Human Non-small Cell Lung Cancer Research Articles

Abstract B168: Identification of STX-241, a CNS-penetrant and mutant-selective EGFR inhibitor with activity on osimertinib-resistant C797x mutations

Abstract Background: EGFR mutations are well validated clinical targets in non-small cell lung cancer (NSCLC). Osimertinib, a highly-selective EGFR mutation-targeting covalent drug, is increasingly used in the first line setting for patients with NSCLC bearing EGFR L858R mutation or exon 19 deletions (ex19del). In a subset of these patients, co-occurring L858R/C797x or ex19del/C797x mutations (“double mutants”) are emerging as an on-target resistance mechanism, necessitating the need for new therapies, particularly in patients with CNS metastases. Materials and methods: STX-241 was tested across a panel of in vitro biochemical, cell signaling, and proliferation assays for potency against EGFR L858R and ex19del single mutants and the corresponding C797S double mutants. Additionally, STX-241 was tested for in vivo activity in mice bearing human NSCLC cell line xenografts NCI-H3255 (L858R) and PC-9 (ex19del), and in a PC-9-derived ex19del/C797S double mutant knock-in xenograft. Free CNS penetration (Kp,uu) was determined in non-tumor bearing mice. Osimertinib and gefitinib (an approved reversible EGFR inhibitor) were used as benchmark molecules across assays. Results: STX-241, an ATP-competitive reversible EGFR inhibitor representing novel chemical matter, demonstrated potent and selective inhibition of recombinant EGFR L858R/C797S mutant protein relative to wild-type, with increased mutant residence time relative to gefitinib. Potent (high picomolar to low nanomolar) inhibition of L858R, ex19del, L858R/C797S, and ex19del/C797S mutants was observed in proliferation assays using engineered Ba/F3 and human NSCLC cell lines. STX-241 demonstrated >150x selectivity for all tested EGFR mutants relative to wild-type EGFR in engineered Ba/F3 cells and human cancer cells. In these assays, STX-241 selectivity exceeded that of the benchmark reversible EGFR inhibitor gefitinib. Strong potency and double mutant selectivity was also observed for STX-241 in pharmacodynamic assays measuring EGFR pathway activation (pEGFR). STX-241 was well tolerated at doses of 15 mg/kg BID or 50 mg/kg QD in mice, where regression of EGFR exon 19 or 21 mutant NSCLC xenografts was observed, concomitant with EGFR pathway suppression. Notably, using an isogenic pair of PC-9 (EGFR ex19del) NSCLC xenografts that differ only by the presence of a C797S mutation, STX-241 demonstrates no drop off in antitumor activity in the presence of C797S. In mouse pharmacokinetic studies measuring free CNS penetration, Kp,uu measurements were comparable to Osimertinib, run in parallel as a benchmark. Conclusions: STX-241 demonstrates strong potency and selectivity against EGFR L858R/C797S and ex19del/C797S double mutants, as well as robust CNS penetrance. These data warrant further exploration of this potential best-in-class inhibitor in the clinic for patients progressing on Osimertinib via C797x mutation. Citation Format: Raymond A Pagliarini, Benjamin C Milgram, Deanna R Borrelli, Erin O'Hearn, Michael R. Huff, Brendon Ladd, Natasja Brooijmans, Weixue Wang, Petr Kuzmic, Angel Guzman-Perez, Darrin D Stuart. Identification of STX-241, a CNS-penetrant and mutant-selective EGFR inhibitor with activity on osimertinib-resistant C797x mutations [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B168.

Noninvasive Monitoring of Immunotherapy in Lung Cancer by Lymphocyte Activation Gene 3 PET Imaging of Tumor-Infiltrating Lymphocytes.

