Nanophase-Separated Block Copolymer Layers Enabling Stable Zinc Metal Batteries.

Aqueous Zn-ion batteries are promising and efficient energy storage systems owing to their low cost, high safety, and satisfactory capacity. However, the instability of Zn metal anodes, caused by dendritic growth and parasitic side reactions, hinders their practical application. In this study, a nanophase-separated block copolymer layer that enhances the reversibility of Zn metal anodes is introduced. This layer consists of two components: a high-performance engineering-plastic-based hydrophobic block exhibiting excellent mechanical properties and chemical stability, and a hydrophilic block that significantly improves the interfacial stability of the anode by selectively permeating Zn ions through the separated nanophase channels. Through an improved electrochemical system and scalable fabrication process, this block copolymer provides a feasible approach for the practical application of Zn metal anodes in aqueous energy storage systems.

Spatially resolved analysis of pancreatic cancer identifies therapy-associated remodeling of the tumor microenvironment.

In combination with cell-intrinsic properties, interactions in the tumor microenvironment modulate therapeutic response. We leveraged single-cell spatial transcriptomics to dissect the remodeling of multicellular neighborhoods and cell-cell interactions in human pancreatic cancer associated with neoadjuvant chemotherapy and radiotherapy. We developed spatially constrained optimal transport interaction analysis (SCOTIA), an optimal transport model with a cost function that includes both spatial distance and ligand-receptor gene expression. Our results uncovered a marked change in ligand-receptor interactions between cancer-associated fibroblasts and malignant cells in response to treatment, which was supported by orthogonal datasets, including an ex vivo tumoroid coculture system. We identified enrichment in interleukin-6 family signaling that functionally confers resistance to chemotherapy. Overall, this study demonstrates that characterization of the tumor microenvironment using single-cell spatial transcriptomics allows for the identification of molecular interactions that may play a role in the emergence of therapeutic resistance and offers a spatially based analysis framework that can be broadly applied to other contexts.

The expression of PD-L1 on tumor-derived exosomes enhances infiltration and anti-tumor activity of αCD3 × αPD-L1 bispecific antibody-armed T cells.

Anti-cluster of differentiation (CD) 3 × α programmed death-ligand 1 (PD-L1) bispecific T-cell engager (BsTE)-bound T-cells (BsTE:T) are a promising new cancer treatment agent. However, the mechanisms of action of bispecific antibody-armed activated T-cells are poorly understood. Therefore, this study aimed to investigate the anti-tumor mechanism and efficacy of BsTE:T. The BsTE:T migration was assessed in vivo and in vitro using syngeneic and xenogeneic tumor models, flow cytometry, immunofluorescence staining, transwell migration assays, microfluidic chips, Exo View R100, western blotting, and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 technology. In murine B16 melanoma, MC38 colon cancer, and human multiple myeloma cells, BsTE:T exhibited superior tumor elimination relative to that of T-cells or BsTE alone. Moreover, BsTE:T migration into tumors was significantly enhanced owing to the presence of PD-L1 in tumor cells and secretion of PD-L1-containing exosomes. Furthermore, increased infiltration of CD44highCD62Llow effector memory CD8+ T-cells into tumors was closely associated with the anti-tumor effect of BsTE:T. Therefore, BsTE:T is an innovative potential anti-tumor therapy, and exosomal PD-L1 plays a crucial role both in vitro and in vivo in the anti-tumor activity of BsTE:T.

Multi-Objective Bayesian Optimization for Laminate-Inspired Mechanically Reinforced Piezoelectric Self-Powered Sensing Yarns.

Piezoelectric fiber yarns produced by electrospinning offer a versatile platform for intelligent devices, demonstrating mechanical durability and the ability to convert mechanical strain into electric signals. While conventional methods involve twisting a single poly(vinylidene fluoride-co-trifluoroethylene)(P(VDF-TrFE)) fiber mat to create yarns, by limiting control over the mechanical properties, an approach inspired by composite laminate design principles is proposed for strengthening. By stacking multiple electrospun mats in various sequences and twisting them into yarns, the mechanical properties of P(VDF-TrFE) yarn structures are efficiently optimized. By leveraging a multi-objective Bayesian optimization-based machine learning algorithm without imposing specific stacking restrictions, an optimal stacking sequence is determined that simultaneously enhances the ultimate tensile strength (UTS) and failure strain by considering the orientation angles of each aligned fiber mat as discrete design variables. The conditions on the Pareto front that achieve a balanced improvement in both the UTS and failure strain are identified. Additionally, applying corona poling induces extra dipole polarization in the yarn state, successfully fabricating mechanically robust and high-performance piezoelectric P(VDF-TrFE) yarns. Ultimately, the mechanically strengthened piezoelectric yarns demonstrate superior capabilities in self-powered sensing applications, particularly in challenging environments and sports scenarios, substantiating their potential for real-time signal detection.

