmn; 6453; 21d = 11222 \u0026plusmn; 7601), anti-VEGF + nab-Paclitaxel (14d = 7600 \u0026plusmn; 4537; 21d = 7900 \u0026plusmn; 5201), and nab-Paclitaxel (14d = 7500 \u0026plusmn; 4380; 21d = 8652 \u0026plusmn; 6308) groups than in untreated control (14d = 9400 \u0026plusmn; 5297; 21d = 13565 \u0026plusmn; 12576) and Paclitaxel (14d = 11900 \u0026plusmn; 3227) groups at all times (14 d - p=0.042 and 21 d - p=0.028).\u0026nbsp;MPE presented a mixed inflammatory infiltrate and macrophages interspersed with malignant cells.\u003c/p\u003e\n\u003cp\u003eLDH levels (U/L)\u0026nbsp;were progressively increased in all groups, especially in the groups Paclitaxel (17000 \u0026plusmn; 372) and saline (16220 \u0026plusmn; 780) on the 14th day after neoplasm induction. The group treated with anti-VEGF + nab-Paclitaxel (5630 \u0026plusmn; 376) presented the lowest levels of LDH compared to other groups (p\u0026lt;0.001).\u003c/p\u003e\n\u003cp\u003eWe did not observe statistical differences in total protein levels in the pleural fluid of treated or the control group. Pleural fluid TNF-\u0026alpha;\u0026nbsp;levels in treated and control mice were indetectable (\u0026lt;15.6 pg/mL). Nevertheless, the levels of VEGF and IL-6 were significantly higher on the 21st day in the control untreated group (p\u0026lt;0.05) (Figure 2).\u003c/p\u003e\n\u003cp\u003eTumor implantation in the pleura was observed in all groups from the 14th day, with the lowest scores in animals that received anti-VEGF + nab-Paclitaxel (Figure 3). On the 21st day, the untreated group showed the maximum degree pleural implantation. Tumor implants were observed in the heart (cardiac muscle and pericardium) after the 14th day, mainly in the untreated group. Metastasis to the kidneys, spleen, and liver were not noted.\u003c/p\u003e\n\u003cp\u003ePleural carcinomatosis was lethal in all groups, with a mortality of 100% after 20 days in the Paclitaxel group, 24 days in the anti-VEGF group, 25 days in the untreated and nab-Paclitaxel groups, and after 30 days in the group that received anti-VEGF + nab-Paclitaxel (p=0.032) (Figure 4).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eLung cancer is a disease with high mortality and represents a challenge for therapeutic approach. Of note, MPE becomes an aggravating factor of the pathology since malignant cells in suspension may fall into the systemic circulation leading to metastasis\u003csup\u003e17,18\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eOur experimental model of neoplasia induced by LLC cells intended to simulate a MPE environment for better understand the pathophysiology of lung cancer implantation in the parietal and visceral pleura, and analyze the evolution of MPE after treatment with intrapleural infusion of paclitaxel, anti-VEGF, nab-paclitaxel, and association of anti-VEGF plus nab-paclitaxel, compared to a control group injected with saline.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAfter intrapleural injection of LLC cells, all animals developed intrapleural tumor implantation with MPE. With the evolution of the disease, we observed progressive weight loss, evidenced from the 14th day after LLC cell implantation.\u003c/p\u003e\n\u003cp\u003eComparing the groups, the more pronounced weight loss after 14 days was in control, anti-VEGF, and Paclitaxel groups, when compared with nab-Paclitaxel and anti-VEGF + nab-Paclitaxel groups. After 21 days, the anti-VEGF + nab-Paclitaxel group showed the lowest weight loss compared to other groups.\u003c/p\u003e\n\u003cp\u003eThese results suggest a less aggressive effect of the tumor in the group treated with the combination of anti-VEGF + nab-Paclitaxel in relation to mobility and metabolic activity with caloric expenditure, promoting a need for food intake with the maintenance of muscle mass, as suggested in previous studies that evaluated weight loss and quality of life in patients with advanced cancer\u003csup\u003e19\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eSimilar to weight loss, the activity of animals evaluated for mobility was temporally decreased with disease progression. The decrease in mobility