Proteogenomic characterization of small cell lung cancer identifies biological insights and subtype-specific therapeutic strategies.

We performed comprehensive proteogenomic characterization of small cell lung cancer (SCLC) using paired tumors and adjacent lung tissues from 112 treatment-naive patients who underwent surgical resection. Integrated multi-omics analysis illustrated cancer biology downstream of genetic aberrations and highlighted oncogenic roles of FAT1 mutation, RB1 deletion, and chromosome 5q loss. Two prognostic biomarkers, HMGB3 and CASP10, were identified. Overexpression of HMGB3 promoted SCLC cell migration via transcriptional regulation of cell junction-related genes. Immune landscape characterization revealed an association between ZFHX3 mutation and high immune infiltration and underscored a potential immunosuppressive role of elevated DNA damage response activity via inhibition of the cGAS-STING pathway. Multi-omics clustering identified four subtypes with subtype-specific therapeutic vulnerabilities. Cell line and patient-derived xenograft-based drug tests validated the specific therapeutic responses predicted by multi-omics subtyping. This study provides a valuable resource as well as insights to better understand SCLC biology and improve clinical practice.

Chemical Proteomics Identifies Druggable Vulnerabilities in a Genetically Defined Cancer.

The transcription factor NRF2 is a master regulator of the cellular antioxidant response, and it is often genetically activated in non-small-cell lung cancers (NSCLCs) by, for instance, mutations in the negative regulator KEAP1. While direct pharmacological inhibition of NRF2 has proven challenging, its aberrant activation rewires biochemical networks in cancer cells that may create special vulnerabilities. Here, we use chemical proteomics to map druggable proteins that are selectively expressed in KEAP1-mutant NSCLC cells. Principal among these is NR0B1, an atypical orphan nuclear receptor that we show engages in a multimeric protein complex to regulate the transcriptional output of KEAP1-mutant NSCLC cells. We further identify small molecules that covalently target a conserved cysteine within the NR0B1 protein interaction domain, and we demonstrate that these compounds disrupt NR0B1 complexes and impair the anchorage-independent growth of KEAP1-mutant cancer cells. Our findings designate NR0B1 as a druggable transcriptional regulator that supports NRF2-dependent lung cancers.

Deciphering the immunopeptidome in vivo reveals new tumour antigens.

Immunosurveillance of cancer requires the presentation of peptide antigens on major histocompatibility complex class I (MHC-I) molecules1-5. Current approaches to profiling of MHC-I-associated peptides, collectively known as the immunopeptidome, are limited to in vitro investigation or bulk tumour lysates, which limits our understanding of cancer-specific patterns of antigen presentation in vivo6. To overcome these limitations, we engineered an inducible affinity tag into the mouse MHC-I gene (H2-K1) and targeted this allele to the KrasLSL-G12D/+Trp53fl/fl mouse model (KP/KbStrep)7. This approach enabled us to precisely isolate MHC-I peptides from autochthonous pancreatic ductal adenocarcinoma and from lung adenocarcinoma (LUAD) in vivo. In addition, we profiled the LUAD immunopeptidome from the alveolar type 2 cell of origin up to late-stage disease. Differential peptide presentation in LUAD was not predictable by mRNA expression or translation efficiency and is probably driven by post-translational mechanisms. Vaccination with peptides presented by LUAD in vivo induced CD8+ T cell responses in naive mice and tumour-bearing mice. Many peptides specific to LUAD, including immunogenic peptides, exhibited minimal expression of the cognate mRNA, which prompts the reconsideration of antigen prediction pipelines that triage peptides according to transcript abundance8. Beyond cancer, the KbStrep allele is compatible with other Cre-driver lines to explore antigen presentation in vivo in the pursuit of understanding basic immunology, infectious disease and autoimmunity.

Development and validation of a supervised deep learning algorithm for automated whole-slide programmed death-ligand 1 tumour proportion score assessment in non-small cell lung cancer.

