Single-cell transcriptome analyses reveal critical roles of RNA splicing during leukemia progression.

Leukemogenesis is proposed to be a multistep process by which normal hematopoietic stem and progenitor cells are transformed into full-blown leukemic cells, the details of which are not fully understood. Here, we performed serial single-cell transcriptome analyses of preleukemic and leukemic cells (PLCs) and constructed the cellular and molecular transformation trajectory in a Myc-driven acute myeloid leukemia (AML) model in mice, which represented the transformation course in patients. We found that the Myc targets were gradually up-regulated along the trajectory. Among them were splicing factors, which showed stage-specific prognosis for AML patients. Furthermore, we dissected the detailed gene network of a tipping point for hematopoietic stem and progenitor cells (HSPCs) to generate initiating PLCs, which was characterized by dramatically increased splicing factors and unusual RNA velocity. In the late stage, PLCs acquired explosive heterogeneity through RNA alternative splicing. Among them, the Hsp90aa1hi subpopulation was conserved in both human and mouse AML and associated with poor prognosis. Exon 4 skipping of Tmem134 was identified in these cells. While the exon skipping product Tmem134ß promoted the cell cycle, full-length Tmem134α delayed tumorigenesis. Our study emphasized the critical roles of RNA splicing in the full process of leukemogenesis.

Regenerative failure of intrahepatic biliary cells in Alagille syndrome rescued by elevated Jagged/Notch/Sox9 signaling.

Despite the robust healing capacity of the liver, regenerative failure underlies numerous hepatic diseases, including the JAG1 haploinsufficient disorder, Alagille syndrome (ALGS). Cholestasis due to intrahepatic duct (IHD) paucity resolves in certain ALGS cases but fails in most with no clear mechanisms or therapeutic interventions. We find that modulating jag1b and jag2b allele dosage is sufficient to stratify these distinct outcomes, which can be either exacerbated or rescued with genetic manipulation of Notch signaling, demonstrating that perturbations of Jag/Notch signaling may be causal for the spectrum of ALGS liver severities. Although regenerating IHD cells proliferate, they remain clustered in mutants that fail to recover due to a blunted elevation of Notch signaling in the distal-most IHD cells. Increased Notch signaling is required for regenerating IHD cells to branch and segregate into the peripheral region of the growing liver, where biliary paucity is commonly observed in ALGS. Mosaic loss- and-gain-of-function analysis reveals Sox9b to be a key Notch transcriptional effector required cell autonomously to regulate these cellular dynamics during IHD regeneration. Treatment with a small-molecule putative Notch agonist stimulates Sox9 expression in ALGS patient fibroblasts and enhances hepatic sox9b expression, rescues IHD paucity and cholestasis, and increases survival in zebrafish mutants, thereby providing a proof-of-concept therapeutic avenue for this disorder.

Intrahepatic cholangiocyte regeneration from an Fgf-dependent extrahepatic progenitor niche in a zebrafish model of Alagille Syndrome.

BACKGROUND AND AIMS: Alagille Syndrome (ALGS) is a congenital disorder caused by mutations in the Notch ligand gene JAGGED1, leading to neonatal loss of intrahepatic duct (IHD) cells and cholestasis. Cholestasis can resolve in certain patients with ALGS, suggesting regeneration of IHD cells. However, the mechanisms driving IHD cell regeneration following Jagged loss remains unclear. Here, we show that cholestasis due to developmental loss of IHD cells can be consistently phenocopied in zebrafish with compound jagged1b and jagged2b mutations or knockdown. APPROACH AND RESULTS: Leveraging the transience of jagged knockdown in juvenile zebrafish, we find that resumption of Jagged expression leads to robust regeneration of IHD cells through a Notch-dependent mechanism. Combining multiple lineage tracing strategies with whole-liver three-dimensional imaging, we demonstrate that the extrahepatic duct (EHD) is the primary source of multipotent progenitors that contribute to the regeneration, but not to the development, of IHD cells. Hepatocyte-to-IHD cell transdifferentiation is possible but rarely detected. Progenitors in the EHD proliferate and migrate into the liver with Notch signaling loss and differentiate into IHD cells if Notch signaling increases. Tissue-specific mosaic analysis with an inducible dominant-negative Fgf receptor suggests that Fgf signaling from the surrounding mesenchymal cells maintains this extrahepatic niche by directly preventing premature differentiation and allocation of EHD progenitors to the liver. Indeed, transcriptional profiling and functional analysis of adult mouse EHD organoids uncover their distinct differentiation and proliferative potential relative to IHD organoids. CONCLUSIONS: Our data show that IHD cells regenerate upon resumption of Jagged/Notch signaling, from multipotent progenitors originating from an Fgf-dependent extrahepatic stem cell niche. We posit that if Jagged/Notch signaling is augmented, through normal stochastic variation, gene therapy, or a Notch agonist, regeneration of IHD cells in patients with ALGS may be enhanced.