Although immunotherapy has revolutionized the entire cancer treatment landscape, small fractions of patients respond to immunotherapy. Early identification of responders may improve patient management during immunotherapy. In this study, we evaluated a PET approach for monitoring immunotherapy in lung cancer by imaging the upregulation of lymphocyte activation gene 3 (LAG-3)-expressing (LAG-3+) tumor-infiltrating lymphocytes (TILs). Methods: We synthesized a LAG-3-targeted molecular imaging probe, [68Ga]Ga-NOTA-C25 and performed a series of invitro and invivo assays to test its specificity. Next, [68Ga]Ga-NOTA-C25 PET was used to monitor immunotherapy in murine lung cancer-bearing mice and in humanized mouse models for assessing clinical translational potential, with confirmation by immunostaining and flow cytometry analysis. Results: [68Ga]Ga-NOTA-C25 PET could noninvasively detect intertumoral differences in LAG-3+ TIL levels in different tumor models. Importantly, in Lewis lung carcinoma tumor models treated with an agonist of a stimulator of interferon genes, [68Ga]Ga-NOTA-C25 PET also detected an immunophenotyping transition of the tumor from "cold" to "hot" before changes in tumor size. Meanwhile, animals carrying "hot" tumor showed more significant tumor inhibition and longer survival than those carrying "cold" tumor. [68Ga]Ga-NOTA-C25 PET also showed markedly higher tumor uptake in immune system-humanized mice carrying human non-small cell lung cancer than immunodeficient models. Conclusion: [68Ga]Ga-NOTA-C25 PET could be used to noninvasively monitor the early response to immunotherapy by imaging LAG-3+ TILs in lung cancer. [68Ga]Ga-NOTA-C25 PET also exhibited excellent translational potential, with great significance for the precise management of lung cancer patients receiving immunotherapy.

EML4-ALK fusion protein in Lung cancer cells enhances venous thrombogenicity through the pERK1/2-AP-1-tissue factor axis

BackgroundAccumulating evidence links the echinoderm microtubule-associated protein-like 4 (EML4)-anaplastic lymphoma kinase (ALK) rearrangement to venous thromboembolism (VTE) in non-small cell lung cancer (NSCLC) patients. However, the corresponding mechanisms remain unclear.MethodHigh-throughput sequencing analysis of H3122 human ALK-positive NSCLC cells treated with ALK inhibitor/ dimethyl sulfoxide (DMSO) was performed to identify coagulation-associated differential genes between EML4-ALK fusion protein inhibited cells and control cells. Sequentially, we confirmed its expression in NSCLC patients’ tissues and in the plasma of a subcutaneous xenograft mouse model. An inferior vena cava (IVC) ligation model was used to assess clot formation potential. Additionally, pathways involved in tissue factor (TF) regulation were explored in ALK-positive cell lines H3122 and H2228. Statistical significance was determined by Student t-test and one-way ANOVA using SPSS.ResultsSequencing analysis identified a significant downregulation of TF after inhibiting EML4-ALK fusion protein activity in H3122 cells. In clinical NSCLC cases, TF expression was increased especially in ALK-positive NSCLC tissues. Meanwhile, H3122 and H2228 with high TF expression exhibited shorter plasma clotting time and higher TF activity versus ALK-negative H1299 and A549 in cell culture supernatant. Mice bearing H2228 tumor showed a higher concentration of tumor-derived TF and TF activity in plasma and the highest adjusted IVC clot weights. Limiting EML4-ALK protein phosphorylation downregulated extracellular regulated protein kinases 1/2 (ERK1/2)-activating the protein-1(AP-1) signaling pathway and thus attenuated TF expression.ConclusionEML4-ALK fusion protein may enhance venous thrombogenicity by regulating coagulation factor TF expression. There was potential involvement of the pERK1/2-AP-1 pathway in this process.

Antitumor activity of pegylated human interferon β as monotherapy or in combination with immune checkpoint inhibitors via tumor growth inhibition and dendritic cell activation

Type I interferons (IFN), especially human IFN alpha (IFNα), have been utilized for antitumor therapy for decades. Human interferon beta (IFNβ) is rarely used for cancer treatment, despite advantages over IFNα in biological activities such as tumor growth inhibition and dendritic cell (DC) activation. The utilization of pegylated human IFNβ (PEG-IFNβ), as monotherapy or in combination with immune checkpoint inhibitors (ICIs) was evaluated in this study through in vivo efficacy studies in syngeneic mouse melanoma, non-small cell lung cancer (NSCLC), and colon adenocarcinoma (COAD) models resistant to immune checkpoint inhibitors (ICIs). In vitro comparative study of PEG-IFNβ and pegylated IFNα-2b was performed in terms of tumor growth inhibition against human melanoma, NSCLC and COAD cell lines and activation of human monocyte-derived DCs (MoDCs). Our data demonstrate that the in vivo antitumor effects of PEG-IFNβ are partially attributable to tumor growth-inhibitory effects and DC-activating activities, superior to pegylated IFNα-2b. Our findings suggest that utilizing PEG-IFNβ as an antitumor therapy can enhance the therapeutic effect of ICIs in ICI-resistant tumors by directly inhibiting tumor growth and induction of DC maturation.

