PurposeEpidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) have been widely used as the standard-of-care first-line treatment for EGFR-mutated non-small cell lung cancer (NSCLC) patients. However, EGFR-TKI resistance has become a major challenge for almost all patients with EGFR-mutated NSCLC. Both amivantamab (EGFR-MET bispecific antibody) and patritumab deruxtecan (HER3 antibody-drug conjugate) have shown promising efficacy in clinical trials for NSCLC resistant to osimertinib. This study aimed to evaluate a novel therapeutic strategy combining amivantamab and patritumab deruxtecan to overcome osimertinib resistance in NSCLC.MethodsThree osimertinib-resistant non-small cell lung cancer cell lines were established in vitro. Changes in relevant targets between pre- and post-resistance states were explored at the RNA and protein levels. Subsequently, the efficacy and safety of combination therapy were verified in vitro and in vivo respectively. Changes in treated mice immune microenvironment post-combination therapy were analyzed by flow cytometry, while bulk-RNA sequencing was conducted on tumor tissues.ResultsWe found that in vitro studies, when combined, amivantamab and patritumab deruxtecan both exhibited a synergistic effect on cell lines that were sensitive or resistant to Osimertinib, and the use of amivantamab increases the expression of HER3 in certain cell lines. Furthermore, the combination therapy polarized macrophages toward the M1 phenotype in vivo, thereby constructing an immune microenvironment unfavorable for tumor growth.ConclusionIn conclusion, we have proposed a new therapeutic strategy for NSCLC after osimertinib resistance. The combined strategy of amivantamab and patritumab deruxtecan highlight a promising therapeutic avenue, warranting future clinical trials to validate safety and efficacy.

AIMS:Lipid-associated macrophages are a specific subpopulation of macrophages that play a crucial role in cancer progression and treatment resistance. However, the functional impact of lipid-associated macrophages in lung adenocarcinoma (LUAD) remains poorly understood. This study aims to investigate the role and underlying mechanisms of lipid-associated macrophages in LUAD liver metastasis and resistance to osimertinib, a third-generation epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI). METHODS:Single-cell RNA sequencing (scRNA-seq) was performed on human lung tumor tissues from patients with primary LUAD and those with LUAD liver metastasis, which identified a novel subpopulation of stabilin-1 (STAB1)+ lipid-associated macrophages. The influence of STAB1+ lipid-associated macrophages on LUAD liver metastasis and osimertinib resistance was evaluated in vitro and in vivo. An in vitro co-culture system was established to investigate the interaction between LUAD cells and lipid-associated macrophages, and the mechanisms were analyzed by RNA-seq, Luminex multi-factor detection, Co-IP, in vivo, and rescue experiments. RESULTS:The subpopulation of STAB1+ lipid-associated macrophages was more abundant in liver metastatic LUAD tumors than in primary tumors. This lipid-associated macrophage subpopulation exhibited a stronger ability of lipid uptake from tumors and lipid droplet accumulation. We found that C-X-C motif ligand 12 (CXCL12) chemokine secreted by liver metastatic LUAD cells was responsible for recruiting circulating monocytes and subsequently inducing their differentiation into STAB1+ lipid-associated macrophages. STAB1 overexpression impaired the phagocytic ability of macrophage towards dying tumor cells by upregulating the signal regulatory protein α (SIRPα)-CD47 "don't eat me" signal. In tumor xenograft models, inhibition of STAB1+ lipid-associated macrophages effectively suppressed LUAD osimertinib resistance and liver metastasis. CONCLUSIONS:Our study demonstrates that STAB1+ lipid-associated macrophages contribute to LUAD liver metastasis and osimertinib resistance by impairing macrophage phagocytosis via the SIRPα-CD47 axis, opening a potential avenue for future research and treatment development.