RhoA-stimulated intra-capillary morphology switch facilitates the arrest of individual circulating tumor cells.

Metastasis is the primary cause of death for most cancer patients. Hematogenous arrest of circulating tumor cells (CTCs) is an essential prerequisite for metastases formation. Using transparent transgenic zebrafish (kdrl:eGFP; Casper), together with resonant laser scanning confocal microscopy, we tracked the fate of CTCs in vivo in the blood circulation for days. We found the intra-capillary morphology-switch (ICMS) of individual CTCs from strip to sphere was necessary for their intravascular arrests. Further genetic and pharmacological inhibition experiments indicated that the RhoA signaling was necessary for ICMS and the arrest of CTCs. At last, we demonstrated that early treatment by a clinically approved RhoA/ROCK inhibitor, Fasudil, could efficiently inhibit the initial arrest of individual CTCs and reduce the incidence of tumor metastasis in both zebrafish and mouse models. These results together indicate that RhoA-stimulated ICMS represents a mechanism for the arrest of individual CTCs, providing a potential target for future treatments of hematogenous metastatic disease.

Efficacy of Lenvatinib, a multitargeted tyrosine kinase inhibitor, on laser-induced CNV mouse model of neovascular AMD.

Neovascular age-related macular degeneration (AMD) is a leading cause of vision loss worldwide. Although intravitreal injection of anti-VEGF antibodies and VEGF Trap have significant clinical benefits, the complications of intravitreal injection, drug resistance and patient compliance still need to be concerned. In this study, the effects of an orally administered multi-targeted tyrosine kinase inhibitor (Lenvatinib, E7080) were evaluated in vitro and in vivo on neovascular AMD mouse model. The results showed that E7080 effectively inhibited the proliferation, migration and tubule formation of human choroidal microvascular endothelial cells (HCMECs), and suppressed the angiogenesis of zebrafish subintestinal vessels without causing malformation. The anti-angiogenic effect of E7080 on the laser-induced choroidal neovascularization (CNV) mouse model by oral administration of 10 mg/kg/day was observed. The fluorescein angiography showed CNV leakage area in treatment group vs control group was 3.407 ±â€¯0.2939 vs 5.202 ±â€¯0.9001 (P = .0133) at day 7th post laser-induced CNV, 1.138 ±â€¯0.4334 vs 3.122 ±â€¯0.3466 (P = .0064) at day 14th, 1.401 ±â€¯0.6577 vs 2.781 ±â€¯0.9815 (P = .00262) at day 21th respectively. Moreover, pharmacokinetics analysis in rat retina showed that E7080 rapidly penetrated the blood-retina barrier to retina through oral administration. The T1/2 in retina was 3.81 ±â€¯0.77 h, the Tmax was 4.60 ±â€¯0.73 h, the AUC0-∞ was 110448.51 ± 18532.51 h*ng/g after a single dose administration analyzed by liquid chromatography-tandem mass spectrometry (LC/MS/MS). In conclusion, our study suggested that orally administered E7080 can be a novel therapeutic strategy for neovascular AMD.

Transdifferentiation of fast skeletal muscle into functional endothelium in vivo by transcription factor Etv2.

