Transcription factor RUNX3 promotes CD8+ T cell recruitment by CCL3 and CCL20 in lung adenocarcinoma immune microenvironment.

Considering the existence of immune-desert in tumor microenvironment, the clinical efficacy of immunotherapy for lung adenocarcinoma is limited. This study aims to investigate the ability of transcription factors in regulating tumor immune microenvironment in lung adenocarcinoma. RNA-seq data were collected from the The Cancer Genome Atlas database. The relationships between transcription factors and immune infiltrates were assessed. Runt-related transcription factor 3 (RUNX3)-associated immune pathways were investigated by the Kyoto Encyclopedia of Genes and Genomes, Gene Ontology, and Gene set enrichment analysis. Upregulated chemokines in the RUNX3-overexpressed cell line were determined by quantitative real-time polymerase chain reaction, western blot, and enzyme-linked immunosorbent assay. These chemokines were further confirmed in RUNX3-downregulated cell lines. Immunochemistry was conducted to determine the expression of RUNX3, CCL3, CCL20, and the numbers of CD8+ T lymphocytes in human lung cancer tissues. Chemokine receptors in CD8+ T cells were explored by flow cytometry and immunofluorescence. T cell recruitment was investigated by transwell assay. After screening 406 transcription factors, RUNX3 was found strongly correlated T cells, cytotoxic lymphocytes, and CD8+ T cells. RUNX3 was associated with a variety of immunomodulators, including LAG3, CTLA-4, PD-1, and TIGIT. More importantly, RUNX3 was involved in immune-related pathways, especially immune cell migration-related pathways. Further investigation exhibited RUNX3 could upregulate CCL3 and CCL20 whose receptors CCR5 and CCR6 were upregulated in CD8+ effector T cells, while downregulation of RUNX3 decreased the expression of CCL3 and CCL20 and the infiltration of CD8+ T cells in RUNX3-downregulated lung cancer cell lines. Immunochemistry exhibited positive correlations of RUNX3 with CCL3 and CD8+ T cells in clinical lung adenocarcinoma samples. The chemotaxis assay proved RUNX3 could promote CD8+ T cell recruitment by upregulating CCL3 and CCL20. This study unearths RUNX3 related molecular mechanisms of tumor immune microenvironment and may reverse the immune-desert condition in lung adenocarcinoma and be combined with immune checkpoint blockade and adoptive cell therapy.

Identification of an immune signature predicting prognosis risk of patients in lung adenocarcinoma.

BACKGROUND: Lung cancer has become the most common cancer type and caused the most cancer deaths. Lung adenocarcinoma (LUAD) is one of the major type of lung cancer. This study aimed to establish a signature based on immune related genes that can predict patients' OS for LUAD. METHODS: The expression data of 976 LUAD patients from The Cancer Genome Atlas database (training set) and the Gene Expression Omnibus database (four testing sets) and 1534 immune related genes from the ImmPort database were used for generation and validation of the signature. The glmnet Cox proportional hazards model was used to find the best gene model and construct the signature. To assess the independently prognostic ability of the signature, the Kaplan-Meier survival analysis and Cox's proportional hazards model were performed. RESULTS: A gene model consisting of 30 immune related genes with the highest frequency after 1000 iterations was used as our signature. The signature demonstrated robust prognostic ability in both training set and testing set and could serve as an independent predictor for LUAD patients in all datasets except GSE31210. Besides, the signature could predict the overall survival (OS) of LUAD patients in different subgroups. And this signature was strongly associated with important clinicopathological factors like recurrence and TNM stage. More importantly, patients with high risk score presented high tumor mutation burden. CONCLUSIONS: This signature could predict prognosis and reflect the tumor immune microenvironment of LUAD patients, which can promote individualized treatment and provide potential novel targets for immunotherapy.

A novel neoadjuvant therapy for early-stage non-small cell lung cancer in a mouse model.