was precocious in the group treated with Paclitaxel (after the 7th day) when compared with the group anti-VEGF + nab-Paclitaxel, indicating a beneficial effect with the combination of these two drugs\u003csup\u003e19\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eIn the daily follow-up to assess survival after neoplasm induction, we observed that pleural carcinomatosis was lethal in all evaluated groups, regardless of treatment, with a mortality rate of 100%. Animal death occurred earlier in the Paclitaxel group (after 20 days), and with 24 days in the anti-VEGF group, 25 days in the untreated and nab-Paclitaxel groups, and with 30 days in the group that received Anti-VEGF + nab-Paclitaxel (p=0.032).\u003c/p\u003e\n\u003cp\u003eIn this experimental study, we observed, an increase in the overall survival of the group treated with anti-VEGF + nab-Paclitaxel, five days higher than the other groups. Our results are consistent with previous results, which indicate that the best treatment for this neoplasm is a combination of the two drugs\u003csup\u003e20\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eA progressive increase in pleural fluid volume was observed in all groups from the 14th day. The Paclitaxel group had a greater pleural fluid volume recovered than all other groups, possibly due to the caustic action of the vehicles castor oil and ethanol used in its formulation. Other chemotherapy drugs are known to be toxic e.g., docetaxel that solubilized in sorbitol (Tween\u0026reg; 80) promotes intense systemic and local adverse effects, and is being routinely administered intravenously and slowly; however, this agent is not recommended for instillation in the pleural space\u003csup\u003e21\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eOur results showed that these agents injected in the pleural space led to an intense inflammatory reaction, promoting the exudation of pleural mesothelial cells and, consequently, an abundant production of pleural effusion.\u003c/p\u003e\n\u003cp\u003ePrevious studies used the same method with Paclitaxel as a sclerosing agent to promote an inflammatory response for pleurodesis induction and for local chemotherapy treatment. The results in relation to pleurodesis seemed promising. However, despite using different concentrations from those used in our work, the adverse effects were the same as those observed by us\u003csup\u003e22,23\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003ePleural fluid volume was significantly lower on the 14th day in all animals receiving anti-VEGF, nab-Paclitaxel, and Anti-VEGF + nab-Paclitaxel. The results found in the targeted therapy with anti-VEGF alone were somewhat satisfactory\u0026nbsp;if compared to the group treated with Paclitaxel, and mainly with the untreated group, suggesting its use as an adjuvant in association with a chemotherapy drug\u003csup\u003e14\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eSimilar to the group treated with anti-VEGF alone, the group that received treatment on the 7th and 14th days with nab-Paclitaxel had lower levels of pleural fluid volume when compared with the groups treated with Paclitaxel and the untreated group on the 14th and 21st days.\u003c/p\u003e\n\u003cp\u003eSuch behavior makes us believe that the action of nanoparticles is responsible for a more effective delivery of the chemotherapy drug to the tumor environment. By providing the tumor cells with albumin carried by the chemotherapy drug Paclitaxel in an encapsulated format, less loss between instillation and absorption by the tumor environment is observed\u003csup\u003e23\u003c/sup\u003e. This effect would promote a more effective chemotherapeutic action in the pleural space, with fewer adverse effects, reducing the tumor mass, the formation of new tumors and, consequently, reducing the number of new vascularization, one of the main causes of the formation of malignant pleural effusion\u003csup\u003e25,26\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eAssociated treatments, chemotherapy, and targeted therapy has shown promising action in reducing pleural fluid volume. The association