Figure S4. ANXA3 has no effect on ferroptosis in macrophages.

Benzo[a]pyrene (BaP), a ubiquitous environmental carcinogen predominantly found in tobacco smoke and air pollution, plays a pivotal role in head and neck squamous cell carcinoma (HNSC) pathogenesis. However, the molecular networks governing BaP-induced toxicity and programmed cell death (PCD) mechanisms in HNSC remain poorly characterized, limiting the development of targeted therapeutic interventions. We systematically analyzed PCD patterns in HNSC using the GSE30784 dataset and identified BaP toxicity targets through comprehensive database mining. Machine learning algorithms, including RF and SVM, were employed to identify core toxic targets. Target validation was performed using the TCGA-HNSC cohort, followed by the construction of a prognostic nomogram. Mechanistic insights were obtained through spatial transcriptomics, single-cell RNA sequencing (scRNA-seq), gene set enrichment analysis (GSEA), molecular docking, and molecular dynamics simulations to elucidate BaP-protein interactions. We identified ten distinct PCD modes significantly dysregulated in HNSC compared to normal tissues. Among 260 putative BaP targets, 24 demonstrated significant associations with PCD pathways in HNSC. Machine learning analysis revealed SERPINE1, TNFRSF10B, and STK3 as core mediators of BaP toxicity, with SERPINE1 emerging as the predominant driver of BaP-induced cellular dysfunction. The integrated nomogram achieved robust performance in cancer risk stratification. Spatial transcriptomic analysis demonstrated preferential enrichment of these targets in malignant epithelial cells, while scRNA-seq revealed cell type-specific expression patterns. GSEA identified enrichment in apoptotic signaling, TGF-β pathway activation, and DNA damage response mechanisms. Molecular docking studies revealed high-affinity binding interactions, with molecular dynamics simulations confirming stable BaP-protein complexes. This integrative multi-omics analysis elucidates the complex molecular architecture underlying BaP-induced toxicity in HNSC, establishing SERPINE1 and STK3 as promising prognostic biomarkers and potential therapeutic targets. Our findings provide mechanistic insights into environmental carcinogen-mediated HNSC pathogenesis and offer a rational framework for developing precision medicine approaches targeting BaP-associated malignancies.

Figure S3. TAMs-derived-exo-ANXA3 inhibits ferroptosis in LSCC cells.

BACKGROUND:Prehypertension (Pre-HTN) is highly prevalent and substantially increases the risk of developing hypertension and cardiovascular disease. Gut microbiota (GM) dysbiosis and altered lipid metabolism are increasingly recognized as critical regulators of blood pressure (BP). Traditional Chinese Medicine (TCM) formulas, such as Qianyang Yuyin Granules (QYYY), offer multi-target interventions, yet their preventive mechanisms in Pre-HTN remain unclear. PURPOSE:This study aimed to investigate the antihypertensive effects of QYYY and elucidate its underlying mechanisms in a prehypertensive rat model. METHODS:Prehypertensive spontaneously hypertensive rats (SHRs) were treated with QYYY for four weeks. Multi-omics analyses, including metagenomics, plasma metabolomics, and transcriptomics, were conducted. Causal involvement of GM was tested using antibiotic-induced pseudo-germ-free SHRs with fecal microbiota transplantation (FMT) from QYYY-treated donors, administered alone or in combination with QYYY. Gut barrier integrity, systemic inflammation, and vascular function were evaluated by histology, immunofluorescence, transmission electron microscopy, and ELISA. RESULTS:QYYY significantly lowered SBP and DBP, reversed GM dysbiosis, normalized the Firmicutes/Bacteroidetes ratio, and modulated differential bacteria including Frisingicoccus and Blautia. These microbial shifts correlated with restoration of lysophosphatidylethanolamines (LPEs), inversely associated with BP, revealing a GM-lipid-BP axis. FMT alone was insufficient, whereas the combination of FMT+QYYY produced the strongest antihypertensive effect, restoring intestinal barrier integrity, enhancing ZO-1 expression, and normalizing Ang-II and NO levels. Transcriptomic analyses suggested PPAR and ROS signaling pathways as potential mechanisms mediating the antihypertensive effect of QYYY. CONCLUSION:QYYY prevents BP elevation in Pre-HTN via synergistic microbiota-dependent and independent mechanisms, offering a comprehensive strategy for early hypertension prevention.

