Malignant Pleural Effusion Volume Research Articles

Immunotherapeutic hydrogel for co-delivery of STAT3 siRNA liposomes and lidocaine hydrochloride for postoperative comprehensive management of NSCLC in a single application

Despite standard treatment for non-small cell lung cancer (NSCLC) being surgical resection, cancer recurrence and complications, such as induction of malignant pleural effusion (MPE) and significant postoperative pain, usually result in treatment failure. In this study, an alginate-based hybrid hydrogel (SOG) is developed that can be injected into the resection surface of the lungs during surgery. Briefly, endoplasmic reticulum-modified liposomes (MSLs) pre-loaded with the signal transducer and activator of transcription 3 (STAT3) small interfering RNA and lidocaine hydrochloride are encapsulated in SOG. Once applied, MSLs strongly downregulated STAT3 expression in the tumor microenvironment, resulting in the apoptosis of lung cancer cells and polarization of tumor-associated macrophages towards the M1-like phenotype. Meanwhile, the release of lidocaine hydrochloride (LID) was beneficial for pain relief and natural killer cell activation. Our data demonstrated MSL@LID@SOG not only efficiently inhibited tumor growth but also potently improved the quality of life, including reduced MPE volume and pain relief in orthotopic NSCLC mouse models, even with a single administration. MSL@LID@SOG shows potential for comprehensive clinical management upon tumor resection in NSCLC, and may alter the treatment paradigms for other cancers.

Intrapleural nano-immunotherapy promotes innate and adaptive immune responses to enhance anti-PD-L1 therapy for malignant pleural effusion.

Malignant pleural effusion (MPE) is indicative of terminal malignancy with a uniformly fatal prognosis. Often, two distinct compartments of tumour microenvironment, the effusion and disseminated pleural tumours, co-exist in the pleural cavity, presenting a major challenge for therapeutic interventions and drug delivery. Clinical evidence suggests that MPE comprises abundant tumour-associated myeloid cells with the tumour-promoting phenotype, impairing antitumour immunity. Here we developed a liposomal nanoparticle loaded with cyclic dinucleotide (LNP-CDN) for targeted activation of stimulators of interferon genes signalling in macrophages and dendritic cells and showed that, on intrapleural administration, they induce drastic changes in the transcriptional landscape in MPE, mitigating the immune cold MPE in both effusion and pleural tumours. Moreover, combination immunotherapy with blockade of programmed death ligand 1 potently reduced MPE volume and inhibited tumour growth not only in the pleural cavity but also in the lung parenchyma, conferring significantly prolonged survival of MPE-bearing mice. Furthermore, the LNP-CDN-induced immunological effects were also observed with clinical MPE samples, suggesting the potential of intrapleural LNP-CDN for clinical MPE immunotherapy.

A Method for Isolating Tumor Cells from Large Volume of Malignant Pleural Effusion and Its Efficacy Evaluation

背景与目的恶性胸腔积液（malignant plural effusion, MPE）是液体活检基因检测的常用标本来源，本研究评估一种从大体积MPE中分离肿瘤细胞方法的分离效果及其在基因检测中的应用前景。方法收集20例伴MPE的晚期肺癌患者一次胸腔积液引流的全部MPE（> 500 mL），联合运用细胞分离介质Percoll和Ficoll分离肿瘤细胞，统计分离情况。对其中既往行组织基因检测的肺腺癌患者，分别使用胸腔积液上清游离DNA（tumor derived DNA from pleural effusion supernatant, etDNA），总细胞DNA及分离肿瘤细胞DNA（DNA from tumor cells in pleural effusion, ETC-DNA）3种成分进行二代基因测序，比较检测情况。结果从MPE中分离得到细胞中位数量8.50×104个（上下四分位间距9.25×103-3.75×105），肿瘤细胞纯度85.50%±5.80%。对10例既往行组织表皮生长因子受体（epidermal growth factor receptor, EGFR）基因检测的患者，etDNA、总细胞DNA及ETC-DNA EGFR基因突变检出率分别为70.00%、50.00%、70.00%。ETC-DNA基因检测阳性率与组织（P > 0.999, kappa=1.000）和etDNA（P > 0.999, kappa=1.000）一致性均良好。etDNA、总细胞DNA、ETC-DNA EGFR基因突变中位丰度分别为16.05%（4.78%-43.06%）、1.09%（0.00%-2.39%）和33.02%（18.50%-76.70%）。ETC-DNA检测丰度倾向高于etDNA，但差异无统计学意义。结论该提取方法可以有效地从大体积MPE中分离出大量高纯度肿瘤细胞，利用提取出的肿瘤细胞进行基因检测可能可以提高基因检测效能，值得进一步研究。

Accumulation of TNFR2-expressing regulatory T cells in malignant pleural effusion of lung cancer patients is associated with poor prognosis.