Background: Lung cancer is the common malignancy with high mortality rate in the world. Even with curative resection for early-stage lung cancer patients, the rate of postoperative recurrence and metastasis is still high. Neoadjuvant nivolumab combined with chemotherapy leads to improved pathological complete response rate and event-free survival in resectable non-small cell lung cancer (NSCLC) patients. However, the neoadjuvant therapy is not only accompanied by grade 3 or above adverse events which resulting in the potential missing out on the window for curative surgery for the patients, but also has low efficacy especially in patients with low programmed death ligand 1 (PD-L1) expression. Hence, it is particularly important to explore innovative ways to inhibit tumour recurrence and metastasis. Methods: In the present study, we investigated whether neoadjuvant therapy with intralesional Rose Bengal (RB) elicited specific immune responses compared with control group, and then the lung cancer mouse model was used to evaluated the immunological mechanism. Results: The secondary Lewis lung cancer cells (LLCs) tumour growth was significantly suppressed by RB intralesional injection into subcutaneous tumour; the formation rate of secondary tumours induced by the B16 melanoma cell injection was 100%. Intralesional RB neoadjuvant therapy before surgical resection exhibited effectively enhanced T central memory cells (Tcm) and T memory stem cells (Tscm) + naïve T cells (Tn) infiltration, elicited stronger cytotoxic T lymphocyte (CTL) responses against LLCs, and displayed markedly higher proportions of splenic lymphocytes that produce tumour necrosis factor-alpha (TNF-α) and interferon-gamma (IFN-γ) upon restimulation in a lung cancer mouse model. Conclusions: Based on our preclinical data, neoadjuvant therapy with intralesional RB injection generated immune memory and prevented the recurrence and metastasis of tumour in a lung cancer mouse model, which provides a new strategy for neoadjuvant treatment of early-stage NSCLC.

Preoperative diameters of aorta influence the remodeling after surgery for type A aortic dissection.

Background: Although current research revealed the preoperative diameter of the aorta is related to aortic remodeling, prognosis should consider the true lumen (TL) and false lumen (FL) respectively too. Methods: The cohort of this retrospective analysis included 161 type A aortic dissection (TAAD) patients who underwent surgery at a single institution from September 1, 2017, to September 1, 2018. Computed tomography angiography (CTA) images were reviewed to assess changes of the diameters of the TL, FL and total aorta at the levels of the stented segment, distal end of the stent, celiac trunk, and below the renal artery. Results: During the study period, positive remodeling was observed in 33 (20.5%) patients. The probability of negative remodeling far from the stent segment was greater than the aorta close to the stent. Only the TL diameter was associated with each levels' changes and underwent significant change (P<0.05). Multivariate analysis identified aortic regurgitation as a risk factor for remodeling in the distal end of the stent. The maximum diameter of the FL was the only risk factor related to the remodeling type (odds ratio =0.10; 95% confidence interval: 0.01-0.51), a maximum diameter of the FL of >1.28 cm was associated with a higher probability of negative remodeling after surgery (specificity =0.994; sensitivity =0.571; area under the receiver operating characteristic curve =0.76). Conclusions: The TL and FL diameters on preoperative CTA images can be used to assess the risk of negative remodeling after surgery.

A Novel In Situ Dendritic Cell Vaccine Triggered by Rose Bengal Enhances Adaptive Antitumour Immunity.

Dendritic cell- (DC-) based vaccination has emerged as a promising antitumour immunotherapy. However, overcoming immune tolerance and immunosuppression in the tumour microenvironment (TME) is still a great challenge. Recent studies have shown that Rose Bengal (RB) can effectively induce immunogenic cell death (ICD) in cancer cells, presenting whole tumour antigens for DC processing and presentation. However, the synergistic antitumour effect of combining intralesional RB with immature DCs (RB-iDCs) remains unclear. In the present study, we investigated whether RB-iDCs have superior antitumour effects compared with either single agent and evaluated the immunological mechanism of RB-iDCs in a murine lung cancer model. The results showed that intralesional RB-iDCs suppressed subcutaneous tumour growth and lung metastasis, which resulted in 100% mouse survival and significantly increased TNF-α production by CD8+ T cells. These effects were closely related to the induction of the expression of distinct ICD hallmarks by RB in both bulk cancer cells and cancer stem cells (CSCs), especially calreticulin (CRT), thus enhancing immune effector cell (i.e., CD4+, CD8+, and memory T cells) infiltration and attenuating the accumulation of immunosuppressive cells (i.e., Tregs, macrophages, and myeloid-derived suppressor cells (MDSCs)) in the TME. This study reveals that the RB-iDC vaccine can synergistically destroy the primary tumour, inhibit distant metastasis, and prevent tumour relapse in a lung cancer mouse model, which provides important preclinical data for the development of a novel combinatorial immunotherapy.

