M2-polarized macrophages promote metastatic behavior of Lewis lung carcinoma cells by inducing vascular endothelial growth factor-C expression

OBJECTIVES:

Tumor-associated macrophages that generally exhibit an alternatively activated (M2) phenotype have been linked to tumor progression and metastasis. However, the role of M2-polarized macrophages in the growth and metastasis of lung adenocarcinoma remains enigmatic. The aim of this study was to explore the effect of M2 macrophages on the proliferation and migration of mouse Lewis lung carcinoma cells and tumor-induced lymphangiogenesis.

METHODS:

Trypan blue staining and the Transwell migration assay were performed to evaluate the effects of activated (M1 or M2) macrophages on the proliferation and migration of Lewis cells. Furthermore, vascular endothelial growth factor-C expression in Lewis cells and nitric oxide secretion from activated macrophages were detected during the co-culture assay. Following treatment with activated macrophages, lymphatic endothelial cells differentiated into capillary-like structures, and the induction of Lewis cell migration was assessed using a two-dimensional Matrigel-based assay.

RESULTS:

In the co-culture Transwell system, the proliferation and migration of Lewis cells were promoted by M2 macrophages. Moreover, the co-culture significantly increased the expression of vascular endothelial growth factor-C by Lewis cells and reduced the secretion of nitric oxide from M2 macrophages, which subsequently led to the capillary morphogenesis of lymphatic endothelial cells. Interestingly, following co-culture with Lewis cells, the function of RAW264.7 cells was polarized toward that of the M2 macrophage phenotype.

CONCLUSION:

M2-polarized macrophages promoted the metastatic behavior of Lewis cells by inducing vascular endothelial growth factor-C expression. Thus, the interruption of signaling between M2 macrophages and Lewis cells may be considered to be a new therapeutic strategy.     

Cribriform pattern in lung invasive adenocarcinoma correlates with poor prognosis in a Chinese cohort

The 2011 International　Association　for　the　Study　of　Lung　Cancer/American　Thoracic　Society/European　Respiratory　Society (IASLC/ATS/ERS) lung adenocarcinoma classification and the following 2015 WHO classification have both been validated for their predictive values of histologic subtypes for prognosis. We sought to investigate the clinicopathological and prognostic significance of the cribriform pattern in lung adenocarcinomas. Histologic subtypes were evaluated in 395 patients who underwent complete resection for invasive lung adenocarcinomas between 2011 and 2013. Cribriform pattern was correlated with clinicopathological factors as well as molecular and survival data. One hundred and thirty cases (33%) were present with cribriform pattern (5–100%; mean?±?SD, 24% ± 22%). Thirty two (8%) of those were reclassified into cribriform predominant tumors. Presence of cribriform pattern (≥5%) was significantly associated with lymph/vascular invasion (P＜0.001), nodal positivity (P = 0.003), higher T stage(P = 0.005) and higher TNM stage (P = 0.001). Cribriform pattern (≥10%) was highly associated with worse disease-free survival (DFS) and overall survival (OS) (mean DFS: 42.6 months, P?<?0.001; mean OS: 64.1 months, P = 0.012). The DFS or OS for cribriform predominant tumors was similar to that for solid or micropapillary tumors. In multivariate analysis, cribriform pattern (≥10%) or cribriform predominant subtype was an independent predictor for DFS. Cribriform pattern was a specific pattern compared to other acinar patterns, presenting with more aggressive behavior. Moreover, presence of cribriform pattern was a strong predictor for worse prognosis and should be considered a high grade pattern. Our study provides further evidence for cribriform pattern to be acknowledged as an independent subtype in the future classification.     

