Interpretable Machine Learning-Aided Optical Deciphering of Serum Exosomes for Early Detection, Staging, and Subtyping of Lung Cancer.

Lung cancer (LC) is the leading cause of cancer-related mortality worldwide, underscoring an urgent need for strategies that enable early detection and phenotypic classification. Here, we conducted a label-free surface-enhanced Raman spectroscopic (SERS) analysis of serum exosomes from 643 participants to elucidate the biochemical deregulation associated with LC progression and the unique phenotypes of different LC subtypes. Iodide-modified silver nanofilms were prepared to rapidly acquire SERS spectra with a high signal-to-noise ratio using 0.5 µL of patient exosomes. We performed interpretable and automated machine learning (ML) analysis of differential SERS features of serum exosomes to build LC diagnostic models, which achieved accuracies of 100% and 81% for stage I lung adenocarcinoma and its preneoplasia, respectively. In addition, the ML-derived exosomal SERS models effectively recognized different LC subtypes and disease stages to guide precision treatment. Our findings demonstrate that spectral fingerprinting of circulating exosomes holds promise for decoding the clinical status of LC, thus aiding in improving the clinical management of patients.

The diagnosis interval influences risk factors of mortality in patients with co-existent active tuberculosis and lung cancer: a retrospective study.

BACKGROUND: Previous studies reported that tuberculosis (TB) is associated with an increased risk of lung cancer or the survival and mortality of lung cancer. However, the impact of coexisting TB on the survival of lung cancer patients was controversial. We aimed to identify risk factors on the survival rate of patients with co-existent active TB and lung cancer. METHODS: One hundred seventy-three patients diagnosed with active TB and lung cancer from January 2016 to August 2021 in Shanghai pulmonary hospital were selected and divided into two groups (≤ 6 months, > 6 months) according to the diagnosis interval between active TB and lung cancer (the order of diagnosis is not considered). The clinical characteristics and survival were analyzed. Univariate and multivariate logistic regression analyses were used to identify the risk factors for overall survival (OS). RESULTS: One hundred seventy-three patients were diagnosed with lung cancer and active TB. The study population exhibited a median age of 64 years, with a majority of 81.5% being male, 58.0% of patients had a history of smoking. Among those involved, 93.6% had pulmonary TB, 91.9% were diagnosed with non-small cell lung cancer (NSCLC), 76.9% were Eastern Cooperative Oncology Group (ECOG) 0-2 and 12.7% were ECOG 3-4. We observed better survival in the > 6 months group compared with the ≤ 6 months group (hazard ratio [HR] 0.456, 95% confidence interval [CI]:0.234-0.889, P = 0.017). The 1-, 3-, and 5- year OS rates were 94.2%, 80.3%, and 77.6%, respectively, in the > 6 months group and 88.3%, 63.8%, and 58.5%, respectively, in the ≤ 6 months group. Surgery (HR 0.193, [95% CI, 0.038-0.097]; P = 0.046) and ECOG Performance Status (HR 12.866, [95% CI, 2.730-60.638]; P = 0.001) were independent prognostic factors in the > 6 months group. CONCLUSIONS: Patients diagnosed with lung cancer and active TB for more than half a year have a significantly better prognosis than those diagnosed within half a year. ECOG Performance Status and surgery might possibly affect the outcomes of patients with co-existent active TB and lung cancer.

Serum fingerprinting by slippery liquid-infused porous SERS for non-invasive lung cancer detection.

Lung cancer (LC) remains the most commonly diagnosed cancer. Timely diagnosis is crucial for improving the clinical outcomes of LC patients. Serum molecular patterns reflect the physiological and pathological status of individuals, and are promising as diagnostic targets for malignancies. Here, we report a spectroscopic method for the rapid identification of LC based on the label-free fingerprinting of clinical serum samples with slippery liquid-infused porous surface-enhanced Raman spectroscopy (SLIPSERS). We first demonstrate the capability of SLIPSERS for the delivery and preconcentration of serum molecules into the SERS hot spots from an evaporating liquid droplet, enabling the acquisition of vibrational fingerprints of serum molecules with only 1 µL of blood serum in minutes. The averaged SLIPSERS signals of the serum sample from a cohort of 33 LC patients and 23 healthy controls reveal both metabolic and biomacromolcular alterations under LC conditions. By analyzing the SLIPSERS data with chemometric methods, we further demonstrate that the SLIPSERS profiling of serum molecular patterns allows the reliable discrimination of LCs from healthy controls. Considering the ease of operation and high efficiency, our SLIPSERS-based serum biopsy method should hold great potential for non-invasive LC diagnosis.

