Malignant gliomas are incurable brain neoplasms with dismal prognoses and near-universal fatality, with minimal therapeutic progress despite billions of dollars invested in research and clinical trials over the last 2 decades. Many glioma studies have utilized disparate histologic and genomic platforms to characterize the stunning genomic, transcriptomic, and immunologic heterogeneity found in gliomas. Single-cell and spatial omics technologies enable unprecedented characterization of heterogeneity in solid malignancies and provide a granular annotation of transcriptional, epigenetic, and microenvironmental states with limited resected tissue. Heterogeneity in gliomas may be defined, at the broadest levels, by tumors ostensibly driven by epigenetic alterations (IDH- and histone-mutant) versus non-epigenetic tumors (IDH-wild type). Epigenetically driven tumors are defined by remarkable transcriptional programs, immunologically distinct microenvironments, and incompletely understood topography (unique cellular neighborhoods and cell–cell interactions). Thus, these tumors are the ideal substrate for single-cell multiomic technologies to disentangle the complex intra-tumoral features, including differentiation trajectories, tumor-immune cell interactions, and chromatin dysregulation. The current review summarizes the applications of single-cell multiomics to existing datasets of epigenetically driven glioma. More importantly, we discuss future capabilities and applications of novel multiomic strategies to answer outstanding questions, enable the development of potent therapeutic strategies, and improve personalized diagnostics and treatment via digital pathology. Epigenetically driven gliomas harbor unique single-cell and spatial profiles;IDH and histone mutations result in distinct microenvironment features;Novel spatial omics technologies present a rich opportunity to study glioma subtypes1 The advent of single-cell sequencing, and more recently, spatial omics technologies, have catalyzed the rate of discovery in brain tumors and represent a promising frontier to inform the development of novel therapies. Taken together, these “multiomic” technologies capture tens of thousands of transcriptional and epigenetic features within individual cells through various sequencing and imaging approaches, allowing for the vast spectrum of cell states and interactions to be studied at an unprecedented resolution.2–4 A growing volume of studies features these techniques along with increasing experimental sophistication and computational power in order to dissect and characterize both the heterogeneity and the common features in the glioma microenvironment. These studies have uncovered core principles of glioma pathogenesis5–7 which motivate promising novel therapeutic strategies for this devastating disease including the identification of actionable targets for differentiation therapy8 and immunotherapy9 among other approaches.Cancers arising from glial cells (gliomas) are nearly universally fatal with limited therapeutic options aside from surgical resection, chemotherapy, and radiotherapy. Glioblastoma (GBM), the most aggressive form, has a 5-year survival of less than 5%.11 Notably, the epigenomic features and unique microenvironmental landscapes  suggest that high-dimensional profiling with multiomic technologies is aptly suited to study such tumors. IDH mutations define a subset of high-grade glioma and >80% of diffuse low-grade gliomas, of which 50-75% progress to become World Health Organization (WHO) grade IV tumors.12 Histone mutations (H3K27 and H3G34) are common in pediatric gliomas, with the H3K27M mutation found in 80% of diffuse intrinsic pontine gliomas,13 as well as in adult midline glioma. The WHO 2021 CNS update incorporates many additional genomic alterations and defines IDH-mutant (Mut) astrocytoma, IDH-Mut oligodendroglioma, and H3K27-altered diffuse midline glioma, among others, as separate entities from IDH-WT glioblastoma, recognizing their distinct clinical and histopathological features.14Many existing studies focus on isocitrate dehydrogenase (IDH)-wild type (WT) glioblastoma, the most common form of high-grade glioma, with many of the seminal studies and single-cell atlases specifically excluding cases with mutations in IDH or core histone proteins.15 In neoplastic cells, this results in the characteristic G-CIMP signature defined by CpG island hypermethylation,16 and 2-HG has far-reaching effects on the tumor microenvironment