Etsrp/Etv2 (Etv2) is an evolutionarily conserved master regulator of vascular development in vertebrates. Etv2 deficiency prevents the proper specification of the endothelial cell lineage, while its overexpression causes expansion of the endothelial cell lineage in the early embryo or in embryonic stem cells. We hypothesized that Etv2 alone is capable of transdifferentiating later somatic cells into endothelial cells. Using heat shock inducible Etv2 transgenic zebrafish, we demonstrate that Etv2 expression alone is sufficient to transdifferentiate fast skeletal muscle cells into functional blood vessels. Following heat treatment, fast skeletal muscle cells turn on vascular genes and repress muscle genes. Time-lapse imaging clearly shows that muscle cells turn on vascular gene expression, undergo dramatic morphological changes, and integrate into the existing vascular network. Lineage tracing and immunostaining confirm that fast skeletal muscle cells are the source of these newly generated vessels. Microangiography and observed blood flow demonstrated that this new vasculature is capable of supporting circulation. Using pharmacological, transgenic, and morpholino approaches, we further establish that the canonical Wnt pathway is important for induction of the transdifferentiation process, whereas the VEGF pathway provides a maturation signal for the endothelial fate. Additionally, overexpression of Etv2 in mammalian myoblast cells, but not in other cell types examined, induced expression of vascular genes. We have demonstrated in zebrafish that expression of Etv2 alone is sufficient to transdifferentiate fast skeletal muscle into functional endothelial cells in vivo. Given the evolutionarily conserved function of this transcription factor and the responsiveness of mammalian myoblasts to Etv2, it is likely that mammalian muscle cells will respond similarly.

[DNA barcoding and its utility in commonly-used medicinal snakes].

Identification accuracy of traditional Chinese medicine is crucial for the traditional Chinese medicine research, production and application. DNA barcoding based on the mitochondrial gene coding for cytochrome c oxidase subunit I (COI), are more and more used for identification of traditional Chinese medicine. Using universal barcoding primers to sequence, we discussed the feasibility of DNA barcoding method for identification commonly-used medicinal snakes (a total of 109 samples belonging to 19 species 15 genera 6 families). The phylogenetic trees using Neighbor-joining were constructed. The results indicated that the mean content of G + C(46.5%) was lower than that of A + T (53.5%). As calculated by Kimera-2-parameter model, the mean intraspecies genetic distance of Trimeresurus albolabris, Ptyas dhumnades and Lycodon rufozonatus was greater than 2%. Further phylogenetic relationship results suggested that identification of one sample of T. albolabris was erroneous. The identification of some samples of P. dhumnades was also not correct, namely originally P. korros was identified as P. dhumnades. Factors influence on intraspecific genetic distance difference of L. rufozonatus need to be studied further. Therefore, DNA barcoding for identification of medicinal snakes is feasible, and greatly complements the morphological classification method. It is necessary to further study in identification of traditional Chinese medicine.

Cathepsin B as a potential prognostic and therapeutic marker for human lung squamous cell carcinoma.

BACKGROUND: The lung squamous cell carcinoma survival rate is very poor despite multimodal treatment. It is urgent to discover novel candidate biomarkers for prognostic assessment and therapeutic targets to lung squamous cell carcinoma (SCC). RESULTS: Herein a two-dimensional gel electrophoresis and ESI-Q-TOF MS/MS-based proteomic approach was used to identify differentially expressed proteins between lung SCC and adjacent normal tissues. 31 proteins with significant alteration were identified. These proteins were mainly involved in metabolism, calcium ion binding, signal transduction and so on. Cathepsin B (CTSB) was one of the most significantly altered proteins and was confirmed by western blotting. Immunohistochemistry showed the correlation between higher CTSB expression and lower survival rate. No statistically significant difference between CTSB-shRNA treated group and the controls was observed in tumor volume, tumor weight, proliferation and apoptosis. However, the CTSB-shRNA significantly inhibited tumor metastases and prolonged survival in LL/2 metastatic model. Moreover, CTSB, Shh and Ptch were up-regulated in patients with metastatic lung SCC, suggesting that hedgehog signaling might be activated in metastatic lung SCC which could affect the expression of CTSB that influence the invasive activity of lung SCC. CONCLUSIONS: These data suggested that CTSB might serve as a prognostic and therapeutic marker for lung SCC.

Investigation on the antibacterial micro-porous titanium with silver nano-particles.

Micro-porous titanium is coated with silver nanoparticles by using a simple chemical reduction method that exhibits excellent antibacterial ability. Scanning electron microscopy (SEM) shows that the silver nanoparticles with average sizes of about 100 nm are formed homogeneously on the micro-porous titanium surface. After the micro-porous Ti coated with silver nano particles is treated by heating, the average size of the silver nano particles is slightly increased, but the nano particles are more uniformly dispersed on the surface of the micro-porous titanium. X-ray diffraction (XRD) indicates that those nanoparticles are metallic silver produced on the micro-porous titanium surface. The samples of micro-porous titanium coated with silver nanoparticles inhibit the growth of Escherichia coli. Our results show that the electrical double layer of the samples play an important role in the antibiosis and this study opens a new window for antibacterial mechanism which may be suitable for the other antibacterial metallic materials.