Targeting the aryl hydrocarbon receptor (AhR) with BAY 2416964: a selective small molecule inhibitor for cancer immunotherapy

BackgroundThe metabolism of tryptophan to kynurenines (KYN) by indoleamine-2,3-dioxygenase or tryptophan-2,3-dioxygenase is a key pathway of constitutive and adaptive tumor immune resistance. The immunosuppressive effects of KYN in the tumor...

Design of novel analogues of t-DPH1 with reduced cytotoxicity, taking the three conserved characteristics of the dermaseptin family as the feasible starting point

In recent years, there has been growing scientific interest in balancing the bioactivity and cytotoxicity of antimicrobial peptides (AMPs) for more comprehensive research and application. In this study, we focused on t-DPH1, a representative member of the dermaseptin family, as a template. We synthesized five analogues were synthesised to to assess the impact of three conserved features of the dermaseptin family (1. the tryptophan residue at 3rd position, 2. the consensus motif at mid-region, and 3. C-terminal amidation) on the bioactivity of t-DPH1 and their potential to reduce cytotoxicity. Our results demonstrated that analogues lacking an amino group at the C-terminus, specifically t-DPH1-NH2, exhibited potent antimicrobial activity against Gram-negative bacteria, including Escherichia coli (E. coli) and Klebsiella pneumoniae (K. pneumoniae). Furthermore, t-DPH1-NH2 retained its robust antiproliferative activity against the human non-small lung cancer cell line H157. Regarding cytotoxicity, t-DPH1-NH2 displayed lower levels of haemolytic activity and cytotoxicity against two normal cell lines. Notably, we employed C. elegans as an in vivo model to assess the cytotoxicity of peptides, and the results revealed no induction of in vivo toxicity by t-DPH1-NH2. Collectively, t-DPH1-NH2 provides valuable insights for further investigation as promising bioactive peptide templates in the development of antibiotic and anticancer prototype drugs. The modifications implemented based on the conserved structure of the peptide family offer a new direction for reducing the cytotoxicity of peptides.

Novel family of [RuCp(N,N)(P)]+ compounds with simultaneous anticancer and antibacterial activity: Biological evaluation and solution chemistry studies

A family of ten novel ruthenium(II)-cyclopentadienyl organometallics of general formula [Ru(η5-C5H5)(N,N)(PPh2(C6H4COOR)][CF3SO3] (1–10) in which (N,N) = 4,4′-R′-2,2′-bipyridyl (R = –H or –CH2CH2OH; R’ = –H, –CH3, –OCH3, –CH2OH, and –CH2-biotin) was prepared from [Ru(η5-C5H5)(PPh2(C6H4COOH))2Cl]. All compounds were fully characterized by means of several spectroscopic and analytical techniques, and the molecular structures of [Ru(η5-C5H5)(PPh2(C6H4COOH))2Cl], 1, 3 and 4 have been additionally studied by single-crystal X-ray diffraction. The anticancer activity of all compounds was evaluated in sensitive and multidrug-resistant counterpart cell lines from human colorectal cancer (Colo 205 and Colo 320) and non-small cell lung cancer NSCLC (A549, NCI–H460 versus NCI–H460/R) as well. Notably, compounds 6 and 7 (R CH2CH2OH and (N,N) = bipy or Me2bipy, respectively) showed antiproliferative effect against both cell lines with high intrinsic selectivity towards cancer cells. The antibacterial activity of all compounds was also evaluated against both Gram negative and Gram positive strains, and some compounds in the series showed potent antibacterial activity against Staphylococcus aureus strains, including the methicillin-resistant MRSA strains.Solution speciation studies revealed that the complexes bearing the PPh2(C6H4COO−) ligand are neutral at physiological pH (7.4) in contrast with their ethylene glycol derivatives that have a permanent positive charge. While all compounds are lipophilic, the difference in the distribution coefficient for neutral and charged complexes is around one order of magnitude. Complexes 6 and 7 exhibited excellent biological activity and were selected for further studies. Spectrofluorometric methods were used to investigate their interaction with biomolecules such as human serum albumin (HSA) and calf thymus DNA (ct-DNA). For these complexes, binding site II of HSA is a possible binding pocket through non-covalent interactions. The release of ethidium from the DNA adduct by the charged complexes proves their interaction with DNA in contrast to the neutral ones.In conclusion, Ru(II)-cyclopentadienyl complexes with 2,2′-bipyridyl-derivatives and an ethylene glycol moiety tethered to the phenylphosphane co-ligand are very promising from a therapeutic perspective, in particular complexes 6 and 7 that display remarkable antibacterial activity with a high anti-proliferative effect against colon and non-small cell lung cancers, both clinically challenging neoplasias in need of effective solutions.

