Endothoracic lymphatic plexus­hemiazygos vein anastomosis for chylothorax complicated with hepatocellular carcinoma: A case report.

For patients with hepatocellular carcinoma and cirrhosis, the rupture of thin lymphatic vessel walls leads to a profuse outflow of lymph fluid. Typically, chyloperitoneum tends to precede the development of chylothorax in patients with cancer. The present study describes the case of a male patient with hepatocellular carcinoma who developed chylothorax without chyloperitoneum. Computed tomography showed lymphatic system developmental abnormalities with a large volume of leaked lymph fluid. Multiple thoracic duct ligations (TDLs) failed, but a side-to-end lymphatic venous anastomosis (LVA) surgery resolved the symptoms. To the best of our knowledge, there are no reports of chylothorax occurrence after cirrhosis further complicated by congenital lymphatic abnormalities in the English-language literature. In conclusion, LVA could be appropriate to treat chylothorax when TDL is ineffective as a remedial or even prophylactic intervention.

Lymph Node Ratio Enhances Predictive Value for Treatment Outcomes in Patients with Non-Small Cell Lung Cancer Undergoing Surgery: A Retrospective Cohort Study.

Purpose: To compare the prognostic value of lymph node ratio (LNR) and pN in patients with non-small cell lung cancer (NSCLC) undergoing surgery. Materials and methods: NSCLC patients were investigated between 2004 and 2015 from the Surveillance, Epidemiology, and End Results databases. The X-tile software was used to determine LNR cut-off values. Kaplan-Meier analysis was employed to assess cancer-specific survival (CSS) and overall survival (OS). Results: The identified cut-off values of LNR were 0.19 and 0.73. Median CSS for LNR1 (LNR < 0.19), LNR2 (0.19 ≤ LNR ≤ 0.73), and LNR3 (LNR > 0.73) were 71, 41, and 17 months. Both LNR2 (HR = 1.46, 95% CI: 1.36-1.57; P < 0.001) and LNR3 (HR = 2.85, 95% CI: 2.58-3.15; P < 0.001) demonstrated poorer median CSS compared to LNR1. Similarly, median OS for LNR1, LNR2, and LNR3 were 50, 35, and 16 months. LNR2 (HR = 1.36, 95% CI: 1.27-1.45; P < 0.001) and LNR3 (HR = 2.60, 95% CI: 2.37-2.85; P < 0.001) exhibited worse median OS compared to LNR1. A revised pN (r-pN) classification incorporating LNR and pN demonstrated superior penalized goodness-of-fit and discriminative ability in predicting CSS and OS compared to both LNR and pN. Conclusion: LNR outperformed pN in predicting CSS and OS in NSCLC patients undergoing surgery, potentially leading to more precise adjuvant treatment decisions.

CEMIP-mediated hyaluronan metabolism facilitates SCLC metastasis by activating TLR2/c-Src/ERK1/2 axis.

Small-cell lung cancer (SCLC) is a highly metastatic and recalcitrant malignancy. Metastasis is the major cause of death in patients with SCLC but its mechanism remains poorly understood. An imbalance of hyaluronan catabolism in the extracellular matrix accelerates malignant progression in solid cancers due to the accumulation of low-molecular-weight HA. We previously found that CEMIP, a novel hyaluronidase, may act as a metastatic trigger in SCLC. In the present study, we found that both CEMIP and HA levels were higher in SCLC tissues than in paracancerous tissues from patient specimens and in vivo orthotopic models. Additionally, high expression of CEMIP was associated with lymphatic metastasis in patients with SCLC, and in vitro results showed that CEMIP expression was elevated in SCLC cells relative to human bronchial epithelial cells. Mechanistically, CEMIP facilitates the breakdown of HA and accumulation of LMW-HA. LMW-HA activates its receptor TLR2, and subsequently recruits c-Src to activate ERK1/2 signalling, thereby promoting F-actin rearrangement as well as migration and invasion of SCLC cells. In addition, the in vivo results verified that depletion of CEMIP attenuated HA levels and the expressions of TLR2, c-Src, and phosphorylation of ERK1/2, as well as liver and brain metastasis in SCLC xenografts. Furthermore, the application of the actin filament inhibitor latrunculin A significantly inhibited the liver and brain metastasis of SCLC in vivo. Collectively, our findings reveal the critical role of CEMIP-mediated HA degradation in SCLC metastasis and suggest its translational potential as an attractive target and a novel strategy for SCLC therapy.

