Malignant Pleural Effusion Formation Research Articles

S100A9 Regulated M1/M2 Macrophage Polarization in Interleukin-10-Induced Promotion of Malignant Pleural Effusion.

Interleukin-10 (IL-10) promotes the formation and development of malignant pleural effusion (MPE). Previous studies have elucidated the pathogenesis from the view of the immune-regulation function of CD4+ T-cells. However, the underlying mechanism is still not fully understood. In this study, our results showed that IL-10 deficiency reduced the percentage of macrophages in mouse MPE and regulated M1/M2 polarization in vivo and in vitro. The migration capacity of tumor cells was suppressed, and apoptosis was promoted when tumor cells were cocultured with MPE macrophages in the absence of IL-10. Messenger RNA sequencing of MPE macrophages showed that S100A9 was downregulated in IL-10-/- mice. Bone marrow-derived macrophages obtained from wild-type mice transfected with S100A9-specific small interfering RNAs (siRNAs) also showed less M2 and more M1 polarization than those from the siRNA control group. Furthermore, downregulation of S100A9 using S100A9-specific siRNA suppressed MPE development, decreased macrophages, and modulated macrophage polarization in MPE in vivo. In conclusion, S100A9 plays a vital role in the process of IL-10 deficiency-mediated MPE suppression by regulating M1/M2 polarization, thus influencing the tumor-migration capacity and apoptosis. This could result in clinically applicable strategies to inhibit the formation of MPE by regulating the polarization of MPE macrophages.

Exosomal miRNA-profiling of pleural effusion in lung adenocarcinoma and tuberculosis.

Pleural effusion (PE) caused by lung cancer is prevalent, and it is difficult to differentiate it from PE caused by tuberculosis. Exosome-based liquid biopsy offers a non-invasive technique to diagnose benign and malignant PE. Exosomal miRNAs are potential diagnostic markers and play an essential role in signal transduction and biological processes in tumor development. We hypothesized that exosomal miRNA expression profiles in PE would contribute to identifying its diagnostic markers and elucidating the molecular basis of PE formation in lung cancer. The exosomes from PE caused by lung adenocarcinoma (LUAD) and pulmonary tuberculosis were isolated and verified by transmission electron microscopy. The exosomal miRNA profiles were identified using deep sequencing and validated with quantitative real-time PCR (qRT-PCR). We performed bioinformatic analysis for differentially expressed miRNAs to explore how exosomal miRNAs regulate pleural effusion. We identified 99 upregulated and 91 downregulated miRNAs in malignant pleural effusion (MPE) compared to tuberculous pleural effusion (TPE). Seven differentially expressed miRNAs (DEmiRNAs) were validated by qRT-PCR, out of which 5 (71.4%) were confirmed through sequencing. Gene Ontology (GO) analysis revealed that most exosomal miRNAs target genes were involved in regulating cellular processes and nitrogen compound metabolism. According to the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis, the exosomal miRNAs target genes were mainly involved in Fc gamma R-mediated phagocytosis, Rap1 signaling pathway, and breast cancer. The hub genes, including ITGAM, FOXO1, MAPK14, YWHAB, GRIN1, and PRF1, were screened through plug-in cytoHubba. The PFR1 was identified as a critical gene in MPE formation using single-cell sequencing analysis. Additionally, we hypothesized that tumor cells affected natural killer cells and promoted the generation of PE in LUAD via the exosomal hsa-miR-3120-5p-PRF1 axis. We identified exosomal miRNA profiles in LUAD-MPE and TPE, which may help in the differential diagnosis of MPE and TPE. Bioinformatic analysis revealed that these miRNAs might affect PE generation through tumor immune response in LUAD. Our results provided a new theoretical basis for understanding the function of exosomal miRNAs in LUAD-MPE.