Single-cell analysis reveals cellular and molecular factors counteracting HPV-positive oropharyngeal cancer immunotherapy outcomes.

BACKGROUND: Oropharyngeal squamous cell carcinoma (OPSCC) induced by human papillomavirus (HPV-positive) is associated with better clinical outcomes than HPV-negative OPSCC. However, the clinical benefits of immunotherapy in patients with HPV-positive OPSCC remain unclear. METHODS: To identify the cellular and molecular factors that limited the benefits associated with HPV in OPSCC immunotherapy, we performed single-cell RNA (n=20) and T-cell receptor sequencing (n=10) analyses of tonsil or base of tongue tumor biopsies prior to immunotherapy. Primary findings from our single-cell analysis were confirmed through immunofluorescence experiments, and secondary validation analysis were performed via publicly available transcriptomics data sets. RESULTS: We found significantly higher transcriptional diversity of malignant cells among non-responders to immunotherapy, regardless of HPV infection status. We also observed a significantly larger proportion of CD4+ follicular helper T cells (Tfh) in HPV-positive tumors, potentially due to enhanced Tfh differentiation. Most importantly, CD8+ resident memory T cells (Trm) with elevated KLRB1 (encoding CD161) expression showed an association with dampened antitumor activity in patients with HPV-positive OPSCC, which may explain their heterogeneous clinical outcomes. Notably, all HPV-positive patients, whose Trm presented elevated KLRB1 levels, showed low expression of CLEC2D (encoding the CD161 ligand) in B cells, which may reduce tertiary lymphoid structure activity. Immunofluorescence of HPV-positive tumors treated with immune checkpoint blockade showed an inverse correlation between the density of CD161+ Trm and changes in tumor size. CONCLUSIONS: We found that CD161+ Trm counteracts clinical benefits associated with HPV in OPSCC immunotherapy. This suggests that targeted inhibition of CD161 in Trm could enhance the efficacy of immunotherapy in HPV-positive oropharyngeal cancers. TRIAL REGISTRATION NUMBER: NCT03737968.

Joint analysis of mutational and transcriptional landscapes in human cancer reveals key perturbations during cancer evolution.

BACKGROUND: Tumors are able to acquire new capabilities, including traits such as drug resistance and metastasis that are associated with unfavorable clinical outcomes. Single-cell technologies have made it possible to study both mutational and transcriptomic profiles, but as most studies have been conducted on model systems, little is known about cancer evolution in human patients. Hence, a better understanding of cancer evolution could have important implications for treatment strategies. RESULTS: Here, we analyze cancer evolution and clonal selection by jointly considering mutational and transcriptomic profiles of single cells acquired from tumor biopsies from 49 lung cancer samples and 51 samples with chronic myeloid leukemia. Comparing the two profiles, we find that each clone is associated with a preferred transcriptional state. For metastasis and drug resistance, we find that the number of mutations affecting related genes increases as the clone evolves, while changes in gene expression profiles are limited. Surprisingly, we find that mutations affecting ligand-receptor interactions with the tumor microenvironment frequently emerge as clones acquire drug resistance. CONCLUSIONS: Our results show that lung cancer and chronic myeloid leukemia maintain a high clonal and transcriptional diversity, and we find little evidence in favor of clonal sweeps. This suggests that for these cancers selection based solely on growth rate is unlikely to be the dominating driving force during cancer evolution.

Immune-restoring CAR-T cells display antitumor activity and reverse immunosuppressive TME in a humanized ccRCC mouse model.