Overexpression of YAP1 in EGFR mutant lung adenocarcinoma prior to tyrosine kinase inhibitor therapy is associated with poor survival

EGFR tyrosine kinase inhibitor (EGFR TKI) is approved as first-line treatment for advanced-stage EGFR mutant lung adenocarcinoma (LADC). Yes-associated protein 1 (YAP1), a main effector of the Hippo pathway, is associated with adverse prognosis and disruption of EGFR TKI modulation of non-small cell lung cancer. In this study, we demonstrated a prognostic role of YAP1 in EGFR mutant LADC and efficacy of EGFR TKI therapy. A total of 63 patients, including 41 with paired lung cancer specimens before and after EGFR TKI therapy and 22 with non-paired lung cancer specimens prior to EGFR TKI, were enrolled for examination. Expression of YAP1 protein was evaluated using immunohistochemistry. Fifteen paired cases (36.6%) with high nuclear YAP1 expression were detected in the pre-EGFR TKI LADC group and 21 paired cases (52.5%) after treatment with EGFR TKI. Nuclear YAP1 expression was significantly upregulated after EGFR TKI therapy (P?=?.002). Fifteen paired cases with high nuclear YAP1 expression before EGFR TKI LADCs showed poorer overall survival (OS) (P?=?.023) and progression-free survival (PFS) (P?=?.041). Among the 63 patients under study, those with high nuclear YAP1 expression before EGFR TKI showed shorter OS (P?=?.038) and PFS (P?<?.001). High nuclear YAP1 expression in cases with acquired EGFR exon 20 T790?M mutant LADCs showed poorer OS (P?<?.001). We demonstrated that YAP1 burden before clinical application of EGFR TKI plays a crucial role in prognosis of EGFR mutant LADC treated using EGFR TKI.     

Gpnmb在小鼠M1和M2型骨髓源性巨噬细胞中表达的差异

目的了解小鼠M1和M2型骨髓源性巨噬细胞（ BMMφs）中非转移性黑色素瘤糖蛋白b（ Gpnmb）表达的差异。方法原代培养小鼠BMMφs,免疫荧光染色F4/80和流式细胞仪检测CD11b鉴定巨噬细胞;用IFN-γ和LPS诱导BMMφs向M1型分化,用IL-4诱导向M2型分化。实时荧光定量PCR检测M1型巨噬细胞标志物（ TNF-α、iNOS）、M2型巨噬细胞标志物（ MMR、Arg-1）和Gpn-mb的mRNA表达;免疫荧光双染色、Western印迹、流式细胞仪检测Gpnmb与MMR的蛋白表达。结果（1）免疫荧光染色结果示BMMφs中F4/80高表达;流式细胞仪检测结果示BMMφs中有（92．7±6．1）％细胞表达CD11b,提示BMMφs培养成功;（2）相对于未分化的M0型BMMφs,TNF-α、iNOS mRNA在M1型BMMφs中高表达（P均＜0．01）,而MMR、Arg-1 mRNA在M2型BMMφs中高表达（P均＜0．01）,提示原代M1、M2型BMMφs分化成功;（3）M2型BMMφs 的Gpnmb mRNA和蛋白表达均较M0型和M1型BMMφs显著增高（P均＜0．01）;免疫荧光双染色及流式细胞仪结果显示,BMMφs中Gpnmb与MMR共表达,在M2型BMMφs中MMR阳性BMMφs有（83．2±9．7）％表达Gpnmb。结论 M2型BMMφs的Gpnmb表达较M1型BMMφs显著增高,提示Gpnmb可能作为鉴别M1、M2型巨噬细胞的标志物,在巨噬细胞的表型分化中起作用。     

Correlation between EZH2 and CEP55 and lung adenocarcinoma prognosis

Objective

Recently, accumulated evidence indicates that the enhancer of zeste homologue 2 (EZH2) is highly expressed in a wide range of cancer types, including NSCLC. The downstream genes regulated by EZH2 were screened using bioinformatics analysis. This study aimed to analyse the correlation between the downstream genes of EZH2 and the prognosis of lung adenocarcinoma.