BackgroundRegulatory T cells (Tregs) may represent a major cellular mechanism in immune suppression by dampening the anti-tumor response in malignant pleural effusion (MPE). Tumor necrosis factor receptor type II (TNFR2) has emerged as a novel identification for the maximally suppressive subset of Tregs in the tumor environment. At present, the significance of TNFR2 expression on Tregs in MPE remains unclear.MethodsThe distribution of TNFR2+cells in Tregs and effector T cells (Teffs) in MPE, peripheral blood (PB), and tuberculosis pleural effusion (TPE) were determined. The associations between TNFR2+Tregs frequencies present in MPE and the clinical and laboratorial characteristics of patients with lung cancer were investigated. The immunosuppressive phenotype of TNFR2+Tregs in MPE was analyzed. The effects of the TNF-TNFR2 interaction on the immunosuppressive function of Tregs was explored. The efficacy of targeting TNFR2 for MPE therapy was examined. The source of TNF in MPE was identified.ResultsWe observed that markedly higher levels of TNFR2 were expressed in MPE Tregs compared with the levels expressed in MPE Teffs, PB Tregs, or in TPE Tregs. The frequencies of TNFR2+Tregs were positively correlated with the number of tumor cells in MPE, as well as the volume of MPE. High frequencies of TNFR2+Tregs in MPE indicated short survival time and poor performance status for MPE patients. Compared to TNFR2-Tregs, TNFR2+Tregs expressed higher levels of immunosuppressive molecules cytotoxic T lymphocyte-associated protein 4 (CTLA-4), programmed cell death-ligand 1 (PD-L1), and replicating marker Ki-67. Consequently, the proportions of interferon gamma (IFN-γ)-producing cytotoxic T lymphocytes (CTLs) were significantly increased after TNFR2 blockade. Furthermore, tumor necrosis factor (TNF), through interaction with TNFR2, enhanced the suppressive capacity of Tregs by up-regulating CTLA-4 and PD-L1 expression. Interestingly, T helper 1 (Th1) and T helper 17 (Th17) cells are the major source of TNF in MPE, suggesting that MPE Teffs may paradoxically promote tumor growth by boosting MPE Treg activity via the TNF-TNFR2 pathway.ConclusionsOur data expanded the immunosuppressive mechanism present in MPE induced by Tregs, and provides novel insight for the diagnosis, disease evaluation, and treatment of MPE patients.

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.

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.

The clinical efficacy and safety of paclitaxel combined with avastin for NSCLC patients diagnosed with malignant pleural effusion.

The current study aimed to investigate the clinical efficacy of paclitaxel combined with avastin for non-small cell lung cancer (NSCLC) patients diagnosed with malignant pleural effusion (MPE). Total of 33 patients diagnosed with NSCLC as well as malignant pleural effusion were included. All of them received paclitaxel (175 mg/m2) and avastin (5 mg/kg). Clinical efficacy was evaluated using the total response rate, overall survival, progression-free survival and changes in MPE volume. Adverse events and rates of toxicities were examined as well. The total response rate reached 77% while the overall survival and the median progression-free survival were respectively 22.2 months and 8.4 months. Toxicities of grade 3-4 consisted of neutropenia in 57% of patients, anemia in 17% of them, febrile neutropenia in 11%, as well as anorexia in 7%. No treatment-correlated deaths were found. Paclitaxel combined with avastin decreased MPE volume and increased survival rate of NSCLC patients via inhibiting vascular endothelial growth factor expression.

Immune Regulation of Toll-Like Receptor 2 Engagement on CD4+ T Cells in Murine Models of Malignant Pleural Effusion.