Bisabosqual A: A novel asparagine synthetase inhibitor suppressing the proliferation and migration of human non-small cell lung cancer A549 cells

Asparagine synthetase (ASNS) is a crucial enzyme for the de novo biosynthesis of endogenous asparagine (Asn), and ASNS shows the positive relationship with the growth of several solid tumors. Most of ASNS inhibitors are analogs of transition-state in ASNS reaction, but their low cell permeability hinders their anticancer activity. Therefore, novel ASNS inhibitors with a new pharmacophore urgently need to be developed. In this study, we established and applied a system for in vitro screening of ASNS inhibitors, and found a promising unique bisabolane-type meroterpenoid molecule, bisabosqual A (Bis A), able to covalently modify K556 site of ASNS protein. Bis A targeted ASNS to suppress cell proliferation of human non-small cell lung cancer A549 cells and exhibited a synergistic effect with L-asparaginase (L-ASNase). Mechanistically, Bis A promoted oxidative stress and apoptosis, while inhibiting autophagy, cell migration and epithelial-mesenchymal transition (EMT), impeding cancer cell development. Moreover, Bis A induced negative feedback pathways containing the GCN2-eIF2α-ATF4, PI3K-AKT-mTORC1 and RAF-MEK-ERK axes, but combination treatment of Bis A and rapamycin/torin-1 overcame the potential drug resistance triggered by mTOR pathways. Our study demonstrates that ASNS inhibition is promising for cancer chemotherapy, and Bis A is a potential lead ASNS inhibitor for anticancer development.

Design, synthesis, and antiproliferative activity of novel 1,2,4-triazole-chalcone compounds

Abstract A series of novel 1,2,4-triazole-chalcone compounds 10a–10s were designed by molecular hybridization strategy and synthesized. The molecular structures of the novel chalcones were characterized by 1H-NMR, 13C-NMR, and HRMS. The anti-proliferative activities of the novel chalcones against four cancer cell lines in vitro were examined by MTT, four tumor cell lines including human colon cancer cell SW620, human non-small cell lung cancer cell A549, human cervical cancer cell HeLa, and human breast cancer cell MCF-7. Compound 10o showed certain selectivity to SW620 cell line, and its IC50 value was 21.55 μM. 10q had good anti-proliferative activity against A549 cells with an IC50 value of 25.58 μM.

KCNAB2 overexpression inhibits human non-small-cell lung cancer cell growth in vitro and in vivo

Non-small-cell lung cancer (NSCLC) accounts for approximately 85% of all lung cancer cases. NSCLC patients often have poor prognosis demanding urgent identification of novel biomarkers and potential therapeutic targets. KCNAB2 (regulatory beta subunit2 of voltage-gated potassium channel), encoding aldosterone reductase, plays a pivotal role in regulating potassium channel activity. In this research, we tested the expression of KCNAB2 as well as its potential functions in human NSCLC. Bioinformatics analysis shows that expression of KCNAB2 mRNA is significantly downregulated in human NSCLC, correlating with poor overall survival. In addition, decreased KCNAB2 expression was detected in different NSCLC cell lines and local human NSCLC tissues. Exogenous overexpression of KCNAB2 potently suppressed growth, proliferation and motility of established human NSCLC cells and promoted NSCLC cells apoptosis. In contrast, CRISPR/Cas9-induced KCNAB2 knockout further promoted the malignant biological behaviors of NSCLC cells. Protein chip analysis in the KCNAB2-overexpressed NSCLC cells revealed that KCNAB2 plays a possible role in AKT-mTOR cascade activation. Indeed, AKT-mTOR signaling activation was potently inhibited following KCNAB2 overexpression in NSCLC cells. It was however augmented by KCNAB2 knockout. In vivo, the growth of subcutaneous KCNAB2-overexpressed A549 xenografts was significantly inhibited. Collectively, KCNAB2 could be a novel effective gene for prognosis prediction of NSCLC. Targeting KCNAB2 may lead to the development of advanced therapies.