DADS suppresses human esophageal xenograft tumors through RAF/MEK/ERK and mitochondria-dependent pathways.

Diallyl disulfide (DADS) is a natural organosulfur compound isolated from garlic. DADS has various biological properties, including anticancer, antiangiogenic, and antioxidant effects. However, the anticancer mechanisms of DADS in human esophageal carcinoma have not been elucidated, especially in vivo. In this study, MTT assay showed that DADS significantly reduced cell viability in human esophageal carcinoma ECA109 cells, but was relatively less toxic in normal liver cells. The pro-apoptotic effect of DADS on ECA109 cells was detected by Annexin V-FITC/propidium iodide (PI) staining. Flow cytometry analysis showed that DADS promoted apoptosis in a dose-dependent manner and the apoptosis rate could be decreased by caspase-3 inhibitor Ac-DEVD-CHO. Xenograft study in nude mice showed that DADS treatment inhibited the growth of ECA109 tumor in both 20 and 40 mg/kg DADS groups without obvious side effects. DADS inhibited ECA109 tumor proliferation by down-regulating proliferation cell nuclear antigen (PCNA). DADS induced apoptosis by activating a mitochondria-dependent pathway with the executor of caspase-3, increasing p53 level and Bax/Bcl-2 ratio, and downregulating the RAF/MEK/ERK pathway in ECA109 xenograft tumosr. Based on studies in cell culture and animal models, the findings here indicate that DADS is an effective and safe anti-cancer agent for esophageal carcinoma.

Identification of immune-related gene signature for non-small cell lung cancer patients with immune checkpoint inhibitors.

Background: The utilization of immune checkpoint inhibitors (ICIs) has become the established protocol for treating advanced non-small cell lung cancer (NSCLC). This work aimed to identify the immune-related gene signature that can predict the prognosis of NSCLC patients receiving ICI treatment. Methods: The ImmPort database was queried to obtain a list of immune-related genes (IRGs). Differentially expressed IRGs in NSCLC patients were identified using the TCGA database. RNA-seq data and clinical information from NSCLC patients receiving immunotherapy were obtained from the GEO database (GSE93157 and ////). A gene signature was generated through multivariate Cox and LASSO regression analyses. The prognostic value and function of this gene signature were thoroughly investigated using comprehensive bioinformatics analyses. Results: A total of 6 prognostic-related genes were identified from 617 differentially expressed genes, and two prognostic-related differentially expressed genes (CAMP and IL17A) were determined to construct gene signature. Our gene signature demonstrated superior performance compared to other clinicopathological parameters in predicting the prognosis of NSCLC patients receiving immunotherapy, with an area under the ROC curve (AUC) of 0.812. Furthermore, immune infiltration analysis indicated that the high-risk group was enriched with resting CD4 T cell memory, while the low-risk group showed a "hot" tumor microenvironment that promotes anti-tumor immunity in NSCLC patients. Conclusion: Gene signatures based on immune-related genes exhibited excellent indicator performance of prognosis and immune infiltration, which has the potential to be an effective biomarker for NSCLC with ICI treatment.

Prognostic role of dynamic changes in inflammatory indicators in patients with non-small cell lung cancer treated with immune checkpoint inhibitors-a retrospective cohort study.