AIMS: Immunohistochemical programmed death-ligand 1 (PD-L1) staining to predict responsiveness to immunotherapy in patients with advanced non-small cell lung cancer (NSCLC) has several drawbacks: a robust gold standard is lacking, and there is substantial interobserver and intraobserver variance, with up to 20% discordance around cutoff points. The aim of this study was to develop a new deep learning-based PD-L1 tumour proportion score (TPS) algorithm, trained and validated on a routine diagnostic dataset of digitised PD-L1 (22C3, laboratory-developed test)-stained samples. METHODS AND RESULTS: We designed a fully supervised deep learning algorithm for whole-slide PD-L1 assessment, consisting of four sequential convolutional neural networks (CNNs), using aiforia create software. We included 199 whole slide images (WSIs) of 'routine diagnostic' histology samples from stage IV NSCLC patients, and trained the algorithm by using a training set of 60 representative cases. We validated the algorithm by comparing the algorithm TPS with the reference score in a held-out validation set. The algorithm had similar concordance with the reference score (79%) as the pathologists had with one another (75%). The intraclass coefficient was 0.96 and Cohen's κ coefficient was 0.69 for the algorithm. Around the 1% and 50% cutoff points, concordance was also similar between pathologists and the algorithm. CONCLUSIONS: We designed a new, deep learning-based PD-L1 TPS algorithm that is similarly able to assess PD-L1 expression in daily routine diagnostic cases as pathologists. Successful validation on routine diagnostic WSIs and detailed visual feedback show that this algorithm meets the requirements for functioning as a 'scoring assistant'.

Brigatinib versus Crizotinib in ALK-Positive Non-Small-Cell Lung Cancer.

BACKGROUND: Brigatinib, a next-generation anaplastic lymphoma kinase (ALK) inhibitor, has robust efficacy in patients with ALK-positive non-small-cell lung cancer (NSCLC) that is refractory to crizotinib. The efficacy of brigatinib, as compared with crizotinib, in patients with advanced ALK-positive NSCLC who have not previously received an ALK inhibitor is unclear. METHODS: In an open-label, phase 3 trial, we randomly assigned, in a 1:1 ratio, patients with advanced ALK-positive NSCLC who had not previously received ALK inhibitors to receive brigatinib at a dose of 180 mg once daily (with a 7-day lead-in period at 90 mg) or crizotinib at a dose of 250 mg twice daily. The primary end point was progression-free survival as assessed by blinded independent central review. Secondary end points included the objective response rate and intracranial response. The first interim analysis was planned when approximately 50% of 198 expected events of disease progression or death had occurred. RESULTS: A total of 275 patients underwent randomization; 137 were assigned to brigatinib and 138 to crizotinib. At the first interim analysis (99 events), the median follow-up was 11.0 months in the brigatinib group and 9.3 months in the crizotinib group. The rate of progression-free survival was higher with brigatinib than with crizotinib (estimated 12-month progression-free survival, 67% [95% confidence interval {CI}, 56 to 75] vs. 43% [95% CI, 32 to 53]; hazard ratio for disease progression or death, 0.49 [95% CI, 0.33 to 0.74]; P<0.001 by the log-rank test). The confirmed objective response rate was 71% (95% CI, 62 to 78) with brigatinib and 60% (95% CI, 51 to 68) with crizotinib; the confirmed rate of intracranial response among patients with measurable lesions was 78% (95% CI, 52 to 94) and 29% (95% CI, 11 to 52), respectively. No new safety concerns were noted. CONCLUSIONS: Among patients with ALK-positive NSCLC who had not previously received an ALK inhibitor, progression-free survival was significantly longer among patients who received brigatinib than among those who received crizotinib. (Funded by Ariad Pharmaceuticals; ALTA-1L ClinicalTrials.gov number, NCT02737501 .).

Infrared spectroscopic imaging study of BV-2 microglia altering tumor cell biological activity and cellular fraction.

Tumor brain metastasis is a severe threat to patients' neurological function, in which microglia may be involved in the process of tumor cell metastasis among nerve cells. Our study focused on the interaction between microglia and breast and lung cancer cells. Changes in the proliferation and migration ability of cocultured tumor cells were examined; synchrotron radiation-based fourier transform infrared microspectroscopy (SR-FTIR) was used to detect changes in the structures and contents of biomolecules within the tumor cells. The experimental results showed that the proliferation and migration ability of tumor cells increased after coculture, and the structures and contents of biological macromolecules in tumor cells changed. The absorption peak positions of the amide Ⅱ and amide Ⅰ bands observed for the four kinds of tumor cells changed, and the absorption intensities were significantly enhanced, indicating changes in the secondary structures and contents of proteins in tumor cells, which may be the root cause of the change in tumor cell characteristics. Therefore, the metabolites of microglia may be involved in the progression of tumor cells in the nervous system. In this study, we focused on the interaction between microglia and tumor cells by using SR-FTIR and provided a new understanding of the mechanism of brain metastasis.