Single-cell mass cytometric analysis of peripheral immunity and multiplex plasma marker profiling of non-small cell lung cancer patients receiving PD-1 targeting immune checkpoint inhibitors in comparison with platinum-based chemotherapy.

The effect of platinum-based chemotherapy (Chem.) and second- or multiple- line immune checkpoint PD-1 blocking therapy by Nivolumab or Pembrolizumab (ICI) was assayed in the peripheral blood of non-small cell lung cancer (NSCLC) patients. Flow cytometry was used to detect NSCLC-related antigen binding IgG antibodies. The Luminex MagPix multiplex bead-based cytokine/chemokine detecting system was used to quantitatively measure 17 soluble markers in the plasma samples. Single-cell mass cytometry was applied for the immunophenotyping of peripheral leukocytes. The incubation of patient derived plasma with human NSCLC tumor cell lines, such as A549, H1975, and H1650, detected NSCLC-specific antibodies reaching a maximum of up to 32% reactive IgG-positive NSCLC cells. The following markers were detected in significantly higher concentration in the plasma of Chem. group versus healthy non-smoker and smoker controls: BTLA, CD27, CD28, CD40, CD80, CD86, GITRL, ICOS, LAG-3, PD-1, PD-L1, and TLR-2. The following markers were detected in significantly higher concentration in the plasma of ICI group versus healthy non-smoker and smoker controls: CD27, CD28, CD40, GITRL, LAG-3, PD-1, PD-L1, and TLR-2. We showed the induction of CD69 and IL-2R on CD4+ CD25+ T-cells upon chemotherapy; the exhaustion of one CD8+ T-cell population was detected by the loss of CD127 and a decrease in CD27. CD19+CD20+, CD79B+, or activated B-cell subtypes showed CD69 increase and downregulation of BTLA, CD27, and IL-2R in NSCLC patients following chemotherapy or ICI. Peripheral immunophenotype caused by chemotherapy or PD-1 blocking was shown in the context of advanced NSCLC.

SERPINE1 and SERPINB7 as potential biomarkers for intravenous vitamin C treatment in non-small-cell lung cancer

High dose intravenous vitamin C (IVC) has been proposed as a pro-oxidant anticancer agent. However, there is a lack of biomarkers that are specific for this treatment. Here, we explored profiles of gene expression responding to IVC treatment in non-small cell lung cancer (NSCLC) cells as an effort for potential biomarker discovery. Genome-wide RNA-seq was performed in human NSCLC cell lines treated with pharmacological concentrations of vitamin C(VitC) for differential expression of genes. The identified genes were analyzed for correlations with patient prognosis using data from the Kaplan-Meier Plotter and the Human Protein Atlas databases. Further, tumor samples from a retrospective study of 153 NSCLC patients were analyzed with immunohistochemistry for expression of targeted genes, and patient prognosis was correlated to these genes. Two genes, namely SERPINE1 and SERPINB7 were found to be downregulated in NSCLC cells following VitC treatment. Combined patient data from the cohort analysis and online databases revealed that these 2 genes presented an unfavorable prognostic prediction of overall survival (OS) in NSCLC patients receiving standard of care. However, high expression level of these 2 genes were associated with prolonged OS in NSCLC patients receiving IVC in addition to standard of care. These data revealed that SERPINE1 and SERPINB7 have the potential to serve as predictive factors indicating favorable responses to IVC treatment in patients with NSCLC. Further validations are warranted.

Investigation of proapoptotic and anticancer effects mediated by intracellular ceramide changing dependent on ARN14974=BOC treatment in human non-small cell lung cancer