Background: Immune checkpoint inhibitors (ICIs) have become one of the standard treatments for non-small cell lung cancer (NSCLC) patients without driver mutations. However, a considerable proportion of patients suffer from severe immune side effects and fail to respond to ICIs. As effective biomarkers, programmed cell death ligand 1 (PD-L1) expression, microsatellite instability (MSI), the tumor mutation burden (TMB) and tumor-infiltrating lymphocytes (TILs) require invasive procedures that place heavy physical and psychological burdens on patients. This study aims to identify simple and effective markers to optimize patient selection through therapeutic decisions and outcome prediction. Methods: This retrospective study comprised 95 patients with metastatic NSCLC who were treated with ICIs either as the standard of care or in a clinical trial. The following data were extracted from the medical records. The baseline and dynamic neutrophil-to-lymphocyte ratio (NLR) and platelet-to-lymphocyte ratio (PLR) were calculated in the present study. Responses were assessed by computed tomography (CT) imaging and classified according to the Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1 every 6-12 weeks during treatment. Results: In total, 95 patients were included in the present study. The median age of patients was 61 years, 83.2% (79/95) patients were male, 62.1% (59/95) were former or current smokers, 66.3% (63/95) had adenocarcinoma, 93.7% (89/95) had stage IV disease, and 87.4% were without molecular alterations. A higher overall response rate (ORR) and prolonged median progression-free survival (PFS) was observed in patients with a lower cycle 3 (C3) NLR [7.7 vs. 5.5 months, hazard ratio (HR): 1.70, 95% confidence interval (CI): 0.90-3.22; P=0.12] and derived NLR (dNLR) (8.2 vs. 5.6 months, HR: 1.67, 95% CI: 0.94-2.97; P=0.08). After two cycles of ICI treatment, patients who had an increased NLR, dNLR, and PLR had a lower ORR and an inferior median PFS than those with a decreased NLR (5.5 vs. 8.5 months, HR: 1.87, 95% CI: 1.09-3.21; P=0.02), dNLR (5.6 vs. 8.4 months, HR: 1.49, 95% CI: 0.87-2.57; P=0.15), and PLR (11.8 vs. 5.5 months, HR: 2.28, 95% CI: 1.32-3.94; P=0.003). Moreover, patients with both an increased NLR and PLR had a worse ORR and median PFS than those with either an increased NLR or PLR, or both an increased NLR and PLR (11.8 vs. 5.5 vs. 5.6 months, P=0.003). In addition, the dynamic changes in the PLR could serve as an independent predictive factor of PFS in NSCLC patients treated with ICIs. Conclusions: Elevated dynamic changes in the NLR and PLR were associated with lower response rates and shorter PFS in the patients with NSCLC treated with ICIs. Our results also highlight the role of dynamic changes in the PLR in identifying patients with NSCLC who could benefit from ICIs.

Activity-Based Self-Enriched SERS Sensor for Blood Metabolite Monitoring.

The monitoring of metabolites in biofluids provides critical clues for disease diagnosis and evaluation. Yet, the quantitative detection of metabolites remains challenging for surface-enhanced Raman spectroscopy (SERS) due to poor reproducibility in preparation and manipulation of SERS nanoprobes. Herein, we develop an activity-based, slippery liquid-infused porous surface SERS (abSLIPSERS) sensor for facile quantification of metabolites with unmodified naked metal nanoparticles (NPs) by integrating biocatalysis-boronate oxidation cascades with SLIPS-driven self-concentration and delivering. Upon mixing the target metabolite with a specific oxidase, a H2O2-sensitive phenylboronate probe, and the naked Au NPs, H2O2 produced from the biocatalytic reaction oxidizes the phenylboronate probe to phenol, resulting in a ratiometric SERS response. Meanwhile, the SLIPS enables the complete enrichment of molecules and NPs within an evaporating liquid droplet, delivering the probes to the SERS-active sites for Raman amplification. Compared with conventional SERS biosensors, abSLIPSERS avoids multistep synthesis and biofunctionalization of nanoprobes, which significantly simplifies the detection workflow and improves the reproducibility. The abSLIPSERS sensor also shows tunable dynamic range beyond 4 orders of magnitude and allows quantifying any other metabolites with specific enzymes. We demonstrate abSLIPSERS sensing of lactate, glucose, and choline in human serum for exploring energy metabolism in lung cancer. This study opens up a new opportunity for future point-of-care testing of circulating metabolites by SERS and will help to facilitate the translation of SERS bioanalysis to clinical settings.

Efficacy of immune checkpoint inhibitors in advanced non-small cell lung cancer harboring BRAF mutations.

Background: Despite immune checkpoint inhibitors (ICI) being widely used to treat patients with advanced non-small cell lung cancer (NSCLC), few studies examine the role of ICI in patients with proto-oncogene B-Raf, serine/threonine kinase (BRAF) mutations. Methods: A retrospective study was conducted for patients with BRAF-mutant NSCLC who received treatment at Shanghai Pulmonary Hospital between 2014 and 2022. Primary end point was progression-free survival (PFS). Secondary end point was best response (RECIST, version 1.1). Results: The study involved a total of 34 patients with 54 treatments recorded. The median PFS for the whole cohort was 5.8 months and the overall objective response rate (ORR) was 24%. Patients who were treated with ICI combined with chemotherapy reported a median PFS of 12.6 months and an ORR of 44%. Those who were treated with non-ICI therapy came with a median PFS of 5.3 months and an ORR of 14%. Specifically, patients had better clinical benefits with first-line ICI-combined therapy. The PFS was 18.5 months whereas that of non-ICI group was 4.1 months. The ORR was 56% in ICI-combined group and 10% in non-ICI cohort. Conclusions: The findings observed an evidential and significant susceptibility to ICIs combined therapy in patients with BRAF-mutant NSCLC, especially in first-line treatment.

