Significance of macrophage infiltration in the prognosis of lung adenocarcinoma patients evaluated by scRNA and bulkRNA analysis.

Purpose: To investigate the significance of macrophage infiltration to the prognosis of lung adenocarcinoma. Methods: R language bioinformatics analysis technology, was used to obtain macrophage infiltration-related module genes through WGCNA (Weighted Gene Co-Expression Network Analysis). Marker genes of macrophage subtypes were identified using single-cell sequencing of lung adenocarcinoma tissue. Risk score models were constructed and validated using external data cohorts and clinical samples. Results: Analysis of cohorts TCGA-LUAD, GSE11969, GSE31210, GSE50081, GSE72094 and GSE8894, revealed a negative correlation between macrophage infiltration and survival. Immunohistochemical analyses of clinical samples were consistent with these data. Based on cell-cluster-markers and TAMs-related-genes, TOP8 genes were obtained (C1QTNF6, CCNB1, FSCN1, HMMR, KPNA2, PRC1, RRM2, and TK1) with a significant association to prognosis. Risk score models including 9 factors (C1QTNF6, FSCN1, KPNA2, GLI2, TYMS, BIRC3, RBBP7, KRT8, GPR65) for prognosis were constructed. The efficacy, stability and generalizability of the risk score models were validated using multiple data cohorts (GSE19188, GSE26939, GSE31210, GSE50081, GSE42127, and GSE72094). Conclusions: Macrophage infiltration negatively correlates with prognosis in patients with lung adenocarcinoma. Based on cell-cluster-markers and TAMs-related-genes, both TOP8 genes (C1QTNF6, CCNB1, FSCN1, HMMR, KPNA2, PRC1, RRM2, TK1) and risk score models using C1QTNF6, FSCN1, KPNA2, GLI2, TYMS, BIRC3, RBBP7, KRT8, GPR65 could predict disease prognosis.

LncRNA MIR9-3HG enhances LIMK1 mRNA and protein levels to contribute to the carcinogenesis of lung squamous cell carcinoma via sponging miR-138-5p and recruiting TAF15.

The aberrantly expressed long non-coding RNAs (lncRNAs) are closely correlated with the malignant progression of cancer cells. In our study, we identified lncRNA MIR9-3 host gene (MIR9-3HG) as the research target and explored its roles in lung squamous cell carcinoma (LUSC). RT-qPCR was conducted to reveal that MIR9-3HG was observably overexpressed in LUSC cells. Functional assays encompassing colony formation, 5-ethynyl-2'-deoxyuridine (EdU) staining, transwell and flow cytometry assays and western blot detecting related proteins demonstrated that MIR9-3HG depletion hampered cell proliferation, migration, invasion and epithelial-mesenchymal transition (EMT) while accelerating cell apoptosis in LUSC. Subcellular fractionation assay were performed to demonstrate that MIR9-3HG was prominently distributed in the cytoplasm of LUSC cells. Luciferase reporter, RNA-binding protein immunoprecipitation (RIP), immunofluorescence (IF), fluorescent in situ hybridization (FISH) and RNA pull down assays were implemented to confirm that MIR9-3HG modulates LIM domain kinase 1 (LIMK1) mRNA and protein levels by sequestering microRNA-138-5p (miR-138-5p) and recruiting TATA-box binding protein associated factor 15 (TAF15) protein. Taken together, our research determined that MIR9-3HG up-regulated LIMK1 mRNA and protein levels to promote LUSC carcinogenesis, which offers a novel insight into mechanisms of LUSC.

LncRNA LINC00649 recruits TAF15 and enhances MAPK6 expression to promote the development of lung squamous cell carcinoma via activating MAPK signaling pathway.

Lung squamous cell carcinoma (LUSC) represents one of the commonest types of lung cancer featured with high morbidity and poor prognosis. Many types of research have documented that long noncoding RNAs (lncRNAs) exert crucial functions in the development of cancers, and LUSC is included. In our study, we aimed at unveiling the mechanism underlying long intergenic nonprotein coding RNA 0649 (LINC00649) in LUSC cells. As a result, LINC00649 was discovered to be with high expression in LUSC cells. Moreover, it was confirmed through functional assays that the knockdown of LINC00649 hindered the occurrence and progression of LUSC. Results of mechanism assays validated that E2F transcription factor 7 (E2F7) was a transcription activator of LINC00649 and induced its up-regulation in LUSC cells. Furthermore, LINC00649 recruited TAF15, which is TATA-box binding protein associated factor 15 to stabilize mitogen-activated protein kinase 6 (MAPK6) expression and activate the transcription of MAPK6, thereby enhancing MAPK6 expression to activate the MAPK signaling pathway. Eventually, results of rescue assays suggested that overexpression of MAPK6 offset the influence of LINC00649 silencing on LUSC progression. In summary, our research determined the E2F7/LINC00649/TAF15/MAPK6/MAPK signaling pathway in regulating LUSC development, which made LINC00649 a potential biomarker for LUSC treatment.