Tumor-associated macrophages,epidermal growth factor receptor correlated with the triple negative phenotype in endometrial endometrioid adenocarcinoma

It has been well established that tumor-associated macrophages (TAMs) play a tumor-promoting role in endometrial endometrioid adenocarcinoma (EEC). However, the association with TAMs and the triple-negative phenotype (TNP) in EEC has not yet been reported. We used immunohistochemistry to examine the expression of CD68, estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor 2 (HER2), and epidermal growth factor receptor (EGFR) in 186 cases of EEC. Fluorescent in situ hybridization (FISH) was also used for HER2 amplification, and the association with TAMs count, EGFR expression, and triple-negative phenotype was analyzed. Twenty-eight of 186 patients (15.05%) had the TNP. It was associated with advanced stage disease (P < 0.0001), high grade disease (P < 0.0001), depth of myometrial invasion (P = 0.003), pelvic lymph node metastasis (P < 0.001), lymphovascular space invasion (P = 0.001), and EGFR expression (P = 0.032). Margin TAMs count was also significantly increased in the TNP-positive group, the EGFR-positive group, and the PR-negative group (P < 0.001, respectively). The TNP was associated with a significantly worse overall survival (OS) (log rank test, P = 0.018). The estimated 5-year OS of patients with TNP was 59.1%, while that without TNP was 78.5%. Multivariate analysis showed high margin TAMs, and the histopathological grades were significantly associated with OS. The TNP in EEC is associated with poor prognostic surgical–pathological factors, worse prognosis, as well as with high margin TAMs and overexpression of EGFR, which may serve as potential targeted therapies for the special phenotype in EEC.     

肺腺癌巨噬细胞M2表型边缘极化效应观察及预后分析

目的:探讨肺腺癌边缘区巨噬细胞M2表型的极化效应、边缘/中心比值及对预后的影响。方法:采用双重免疫组化技术,观察巨噬细胞CD163+/CD68+表型(M2表型)在49例原位肺腺癌(AIS)、11例微小浸润性腺癌(MIA)、57例浸润性腺癌(IA)边缘区及中心区的分布规律和差异,探讨巨噬细胞边缘极化效应及边缘/中心比值在肺腺癌进程中的作用及机制。采用单因素Kaplan-Meier生存曲线分析及多变量Cox生存分析探讨M2表型边缘极化状态与预后的关系。结果:肺腺癌边缘区M2型巨噬细胞较中心区域呈现极性聚集,具有显著差异(P0.01)。根据中位数分高、低极化组,低极化组AIS中M2型巨噬细胞计数值与MIA及IA比较未见明显差别,但其在高、极化组AIS中的计数值依次低于MIA和IA,差异有统计学意义(均P0.01)。单因素Kaplan-Meier生存曲线分析及log-rank检验结果显示边缘区巨噬细胞M2表型数量及边缘/中心比值与生存时间呈负相关(2=44.71,P0.01;2=21.75,P0.01)。多变量Cox生存分析表明M2表型边缘高极化状态和边缘/中心比值是独立的预后危险因素(P0.01)。结论:巨噬细胞M2表型在肺腺癌边缘区存在边缘极化效应,其边缘极化状态和边缘/中心比值是独立的预后危险因素,因此术前穿刺判断边缘极化状态或术后活检检测M2型巨噬细胞型边缘/中心比值可能是评估预后的一种有效方法。     

Correlated low IGF2BP1 and FOXM1 expression predicts a good prognosis in lung adenocarcinoma

IGF2BP1 and FOXM1 are shown to be critical in the regulation of caner progression. However, the prognostic value of IGF2BP1 in lung adenocarcinoma and its relationship with FOXM1 still remains unclear. In this study, the expression and biological significance of both IGF2BP1 and FOXM1 were evaluated in 188 lung adenocarcinoma, at mRNA and protein levels. We showed that mRNA and protein levels of IGF2BP1 and FOXM1 were upregulated in lung adenocarcinoma compared to adjacent non-cancerous tissues. High IGF2BP1 expression was correlated with a poor prognosis for lung adenocarcinoma patients. Moreover, IGF2BP1 expression was positively associated with FOXM1 expression. Meanwhile, the findings indicated that low IGF2BP1 combined with low FOXM1 expression, was negatively correlated with pathological stage and lymph node metastasis, predicted good outcomes for lung adenocarcinoma patients. Additionally, low IGF2BP1 and FOXM1 expression status, is an independent prognostic factor for lung adenocarcinoma after surgical resection. We demonstrate that low IGF2BP1 and FOXM1 expression can serve as a potential factor for the clinical diagnosis and prognosis of lung adenocarcinoma, and targeted inhibition of IGF2BP1 and FOXM1 might be an alternative strategy for the management of lung adenocarcinoma.     