Small cell lung cancer transformation and tumor heterogeneity after sequential targeted therapy and immunotherapy in EGFR-mutant non-small cell lung cancer: A case report.

Background: Histological transformation from non-small cell lung cancer (NSCLC) to small cell lung cancer (SCLC) is one of mechanisms of the acquired resistance to epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitors (TKI). However, SCLC transformation and tumor heterogeneity have never been reported in sequential targeted therapy and immunotherapy. Case presentation: Here, we described a patient with advanced EGFR-mutant NSCLC, who received erlotinib and underwent the resistance with EGFR T790M (-). The patient then received chemotherapy plus immunotherapy of programmed cell death 1 (PD-1) inhibitor, encountered progression with pathological transformation from NSCLC to SCLC that was overcome by chemotherapy of etoposide plus carboplatin (EC) with the main lesion significantly shrinking while metastatic nodules increasing. The pathology of the metastatic nodule showed NSCLC with EGFR T790M (+). Based on the tumor heterogeneity, EC chemotherapy combined with osimertinib was used, and patients responded well. The patient experienced four lung biopsies in all, which helped to provide the patient with precise treatment. Conclusions: This case suggested that SCLC transformation and tumor heterogeneity should be paid attention to when disease progression occurred in advanced NSCLC whether receiving targeted therapy or immunotherapy.

Iron Oxide Nanoparticles as Autophagy Intervention Agents Suppress Hepatoma Growth by Enhancing Tumoricidal Autophagy.

The combined treatment with nanoparticles and autophagy inhibitors, such as chloroquine (CQ) and hydroxychloroquine (HCQ), is extensively explored for cancer therapy. However, the toxicity of autophagy inhibitors and their unselective for tumoricidal autophagy have seriously hindered the application of the combined treatment. In this study, a carboxy-functional iron oxide nanoparticle (Fe2O3@DMSA) is designed and identified to significantly exert an antitumor effect without adding CQ or HCQ. Further investigation indicates that the effective inhibition effect of Fe2O3@DMSA alone on hepatoma growth is triggered by inhibiting the fusion of autophagosomes and lysosomes to enhance tumoricidal autophagy, which is induced by intracellular iron-retention-induced sustained reactive oxygen species (ROS) production. Furthermore, in two hepatoma-bearing mouse models, Fe2O3@DMSA alone effectively suppresses the growth of tumors without obvious toxic side effects. These studies offer a promising strategy for cancer therapy.

A Biomimetic Plasmonic Nanoreactor for Reliable Metabolite Detection.

Reliable monitoring of metabolites in biofluids is critical for diagnosis, treatment, and long-term management of various diseases. Although widely used, existing enzymatic metabolite assays face challenges in clinical practice primarily due to the susceptibility of enzyme activity to external conditions and the low sensitivity of sensing strategies. Inspired by the micro/nanoscale confined catalytic environment in living cells, the coencapsulation of oxidoreductase and metal nanoparticles within the nanopores of macroporous silica foams to fabricate all-in-one bio-nanoreactors is reported herein for use in surface-enhanced Raman scattering (SERS)-based metabolic assays. The enhancement of catalytical activity and stability of enzyme against high temperatures, long-time storage or proteolytic agents are demonstrated. The nanoreactors recognize and catalyze oxidation of the metabolite, and provide ratiometric SERS response in the presence of the enzymatic by-product H2O2, enabling sensitive metabolite quantification in a "sample in and answer out" manner. The nanoreactor makes any oxidoreductase-responsible metabolite a candidate for quantitative SERS sensing, as shown for glucose and lactate. Glucose levels of patients with bacterial infection are accurately analyzed with only 20 µL of cerebrospinal fluids, indicating the potential application of the nanoreactor in vitro clinical testing.

Author Correction: PLGA-based dual targeted nanoparticles enhance miRNA transfection efficiency in hepatic carcinoma.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Autophagy-deficient Kupffer cells promote tumorigenesis by enhancing mtROS-NF-κB-IL1α/ß-dependent inflammation and fibrosis during the preneoplastic stage of hepatocarcinogenesis.