Treatment of malignant pleural effusion in non-small cell lung cancer with VEGF-directed therapy

Background Vascular endothelial growth factor (VEGF) is a critical regulator of malignant pleural effusion (MPE) in non-small-cell lung cancer (NSCLC). Bevacizumab (BEV) and apatinib (APA) are novel VEGF blockers that inhibit lung cancer cell proliferation and the development of pleural effusion. Methods In this study, we established Lewis lung cancer (LLC) xenograft mouse models to compare the therapeutic effect of APA and BEV in combination with cisplatin (CDDP) against MPE. The anti-tumour and anti-angiogenic effects of this combination therapy were evaluated by 18F-FDG PET/CT imaging, TUNEL assay and Immunohistochemistry. Results The triple drug combination significantly prolonged the overall survival of the tumour-bearing mice by reducing MPE and glucose metabolism and was more effective in lowering VEGF/soluble VEGFR-2 levels in the serum and pleural exudates compared to either of the monotherapies. Furthermore, CDDP + APA + BEV promoted in vivo apoptosis and decreased microvessel density. Conclusions Mechanistically, LLC-induced MPE was inhibited by targeting the VEGF-MEK/ERK pathways. Further studies are needed to establish the synergistic therapeutic effect of these drugs in NSCLC patients with MPE. KEY MESSAGES Combined treatment of MPE with apatinib, bevacizumab and cisplatin can prolong the survival time of mice, reduce the content of MPE, decrease the SUVmax of thoracic tumour tissue, down-regulate the content of VEGF and sVEGFR-2 in serum and pleural fluid, and promote the apoptosis of tumour cells. Angiogenesis and MPE formation can be inhibited by down-regulation of HIF-1α, VEGF, VEGFR-2, MEK1 and MMP-2 molecular signalling pathway proteins.

CSF1/CSF1R signaling mediates malignant pleural effusion formation.

Malignant pleural effusion (MPE) is an incurable common manifestation of many malignancies. Its formation is orchestrated by complex interactions among tumor cells, inflammatory cells, and the vasculature. Tumor-associated macrophages present the dominant inflammatory population of MPE, and M2 macrophage numbers account for dismal prognosis. M2 polarization is known to be triggered by CSF1/CSF1 receptor (CSF1R) signaling. We hypothesized that CSF1R+ M2 macrophages favor MPE formation and could be therapeutically targeted to limit MPE. We generated mice with CSF1R-deficient macrophages and induced lung and colon adenocarcinoma–associated MPE. We also examined the therapeutic potential of a clinically relevant CSF1R inhibitor (BLZ945) in lung and colon adenocarcinoma–induced experimental MPE. We showed that CSF1R+ macrophages promoted pleural fluid accumulation by enhancing vascular permeability, destabilizing tumor vessels, and favoring immune suppression. We also showed that CSF1R inhibition limited MPE in vivo by reducing vascular permeability and neoangiogenesis and impeding tumor progression. This was because apart from macrophages, CSF1R signals in cancer-associated fibroblasts leading to macrophage inflammatory protein 2 secretion triggered the manifestation of suppressive and angiogenic properties in macrophages upon CXCR2 paracrine activation. Pharmacological targeting of the CSF1/CSF1R axis can therefore be a vital strategy for limiting MPE.

A Retrospective Analysis of the Palliative Surgical Treatment in Patients with Malignant Pleural Effusion

Abstract Introduction: The formation of malignant pleural effusion (MPE) is a clinical manifestation of an advanced malignancy or its dissemination. The focus of treatment is primarily palliative and aimed at relieving symptoms, especially dyspnoea. Material and Methods: Clinical data from patients who were hospitalized at the Clinic of Thoracic Surgery, JFMED CU and Martin University Hospital, in the years 2015–2019 were retrospectively explored and statistically analyzed based on their medical records. Results: From the group of patients with proven MPE (n=67), 32 patients were male (48%) and 35 were female (52%). The mean age was 62.3 years (65.4 for males and 59.4 for females). The three most common primary malignancies were lung cancer (n=24), breast cancer (n=14), and kidney cancer (n=6). In 38 patients with MPE a talc pleurodesis via VATS was performed, with a median survival of 341 days (95% CI 256–859). Drainage following the talc slurry pleurodesis was performed in 10 patients with a median survival of 91.5 days (95% CI 64-NA). Ten patients with MPE underwent drainage only. The overall median survival time after all types of surgical interventions was 301 days (95% CI 207-389 days). Conclusion: Management of MPE depends on the patient´s prognosis. A definitive intervention is required in patients with a long-term survival, while in patients with a short life expectancy procedures leading to the shortest hospital stay are preferred. Videothoracoscopic procedures with pleurodesis represent an effective treatment for patients with symptomatic MPE with a good performance status, presence of lung re-expansion following pleural drainage or expected survival.