High expression of Ras-specific guanine nucleotide-releasing factor 2 (RasGRF2) in lung adenocarcinoma is associated with tumor invasion and poor prognosis

The expression of Ras-specific guanine nucleotide-releasing factor 2 (RasGRF2) in lung adenocarcinomas was examined using immunohistochemistry in relation to clinicopathological characteristics and prognosis. In comparison to low expression, high expression of RasGRF2 was more closely associated with poor prognosis. Interestingly, expression of phosphorylated epithelial cell transforming 2 (pECT2), which – like RasGRF2 – is also a guanine-nucleotide exchange factor, was also associated with prognosis, and patients with high expression of both RasGRF2 and pECT2 had a much poorer outcome than those who were negative for both.     

Concentrations of SP-A and HSP70 are associated with polarization of macrophages in pleural effusions of non-small cell lung cancer

Damage-associated molecular pattern (DAMP) molecules can initiate an immune response through Toll-like receptors (TLRs). DAMPs are released from cells as a response to the extracellular danger and can be by-products of tissue damage. In cancer microenvironment necrotic cells release debris which has potency to become DAMPs. Non-small cell lung cancer (NSCLC) is often accompanied by pleural effusion (PE), which contains a variety of DAMPs. Surfactant protein A (SP-A) and heat shock protein 70 (Hsp70) are important DAMPs in the respiratory tract.The aim of this study was to determine a correlation between SP-A or Hsp70 and development of PE in the course of NSCLC. Moreover, we aimed to determine relationships between DAMPs and certain humoral factors associated with formation and persistence of PE as well as pleural-residing macrophages. In 34 PE samples, we estimated concentration of SP-A, Hsp70, IL-6, IL-18, G-CSF, M-CSF, SCF, SDF1α, VEGF as well as the fraction of macrophages and their pattern of polarization.We have found correlations between the concentration of the SP-A and Hsp70 and the percentage of PE-derived macrophages, also between concentrations of SP-A and Hsp70, and cytokines which participate in inflammation and processes involved in remodeling of extracellular matrix (ECM). Our data indicate an important role of SP-A during the development of PE associated with NSCLC. We suggest that measurement of concentration level of SP-A can be helpful in the course of diagnosis of malignant PE associated with NSCLC.     

The urokinase-type plasminogen activator and inhibitors in resectable lung adenocarcinoma

BackgroundThe urokinase-type plasminogen activator (uPA) system is closely related to the occurrence and progression of cancer in many aspects. Previous studies demonstrated that the conclusions about the prognosis value of uPA, plasminogen activator inhibitor 1 (PAI-1) and plasminogen activator inhibitor 2 (PAI-2) in lung cancer are controversial, so this study was performed for the exploration of the predictive effect of uPA, PAI-1 and PAI-2 on the overall survival (OS) of resectable pulmonary adenocarcinoma patients.MethodsUPA, PAI-1 and PAI-2 expression levels were assayed by immunohistochemical staining based on tissue microarray (TMA) that is composed of formalin-fixed paraffin-embedded specimens from 84 resectable lung adenocarcinoma patients from July 2004 to June 2009. The relationship of IHC, mRNA expression levels of three molecules were investigated respectively. The three molecules’ relationship with clinicopathologic parameters and OS was explored by Chi-square, Kaplan-Meier, and Cox regression analyses. The Cancer Genome Atlas (TCGA) database was used to analyze differential gene expressions of RNA-sequencing data of pulmonary adenocarcinoma and normal tissues, and Kaplan-Meier methods were adopted to confirm the prognostic value of uPA, PAI-1 and PAI-2 in resectable lung adenocarcinoma in TCGA database and the R package MethylMix was used to conduct an analysis integrating methylation data and gene expression of RNA-sequencing data based on TCGA.ResultsUPA, PAI-1 and PAI-2 had much higher IHC expression levels in tumor than those in the normal tissues (uPA, Z = -10.511; PAI-1, Z = -4.836; PAI-2, Z = -6.794; all P < 0.0001). High DNA methylation level of gene uPA resulted in the decrease of its expression. In addition, expression level of PAI-2 was positively associated with tumor size (χ² = 8.372, P = 0.004). Multivariate analyses showed TNM stage III was an independent adverse prognostic factor (hazard ratio = 3.736, 95 % confidence interval = 1.097–12.72, P = 0.035). Kaplan-Meier method revealed that uPA, PAI-1 and PAI-2 expression levels were not related to the OS for 84 resectable lung adenocarcinoma patients. According to TCGA data, PAI-1 expression level was identified as a potential adverse predictor for prognosis of resectable lung adenocarcinoma (Gehan-Breslow-Wilcoxon test, P = 0.025).ConclusionsOur data show that, the expression levels of uPA, PAI-1 and PAI-2 are significantly up-regulated in resectable lung adenocarcinoma. Besides, this study highlights PAI-1 as a latent adverse prognostic factor in resectable adenocarcinoma of lung.     