such as its ability to suppress antitumor T cell activity.17 The most common histone mutation (H3K27M) results in epigenetic dysregulation via global H3K27me3 reduction through PRC2 inhibition among other mechanisms.13 This epigenetic dysregulation allows for the accumulation of potent oncogenic genomic alterations  and malignant transformation. We collectively refer to these glioma subtypes (IDH/H3 mutant) as epigenetically driven gliomas.IDH and histone mutations are mutually exclusive, though both create unique, non-stochastic global epigenetic dysregulation resulting in cellular reprogramming and oncogenesis. Activating mutations in IDH (most commonly IDH1-R132H) produce the oncometabolite 2-hydroxygluatarate (2-HG), which inhibits DNA and histone demethylases, among other oncogenic epigenomic changes such as PDGFRA activation.The current review summarizes mechanistic insights gleaned from single-cell and spatial multiomic datasets and analyses of epigenetically driven gliomas  and prop6 Verhaak et al. were the first to describe a trancriptomically defined GBM classification scheme based on bulk RNA-sequencing, yielding four unique molecular subtypes: Proneural, neural, classical, and mesenchymal.18 Notably, IDH-mutant (Mut) tumors displayed exclusively proneural or neural signatures. More recently, single-cell sequencing has demonstrated that individual neoplastic cells fall along a spectrum of differentiation. Profiling of 6 IDH-Mut oligodendrogliomas by Tirosh et al. identified a cellular hierarchy in which proliferating stem-like cells give rise to astrocytic (AC) and oligodendrocytic (OC) tumor cells. This hierarchy was shown to be independent of tumor subclonality, such that derivatives of a single progenitor cell stochastically traverse either an AC or OC lineage.19Gliomas are fundamentally characterized by deranged cellular differentiation, with cell states that recapitulate the trajectory of the developing brain.TP53 and ATRX mutations) and IDH-Mut oligodendrogliomas .20 This study discovered multiple salient findings that were shared across IDH-A and IDH-O gliomas. For example, intra-subtype cellular lineage trajectories displayed nearly fixed ratios, and higher-grade tumors exhibited a greater proportion of cycling/undifferentiated cells. IDH-WT tumors differed markedly in their lineage composition, with a shift towards the AC-like lineage. Interestingly, whereas AC-like and OC-like lineage states are inversely correlated and mutually repressive in IDH-O tumors, IDH-A tumors feature multiple lineage identities within individual cells, suggesting that IDH-A represents an intermediate transcriptional state between IDH-O and IDH-WT glioma.This work was expanded by Venteicher et al. with a larger cohort of 16 patients across IDH-Mut astrocytomas -like, neural progenitor (NPC)-like, and mesenchymal), and experimentally demonstrated plasticity between these states using a lineage-tracing system.5 While the proneural to mesenchymal axis primarily captures the spectrum of neoplastic cell states in IDH-WT glioma,22 a distinctly mesenchymal state has not yet been described in de novo IDH-Mut tumors. However, integrated atlases of multiple datasets can greatly increase the power to uncover novel cell phenotypes10 and are much needed to corroborate the idea of transcriptional plasticity in astrocytic IDH-Mut gliomas. Importantly, a large study of 86 primary-recurrent patient-matched paired samples of IDH-WT GBM revealed a shift from proneural towards mesenchymal phenotypes using scRNA-seq.22 Through single-cell deconvolution of bulk RNA-seq, an analysis of 304 longitudinal patients demonstrated that IDH-Mut gliomas exhibit distinct cell-type changes during progression, tending to maintain a proneural phenotype and displaying an increase in stem-like neoplastic cells and a decrease in differentiated-like neoplastic cells.23 However, an analogous study at the single-cell level has not yet been undertaken.Wang et al. similarly identified three clusters of tumor cells across IDH-Mut gliomas, termed IDH-A, IDH-O, and IDH-S (stem-like) cells.24 Another report with scRNA-seq of 2 IHD-Mut gliomas and 16 IDH-WT gliomas presented tumor cells stratified by: Epithelial-to-mesenchymal transition, MYC-targets, interferon-gamma response, and hypoxia signatures.9 In IDH-Mut glioma, sequencing of 576 SOX2+ sorted cells (putative glioma stem cells) across 6 patients uncovered rare populations with differential metabolic properties that are independent