Expression and Predictive Significance of FHL1 and SLIT3 in Surgically Resected Lung Adenocarcinoma.

OBJECTIVE: Lung adenocarcinoma (LUAD) is the most common type of lung cancer. However, predictive biomarkers for early efficacy and prognosis evaluation in patients with surgically resected LUAD are not completely explained. METHODS: Differentially expressed genes (DEGs), gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) were identified by RNA sequencing (RNA-Seq) between thirteen LUAD tissues and five normal lung tissues. The expression of DEGs was confirmed by qRT-PCR and a validated cohort from GEPIA. Protein-protein interaction (PPI) network of the top 5% DEGs was constructed by STRING and visualized in Cytoscape. Immunofluorescence results were acquired from clinical specimens from LUAD patients. The expression of FHL1 was analyzed by ImageJ. Survival analysis was performed using the GEPIA dataset. RESULTS: Consistent with the RNA-Seq data, validation of DEGs expression by qRT-PCR and GEPIA cohort showed that FHL1 and SLIT3 were down-regulated in LUAD patient tissues compared with non-tumor tissues. Moreover, FHL1 was significantly reduced in LUAD cell lines compared to the bronchial epithelium cell line (P < 0.01). However, SLIT3 was elevated in A549 and H1299 cells (wide type EGFR) (P < 0.05) while decreased in HCC827 and PC9 cells (mutant EGFR) compared to BESA-2B cells (P < 0.01). PPI network revealed the most significant cluster with 10 nodes and 43 edges. Immunofluorescent staining also showed that the expression of FHL1 was lower in LUAD tissues compared with that in normal lung tissues (P < 0.01). The expressions of SLIT3 and FHL1 were positively correlated. Specifically, the higher expression level of SLIT3 and FHL1 independently predicted a better prognosis (P < 0.01 or P < 0.05). CONCLUSION: Our findings provide two novel candidates, FHL1 and SLIT3, for prognostic evaluation and treatments after surgery.

Postoperative radiotherapy in pIIIA-N2 non-small cell lung cancer after complete resection and adjuvant chemotherapy: A meta-analysis.

BACKGROUND: This study aimed to evaluate the effect of postoperative radiotherapy (PORT) in patients with pIIIA-N2 non-small cell lung cancer after complete resection and adjuvant chemotherapy. METHODS: Electronic databases (PubMed, Web of Science databases, Embase, and the Cochrane Central Register of Controlled Trials) were systematically searched to extract randomized control trials comparing PORT with observation in pIIIA-N2 non-small cell lung cancer patients until October 2021. Main outcomes were disease-free survival (DFS), overall survival (OS), and local recurrence. RESULTS: Three-phase 3 randomized control trials involving 902 patients were included: 455 patients in the PORT group and 447 patients in the observation group. The methodological quality of the 3 randomized control trials were high quality. The pooled analysis revealed that PORT decreased local recurrence rate (odds ratio = 0.56, 95% confidence interval [CI]: 0.40-0.76). However, PORT did not improve median DFS (hazard ratio = 0.84, 95% CI: 0.71-1.00) and OS (hazard ratio = 1.02, 95% CI: 0.68-1.52). CONCLUSIONS: PORT decreased the incidence of local recurrence. However, PORT did not improve DFS and OS.

External validation of the eighth edition of the TNM classification for lung cancer in small cell lung cancer.

OBJECTIVES: The newly released eighth edition of the American Joint Committee on Cancer TNM staging system for lung cancer seeks to improve prognostic accuracy but lacks external validation for small cell lung cancer (SCLC). Moreover, previous studies posed a few questions concerning survival differences for patients with specific site N3 node involvement or single-site metastasis (SSM) in different distant organs. The aim of this study was to validate the eighth edition of the TNM classification for SCLC in an independent multi-institutional cohort from China and answer the questions raised by the previous research. METHODS: Patients with SCLC from four Chinese cancer centers between 2009 and 2019 were reclassified according to the seventh and eighth edition of the TNM classification. Survival was estimated using the Kaplan-Meier method. Comparisons between adjacent categories and stage groups were performed using Cox proportional hazard regression. R2 statistics were calculated to evaluate the discriminating performance of editions. RESULTS: Of 3384 enrolled cases, 3358 had clinical stage, 537 had pathological stage, and 511 had both. Progressive deterioration of survival was observed with advancing of TNM categories and stages both in the seventh and the eighth edition. The eighth edition stages had a higher R2 statistic than the seventh edition (0.207 versus 0.197). Newly defined categories M1b and M1c and stages IIIC, IVA and IVB in the eighth edition discriminated groups with significantly different prognosis. Patients with N3 contralateral supraclavicular nodes had a significantly worse prognosis than those without (p = 0.032). For patients with single-site metastasis, liver involvement showed a worse prognosis compared to brain involvement (p = 0.030). CONCLUSIONS: Our study provided an external validation of the eighth edition of the TNM classification for lung cancer in Chinese patients with SCLC, and confirmed its improved prognostic accuracy compared with the seventh edition. Patients with N3 and M1b might represent heterogeneous populations that warrant further research.