INTRODUCTION:Acquired resistance to third-generation EGFR-tyrosine kinase inhibitors (EGFR-TKIs) is a major challenge in NSCLC, with approximately 50% of cases lacking precise resistance mechanisms. This study investigates the immunosuppressive tumor microenvironment (TME) driving resistance and develops a novel triple-combination therapy to restore T-cell antitumor activity. METHODS:Single-cell RNA sequencing and multicolor fluorescence staining were performed on NSCLC patient samples to analyze TME changes post-EGFR-TKI resistance. A triple therapy combining BC3448 (EGFR and CD3 bispecific T-cell engager), tucidinostat (histone deacetylase inhibitor), and WBP3425 (4-1BB agonist) was tested using in vitro co-culture assays, syngeneic cell-derived xenograft models in humanized NOG-EXL mice, and multi-omics analyses. RESULTS:Single-cell RNA sequencing revealed reduced T-cell infiltration or activation and increased immunosuppressive myeloid cells in resistant NSCLC. BC3448 monotherapy activated T cells and induced tumor cell apoptosis in vitro but was limited in vivo because of myeloid-driven immunosuppression. The triple therapy significantly enhanced tumor regression in osimertinib-resistant models (tumor growth inhibition >70%, p < 0.001), promoted CD8+ effector T-cell differentiation, and suppressed Tregs and M2 macrophages. CD40-CD40L axis activation between T cells and monocyte-derived macrophages was critical for TME remodeling, with spatial profiling revealing increased CD40L+ T-cell and CD40+ macrophage proximity, correlating with higher IFN-γ and reduced angiogenesis. A durable response to BC3448 monotherapy was observed in an immunotherapy-resistant patient with NSCLC (>2 y of stable disease), presenting a translational potential of this approach. CONCLUSIONS:This study establishes a novel triple therapy that overcomes the limitations of bispecific T-cell engagers in cold and immunosuppressive TMEs and provides an immunomodulatory approach to addressing third-generation EGFR-TKI resistance.

Pulmonary nodules are common. It is important to identify novel noninvasive biomarkers for the diagnosis and prognosis of malignant lung nodules. We retrospectively collected preoperative plasma samples at Shanghai Pulmonary Hospital and measured the 24 cytokines involved in the development of lung cancer using electrochemiluminescence multiplex assays. Univariable and multivariable analysis was utilized to estimate relationships between the cytokines with malignant nodules. The model was constructed by logistic regression analysis. The cytokines associated with postoperative recurrence were analyzed and validated in an independent cohort. Receiver operating characteristic (ROC) curve analysis was applied to evaluate the performance. The counts of circulating tumor cells identified by ligand-targeted PCR in the preoperative period failed to distinguish benign from malignant nodules. After adjustment for age, gender and smoking status, IL-4 and GM-CSF were associated with decreased odds of malignant nodules (OR 0.27, 95% CI 0.07–1.02; OR 0.20, 95% CI 0.05–0.75, respectively). The plasma cytokine panel (IL-17A, IL-4 and GM-CSF) was constructed for the preoperative diagnosis of malignant nodules with an AUC of 0.73. TSLP showed significantly differential expression between recurrence patients and recurrence-free patients (p < 0.05). The corresponding ROC curve yielded an AUC of 0.844 in the screening set and 0.847 in the validation set. Taken together, we constructed a plasma cytokine panel (IL-17A, IL-4 and GM-CSF) for the preoperative diagnosis of malignant nodules, and found that TSLP could be as a recurrence predictor in resected NSCLC patients.

Highly sensitive and specific detection of single nucleotide polymorphisms (SNPs) in genomic DNA is of great significance in disease diagnosis and personalized medicine. Here, we developed a fluorescent method for highly specific detection of SNPs in mixed-sequence KRAS double-stranded DNA (dsDNA) under physiological conditions by combining peptide nucleic acid (PNA)-directed padlock probe assembly with rolling circle amplification (RCA). The core of this approach involves the selective opening of dsDNA using an acridine-linked cyclopentane PNA (Acr-PNAcyp4), followed by hybridization of a padlock probe and subsequent RCA. The resulting RCA products are then detected through hybridization with PNA molecular beacon (PNA MB) probes. Making use of the high specificity of PNA-mediated strand invasion (PMSI) and ligation reactions in conjunction with the robust amplification capability of RCA technology, the method achieves precise detection of target dsDNA at concentrations as low as 0.66 fM, enabling effective discrimination among different mutation types. Furthermore, the approach has been successfully applied to the detection of KRAS mutations in colorectal cancer cells, confirming its practical utility in biological samples. This method does not require sophisticated thermal cycling equipment, highlighting its potential for use in clinical settings and resource-limited environments for the detection of low-abundance disease-related mutations.