of underlying differentiation states.25Differentiation states alone may not adequately capture important  cell populations or explain physiologic activity in glioma cells. One report in IDH-WT glioma proposed an scRNA-seq-based classification scheme of 4 states based on both differentiation and metabolic pathway expression: Glycolytic/plurimetabolic, mitochondrial, neuronal, and proliferative/progenitor.26 a recent analysis of an integrated scRNA-seq dataset of 4 IDH-WT and 3 IDH-Mut tumors suggests that IDH-Mut tumors harbor unique GABAergic neuron-like neoplastic cells which are marked by SOX11.27 Functional validation using a patch-clamp demonstrated that these cells uniquely exhibited neurophysiological properties. Taken together, these findings suggest that IDH-Mut tumors possess distinct metabolic and electrophysiologic properties that correspond to unique clinical and pathological features.Lastly, following the seminal work by Venkatesh et al. demonstrating that glioma cell synaptic integration promotes tumor progression,28 However, a later study of 22 IDH-A/O gliomas  recapitulated the prior three scRNA-seq-based cell states -like).29 Remarkably, this study identified increased open chromatin epigenomic states in IDH-A tumors, and linked astrocytic/oligodendrocytic phenotypes to transcription factor induction by secondary mutations . A recent single-cell multiomic analysis by Wei et al. suggested that both IDH-A and IDH-O gliomas resemble early stages of the oligodendrocyte lineage with a differentiation block due to hypermethylation and suppression of essential regulators of the myelination program.30 This analysis further elucidates the fundamental mechanistic role of IDH mutations across IDH-Mut astrocytomas and oligodendrogliomas and motivates a potential differentiation therapy approach for IDH-Mut glioma. Importantly, these studies emphasize the need for multiomic interrogation and epigenetic profiling of IDH-Mut glioma.An initial single-nucleus assay for transposase accessible chromatin using sequencing (snATAC-seq) study of IDH-Mut gliomas consisting of only 336 cells corroborated stratification of IDH-Mut gliomas by the IDH-A/O classification.31 They identified clear separation of methylation patterns between IDH-Mut and IDH-WT tumors and identified more granular localization of epigenomic dysregulation, including unique enhancer hypermethylation with differentiation to AC and OC-like cells and decoupling of DNA methylation and expression at distinct genomic loci related to expression of cell cycle and proliferation genes. A separate multiomic study of ~1000 single-cell DNA methylomes and concomitant scRNA-seq similarly found that methylation patterns stratify IDH-Mut and IDH-WT cells, while superimposition of methylation and transcriptomic patterns identified unique cell states representative of distinct physiologic events, namely the cellular response to hypoxia.32Lastly, novel methods of single-cell DNA methylation profiling are able to capture the dysregulated DNA methylation unique to IDH-Mut gliomas. Chaligne et al. applied multimodal single-cell reduced-representation bisulfite methylation sequencing (RRBS) + scRNA-seq + targeted genotyping in ~800 IDH-Mut and ~800 IDH-WT cells across 14 patients.n = 6 patients).33 Notably, these studies inferred that the cell of origin/progenitor state in H3K27M-Mut glioma more closely resembled oligodendrocyte progenitor cells (OPCs), while IDH-Mut glioma progenitors resembled neural progenitor cells. Comparison of H3K27M-Mut glioma and IDH-Mut glioma reveal a higher fraction of non-proliferating OC-like cells in H3K27M-Mut glioma.34 These findings strongly imply that H3K27M histone mutations specifically induce a differentiation block along the OPC-OC axis and maintain a source of OPC-like progenitors to repopulate the tumor.34 Importantly, this work ascribes a unique functional role of OPCs to the maintenance of H3K27M-Mut gliomas.The characterization of histone-mutated gliomas has especially benefited from multiomic analyses. H3K27M-mutant gliomas were initially profiled at the single-cell level by Filbin et al., who identified a similar trajectory to that of IDH-Mut glioma involving proliferating stem cell-like cells which self-renew and differentiate into AC-like and OC-like cells  than IDH-O tumors,20 and are enriched for monocytic-lineage cells from circulation. Interestingly, myeloid cells in IDH-O tumors are mostly comprised of microglia.29 In H3K27M-Mut gliomas, microglial