Expression of stabilin-1 in M2 macrophages in human granulomatous disease and melanocytic lesions

Abstract: Stabilin-1 is an endocytotic scavenger receptor, specifically expressed by non-continuous sinusoidal endothelial cells in the liver, spleen and lymph nodes and by M2 or alternatively activated macrophages in human malignancies. We analysed paraffin-embedded tissue of melanocytic lesions and granulomatous diseases for stabilin-1 expression, using the human/murine RS1 antibody. The specificity of the RS1 staining was confirmed in a knockout model, as only M2-like tumor-associated macrophages and vessels of a B16F10 melanoma in wild type mice stained positive; while staining of tumor-associated macrophages and vessels originating from stabilin-1 deficient mice remained negative for stabilin-1 specific antibody RS1. In human specimens, the RS1 antibody stained tumor-associated macrophages in all pathological stages of melanoma. In addition, five cases of juvenile xanthogranulomas and one case of necrobiotic xanthogranuloma were strongly stabilin-1 positive, while Th-1 cytokine dominated granulomatous diseases such as sarcoidosis and granulomatous leprosy were negative. Stabilin-1 positive vessels were found in all analysed non-Langerhans cell histiocytoses and melanocytic lesions. No stabilin-1 positive vessels were present in any other granulomatous diseases.     

Proportion of goblet cell is associated with malignant potential in invasive mucinous adenocarcinoma of the lung

Invasive mucinous adenocarcinoma (IMA) is a newly classified variant of lung adenocarcinoma. The aim of this study was to examine the correlation between the proportion of goblet cells and the clinicopathological characteristics of IMA. Ninety‐nine patients with stage I IMA were included in this study. We estimated prognostic impact of goblet cell proportion. We classified them into two groups: the cases with a high goblet cell proportion (HGP, goblet cell proportion ≥80%) and the cases with a low goblet cell proportion (LGP, goblet cell proportion ≤30%), and compared the expression levels of five cancer progression markers and the number of tumor‐promoting stromal cells between the two groups. Univariate and multivariate analysis revealed that the goblet cell proportion was a prognostic factor for recurrence free survival (P < 0.01) and overall survival (P = 0.01). The expression levels of the cancer stem cell‐related marker, ALDH‐1, and proliferation‐related marker, geminin were significantly higher in the LGP group than in the HGP group. CD204+ tumor‐associated macrophages were significantly more in the LGP stroma than the HGP stroma. Our current study indicated that the proportion of goblet cells was correlated with the malignant potential in surgically resected IMA.     