Sfingolipidler hücre membran yapısında bulunan ve kanser hücresinde sinyalleşme işlevlerini kontrol ederek büyüme, proliferasyon, göç, metastaz gibi biyolojik fonksiyonları düzenlemekte olan moleküllerdir. Kanser tedavisinde son yıllardaki araştırmalar, kanser hücrelerindeki sfingolipid sinyalleşmesini hedef alan, yenilikçi moleküler ve farmakolojik araçlar kullanılarak tümör büyümesinin ve kemoterapi, radyoterapi ve/veya immünoterapiye yanıtta sfingolipidlerin ve alt hedeflerinin rolü hakkında mekanik ayrıntılar bulmayı amaçlamıştır.
 Akciğer kanseri ülkemizde ve dünyada en sık görülen kanser türlerindendir. Kanser kaynaklı ölümlerin önemli bir kısmı akciğer kanserine bağlıdır. Akciğer kanseri için yeni ve etkili tedavi yöntemlerinin geliştirilmesine her gün daha çok ihtiyaç duyulmaktadır.
 Bu çalışmada, ARN14974=BOC seramidaz inhibitörünün katı lipit nanopartikül formunun sentezlenmesi ile seramidin hücre içi seviyesini arttırarak hücrenin yaşamını ve proliferasyonunu sağlayan ve apoptozu inhibe eden seramidaz-1-fosfatın oluşumunu engelleyerek yeni ve etkili bir tedavi yaklaşımının küçük hücreli dışı akciğer kanseri hücre hattı A549 hücrelerinde araştırılması amaçlanmıştır.
 Çalışma sonuçları ARN14974=BOC’un ve nanopartikül formunun A549 hücrelerinde sitotoksik ve antiproliferatif etkileri tespit edilmiş ve bu hücrelerde apoptozu gösteren morfolojik ve ince yapısal değişiklikler saptanmıştır. Bu moleküllerin A549 hücrelerinde reaktif oksijen türlerinin artması ve hücre döngüsünün durdurulmasına neden olarak apoptozu tetiklemiştir. Bu sonuçlar, insan küçük hücreli dışı akciğer kanseri hücrelerinde hücre ölümünün sentezlenen ARN14974=BOC’un nanopartikül formu tarafından daha etkin bir biçimde gerçekleştirildiğini göstermiştir.

Use of recombinant microRNAs as antimetabolites to inhibit human non-small cell lung cancer.

During the development of therapeutic microRNAs (miRNAs or miRs), it is essential to define their pharmacological actions. Rather, miRNA research and therapy mainly use miRNA mimics synthesized invitro. After experimental screening of unique recombinant miRNAs produced invivo, three lead antiproliferative miRNAs against human NSCLC cells, miR-22-3p, miR-9-5p, and miR-218-5p, were revealed to target folate metabolism by bioinformatic analyses. Recombinant miR-22-3p, miR-9-5p, and miR-218-5p were shown to regulate key folate metabolic enzymes to inhibit folate metabolism and subsequently alter amino acid metabolome in NSCLC A549 and H1975cells. Isotope tracing studies further confirmed the disruption of one-carbon transfer from serine to folate metabolites by all three miRNAs, inhibition of glucose uptake by miR-22-3p, and reduction of serine biosynthesis from glucose by miR-9-5p and -218-5p in NSCLC cells. With greater activities to interrupt NSCLC cell respiration, glycolysis, and colony formation than miR-9-5p and -218-5p, recombinant miR-22-3p was effective to reduce tumor growth in two NSCLC patient-derived xenograft mouse models without causing any toxicity. These results establish a common antifolate mechanism and differential actions on glucose uptake and metabolism for three lead anticancer miRNAs as well as antitumor efficacy for miR-22-3p nanomedicine, which shall provide insight into developing antimetabolite RNA therapies.

Efficient detection of single circulating tumor cell in blood using Raman mapping based on Aptamer-SERS bio-probe coupled with micropore membrane filtration

Rapid and accurate detection of rare circulating tumor cells (CTCs) in human blood still remains a challenge. We present a surface enhanced Raman spectroscopy (SERS) method based on aptamer-SERS bio-probe recognition coupled with micropore membrane filtration capture for the detection of CTCs at single cell level. The parylene micropore membrane with optimized micropore size installed on a filtration holder could capture bio-probe labeled CTCs by gravity in less than 10 s, and only with very less white blood cells (WBCs) residual. In order to facilitate the synthesis of the aptamer-SERS bio-probe, ethyl acetate dehydration method was established. The bio-probe can be rapidly synthesized within 2 h by binding SH-aptamer to 4- mercaptobenzoic acid (4-MBA) modified AuNPs with the help of ethyl acetate. The SERS bio-probe with selected specific aptamer could distinguish single human non-small cell lung cancer A549 cells from residual WBCs on membrane efficiently and reliably based on their Raman signal intensity difference at 1075 cm−1. Through the filter membrane coupled with aptamer-SERS bio-probe system, even 20 A549 cells in blood solution simulating CTCs sample can be detected, which the recovery rate and recognition rate are more than 90%. This method is rapid, reliable and cost-effective, which indicates a good prospect in clinical application for CTCs detection.

