Supplementary Table 1. qRT-PCR primers of cholesterol biosynthesis genes

BACKGROUND:Cardiac fibroblasts (CFs) are essential for cardiac morphogenesis and homeostasis. We investigated whether transcription factor EB (TFEB) directly targeted and suppressed the activation of CFs, aiming to elucidate its underlying pathological mechanism from the perspectives of gene and cell therapy. METHODS:Following myocardial infarction (MI) induction, we performed transcriptome sequencing of CFs isolated from R26-LSL-TFEB+/+; Acta2-cre (n = 3) and R26-LSL-TFEB+/+ (n = 3) mice. Differential gene expression and functional enrichment analyses were conducted using R software. The binding between TFEB and Thrombospondin-1 (Thbs1) was validated by ChIP-qPCR assay. CFs were extracted from adult mice in the R26-LSL-TFEB+/+; Acta2-cre and R26-LSL-TFEB+/+ groups. Protein expressions of integrin, CD47, CD36, Thbs1, p-paxillin, vinculin, P-FAK, and α- SMA were detected by Western blot. Cell migration was assessed by the wound healing and Transwell assays. RESULTS:TFEB modulated the expression of a broad spectrum of RNAs associated with the transformation of CFs. Pathway analysis revealed significant enrichment in pathways related to extracellular matrix (ECM) receptor interaction and focal adhesion (FA). Notably, both mRNA and protein levels of Thbs1 were markedly elevated in TFEB-overexpressing CFs. Integrated computational prediction and chromatin immunoprecipitation assays identified that TFEB directly bound to the promoter region of Thbs1. This binding was associated with downstream modulation of its receptor network and a concomitant reduction in FA complex activation at the protein level. These findings positioned Thbs1 as a key transcriptional target through which TFEB regulated ECM-related signaling and cellular adhesion dynamics in CFs. DISCUSSION:The current findings showed that the modulation of Thbs1 and associated FA signaling was a mechanism through which TFEB overexpression exerted its anti-fibrotic effects on CFs. This highlighted the TFEB-Thbs1 axis as a potential novel target for developing therapeutic strategies to mitigate cardiac fibrosis. CONCLUSIONS:This study suggested that the protective effect of TFEB against MI injury was associated with the Thbs1/FA signaling pathway, providing a novel potential therapeutic target for cardiac fibrosis.

ABSTRACT Drugs induce coordinated phenotypic changes across multiple modalities, including transcriptional reprogramming and cellular morphological remodeling. Predicting these drug-induced modality changes is central to drug discovery, mechanism-of-action studies and precision therapeutics, however, prediction performance depends critically on how both drug compounds and cellular states are represented. Despite rapid advances in drug molecular and gene representation methods, a systematic evaluation of these methods remains lacking. Herein, we introduce MVCBench, a comprehensive benchmarking framework for evaluating drug molecular and gene representation methods in predicting drug-induced multimodal virtual cell (MVC) phenotypes. MVCBench leverages large-scale transcriptomic and high-content imaging data and systematically evaluates 24 representation methods (12 drug molecular and 12 gene representation methods) across nearly 1.1 million drug-induced profiles, under both in-distribution and out-of-distribution settings spanning unseen compounds, cell lines, assay plates and datasets. Our benchmarking reveals a pronounced modality-dependent asymmetry: advanced drug molecular representations substantially improve the prediction of drug-induced morphological phenotypes but provide only limited gains for gene expression prediction relative to classical fingerprints, whereas task-specific gene representations outperform general-purpose foundation models in predicting drug-induced transcriptomic responses. Predictive performance also deteriorates sharply under distribution shift, highlighting persistent challenges in cross-dataset and cross-platform generalization. We further show that integrating transcriptomic and morphological modalities consistently improves prediction accuracy, and derive practical design principles for MVC architectures, including modality-aware loss calibration and fusion strategies. Together, MVCBench provides a systematic foundation for evaluating representation methods and offers guidance for developing robust MVC models of drug-induced cellular responses.