Identification of a novel therapeutic candidate, NRK, in primary cancer-associated fibroblasts of lung adenocarcinoma microenvironment.

PURPOSE: Lung adenocarcinoma (LUAD) accounts for approximately half of patients in lung cancer. Cancer-associated fibroblasts (CAFs) are the major component in the tumor microenvironment (TME). Targeting CAFs is a promising therapeutic strategy for cancer treatment. However, therapeutic targets of CAFs in LUAD remains largely unclear. METHODS: Seven CAFs and nine normal fibroblasts (NFs) were isolated from tumor and paratumor tissues of LUAD patients undergoing surgery, respectively. RNA-seq and bioinformatics analysis were performed to identify the differentially expressed genes (DEGs) and their functions in CAFs compared with NFs. DEGs of ten overlaying were obtained from RNA-seq, our previously reported lncRNA microarray and public datasets (E-MTAB-6149, E-MTAB-6653) and validated by RT-qPCR. Nik-related kinase (NRK) was further validated by RT-qPCR, immunofluorescence (IF), Western Blot (WB) in vitro, and in Cancer Cell Line Encyclopedia (CCLE) database. Survival analysis was performed on Kaplan-Meier plotter. RESULTS: A total of 1799 DEGs were identified, including 650 upregulated DEGs and 1149 downregulated DEGs. The upregulated and downregulated DEGs were mostly enriched in extracellular matrix (ECM) functions and in glycolysis/gluconeogenesis pathways. Interestingly, NRK was the most significantly upregulated overlaying DEGs which was rarely associated with CAFs before. NRK was predominantly expressed in CAFs, but weakly expressed in NFs, normal lung bronchial epithelial cell line BEAS-2B, LUAD cell lines A549 and H1299, as well as in the majority of 191 lung cancer cell lines including LUAD. Moreover, elevated NRK predicted poor survival in LUAD patients. CONCLUSION: Here, we first report that NRK is significantly elevated in LUAD-associated CAFs and may function as a promising therapeutic target for cancer combination treatment. Besides, modulation of ECM and glycolysis/gluconeogenesis pathways may be an efficient approach to alter CAFs functionality in LUAD.

Expert consensus on resection of chest wall tumors and chest wall reconstruction.

Chest wall tumors are a relatively uncommon disease in clinical practice. Most of the published studies about chest wall tumors are usually single-center retrospective studies, involving few patients. Therefore, evidences regarding clinical conclusions about chest wall tumors are lacking, and some controversial issues have still to be agreed upon. In January 2019, 73 experts in thoracic surgery, plastic surgery, science, and engineering jointly released the Chinese Expert Consensus on Chest Wall Tumor Resection and Chest Wall Reconstruction (2018 edition). After that, numerous experts put forward new perspectives on some academic issues in this version of the consensus, pointing out the necessity to further discuss the points of contention. Thus, we conducted a survey through the administration of a questionnaire among 85 experts in the world. Consensus has been reached on some major points as follows. (I) Wide excision should be performed for desmoid tumor (DT) of chest wall. After excluding the distant metastasis by multi-disciplinary team, solitary sternal plasmacytoma can be treated with extensive resection and adjuvant radiotherapy. (II) Wide excision with above 2 cm margin distance should be attempted to obtain R0 resection margin for chest wall tumor unless the tumor involves vital organs or structures, including the great vessels, heart, trachea, joints, and spine. (III) For patients with chest wall tumors undergoing unplanned excision (UE) for the first time, it is necessary to carry out wide excision as soon as possible within 1-3 months following the previous surgery. (IV) Current Tumor Node Metastasis staging criteria (American Joint Committee on Cancer) of bone tumor and soft tissue sarcoma are not suitable for chest wall sarcomas. (V) It is necessary to use rigid implants for chest wall reconstruction once the maximum diameter of the chest wall defect exceeds 5 cm in adults and adolescents. (VI) For non-small cell lung cancer (NSCLC) invading the chest wall, wide excision with neoadjuvant and/or adjuvant therapy are recommended for patients with stage T3-4N0-1M0. As clear guidelines are lacking, these consensus statements on controversial issues on chest wall tumors and resection could possibly serve as further guidance in clinical practice during the upcoming years.