One of the major barriers that have restricted successful use of chimeric antigen receptor (CAR) T cells in the treatment of solid tumors is an unfavorable tumor microenvironment (TME). We engineered CAR-T cells targeting carbonic anhydrase IX (CAIX) to secrete anti-PD-L1 monoclonal antibody (mAb), termed immune-restoring (IR) CAR G36-PDL1. We tested CAR-T cells in a humanized clear cell renal cell carcinoma (ccRCC) orthotopic mouse model with reconstituted human leukocyte antigen (HLA) partially matched human leukocytes derived from fetal CD34+ hematopoietic stem cells (HSCs) and bearing human ccRCC skrc-59 cells under the kidney capsule. G36-PDL1 CAR-T cells, haploidentical to the tumor cells, had a potent antitumor effect compared to those without immune-restoring effect. Analysis of the TME revealed that G36-PDL1 CAR-T cells restored active antitumor immunity by promoting tumor-killing cytotoxicity, reducing immunosuppressive cell components such as M2 macrophages and exhausted CD8+ T cells, and enhancing T follicular helper (Tfh)-B cell crosstalk.

Therapy-associated remodeling of pancreatic cancer revealed by single-cell spatial transcriptomics and optimal transport analysis.

In combination with cell intrinsic properties, interactions in the tumor microenvironment modulate therapeutic response. We leveraged high-plex single-cell spatial transcriptomics to dissect the remodeling of multicellular neighborhoods and cell-cell interactions in human pancreatic cancer associated with specific malignant subtypes and neoadjuvant chemotherapy/radiotherapy. We developed Spatially Constrained Optimal Transport Interaction Analysis (SCOTIA), an optimal transport model with a cost function that includes both spatial distance and ligand-receptor gene expression. Our results uncovered a marked change in ligand-receptor interactions between cancer-associated fibroblasts and malignant cells in response to treatment, which was supported by orthogonal datasets, including an ex vivo tumoroid co-culture system. Overall, this study demonstrates that characterization of the tumor microenvironment using high-plex single-cell spatial transcriptomics allows for identification of molecular interactions that may play a role in the emergence of chemoresistance and establishes a translational spatial biology paradigm that can be broadly applied to other malignancies, diseases, and treatments.

ERdj5 protects goblet cells from endoplasmic reticulum stress-mediated apoptosis under inflammatory conditions.

Endoplasmic reticulum stress is closely associated with the onset and progression of inflammatory bowel disease. ERdj5 is an endoplasmic reticulum-resident protein disulfide reductase that mediates the cleavage and degradation of misfolded proteins. Although ERdj5 expression is significantly higher in the colonic tissues of patients with inflammatory bowel disease than in healthy controls, its role in inflammatory bowel disease has not yet been reported. In the current study, we used ERdj5-knockout mice to investigate the potential roles of ERdj5 in inflammatory bowel disease. ERdj5 deficiency causes severe inflammation in mouse colitis models and weakens gut barrier function by increasing NF-κB-mediated inflammation. ERdj5 may not be indispensable for goblet cell function under steady-state conditions, but its deficiency induces goblet cell apoptosis under inflammatory conditions. Treatment of ERdj5-knockout mice with the chemical chaperone ursodeoxycholic acid ameliorated severe colitis by reducing endoplasmic reticulum stress. These findings highlight the important role of ERdj5 in preserving goblet cell viability and function by resolving endoplasmic reticulum stress.

scHumanNet: a single-cell network analysis platform for the study of cell-type specificity of disease genes.

A major challenge in single-cell biology is identifying cell-type-specific gene functions, which may substantially improve precision medicine. Differential expression analysis of genes is a popular, yet insufficient approach, and complementary methods that associate function with cell type are required. Here, we describe scHumanNet (https://github.com/netbiolab/scHumanNet), a single-cell network analysis platform for resolving cellular heterogeneity across gene functions in humans. Based on cell-type-specific gene networks (CGNs) constructed under the guidance of the HumanNet reference interactome, scHumanNet displayed higher functional relevance to the cellular context than CGNs built by other methods on single-cell transcriptome data. Cellular deconvolution of gene signatures based on network compactness across cell types revealed breast cancer prognostic markers associated with T cells. scHumanNet could also prioritize genes associated with particular cell types using CGN centrality and identified the differential hubness of CGNs between disease and healthy conditions. We demonstrated the usefulness of scHumanNet by uncovering T-cell-specific functional effects of GITR, a prognostic gene for breast cancer, and functional defects in autism spectrum disorder genes specific for inhibitory neurons. These results suggest that scHumanNet will advance our understanding of cell-type specificity across human disease genes.

Obesity Exacerbates Coxsackievirus Infection via Lipid-Induced Mitochondrial Reactive Oxygen Species Generation.