Studies in lung cancer cytokine proteomics: a review.

INTRODUCTION: Proteins are molecules that have role in the progression of the diseases. Proteomics is a tool that can play an effective role in identifying diagnostic and therapeutic biomarkers for lung cancer. Cytokines are proteins that play a decisive role in activating body's immune system in lung cancer. They can increase the growth of the tumor (oncogenic cytokines) or limit tumor growth (anti-tumor cytokines) by regulating related signaling pathways such as proliferation, growth, metastasis, and apoptosis. AREAS COVERED: In the present study, a total of 223 papers including 196 research papers and 27 review papers, extracted from PubMed and Scopus and published from 1997 to present, are reviewed. The most important involved-cytokines in lung cancer including TNF-α, IFN- γ, TGF-ß, VEGF and interleukins such as IL-6, IL-17, IL-8, IL-10, IL-22, IL-1ß and IL-18 are introduced. Also, the pathological and biological role of such cytokines in cancer signaling pathways is explained. EXPERT OPINION: In lung cancer, the cytokine expression changes under the physiological conditions of the immune system, and inflammatory cytokines are associated with the progression of lung cancer. Therefore, the cytokine expression profile can be used in the diagnosis, prognosis, prediction of therapeutic responses, and survival of patients with lung cancer.

Decreased sphingomyelin (t34:1) is a candidate predictor for lung squamous cell carcinoma recurrence after radical surgery: a case-control study.

BACKGROUND: To reduce disease recurrence after radical surgery for lung squamous cell carcinomas (SQCCs), accurate prediction of recurrent high-risk patients is required for efficient patient selection for adjuvant chemotherapy. Because treatment modalities for recurrent lung SQCCs are scarce compared to lung adenocarcinomas (ADCs), accurately selecting lung SQCC patients for adjuvant chemotherapy after radical surgery is highly important. Predicting lung cancer recurrence with high objectivity is difficult with conventional histopathological prognostic factors; therefore, identification of a novel predictor is expected to be highly beneficial. Lipid metabolism alterations in cancers are known to contribute to cancer progression. Previously, we found that increased sphingomyelin (SM)(d35:1) in lung ADCs is a candidate for an objective recurrence predictor. However, no lipid predictors for lung SQCC recurrence have been identified to date. This study aims to identify candidate lipid predictors for lung SQCC recurrence after radical surgery. METHODS: Recurrent (n = 5) and non-recurrent (n = 6) cases of lung SQCC patients who underwent radical surgery were assigned to recurrent and non-recurrent groups, respectively. Extracted lipids from frozen tissue samples of primary lung SQCC were analyzed by liquid chromatography-tandem mass spectrometry. Candidate lipid predictors were screened by comparing the relative expression levels between the recurrent and non-recurrent groups. To compare lipidomic characteristics associated with recurrent SQCCs and ADCs, a meta-analysis combining SQCC (n = 11) and ADC (n = 20) cohorts was conducted. RESULTS: Among 1745 screened lipid species, five species were decreased (≤ 0.5 fold change; P < 0.05) and one was increased (≥ 2 fold change; P < 0.05) in the recurrent group. Among the six candidates, the top three final candidates (selected by AUC assessment) were all decreased SM(t34:1) species, showing strong performance in recurrence prediction that is equivalent to that of histopathological prognostic factors. Meta-analysis indicated that decreases in a limited number of SM species were observed in the SQCC cohort as a lipidomic characteristic associated with recurrence, in contrast, significant increases in a broad range of lipids (including SM species) were observed in the ADC cohort. CONCLUSION: We identified decreased SM(t34:1) as a novel candidate predictor for lung SQCC recurrence. Lung SQCCs and ADCs have opposite lipidomic characteristics concerning for recurrence risk. TRIAL REGISTRATION: This retrospective study was registered at the UMIN Clinical Trial Registry ( UMIN000039202 ) on January 21, 2020.

Role of Synaptophysin, Chromogranin and CD56 in adenocarcinoma and squamous cell carcinoma of the lung lacking morphological features of neuroendocrine differentiation: a retrospective large-scale study on 1170 tissue samples.