transcriptional signatures are observed in pediatric patients, while adult patients exhibit more macrophage signatures.33 In all cases, a spectrum of macrophage to microglia phenotypes is observed, indicating that a binary classification of bone-marrow-derived macrophages and resident microglia as previously defined44 may not be sufficient to characterize these cells. However, considering that immune populations in gliomas are rare in general  and especially depleted in epigenetically driven gliomas compared to IDH-WT gliomas, single-cell profiling of bulk tumors is generally only able to profile enough cells for a broad characterization.Single-cell omics approaches have emerged as a crucial tool for dissecting the tumor-immune microenvironment (TIME). It has been well established that the TIME of epigenetically driven gliomas differs from that of IDH-WT gliomas in key ways. Notably, IDH-Mut tumors exhibit immune-cold signatures characterized by a general paucity of immune cells.45 Recently, the largest profiling study to date by Gupta et al. characterized the immune cells of 18 gliomas across IDH mutation and recurrence status, and identified a unique subset of metabolically enriched microglia that were restricted to IDH-Mut gliomas.46 Moreover, they similarly identified a population of TREM2+ myeloid cells and showed in a mouse model that these cells mediate antitumor immunity in glioma, in contrast to the immunosuppressive role of TREM2 reported in other cancers.46 Perhaps more than for other cellular components of glioma, it is evident that single-cell multiomic analyses are desperately needed for lucid evaluation of the immune compartment.Sorting of CD45+ cells followed by scRNA-seq enables finer dissection of immune cell types, and recent reports demonstrate that the IDH-Mut TIME is distinct from that of IDH-WT glioma. First, profiling of enriched myeloid cells across 4 IDH-WT and 3 IDH-Mut grade IV gliomas identified a predominance of TREM2+ macrophages in IDH-Mut glioma along with fewer monocytes and non-inflammatory macrophages.47 In a second study of immune cell sequencing of both mouse and human glioma , Alghamri et al. identified that G-CSF secreted by stem cell-like cells in IDH-Mut glioma (detectable in human serum samples) induces proliferation of non-immunosuppressive granulocytes, and demonstrated the therapeutic potential of G-CSF in a mouse glioma model.48 While IDH-Mut glioma is characterized by an immunosuppressive microenvironment, multiomic analyses have the potential to uncover targetable interactions for immunomodulatory therapeutics.In contrast to studies suggesting predominantly unidirectional roles for IDH-Mut myeloid cells, additional studies have revealed distinct immunosuppressive and inflammatory interactions between myeloid cells and tumor cells in IDH-Mut tumors. By profiling 8 IDH-WT/6 IDH-Mut tumors along with in vitro and in vivo studies, Friedrich et al. directly implicated the oncometabolite 2-hydroxyglutarate in modulating macrophage tryptophan metabolism, resulting in increased immunosuppression in IDH-Mut tumors. They subsequently demonstrated that targeting tryptophan metabolism can reverse this immunosuppression.49 Importantly, their key finding that the CD161 receptor inhibits T cell-mediated killing of glioma is relevant independent of IDH status.In addition to myeloid cells, a glioma T cell atlas from 16 IDH-WT and 15 IDH-Mut gliomas by Mathewson et al. demonstrates important differences in the lymphoid populations. Notably, while both tumor types shared a similar composition of infiltrating T cell subsets, both CD4+ and CD8+ T cell populations were greater in number and had greater cytotoxicity, interferon, and cellular stress programs in IDH-WT glioma.Overall, single-cell studies have revealed that IDH-Mut glioma harbors a complex TIME with both immunosuppressive and immune activating factors that drive the clinical course of the disease and may yield therapeutic opportunities. Notably, there is a paucity of single-cell immune data for histone-mutant gliomas, with many open questions as to the mechanisms of immune evasion in those glioma subtypes.50 revealed localized shifts in immune cell composition in GBM explants under immunotherapy,51 low-plex RNA-FISH directly visualized paracrine signaling between neoplastic cells and adjacent nonmalignant glia,22 and spatial RNA-sequencing identified spatially distinct transcriptional programs and an immunosuppressive tumor-myeloid niche.52 