Supplementary Tables S1-S5 and Data

OBJECTIVE:To investigate the effect of Qianyang Yuyin granule (QYYY) on AngII-induced hypertensive cardiac remodeling, focusing on the role of pyruvate kinase isozyme M2 (PKM2) mediated glycolysis. METHODS:A hypertensive mouse model was established in male C57BL/6 mice by continuous infusion of angiotensin II (AngII; 1000 ng·kg-1·min-1). Mice were administered varying doses of QYYY, with sacubitril/ valsartan (Sac/Val) serving as the positive control. Parameters evaluated included blood pressure, cardiac function, hypertrophy, fibrosis, inflammation, and apoptosis. The metabolic profile of myocardial tissue was analyzed using ultra performance liquid chromatography tandem mass spectrometry. Additionally, the involvement of the hypoxia-inducible factor 1-alpha (HIF-1α)/PKM2 signaling pathway was examined by Western blotting and immunohistochemistry. RESULTS:QYYY significantly lowered blood pressure, attenuated cardiac hypertrophy and fibrosis, reduced serum levels of inflammatory factors tumor necrosis factor-α and tumor necrosis factor-β, and decreased activation of phospho-NF-kappa B p65 pathway in cardiac tissue of hypertensive mice. Metabolomic analysis indicated that QYYY ameliorated cardiometabolic dysfunction, primarily associated with energy and amino acid metabolism, involving modulation of the HIF-1α/PKM2-mediated glycolytic pathway. CONCLUSION:QYYY effectively improves cardiac remodeling in hypertensive mice, potentially through inhibition of the PKM2-mediated glycolytic signaling pathway.

Figure S1. Proteome profiling in LSCC tissue.

Figure S2. ANXA3 promotes M2-like polarization of macrophages by activating the AKTGSK3β-β-catenin pathway.

Pancreatic ductal adenocarcinoma (PDAC) remains a formidable challenge due to its late diagnosis and intrinsic treatment resistance, exacerbates by its development from chronic inflammation to cancer transition (ICT). Here, this investigation aims to develop and evaluate ABSi-148, a novel near-infrared (NIR) agent targeting hypoxic carbonic anhydrase IX (CA IX), for its potential applications in ICT imaging and even PDAC treatment. ABSi-148 is synthesized from 4-(2-Aminoethyl) benzene sulfonamide (ABS), a sulfonamide derivative, conjugating with MHI-148 dye with merits of exceptional NIR-emitting traits, high biocompatibility, and deep tissue penetration imaging capability. It selectively accumulates in CoCl2-induced pancreatic stellate cells and pancreatic cancer cells via binding with transmembrane CA IX in vitro. Meanwhile, ABSi-148 effectively visualizes the early pancreatic lesion, and its long-term administration inhibits the progression of hypoxia-related fibrosis involved in pancreatic intraepithelial neoplasias (PanINs), and even PDAC progression in vivo. Besides, ABSi-148 monitors treatment efficacy and localizes hypoxic tumor regions, enhancing survival in tamoxifen combined with caerulein-induced KPC mice. Overall, ABSi-148 emerges as a theranostic NIR agent for precise diagnosis and targeted therapy in ICT of PDAC, promising to alleviate tumor progression and enhancing outcomes.

ABSTRACT:Integration of torque teno mini virus (TTMV) generating the TTMV::RARA (retinoic acid receptor α) fusion represents a newly recognized subtype of acute promyelocytic leukemia (APL) that merits detailed investigation. We present, to our knowledge, the first comprehensive characterization of its epidemiologic profile, clinical presentation, virologic characteristics, and underlying molecular mechanisms. Our findings indicate that TTMV::RARA is more prevalent in pediatric patients and represents the second most common retinoic acid receptor fusion after PML::RARA. Affected patients exhibit a high incidence of extramedullary involvement, particularly myeloid sarcoma. Cytogenetic abnormalities involving i(17)(q10) or 7q22 were identified in 52.0% of cases, largely in a mutually exclusive manner. Co-occurring mutations in epigenetic regulators were present in 76.9% of patients. Although most patients achieved initial remission, relapse was common and associated with rapid acquisition of all-trans retinoic acid (ATRA)-resistant mutation and secondary chemoresistance. Venetoclax-containing regimens demonstrated encouraging clinical efficacy. Phylogenetic analysis indicated that patient-derived TTMV strains clustered into a distinct clade. TTMV integration consistently occurred within RARA intron 2, involving a consensus fragment of 510 to 610 base pairs encompassing the viral promoter and open reading frame 2 (ORF2) N terminus, likely mediated by microhomology-driven recombination. Tandem RUNX1-binding motifs within the integrated viral promoter may underlie the myelotropism of these TTMV strains and facilitate transcriptional activation of TTMV::RARA. The chimeric protein retains at least the first 56 N-terminal residues of ORF2 and remains transcriptionally responsive to pharmacological concentrations of ATRA. These findings establish TTMV::RARA-APL as a distinct leukemia entity, laying the foundation for future studies on virus-mediated leukemogenesis and therapeutic strategies.