Knockdown of DLK4 inhibits non-small cell lung cancer tumor growth by downregulating CKS2.

Non-small cell lung cancer (NSCLC) accounts for 85% of all lung cancer cases and is considered as the most common type of cancer. DLX4 was originally identified as a β-globin gene suppressor in red blood cells, which plays critical roles in several types of cancers. However, the role and related mechanism of DLX4 in NSCLC are still unclear. The study aimed to uncover the expression of DLX4 in human NSCLC cells and tissues, reveal its possible role in NSCLC, and investigate the underlying mechanisms. Immunoblot and TCGA database were used to detect the expression of DLX4 in human NSCLC cells and tissues. CCK-8, colony formation, and FCM assays were conducted to detect the effects of DLX4 on the viability and cell cycle of NCI-H2170 and A549 cells. Immunoblot assays were further performed to investigate the possible mechanism underlying DLX4 affecting the growth of NSCLC. We revealed that knockdown of DLX4 inhibited NSCLC cell proliferation. We further revealed that DLX4 knockdown induced the NSCLC cell cycle arrest. Our results further showed that downregulation of DLX4 suppressed YB-1 expression, which further suppressed CKS2 expression, thereby suppressing tumor growth of NSCLC. In conclusion, DLX4 has the potential to serve as a promising drug for NSCLC treatment.

Kaempferol promotes non-small cell lung cancer cell autophagy via restricting Met pathway

BackgroundKaempferol is extracted from Hedyotis diffusa, exerting an obvious anti-cancer effect. Here in the present study, we explored the anti-cancer effects and mechanism of kaempferol in non-small cell lung cancer cell (NSCLC). PurposeOur objective is to figure out the molecular mechanism by which kaempferol promotes autophagy in NSCLC cells. Study designA549 and H1299 NSCLC cell lines were used for in vitro experiments. And BALB/c nude mice of NSCLC were used to perform in vivo experiments. MethodsFor in vitro experiments, CCK-8 and EdU assay was used to observe the effect of kaempferol on NSCLC cell proliferation. Confocal microscopy of mCherry-EGFR-LC3 assay and electron microscopy assay were used to detect NSCLC cell autophagy. Protein expression was determined using Western blot, and mRNA expression was determined using qRT-PCR. Flow cytometry was performed to detect the cell apoptosis. For in vivo experiments, a subcutaneously implanted tumor model in BALB/C nude mice was performed using human NSCLC cell line A549-Luc. The kaempferol effect on NSCLC mice model was detected by measuring the tumor weight and bioluminescence intensity. Immunohistochemistry was done to measure the key protein expression from mice tumor tissues. ResultsOur results confirmed that kaempferol inhibited NSCLC cell proliferation significantly. And it promoted NSCLC cell autophagy, leading to NSCLC cell death. Interestingly, Met-was greatly inhibited at both protein and mRNA levels. Meanwhile, PI3K/AKT/mTOR signaling pathway was inhibited accordingly. Furthermore, overexpressing Met-reversed the effect of kaempferol on NSCLC cell viability and cell autophagy with significance. Finally, the above effect and pathway were validated using the xenograft model. ConclusionKaempferol may exert its anti-NSCLC effect by promoting NSCLC cell autophagy. Mechanistically, Met-and its downstream PI3K/AKT/mTOR signaling pathway were involved in the process, which provides a novel mechanism how kaempferol functions in inhibiting NSCLC.

The IL-25/ILC2 axis promotes lung cancer with a concomitant accumulation of immune-suppressive cells in tumors in humans and mice.