Coxsackievirus B3 (CVB3) infection causes acute pancreatitis and myocarditis. However, its pathophysiological mechanism is unclear. Here, we investigated how lipid metabolism is associated with exacerbation of CVB3 pathology using high-fat diet (HFD)-induced obese mice. Mice were intraperitoneally inoculated with 1×106 pfu/mouse of CVB3 after being fed a control or HFD to induce obesity. Mice were treated with mitoquinone (MitoQ) to reduce the level of mitochondrial ROS (mtROS). In obese mice, lipotoxicity of white adipose tissue-induced inflammation caused increased replication of CVB3 and mortality. The coxsackievirus adenovirus receptor increased under obese conditions, facilitating CVB3 replication in vitro. However, lipid-treated cells with receptor-specific inhibitors did not reduce CVB3 replication. In addition, lipid treatment increased mitochondria-derived vesicle formation and the number of multivesicular bodies. Alternatively, we found that inhibition of lipid-induced mtROS decreased viral replication. Notably, HFD-fed mice were more susceptible to CVB3-induced mortality in association with increased levels of CVB3 replication in adipose tissue, which was ameliorated by administration of the mtROS inhibitor, MitoQ. These results suggest that mtROS inhibitors can be used as potential treatments for CVB3 infection.

Bispecific Antibody-Bound T Cells as a Novel Anticancer Immunotherapy.

Chimeric antigen receptor T (CAR-T) cell therapy is one of the promising anticancer treatments. It shows a high overall response rate with complete response to blood cancer. However, there is a limitation to solid tumor treatment. Additionally, this currently approved therapy exhibits side effects such as cytokine release syndrome and neurotoxicity. Alternatively, bispecific antibody is an innovative therapeutic tool that simultaneously engages specific immune cells to disease-related target cells. Since programmed death ligand 1 (PD-L1) is an immune checkpoint molecule highly expressed in some cancer cells, in the current study, we generated αCD3xαPD-L1 bispecific antibody (BiTE) which can engage T cells to PD-L1+ cancer cells. We observed that the BiTE-bound OT-1 T cells effectively killed cancer cells in vitro and in vivo. They substantially increased the recruitment of effector memory CD8+ T cells having CD8+CD44+CD62Llow phenotype in tumor. Interestingly, we also observed that BiTE-bound polyclonal T cells showed highly efficacious tumor killing activity in vivo in comparison with the direct intravenous treatment of bispecific antibody, suggesting that PD-L1-directed migration and engagement of activated T cells might increase cancer cell killing. Additionally, BiTE-bound CAR-T cells which targets human Her-2/neu exhibited enhanced killing effect on Her-2-expressing cancer cells in vivo, suggesting that this could be a novel therapeutic regimen. Collectively, our results suggested that engaging activated T cells with cancer cells using αCD3xαPD-L1 BiTE could be an innovative next generation anticancer therapy which exerts simultaneous inhibitory functions on PD-L1 as well as increasing the infiltration of activated T cells having effector memory phenotype in tumor site.

Quiescent cancer cells resist T cell attack by forming an immunosuppressive niche.

Immunotherapy is a promising treatment for triple-negative breast cancer (TNBC), but patients relapse, highlighting the need to understand the mechanisms of resistance. We discovered that in primary breast cancer, tumor cells that resist T cell attack are quiescent. Quiescent cancer cells (QCCs) form clusters with reduced immune infiltration. They also display superior tumorigenic capacity and higher expression of chemotherapy resistance and stemness genes. We adapted single-cell RNA-sequencing with precise spatial resolution to profile infiltrating cells inside and outside the QCC niche. This transcriptomic analysis revealed hypoxia-induced programs and identified more exhausted T cells, tumor-protective fibroblasts, and dysfunctional dendritic cells inside clusters of QCCs. This uncovered differential phenotypes in infiltrating cells based on their intra-tumor location. Thus, QCCs constitute immunotherapy-resistant reservoirs by orchestrating a local hypoxic immune-suppressive milieu that blocks T cell function. Eliminating QCCs holds the promise to counteract immunotherapy resistance and prevent disease recurrence in TNBC.

The importance of enhancer methylation for epigenetic regulation of tumorigenesis in squamous lung cancer.