Toll-like receptor (TLR) 2 has a well-known role in sensing multiple ligands that include microbial products, endotoxin, and some extracellular matrix molecules; however, its role in the development of malignant pleural effusion (MPE) remains unknown. We performed the present study to explore the impact of TLR2 signaling on the development of MPE and to define the underlying mechanisms by which TLR2 works. Development of MPE was compared between TLR2-/- and wild-type (WT) mice. The effect of TLR2 on differentiation of T helper type 17 (Th17), Th9, and Th2 cells in MPE was explored. The mechanisms of TLR2 on survival of mice bearing MPE were also investigated. MPE volume in TLR2-/- mice was lower than that in WT mice, and the survival of TLR2-/- mice bearing MPE was longer than that of WT mice. TLR2 deficiency increased, and TLR2 activation decreased, Th17 cells in MPE, whereas TLR2 signaling showed the contrary effects on Th2 cells. Th9 cells were increased in MPE of TLR2-/- mice but were not influenced by TLR2 signaling. Intraperitoneal injection of anti-IL-17 monoclonal antibody (mAb), anti-IL-9 mAb, or recombinant mouse IL-4 accelerated the death of TLR2-/- mice bearing MPE, and intraperitoneal injection anti-IL-17 mAb in TLR2-/- mice was associated with a significantly shorter survival time than in WT mice. We have demonstrated, for the first time, that TLR2 signaling promotes the development of MPE and accelerates the death of mice bearing MPE by directly suppressing Th17 cell differentiation and directly promoting Th2 cell differentiation, and also by indirectly suppressing Th9 cell differentiation via an IL-17-dependent mechanism.

Icmt inhibition exerts anti-angiogenic and anti-hyperpermeability activities impeding malignant pleural effusion.

Small GTPases are pivotal regulators of several aspects of tumor progression. Their implication in angiogenesis, vascular permeability and tumor-associated inflammatory responses is relevant to the pathobiology of Malignant Pleural Effusion (MPE). Inhibition of isoprenylcysteine carboxylmethyltransferase (Icmt) abrogates small GTPase activation. We therefore hypothesized that cysmethynil, an Icmt inhibitor would limit pleural fluid accumulation in two models, a lung-adenocarcinoma and a mesothelioma-induced MPE. Cysmethynil significantly reduced MPE volume in both models and tumor burden in the adenocarcinoma model. It inhibited pleural vascular permeability and tumor angiogenesis in vivo and reduced endothelial cell proliferation, migration and tube formation in vitro. Cysmethynil also promoted M1 anti-tumor macrophage homing in the pleural space in vivo, and inhibited tumor-induced polarization of macrophages towards a M2 phenotype in vitro. In addition, the inhibitor promoted adenocarcinoma cell apoptosis in vivo. Inhibition of small GTPase might thus represent a valuable strategy for pharmacotherapy of MPE.

Therapeutical effects of pleural injecting recombinant human endostain to malignant pleural effusion nude mice model

背景与目的恶性胸腔积液（malignant pleural effusion, MPE）临床预后不佳，胸腔内抗血管治疗可能对恶性胸腔积液具有治疗作用，本研究旨在探讨胸腔内注射重组人血管内皮抑素、顺铂、重组人血管内皮抑素联合顺铂对裸鼠恶性胸腔积液的治疗作用。方法BALB/c裸鼠胸膜腔内注射Lewis肺癌细胞（Lewis lung cancer cell, LCC）构建恶性胸腔积液模型，造模后分别胸腔内注射重组人血管内皮抑素（E）、顺铂（P）以及重组人血管内皮抑素联合顺铂（EP）并分析各组裸鼠胸腔积液量、胸膜肿瘤微血管密度（micro vessel density, MVD）以及血管生成、凋亡相关基因的表达变化。结果重组人血管内皮抑素及重组人血管内皮抑素联合顺铂胸腔内注射可以使裸鼠MPE量减少，且与裸鼠胸腔肿瘤组织MVD下降呈正相关；且重组人血管内皮抑素及重组人血管内皮抑素联合顺铂胸腔内注射后，MPE裸鼠胸腔肿瘤组织血管内皮生长因子（Vescular epidermal growth factor-α, VEGF-α）表达下降、低氧诱导因子-α（hypoxia induced factor-1, HIF1-α）表达升高。结论胸腔内注射LLC细胞可成功制作裸鼠MPE模型。重组人血管内皮抑素裸鼠胸膜腔内注射对MPE裸鼠具有治疗作用，其治疗作用可能是通过下调VEGF-α，抑制肿瘤新生血管生成，下调微血管密度而达成的。

Efficacy of Arsenic Trioxide in the Treatment of Malignant Pleural Effusion Caused by Pleural Metastasis of Lung Cancer.