Background MiRNA expression profiles may serve as valuable biomarkers for early cancer diagnosis. However, the detection of miRNA remains greatly challenging due to its shorter length and lower abundance in cells. Electrochemical biosensors based on DNA nanostructures have attracted significant attention due to their improved capture efficiency, high sensitivity, and ease of miniaturization, but the complex construction process, non-specific interactions among DNA probes, and their low stability continue to severely restrict their widespread application in clinical diagnostics. Therefore, developing robust and sensitive methods for miRNA analysis is still of great value. Results We describe an ultra-sensitive electrochemical biosensor for miRNA by integrating the PNA-DNA2 three-way junction (3WJ) nanostructure with target-recycling catalytic hairpin assembly (CHA) and hybridization chain reaction (HCR) dual cascade isothermal amplification. The target miRNA triggers CHA amplification, resulting in the generation of substantial quantities of double-stranded DNA (dsDNA) products. These dsDNA are subsequently captured by PNA probes immobilized on the electrode surface, leading to the formation of a densely packed layer of PNA-DNA2 3WJ nanostructure. Subsequently, the single-stranded termini of the two branches extending from 3WJ function as promoters to initiate the HCR, resulting in the formation of a layer of intricately intertwined branched long dsDNA molecules, which could adsorb substantial quantities of [Ru(NH3)6]3+, thereby significantly amplifying the electrochemical signal. This electrochemical biosensor exhibits exceptional sensitivity towards miRNA-21, achieving a detection limit as low as 2.9 aM while effectively discriminating single base mutations. Significance This represents the first electrochemical system for miRNA detection by integrating the PNA-DNA2 3WJ with CHA-HCR dual cascade isothermal amplification. The robust, specific and ultrasensitive feature makes cancer cell miRNA monitoring possible, suggesting its great application prospect as a promising sensing platform for monitoring various miRNA biomarkers in cancer diagnostics.

The phosphorylation of nucleic acids mediated by 5′-polynucleotide kinase (PNK) exerts a crucial regulatory function in a wide range of significant cellular activities. Nevertheless, the current approaches for detecting PNK require expensive labeled probes and complex instrumentation, making it impossible to achieve real-time, on-site, and rapid analysis. Here, we take T4 PNK as a model and establish a novel colorimetric strategy for the detection of PNK activity and its inhibition by means of a coupled enzyme-assisted cyclic strand displacement amplification (SDA) and peptide nucleic acid (PNA)-gold nanoparticle (AuNP) based platform. The inspiration for this innovative strategy comes from the high stability, strong binding ability, and potent regulatory effect of PNA probes on AuNPs. Under the catalysis of PNK, the 5′-hydroxyl end of the hairpin-shaped DNA (hpDNA) is initially phosphorylated and subsequently digested by λ exonuclease (λ exo). This results in the release of a single-stranded DNA, which serves as a triggering factor to initiate the strand displacement reaction (SDR). The replaced PNA probe adheres to the surface of AuNPs, inducing their aggregation and causing a remarkable color change. Meanwhile, the double-stranded SDR product releases the SDR trigger with the aid of a nicking enzyme, triggering the next round of the SDR cycle and achieving highly efficient and controllable signal amplification. This assay is simple to operate and does not require bulky and expensive instruments or complex labeled probes. Compared with existing colorimetric methods, the detection sensitivity has been greatly improved, reaching 3.52 × 10−4 U/mL. Additionally, the method has demonstrated satisfactory results when applied to intricate biological matrices and the screening of T4 PNK inhibitors. Therefore, the proposed strategy holds significant potential for real-time analysis, high-throughput detection, and PNK-related drug screening.

BackgroundLung adenocarcinoma (LUAD) represents the most common form of lung cancer, contributing to significant global mortality. Metabolic reprogramming in tumor cells has been increasingly recognized as a hallmark of tumorigenesis, contributing to an immunosuppressive microenvironment. Given the promising prediction value of metabolism-related genes in LUAD, this study aims to explore the role of MS4A7, a member of the MS4A gene family, in LUAD prognosis and immune microenvironment dynamics.MethodsA prognostic signature for LUAD was developed using the LASSO-Cox regression algorithm with RNA-seq data from 500 LUAD patients in The Cancer Genome Atlas database. Genes with differential expression linked to metabolic pathways were identified, and 20 genes were included to develop a risk signature. Further functional enrichment analysis was conducted to compare the biological pathways activated in high-risk versus low-risk groups. Single-cell RNA sequencing was employed to identify the expression profile and role of MS4A7 in different macrophage populations within the LUAD.ResultsThe constructed prognostic model displayed high predictive accuracy, outperforming single gene-based predictions. High-risk patients exhibited significantly poorer survival outcomes. Pathway enrichment analysis revealed dysregulated metabolic pathways in high-risk patients, including activation of glycolysis, mTORC1 signaling, and ROS production. Single-cell RNA sequencing revealed that MS4A7 expression was predominantly found in macrophage populations, with high expression localized in MS4A7+ macrophages. These macrophages exhibited distinct metabolic reprogramming and key immune functions, particularly in crosstalk with T cells and neutrophils.ConclusionThe MS4A7 gene plays a critical role in LUAD prognosis, particularly through its involvement in immune modulation within the TME. MS4A7+ macrophages, characterized by distinct metabolic reprogramming and immune interactions, are pivotal in shaping LUAD progression and immune response. The findings highlight the potential of MS4A7 as a novel prognostic biomarker and therapeutic target for LUAD. Further investigation into the metabolic and immune regulatory mechanisms of MS4A7+ macrophages could offer new insights into LUAD treatment strategies.