IL-10 promotes malignant pleural effusion by regulating TH 1 response via an miR-7116-5p/GPR55/ERK pathway in mice.

IL-10, produced by a wide variety of cells, is a highly pleiotropic cytokine that plays a critical role in the control of immune responses. However, its regulatory activity in tumor immunity remains poorly understood. In this study, we report that IL-10 deficiency robustly suppressed the formation of malignant pleural effusion (MPE) and significantly enhanced miR-7116-5p expression in pleural CD4+ T cells. We demonstrated that miR-7116-5p suppressed IL-10-mediated MPE formation by inhibiting pleural vascular permeability as well as tumor angiogenesis and tumor growth. IL-10 promoted MPE formation by suppressing miR-7116-5p that enhances TH 1 response. We identified Gprotein-coupled receptor 55 (GPR55) as a potential target of miR-7116-5p, and miR-7116-5p promoted TH 1 cell function by downregulating GPR55. Moreover, GPR55 promoted MPE formation by inhibiting TH 1 cell expansion through the ERK phosphorylation pathway. These results uncover an IL-10-mediated pathway controlling TH 1 cells and demonstrate a central role for miR-7116-5p/GPR55/ERK signaling in the physiological regulation of IL-10-driven pro-malignant responses.

Chinese medicine Xiaoshui decoction inhibits malignant pleural effusion in mice and mediates tumor-associated macrophage polarization by activating autophagy.

Xiaoshui decoction (XSD) is a traditional Chinese medicine compound prescription that has been shown to reinforce the spleen and remove the fluid retention, while being widely used in the treatment of malignant pleural effusion (MPE). We previously reported that XSD alleviates symptoms and improves the quality of life in patients with MPE; however, the mechanism employed by XSD on MPE has not yet been elucidated. To investigate the role and mechanism of XSD in inhibiting the development of MPE, and in regulating macrophage polarization in vitro and in vivo. A murine MPE model was used to study the effect of XSD on MPE. Mice with MPE were randomly allocated to a control group and XSD-low-dose (1.144g/mL), XSD-middle-dose (2.288g/mL), XSD-high-dose (4.576g/mL), or cisplatin groups. RAW264.7cells were induced to form tumor-associated macrophages (TAMs) as well as M1 and M2 macrophages using different conditioned media in vitro. XSD effectively inhibited MPE formation, reduced pleural permeability and angiogenesis, and prolonged mice survival. Particularly, XSD treatment induced the polarization of TAMs to the M1 phenotype in MPE. Moreover, in-vitro XSD remarkably promoted the expansion of M1 macrophages and reduced M2 macrophages by enhancing autophagy. XSD inhibits MPE development and regulates macrophage polarization by activating autophagy, indicating that XSD may serve as a novel option for integrative MPE therapies.

IL-17A-Producing γδT Cells Inhibit the Formation of Malignant Pleural Effusions.