Group 2 innate lymphoid cells (ILC2) can be activated by interleukin (IL)-33 or IL-25. IL-25-activated ILC2 cells help protect the host against helminth infection while exacerbating allergic-like inflammation and tissue damage in the lung. In the context of cancer, IL-33-activated ILC2 cells were found to bear anti-tumoral functions in lung cancer while IL-25-activated ILC2 cells promoted tumorigenesis in colorectal cancer. The role of IL-25-activated ILC2 cells in lung cancer remains to be addressed. We examined the overall survival of human non-small cell lung cancer (NSCLC) patients according to IL25 expression as well as the distribution of ILC2 cells and regulatory T cells (Tregs) in various NSCLC patient tissues and peripheral blood (PB) of healthy donors (HDs). We analyzed the effect of adoptive transfer of IL-25-activated ILC2 cells on tumor growth, metastasis and survival in a heterotopic murine model of lung cancer. We report that human NSCLC patients with high IL-25 expression have reduced overall survival. Moreover, NSCLC patients bear increased frequencies of ILC2s compared to HDs. Frequencies of Tregs were also increased in NSCLC patients, concomitantly with ILC2s. In mice bearing heterotopic lung cancer, adoptive transfer of IL-25-activated ILC2s led to increased tumor growth, increased metastasis and reduced survival. The frequencies of monocytic myeloid-derived suppressor cells (M-MDSCs) were found to be increased in the tumors of mice that received ILC2s as compared to controls. Overall, our results indicate that the IL-25/ILC2 axis promotes lung cancer potentially by recruiting immune-suppressive cells to the tumors both in humans and in mice, and that it may therefore represent a suitable novel target for NSCLC immunotherapeutic development.

Characterization of chemoresistant human non-small cell lung cancer cells by metabolic and lipidomic profiling.

Lung cancer is one of the most malignant cancers and the leading cause of cancer-related deaths worldwide, while acquired chemoresistance would represent a major problem in the treatment of non-small cell lung cancer (NSCLC) because of the reduced treatment effect and increased rates of recurrence. To establish the chemoresistant NSCLC cells, doxorubicin was treated to A549 cells over 3 months at gradually increasing concentrations from 0.03 to 0.5 µM. Real-time PCR and Western blotting were employed for investigating mRNA and protein expression of the glutathione peroxidase (GPX) protein family and multidrug resistance protein 1 (MRP1) in A549 and A549/CR cells. We also employed gas chromatography mass-spectrometry and nano electrospray ionization mass-spectrometry coupled with multivariate statistical analysis to characterize the unique metabolic and lipidomic profiles of chemoresistant NSCLC cells in order to identify potential therapeutic targets. Reactive oxygen species levels were decreased, and mRNA and protein levels of GPX2 and multidrug resistance protein 1 (MRP1) were increased in A549/CR. We identified 87 metabolites and intact lipid species in A549 and A549/CR. Among these metabolites, lactic acid, glutamic acid, glycine, proline, aspartic acid, succinic acid, and ceramide, alongside the PC to PE ratio, and arachidonic acid-containing phospholipids were suggested as characteristic features of chemoresistant NSCLC cells (A549/CR). This study reveals characteristic feature differences between drug-resistance NSCLC cells and their parental cells. We suggest potential therapeutic targets in chemoresistant NSCLC. Our results provide new insight into metabolic and lipidomic alterations in chemoresistant NSCLC. This could be used as fundamental information to develop therapeutic strategies for the treatment of chemoresistant NSCLC patients.

FOXA1 prolongs S phase and promotes cancer progression in non-small cell lung cancer through upregulation of CDC5L and activation of the ERK1/2 and JAK2 pathways.

Non-small cell lung cancer (NSCLC) causes high mortality worldwide; however, its molecular pathways have not been fully investigated. The relationship between FOXA1 and CDC5L as well as their roles in NSCLC have not been comprehensively studied. Clinical tissues were collected from 78 NSCLC patients for clinical studies. The BEAS-2B human normal lung epithelial cell line and the A549, Calu-3, H526 and H2170 human NSCLC cell lines were used for in vitro studies. sh-FOXA1 and oe-CDC5L constructs were used to generate knockdown and overexpression models, respectively. The CCK-8 assay was used to analyze cell viability. The cell cycle and apoptosis were evaluated by flow cytometry analysis. The relationship between FOXA1 and CDC5L was demonstrated using dual-luciferase and ChIP assays. Gene levels were examined via immunohistochemistry, qRT-PCR and western blot analysis. FOXA1 levels were increased in NSCLC clinical tissues and cell lines. Depletion of FOXA1 increased the apoptosis rate and increased the proportion of cells in G2/M phase. In addition, we demonstrated that FOXA1 was directly bound to the promoter of CDC5L and that depletion of FOXA1 inhibited CDC5L expression. Overexpression of CDC5L induced ERK1/2 phosphorylation, induced JAK2 phosphorylation, inhibited cell apoptosis, prolonged S phase, and significantly reversed the effects of FOXA1 knockdown on the progression of NSCLC. The present study demonstrated that FOXA1 prolongs S phase and promotes NSCLC progression through upregulation of CDC5L and activation of the ERK1/2 and JAK2 pathways.