BACKGROUND: Synaptophysin, chromogranin and CD56 are recommended markers to identify pulmonary tumors with neuroendocrine differentiation. Whether the expression of these markers in pulmonary adenocarcinoma and pulmonary squamous cell carcinoma is a prognostic factor has been a matter of debate. Therefore, we investigated retrospectively a large cohort to expand the data on the role of synaptophysin, chromogranin and CD56 in non-small cell lung cancer lacking morphological features of neuroendocrine differentiation. METHODS: A cohort of 627 pulmonary adenocarcinomas (ADC) and 543 squamous cell carcinomas (SqCC) lacking morphological features of neuroendocrine differentiation was assembled and a tissue microarray was constructed. All cases were stained with synaptophysin, chromogranin and CD56. Positivity was defined as > 1% positive tumor cells. Data was correlated with clinico-pathological features including overall and disease free survival. RESULTS: 110 (18%) ADC and 80 (15%) SqCC were positive for either synaptophysin, chromogranin, CD56 or a combination. The most commonly positive single marker was synaptophysin. The least common positive marker was chromogranin. A combination of ≤2 neuroendocrine markers was positive in 2-3% of ADC and 0-1% of SqCC. There was no significant difference in overall survival in tumors with positivity for neuroendocrine markers neither in ADC (univariate: P = 0.4; hazard ratio [HR] = 0.867; multivariate: P = 0.5; HR = 0.876) nor in SqCC (univariate: P = 0.1; HR = 0.694; multivariate: P = 0.1, HR = 0.697). Likewise, there was no significant difference in disease free survival. CONCLUSIONS: We report on a cohort of 1170 cases that synaptophysin, chromogranin and CD56 are commonly expressed in ADC and SqCC and that their expression has no impact on survival, supporting the current best practice guidelines.

PD-(L)1 inhibitors as single-agent or in combination with chemotherapy for advanced, PD-L1-high non-small cell lung cancer: a meta-analysis.

Introduction: The best treatment for advanced, PD-L1-high non-small-cell lung cancer remains a debated issue. Methods: A meta-analysis of randomized clinical trials (RCTs) was performed to compare the efficacy and safety of PD-(L)1 inhibitors alone or plus chemotherapy (CT) for advanced, PD-L1-high non-small-cell lung cancer. Results: 14 RCTs were included. The combination of a PD-(L)1 inhibitor with CT resulted in the improvement of progression-free survival (HR: 0.59; 95% CI: 0.43-0.79; p = 0.0005) and objective response rate (RR: 1.66; 95% CI: 1.14-2.42; p = 0.008). No overall survival difference was documented (HR: 0.99; 95% CI: 0.77-1.27; p = 0.95). The risk of grade ≥3 treatment-related adverse events was significantly reduced with immune-checkpoint inhibitor single-agent therapy compared with immune-checkpoint inhibitors plus CT (RR: 0.38; 95% CI: 0.32-0.45; p = 0.00001). Conclusion: The combination of a PD-(L)1 inhibitor and CT appears to be associated with improved PFS and ORR, but similar OS, compared with PD-(L)1 inhibitor single-agent therapy in patients with PD-L1-high non-small-cell lung cancer.

Distinct transcriptional programs of SOX2 in different types of small cell lung cancers.

SOX2 is recognized as an oncogene in human small cell lung cancer (SCLC), which is an aggressive neuroendocrine (NE) tumor. However, the role of SOX2 in SCLC is not completely understood, and strategies to selectively target SOX2 in SCLC cells remain elusive. Here, we show, using next-generation sequencing, that SOX2 expressed in the ASCL1-high SCLC (SCLC-A) subtype cell line is dependent on ASCL1, which is a lineage-specific transcriptional factor, and is involved in NE differentiation and tumorigenesis. ASCL1 recruits SOX2, which promotes INSM1 and WNT11 expression. Immunohistochemical studies revealed that SCLC tissue samples expressed SOX2, ASCL1, and INSM1 in 18 out of the 30 cases (60%). Contrary to the ASCL1-SOX2 signaling axis controlling SCLC biology in the SCLC-A subtype, SOX2 targets distinct genes such as those related to the Hippo pathway in the ASCL1-negative, YAP1-high SCLC (SCLC-Y) subtype. Although SOX2 knockdown experiments suppressed NE differentiation and cell proliferation in the SCLC-A subtype, they did not sufficiently impair the growth of the SCLC-Y subtype cell lines in vitro and ex vivo. The present results support the importance of the ASCL1-SOX2 axis as a main subtype of SCLC, and suggest the therapeutic potential of targeting the ASCL1-SOX2 axis.