Immune checkpoint inhibitors (ICIs) have significant therapeutic effects but can also cause fatal lung injury. However, the lack of mouse animal models of ICI-related lung injury (ICI-LI) has limited the in-depth exploration of its pathogenesis. In clinical practice, underlying lung diseases increase the risk of lung injury. Thus, we used a mouse model of lung injury induced by bleomycin (BLM) and then administered anti-programmed cell death 1 (aPD-1) antibodies to induce ICI-LI. Compared with the BLM group, the aPD-1 + BLM group presented more significant weight loss, greater levels of lung inflammation and fibrosis, and decreased lung function. In this ICI-LI model, high levels of caspase-3/gasdermin E (GSDME) were detected in the lung tissue of mice, and the JNK inhibitor SP600125 mitigated lung damage by inhibiting GSDME-mediated pyroptosis. Consistent with the findings in the animal model, immunofluorescence and RNA sequencing of lung tissue from ICI-LI patients revealed upregulation of the expression of genes related to the GSDME-related pyroptosis pathway. Our results suggest that GSDME-mediated pyroptosis may be associated with the pathogenesis of ICI-LI, indicating that targeting GSDME could be a potential therapeutic strategy for treating ICI-LI.

Background: Laryngeal squamous cell carcinoma (LSCC), the predominant histological subtype of laryngeal cancer with a poor diagnosis, requires further exploration of its molecular mechanisms and potential therapeutic targets. Methods: The expression of MNAT1 in LSCC was detected by western blotting and IHC. EDU analysis, colony formation assay, scratch assay, transwell assay and flow cytometry were used to detect cell proliferation, migration, invasion and apoptosis. The downstream genes of MNAT1 were predicted by RNA-seq. The interaction between MNAT1 and GDF15 was verified by Co-immunoprecipitation assay. The effect of MNAT1 on mitochondrial activity in LSCC cells was determined by ROS, JC-1, and lysosomal mitochondrial activity. The effect of MNAT1 and GDF15 on tumor growth of drug-resistant cells was evaluated in vivo. Results: MNAT1 was highly expressed in LSCC tissues. After MNAT1-knockdown, the proliferation, migration and invasion of LSCC cells were inhibited, the level of apoptosis was significantly increased, and the resistance to cisplatin was decreased. MNAT1 interacts with GDF15. MNAT1 affects cell proliferation, migration and invasion through GDF15, and further affects mitochondrial apoptosis through AMPK pathway. In addition, MNAT1-knockdown and GDF15-knockdown reduced the tumor growth rate and enhanced the sensitivity of cisplatin in vivo. Conclusions: MNAT1 promoted GDF15-mediated changes in AMPK pathway to affect mitochondrial apoptosis, which reveals the progression of LSCC and the mechanism of chemotherapy resistance, providing a new understanding of the mechanism of mitochondrial apoptosis and chemotherapy resistance in LSCC.