of drugs demonstrated the contribution of each one, nab-Paclitaxel in tumor mass reduction by acting on cell division, through a more effective delivery and a greater concentration of nano-particles, and anti-VEGF in the reduction of neovascularization and permeability by blocking VEGFR\u003csup\u003e27\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eIn the group treated only with Paclitaxel, the results found in relation to the levels of pleural fluid suggest an intense inflammatory activity caused by the vehicle used in its formulation\u003csup\u003e22,23\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eMalignant pleural effusion is characterized by a large number of nucleated cells. These cells are recruited after injury suffered by the pleural mesothelium in response to the production of cytokines and by tumor cells in large quantities\u003csup\u003e28,29\u003c/sup\u003e. In our study, we observed this characteristic with an intense inflammatory infiltrate with a high number of neutrophils, lymphocytes, macrophages, and abundant neoplastic cells.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eVEGF levels in all groups showed a progressive increase following the evolution of the disease. On the 21st day, the saline group presented with three times the value of the treated with anti-VEGF + nab-Paclitaxel group. These results suggest that neovascularization of the tumor environment is necessary for neoplastic cell nutrition and tumor establishment.\u003c/p\u003e\n\u003cp\u003eWe observed that IL-6 detected in pleural fluid in our experimental model showed the same behavior as VEGF production. IL-6 is secreted by various cell populations within MPE, including cancer cells, macrophages, and pleural mesothelial cells. The levels of this cytokine increased slightly in all groups, except in the group treated with Paclitaxel at 14 days and the control group at 21 days, which could indicate a progression of the local inflammatory reaction in response to the pleural injury by the developing tumors.\u003c/p\u003e\n\u003cp\u003eThe group treated with Anti-VEGF + nab-Paclitaxel had the lowest levels of IL-6 at all times compared to other groups, allowing us to consider the beneficial effects of these associated drugs in decreasing the inflammatory response.\u003c/p\u003e\n\u003cp\u003eIn all study groups, we found tumor implants in the pleura and the presence of loose tumors in the pleural space. Extensive tumor masses formed bridges between the lung parenchyma and the rib cage and infiltrated neighboring anatomical structures, including the chest wall, mediastinum, and diaphragm. All groups had high implant scores in the visceral and parietal pleura on the 21\u003csup\u003est\u003c/sup\u003e day, corroborating our previous study\u003csup\u003e16\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eRegarding the histological evaluations of tumor implants in different organs, we observed metastases only in the pericardium and in the cardiac muscle. Although Lam \u003cem\u003eet al.\u003c/em\u003e described that the incidence of metastatic tumors involving the heart is rarely diagnosed, their occurrence can vary from 1.2 to 19.1%. Furthermore, Desai et al.\u003csup\u003e30\u003c/sup\u003e described that, despite the low probability, the occurrence of cardiac metastases is closely linked to tumor progression and disease evolution\u003csup\u003e30\u003c/sup\u003e.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn this experimental murine model of malignant pleural effusion with lung adenocarcinoma cells, intrapleural treatment with paclitaxel loaded with lipid nanoparticles associated with anti-VEGF significantly increased survival time, decreased weight loss and mobility, pleural fluid volume, and, in addition, reduced tumor implant scores.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe methods and procedures experimental were approved by the University Ethics Committee (CEUA 1216/2019).