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The detection of low-abundance mutation genes of the epidermal growth factor receptor (EGFR) exon 21 (EGFR L858R) plays a crucial role in the diagnosis of non-small cell lung cancer (NSCLC), as it enables early cancer detection and facilitates the development of treatment strategies. A detection platform was developed by combining the MscI restriction enzyme with the recombinase-aided isothermal amplification (RAA) technique (MRE-RAA). During the RAA process, "TGG^CCA" site of the wild-type genes was cleaved by the MscI restriction enzyme, while only the low-abundance mutation genes underwent amplification. Notably, when the RAA product was combined with CRISPR-Cas system, the sensitivity of detecting the EGFR L858R mutation increased by up to 1000-fold for addition of the MscI restriction enzyme. This achievement marked the first instance of attaining an analytical sensitivity of 0.001%. Furthermore, a disk-shaped microfluidic chip was developed to automate pretreatment while concurrently analyzing four blood samples. The microfluidic features of the chip include DNA extraction, MRE-RAA, and CRISPR-based detection. The fluorescence signal is employed for detection in the microfluidic chip, which is visible to the naked eye upon exposure to blue light irradiation. Furthermore, this platform has the capability to facilitate early diagnosis for various types of cancer by enabling high-sensitivity detection of low-abundance mutation genes., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier B.V.)

Significance: Sialylated cancer IgG activates c-Met-SOX2 signaling to promote stemness properties in cancer cells and can be targeted to suppress tumor growth., (©2023 American Association for Cancer Research.)

Recent advances in intratracheal delivery strategies have sparked considerable biomedical interest in developing this promising approach for lung cancer diagnosis and treatment. However, there are very few relevant studies on the behavior and mechanism of imaging nanoparticles (NPs) after intratracheal delivery. Here, we found that nanosized perfluoro-15-crown-5-ether (PFCE NPs, ∼200 nm) exhibite significant 19 F-MRI signal-to-noise ratio (SNR) enhancement than perfluorooctyl bromide (PFOB NPs) up to day 7 after intratracheal delivery. Alveolar macrophages (AMs) engulf PFCE NPs, become PFCE NPs-laden AMs, and then migrate into the tumor margin, resulting in increased tumor PFCE concentration and 19 F-MRI signals. AMs-mediated translocation of PFCE NPs to lung draning lymph nodes (dLNs) decreases the background PFCE concentration. Our results shed light on the dynamic AMs-mediated translocation of intratracheally delivered PFC NPs for effective lung tumor visualization and reveal a pathway to develop and promote the clinical translation of an intratracheal delivery-based imaging strategy.

Background: Non-small cell lung cancer (NSCLC) patients with primary tumors and liver metastases have substantially reduced survival. Since mesenchymal-epithelial transition factor (MET) plays a significant role in the molecular mechanisms of advanced NSCLC, small molecule MET inhibitor capmatinib (INC280) hold promise for clinically NSCLC treatment. However, the major obstacles of MET-targeted therapy are poor drug solubility and off-tumor effects, even oral high-dosing regimens cannot significantly increase the therapeutic drug concentration in primary and metastatic NSCLC., Methods: We developed a multirooted delivery system INC280-PFCE nanoparticles (NPs) by loading INC280 into perfluoro-15-crown-5-ether for improving MET-targeted therapy. Biodistribution and anti-MET/antimetastatic effects of NPs were validated in orthotopic NSCLC and NSCLC liver metastasis models in a single low-dose. The efficacy of INC280-PFCE NPs was also explored in human NSCLC specimens., Results: INC280-PFCE NPs exhibited excellent antitumor ability in vitro. In orthotopic NSCLC models, sustained release and prolonged retention behaviors of INC280-PFCE NPs within tumors could be visualized in real-time by 19 F magnetic resonance imaging ( 19 F-MRI), and single pulmonary administration of NPs showed more significant tumor growth inhibition than oral administration of free INC280 at a tenfold higher dose. Furthermore, a single low-dose INC280-PFCE NPs administered intravenously suppressed widespread dissemination of liver metastasis without systemic toxicity. Finally, we verified the clinical translation potential of INC280-PFCE NPs in human NSCLC specimens., Conclusions: These results demonstrated high anti-MET/antimetastatic efficacies, real-time MRI visualization and high biocompatibility of NPs after a single low-dose., (© 2022. The Author(s).)