Hippo pathway activity is predictive for poor cancer immunotherapy outcome

Castration-resistant prostate cancer (CRPC) lethality arises from epigenetic-driven resistance to androgen deprivation therapy (ADT). Here, we uncover a compensatory epigenetic switch between DNA methylation and H3K27me3-mediated repression as a critical barrier to epigenetic therapy in CRPC. Integrative multiomics analyses reveal that DNMT inhibitors (DNMTis) trigger EZH2-dependent H3K27me3 accumulation at the ADAMTS1 locus-a master collagenase essential for extracellular matrix (ECM) remodeling-perpetuating fibrotic niche formation and therapy resistance. Dual targeting of DNMTs and EZH2 disrupts this epigenetic plasticity, synergistically reactivating ADAMTS1 to degrade collagen-rich stroma, suppress FAK/MAPK mechanotransduction signaling, and reverse epithelial-mesenchymal transition (EMT). Crucially, in immunocompetent models, this strategy achieves >90% tumor suppression and reverses immunosuppression by enhancing cytotoxic CD8+ T cell infiltration 11.4-fold while depleting immunosuppressive macrophages and Tregs. Mechanistically, dual therapy inactivates the FAK/MAPK/EMT axis via ADAMTS1-mediated ECM degradation, overcoming stromal-mediated resistance. Our work establishes epigenetic-ECM coevolution as a hallmark of CRPC and provides a rationally designed combination therapy to dismantle the therapy-resistant niche.

Supplementary Table 6. The independent replicates of Figure 6B

Background Colorectal carcinoma (CRC) is one of the most common malignant tumors, and its main cause of death is tumor metastasis. RNA N 6 -methyladenosine (m 6 A) is an emerging regulatory mechanism for gene expression and methyltransferase-like 3 (METTL3) participates in tumor progression in several cancer types. However, its role in CRC remains unexplored. Methods Western blot, quantitative real-time PCR (RT-qPCR) and immunohistochemical (IHC) were used to detect METTL3 expression in cell lines and patient tissues. Methylated RNA immunoprecipitation sequencing (MeRIP-seq) and transcriptomic RNA sequencing (RNA-seq) were used to screen the target genes of METTL3. The biological functions of METTL3 were investigated in vitro and in vivo. RNA pull-down and RNA immunoprecipitation assays were conducted to explore the specific binding of target genes. RNA stability assay was used to detect the half-lives of the downstream genes of METTL3. Results Using TCGA database, higher METTL3 expression was found in CRC metastatic tissues and was associated with a poor prognosis. MeRIP-seq revealed that SRY (sex determining region Y)-box 2 (SOX2) was the downstream gene of METTL3. METTL3 knockdown in CRC cells drastically inhibited cell self-renewal, stem cell frequency and migration in vitro and suppressed CRC tumorigenesis and metastasis in both cell-based models and PDX models. Mechanistically, methylated SOX2 transcripts, specifically the coding sequence (CDS) regions, were subsequently recognized by the specific m 6 A “reader”, insulin-like growth factor 2 mRNA binding protein 2 (IGF2BP2), to prevent SOX2 mRNA degradation. Further, SOX2 expression positively correlated with METTL3 and IGF2BP2 in CRC tissues. The combined IHC panel, including “writer”, “reader”, and “target”, exhibited a better prognostic value for CRC patients than any of these components individually. Conclusions Overall, our study revealed that METTL3, acting as an oncogene, maintained SOX2 expression through an m 6 A-IGF2BP2-dependent mechanism in CRC cells, and indicated a potential biomarker panel for prognostic prediction in CRC.

Objective:Fine particulate matter (PM2.5) within polluted air is a significant health risk and a strong oxidant. Even exposure to low-level PM2.5 (at or below the WHO standard) is linked to neuroinflammatory, oxidative stress and the development of neurological disorders in the long term. This study aimed to investigate the prophylactic effects of antioxidant supplementation on mitigating sub-chronic exposure to low-level PM2.5-induced brain pathology in mice. Materials and methods:Mice were exposed to traffic-derived PM2.5 (5 μg/day) collected from a highway in Sydney, Australia, once daily for 3 weeks, while receiving antioxidant, vitamin C (10 mM) or N-acetylcysteine (NAC, 40 mM), via drinking water. Oxidative stress and neuronal loss were assessed across different brain regions. In vitro experiments were conducted to evaluate neuronal integrity and mitochondrial function by VC and NAC treatment in PM2.5-exposed primary neurons. Results:Sub-chronic PM2.5 exposure increased lipid peroxidation and reduced neurofilament density in the cortex, hippocampus, and thalamus, which were mitigated by VC and NAC supplementation. Although oxidative stress (ROS accumulation and increase in 4-HNE) was prevented in all brain regions by VC and NAC, neurofilament loss remained. In vitro, VC and NAC reduced mitochondrial ROS production, which in turn improved neuronal and synaptic survival, suggesting mitochondria-dependent oxidative stress plays a central role in PM2.5-induced neurotoxicity at least in the cortex and hippocampus. Conclusion:Mitochondria-dependent oxidative stress is a key mechanism underlying PM2.5-induced neurotoxicity, which can be attenuated by VC and NAC supplementation. This highlights a potential prophylactic strategy to partially protect the brain from polluted air.