INTRODUCTION:Accumulation of regulatory T (Treg) cells, an immunosuppressive population, limits the efficacy of immunotherapy in NSCLC. C-C motif chemokine receptor 8 (CCR8) is selectively expressed in tumor-infiltrating Treg cells and is, therefore, considered an ideal target. METHODS:The efficacy and safety of anti-CCR8 monotherapy and its combination with programmed cell death protein-1 (PD1) inhibitor were evaluated in four NSCLC-bearing mice models. To track the dynamic changes in tumor microenvironment, we performed the single-cell RNA sequencing, the single-cell T-cell receptor sequencing analysis, the flow cytometry, the multi-color immunofluorescence, and the Luminex assay on tumors after three, seven, 14, and 21 days of different treatment regimens. Then, in vitro and in vivo experiments were applied to validate our findings and explore molecular mechanisms of the synergistic effects. RESULTS:Across four NSCLC-bearing mice models, the combination of CCR8 antibody and PD1 inhibitor significantly reduced tumor growth (p < 0.05) without obvious mouse body weight drops and systemic cytokine storm. The anti-CCR8 therapy synergizes with PD1 blockade by remodeling the tumor microenvironment and disrupting CCR8+Treg-C-C motif chemokine ligand 5 (CCL5)+ dendritic cells (DC) interaction. Mechanistically, therapeutic depletion of CCR8+Treg cells combined with PD1 inhibitor extremely increased interleukin-12 secretion by the Janus kinase-signal transducer and activator of transcription (JAK-STAT) pathway activation on CCL5+ DCs, thereby promoting cytotoxic activity of CD8+ T cells. The therapeutic potential of the CCR8 antibody LM-108 in combination with immunotherapy was observed in clinical patients with advanced NSCLC. CONCLUSION:Overall, CCR8 expression on tumor-infiltrating Treg cells is correlated with immunosuppressive function on DCs and CD8+ T cells, thus impeding antitumor immunity.

e20669 Background: Immune checkpoint inhibitors (ICIs) pushed therapeutic strategy of NSCLC into a new era. Nevertheless, the clinical benefit of ICI therapy in EGFR-driven NSCLC patients, especially patients resistant to EGFR-TKI was unsatisfactory. Immunosuppressive tumor microenvironment (TME) after EGFR TKI treatment proved huge influence on the efficacy of ICIs. Hence, investigation of the mechanism underlying the formation of suppressive TME and exploration of its potential intervention was the urgent requirement for EGFR-TKI resistant NSCLC research. Methods: ZEB2 expression in human NSCLC cell lines and NSCLC patients’ samples were assessed utilizing qRT-PCR, western blotting, and immunohistochemistry (IHC). The effects of EGFR-TKI resistance and ZEB2 on TAMs polarization were investigated via qRT-PCR analysis of M1/M2-like phenotypic biomarkers and flow cytometry. The modulation of ZEB2 on cytokines’ secretion was evaluated through qRT-PCR, ELISA and MSD electrochemiluminescence. The direct influence of ZEB2 on promoter regions of cytokines was explored using dual luciferase reporter assay. The regulation of signaling pathway on ZEB2 was analyzed by correlative analysis using LUAD data from TCGA database, the results were then consolidated using western blotting. CDX mouse models, established by injecting PC9 and HCC827 cells after intervention subcutaneously into the BALB/c nude mice, were used to further confirm the influence of EGFR-TKI resistance and ZEB2 on TAMs polarization. Results: It was elucidated that EGFR-TKI resistance induced M2 polarization and inhibited M1 polarization of TAMs, which was critical for the formation of immunosuppressive TME. ZEB2 was found to be upregulated in EGFR-TKI resistant NSCLC in vitro, in vivo and in silicon. Based on these results, ZEB2 upregulation could induce M2 polarization and impede M1 polarization of TAMs, demonstrating its essential role in the influence of EGFR-TKI resistant NSCLC on TAMs polarization. Besides, ZEB2 overexpression was dependent on PI3K-Akt signaling pathway which was upregulated after EGFR-TKI resistance in NSCLC. Finally, the mechanism underlying ZEB2’s regulation on TAMs polarization was proved to be associated with cytokines’ secretion. Apart from its inducement on TGF-β1 secretion, ZEB2 could directly bind to the promoter region of CSF-1 to elevate its secretion. Conclusions: ZEB2, which was upregulated in EGFR-TKI resistant NSCLC on a PI3K-Akt signaling pathway dependent manner, could induce M2 polarization and hinder M1 polarization via elevating the secretion of CSF-1 and TGF-β1. Devoting to improving the efficacy of ICI therapy, ZEB2 could become a potential target for intervention to modulate TME after the development of EGFR-TKI resistance.