Low accumulation of anticancer drugs in tumors and serious systemic toxicity remain the main challenges to the clinical efficiency of pharmaceuticals. Pulmonary delivery of nanoscale-based drug delivery systems offered a strategy to increase antitumor activity with minimal adverse exposure. Herein, we report an osimertinib-loaded perfluoro-15-crown-5-ether (AZD9291-PFCE) nanoemulsion, through intratracheal and intravenous delivery, synergizes with 19 F magnetic resonance imaging ( 19 F MRI)-guided low-intensity focused ultrasound (LIFU) for lung cancer therapy. Pulmonary delivery of AZD9291-PFCE nanoemulsion in orthotopic lung carcinoma models achieves quick distribution of the nanoemulsion in lung tissues and tumors without short-term and long-term toxic effects. Furthermore, LIFU can trigger drug release from the AZD9291-PFCE nanoemulsion and specifically increases tumor vascular and tumor tissue permeability. 19 F MRI was applied to quantify nanoemulsion accumulation in tumors in real time after LIFU irradiation. We validate the treatment effect of AZD9291-PFCE nanoemulsion in resected human lung cancer tissues, proving the translational potential to enhance clinical outcomes of lung cancer therapy. Thus, this work presents a promising pulmonary nanoemulsion delivery system of osimertinib (AZD9291) for targeted therapy of lung cancer without severe side effects.

Circular RNAs hold significant regulatory functions during various tumors. However, the exact hsa_circ_0041268 roles in non-small cell lung cancer (NSCLC) along with regulatory mechanism are unknown. In this study, RT-qPCR was used to perceive hsa_circ_0041268 expressions in NSCLC cell lines. Our team constructed small interfering RNA for hsa_circ_0041268. NSCLC cell proliferation, migration, and tumorigenesis in nude mice were assayed to confirm hsa_circ_0041268 activities in NSCLC cells. We then used bioinformatics and luciferase reporter analyses to characterize the hsa_circ_0041268 downstream targets. The result shows that the expressions of hsa_circ_0041268 incremented in NSCLC cell lines and hsa_circ_0041268 downregulation decreased cell proliferation and migration. ROCK1 and miR-214-5p were hsa_circ_0041268 downstream targets. miR-214-5p downregulation or ROCK1 overexpression restored migration and proliferation abilities after hsa_circ_0041268 silencing. ROCK1 overexpression renovated migration and proliferation abilities after miR-214-5p overexpression. In vivo investigations confirmed that hsa_circ_0041268 downregulation inhibited tumor formation and metastasis in nude mice xenografts. Together, results demonstrated that hsa_circ_0041268 acted as tumor promoter through novel hsa_circ_0041268/miR-214-5p/ROCK1 axis, which highlighted its potential as NSCLC therapeutic agent., (© 2022 The Authors. Journal of Clinical Laboratory Analysis published by Wiley Periodicals LLC.)

Inefficient tumor accumulation and penetration remain as the main challenges to therapy efficacy of lung cancer. Local delivery of smart nanoclusters can increase drug penetration and provide superior antitumor effects than systemic routes. Here, we report self-assembled pH-sensitive superparamagnetic iron oxide nanoclusters (SPIONCs) that enhance in situ ferroptosis and apoptosis with radiotherapy and chemodynamic therapy. After pulmonary delivery in orthotopic lung cancer, SPIONCs disintegrate into smaller nanoparticles and release more iron ions in an acidic microenvironment. Under single-dose X-ray irradiation, endogenous superoxide dismutase converts superoxide radicals produced by mitochondria to hydrogen peroxide, which in turn generates hydroxyl radicals by the Fenton reaction from iron ions accumulated inside the tumor. Finally, irradiation and iron ions enhance tumor lipid peroxidation and induce cell apoptosis and ferroptosis. Thus, rationally designed pulmonary delivered nanoclusters provide a promising strategy for noninvasive imaging of lung cancer and synergistic therapy.