Exploration of Matrix Effects in Laser Ablation Inductively Coupled Plasma Mass Spectrometry Imaging of Cisplatin-Treated Tumors.

The use of a low aerosol dispersion ablation chamber within a laser ablation inductively coupled plasma mass spectrometer (LA-ICP-MS) setup allows for high-resolution, high-speed imaging of the distribution of elements within a sample. Here we show how this enhanced capability creates new analytical problems and solutions. We report the distribution of platinum at the cellular level in non-small cell lung cancer (NSCLC) explant models after treatment with clinically relevant doses of cisplatin. This revealed for the first time a correlation between the platinum signal and the presence of carbon deposits within lung tissue. We show how complementary ion beam analysis techniques, particle-induced X-ray emission (PIXE) and elastic backscattering spectrometry (EBS), can be used to explore potential matrix effects in LA-ICP-MS data. For these samples, it was confirmed that the enhancement was unlikely to have resulted from a matrix effect alone. Thus, the presence of carbon deposits within tissue has potential implications for the effective distribution of the cisplatin drug.

Metal-Free Photoinduced Atom Transfer Radical Polymerization for Highly Sensitive Detection of Lung Cancer DNA.

Convenient and sensitive detection of biomolecules is of great significance to disease diagnosis. In this work, a metal-free photoinduced atom transfer radical polymerization (photoATRP) by a reductive quenching pathway as a novel strategy is applied to achieve lung cancer DNA detection. Thiolated PNA is exploited to specifically recognize target DNA, and the initiator of photoATRP is linked to the electrode surface via phosphate-Zr4+ -carboxylate. Under the excitation of blue light, the reductive quenching pathway is activated with eosin Y (EY) as photoredox catalyst and N,N,N',N'',N'-pentamethyldiethylenetriamine (PMDETA) as electron donor, and numerous polymeric chains are formed. Under optimal conditions, the linear range of this strategy is from 0.1 pm to 10 nm (R2 =0.989) with a limit of detection (LOD) of 1.4 fm (14 zmol in 10 µL). The variety of possible light sources for photoATRP and simple operation endow this biosensor with great potential for practical applications.

Comprehensive analysis of spread through air spaces in lung adenocarcinoma and squamous cell carcinoma using the 8th edition AJCC/UICC staging system.

BACKGROUND: This study aimed to comprehensively investigate the effect of spread through air spaces (STAS) on clinicopathologic features, molecular characteristics, immunohistochemical expression, and prognosis in lung adenocarcinomas (ADC) and squamous cell carcinomas (SQCC) based on the 8th edition AJCC/UICC staging system. METHODS: In total, 303 ADC and 121 SQCC cases were assessed retrospectively. Immunohistochemical staining was performed for E-cadherin, vimentin, Ki67, survivin, Bcl-2, and Bim. Correlations between STAS and other parameters were analyzed statistically. RESULTS: STAS was observed in 183 (60.4%) ADC and 39 (32.2%) SQCC cases. In ADC, the presence of STAS was associated with wild-type EGFR, ALK and ROS1 rearrangements, low E-cadherin expression, and high vimentin and Ki67 expression. In SQCC, STAS was associated with low E-cadherin expression and high vimentin and survivin expression. Based on univariate analysis, STAS was associated with significantly shorter disease-free survival (DFS) and overall survival (OS) in ADC. In SQCC, STAS tended to be associated with shorter OS. By multivariate analysis, STAS was an independent poor prognostic factor in ADC for DFS but not OS. Stratified analysis showed that STAS was correlated with shorter DFS for stage I, II, IA, IB, and IIA ADC based on univariate analysis and was an independent risk factor for DFS in stage I ADC cases based on multivariate analysis. CONCLUSIONS: Our findings revealed that STAS is an independent negative prognostic factor for stage I ADC using the new 8th edition AJCC/UICC staging system. Stage I patients with STAS should be followed up more closely and might need different treatment strategies.