PD-1 blockade plus chemotherapy has become the first-line standard of care for patients with advanced non-small-cell lung cancer (NSCLC) without oncogenic drivers. Oncogenic-driven advanced NSCLC showed limited response to PD-1 blockade monotherapy or chemotherapy alone. Whether NSCLC patients with oncogenic drivers could benefit from PD-1 blockade plus chemotherapy remains undetermined. Three hundred twelve NSCLC patients with at least one oncogenic driver alteration received PD-1 plus chemotherapy or each monotherapy were retrospectively identified. Objective response rate (ORR), progression-free survival (PFS), and overall survival (OS) were compared to evaluate the therapeutic outcomes differences among patients with different oncogenic drivers. One hundred sixty-two patients received PD-1 blockade plus chemotherapy, 57 received PD-1 blockade monotherapy and 93 received chemotherapy alone were included. Oncogenic driver mutations including KRAS (31.4

Recurrent Clostridioides difficile infection (rCDI) is a global health threat that has received considerable attention. Berberine (BBR), a natural pentacyclic isoquinoline alkaloid, has been used as a cost-effective treatment for intestinal infections in Asia for many years. However, the effect of BBR on rCDI is not clear. The efficacy and underlying mechanisms of BBR were evaluated in a vancomycin-dependent rCDI mouse model and an intestinal organoids model. The study findings showed that BBR treatment alleviated the severity of infection and increased survival rate in rCDI mice. Mechanistically, BBR alleviated intestinal epithelial damage with higher Occludin expression, suppressed some inflammatory pathways and reduced the level of inflammatory factors in both the caecum and serum. Moreover, 16S rRNA sequencing analysis indicated that BBR reshaped the gut microbiota by increasing the abundance of Firmicutes and reducing the abundance of Proteobacteria. At genus level, BBR treatment increased levels of Blautia and Bilophila, and reduced levels of Proteus. In addition, acetic acid, one of the short-chain fatty acids (SCFAs), was also increased after BBR treatment in rCDI mice. Collectively, BBR exerted a protective effect in rCDI via multiple underlying mechanisms and is a potential drug candidate for alleviating rCDI, but further research is needed in this area.

Toehold-mediated strand displacement (TMSD) forms the molecular basis of most dynamic DNA nanodevices to date and provides one of the most promising strategies for microRNA (miRNA) profiling at the single cell level. However, traditional TMSD methods are susceptible to false positive identification of wild-type templates for miRNA variants with small thermodynamic differences. Furthermore, the low detection sensitivity and poor stability in the biological environment also bring significant challenges in applying this technology to single-cell miRNA analysis. Herein, we develop a peptide nucleic acid (PNA)-based blocker displacement amplification (PBDA) system that utilizes TMSD and in situ rolling circle amplification (RCA) strategies to enable highly specific and sensitive in situ imaging of single-cell miRNA. PNA, due to its exceptional stability, strong affinity, and sequence selectivity for complementary strands, can serve as an effective blocker to bind with the RCA template. This significantly enhances the detection system’s ability to discriminate single-base mismatches and improves its capability to identify specific target miRNAs. Moreover, in situ target-primed RCA significantly enhances the detection sensitivity of the detection system towards the target miRNA, achieving a detection limit as low as 0.18 fM. Therefore, this study presents a robust and efficient solution for the in-situ analysis of single-cell miRNAs.