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors have no conflicts of interest to declare\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe São Paulo Research Foundation (FAPESP) # 2013/13295-9\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors' contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCSRS: Execution of experimental techniques; revision of manuscript and references;\u003c/p\u003e\n\u003cp\u003eLRT: Interpretation of the data and writing the manuscript;\u003c/p\u003e\n\u003cp\u003eKRP: Execution of experimental and laboratorial techniques;\u003c/p\u003e\n\u003cp\u003ePFBC: Contributed to the conception and design of the study;\u003c/p\u003e\n\u003cp\u003eVAA: Execution of experimental and biochemical and cytological analysis;\u003c/p\u003e\n\u003cp\u003eRKSB: Execution of experimental and statistical analysis;\u003c/p\u003e\n\u003cp\u003eACR: Laboratorial techiques revision of the manuscript;\u003c/p\u003e\n\u003cp\u003eEM: Contributed to the conception and design of the study and revision of manuscript;\u003c/p\u003e\n\u003cp\u003eRLM: Pathological analysis and critical revision of the manuscript for important intellectual content;\u003c/p\u003e\n\u003cp\u003eMMPA: conception and design of the study, acquisition and interpretation of the data, statistical analysis, writing and final review of the manuscript.\u003c/p\u003e\n\u003cp\u003eAll authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003ePorcel JM, Gasol A, Bielsa S, Civit C, Light RW, Salud A. 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J Cancer Res Clin Oncol. 2020;146:631\u0026ndash;645.\u003c/li\u003e\n\u003cli\u003eAcencio MMP, Puka J, Alvarenga VA, Martins V, de Carvalho ML, Marchi E, et al. Intrapleural targeted therapies (anti-VEGF and anti-EGFR) in the model of malignant pleural effusion. Oncotarget. 2017;8:105093\u0026ndash;105102.\u003c/li\u003e\n\u003cli\u003eJantz MA, Antony VB. Pathophysiology of the pleura. Respiration. 2008;75:121\u0026ndash;133.\u003c/li\u003e\n\u003cli\u003eAcencio MM, Puka J, Marchi E, Antonangelo L, Terra RM, Vargas FS, et al. A modified experimental model of malignant pleural disease induced by lung Lewis carcinoma (LLC) cells. J Transl Med. 2015;15:302.\u003c/li\u003e\n\u003cli\u003ePorcel J, Esquerda A, Vives M, Bielsa S. Etiology of pleural effusions: analysis of more than 3,000 consecutive thoracenteses. Arch Bronconeumol. 2014;50:161\u0026ndash;165.\u003c/li\u003e\n\u003cli\u003eJunior CTS, Marchi E, Teixeira LR. 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Expert Opin Pharmacother. 2008;9:2603\u0026ndash;2616.\u003c/li\u003e\n\u003cli\u003ePerng RP, Chen YM, Wu MF, Chou KC, Lin WC, Liu JM, et al. Phase II trial of intrapleural paclitaxel injection for non-small-cell lung cancer patients with malignant pleural effusions. Respir Med. 1998;92:473\u0026ndash;479.\u003c/li\u003e\n\u003cli\u003eLombardi G, Nicoletto MO, Gusella M, Fiduccia P, Dalla Palma M, Zuin A, et al. Intrapleural paclitaxel for malignant pleural effusion from ovarian and breast cancer: a phase II study with pharmacokinetic analysis. Cancer Chemother Pharmacol. 2012;69:781\u0026ndash;787.\u003c/li\u003e\n\u003cli\u003eWang J, Li Y, Nie G. Multifunctional biomolecule nanostructures for cancer therapy. Nat Rev Mater. 2021;19:1\u0026ndash;18.\u003c/li\u003e\n\u003cli\u003eGradishar WJ. Albumin-bound paclitaxel: a next-generation taxane. Expert Opin Pharmacother. 2006;7:1041\u0026ndash;1053.\u003c/li\u003e\n\u003cli\u003eGradishar WJ, Tjulandin S, Davidson N, Shaw H, Desai N, Bhar P, et al. Phase III trial of nanoparticle albumin-bound paclitaxel compared with polyethoxylated castor oil-based paclitaxel in women with breast cancer. J Clin Oncol. 2005;23:7794\u0026ndash;7803.\u003c/li\u003e\n\u003cli\u003eHao X, Zhu Y, Mu Y, Wang S, Li J, Xing P. Nab-paclitaxel in combination with Bevacizumab in patients with non-squamous non-small cell lung cancer after failure of at least one prior systemic regimen. J Cancer. 2020 Sep 11;6421\u0026ndash;6428.\u003c/li\u003e\n\u003cli\u003eAntonagelo L. In: Vargas FS, Teixeira LR, Marchi E, editors. Derrame Pleural. S\u0026atilde;o Paulo: Editora Roca; 2004. p. 125\u0026ndash;142.\u003c/li\u003e\n\u003cli\u003eKerkar SP, Restifo NP. Cellular constituents of immune escape within the tumor microenvironment. Cancer Res. 2012;72:3125\u0026ndash;3130.