A retrospective study of thoracic endovascular aortic repair timing in patients with uncomplicated type B dissection who have a smoking history.

Objective: To determine the optimal timing of thoracic endovascular aortic repair (TEVAR) for patients with uncomplicated type B dissections who have a smoking history. Methods: Data from 308 consecutive patients with uncomplicated type B dissections, who have a smoking history and onset-to-TEVAR time within 90 days, were analyzed. The patients were divided into two groups: Acute and subacute phases. Univariate and multivariate regression analyses were performed. Smooth curve fitting and threshold analysis were performed to characterize the relationship between the onset-to-TEVAR time and follow-up deaths. Results: There were no significant differences between the two groups. Smooth curve fitting and threshold effect analysis showed that if early TEVAR was performed within 9.4 days from onset, there was better long-term survival and there was no significant difference after 9.4 days. Conclusion: By studying the relationship between onset-to-TEVAR time and all-cause mortality, we found that early TEVAR may have a lower all-cause mortality rate during follow-up in uncomplicated type B dissection patients who have a smoking history and within 90 days from onset.

Development of a nomogram for preoperative prediction of lymph node metastasis in non-small cell lung cancer: a SEER-based study.

BACKGROUND: Lymph node dissection is an important part of lung cancer surgery. Preoperational evaluation of lymph node metastases decides which dissection pattern should be chosen. The present study aimed to develop a nomogram to predict lymph node metastases on the basis of clinicopathological features of non-small cell lung cancer (NSCLC) patients. METHODS: A total of 35,138 patients diagnosed with NSCLC from 2010-2015 were selected from the Surveillance, Epidemiology, and End Results (SEER) database. Patients were randomly divided into training cohort and validation cohort. Possible risk factors were included and analyzed by logistic regression models. A nomogram was then constructed and validated. RESULTS: 21.83% of all patients were confirmed with positive lymph node metastasis. Age at diagnosis, sex, stage, T status, tumor size, grade and laterality were identified as predicting factors for lymph node involvement. These variables were included to build the nomogram. The AUC of the model was 0.696 (95% CI, 0.617 to 0.775). The model was further validated in the validation set with AUC 0.693 (95% CI, 0.628 to 0.758). The model presented with good prediction accuracy in both training cohort and validation cohort. CONCLUSIONS: We developed a convenient clinical prediction model for regional lymph node metastases in NSCLC patients. The nomogram will help physicians to determine which patients will receive the most benefit from lymph node dissection.

Impact of the homogeneous and heterogeneous risk factors on the incidence and survival outcome of bone metastasis in NSCLC patients.

PURPOSE: Bone metastases (BM) is reported as the most frequent distant metastasis in non-small cell lung cancer (NSCLC), but the risk factors for the incidence and prognosis of BM patients in NSCLC have not been extensively elucidated. This study aimed to find risk factors to predict BM patients' morbidity and survival outcome in NSCLC. METHODS: 63,505 patients of NSCLC in the Surveillance, Epidemiology and End Results database diagnosed from 2010 to 2015 were used to analyze risk factors for developing BM by conducting multivariable logistic regression. Of these patients, 6152 and 5664 BM patients diagnosed between 2010 and 2014 were selected to investigate predictive factors for BM overall survival (OS) and cancer-specific survival (CSS) using the multivariable Cox proportional hazards regression. RESULTS: There were overall 7486 (11.79%) BM patients in NSCLC. The homogeneous risk factors for BM patients' morbidity and survival outcome included male, higher T stage, lymph node involvement, poor differentiation grade, brain metastases, and liver metastases. Married status, adenocarcinoma type and lung metastases were positively correlated with BM incidence, while older age, white race, unmarried status, and SCC and other NSCLC types could predict poor OS and CSS of BM in NSCLC. CONCLUSIONS: The homogeneous and heterogeneous risk factors for morbidity and survival outcome of BM patients could help physicians in more precise and individualized screening and therapies for BM patients in NSCLC.