Background: MicroRNAs can regulate tumor metastasis either as oncomiRs or suppressor miRNAs. Here, we investigated the role of microRNA 224 (miR-224) in lymphatic metastasis of non-small-cell lung cancer (NSCLC)., Methods: The expression of miR-224 was demonstrated by a validation cohort of 156 lung cancer patients (77 cases with lymphatic metastasis) by quantitative polymerase chain reaction (qPCR). In vitro and in vivo experiments were performed to study the malignant phenotype after upregulation and inhibition of miR-224 expression. Furthermore, the direct target genes of miR-224 were determined by a luciferase reporter assay., Results: First, miR-224 was identified as a highly expressed miRNA in tumor tissues with lymphatic metastasis, with an area under the curve (AUC) of 0.57 as determined by qPCR analysis of a validation cohort of 156 lung cancer patients. Then, in vitro and in vivo experiments indicated that forced expression of miR-224 in H1299 cells promoted not only cell viability, plate colony formation, migration and invasion in vitro but also tumor growth and lung metastasis in vivo. Consistently, inhibition of miR-224 suppressed malignant characteristics both in vitro and in vivo. Moreover, molecular mechanistic research suggested that miR-224 enhanced NSCLC by directly targeting the tumor suppressor angiopoietin-like protein (ANGPTL)., Conclusions: Overall, the collective findings demonstrate that miR-224 is a potential biomarker for the prediction of lymphatic metastasis of NSCLC.

The epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitors (TKIs) have revolutionized the treatment of non-small cell lung cancer (NSCLC). EGFR-TKI positron emission tomography (PET) probes based on the central quinazoline core show great potential for NSCLC diagnosis, and pre-clinical and clinical therapy monitoring. In our previous research, anilino quinazoline based PET probe, N-(3-chloro-4-fluorophenyl)-7-(2-(2-(2-(2- 18 F-fluoroethoxy) ethoxy) ethoxy) ethoxy)-6-methoxyquinazolin-4-amine ( 18 F-MPG), have been developed, and it has been successfully demonstrated to be a powerful non-invasive imaging tool for differentiating EGFR mutation status and stratifying NSCLC patients for EGFR-TKI treatment in a clinical study (n = 75 patients). Moreover, it has been found that 18 F-MPG shows excellent tumor targeting performance and good pharmacokinetic characteristics in NSCLC patients. These results motivate us to investigate the cancer treatment efficacy of non-radioactive F-MPG and its analogue N-(3-chloro-4-fluorophenyl)-7-(2-(2-(2-(2-hydroxyethoxy)ethoxy) ethoxy) ethoxy)-6-methoxyquinazolin-4-amine (OH-MPG) in vitro and in small animal models. Our studies revealed that both F-MPG and OH-MPG displayed high therapeutic effect to NSCLC cells (IC 50 = 5.3 nM and 2.0 nM to HCC827 cells for F-MPG and OH-MPG, respectively). More importantly, compared with a standard EGFR-TKI, 4-(3-bromoanilino)-6,7-dimethoxyquinazoline (PD153035), F-MPG and OH-MPG showed stronger tumor inhibition in preclinical models. Furthermore, the treatment efficacy of F-MPG or OH-MPG monitored by 18 F-FDG-PET indicated that tumor uptake in treated groups was significantly decreased. Ex vivo experiments showed that the levels of serum biomarkers and pathological changes in the liver were significantly reduced in the F-MPG and OH-MPG group, compared to PD153035 treated group. In conclusion, EGFR targeted F-MPG and OH-MPG exhibit promising anti-tumor activity with limited liver damage, thus representing promising drug candidates for further investigation for combating the deadly NSCLC.