Tumors are ecological systems shaped by continuous exchange with surrounding cells. The transfer of functional mitochondria, which reprograms malignant behavior, introduces a distinct layer to this ecology. Cancer evolution may proceed not solely through mutation and selection but also through the horizontal assimilation of organellar traits acquired from neighboring cells.

Chemically induced proximity is a powerful modality for manipulating protein function. Most of the effort in this field has focused on targeted protein degradation, but recruitment of other types of post-translational modification enzymes to a target protein is also of interest. To construct such reagents, one would ideally like to have ligands that engage the enzyme without inhibiting its activity. In this study, we describe a screening platform for the discovery of noninhibitory macrocyclic ligands for a protein tyrosine phosphatase, using PTP1B as an exemplary model target. This workflow involves sequential screens of small libraries of bead-displayed macrocycles in which only one position of the macrocycle is varied in each round of screening while the others are held as invariant placeholders. The beads co-display a high KM substrate for the phosphatase, allowing ligand-dependent recruitment of the enzyme to the bead surface to be coupled to dephosphorylation of the co-displayed substrate. This is detected by staining with a labeled anti-phosphotyrosine antibody. Finally, we demonstrate that the same general approach can be applied to proteins lacking enzymatic activity by screening against biotin ligase-target protein fusions and employing a proximity labeling-like assay to register screening hits.

Advancements in multi-omics research have demonstrated the potential of integrating human microbiome and metabolomics data to better understand physiological processes and improve prediction accuracy in studies of human health. While conventional models utilizing single-omics data provide valuable perspectives, they often fail to capture the complexity of biological systems. Recent developments in supervised contrastive learning frameworks have enhanced predictive performance for categorical responses, yet limitations persist in extending these methods to continuous outcomes. A robust model capable of addressing these gaps could significantly enhance multi-omics predictions and provide new insights into complex biological interactions. Here, we present MB-SupCon-cont, a novel supervised contrastive learning framework designed for both categorical and continuous responses in multi-omics data. MB-SupCon-cont improves prediction accuracy by incorporating a generalized contrastive loss function that defines similarity and dissimilarity for continuous responses using three distance-based weighting methods. Through simulation studies and two real-world datasets for Type 2 Diabetes (T2D) and High-Fat Diet (HFD), we demonstrate that MB-SupCon-cont consistently achieves lower prediction errors than tuned conventional models, canonical correlation analysis, and autoencoder baselines, with most reaching statistical significance. We further provide a validation-based rule for selecting the weighting method and show that the learned embeddings align more closely with the response and recover known microbe and metabolite associations. The framework also provides superior representation learning and improves data visualization in lower-dimensional spaces. These findings suggest that MB-SupCon-cont is a powerful tool for general multi-omics prediction and may have broad applicability in biomedical research.

Supplementary Figure 2. Gene expression in tumors and gating strategy

T cells are the central players in antitumor immunity, and effective tumor killing depends on their ability to infiltrate into the tumor microenvironment (TME) while maintaining normal cytotoxicity. However, late-stage tumors develop immunosuppressive mechanisms that impede T cell movement and induce exhaustion. Investigating T cell migration in human tumors in vivo could provide insights into tumor immune escape, although it remains a challenging task. In this study, we developed ReMiTT, a computational method that leverages spatial transcriptomics data to track T cell migration patterns within tumor tissue. Applying ReMiTT to multiple tumor samples, we identified potential migration trails. On these trails, chemokines that promote T cell trafficking displayed an increasing trend. Additionally, we identified key genes and pathways enriched on these migration trails, including those involved in cytoskeleton rearrangement, leukocyte chemotaxis, cell adhesion, leukocyte migration, and extracellular matrix remodeling. Furthermore, we characterized the phenotypes of T cells along these trails, showing that the migrating T cells are highly proliferative. Our findings introduce an approach for studying T cell migration and interactions within the TME, offering valuable insights into tumor-immune dynamics.