Effect of clinicopathologic features on survival of patients with thymic carcinomas and thymic neuroendocrine tumors: A population-based analysis.

BACKGROUND: Thymic carcinomas (TCs) and thymic neuroendocrine tumors (TNETs) are aggressive cancers with poor survival outcome and limited investigation. This study is to investigate clinicopathologic features on TC and TNET patients' prognosis of a large cohort. MATERIALS AND METHODS: The Surveillance, Epidemiology, and End Results database were used to identify a total of 362 TC and TNET patients with documented clinicopathologic features we investigated. The characteristics and overall survival of the TC and TNET patients were studied. RESULTS: Two hundred and forty TC and 122 TNET patients were identified. For the entire cohort of TC and TNET, histologic type (P < 0.001), tumor size (P = 0.015), Masaoka-Koga stage (P = 0.008), regional node positive (P = 0.004), surgery of primary site (P < 0.001), lymph node surgery (P = 0.013), and chemotherapy (P = 0.001) were considered as significant clinicopathologic features that could affect prognosis of TC and TNET patients in univariate analysis. More importantly, histologic type (P < 0.001), regional nodes positive (P = 0.03) and surgery of primary site (P < 0.001) were able to independently predict overall survival of those patients. In addition, for the cohort of TC, we found that regional nodes positive (P = 0.034) and surgery of primary site (P = 0.001) could be independent predictors of TC patients' survival. CONCLUSION: Regional nodes detection is essential for TC and TNET patients. Surgery of primary site is the preferred primary treatment for those patients.

REG4 is an indicator for KRAS mutant lung adenocarcinoma with TTF-1 low expression.

OBJECTIVES: Recent research has classified lung adenocarcinoma patients with KRAS mutation into three subtypes by co-occurring genetic events in TP53 (KP subgroup), STK11/LKB1 (KL subgroup) and CDKN2A/B inactivation plus TTF-1 low expression (KC subgroup). The aim of this study was to identify valuable biomarkers by searching the candidate molecules that contribute to lung adenocarcinoma pathogenesis, especially KC subtype. MATERIALS AND METHODS: We analyzed the publicly available database and identified the candidate REG4 using the E-GEOD-31210 dataset, and then confirmed by TCGA dataset. In addition, an independent cohort of 55 clinical samples was analyzed by quantitative real-time PCR analysis. Functional studies and RNA sequencing were performed after silencing the REG4 expression. RESULTS: REG4, an important regulator of gastro-intestinal carcinogenesis, was highly expressed in KRAS mutant lung adenocarcinoma with low expression of TTF-1 (KC subtype). The results were validated both by gene expression analysis and immunohistochemistry study in an independent 55 clinical samples from Fudan University Shanghai Cancer Center. Further in vitro and in vivo functional assays revealed silencing REG4 expression significantly reduces cancer cell proliferation and tumorigenesis. Moreover, RNA sequencing and GSEA analysis displayed that REG4 knockdown might induce cell cycle arrest by regulating G2/M checkpoint and E2F targets. CONCLUSION: Our results indicate that REG4 plays an important role in KRAS-driven lung cancer pathogenesis and is a novel biomarker of lung adenocarcinoma subtype. Future studies are required to clarify the underlying mechanisms of REG4 in the division and proliferation of KC tumors and its potential therapeutic value.

A novel strategy of EGFR­TKI combined chemotherapy in the treatment of human lung cancer with EGFR­sensitive mutation.