Background: Folate receptors (FRs) hold great potential as important diagnostic and prognostic biological marker for cancer., Purpose: To assess the targeted capability of the FR-targeted perfluorocarbon (PFC) nanoparticles and to assess in vivo the relationship between FR expression and tumor proliferation with fluorine-19 ( 19 F) MR molecular imaging., Study Type: Prospective animal cancer model., Animal Model: H460 (n = 14) and A549 (n = 14) nude mice subcutaneous tumor models., Field Strength: 9.4T, 1 H and 19 F RARE sequences., Assessment: Intracellular uptake of the PFC nanoparticles was tested in H460 and A549 cell lines. 19 F MRI of H460 and A549 subcutaneous tumors was performed following intravenous injection of PFC nanoparticles. The concentration of PFC in tumors were compared. 3'-Deoxy-3'- 18 F-fluorothymidine ( 18 F-FLT) positron emission tomography / computed tomography (PET/CT) imaging, Ki-67, and proliferating cell nuclear antigen (PCNA) staining were performed to confirm tumor proliferation., Statistical Tests: One-way or two-way analysis of variance. P < 0.05 was considered a significant difference., Results: The diameter of the FR-targeted nanoparticles was 108.8 ± 0.56 nm, and the zeta potential was -58.4 ± 10.8 mV. H460 cells incubated with FR-targeted nanoparticles showed ∼59.87 ± 3.91% nanoparticles-labeled, which is significantly higher than the other groups (P < 0.001). The PFC concentration in H460 tumors after injection with FR-targeted nanoparticles was 4.64 ± 1.21, 8.04 ± 1.38, and 9.16 ± 2.56 mmol/L at 8 hours, 24 hours, and 48 hours, respectively (P < 0.05 compared to others). The ratio of 18 F-FLT uptake for H460 and A549 tumors was 3.32 ± 0.17 and 1.48 ± 0.09 (P < 0.05), and there was more Ki-67 and PCNA in H460 tumor than A549. DATA CONCLUSION: 19 F MRI with FR-targeted PFC nanoparticles can be used in differentiating of FR-positive and FR-negative tumors, and further, in evaluation of the two cancer models proliferation., Level of Evidence: 1 Technical Efficacy: Stage 2 J. Magn. Reson. Imaging 2018;48:1617-1625., (© 2018 International Society for Magnetic Resonance in Medicine.)

Aim: To demonstrate the feasibility of intratracheal administration in orthotopic lung cancer model with 19 F MRI., Materials & Methods: α v β 3 -integrin targeting ability of the perfluorocarbon (PFC) nanoparticles was tested. Orthotopic lung cancer model was established in rabbits under computed tomography guidance. α v β 3 -targeted PFC nanoparticles were administrated intratracheally or intravenously, and 19 F MRI was performed before and up to 24 h after administration., Results: The targeted PFC nanoparticles could bind with α v β 3 -integrin. PFC concentrations in the tumors of intratracheal group after administration were significantly higher than intravenous group., Conclusion: Intratracheal administration of PFC nanoparticles was shown to be feasible and efficacious. 19 F MRI with α v β 3 -targeted PFC nanoparticles provided quantitative assessment of nanoparticles distribution and tumor angiogenesis.

Eighty percent of lung cancers originate as subtle premalignant changes in the airway mucosal epithelial layer of bronchi and alveoli, which evolve and penetrate deeper into the parenchyma. Liquid-ventilation, with perfluorocarbons (PFC) was first demonstrated in rodents in 1966 then subsequently applied as lipid-encapsulated PFC emulsions to improve pulmonary function in neonatal infants suffering with respiratory distress syndrome in 1996. Subsequently, PFC nanoparticles (NP) were extensively studied as intravenous (IV) vascular-constrained nanotechnologies for diagnostic imaging and targeted drug delivery applications. Methods: This proof-of-concept study compared intratumoral localization of fluorescent paramagnetic (M) PFC NP in the Vx2 rabbit model using proton ( 1 H) and fluorine ( 19 F) magnetic resonance (MR) imaging (3T) following intratracheal (IT) or IV administration. MRI results were corroborated by fluorescence microscopy. Results: Dynamic 1 H-MR and 19 F-MR images (3T) obtained over 72 h demonstrated marked and progressive accumulation of M-PFC NP within primary lung Vx2 tumors during the first 12 h post IT administration. Marked 1 H and 19 F MR signal persisted for over 72 h. In contradistinction, IV M-PFC NP produced a modest transient signal during the initial 2 h post-injection that was consistent circumferential blood pool tumor enhancement. Fluorescence microscopy of excised tumors corroborated the MR results and revealed enormous intratumor NP deposition on day 3 after IT but not IV treatment. Rhodamine-phospholipid incorporated into the PFC nanoparticle surfactant was distributed widely within the tumor on day 3, which is consistent with a hemifusion-based contact drug delivery mechanism previously reported. Fluorescence microscopy also revealed similar high concentrations of M-PFC NP given IT for metastatic Vx2 lung tumors. Biodistribution studies in mice revealed that M-PFC NP given IV distributed into the reticuloendothelial organs, whereas, the same dosage given IT was basically not detected beyond the lung itself. PFC NP given IT did not impact rabbit behavior or impair respiratory function. PFC NP effects on cells in culture were negligible and when given IV or IT no changes in rabbit hematology nor serum clinical chemistry parameters were measured. Conclusion: IT delivery of PFC NP offered unique opportunity to locally deliver PFC NP in high concentrations into lung cancers with minimal extratumor systemic exposure., Competing Interests: Competing Interests: The authors have declared that no competing interest exists.