BACKGROUND:The combination of immune checkpoint inhibitors and antiangiogenic drugs has shown promising efficacy in advanced hepatocellular carcinoma (HCC). However, tumor regression and progression-free survival (PFS) vary considerably among patients receiving this therapy. AIM:To identify predictive biomarkers in HCC patients treated with sintilimab (programmed cell death protein-1 inhibitor) plus lenvatinib (tyrosine kinase inhibitor). METHODS:In this single-center study in China, patients with unresectable HCC received sintilimab every 21 days and daily oral lenvatinib. Treatment response was assessed by modified response evaluation criteria in solid tumors. Tumor biopsies underwent RNA sequencing, immune microenvironment profiling, and whole-exome sequencing. Differentially expressed genes (DEGs) and immune cell subsets between response groups were identified, followed by survival analyses. All potential predictors of PFS, together with clinical variables, were included in Cox regression to identify independent prognostic factors. RESULTS:Between August 2019 and November 2021, 33 patients with hepatitis-B-virus-related HCC were enrolled; by January 2024, 13 had undergone potentially curative surgery or ablation. RNA sequencing identified 94 DEGs between responders (n = 22) and non-responders (n = 11) using Fisher's exact test or Wilcoxon rank-sum test (all P < 0.05). High long intergenic non-protein coding RNA 01554 (LINC01554) and whirlin expression were associated with longer PFS in Kaplan-Meier analysis (P < 0.05). DEG-immune cell analysis showed positive correlations with pro-B and plasma cells in responders, and negative correlations with CD4+ central memory T (Tcm), T helper 1, and natural killer T cells in non-responders; none significantly predicted PFS, although CD4+ Tcm cells approached significance (P < 0.10). Whole-exome sequencing revealed Fanconi anemia complementation group D2 mutations enriched in non-responders (P < 0.05), while cut-like homeobox 1 mutations predicted poorer PFS (P = 0.011). Cox regression identified solitary tumor [P = 0.02, hazard ratio (HR) = 0.31], high LINC01554 (P = 0.01, HR = 0.16), and elevated CD4+ Tcm cells (P = 0.05, HR = 0.29) as independent predictors of prolonged PFS. CONCLUSION:Sintilimab plus lenvatinib showed heterogeneous efficacy in HCC. High LINC01554 expression, elevated CD4+ Tcm cells, and solitary tumors may serve as predictive biomarkers for prolonged disease control.

BACKGROUND:Life's Essential 8 (LE8) is known to have a negative correlation with biological aging, while the relationship between the Life's Crucial 9 (LC9) score, which includes mental health, and biological aging remains to be further investigated. METHODS:We obtained data from two national cohorts, the UK Biobank and National Health and Nutrition Examination Survey (NHANES), to analyze the association between LC9 and biological aging. Biological aging was assessed using PhenoAge and KDMAge, with gender, race, and other indicators included as covariates. We applied linear regression models and restricted cubic splines (RCS) to analyze and describe the relationship. Furthermore, we explored the mediating role of oxidative stress and inflammation in the association between LC9 and biological aging. Subgroup analyses were conducted using multiple linear regression models, and differences between subgroups were assessed through interaction p-value tests. Sensitivity analyses were subsequently performed, followed by an exploration of the underlying mechanisms. RESULTS:In this study, the UK Biobank cohort included 46,599 participants, with 44,973 participants having complete data for all covariates, LC9, and the necessary calculations for PhenoAge and KDMage. In the NHANES cohort, these numbers were 11,726 and 5,936, respectively. In the UK Biobank cohort, a significant association was found between the LC9 score and PhenoAge (β = -2.484, p < 0.001), with similar results observed for KDMage (β = -7.987, p < 0.001). Similar findings were observed in the NHANES cohort, with significant associations between the LC9 score and both PhenoAge (β = -5.327, p < 0.001) and KDMAge (β = 11.826, p < 0.001). These findings align with previous research suggesting that higher LC9 scores are associated with slower biological aging. After multivariable adjustment, an "inverse L-shaped" relationship was observed (non-linear P < 0.001). In the mediation analysis, oxidative stress and inflammation showed significant mediating effects between LC9 and both PhenoAge and KDMage (p < 0.001 for both). In the subgroup analysis, the LC9 score showed broad applicability, particularly among male participants aged over 60 years. CONCLUSION:This cohort study suggests that higher LC9 scores are associated with slower biological aging. In addition to emphasizing diet and lifestyle habits, the role of mental health in biological aging should not be overlooked.

In this work, we have developed a Sanger's reagent-based photocage, LNDA-NBD-Sanger, which releases the caged COX-2 inhibitor, lenalidomide (LNDA), under 400 nm UV irradiation while producing a fluorescent signal from the activated nitrobenzoxadiazole (NBD) derivative, realizing the monitoring of LNDA release in cancer cells and light-controlled anti-cancer therapy.