Development of a lateral flow aptamer assay strip for facile identification of theranostic exosomes isolated from human lung carcinoma cells.

Exosomes are Extracellular Vesicles (EV) that own unique structural features and functions and have gradually become the hot research spot in recent years. The tumor-derived exosomes contain various types of useful biological information, and medical identification of exosomes relied on the specific characterization of membrane surface proteins. In this study, in order to rapidly identify non-small cell lung cancer (NSCLC)-derived exosomes, based on an aptamer against CD63 protein on exosome membrane, a low cost lateral flow aptamer assay (LFAA) test strip using nanogold particles as visualization probes was successfully developed for facile identification of A549 exosomes isolated from human lung carcinoma cells diluted from 6.4 × 109 particles/mL herein.

A density functional theory study on the interaction of toluene with transition metal decorated carbon nanotubes: a promising platform for early detection of lung cancer from human breath.

In this study, single-wall carbon nanotubes (SWCNTs) decorated by platinum-group transition metals (Pt, Pd, Rh, or Ru) were introduced as promising nanosensors for the detection of toluene, an important biomarker in the exhaled breath of the lung cancer patients. First-principle calculations based on density functional theory (DFT) was employed to scrutinize the impact of an individual toluene gas molecule on the structural, electronic, and magnetic properties of pristine and metal decorated SWCNTs. It was discovered that toluene is physisorbed on the pristine SWCNT through the interaction of the π orbitals of the carbon atoms in the toluene and the nanotube. Decoration of the SWCNT with metal atoms enhanced the adsorption energies significantly by means of strong overlapping between d orbital of the metal atoms and p orbital of C atoms in the benzene ring of toluene. Investigations showed that toluene is strongly chemisorbed on Rh- and Ru-SWCNT systems via strong covalent bonds with the superior response (-96.98% and -99.98%, respectively), and moderately chemisorbed on Pt-SWCNTs (-27.3%) and Pd-SWCNTs (61.60%). Our findings propose metal decorated SWCNT molecular sensors are attractive candidates for the detection of toluene and other lung cancer biomarkers in the exhaled breath of the lung cancer patients.

CircAKT3 inhibits glycolysis balance in lung cancer cells by regulating miR-516b-5p/STAT3 to inhibit cisplatin sensitivity.

OBJECTIVE: Lung cancer was one of the most deadly cancers around the world. Circular RNA AKT3 (CircAKT3) was highly expressed in lung cancer and could inhibit cell proliferation, but there were few studies on the mechanism of specific regulation of drug resistance. Therefore, we aimed to provide new ideas and perspectives for the role of circAKT3 in the mechanism of tumor resistance. METHODS: The levels of circAKT3, miR-516b-5p and STAT3 in lung cancer tissues and cells were examined using quantitative real-time polymerase chain reaction (qRT-PCR) or western blot assays. 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay was used to examine the sensitivity of cells treated under different conditions to cisplatin (DDP). A glucose assay kit and lactate assay kit were used to assess glycolysis and lactate production of cells treated with different plasmids and 2-deoxy-glucose (2-DG). Western blot analysis was used to detect the expression level of the hypoxia-inducible factor (HIF-1α) in A549 and H1299 cells. Starbase 3.0 predicted a targeted relationship between circAKT3 and miR-516b-5p, STAT3 and miR-516b-5p, and the relationship was proved by a dual-luciferase reporter assay. Knockdown of circAKT3 was used to study the effects of circAKT3 on tumor development in vivo. RESULTS: The levels of circAKT3 and STAT3 were upregulated, miR-516b-5p was downregulation in lung cancer tissues and cells. Functionally, circAKT3 knockdown improved cell sensitivity to DDP, and repressed glycolysis in lung cancer cells. Meanwhile, inhibition of HIF-1α-dependent glycolysis attenuated the circAKT3-induced increase of chemo-resistance in A549 cells. Mechanistically, miR-516b-5p was found to possess some binding sites with circAKT3. Noticeably, the inhibitory action of circAKT3 knockdown on DDP resistance and glycolysis was overturned through inhibitor of miR-516b-5p in lung cancer cells. Furthermore, besides, circAKT3 knockdown suppressed lung tumor cell growth by the miR-516b-5p/STAT3 axis in vivo. CONCLUSIONS: CircAKT3 inhibit cisplatin sensitivity of lung cancer cells at least partly through regulating miR-516b-5p/STAT3 axis-mediated glycolysis balance, providing a possible long noncoding RNA -targeted therapy for lung cancer.