The discovery of epidermal growth factor receptor (EGFR)­sensitive mutations in non­small cell lung cancer (NSCLC) and the successful clinical application of EGFR tyrosine kinase inhibitors (TKIs) have changed the regimen of lung cancer therapy from traditional cytotoxic chemotherapy to molecular­targeted cancer therapy. However, the main limitation of EGFR­TKI therapy is the heterogeneity of lung cancer harboring EGFR­sensitive mutations. In addition, the synergistic effect of the administration of chemotherapy and EGFR­TKIs, combined with tumor heterogeneity, on NSCLC remains unclear. The present study aimed to investigate the optimal schedule for combined treatment with paclitaxel/gemcitabine and gefitinib in co­cultured NSCLC cell lines, in which PC9 cells were mixed with A549 cells at 0:1, 1:19, 1:3, 1:1, 3:1 and 1:0 ratios, and clarified the associated mechanisms. The mixed cells were used to simulate the tumor heterogeneity in the human cancer environment and to define the differential anti­proliferative effects of nine schedules of paclitaxel/gemcitabine and gefitinib, based on cell cycle distribution. We confirmed that gefitinib arrested PC9 cell growth, mainly at the G1 phase, at 24 h regardless of low or high concentration. After a 24­h culture in gefitinib­free medium, the cell cycle returned to its normal state. Paclitaxel and gemcitabine induced G2/M phase and S phase arrest at 72 h, respectively. The anti­proliferative effect of paclitaxel/gemcitabine followed by gefitinib resulted in the optimum anti­proliferative activity compared with the other seven schedules, which was not affected by tumor heterogeneity. Cell cycle­dependent synergism may contribute to this effect. Our results are in accordance with most of the existing clinical trials, and could provide a potential treatment option for patients with advanced NSCLC and for the ongoing clinical investigation of the sequential treatment of NSCLC.

Protective immunity elicited by measles vaccine exerts anti-tumor effects on measles virus hemagglutinin gene-modified cancer cells in a mouse model.

PURPOSE: Measles vaccine is widely used in China to prevent the measles virus (MV) infection. People immunized with measles vaccine can obtain long-term protective immunity. Measles virus surface glycoprotein hemagglutinin (H) can also induce MV-specific immune responses. However, little is known about whether the existence of the protective immune system against MV in the host can exert anti-tumor effects and whether the MV-H gene can serve as a therapeutic gene. METHODS: We first vaccinated mice with measles vaccine, then inoculated them with MV-H protein-expressing tumor cells and observed the rate of tumor formation. We also treated mice with H protein-expressing tumor cells with measles vaccine and assessed tumor size and overall survival. RESULTS: Active vaccination using measles vaccine not only protected mice from developing tumors, but also eradicated established tumors. Measles vaccine elicited H-specific IFN-γ, TNF-α and granzyme B-producing CD8+ T cells and increased cytotoxic T lymphocyte (CTL) activity specific for H antigen, which provided a strong therapeutic benefit against H protein-expressing tumors. In addition, measles vaccine decreased the population of myeloid-derived suppressor cells (MDSCs) and regulatory T cells (Tregs). CONCLUSIONS: Our study demonstrated that tumor cells expressing H protein could activate the immune memory response against MV, which exerted specific anti-tumor effects, and indicated that the MV-H gene can be used as a potential therapeutic gene for cancer gene therapy.

Adeno-associated virus-mediated osteoprotegerin gene transfer protects against joint destruction in a collagen-induced arthritis rat model.

OBJECTIVE: To evaluate the in vivo joint protection effect of recombinant adeno-associated virus-mediated gene transfer of human osteoprotegerin (rAAV-hOPG). METHODS: Collagen-induced arthritis (CIA) rat model was established. CIA rats were randomly divided into three groups: CIA control group (PBS), rAAV-EGFP (enhanced green fluorescent protein) group and rAAV-hOPG (100 µL/d) group, which received corresponding intra-articular injection treatment. The thickness of the palms and soles, arthritis index, radiological score, pathological score, bone damage factor and protein expression of inflammatory factors were measured and compared with normal control group rats. RESULTS: Positive fluorescence of frozen section confirmed that rAAV-hOPG was efficiently transduced into the synovial tissues of test rats. In rAAV-hOPG group compared with CIA control group, the radiological score was 30.18% lower (P<0.05); the expression of OPG protein was 93.41% higher (P<0.05); the expression of matrix metalloproteinase-3 (MMP-3) protein was 35.38% lower (P<0.05); however, the expression of IL-1ß was not significant; the scores of pannus and inflammation in rAAV-hOPG group have no significant difference. CONCLUSION: These results suggest that adeno-associated virus-mediated transfer of human osteoprotegerin is effectively transducted into the synovial tissues of CIA model, and protects against articular cartilage and bone destruction, but has no obvious efficiency on inflammation. The results also demonstrate that gene transfer using rAAV-hOPG may be a feasible and effective therapeutic candidate to treat or prevent joint destruction in inflammatory arthritis.