ETHNOPHARMACOLOGICAL RELEVANCE:Qianyang Yuyin granules (QYYY) have been used clinically to treat hypertension for over two decades. Previous clinical trials have shown that QYYY can improve vascular elastic function in hypertensive patients. However, the underlying pharmacological mechanism is unclear. AIM OF THE STUDY:To elucidate the effects and mechanisms of QYYY on vascular remodeling using a multidisciplinary approach that includes network pharmacology, proteomics, and both in vitro and in vivo experiments. MATERIALS AND METHODS:The main components of QYYY were identified using ultra-high-performance liquid chromatography and high-resolution mass spectrometry. Network pharmacology and molecular docking were employed to predict QYYY's primary active ingredients, potential therapeutic targets and intervention pathways in hypertensive vascular remodeling. We induced hypertension in male C57BL/6 mice by infusing angiotensin II (Ang II) via osmotic minipumps, and performed pre-treatment with QYYY or Sacubitril/valsartan (Entresto). Blood pressure was monitored in vivo, followed by the extraction of aortas to examine pathological structural changes and alterations in protein expression patterns. The expression and location of proteins involved in the HIF-1α/TWIST1/P-p65 signaling pathway were investigated, as well as markers of vascular smooth muscle cells (VSMCs) phenotypic switch. In vitro, we studied the effects of QYYY water extract on Ang II-stimulated human aortic VSMCs. We investigated whether QYYY could affect the HIF-1α/TWIST1/P-p65 signaling pathway, thereby ameliorating apoptosis, autophagy, and phenotype switch in VSMCs. RESULTS:We identified 62 main compounds in QYYY, combined with network pharmacology, speculated 827 potentially active substances, and explored 1021 therapeutic targets. The KEGG pathway analysis revealed that the mechanisms of action associated with QYYY therapy potentially encompass various biological processes, including metabolic pathways, TNF signaling pathways, apoptosis, Ras signaling pathways, HIF-1 signaling pathways, autophagy-animal pathways. In hypertensive mice, QYYY restored abnormally elevated blood pressure, vascular remodeling, and inflammation with a dose-response relationship while altering abnormal protein patterns. In vitro, QYYY could inhibit abnormal proliferation, migration, intracellular Ca2+ accumulation and cytoskeletal changes of VSMCs. It improved mitochondrial function, reduced ROS levels, stabilized membrane potential, prevented cell death, and reduced overproduction of TGF-β1, TNF-a, and IL-1β. CONCLUSION:QYYY may be able to inhibit the overactivation of the HIF-1α/TWIST1/P-p65 signaling pathway, improve the phenotypic switch, and balance apoptosis and autophagy in VSMCs, thereby effectively improving vascular remodeling caused by hypertension.

Cell cycle protein E2 (CCNE2) is a member of the Cyclin family, known for driving tumor cell proliferation and invasion. However, the mechanism of its action in head and neck squamous cell carcinoma (HNSCC) remains unclear. The aim of this study is to investigate the relationship between CCNE2 and cisplatin resistance and survival prognosis of head and neck squamous cell carcinoma. We performed transcriptomic sequencing of HNSCC and HNSCC/DDP. Kaplan-Meier analysis and COX regression analysis were used to evaluate the relationship between CCNE2 expression and survival prognosis of HNSCC patients. Multiple potential biological functions of CCNE2 in HNSCC were identified using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG). Single-sample gene set enrichment analysis (ssGSEA) was used to explore tumor immune infiltration. The potential mechanism of CCNE2 was explored by molecular docking and immunoprecipitation. Cell migration, cell invasion and cell proliferation assays were used to investigate the mechanism of CCNE2 in HNSCC. CCNE2 is up-regulated in HNSCC tissues and cell lines and is associated with poor prognosis. The high expression of CCNE2 in HNSCC is associated with clinical significance. GO and KEGG analysis showed that ccne2 related genes may be involved in the regulation of DNA double-strand break repair and DNA metabolic process. CCNE2 expression was positively correlated with the infiltration levels of helper T cells, Tcm cells and Th2 cells, and negatively correlated with the infiltration levels of DC, neutrophils and pDC. CCNE2 regulates the invasion, migration and proliferation of HNSCC cells by targeting MNAT1. CCNE2 also altered cisplatin resistance in HNSCC/DDP. CCNE2 may be an independent prognostic biomarker of HNSCC through MNAT1, which provides new ideas for cisplatin resistance and therapeutic targets of HNSCC.