Autophagy serves a critical function in the pathogenesis, response to therapy and clinical outcome in cancers. Although a recent report showed a role of iASPP in suppressing autophagy, its potential activity as a regulator of autophagy has not been investigated in lung cancer. Here we investigated the potential function and molecular mechanism of iASPP in mediating autophagy in human non-small-cell lung cancer. Our data suggested that forced expression of iASPP triggered autophagic flux, while inhibition of iASPP suppressed autophagy at the autophagsome formation stage in vitro. Furthermore, in vivo overexpression of iASPP in SCID/NOD mice promoted tumorigenesis and autophagy, with an increase in the conversion from LC3-I to LC3-II. The effects of iASPP were mediated through activation of mTOR pathway. Finally, cytoplasmic iASPP expression was upregulated in lung cancer patients, and was identified as an independent poor prognostic factor for lung cancer-specific death in patient samples. Taken together, our data showed that iASPP could promote tumor growth by increasing autophagic flux, and iASPP could serve as a poor prognostic factor and a potential therapeutic target in lung cancer.

To identify specific circulating microRNAs that were associated with the lymphatic metastasis in lung cancer, we performed miRNA microarray analysis of lymph node with and without metastasis from five lung cancer patients. Top six differentially expressed miRNAs were selected for further validation. A training cohort of 26 patients with lung cancer was firstly recruited and the selected miRNAs in the plasma samples were investigated. miRNA-422a, with highest diagnostic accuracy in lymphatic metastasis was identified (AUC, area under the receiver operating characteristic curve, 0.744; 95%CI, 0.570-0.918). The diagnostic value of miR-422a was also demonstrated by a validation cohort of 51 lung cancer patients (AUC, 0.880; 95%CI, 0.787-0.972). Moreover, a high diagnostic value was also observed after integrated analysis of training and validation cohorts (AUC, 0.792; 95%CI, 0.688-0.896). The odds ratio of high miR-422a expression for lymphatic metastasis in lung cancer was 13.645 (95%CI, 2.677-69.553) after adjustment of the potential confounding factors. Furthermore, we predicted the target genes of miR-422a by combining the online database, miRcords, and the data from GEO and TCGA. Sixty-one target genes of miR-422a that might be involved in lymphatic metastasis in lung cancer were identified. And GO analysis suggested multiple target genes relatively concentrated in the biological processes of apoptosis, transport, and protein phosphorylation.

This study is to investigate the relationship between ectonucleoside triphosphate diphosphohydrolase 5 (ENTPD5) expression and lung cancer clinicopathological factors, and the impact of ENTPD5 on lung cancer cell functions. Lung cancer specimens and matched adjacent normal tissues were obtained from patients without any preoperative radiotherapy or chemotherapy. Knockdown of ETNPD5 expression led to significantly decreased lung cancer cell growth rate, markedly increased apoptosis and the ability to repair, and significantly reduced invasion. Gene chip tests showed that knockdown of ENTPD5 expression caused more Caspase expression. Quantitative real-time polymerase chain reaction showed that the Caspase 3 expression was significantly increased after the knockdown of ENTPD5. In addition, immunohistochemistry showed that the tumor growth marker, proliferating cell nuclear antigen, was significantly reduced in the knockdown model. Tumorigenicity assay and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay showed that the apoptosis of lung cancer cells was increased in the knockdown model. Our results suggest that ENTPD5 affects lung cancer apoptosis via Caspase 3 pathway, and can be potentially used to monitor prognosis or to guide appropriate therapeutic regimens.