Evosep One Enables Robust Deep Proteome Coverage Using Tandem Mass Tags while Significantly Reducing Instrument Time.

The balance between comprehensively analyzing the proteome and using valuable mass spectrometry time is a genuine challenge in the field of proteomics. Multidimensional fractionation strategies have significantly increased proteome coverage, but often at the cost of increased mass analysis time, despite advances in mass spectrometer acquisition rates. Recently, the Evosep One liquid chromatography system was shown to analyze peptide samples in a high-throughput manner without sacrificing in-depth proteomics coverage. We demonstrate the incorporation of Evosep One technology into our multiplexing workflow for analysis of tandem mass tag (TMT)-labeled nonsmall cell lung carcinoma (NSCLC) patient-derived xenografts (PDXs). By the use of a 30 samples per day Evosep workflow, >12 000 proteins were identified in 48 h of mass spectrometry time, which is comparable to the number of proteins identified by our conventional concatenated EASY-nLC workflow in 60 h. Shorter Evosep gradient lengths reduced the number of protein identifications by 10% while decreasing the mass analysis time by 50%. This Evosep workflow will enable quantitative analysis of multiplexed samples in less time without conceding depth of proteome coverage.

Methyl DNA phosphate adduct formation in lung tumor tissue and adjacent normal tissue of lung cancer patients.

The formation of methyl DNA adducts is a critical step in carcinogenesis initiated by the exposure to methylating carcinogens. Methyl DNA phosphate adducts, formed by methylation of the oxygen atoms of the DNA phosphate backbone, have been detected in animals treated with methylating carcinogens. However, detection of these adducts in human tissues has not been reported. We developed an ultrasensitive liquid chromatography-nanoelectrospray ionization-high resolution tandem mass spectrometry method for detecting methyl DNA phosphate adducts. Using 50 µg of human lung DNA, a limit of quantitation of two adducts/1010 nucleobases was achieved. Twenty-two structurally unique methyl DNA phosphate adducts were detected in human lung DNA. The adduct levels were measured in both tumor and adjacent normal tissues from 30 patients with lung cancer, including 13 current smokers and 17 current non-smokers, as confirmed by measurements of urinary cotinine and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol. Levels of total methyl DNA phosphate adducts in normal lung tissues were higher in smokers than non-smokers, with an average of 13 and 8 adducts/109 nucleobases, respectively. Methyl DNA phosphate adducts were also detected in lung tissues from untreated rats with steady-state levels of 5-7 adducts/109 nucleobases over a period of 70 weeks. This is the first study to report the detection of methyl DNA phosphate adducts in human lung tissues. The results provide new insights toward using these DNA adducts as potential biomarkers to study human exposure to environmental methylating carcinogens.

Applicability of Phenylhydrazine Labeling for Structural Studies of Fucosylated N-Glycans.

Fucosylation is a common modification, and its site in glycans refers to different normal and pathological processes. Despite intensive research, there is still a lack of methods to discriminate unambiguously the fucose position in one-step. In this work, we propose utility of phenylhydrazine (PHN) labeling for structural studies of fucosylated N-glycans by tandem MALDI mass spectrometry (MS) in the positive ion mode. PHN-tag influences the production of specific ion types, and the MS/MS fragmentation pattern provides useful structural information. All types of core fucosylated N-glycans have produced two abundant ions consistent with B- and C-glycosidic cleavages corresponding to the loss of the FucGlcNAcPHN residue with a mass 457 and 441 Da from the parent ions. These types of fragment ions in N-glycans without a core fucose were associated with the loss of the GlcNAcPHN unit (311 and 295 Da), and fucose cleavage followed the loss of the chitobiose residue. Since diagnostic useful cleavages produce peaks with significant intensities, this approach is also beneficial for rapid recognition of antenna from core fucosylation in glycans detected with low abundances. Moreover, in multifucosylated glycans, this type of labeling allows to distinguish how many fucose residues are on the specific antenna and provides additional information on the topology of N-glycans, such as type of antennarity or identification of bisecting moiety. The practical applicability of the approach is demonstrated on the analysis of multifucosylated N-glycans detected with lower abundances in lung cancer samples.