Spatial proteomics enables direct spatial immunophenotyping, correlation with protein stains used in routine neuropathology, quantification of extracellular features, and specific identification of neoplastic cells through the detection of mutant IDH or histone proteins when applicable. However, these approaches can only quantify up to around 60 targets. Spatial transcriptomic approaches allow for the precise quantification of hundreds to thousands of RNA targets, yet are less equipped to study extracellular features and are less translationally applicable.Spatial profiling technologies are still in their infancy, but present unprecedented opportunities to study the tumor microenvironment in gliomas. Paired with robust computational approaches, these techniques can identify ligand-receptor interactions, characterize specialized cellular neighborhoods, and uncover spatially defined biomarkers. Current work is limited thus far, especially with regard to epigenetically driven tumors. However, recent work in IDH-WT tumors has demonstrated the utility of these approaches, despite limited sample sizes. For example, spatial proteomics using co-detection by indexing (CODEX)53 The analysis of IDH-Mut tumors was limited to recapitulating key findings about immune infiltration, notably that NK cells and macrophages are reduced in IDH-Mut tumors. These limitations are primarily due to the use of a broad antibody panel with limited resolution to discern tumor cell states. Current spatial proteomic analysis of histone-mutant tumors is quite minimal, with published datasets limited to a 24-plex cyclic immunofluorescence panel (CyCIF)54 on 11 H3K27M-Mut tissue microarray cores54 and an 8-plex CODEX panel in 4 H3K27M-Mut gliomas.35In the largest spatial profiling effort to date, Karimi et al. applied imaging mass cytometry, which employs metal-tagged antibodies followed by precise laser ablation and quantification. They measured 42 protein targets, profiling 389 images across 139 HGGs and 46 brain metastases, including 19 images from IDH-Mut glioma.55 seqFISH,56 GeoMx,57 Slide-seq,58 and the 10x Genomics platforms, Visium and Xenium. The largest spatial transcriptomics dataset for IDH-Mut tumors was generated by Kuchroo et al. using the 10x Genomics Visium platform and includes 10 IDH-Mut astrocytomas across WHO grades II–IV.59 Through a novel ligand-receptor simulation analysis, they enforced the significance of the previously established SPP1-CD44 signaling axis in glioma and identified that genes related to hypoxia regulation and antigen presentation are directly associated with this ligand-receptor interaction.59Spatial transcriptomics can be accomplished via hybridization of fluorescent oligonucleotide probes with direct visualization, or via RNA-capture and next-generation sequencing of regions (“spots”) on a microfluids platform. The former allows for high resolution and high accuracy at the single-cell level but requires a customized gene panel limited to several hundred targets. The latter allows for sequencing of the whole transcriptome, but is currently limited to a resolution of 1 to 10 cells per spot and thus requires computational deconvolution approaches, although it will likely achieve true single-cell resolution in the near future. There is a growing range of methods including MERFISH,60 which utilizes the amplification of barcoded probes, on 16 regions across 14 patients with a 116 cell-type-specific gene panel.35 Interestingly, they found that AC-like cells constitute the major malignant cell compartment when assessed via HybISS, challenging the consistent finding by scRNA-seq that OPC-like stem cells are the major cell type in H3K27M-Mut glioma. The authors hypothesize that cell dissociation during sequencing protocols  could differentially damage AC-like cells, enforcing the need for orthogonal single-cell profiling techniques. Notably, this observation does not challenge the key hypothesis that there is a differentiation block to the OC-like state. In addition, the authors identified a pattern of colocalization between vascular cells and mesenchymal cells as well as a niche of cycling OPC-like and OC-like cells surrounded by non-proliferating AC-like cells.As described earlier, Liu et al. complemented their single-cell RNA and ATAC atlas of H3K27M-mutant gliomas with spatial transcriptomics via hybridization-based in situ sequencing (HybISS),n = 3), IDH-Mut (n = 2), and H3K27M-Mut (n = 5) glioma.61 They applied 10x Genomics Visium