BACKGROUND:The pathogenesis of neuropathic pain is complex and lacks effective clinical treatment strategies. Medical plants and herbal extracts from traditional Chinese medicine with multi-target comprehensive effects have attracted great attention from scientists. PURPOSE:To investigate the pharmacological active components and mechanism underlying the anti-neuralgia effect of classic analgesic formulas Duhuo Jisheng Mixture (DJM). STUDY DESIGN AND METHODS:Chronic Constriction Injury (CCI) surgery was used to assess the efficiency of DJM in treating neuropathic pain. Forty-two C57BL/6 mice were evenly divided into seven groups: sham-operated, sham treatment with DJM (20 ml/kg), CCI treatment with DJM (0, 10, 20, or 40 ml/kg) and CCI treatment with Morphine (3 mg/kg). DJM irrigation stomach was implemented from post-operative day (POD) 0 to POD 21. Paw withdrawal threshold and Thermal latency were conducted on POD 0, 1, 3, 5, 7, 14 and 21. On POD 14, eEPSP in spinal dorsal horn neurons was recorded by patch-clamp. mRNA transcription in spinal cord was also monitored to screen molecular targets and signaling pathways on POD 14. To further validate the influence of DJM on the S1PR1 signaling pathway, 1 mg/kg W146 was infused into mice intrathecally and delivered to the cerebral spinal fluid through one 30 G needle with a whole 20 μl intervertebral between the L5 and L6. Furthermore, the active ingredients of DJM were identified through LC/MS equipment and predicted through Molecular Docking. RESULTS:We demonstrated that DJM treatment reversed CCI-induced pain sensitivity and the excitability of neurons, decreased up-regulation of astrocyte S1PR1, inhibited astrocyte activation, and further inhibited the expression of Purinergic P2Y1R and its downstream molecule p-JNK in mouse spinal cord, as well as the release of inflammatory factors. Interestingly, due to the regulatory role of astrocytes on neurons, the effects of DJM on astrocytes ultimately manifest in pain-effector neurons, resulting in decreased p-ERK, p-CREB, and pain-marking protein c-fos in neurons. S1PR1 Antagonist W146 possessed an equivalent analgesic effect as DJM and inhibited S1PR1 and c-fos expression. According to LC/MS analysis results, a total of 33 active ingredients were screened, of which 20 active ingredients had good binding activity with S1PR1 and were considered to be the main active ingredients for analgesia. CONCLUSION:This study is the first to clarify the effect and molecular mechanism of DJM for anti-neuralgia in particular, which confirms the clinical value of DJM in relieving neuropathic pain. Furthermore, this study innovatively identifies the potential pharmacological components of DJM through LC/MS and Bioinformatics technology, which forms a framework for people's understanding of DJM treatment for neuropathic pain and provides a sufficient theoretical basis of DJM for clinical application.

Fluorescent silver nanoclusters, particularly DNA-templated silver nanoclusters (DNA-AgNCs), have garnered significant attention owing to their tunable fluorescence properties and excellent biocompatibility. Here we demonstrate that thiol-functionalized peptide nucleic acid (SH-PNA) with mixed base composition can significantly enhance the fluorescence emission intensity of C12-AgNCs. This fluorescence enhancement induced by PNA can be effectively modulated through the hybridization of PNA with specific DNA sequences. Based on this finding, we have developed a fluorescence biosensor for sensitive and specific detection of DNA utilizing PNA-AgNCs probes, which enables highly efficient detection of single-base mutations in the TP53 gene fragment. Under optimal conditions, the fluorescence of the PNA-AgNCs probe exhibits a good linear relationship with the concentration of target DNA, achieving a detection limit of 1.3 nM and demonstrating inherent high specificity for single-base mutations. Regardless of the mismatch type, this method allows for the screening of mutant genes within 45 min. Furthermore, this detection strategy is operationally simple and has been successfully applied to the analysis of single-base mismatches in TP53 DNA from NCI-H661 lung cancer cells. In principle, by simply modifying the sequence of the PNA probe, this detection strategy can be readily extended to the detection of other nucleic acids.