Tumor-associated macrophages (TAMs) arise from monocytes and represent major contributors to the immunosuppressive microenvironment of solid tumors. However, the environmental cues that govern TAM differentiation and immunosuppressive activity remain incompletely understood. Here we demonstrate that hepatocellular carcinoma (HCC) cells secrete extracellular vesicles (EVs) that are preferentially taken up by monocytes, inducing their differentiation to TAMs characterized by a distinct immune-inhibitory signature. Mechanistically, HCC-derived EVs encapsulate the lipogenic enzyme ATP-citrate lyase (ACLY), promote palmitate biosynthesis in targeted monocytes, thereby enhancing the S-palmitoylation and stability of multiple immune checkpoint proteins. To validate this, we synthesized liposomal vesicles (LVs) decorated with an EV-marker protein CD81, which mimicked the targeting specificity of endogenous EVs for monocytes and differentiated macrophages. When loaded with ACLY proteins as interior cargo, these LVs were sufficient to induce immunosuppressive TAMs and promote HCC progression. Conversely, CD81-decorated LVs encapsulating the ACLY inhibitor SB204990 markedly reduced the TAM-mediated immunosuppressive activity, leading to restrained HCC progression. Importantly, we further demonstrated that targeting EV-transferred, TAM-specific ACLY represents a promising strategy to enhance immunotherapeutic efficacy without notable side effects, particularly when combined with anti-PD-1/PD-L1 antibodies for HCC treatment.

With the global surge in diabetes mellitus prevalence, diabetic osteoporosis (DOP) has become a pressing public health challenge because it markedly increases fracture risk and impairs bone repair capacity. The bone microenvironment, a dynamic ecosystem that maintains skeletal integrity, undergoes profound pathological changes under chronic hyperglycemia: Excessive reactive oxygen species production induced by high glucose and accumulation of advanced glycation end products together trigger oxidative stress and chronic low-grade inflammation in the bone microenvironment (e.g. , activation of the nuclear factor-kappa B signaling pathway). These processes, by inhibiting osteoblast differentiation, enhancing osteoclast activity, and damaging microvascular supply, disrupt the coupled balance of bone remodeling. This article aims to systematically review the effects of hyperglycemia on the bone microenvironment and its molecular mechanisms, clarify the pathological basis of DOP, and provide a theoretical foundation for identifying new targets for prevention, treatment, and clinical intervention.

Methionine metabolism generates the substrate S-adenosylmethionine (SAM), which regulates epigenetic modifications crucial for various cellular processes, particularly tumorigenesis. However, whether methionine metabolism involves epigenetic mechanisms independent of SAM and what roles such mechanisms play in tumorigenesis remain unclear. We show here that the adenosylhomocysteinase (AHCY)–adenosine complex increases mRNA m6A levels in a non-global manner, promoting fatty acid synthesis and tumorigenesis. Adenosine increases mRNA m6A levels by binding to the methionine metabolism enzyme AHCY to form a complex, rather than depending on adenosine receptors. The AHCY–adenosine complex facilitates AHCY dimerization, with adenosine being crucial for dimer stability. AHCY dimers hinder the binding of fat mass and obesity-associated protein (FTO) at the Q86 site to RNA containing the VWDRACH motif, increasing m6A levels and upregulating lipogenesis genes, especially ACACA and SCD1, thus leading to reprogramming of lipid metabolism. Conversely, AHCY mutants that have lost dimerization or FTO-binding ability but retain hydrolase activity suppress lipogenesis and tumor growth without significantly affecting methionine catabolism mediated by AHCY. Loss of AHCY in mice and disruption of AHCY dimerization in tumor cells and patient-derived xenograft models restricted tumor growth. Our findings demonstrate a key SAM-independent link between methionine metabolism and mRNA m6A modification that affects demethylase substrate specificity. This novel link between the methionine cycle and lipid metabolism suggests new strategies for anticancer therapy.

BACKGROUND:S-adenosylhomocysteine (SAH) is a novel risk factor of cardiovascular disease. SAHH (S-adenosylhomocysteine hydrolase) inhibition leads to SAH accumulation and promotes the formation and development of atherosclerosis. However, the role and underlying mechanism of SAHH in the stability of advanced atherosclerotic plaque are still unknown. METHODS:ApoE-deficient mice with heterozygous SAHH knockout or vascular smooth muscle cell (VSMC)-specific or macrophage-specific SAHH knockout were used to investigate the effect of SAHH deficiency on atherosclerotic plaque stability. RESULTS:Heterozygote or VSMCs-specific but not macrophage-specific knockout of SAHH induced VSMC phenotype switching and decreased plaque stability. Whole-genome bisulfite sequencing and RNA sequencing showed that hypomethylation and upregulation of KLF4 (Krüppel-like factor 4) might be associated with SAHH deletion-induced VSMC phenotype switching. Mechanistically, SAHH deficiency resulted in SAH accumulation and induced KLF4 upregulation by inhibiting DNMT (DNA methyltransferase) 3b and leading to hypomethylation of the KLF4 promoter. Furthermore, SAHH deletion reactivated OCT4 (octamer-binding transcription factor 4)-mediated VSMCs migration in a KLF4-dependent manner via TET (tetmethylcytosine dioxygenase) 1-mediated hydroxymethylation of OCT4 promoter. In addition, SAHH deletion resulted in downregulation of TET2-mediated hydroxymethylation of the KLF4 promoter via inhibition of AMPK (AMP-activated protein kinase). Finally, decreased SAHH activity and elevated SAH levels were associated with unstable atherosclerotic plaque in patients with coronary artery disease or ischemic stroke. CONCLUSIONS:These results suggest that SAHH deficiency decreased the stability of atherosclerotic plaques and induced VSMC phenotype switching via epigenetic upregulation of KLF4 and OCT4.