A novel metastatic promoter CEMIP and its downstream molecular targets and signaling pathway of cellular migration and invasion in SCLC cells based on proteome analysis.

PURPOSE: Metastasis is an unavoidable event happened among almost all small cell lung cancer (SCLC) patients. However, the molecular driven factors have not been elucidated. Recently, a novel hydrolase called cell migration inducing hyaluronidase (CEMIP) triggered both migration and invasion in many tumors but not SCLC. Therefore, in this study, we verified that CEMIP promoted migration and invasion in SCLC and applied proteomics analysis to screen out potential target profiles and the signaling pathway related to CEMIP regulation. METHOD: Immunofluorescence was conducted to exam the expression of CEMIP on SCLC and paired adjacent normal tissues among enrollment. RT-qPCR and Western blot (WB) assays were conducted to valuate cellular protein and mRNA expression of CEMIP and EMT markers. Lentivirus-CEMIP-shRNAs and CEMIP plasmid were used for expression manipulating. Changes of cellular migration and invasion were tested through transwell assays. Tandem Mass Tag (TMT) peptide labeling coupled with LC-MS/MS was used for quantifying proteins affected by reducing expression of CEMIP on H446 cells. RESULTS: The expression of CEMIP showed 1.64 ± 0.16-fold higher in SCLC tissues than their normal counterpart. Decreasing the expression of CEMIP on SCLC cells H446 regressed both cellular migration and invasion ability, whereas the promoting cellular migration and invasion was investigated through over-expressing CEMIP on H1688. Proteomic and bioinformatics analysis revealed that total 215 differentially expressed proteins (DEPs) that either their increasing or decreasing relative expression met threshold of 1.2-fold changes with p value ≤ 0.05. The dramatic up-regulated DEPs included an unidentified peptide sequence (encoded by cDNA FLJ52096) SPICE1 and CRYAB, while the expression of S100A6 was largely down-regulated. DEPs mainly enriched on caveolae of cellular component, calcium ion binding of biological process and epithelial cell migration of molecular function. KEGG enrichment indicated that DEPs mainly exerted their function on TGF-ß, GABAergic synapse and MAPK signaling pathway. CONCLUSION: It is the first report illustrating that CEMIP might be one of the metastatic triggers in SCLC. And also, it provided possible molecular mechanism cue and potential downstream target on CEMIP-induced cellular migration and invasion on SCLC.

NFE2L2/KEAP1 Mutations Correlate with Higher Tumor Mutational Burden Value/PD-L1 Expression and Potentiate Improved Clinical Outcome with Immunotherapy.

BACKGROUND: Abnormalities in the KEPA1-NRF2 pathway have a role in cancer progression, metastasis, and resistance to chemo- and radiotherapies. Persistent activation of NRF2 associates with poor prognosis across different cancer types. However, the beneficial therapeutic strategy to harness this pathway in cancer remains unclear. This study aimed to investigate the clinical outcome with immunotherapy in NFE2L2/KEAP1 mutant population. MATERIALS AND METHODS: We investigated the correlation between NFE2L2/KEAP1 mutations and tumor mutational burden (TMB) and programmed death-ligand 1 (PD-L1) expression status to identify the therapeutic vulnerability. For this purpose, relevance analysis with TMB value was performed in 9,040 patients with cancer, and relevance analysis with PD-L1 expression was performed in 3,457 patients. The Memorial Sloan Kettering Cancer Center (MSKCC) database and real-world evidence were used to assess the immunotherapy response in NFE2L2/KEAP1 mutant subsets. RESULTS: NFE2L2/KEAP1 mutations occurred in various cancers, and the highest mutation incidences occurred in lung squamous cell carcinoma (LUSC) at 19.16% (NFE2L2) and 10.31% (KEAP1). We confirmed that higher TMB value and PD-L1 expression were associated with NFE2L2/KEAP1 mutations compared with wild-type, especially in non-small lung cancer. MSKCC database analysis showed the improved survival of patients with NFE2L2/KEAP1 mutant with immunotherapy compared with other treatments (median overall survival 22.52 VS 12.89, p = .0034). Real-world evidence further confirmed the efficacy of immunotherapy in the mutant population. CONCLUSION: Our study revealed that patients with NFE2L2/KEAP1 mutant could achieve improved outcomes from immunotherapy than the other treatments. These findings may broaden the application of immune checkpoint blockade to patients harboring NFE2L2/KEAP1 mutations. IMPLICATIONS FOR PRACTICE: NFE2L2/KEAP1 alterations occur frequently in multiple cancer types and are associated with poor prognosis; however, the efficacious strategy to inhibit this pathway in cancer is poorly understood. This study was designed to analyze the mutational characteristics of NFE2L2/KEAP1 alterations in 9,243 Chinese patients. The highest mutation incidences occurred in lung squamous cell carcinoma at 19.16% (NFE2L2) and 10.31% (KEAP1). Relevance analysis showed the NFE2L2/KEAP1 mutant subsets were associated with higher tumor mutational burden value and programmed death-ligand 1 expression. Clinical data further confirmed NFE2L2/KEAP1 mutations correlate with improved outcome with immunotherapy. These findings suggest the clinical application of immunotherapy in the NFE2L2/KEAP1 mutant population.