The aim of the study was to investigate the mechanism of arsenic trioxide (As2O3) in the treatment of malignant pleural effusion (MPE) caused by pleural metastasis of lung cancer. A mouse model of MPE caused by pleural metastasis of lung cancer was first established, and As2O3 was then intraperitoneally injected to treat the MPE. Mice treated with bevacizumab and bleomycin were included as positive controls, and placebo equivalents were also used as negative controls. The effects of As2O3 on MPE volume, pleural vessel density, vascular permeability, expression of angiogenic function-related factors, including vascular endothelial growth factor (VEGF) and tumor necrosis factor alpha (TNF-α), as well as nuclear factor-κB (NF-κB) activity in pleural carcinomatosis, were observed. Intraperitoneal injection of As2O3 reduced the volume of MPE and decreased vascular density and permeability in pleural metastatic nodules in a dose-dependent manner. Moreover, dose-dependent decreases in VEGF and TNF-α expression in MPE, and NF-κB activity in pleural carcinomatosis, were also found after As2O3 treatment. We showed that As2O3 can down-regulate VEGF expression via inhibition of NF-κB, and decrease vascular density and permeability in pleural metastatic nodules, thereby eliciting its effects on MPE caused by pleural metastasis of lung cancer. Our results provide a foundation for an As2O3-based clinical treatment program.

Establishment of a malignant pleural effusion mouse model with lewis lung carcinoma cell lines expressing enhanced green fluorescent protein.

Malignant pleural effusion (MPE) is a poor prognostic factor in patients with advanced lung cancer. The aim of this study is to establish a mouse model of MPE using Lewis lung carcinoma (LLC) cell lines expressing enhanced green fluorescent protein (EGFP). The mouse model was created by injecting LLC-EGFP cells directly into the pleural cavity of nude mice under the guidance of stereomicroscope and then mice were sacrificed periodically. The dynamic growth and metastasis of tumor cells were screened using in vivo fluorescence imaging. The remaining mice were subjected to transverse computed tomography (CT) periodically to analyze the rate of MPE formation. The survival rate and tumor metastasis were also observed after modeling. Pleural fluid was gently aspirated using a 1 mL syringe and its volume was measured. When two or more mice bore MPE at the same time, we calculated the average volume. The correlation of MPE with the integrated optical density (IOD) were analyzed. Four days after the inoculation of LLC-EGFP cells, green fluorescence was observed by opening the chest wall. The tumor formation rate was 100%, and the IOD gradually increased after inoculation. The metastatic foci were mediastinal, contralateral pleural and pericardial. The metastasis rates were 87%, 73%, and 20%, respectively. CT imagings revealed that the rates of MPE formation on days 7, 14 and 21 were 13%, 46%, and 53%. The mean survival time of nude mice was 28.8 days. The average MPE volume increased obviously on day 10 and peaked on day 16 with a value of 0.5 mL. The MPE volume and IOD were significantly correlated (r=0.91, P<0.0001). This study was the first to establish a mouse model of MPE by injecting LLC-EGFP into the pleural cavity under the guidance of a stereomicroscope. The model can enable dynamic observations of the biological behavior of tumor cells in the pleural cavity. It might be helpful for basic research on advanced lung cancer as well as anti-tumor drug development.

Potential role of AQP1 and VEGF in the development of malignant pleural effusion in mice

Recent studies have revealed the important role of aquaporin-1 (AQP1) in tumor cell migration and angiogenesis. However, the function of AQP1 in malignant pleural effusion has not been well characterized. We established a mouse model to examine the role of AQP1 and vascular endothelial growth factor (VEGF) in the development of malignant pleural effusion. We showed that elevated expressions of AQP1 mRNA and VEGF protein were associated with increased volume of malignant pleural effusion. These results suggest that AQP1 and VEGF play important roles in the development of malignant pleural effusion in mice, which may help us find new strategies for the prevention and treatment of malignant pleural effusion.