γδT cells are an important source of IL-17A and play an anti- or protumor role depending on the surrounding microenvironment. The precise role of γδT cells in the development of malignant pleural effusions (MPE) remains unknown. Using flow cytometry, we analyzed the distribution and differentiation of γδT cells in wild-type (WT) and IL-10-∕- mice. We carefully elucidated the influence of γδT cells on the formation of MPE by depleting γδT cells from WT, IL-10-∕-, and IL-17a-∕- mice. The distribution of γδT17 cells in human MPE and peripheral blood was also determined. Our data showed that both γδT cells and IL-17A-producing γδT (γδT17) cells accumulated in murine MPE, and IL-10 deficiency enhanced their accumulation. γδT cells were the main source of IL-17A in MPE for both WT and IL-10-∕- mice. IL-10 inhibited the chemotactic response of γδT cells to MPE and the proliferation of these cells. IL-10 suppressed γδT cell secretion of IL-17A via RORγt. The ablation of γδT cells accelerated MPE accumulation in both WT and IL-10-∕- mice, but it did not influence MPE accumulation in IL-17a-∕- mice. Patients with higher frequencies of γδT17 cells had significantly longer survival times than patients with lower frequencies of γδT17 cells. Taken together, our data demonstrate that γδT17 cells play an inhibitory role in the progression of MPE, and the accumulation of γδT17 cells in MPE is suppressed by IL-10.

Progress of Bevacizumab in Malignant Pleural Effusion Caused by Non-small Cell Lung Cancer

肺癌是全球范围内发病率最高的恶性肿瘤，晚期肺癌所致的恶性胸腔积液（malignant pleural effusion, MPE）严重影响患者的生活质量和预后。MPE的治疗手段包括胸腔穿刺术、胸膜固定术、胸腔埋管引流、胸腔内灌注治疗等。血管内皮生长因子（vascular endothelial growth factor, VEGF）及其受体是一组影响血管生成的重要配体和受体，是控制血管生成的主要因素，在MPE的形成中发挥重要作用。贝伐珠单抗是一种重组的人源化VEGF单克隆抗体，可与内源性VEGF竞争性结合VEGF受体，抑制新血管生成以及降低血管通透性，阻碍胸腔积液形成，延缓肿瘤发展进程。本综述旨在讨论贝伐珠单抗治疗非小细胞肺癌所致恶性胸腔积液的研究进展，探讨贝伐珠单抗临床应用的方法、疗效、安全性以及未来的发展方向。

IL-10 promotes malignant pleural effusion in mice by regulating TH 1- and TH 17-cell differentiation and migration.

The role of IL-10 in malignant pleural effusion (MPE) remains unknown. By using murine MPE models, we observed that an increase in pleural IL-10 was a significant predictor of increased risk of death. We noted that TH 1- and TH 17-cell content in MPE was higher in IL-10-/- mice than in WT mice, and IL-10 deficiency promoted differentiation into TH 1 but not into TH 17 cells. A higher fraction of TH 1 and TH 17 cells in the MPE of IL-10-/- mice expressed CXCR3 compared with WT mice. We also demonstrated that Lewis lung cancer and colon adenocarcinoma cells secreted large amounts of CXCL10, a ligand of CXCR3, which induced the migration of TH 1 and TH 17 cells into the MPE, and IFN-γ could promote this signaling cascade. Furthermore, intrapleural injection of mice with CXCL10-deficient tumor cells led to decreased TH 1- and TH 17-cell content in MPE, increased MPE volume, and reduced survival of MPE-bearing mice. Taken together, we demonstrated that IL-10 deficiency promoted T-cell differentiation into TH 1 cells and upregulated the CXCR3-CXCL10 signaling pathway that recruits TH 1 and TH 17 cells into MPE, ultimately resulting in decreased MPE formation and longer survival time of mice-bearing MPE.

Nitric oxide suppresses formation of malignant pleural effusion

In the present study, we were prompted to investigate the effect of NO signaling on the formation of malignant pleural effusion (MPE) and determined whether NO affects Th1/Th17 response in MPE. The development of MPE and survival of MPE mice were compared between wild-type and NOS2 ‒/‒ mice. The effect of NOS2 on vascular permeability of pleura and differentiation of Th1 and Th17 in MPE was also explored. Our current data show that production of NO is significantly enhanced in MPE as compared with peripheral circulation, and that nitric oxide synthase-2 (NOS2) deficiency increases pleural vascular permeability and thus promotes the development of MPE, and reduces the survival of mice bearing MPE. NOS2 deficiency inhibits Th1 cell differentiation and promotes Th17 cell differentiation. Our data provide the first evidence that NO inhibits MPE formation by inhibiting fluid extravasation into the pleural space and by promoting Th1 cell differentiation and suppressing Th17 cell differentiation. Overall, our results shed light on the mechanisms by which NO suppresses MPE formation, providing a promising therapeutic strategy in the management of MPE.