[DNA barcoding of COI gene in some medicinal animals of Lacertilia].

To identify some medicinal animals of Lacertilia, in total 59 individuals belonging to 12 species 7 genera 3 families, we used the universal barcoding primers to sequence these species, compared with other homologous sequences (564 bp) obtaining from the GenBank and finally constructed phylogenetic trees using Neighbor-joining, Maximum parsimony and Bayesian inference, respectively. As a result, the mean content of G + C (46.5%) was lower than that of A + T (53.5%). As calculated by Kimera-2-parameter model, the whole individuals mean distance for interspecies and intraspecies was 35. 5% and 1.7%, respectively. The mean distance for interspecies was 21 times as much as that for intraspecies. The mean distance for intraspecies of Gekko swinhonis, Hemidactylus frenatus and G. gecko was greater than 2%, respectively. Further analyses suggested that geographical groups of the three species might be of different subSpecies, even species. Of course, incorporating morphological characters and other unlinked genetic markers in future studies will offer further insights into the divergence. On the basis of phylogenetic trees constructed by COI, our results indicated that the taxonomy of the category (family, genus, and species) by DNA barcoding is consistent with morphological characters. Therefore, DNA barcoding is a useful tool for both identification and phylogeny of medicinal animals of Lacertilia, particularly for nonprofessor identifying authentication of Chinese crude drugs of these species.

Lichenoid mucocutaneous reactions associated with sintilimab therapy in a non-small cell lung adenocarcinoma patient: case report and review.

The immune checkpoint inhibitor (ICI), anti-programmed cell death receptor-1 (PD-1) antibody, has gained widespread approval for treating various malignancies. Among the immune-related adverse reactions (irAEs) during ICI treatment, the lichenoid reaction is noteworthy. Sintilimab, a new PD-1 inhibitor, has secured approval in China for treating refractory non-Hodgkin's lymphoma, and phase I/II clinical trials for other solid tumors are ongoing both domestically and abroad. This paper presents a case of a mucocutaneous lichenoid reaction associated with sintilimab therapy, its diagnosis, and management. Our study, using multiplex immunofluorescence staining, reveals localized infiltration of CD4+ and CD8+ T lymphocytes in the subepithelial lamina propria region with upregulated PD-1 expression, implying an association between PD-1 expression upregulation and lichenoid reactions provoked by PD-1 monoclonal antibody. We provide a summary of clinical characteristics and treatment guidelines for lichenoid reactions induced by ICIs from previous reports, highlighting the success of a combined therapeutic regimen of oral antihistamines and topical corticosteroids in controlling symptoms without interrupting ICI treatment.

Zebrafish functional xenograft vasculature platform identifies PF-502 as a durable vasculature normalization drug.

Tumor vasculature often exhibits disorder and inefficiency. Vascular normalization offers potential for alleviating hypoxia and optimizing drug delivery in tumors. However, identifying effective agents is hindered by a lack of robust screening. We aimed to establish a comprehensive method using the zebrafish functional xenograft vasculature platform (zFXVP) to visualize and quantify tumor vasculature changes. Employing zFXVP, we systematically screened compounds, identifying PF-502 as a robust vascular normalization agent. Mechanistic studies showed PF-502 induces endothelial cell-cycle arrest, streamlines vasculature, and activates Notch1 signaling, enhancing stability and hemodynamics. In murine models, PF-502 exhibited pronounced vascular normalization and improved drug delivery at a sub-maximum tolerated dose. These findings highlight zFXVP's utility and suggest PF-502 as a promising adjunctive for vascular normalization in clinical settings.

[Prime study of Gekko gecko in HPLC fingerprint].

OBJECTIVE: To establish HPLC fingerprint of Gekko gecko. METHODS: The relative retention time and relative peak area of exteacts of Gekko gecko were determine by HPLC to confirm proper chromatographic condition and obtain the data. RESULTS: Better distribution of relative retention time and relative peak area were shown under the chromatographic condition and the HPLC fingerprint was established. CONCLUSION: The established HPLC fingerprints of Gekko gecko can be used to identify Gekko gecko and its quality control.