[Effect of transcutaneous electrical acupoint stimulation on venous thrombosis after lung cancer surgery: a randomized controlled trial].

OBJECTIVE: To observe the effect of transcutaneous electrical acupoint stimulation (TEAS) on venous thrombosis and quality of life after lung cancer surgery, basing on the conventional nursing and early functional exercise. METHODS: A total of 120 patients diagnosed as non-small cell lung cancer (NSCLC) and received radical resection of lung cancer surgery for the first time were randomized into a conventional nursing group, a rehabilitation training group and a TEAS group, 40 cases in each group. Conventional nursing was adopted in the conventional nursing group. Conventional nursing combined with early functional exercise were adopted in the rehabilitation training group, the exercise was taken 20 min each time, once in both morning and afternoon for 5 days. On the basis of the treatment in the rehabilitation training group, TEAS was applied at Zusanli (ST 36), Xuehai (SP 10), Sanyinjiao (SP 6), etc. in the TEAS group, with disperse-dense wave in frequency of 30 Hz/100 Hz and tolerable intensity, 30 min each time, once in both morning and afternoon for 5 days. The incidence of venous thrombosis in each group was observed at the 5th day after surgery. Before surgery and at the 5th day after surgery, the Caprini thrombus risk assessment was performed, the Karnofsky performance status (KPS) scale and the functional assessment of cancer therapy-lung (FACT-L) were used to evaluate the quality of life. RESULTS: At the 5th day after surgery, no thrombosis was found in the TEAS group, the incidence of venous thrombosis in the TEAS group was lower than 15.0% (6/40) in the conventional nursing group (P<0.05). At the 5th day after surgery, the Caprini scores were increased in the 3 groups (P<0.01), while that in the TEAS group was lower than the conventional nursing group (P<0.05); the KPS scores were decreased in the 3 groups (P<0.01), while those in the TEAS group and the rehabilitation training group were higher than the conventional nursing group (P<0.01, P<0.05); the total scores and the subitem scores of FACT-L were decreased in the 3 groups (P<0.05), while the total score of FACT-L and the subitem score of lung cancer specificity in the TEAS group were higher than those in the conventional nursing group (P<0.05). CONCLUSION: On the basis of the conventional nursing and early functional exercise, TEAS can reduce the incidence of venous thrombosis, effectively prevent thrombosis and improve quality of life.

Diagnosis of NUT carcinoma of lung origin by next-generation sequencing: case report and review of the literature.

NUT carcinoma (NC) is an aggressive squamous tumor characterized by NUT gene rearrangement, and the most common fusion form is BRD4-NUT. However, NC diagnosis is difficult for its rareness and often being confused with a variety of poorly differentiated tumors. A 21-year-old Chinese woman was referred to our hospital for cough and intermittent fever. Chest computed tomography (CT) imaging revealed a left lobe hilar mass. Fiberoptic bronchoscopy results showed that tumor cells were poorly differentiated. In combination with immunohistochemistry staining, she was misdiagnosed with Ewing's sarcoma/primitive neuroectodermal tumor. Next-generation sequencing (NGS) revealing BRD4-NUT fusion, and NUT immunohistochemistry confirmed the diagnosis of NC. Subsequently, left pneumonectomy and lymph node dissection were performed, and the patient received pemetrexed and lobaplatin treatment. NGS technology played an important role in NC diagnosis in this case, and it may have clinical use for rare cancer diagnosis and guidance of potential targeted therapies.