Bone metastasis is a significant challenge in the treatment of advanced breast cancer, with current treatments mainly providing symptom relief without addressing the osteolytic cycle driven by tumor cells and osteoclasts, which leads to continuous bone destruction and tumor progression. Pamidronate (APD), a nitrogen-containing bisphosphonate, has shown potential in managing osteolytic lesions by inhibiting osteoclast activity. However, its clinical application is hindered by rapid systemic clearance and off-target effects. Herein, we developed a multifunctional injectable hydrogel (CHA) by covalently conjugating APD to enhance localized delivery, reduce toxicity, and target both tumor progression and bone degradation to disrupt osteolytic cycle. The CHA hydrogel induces membrane calcification in tumor cells, forming a mineralized layer that impairs nutrient exchange and suppresses tumor growth. Concurrently, CHA modulates the bone microenvironment by downregulating PTHrP expression, inhibiting osteoclastogenesis, and promoting osteogenesis through the upregulation of OPG and RUNX2. Both in vitro and in vivo experiments demonstrated that CHA significantly inhibited tumor growth, prevented bone loss, and facilitated bone regeneration. Moreover, CHA exhibited excellent biocompatibility with no observed systemic toxicity. These results underscore the promise of CHA as a clinically translatable therapeutic strategy for the treatment of osteolytic bone metastases.

Lactate has evolved from being regarded as a byproduct of glycolysis to a pivotal regulator of cancer metabolism and signaling. The Warburg effect underscores how elevated lactate production meets the biosynthetic demands of highly proliferative cancer cells, while shaping an immunosuppressive tumor microenvironment (TME) that supports cancer growth and metastasis. The discovery of lactylation, a novel post-translational modification, has further expanded the conceptual landscape, revealing how lactate serves as both a metabolite and a signaling molecule that couples metabolic reprogramming with gene regulation. This review delineates how lactate dynamically shuttles through the TME and boosts cancer malignancy, including proliferation, metastasis, drug resistance, and immune evasion. Also, this review integrates and discusses how lactate-driven lactylation bridges metabolic and epigenetic control. Furthermore, emerging therapeutic strategies targeting lactate metabolism and lactylation are summarized, revealing their promise in cancer immunotherapy. Collectively, a comprehensive perspective is provided on the multifaceted roles of lactate and lactylation in cancer biology and, more importantly, highlights potential translational avenues for clinical applications.

Given the crucial role of tumor-associated macrophages (TAMs) in cancer development, nano-immunotherapy targeting TAMs represents a pivotal strategy for reversing the immunosuppressive tumor microenvironment (TME) and inhibiting cancer progression. Nano-drug delivery systems have evolved into multimodal platforms that utilize multi-drug synergy and integrated physical energy interventions, showing considerable potential for enhancing drug targeting and biocompatibility. The core objective of TAM-targeted nano-immunotherapy lies in the precise regulation and functional remodeling of TAMs, as well as the synergistic reversal of the immunosuppressive TME. This review addresses the knowledge gaps and challenges in nano-immunotherapy targeting TAMs and explores recent advances in TAM-targeted immunotherapy using various strategies and nanocarriers, with particular emphasis on the relevant cell surface receptors and downstream signaling pathways. Nano-immunotherapeutic strategies targeting TAMs include reprogramming TAMs, influencing TAM polarization, regulating TAM-secreted mediators, inducing TAM exhaustion, enhancing TAM phagocytosis, modulating TAM metabolism, inhibiting TAM recruitment, and blocking PD-L1 expression on TAMs. The innovations of nanocarriers involve the entire chain intelligent design from inorganic nanocrystals, organic polymers to organic-inorganic hybrid systems, and the precise "Trojan horses" constructed using biodegradable polymers and biomimetic membrane vesicles. Intelligent nanorobots targeting TAMs, as autonomous diagnostic and therapeutic systems integrating sensing, decision-making, and execution, represent an effective approach to enhancing the precision and intelligence of anti-tumor strategies.