SMRT sequencing of the full-length transcriptome of Gekko gecko.

Tokay Gecko (Gekko gecko) is a rare and endangered medicinal animal in China. Its dry body has been used as an anti-asthmatic agent for two thousand years. To date, the genome and transcriptome of this species remain poorly understood. Here, we adopted single molecule real-time (SMRT) sequencing to obtain full-length transcriptome data and characterized the transcriptome structure. We identified 882,273 circular consensus (CCS) reads, including 746,317 full-length nonchimeric (FLNC) reads. The transcript cluster analysis revealed 212,964 consensus sequences, including 203,994 high-quality isoforms. In total, 111,372 of 117,888 transcripts were successfully annotated against eight databases (Nr, eggNOG, Swiss-Prot, GO, COG, KOG, Pfam and KEGG). Furthermore, 23,877 alternative splicing events, 169,128 simple sequence repeats (SSRs), 10,437 lncRNAs and 7,932 transcription factors were predicted across all transcripts. To our knowledge, this report is the first to document the G. gecko transcriptome using SMRT sequencing. The full-length transcript data might accelerate transcriptome research and lay the foundation for further research on G. gecko.

FNDC5 Causes Resistance to Sorafenib by Activating the PI3K/Akt/Nrf2 Pathway in Hepatocellular Carcinoma Cells.

In this study, we aimed to reveal the resistance mechanism of hepatocellular carcinoma (HCC) cells to sorafenib by exploring the effect of FNDC5 on sorafenib-induced ferroptosis in HCC cells. We compared the expression level of FNDC5 between sorafenib-resistant and sorafenib-sensitive HCC cell lines and the level of ferroptosis between the groups after treatment with sorafenib. We knocked down FNDC5 in drug-resistant cell lines and overexpressed it in sorafenib-sensitive HCC cell lines to further demonstrate the role of FNDC5 in sorafenib-induced ferroptosis. Using PI3K inhibitors, we revealed the specific mechanism by which FNDC5 functions. In addition, we verified our findings obtained in in vitro experiments using a subcutaneous tumorigenic nude mouse model. The findings revealed that FNDC5 inhibits sorafenib-induced ferroptosis in HCC cells. In addition, FNDC5 activated the PI3K/Akt pathway, which in turn promoted the nuclear translocation of Nrf2 and increased the intracellular antioxidant response, thereby conferring resistance to ferroptosis. Our study provides novel insights for improving the efficacy of sorafenib.

The Hematopoietic Function of Medicinal Wine Maoji Jiu Revealed in Blood Deficiency Model Rats.

Maoji Jiu (MJ), a medicinal wine, has been used commonly by the Chinese to enrich and nourish the blood. In this study, the aim is to examine the hematopoietic function of MJ and investigate its hematopoietic regulation mechanism. Thirty-six female Sprague-Dawley rats (200 ± 20 g) were randomly divided into six groups with six rats in each group. The blood deficiency model was induced by injecting hypodermically with N-acetylphenylhydrazine (APH) and injecting intraperitoneally with cyclophosphamide (CTX), and treatment drugs were given by oral gavage twice a day for continuous 10 days from the start of the experiments. The administration of MJ improved the levels of white blood cells (WBCs), red blood cells (RBCs), hemoglobin (HGB), and hematocrit (HCT) in the blood deficiency model rats. Hematopoietic effect involves regulating the antioxidant activity in the liver and the levels of Bcl-2, Bax, erythropoietin (EPO), transforming growth factor-beta-1 (TGF-ß1), and macrophage colony-stimulating factor (M-CSF) mRNA in spleen tissues to enhance extramedullary hematopoiesis. This study suggests that MJ has a beneficial effect on blood deficiency model rats.

The inhibition of protein translation promotes tumor angiogenic switch.