Cancer-Associated Fibroblasts Accelerate Malignant Progression of Non-Small Cell Lung Cancer via Connexin 43-Formed Unidirectional Gap Junctional Intercellular Communication.

BACKGROUND/AIMS: Gap junctions, which are assembled by connexins, can directly connect the cytoplasm of adjacent cells and enable gap junctional intercellular communication (GJIC) as well as metabolic coupling between neighboring cells. Here, we investigated the role of connexin 43 (Cx43) and its derived GJIC in the interplay between non-small cell lung cancer (NSCLC) cells and cancer-associated fibroblasts (CAFs). METHODS: CAFs and NSCLC cells were co-cultured with direct contact and separated using flow cytometry. Glucose uptake, lactate production, and the expression and activity of PKM-2 and LDH-A in sorted CAFs were measured by a colorimetric assay, western blotting, and enzyme-linked immunosorbent assay (ELISA). Meanwhile, E-cadherin and N-cadherin expression and the migration and invasion of sorted NSCLC cells were detected by western blotting, wound width, and Transwell assays. Pyruvate, acetyl-CoA, and citric acid levels, ATP levels, and LDH-B and α-KG activity in sorted NSCLC cells were determined by a colorimetric or fluorometric assay and ELISA, respectively. Functional GJIC between cells and the subcellular location of connexins were detected by a "Parachute" assay and immunofluorescence. Levels of α-SMA, Cx43, and LDH-B in tissue from patients with NSCLC were determined by immunohistochemistry. RESULTS: Cx43 accumulated in the plasma membrane, which favored the assembly of asymmetric unidirectional GJIC from CAFs to NSCLC cells. CAFs underwent increased aerobic glycolysis and promoted the epithelial-mesenchymal transition, migration, and invasion of NSCLC cells. In contrast, NSCLC cells experienced enhanced oxidative phosphorylation upon CAF stimulation, with an increase in ATP generation and thereby activation of the PI3K/Akt and MAPK/ERK pathways. Metabolic coupling between CAFs and NSCLC cells was under the strict control of Cx43-formed unidirectional GJIC. Patients with high tri-expression of α-SMA, Cx43, and LDH-B had the shortest overall survival and relapse-free survival compared with those with individual overexpression or high bi-expression. CONCLUSION: Cx43-formed unidirectional GJIC plays a critical role in mediating close metabolic cooperation between CAFs and NSCLC cells to support the malignant progression of NSCLC.

Identification of side population cells in human lung adenocarcinoma A549 cell line and elucidation of the underlying roles in lung cancer.

The present study aimed to isolate and characterize side population (SP) cells in the human lung cancer A549 cell line, and elucidate the molecular mechanism of SP cells underlying lung cancer. The SP and non-SP (NSP) cells in A549 cells were isolated and their differentiation was analyzed by fluorescence-activated cell sorting. An in vitro plate clone assay, Matrigel® Transwell assay and chemoresistance analysis of the sorted SP and NSP cells were performed. In addition, the sorted SP and NSP cells were injected into BALB/c nude mice to detect their tumorigenic potential in vivo. The expression of ATP-binding cassette sub-family G member 2 (ABCG2) in transplanted tumors was detected by immunohistochemistry. The SP and NSP cells were successfully isolated. The results demonstrated that SP cells accounted for 1.09% of live A549 cells. SP cells produced SP and NSP cells, while NSP cells only produced NSP cells. In addition, SP cells formed more colonies, exhibited improved invasive ability and increased levels of chemoresistance compared with NSP cells in vitro. SP cells demonstrated a higher tumorigenic potential in BALB/c nude mice, and the number of ABCG2-positive cells in the SP xenograft tumors were significantly increased compared with that in the NSP xenograft tumors. The present study indicated that SP cells isolated from the human lung cancer A549 cell line demonstrated increased tumorigenicity, and improved invasive ability and chemoresistance compared with NSP cells. In addition, detection of ABCG2 expression may assist in predicting the chemotherapeutic outcome of patients, and serve as a target for treating lung cancer.

Efficient targeted tumor imaging and secreted endostatin gene delivery by anti-CD105 immunoliposomes.