Activated naïve B cells promote development of malignant pleural effusion by differential regulation of TH1 and TH17 response.

Inflammatory signaling networks between tumor cells and immune cells contribute to the development of malignant pleural effusion (MPE). B cells have been found in MPE; however, little is known about their roles there. In the present study, by using mouse MPE models, we noted that although the total B cells in MPE were decreased as compared with the corresponding blood and spleen, the percentage of activated naïve B cells expressing higher levels of CD80, CD86, myosin heavy chain-II, CD44, CD69, and programmed cell death-ligand 1 (PD-L1) molecules were increased in wild-type mouse MPE. Compared with wild-type mice, decreased T helper (TH)1 cells and increased TH17 cells were present in B cell-deficient mouse MPE, which paralleled to the reduced MPE volume and longer survival time. Adoptive transfer of activated naïve B cells into B cell-deficient mice was able to increase TH1 cells and decrease TH17 cells in MPE and shorten the survival of mice bearing MPE. Furthermore, we demonstrated that activated naïve B cells inhibited TH17-cell expansion via the PD-1/PD-L1 pathway and promoted naïve CD4+ T-cell differentiation into TH1/TH17 cells through secreting IL-27/IL-6 independent of the PD-1/PD-L1 pathway. Collectively, our data uncovered a mechanism by which naïve B cells promote MPE formation by regulating TH1/TH17 cell responses, making these B cells an attractive target for therapeutic intervention in the fight against cancer.

Myeloid-derived interleukin-1\u03b2 drives oncogenic KRAS-NF-\u03ba\u0392 addiction in malignant pleural effusion

Malignant pleural effusion (MPE) is a frequent metastatic manifestation of human cancers. While we previously identified KRAS mutations as molecular culprits of MPE formation, the underlying mechanism remained unknown. Here, we determine that non-canonical IKKα-RelB pathway activation of KRAS-mutant tumor cells mediates MPE development and this is fueled by host-provided interleukin IL-1β. Indeed, IKKα is required for the MPE-competence of KRAS-mutant tumor cells by activating non-canonical NF-κB signaling. IL-1β fuels addiction of mutant KRAS to IKKα resulting in increased CXCL1 secretion that fosters MPE-associated inflammation. Importantly, IL-1β-mediated NF-κB induction in KRAS-mutant tumor cells, as well as their resulting MPE-competence, can only be blocked by co-inhibition of both KRAS and IKKα, a strategy that overcomes drug resistance to individual treatments. Hence we show that mutant KRAS facilitates IKKα-mediated responsiveness of tumor cells to host IL-1β, thereby establishing a host-to-tumor signaling circuit that culminates in inflammatory MPE development and drug resistance.

381P - Clinical study of apatinib in the treatment of malignant pleural effusion and malignant celiac effusion