Background and Objective:Pulmonary sarcomatoid carcinoma (PSC) accounts for approximately 0.5% of non-small cell lung cancer (NSCLC) cases and is thus a rare subtype. Highly aggressive and hard to detect early, PSC responds poorly to surgery, radiotherapy, and chemotherapy. Therefore, this review aims to synthesize current evidence on its pathogenesis and emerging therapeutic strategies, to improve clinical management. Methods:We searched PubMed for original studies, reviews, clinical trials, and case reports on PSC published until 2025. Moreover, data from ClinicalTrials.gov and major academic conference proceedings were examined for inclusion in this narrative review. Key Content and Findings:The core pathophysiology of PSC is epithelial-mesenchymal transition (EMT), a process that drives biphasic differentiation of tumor cells and remodels the tumor microenvironment (TME), thereby promoting high invasiveness and treatment resistance. Therapeutically, although targetable mutations such as MET exon 14 skipping are relatively frequent in PSC, the efficacy of targeted agents is generally inferior to that for other NSCLC subtypes. Notably, the tumor immune microenvironment of PSC features significant immune cell infiltration and high programmed death-ligand 1 (PD-L1) expression, leading to a generally better response to immune checkpoint inhibitors (ICIs). Consequently, immunotherapy combined with chemotherapy or antiangiogenic agents has emerged as a productive therapeutic strategy. Conclusions:Precision therapy for PSC, particularly immunotherapy-based combination strategies, has demonstrated transformative potential. However, further efforts in this field should involve clarifying the relevant EMT and tumor heterogeneity mechanisms, optimizing existing treatment regimens, and conducting targeted clinical trials, as these measures may advance individualized precision therapy for patients with PSC and improve their outcomes.

Central and peripheral extensive-stage small-cell lung cancer (ES-SCLC) are reported to be two distinct tumor entities, but their responses to the front-line therapies and underlying biological mechanisms remain elusive. In this study, we first compared the outcomes of central and peripheral ES-SCLC receiving front-line chemotherapy or chemo-immunotherapy with a cohort of 265 patients. Then we performed single-cell RNA sequencing (scRNA-seq) on nine treatment-naïve ES-SCLC samples to investigate potential mechanisms underlying the response differences. Under chemotherapy, the peripheral type had a lower objective response rate (44.8% vs. 71.2%, p = 0.008) and shorter progression-free survival (median 3.4 vs. 5.1 months, p = 0.001) than the central type. When comparing chemo-immunotherapy with chemotherapy, the peripheral type showed a greater potential to reduce progression (HR, 0.18 and 0.52, respectively) and death (HR, 0.44 and 0.91 respectively) risks than the central type. Concerning the scRNA-seq data, the peripheral type was associated with chemo-resistant and immune-responsive tumoral and microenvironmental features, including a higher expression level of MYC-Notch-non-neuroendocrine (MYC-Notch-non-NE) axis and a more potent antigen presentation and immune activation status. Our results revealed that central and peripheral ES-SCLC had distinct responses to front-line treatments, potentially due to differential activation statuses of the MYC-Notch-non-NE axis.

Background:BIRC5, also known as survivin, is the smallest but functionally most complex member of the inhibitor of apoptosis protein (IAP) family and plays an important role in tumorigenesis, recurrence, and chemoresistance, this study aimed to investigate its impact on the clinical and tumor microenvironmental features of lung adenocarcinoma (LUAD), together with its prognostic values. Methods:Clinical and transcriptomic data of 535 LUAD samples, 59 normal lung, and 54 patients with non-small cell lung cancer (NSCLC) received immune checkpoint blockades (ICBs) were analyzed. Immune infiltration analysis was conducted to uncover the relationship between tumor microenvironmental features and BIRC5 expression level. The prognostic values of BIRC5 were also evaluated with log-rank test and Cox regression analysis. Results:LUAD had a significantly higher BIRC5 expression level than normal lung tissues. The elevated BIRC5 expression was markedly associated with unfavorable clinical outcomes. Transcriptomic and single-cell sequencing data analysis revealed that tumors with high BIRC5 expression was correlated with multiple pathways' enrichment. Immune infiltration analysis indicated a negative correlation between BIRC5 expression and infiltration levels of CD8+ T cells, dendritic cells (DCs), and natural killer (NK) cell in LUAD, but a positive correlation was observed between BIRC5 expression and regulatory T cells (Tregs) infiltrations. Importantly, NSCLC patients received ICB with high BIRC5 expression had dramatically shorter progression-free survival (PFS; 1.2 vs. 4.5 months; P=0.01) and overall survival (OS; 3.1 vs. 12.7 months; P=0.005) than those with low BIRC5 expression. Conclusions:These findings suggested that high BIRC5 expression was associated with DNA damage/repair, cell invasion and proliferation related pathways enrichment and increased Tregs infiltration, which would result in inferior outcomes in NSCLC received ICB.