As a cellular degradation mechanism, autophagy exerts crucial and complicated effects on HCC development. Liver non-parenchymal cells, including hepatic resident macrophage Kupffer cells, also play important roles in this process. However, most associated studies have focused on the influence of the autophagy level in hepatic cells and HCC cells, but not liver non-parenchymal cells. Based on our previous study, we confirmed that Atg5 silence in the liver during the preneoplastic stage facilitated liver fibrosis, inflammation and, ultimately, tumorigenesis. We further found that autophagy deficiency promotes the production of inflammatory and fibrogenic factors in macrophages. Moreover, Kupffer cell depletion rescued the tumor-promoting effect of autophagy deficiency during the preneoplastic stage. In autophagy-deficient macrophages, mitochondrial ROS mediated inflammation- and fibrosis-promoting effects by increasing IL1α/ß production via enhancing NF-κB-associated pathways. Both blocking of mitochondrial ROS and blocking the IL1 receptor stopped the promotion of fibrosis, inflammation and tumorigenesis resulting from Atg5 knockdown during the preneoplastic stage. In conclusion, autophagy-deficient Kupffer cells promote liver fibrosis, inflammation and, finally, hepatocarcinogenesis during the preneoplastic stage by enhancing mitochondrial ROS- NF-κB-IL1α/ß pathways.

PLGA-based dual targeted nanoparticles enhance miRNA transfection efficiency in hepatic carcinoma.

Hepatic carcinoma (HCC) is a lethal disease associated with high morbidity and poor prognosis. Recently years, gene therapies have offered novel modalities to improve the prognosis of HCC patients. MicroRNA-99a (miR-99a) is frequently down-regulated in HCC, where it acts as a tumor suppressor. Therefore, we constructed monomethoxy (polyethylene glycol)-poly(D,L-lactide-co-glycolide)-poly(L-lysine)-lactobionic acid- anti-vascular endothelial growth factor antibody (mPEG-PLGA-PLL-LA/VEGFab or PEAL-LA/VEGFab) nanoparticles (NPs) with highly specific targeting properties as carriers to restore the expression of miR-99a both in vitro and in vivo, to inhibit HCC progression. In vitro, PEAL-LA/VEGFab NPs showed more efficient delivery of miR-99a to HepG2 cells than the conventional transfection reagent LipofectamineTM2000 (Lip2000). The higher delivery efficiency associated with PEAL-LA/VEGFab NPs consequently resulted in down-regulation of target genes and suppression of the proliferation, migration and invasion of HepG2 cells. In vivo, miR-99a-PEAL-LA/VEGFab NPs inhibited tumor xenograft growth in HCC-bearing mice without causing obvious systemic toxicity. Our results demonstrate that PEAL-LA/VEGFab NPs selectively and effectively deliver miR-99a to HCC cells based on the double-targeting character of these nanoparticles, thereby offering potential for translation into effective clinical therapies for HCC.

Size-dependent cytotoxicity of Fe3O4 nanoparticles induced by biphasic regulation of oxidative stress in different human hepatoma cells.

The application of Fe3O4 nanoparticles (NPs) has made great progress in the diagnosis of disease and in the drug delivery system for cancer therapy, but the relative mechanisms of potential toxicity induced by Fe3O4 have not kept pace with its development in the application, which has hampered its further clinical application. In this article, we used two kinds of human hepatoma cell lines, SK-Hep-1 and Hep3B, to investigate the cytotoxic effects and the involved mechanisms of small Fe3O4 NPs with different diameters (6 nm, 9 nm, and 14 nm). Results showed that the size of NPs effectively influences the cytotoxicity of hepatoma cells: 6 nm Fe3O4 NPs exhibited negligible cytotoxicity and 9 nm Fe3O4 NPs affected cytotoxicity via cellular mitochondrial dysfunction and by inducing necrosis mediated through the mitochondria-dependent intracellular reactive oxygen species generation. Meanwhile, 14 nm Fe3O4 NPs induced cytotoxicity by impairing the integrity of plasma membrane and promoting massive lactate dehydrogenase leakage. These results explain the detailed mechanism of different diameters of small Fe3O4 NPs-induced cytotoxicity. We anticipate that this study will provide different insights into the cytotoxicity mechanism of Fe3O4 NPs, so as to make them safer to use in clinical application.