Background: Malignant pleural effusion and malignant celiac effusion is a severe complication of the advanced malignant tumor, The formation of malignant pleural effusion and malignant celiac effusion relied on VEGF growth factor.Apatinib blocking vascular endothelial growth factor (VEGF) combining with the receptor and blocking signal transduction pathway, thereby inhibiting tumor and the formation of new blood vessels, inhibit the formation of the malignant pleural effusion and malignant celiac effusion. We conducted the study to exploring the single-agent oral path of Apatinib for the treatment of malignant pleural effusion and malignant celiac effusion of the clinical curative effect. Methods: Patients were eligible for inclusion in the study if they had histologically confirmed malignancies, with malignant pleural effusion and malignant celiac effusion. Eastern Cooperative Oncology Group (ECOG) performance status of 0-2. Study Objective: Clinical curative effect. Study Design and Treatment: This study recruited 13 patients with histologically confirmed malignancies, and the patients with malignant pleural effusion and malignant celiac effusion from January 2016 to October 2016. All patients had an Eastern Cooperative Oncology Group (ECOG) performance status of 0-2. All patients were given the oral drug Apatinib, the doses of oral given is 500 mg or 425 mg everyday until progressive disease (PD) or intolerable toxicity. Assessment and Statistical analysis: The assessmet were determined according to the World Health Organization (WHO) criteria. The ORR and DCR analyses were based on frequencies. Results:Table381P Baseline patient characteristicsCharacteristic Apatinib(n = 13)Age(years)Median(range) 58(44-84) Sex Male 7(53.85) Female 6(46.15) EGOG PS 0 2(15.38) 1 6(46.15) 2 5(38.46) Carcinoma Lung cancer 6(46.15) Pancreatic cancer 4(30.77) Breast cancer 2(15.38) Thymic cancer 1(7.7)Efficacy: Apatinib is effective for the treatment of malignant pleural effusion and malignant celiac effusion: CR 5 cases, PR 5 cases, SD 3 cases, overall-response rate (ORR)76.92%, disease-control rate (DCR)100%. Open table in a new tab Efficacy: Apatinib is effective for the treatment of malignant pleural effusion and malignant celiac effusion: CR 5 cases, PR 5 cases, SD 3 cases, overall-response rate (ORR)76.92%, disease-control rate (DCR)100%. Conclusions: Apatinib is a safe, feasible way in the treatment of malignant pleural effusion and malignant celiac effusion. Relying on the simple method and favourable rencent curative effect. Apatinib provides a new way for the treatment of malignant pleural effusion and malignant celiac effusion that deserves further research. Legal entity responsible for the study: GuoXin Ma Funding: None Disclosure: All authors have declared no conflicts of interest.

Abstract 1815: Extracellular vesicles derived from lung cancer cell may contribute to the formation of malignant pleural effusion via activating Stat3/Tissue factor/VEGF

Abstract Malignant pleural effusion (MPE) is a poor prognostic sign for patients with non-small-cell lung cancer (NSCLC) and is noted around 15% at the time of initial diagnosis and 50% in their later courses. Previously, we have shown that IL-6/Stat3/tissue factor (TF)/VEGF pathway enhances lung cancer angiogenesis, metastasis and the generation of MPE. In the current study, we demonstrated that EVs derived from lung cancer cells may also contribute MPE formation. We first used ultra-filtration (UF) method as well as the classical ultra-centrifugation (UC) method to isolate EV from lung cancer cell line and demonstrated that the EVs isolated by UF method were characteristic with exosome marker in Western Blotting (Alix, CD63, and Tsg101), morphology at Transmission electron microscopy (TEM) and size in Nanosight system (30 to 150 nm). After labeling these EVs with the green fluorescent dye Cellvue, EVs uptake in AS2 cell can be imaged by the fluorescent microscopy. After the EV uptake of recipient cell, the oncogenic signal such as Stat3 and AKT were activated with increasing phosphorylation at Western blotting comparing to those adding serum free medium. Using the ELISA method, we found the cargos in these EV were enriched in IL6. We also found these cargo derived from EVs, containing high VEGF and TF, induced more prominent extravasation than serum free medium did in Miles assays. In clinical sample, we also isolated cancer cell from MPE and the purity of the cells was confirmed by immunofluorescent staining with specific TTF-1expression of lung adenocarcinoma. After short-term culture, EV from MPE cancer cell have the characteristic of exosome marker and induced more prominent extravasation than serum free medium as EV from AS2 cell did. And we also isolated EV direct from MPE and pleural effusion from congestive heart failure (CHF) patients. And we found EV derived from MPE induced more prominent extravasation than those derived from CHF in Miles assays. In conclusion, we demonstrated the UF is clinical assessable method for isolating EV from body fluid and EV derived from lung cancer cell and MPE may contribute oncogenic and angiogenic signal transmission accelerating the formation of MPE. Citation Format: Chien-Chung Lin, Wei-Lun Huang, Wu-Chou Su. Extracellular vesicles derived from lung cancer cell may contribute to the formation of malignant pleural effusion via activating Stat3/Tissue factor/VEGF [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1815. doi:10.1158/1538-7445.AM2017-1815