\u003c/li\u003e\n\u003cli\u003eDesai MY, Mankad S. Extension of bronchogenic carcinoma through pulmonary vein into the left atrium detected by echocardiography. Echocardiography. 2004;21:189\u0026ndash;191.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-cancer","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bcan","sideBox":"Learn more about [BMC Cancer](http://bmccancer.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/bcan/default.aspx","title":"BMC Cancer","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"malignant pleural effusion, nanoparticles, Vascular Endothelial Growth Factor, experimental model","lastPublishedDoi":"10.21203/rs.3.rs-5389405/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5389405/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground: \u003c/strong\u003eMalignant pleural effusion (MPE) signals a disease with poor prognosis, high morbid-mortality. Studies of MPE in murine models have reproduced its formation and metastases, and have propose therapies for its control. Drugs associated with nanoparticles (nab) can reduce toxicity and increase action on the tumor cell. Our objective was to evaluate nab-Paclitaxel associated or not with intrapleural anti-VEGF in model of MPE.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods:\u003c/strong\u003e Intrapleural LLC-cells were injected in 250 mice. After 7 days, mice received weekly intrapleurally Paclitaxel, nab-Paclitaxel, anti-VEGF, nab-Paclitaxel+anti-VEGF or Saline (untreated). Twenty animals from each group weekly were weighed, evaluated for mobility and followed until death. Ten animals from each group were euthanized on 7th, 14th or 21st day for evaluation of pleural fluid volume, cytology, LDH, IL-6, VEGF, TNF-α and histological analysis.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults:\u003c/strong\u003e Pleural carcinomatosis was lethal in all groups, with longer survival in nab-Paclitaxel+anti-VEGF group. Pleural fluid was greater in untreated and Paclitaxel groups, with the lowest levels in nab-Paclitaxel+anti-VEGF group. Tumor implantation in pleura was observed in all groups with the lowest scores in animals that received nab-Paclitaxel+anti-VEGF.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions:\u003c/strong\u003e In this experimental model of MPE, intrapleural nab-Paclitaxel associated with anti-VEGF significantly increased survival time, reduced pleural fluid volume and pleural implants.\u003c/p\u003e","manuscriptTitle":"Effect of Intrapleural Anti-Vascular Endothelial Growth Factor (VEGF) Associated With NAB Paclitaxel in a Murine Model of Malignant Pleural Effusion","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-11-19 17:08:48","doi":"10.21203/rs.3.rs-5389405/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-11-07T06:42:06+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-11-07T05:38:03+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-11-05T13:29:05+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Cancer","date":"2024-11-04T15:36:23+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-cancer","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bcan","sideBox":"Learn more about [BMC Cancer](http://bmccancer.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/bcan/default.aspx","title":"BMC Cancer","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"bfd41016-9c72-451a-8957-97894e0f8303","owner":[],"postedDate":"November 19th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-08-04T16:41:42+00:00","versionOfRecord":{"articleIdentity":"rs-5389405","link":"https://doi.org/10.1186/s12885-025-14622-x","journal":{"identity":"bmc-cancer","isVorOnly":false,"title":"BMC Cancer"},"publishedOn":"2025-08-02 16:05:22","publishedOnDateReadable":"August 2nd, 2025"},"versionCreatedAt":"2024-11-19 17:08:48","video":"","vorDoi":"10.1186/s12885-025-14622-x","vorDoiUrl":"https://doi.org/10.1186/s12885-025-14622-x","workflowStages":[]},"version":"v1","identity":"rs-5389405","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5389405","identity":"rs-5389405","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}