BACKGROUND: Anti-CD105 mAb-conjugated immunoliposomes, loaded with secreted mouse endostatin gene, were developed for targeted tumor imaging and antiangiogenic gene therapy. METHODS: The liposomes were investigated for size, zeta-potential, lipid content, antibody binding ability, and pcDNA loading capacity. The ability of immunoliposomes to target tumor-derived endothelial cells and perform gene transfer in vitro was measured and their basic biocompatibility was evaluated. A nude mouse/breast cancer xenograft model was used to examine the tumor internalization of fluorescent-labeled liposomes and the clinical potential of immnuoliposomes loaded with pcDNA3.1-CSF1-endostatin. RESULTS: Loaded immunoliposomes were homogenously distributed with a well-defined spherical shape and bilayer, diameter of 122 ± 11 nm, and zeta potential + 1.40 mV. No significant differences were observed in body weight, liver index, oxidative stress, or liver and kidney function in mice after liposomes exposure. The addition of CD105 mAb to liposomes conferred the ability to target tumor-derived endothelial cells in vitro and in vivo. Systemic intravenous administration of fluorescent immunoliposomes in the xenograft model resulted in selective and efficient internalization in tumor vasculature. Treatment of mice with pcDNA3.1-CSF1-endostatin-loaded immunoliposomes suppressed tumor growth by 71%. CONCLUSIONS: These data demonstrate the advantages of using anti-CD105 mAb-conjugated immunoliposomes to enhance tumor targeting, imaging, and gene transfer applications.

An Analysis of EGFR Mutations among 1506 Cases of Non-Small Cell Lung Cancer Patients in Guangxi, China.

An association between epidermal growth factor receptor (EGFR) and clinical characteristics of non-small cell lung cancer (NSCLC) was reported ten years ago. In addition, a different type of relationship was seen in different ethic races. However, the relationship between these factors is not well understood in the Guangxi province. Up to now, there are only very limited data on the association of TTF1/EGFR protein positivity and EGFR mutation status in NSCLC. This study aims to investigate the role of EGFR gene mutation status on the clinical characteristics and the relationship with TTF-1/EGFR protein positivity of patients with NSCLC in Guangxi, China. 1506 samples from different patients with NSCLC were detected by amplification refractory mutation system for 29 hotspot mutations. Analysis of the relationship between clinical characteristics and EGFR mutation status was performed by using the crosstabs Chi-square and SPSS 21.0 software. Of 1506 samples, 537 (35.7%) revealed tyrosine kinase inhibitor (TKI) sensitive EGFR mutations with 27 (1.8%) cases harboring TKI resistant EGFR mutations or union co-existing EGFR-TKIs sensitive mutations. EGFR-TKIs sensitive mutations were not significantly associated with age and TNM-M stage (P = 0.863; P = 0.572, respectively). However, they were significantly associated with p-stage, TNM-T stage and TNM-N stage (P = 0.011, P < 0.001, P = 0.036, respectively). Immunohistochemical studies revealed that TTF-1 and EGFR protein expression level were all associated with EGFR mutation status (P < 0.001, P = 0.002, respectively). Of the 537 EGFR-TKIs sensitive mutation cases, the rates of exon 19-del, 18 G719X point, exon 21 L858R and L861Q points were 54.6, 0.9, 42.3 and 0.9%, respectively. EGFR TKI-sensitive mutations commonly occur in female, non-smoking and adenocarcinoma patients. The p-stage, TNM-T stage, TNM-N stage, EGFR and TTF-1 protein expression levels have close relationships with EGFR mutation status.

Excision repair cross complementation group 1 is a chemotherapy-tolerating gene in cisplatin-based treatment for non-small cell lung cancer.

This study aimed to evaluate the biological functions of excision repair cross complementation goup 1 (ERCC1) in cell proliferation, cell cycle, invasion and cisplatin response of non-small cell lung cancer (NSCLC) cells. Firstly, ERCC1 gene was successfully transfected into H1299 cells by gene cloning and transfection techniques. Then, cell proliferation was determined with the cell growth curve and colony-forming assays. Flow cytometry (FCM) was employed to investigate the cell cycle distribution. The ability of cell invasion was estimated by means of Matrigel invasion assays. Response of NSCLC cells to cisplatin was detected utilizing MTT assays, and the intracellular drug concentrations were determined by the high performance liquid chromatography (HPLC) analysis. Expression of the two cell membrane proteins, P-glycoprotein (P-gp) and multidrug resistance-associated protein (MRP), was also evaluated utilizing FCM technique. By contrast, ERCC1 expression in the NSCLC A549 cells was silenced by small interfering RNA (siRNA) through RNAi technique. In addition, the cytotoxic effect of cisplatin on A549 cells was detected by MTT assays. In the present study, the results demonstrated that ERCC1 had no effect on cell proliferation, cell cycle and the ability of invasion, but showed significant impact on cisplatin response of the NSCLC H1299 cells. Furthermore, siRNA-induced suppression of ERCC1 evidently enhanced sensitivity to cisplatin of NSCLC A549 cells. Therefore, it is confirmed that ERCC1 is a chemotherapy-tolerating gene and a promising predictor in tailoring chemotherapy of NSCLC.