Lung squamous cell carcinoma (LUSC) is a subtype of non-small cell lung cancer (NSCLC). LUSC occurs at the bronchi, shows a squamous appearance, and often occurs in smokers. To determine the epigenetic regulatory mechanisms of tumorigenesis, we performed a genome-wide analysis of DNA methylation in tumor and adjacent normal tissues from LUSC patients. With the Infinium Methylation EPIC Array, > 850,000 CpG sites, including ~350,000 CpG sites for enhancer regions, were profiled, and the differentially methylated regions (DMRs) overlapping promoters (pDMRs) and enhancers (eDMRs) between tumor and normal tissues were identified. Dimension reduction based on DMR profiles revealed that eDMRs alone and not pDMRs alone can differentiate tumors from normal tissues with the equivalent performance of total DMRs. We observed a stronger negative correlation of LUSC-specific gene expression with methylation for enhancers than promoters. Target genes of eDMRs rather than pDMRs were found to be enriched for tumor-associated genes and pathways. Furthermore, DMR methylation associated with immune infiltration was more frequently observed among enhancers than promoters. Our results suggest that methylation of enhancer regions rather than promoters play more important roles in epigenetic regulation of tumorigenesis and immune infiltration in LUSC.

Dysregulation of TFH-B-TRM lymphocyte cooperation is associated with unfavorable anti-PD-1 responses in EGFR-mutant lung cancer.

Patients with non-small cell lung cancer (NSCLC) with epidermal growth factor receptor (EGFR) mutations exhibit an unfavorable response to PD-1 inhibitor through unclear mechanisms. Hypothesizing that EGFR mutations alter tumor-immune interactions, we compare tumor-infiltrating lymphocytes between EGFR mutant (EGFR-MT) and wild type (EGFR-WT) tumors through single-cell transcriptomic analysis. We find that B cells, CXCL13-producing follicular helper CD4+ T (TFH)-like cells, and tissue-resident memory CD8+ T (TRM)-like cells decreased in EGFR-MT tumors. The NOTCH-RBPJ regulatory network, which is vital for persistence of TRM state, is perturbed, and the interactions between TFH and B cells through the CXCL13-CXCR5 axis disappear in EGFR-MT tumors. Notably, the proportion of TRM-like cells is predictive for anti-PD-1 response in NSCLC. Our findings suggest that the impairment of TFH-B-TRM cooperation in tertiary lymphoid structure formation, accompanied by the dysregulation of TRM homeostasis and the loss of TFH-B crosstalk, underlies unfavorable anti-PD-1 response in EGFR-MT lung tumors.

Antiviral Activity of Chrysin against Influenza Virus Replication via Inhibition of Autophagy.

Influenza viruses cause respiratory infections in humans and animals, which have high morbidity and mortality rates. Although several drugs that inhibit viral neuraminidase are used to treat influenza infections, the emergence of resistant viruses necessitates the urgent development of new antiviral drugs. Chrysin (5,7-dihydroxyflavone) is a natural flavonoid that exhibits antiviral activity against enterovirus 71 (EV71) by inhibiting viral 3C protease activity. In this study, we evaluated the antiviral activity of chrysin against influenza A/Puerto Rico/8/34 (A/PR/8). Chrysin significantly inhibited A/PR/8-mediated cell death and the replication of A/PR/8 at concentrations up to 2 µM. Viral hemagglutinin expression was also markedly decreased by the chrysin treatment in A/PR/8-infected cells. Through the time course experiment and time-of-addition assay, we found that chrysin inhibited viral infection at the early stages of the replication cycle. Additionally, the nucleoprotein expression of A/PR/8 in A549 cells was reduced upon treatment with chrysin. Regarding the mechanism of action, we found that chrysin inhibited autophagy activation by increasing the phosphorylation of mammalian target of rapamycin (mTOR). We also confirmed a decrease in LC3B expression and LC3-positive puncta levels in A/PR/8-infected cells. These results suggest that chrysin exhibits antiviral activity by activating mTOR and inhibiting autophagy to inhibit the replication of A/PR/8 in the early stages of infection.

Systems biology analysis identifies TNFRSF9 as a functional marker of tumor-infiltrating regulatory T-cell enabling clinical outcome prediction in lung cancer.