Mutant KRAS promotes malignant pleural effusion formation

Malignant pleural effusion (MPE) is the lethal consequence of various human cancers metastatic to the pleural cavity. However, the mechanisms responsible for the development of MPE are still obscure. Here we show that mutant KRAS is important for MPE induction in mice. Pleural disseminated, mutant KRAS bearing tumour cells upregulate and systemically release chemokine ligand 2 (CCL2) into the bloodstream to mobilize myeloid cells from the host bone marrow to the pleural space via the spleen. These cells promote MPE formation, as indicated by splenectomy and splenocyte restoration experiments. In addition, KRAS mutations are frequently detected in human MPE and cell lines isolated thereof, but are often lost during automated analyses, as indicated by manual versus automated examination of Sanger sequencing traces. Finally, the novel KRAS inhibitor deltarasin and a monoclonal antibody directed against CCL2 are equally effective against an experimental mouse model of MPE, a result that holds promise for future efficient therapies against the human condition.

The role of microRNA-93 regulating angiopoietin2 in the formation of malignant pleural effusion.

The biological roles of miRNAs in the development of malignant pleural effusion (MPE) are unclear. In this study, the miRNA microarray analysis was performed in two different prognosis groups of lung adenocarcinoma patients. Expression profiles of miRNAs in MPEs were identified. With the help of quantification PCR, we confirmed the expression differences of miRNAs and further analyzed their biological functions and relative target genes in vitro. The target gene of miR‐93 was estimated by online database, and also, the protein was tested. The target gene and the binding sites of specific miRNA were estimated by online database. The combining capacity of binding sites was verified by luciferase reporter gene assay, and the target gene protein was tested by western blot. We detected 107 miRNAs with expression differences (n = 10) and confirmed significant expression differences in miR‐93 and miR‐146a in two groups of patients (n = 84). By manipulating miR‐93 expression of human lymphatic endothelial cells (HLEC) and human umbilical vein endothelial cells (HUVEC), we discovered that high expression of miR‐93 inhibited migration, proliferation, and angiogenesis. And also, miR‐93 increased not only apoptosis, but also G1 phase cell block. By using luciferase reporter gene assay and western blot, we confirmed that angiopoietin2 (Ang2) was the target of miR‐93. The data showed that miR‐93 has an inhibiting effect on pleural effusion. By targeting Ang2, miR‐93 regulates angiogenesis and lymphangiogenesis and plays a role in pathogenetic mechanism of MPE. MiR‐93/Ang2 may shed light on potential new targets in cancer treatment.

Interleukin-17 inhibits development of malignant pleural effusion via interleukin-9-dependent mechanism.

Th17 and Th9 cells have been demonstrated to possess immune regulatory functions in malignant pleural effusion (MPE). However, whether IL-17 can affect differentiation and function of Th9 cells in MPE remains unknown. The objective of the present study was to explore the impact of IL-17 on the in vivo differentiation of Th9 cells in relation to Th2 cells in a murine model of MPE, and to explore whether IL-17 inhibits MPE formation via IL-9‒dependent mechanism. It was found that Th9 and Th2 cells were decreased in MPE from IL-17 -/- mice as compared with wild type mice. IL-17 deficiency inhibited Th9 and Th2 cell differentiation via suppressing transcription factors IRF4 and GATA-3, respectively. IL-17 deficiency enhanced MPE formation by promoting angiogenesis and proliferation of pleural tumors, and thus accelerated the death of mice bearing MPE. The in vivo administration of anti-IL-9 neutralizing mAb accelerated the death of WT mice; whereas administration of exogenous IL-9 improved the survival of IL-17 -/- mice. Our data provide the first definitive evidence that IL-17 promotes the differentiation of Th9 and Th2 cells in MPE. Our findings also demonstrate that IL-17 inhibits the formation of MPE and improves the survival of mice bearing MPE via an IL-9-dependent mechanism.