Long non-coding RNA LALTOP promotes non-small cell lung cancer progression by stabilizing topoisomerase IIα mRNA.

The long noncoding RNAs (lncRNAs) have been shown to actively participate in various biological processes including cancer progression. However, most lncRNAs still have undefined functions. In current work, we identified a novel lncRNA named LALTOP which displayed an oncogenic function in non-small cell lung cancer (NSCLC). LALTOP expression is increased in NSCLC tissues and cell lines. Moreover, LALTOP strongly promoted proliferation and migration of A549 and H1793 cells. RNA-RNA interaction assay showed that LALTOP bound and stabilized topoisomerase II alpha (Top2α) mRNA. Positive correlation can be found between LALTOP and Top2α mRNA expressions in clinical specimens. ASOs targeting LALTOP could markedly inhibit malignant phenotypes of NSCLC. Collectively, LALTOP may serve as an oncogenic lncRNA and enhances NSCLC progression. Targeting LALTOP has therapeutic potential for eradicating lung cancer cells.

Thioridazine inhibits autophagy and sensitizes glioblastoma cells to temozolomide.

Glioblastoma multiforme (GBM) has a poor prognosis with an overall survival of 14-15 months after surgery, radiation and chemotherapy using temozolomide (TMZ). A major problem is that the tumors acquire resistance to therapy. In an effort to improve the therapeutic efficacy of TMZ, we performed a genome-wide RNA interference (RNAi) synthetic lethality screen to establish a functional gene signature for TMZ sensitivity in human GBM cells. We then queried the Connectivity Map database to search for drugs that would induce corresponding changes in gene expression. By this approach we identified several potential pharmacological sensitizers to TMZ, where the most potent drug was the established antipsychotic agent Thioridazine, which significantly improved TMZ sensitivity while not demonstrating any significant toxicity alone. Mechanistically, we show that the specific chemosensitizing effect of Thioridazine is mediated by impairing autophagy, thereby preventing adaptive metabolic alterations associated with TMZ resistance. Moreover, we demonstrate that Thioridazine inhibits late-stage autophagy by impairing fusion between autophagosomes and lysosomes. Finally, Thioridazine in combination with TMZ significantly inhibits brain tumor growth in vivo, demonstrating the potential clinical benefits of compounds targeting the autophagy-lysosome pathway. Our study emphasizes the feasibility of exploiting drug repurposing for the design of novel therapeutic strategies for GBM.

Surgical debulking promotes recruitment of macrophages and triggers glioblastoma phagocytosis in combination with CD47 blocking immunotherapy.

Surgical resection is a standard component of treatment in the clinical management of patients with glioblastoma multiforme (GBM). However, experimental therapies are rarely investigated in the context of tumor debulking in preclinical models. Here, a surgical debulking GBM xenograft model was developed in nude rats, and was used in combination with CD47 blocking immunotherapy, a novel treatment strategy that triggers phagocytosis of tumor cells by macrophages in diverse cancer types including GBM. Orthotopic patient-derived xenograft tumors expressing CD47 were resected at 4 weeks after implantation and immediately thereafter treated with anti-CD47 or control antibodies injected into the cavity. Debulking prolonged survival (median survival, 68.5 vs 42.5 days, debulking and non-debulking survival times, respectively; n = 6 animals/group; P = 0.0005). Survival was further improved in animals that underwent combination treatment with anti-CD47 mAbs (median survival, 81.5 days vs 69 days, debulking + anti-CD47 vs debulking + control IgG, respectively; P = 0.0007). Immunohistochemistical staining of tumor sections revealed an increase in recruitment of cells positive for CD68, a marker for macrophages/immune cell types, to the surgical site (50% vs 10%, debulking vs non-debulking, respectively). Finally, analysis of tumor protein lysates on antibody microarrays demonstrated an increase in pro-inflammatory cytokines, such as CXCL10, and a decrease in angiogenic proteins in debulking + anti-CD47 vs non-debulking + IgG tumors. The results indicated that surgical resection combined with anti-CD47 blocking immunotherapy promoted an inflammatory response and prolonged survival in animals, and is therefore an attractive strategy for clinical translation.

Role of medical thoracoscopy in the treatment of tuberculous pleural effusion.