Regulatory T cells (Tregs) are enriched in the tumor microenvironment and play key roles in immune evasion of cancer cells. Cell surface markers specific for tumor-infiltrating Tregs (TI-Tregs) can be effectively targeted to enhance antitumor immunity and used for stratification of immunotherapy outcomes. Here, we present a systems biology approach to identify functional cell surface markers for TI-Tregs. We selected differentially expressed genes for surface proteins of TI-Tregs and compared these with other CD4+ T cells using bulk RNA-sequencing data from murine lung cancer models. Thereafter, we filtered for human orthologues with conserved expression in TI-Tregs using single-cell transcriptome data from patients with non-small cell lung cancer (NSCLC). To evaluate the functional importance of expression-based markers of TI-Tregs, we utilized network-based measure of context-associated centrality in a Treg-specific coregulatory network. We identified TNFRSF9 (also known as 4-1BB or CD137), a previously reported target for enhancing antitumor immunity, among the final candidates for TI-Treg markers with high functional importance score. We found that the low TNFRSF9 expression level in Tregs was associated with enhanced overall survival rate and response to anti-PD-1 immunotherapy in patients with NSCLC, proposing that TNFRSF9 promotes immune suppressive activity of Tregs in tumor. Collectively, these results demonstrated that integrative transcriptome and network analysis can facilitate the discovery of functional markers of tumor-specific immune cells to develop novel therapeutic targets and biomarkers for boosting cancer immunotherapy.

Tumor-Infiltrating Regulatory T-cell Accumulation in the Tumor Microenvironment Is Mediated by IL33/ST2 Signaling.

Regulatory T cells (Treg) are enriched in the tumor microenvironment (TME) and suppress antitumor immunity; however, the molecular mechanism underlying the accumulation of Tregs in the TME is poorly understood. In various tumor models, tumor-infiltrating Tregs were highly enriched in the TME and had significantly higher expression of immune checkpoint molecules. To characterize tumor-infiltrating Tregs, we performed bulk RNA sequencing (RNA-seq) and found that proliferation-related genes, immune suppression-related genes, and cytokine/chemokine receptor genes were upregulated in tumor-infiltrating Tregs compared with tumor-infiltrating CD4+Foxp3- conventional T cells or splenic Tregs from the same tumor-bearing mice. Single-cell RNA-seq and T-cell receptor sequencing also revealed active proliferation of tumor infiltrating Tregs by clonal expansion. One of these genes, ST2, an IL33 receptor, was identified as a potential factor driving Treg accumulation in the TME. Indeed, IL33-directed ST2 signaling induced the preferential proliferation of tumor-infiltrating Tregs and enhanced tumor progression, whereas genetic deletion of ST2 in Tregs limited their TME accumulation and delayed tumor growth. These data demonstrated the IL33/ST2 axis in Tregs as one of the critical pathways for the preferential accumulation of Tregs in the TME and suggests that the IL33/ST2 axis may be a potential therapeutic target for cancer immunotherapy.

Genome-wide identification of differentially methylated promoters and enhancers associated with response to anti-PD-1 therapy in non-small cell lung cancer.

Although approved programmed cell death protein (PD)-1 inhibitors show durable responses, clinical benefits to these agents are only seen in one-third of patients in most cancer types. Therefore, strategies for improving the response to PD-1 inhibitor for treating various cancers including non-small cell lung cancer (NSCLC) are urgently needed. Compared with genome and transcriptome, tumor DNA methylome in anti-PD-1 response was relatively unexplored. We compared the pre-treatment methylation status of cis-regulatory elements between responders and non-responders to treatment with nivolumab or pembrolizumab using the Infinium Methylation EPIC Array, which can profile ~850,000 CpG sites, including ~350,000 CpG sites located in enhancer regions. Then, we analyzed differentially methylated regions overlapping promoters (pDMRs) or enhancers (eDMRs) between responders and non-responders to PD-1 inhibitors. We identified 1007 pDMRs and 607 eDMRs associated with the anti-PD-1 response. We also identified 1109 and 1173 target genes putatively regulated by these pDMRs and eDMRs, respectively. We found that eDMRs contribute to the epigenetic regulation of the anti-PD-1 response more than pDMRs. Hypomethylated pDMRs of Cytohesin 1 Interacting Protein (CYTIP) and TNF superfamily member 8 (TNFSF8) were more predictive than programmed cell death protein ligand 1 (PD-L1) expression for anti-PD-1 response and progression-free survival (PFS) and overall survival (OS) in a validation cohort, suggesting their potential as predictive biomarkers for anti-PD-1 immunotherapy. The catalog of promoters and enhancers differentially methylated between responders and non-responders to PD-1 inhibitors presented herein will guide the development of biomarkers and therapeutic strategies for improving anti-PD-1 immunotherapy in NSCLC.