Casticin induces caspase-mediated apoptosis via activation of mitochondrial pathway and upregulation of DR5 in human lung cancer cells.

OBJECTIVE: To assess if casticin induces caspase-mediated apoptosis via activation of mitochondrial pathway and upregulation of DR5 in human lung cancer cells. METHODS: Human non-small-cell lung carcinoma cell lines H460, A549 and H157 were cultured in vitro. The cytotoxic activities were determined using MTT assay. The apoptotic cells death was examined by flow cytometry using PI staining and DNA agarose gel electrophoresis. The activities of caspase-3, -8 and -9 were measured via ELISA. Cellular fractionation was determined by flow cytometry to assess release of cytochrome c and the mitochondrial transmembrane potential. Bcl-2/Bcl-XL/XIAP/Bid/DR5 and DR4 proteins were analyzed using western blot. RESULTS: The concentrations required for a 50% decrease in cell growth (IC(50)) ranged from 1.8 to 3.2 µM. Casticin induced rapid apoptosis and triggered a series of effects associated with apoptosis by way of mitochondrial pathway, including the depolarization of the mitochondrial membrane, release of cytochrome c from mitochondria, activation of procaspase-9 and -3, and increase of DNA fragments. Moreover, the pan caspase inhibitor zVAD-FMK and the caspase-3 inhibitor zDEVD-FMK suppressed casticin-induced apoptosis. In addition, casticin induced XIAP and Bcl-XL down-regulation, Bax upregulation and Bid clearage. In H157 cell line, casticin increased expression of DR5 at protein levels but not affect the expression of DR4. The pretreatment with DR5/Fc chimera protein effectively attenuated casticin-induced apoptosis in H157 cells. No correlation was found between cell sensitivity to casticin and that to p53 status, suggesting that casticin induce a p53-independent apoptosis. CONCLUSIONS: Our results demonstrate that casticin induces caspase-mediated apoptosis via activation of mitochondrial pathway and upregulation of DR5 in human lung cancer cells.

[Isolation and identification of side population cells in human lung adenocarcinoma cell line A549].

OBJECTIVE: To isolate and characterize the side population cells (SP cells) in the lung adenocarcinomas cell line A549. METHODS: The protein expression of ABCG2 in human lung adenocarcinoma cell line A549 was detected by immunohistochemistry. SP and NSP cells in the cell line A549 were isolated by FACS, and their differentiation was analysed. ABCG2 expression in the two cell subsets was detected by RT-PCR. The cell growth curves, cell division indexes, cell cycles, plate clone formation tests, migration and invasion assays, chemotherapeutic susceptibility tests, tests of the intracellular drug levels, and the tumor cell implantation experiments on nude mice were applied to study the biological properties of the two cell subsets. The expression of ABCG2 in the transplanted tumor in nude mice was detected by immunohistochemistry and RT-PCR. RESULTS: The positive rate of ABCG2 expression in the A549 cells by immunohistochemistry was 2.13%. SP and NSP cells were isolated by FACS. The SP cells could produce both SP and NSP cells, while NSP cells only produced NSP cells. SP cells expressed ABCG2, but NSP cells did not. The proliferation and migration abilities of the two cell subsets were similar, but the invasion and tumorigenic ability of SP cells was significantly higher than that of NSP cells. The susceptibilities to DDP and its intracellular levels of the two cell subsets were similar, but the susceptibilities to 5-FU, VP16, NVB and GEM and their intracellular levels of NSP cells were significantly higher than those of the SP cells. CONCLUSIONS: SP cells in the human lung adenocarcinomas cell line A549 is enriched with tumor stem cells. An effective way to get lung adenocarcinomas stem cells is to isolate SP cells by FACS.