Chemo-immunotherapy is the current first-line treatment for patients with extensive-stage small cell lung cancer (ES-SCLC), but survival benefits are modest. We aimed to evaluate the safety, antitumor activity and biomarkers of first-line camrelizumab and apatinib plus chemotherapy in untreated ES-SCLC patients. In this single-arm trial (ClinicalTrials.gov NCT05001412), eligible patients received 2 cycles of etoposide and carboplatin (EC) as induction treatment followed by 2–4 cycles of camrelizumab, apatinib plus EC, then maintenance camrelizumab plus apatinib. Primary endpoint was safety. Secondary endpoints included objective response rate (ORR), duration of response, progression-free survival (PFS), and overall survival (OS). Targeted sequencing and whole transcriptome sequencing were performed to explore biomarkers. All enrolled 40 patients were treated and analyzed for safety. During the entire treatment, treatment-emergent adverse events (TEAEs) occurred in 40 patients (100%), and 30 (75.0%) were grade ≥3. The most common grade ≥3 TEAEs were neutropenia (35.0%), anemia (15.0%) and increased alanine aminotransferase (15.0%). No treatment-related deaths occurred. Among 36 evaluable patients, ORR was 88.9% (95% CI: 73.9%–96.9%), median PFS was 7.3 months (95% CI: 6.6–9.2) and median OS was 17.3 months (11.8-not reached). Mutations in RB1, high levels of tumor mutation burden, natural killer cells, and interferons, and low levels of cancer-associated fibroblasts, correlated with prolonged PFS. Induction chemotherapy followed by camrelizumab, apatinib plus EC demonstrated acceptable safety and promising antitumor activity in untreated ES-SCLC patients. The identified biomarkers need further validation. Trial Registration ClinicalTrials.gov Identifier: NCT05001412.

BackgroundGastrointestinal tract cancer (GIC), including oesophageal cancer (EC), gastric cancer (GC) and colorectal cancer (CRC), is characterised with high global incidence and mortality rates, with similar tumourigenic processes. However, the common and heterogeneous molecular features among GIC at single-cell level remain poorly characterised.MethodsSingle-cell RNA-seq data of more than one million high-quality annotated cells from 577 specimens, including 121 ECs, 182 GCs and 254 CRCs, were integrated to systematically decipher the heterogeneous characteristics of GIC. Non-negative matrix factorisation (NMF) was employed to identify epithelial cell meta-programs (MPs), and cell-cell communication analysis was conducted to investigate regulatory interactions between the tumour microenvironment (TME) and these MPs. Additionally, cell lineage inference analysis was performed to identify metaplastic signatures in EC and GC.ResultsWe identified 24 consensus MPs from epithelial cells and 42 distinct subtypes from non-epithelial cells thus offering a comprehensive overview of heterogeneous characteristic in GIC. Notably, we observed that EC exhibited unique features, including heightened activity in stress-related programs and a more exhausted TME, enriched with CD4+ Tregs and CD8+ exhausted T cells. In contrast, epithelial cells in GC displayed increased expression of epithelial-mesenchymal transition (EMT)-related signatures and an activated immune phenotype, marked by enrichment of NK cells and CD8+ effector T cells. Moreover, samples with metaplastic signatures in GC and EC showed similarities to CRC, including elevated expression of metabolism-associated signatures and an abundance of CD4+ helper-like T cells. Finally, we identified the potential regulatory roles of the TME in shaping epithelial cell behaviour.ConclusionsOur findings provide insights into the common and specific cellular and molecular patterns associated with GIC tumourigenesis and TME remodelling. We also elucidate the similarity between GC/EC with metaplastic signature and CRC, which advancing our understanding of these malignancies.Key points A comprehensive single-cell atlas of gastrointestinal tract cancer (GIC) was constructed. GICs exhibit distinct epithelial features and specific tumour microenvironment (TME) patterns, forming diverse niches. GC and EC exhibiting metaplastic features show elevated metabolism-associated signatures and share similarities with CRC.