The 'angiogenic switch' is critical for tumor progression. However, the pathological details and molecular mechanisms remain incompletely characterized. In this study, we established mammal xenografts in zebrafish to visually investigate the first vessel growth (angiogenic switch) in real-time, by inoculating tumor cells into the perivitelline space of live optically transparent Transgenic (flk1:EGFP) zebrafish larvae. Using this model, we found that hypoxia and hypoxia-inducible factor (HIF) signaling were unnecessary for the angiogenic switch, whereas vascular endothelial growth factor A gene (Vegfa) played a crucial role. Mechanistically, transcriptome analysis showed that the angiogenic switch was characterized by inhibition of translation, but not hypoxia. Phosphorylation of eukaryotic translation initiation factor 2 alpha (Eif2α) and the expression of Vegfa were increased in the angiogenic switch microtumors, and 3D tumor spheroids, and puromycin-treated tumor cells. Vegfa overexpression promoted early onset of the angiogenic switch, whereas Vegfa knockout prevented the first tumor vessel from sprouting. Pretreatment of tumor cells with puromycin promoted the angiogenic switch in vivo similarly to Vegfa overexpression, whereas Vegfa knockdown suppressed the increase. This study provides direc and dynamic in vivo evidences that inhibition of translation, but not hypoxia or HIF signaling promotes the angiogenic switch in tumor by increasing Vegfa transcription.

Clinically relevant orthotopic xenograft models of patient-derived glioblastoma in zebrafish.

An accurate prediction of the intracranial infiltration tendency and drug response of individual glioblastoma (GBM) cells is essential for personalized prognosis and treatment for this disease. However, the clinical utility of mouse patient-derived orthotopic xenograft (PDOX) models remains limited given current technical constraints, including difficulty in generating sufficient sample numbers from small tissue samples and a long latency period for results. To overcome these issues, we established zebrafish GBM xenografts of diverse origin, which can tolerate intracranial engraftment and maintain their unique histological features. Subsequent single-cell RNA-sequencing (scRNA-seq) analysis confirmed significant transcriptional identity to that of invading GBM microtumors observed in the proportionally larger brains of model animals and humans. Endothelial scRNA-seq confirmed that the zebrafish blood-brain barrier is homologous to the mammalian blood-brain barrier. Finally, we established a rapid and efficient zebrafish PDOX (zPDOX) model, which can predict long-term outcomes of GBM patients within 20 days. The zPDOX model provides a novel avenue for precision medicine of GBM, especially for the evaluation of intracranial infiltration tendency and prediction of individual drug sensitivity.

Dissecting the genetic and microenvironmental factors of gastric tumorigenesis in mice.

Gastric cancer (GC) is one of the most frequent and lethal malignancies in the world. However, our understanding of the mechanisms underlying its initiation and progression is limited. Here, we generate a series of primary GC models in mice with genome-edited gastric organoids, which elucidate the genetic drivers for sequential transformation from dysplasia to well-differentiated and poorly differentiated GC. Further, we find that the orthotopic GC, but not the subcutaneous GC even with the same genetic drivers, display remote metastasis, suggesting critical roles of the microenvironment in GC metastasis. Through single-cell RNA-seq analyses and functional studies, we show that the interaction between fibronectin 1 on stomach-specific macrophages and integrin a6ß4 on GC cells promotes remote metastases. Taken together, our studies propose a strategy to model GC and dissect the genetic and microenvironmental factors driving the full-range gastric tumorigenesis.

KMT2C deficiency promotes small cell lung cancer metastasis through DNMT3A-mediated epigenetic reprogramming.

Small cell lung cancer (SCLC) is notorious for its early and frequent metastases, which contribute to it as a recalcitrant malignancy. To understand the molecular mechanisms underlying SCLC metastasis, we generated SCLC mouse models with orthotopically transplanted genome-edited lung organoids and performed multiomics analyses. We found that a deficiency of KMT2C, a histone H3 lysine 4 methyltransferase frequently mutated in extensive-stage SCLC, promoted multiple-organ metastases in mice. Metastatic and KMT2C-deficient SCLC displayed both histone and DNA hypomethylation. Mechanistically, KMT2C directly regulated the expression of DNMT3A, a de novo DNA methyltransferase, through histone methylation. Forced DNMT3A expression restrained metastasis of KMT2C-deficient SCLC through repressing metastasis-promoting MEIS/HOX genes. Further, S-(5'-adenosyl)-L-methionine, the common cofactor of histone and DNA methyltransferases, inhibited SCLC metastasis. Thus, our study revealed a concerted epigenetic reprogramming of KMT2C- and DNMT3A-mediated histone and DNA hypomethylation underlying SCLC metastasis, which suggested a potential epigenetic therapeutic vulnerability.