Aim: Immune checkpoint inhibitors (ICIs) have revolutionized the treatment approach for NSCLC. However, the effectiveness of ICI therapy in patients with EGFR-driven NSCLC, particularly those resistant to EGFR-TKI, has been disappointing. The immunosuppressive tumor microenvironment (TME) following EGFR-TKI therapy has been proved to significantly affected the effectiveness of ICIs. Therefore, studying the mechanism behind the development of a suppressive TME and exploring potential interventions is crucial for research on EGFR-TKI-resistant NSCLC. Methods: ZEB2 levels were quantified in human NSCLC cell lines and in tumor specimens from NSCLC patients by quantitative RT-PCR (qRT-PCR), WB, and immunohistochemical staining. To examine how ZEB2 affected macrophage polarization, M1/M2 marker profiles were measured with qRT-PCR and flow cytometry. Changes in cytokine production triggered by altered ZEB2 expression were determined with qRT-PCR, ELISA, and Meso Scale Discovery electrochemiluminescence assays. The direct binding of ZEB2 to cytokine-gene promoters was tested using a dual-luciferase reporter system. Upstream regulatory pathways were investigated by correlating LUAD transcriptomic data from TCGA with ZEB2 expression and validating key findings via western blotting. Finally, cell-derived xenograft (CDX) models were generated by subcutaneously implanting pre-treated PC9 or HCC827 cells into BALB/c nude mice to verify the impact of EGFR-TKI resistance and ZEB2 on tumor-associated macrophage (TAM) polarization in vivo. Results: It was elucidated that EGFR-TKI resistance upregulated the M2 polarization biomarkers, Arg-1 (PC9-GR: P < 0.01; HCC827-GR: P < 0.05) and IL4 (PC9-GR: P < 0.01; HCC827-GR: P < 0.01), while downregulated the M1 polarization biomarkers, TNF-α (PC9-GR: P < 0.01; HCC827-GR: P < 0.01), IL1β (PC9-GR: P < 0.01; HCC827-GR: P < 0.01), and IL6(PC9-GR: P < 0.001; HCC827-GR: P < 0.001) in NSCLC cell lines. Meanwhile, CD206+ TAMs (PC9-GR: P < 0.05; HCC827-GR: P < 0.01) were increased and CD86+ TAMs (PC9-GR: P < 0.05; HCC827-GR: P < 0.05) were decreased in both EGFR-TKI-resistant mice models. Apart from the formation of suppressive TME, ZEB2 was found to be upregulated in PC9-GR (qRT-PCR: P < 0.0001; WB: P < 0.05) and HCC827-GR (qRT-PCR: P < 0.0001; WB: P < 0.05) cells. The same trend was also noticed in clinical samples, with ZEB2 upregulated after gefitinib resistance in NSCLC patients (P < 0.0001). Based on these findings, ZEB2 knockdown was proved to downregulate Arg-1 (PC9-GR: P < 0.01; HCC827-GR: P < 0.05) and IL4 (PC9-GR: P < 0.01; HCC827-GR: P < 0.001), while upregulate the TNF-α (PC9-GR: P < 0.0001; HCC827-GR: P < 0.0001), IL1β (HCC827-GR: P < 0.001), and IL6 (PC9-GR: P < 0.01; HCC827-GR: P < 0.001), indicating its role in M1/M2 polarization in both EGFR-TKI-resistant NSCLC cell lines. The downregulation of CD206+ TAMs (PC9-GR: P < 0.05; HCC827-GR: P < 0.01) and the upregulation of CD86+ TAMs (PC9-GR: P < 0.001; HCC827-GR: P < 0.05) also demonstrated the reversion of suppressive TME after ZEB2 knockout in EGFR-TKI-resistant mice models. Additionally, after the intervention of MK2206, which was an Akt inhibitor, ZEB2 expression was suppressed at both low (PC9-GR: P < 0.001; HCC827-GR: P < 0.001) and high concentrations (PC9-GR: P < 0.001; HCC827-GR: P < 0.0001). Finally, the mechanism underlying ZEB2's regulation on TAM polarization was proved to be associated with cytokine secretion. According to the results of ELISA, apart from its inducement on TGF-β1 secretion (PC9-GR: P < 0.0001; HCC827-GR: P < 0.0001), ZEB2 could directly bind to the promoter region of CSF-1 to elevate its secretion (PC9-GR: P < 0.0001; HCC827-GR: P < 0.0001). Conclusion: In EGFR-TKI-resistant NSCLC, activation of the PI3K-Akt cascade drove a marked rise in ZEB2 expression. The elevated ZEB2 increased CSF-1 and TGF-β1 release, steering macrophages toward an M2 phenotype while impeding M1 polarization. Accordingly, suppressing ZEB2 had the potential to reshape the TME and enhance the effectiveness of ICIs once EGFR-TKI resistance had emerged.