Comprehensive analysis of tumor immune infiltration associated with endogenous competitive RNA networks in lung adenocarcinoma

Cancer immunotherapy has achieved unprecedented success in the treatment of cancer. However, different patients have different responses to immunotherapy. More and more studies have shown that tumor immune heterogeneity has an important influence on the prognosis of cancer. Therefore, understanding the clinical impact of tumor immune infiltration and the regulatory mechanism of RNA molecules is crucial for exploring the pathogenesis of lung adenocarcinoma (LUAD) and the development of immunotherapy protocols.The endogenous competitive RNA hypothesis provides new ideas for studying immune heterogeneity. Therefore, by using the method of immune genomics, this article explores the relationship between immune infiltration and prognosis of patients with lung adenocarcinoma, and found that B-cell immune infiltration highly affects the survival of patients. Through differential analysis, differential mRNAs, lncRNAs and miRNAs were extracted, and 318 differentially expressed mRNAs related to B cell immunity were screened by correlation analysis, and prognosis of patients with COX risk regression model was predicted and analyzed. Through multiple database searches, an immune-related ceRNA regulatory network was constructed, containing 3 key mRNAs, 4 miRNAs, and 50 lncRNAs. Three mRNAs and most miRNAs, lncRNAs, are significantly associated with LUAD prognosis. Bioinformatics analysis of the network showed that LINC00337 may up-regulate the expression of PBK and KIF23 through competitive binding of has-mir-373 and has-mir-519d. The competitive binding of has-mir-373 and has-mir-372 can up-regulate the expression of SLC7A11. The interaction between these RNAs may have an important regulatory role in the immune infiltration in lung adenocarcinoma, thereby affecting the patient's prognosis and immunotherapy efficacy.     

High PITX1 expression in lung adenocarcinoma patients is associated with DNA methylation and poor prognosis

Background

Pituitary homeobox 1 (PITX1) is a member of the PITX gene family which is vital to proper development of early embryo. However, the relationship of PITX1 expression and overall survival (OS) in non-small cell lung cancer (NSCLC) is not clear.

Plakophilin-2 accelerates cell proliferation and migration through activating EGFR signaling in lung adenocarcinoma

BackgroundPlakophilin 2 (PKP2), encodes a plakophilin protein that belongs to the member of desmosomal proteins. It has been reported that high expression of PKP2 is associated with several types of cancer in humans. However, the role of PKP2 in lung cancer remains obscure.MethodsPKP2 expression was investigated in non-small cell lung cancer (NSCLC) tissues and non-tumor tissues by performing immunohistochemistry on a tissue microarray and using The Cancer Genome Atlas (TCGA) database. Kaplan-Meier survival analysis and multivariate Cox-regression analysis were performed to identify the clinical significance of PKP2. 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS), colony formation, Transwell and xenograft tumor growth/ metastasis assays were conducted to evaluate the biological function of PKP2 in vitro and in vivo. Gene set enrichment analysis (GSEA), WB and immunoprecipitation (IP) assay were utilized to explore the potential downstream signaling pathway and molecule mechanism of PKP2 in lung adenocarcinoma (LUAD).ResultsAnalysis of PKP2 expression and clinicopathological parameters reveals a significant correlation of PKP2 expression with gender (n = 1020, P < 0.001) and histological type (n = 1020, P < 0.001). Subsequently, our results demonstrated that high PKP2 expression is not associated with poor survival in different gender of lung cancer patients, and is an unfavorable and independent prognostic biomarker for LUAD patients, but not for LUSC patients. Gene set enrichment analysis (GSEA) revealed that PKP2 expression is positively associated with EGFR signaling in LUAD. Further, in vitro and in vivo assays revealed that PKP2 promotes cell proliferation, migration and invasion through activating EGFR signaling pathway in LUAD cells.ConclusionOur study provides the basis for further investigation of the function and molecular mechanism by which upregulation of PKP2 promotes the development and progression of LUAD. PKP2 may serve as a potential target for anticancer therapies.     

Origin of the T790M mutation and its impact on the clinical outcomes of patients with lung adenocarcinoma receiving EGFR-TKIs

Objective

Recently, a low frequency of de novo T790M mutations existing in tumor tissues before TKIs therapy has been reported. However, the origin of T790M and its impact on clinical outcomes is still being debated. This study aimed to use highly sensitive methods to detect T790M before and after TKIs therapy and investigated the correlation of T790M with clinical prognosis.