BACKGROUND: Fibrous tuberculous pleural effusion (TPE) represents common disease in tuberculous clinic. Medical thoracoscopy has been used to treat pleural empyema and shown promising outcomes, but data of its use in multiloculated and organized TPE remains limited to know. METHODS: The study was performed on 430 cases with TPE. The cases were divided into free-flowing, multiloculated effusion and organized effusion group. Each group was subdivided into two or three types of therapeutic approaches: ultrasound guided pigtail catheter, large-bore tube chest drainage and medical thoracoscopy. Patients with multiloculated or organized effusions received streptokinase, introduced into the pleural cavity via chest tubes. The successful effectiveness of the study was defined as duration of chest drainage, time from treatment to discharge days and no further managements. RESULTS: Patients with organized effusion were older than those with free-flowing effusion and incidence of organized effusion combined with pulmonary tuberculosis (PTB) was higher than those of multiloculated effusion and free-flowing effusion respectively. Positive tuberculosis of pleural fluid culture was higher in organized effusion than that in free-flowing effusion. Sputum positive for acid-fast bacillus (AFB) in organized effusion was higher than that in multiloculated effusion and free-flowing effusion. Medical thoracoscopy showed significant efficacy in the group of multiloculated effusion and organized effusion but free-flowing effusion. No chronic morbidity and mortality related to complications was observed. CONCLUSIONS: Medical thoracoscopy was a safe and successful method in treating multiloculated and organized TPE.

A co-culture model with brain tumor-specific bioluminescence demonstrates astrocyte-induced drug resistance in glioblastoma.

BACKGROUND: Although several studies suggest that stromal fibroblasts mediate treatment resistance in several cancer types, little is known about how tumor-associated astrocytes modulate the treatment response in brain tumors. Since traditionally used metabolic assays do not distinguish metabolic activity between stromal and tumor cells, and since 2-dimensional co-culture system does not recreate the formidable complexity of the microenvironment within 3-dimensional structures such as solid tumor tissue, we instead established a glioblastoma (GBM) cell-specific bioluminescent assay for direct measurements of tumor cell viability in the treatment of clinical relevant drugs. METHODS: Using lentiviral transfection, we established a panel of human GBM cell lines constitutively expressing a fusion transgene encoding luciferase and the enhanced green fluorescence protein (eGFP). We then initiated co-cultures with immortalized astrocytes, TNC-1, and the eGFP/Luc GBM cell lines. Next, we treated all eGFP/Luc GBM cell lines with Temozolomide (TMZ) or Doxorubicin, comparing co-cultures of glioblastoma (GBM) cells and TNC-1 astrocytes with mono-cultures of eGFP/Luc GBM cells. Cell viability was quantitated by measuring the luciferase expression. RESULTS: Titration experiments demonstrated that luciferase expression was proportional to the number of eGFP/Luc GBM cells, whereas it was not influenced by the number of TNC-1 cells present. Notably, the presence of TNC-1 astrocytes mediated significantly higher cell survival after TMZ treatment in the U251, C6, A172 cell lines as well as the in vivo propagated primary GBM tumor cell line (P3). Moreover, TNC-1 astrocytes mediated significantly higher survival after Doxorubicin treatment in the U251, and LN18 glioma cell lines. CONCLUSION: Glioma cell-specific bioluminescent assay is a reliable tool for assessment of cell viability in the brain tumor cell compartment following drug treatment. Moreover, we have applied this assay to demonstrate that astrocytes can modulate chemo sensitivity of GBM tumor cells. These effects varied both with the cell line and cytotoxic drug that were used, suggesting that several mechanisms may be involved.

A novel GFP nude rat model to investigate tumor-stroma interactions.

BACKGROUD: A key strategy for the study of the tumor microenvironment is to implant human tumor cells in an immunodeficient rodent strain ubiquitously expressing a fluorescent marker. Here, a novel nude rat expressing a green fluorescent protein (GFP) transgene was established and engrafted with primary human tumor tissue in order to separate tumor from stromal cell populations for subsequent molecular analysis. METHODS: SD-TG (GFP) 2BalRrrc transgenic rats were crossed with HsdHan™: rnu/rnu Rowett nude rats to develop a GFP expressing immunocompromised rat. PCR and flow cytometry were used to follow the GFP genotype and phenotype in newborns. After three to four generations, animals were implanted with 4 T1 dsRed murine breast cancer cells or primary human glioblastoma (GBM) biopsies to generate xenografts for subsequent separation by fluorescence-activated cell sorting (FACS). RESULTS: Fluorecence microscopy and reverse transcription-PCR demonstrated that GFP, under the control of the human ubiquitin C promoter, was stably maintained and expressed in diverse organs over several generations. Immunophenotyping of blood samples by flow cytometry confirmed that the immunodeficient features of the parental rat strain, HsdHan™: rnu/rnu, were retained in the GFP nude rat. Both the murine cell line and human GBM biopsies engrafted, and stromal cell populations were isolated from dissociated xenografts by FACS to > 95% purity. CONCLUSIONS: A GFP transgene was stably introduced into a nude rat background in which human and murine cancer cells successfully engrafted. This animal strain provides a novel in vivo system for detailed cellular and molecular characterization of tumor-stroma interactions.