Therapeutic effect of IGF-1R-targeting inhibitor (TAE226) on malignant pleural effusion in nude mice

To study the therapeutic effect of IGF-1R inhibitor TAE226 on malignant pleural effusion (MPE) in nude mice. Human lung carcinoma A549 cells were injected into the pleural cavity of nude mice to establish MPE model. The mice were randomly divided into model group and treatment group, and were orally administered with distilled water and TAE226 (20 mg/kg) in the same volume, respectively. The volume of pleural effusion and tumor weight of the two groups were observed. HE staining was used to reveal the histological changes and enzyme-linked immunosorbent assay (ELISA) was used to detect the IGF-1R protein expression. IGF-1R mRNA level in the tumor tissue was determined by RT-PCR. Microvessel density (MVD) and cell proliferation index (PI) were assessed by immunohistochemical analysis. The protein expression levels of IGF-1R, p-IGF-1R, PI3K and p-PI3K in the tumor tissue were determined by Western blotting. The volumes of pleural effusion were (241.4±89.7) μl and (121.7±78.8) μl in the model and treatment groups, respectively (P<0.05). The tumor weight of treatment group was (316.7±186.3) mg, significantly lower than that of the model group (671.4±281.4) mg (P<0.05). RT-PCR analysis showed that IGF-1R mRNA level was 0.914±0.029 in the treatment group, significantly lower than that of the model group (1.152±0.037, P<0.01). The ELISA data revealed that IGF-1R protein expression level of the model group was significantly higher than that of the treatment group [(41.0±4.7) μg/L vs. (24.0±3.1) μg/L, P<0.01]. Immunohistochemical analysis showed that there were significant differences between MVD and PI in the model and treatment groups [MVD, 34.75±3.49 vs. 22.25±3.63; PI, (75.25±7.15)% vs. (45.75±5.12)%; P<0.01 for both). Western blot data showed that IGF-1R and PI3K protein expression levels were not significantly different between the model and treatment groups (1.03±0.33 vs. 0.98±0.37 and 1.05±0.28 vs. 0.98±0.19), respectively (P>0.05), but p-IGF-1R and p-PI3K protein expression levels had significant differences between the two groups (1.08±0.10 vs. 0.51±0.08 and 1.12±0.09 vs. 0.86±0.09), respectively (P<0.01 for both). The IGF-1R inhibitor can effectively inhibit the formation of malignant pleural effusion. Its mechanism may be related to the suppression of tumor cell proliferation, invasion and angiogenesis through inhibition of PI3K signaling. TAE226 treatment may be a potential therapeutic regimen of treating malignant pleural effusion.

Toll-like receptor 4 signaling inhibits malignant pleural effusion by altering Th1/Th17 responses.

Toll-like receptor 4 (TLR4) is involved in multiple malignancies; however, the role of TLR4 in the pathogenesis of malignant pleural effusion (MPE) remains unknown. The objectives of this study were to explore the impact of TLR4 signaling on the development of MPE in a murine model and to define the underline mechanisms by which TLR works. Development of MPE as well as proliferation and angiogenesis of pleural tumor were determined in TLR4(-/-) and wild type mice. Differentiation of Th1 and Th17 cells as well as their signal transductions was explored. The effects of TLR4 signaling on survival of mice bearing MPE were also investigated. Compared with wild type mice, Th1 cells were augmented, and Th17 cells were suppressed in MPE from TLR4(-/-) mice. The in vitro experiments showed that TLR4 deficiency promoted Th1 cell differentiation via enhancing STAT1 pathway and inhibited Th17 cell differentiation via suppressing STAT3 pathway. TLR4 deficiency promoted MPE formation and, thus, accelerated the death of mice bearing MPE, whereas intraperitoneal injection of anti-IFN-γ mAb or recombinant mouse IL-17 protein into TLR4(-/-) mice was associated with improved survival. Our data provides the first definitive evidence of a role for TLR4 signaling in protective immunity in the development of MPE. Our findings also demonstrate that TLR4 deficiency promotes MPE formation and accelerates mouse death by enhancing Th1 and suppressing Th17 response.