spatial transcriptomics and identified four spatially defined gene expression modules conserved across samples representing hypoxic, vascular, invasive, and tumor core niches. They found that OPCs were most abundant in the tumor core but were present in all niches. Importantly, they found that radial glia-like cells were most enriched in the neuron-rich invasive niche in H3K27M-Mut and IDH-WT samples but in the IDH-Mut samples, these cells were most enriched in the hypoxic niche. They also found that invasive growth of these radial glia is stimulated by FAM20C within the invasive niche.61A single study thus far, by Ren et al., integrates spatial profiling for IDH-WT  that underlie tumor progression and resistance to therapy. In accordance with this principle, Panditharatna et al. identified via a CRISPR screen that OPC-like GSCs specifically maintain H3K27M-Mut glioma via the SMARCA4 gene regulatory network and the BRG1-BAF complex.65 SMARCA4 or BRG1-directed therapy depleted the OPC-like cell population, increased AC-like differentiation, and improved survival in a mouse model.Understanding the intricate complexities of tumor cell states and their interactions with immune and stromal components is crucial to developing desperately needed treatments. The integration of multiomic analyses across a range of modalities is an indispensable tool for the identification of therapeutic targets directed at both tumor-intrinsic and tumor-extrinsic (microenvironment) compartments of epigenetic gliomas. For example, pushing the cells towards a particular state could unlock a specific vulnerability which could be exploited via a combination therapy, a strategy that has been proposed as “differentiation therapy.”22 principles of tumor progression have not been fully characterized in epigenetically driven subtypes at the single-cell level. A report of patients with IDH-Mut oligodendroglioma under therapy with the mutant-IDH inhibitor ivosidenib  revealed a shift towards differentiated AC-like states and away from undifferentiated and proliferating cells through scRNA-seq profiling.66 Given the current clinical trials inhibiting other epigenetic regulators such as EZH2 and HDAC in gliomas,63 single-cell characterization of subsequent studies would greatly improve our understanding of epigenetic dysregulation and glioma cell differentiation. For example, through the computational inference of tumor subclones via novel methods which infer copy number variations in single-cell multiomics data,67 one can identify subclones that are evasive or resistant to therapy in order to predict treatment response and identify targeted combinatorial therapeutic strategies.Single-cell approaches also provide strong insight into the effects of tumor therapy, which can identify biomarkers of therapy response and elucidate mechanisms of disease progression. While IDH-WT tumors have been shown to undergo a shift to the mesenchymal state under standard therapy,68 As described earlier,47 tumor-associated myeloid cells are a promising target for therapeutic intervention, and additional profiling will elucidate how these cells respond to immunotherapy in clinical trial contexts.Finally, single-cell profiling of immune cells under therapy would elucidate processes of local and systemic immune modulation and provide insight into how to improve the efficacy of immunotherapies. Profiling of myeloid cells from the CSF of four patients undergoing GD2-CAR T cell therapy for H3K27M-Mut glioma, a promising early trial, revealed populations characterized by differential interferon response over time.2 The promising technologies of spatial ATAC69 and spatial CUT&Tag70 will vastly improve our understanding of the interplay between the tumor microenvironment and epigenetic reprogramming. These approaches utilize a microfluidics system to capture a range of epigenetic features at a resolution of up to 20 μm. In addition, new spatial transcriptomic technologies such as MERFISH, GeoMx, and Xenium can profile up to thousands of genes at subcellular resolution in brain tissue.55 MERFISH has been applied with a limited panel in IDH-WT glioma,64 but most of these technologies have not yet been utilized to study epigenetically driven gliomas.Single-cell and spatial profiling have revealed fundamental pathological processes that define cell-intrinsic oncogenic mechanisms and microenvironment dysregulation in epigenetically driven gliomas . The rap71 are needed to understand the influence of age on glioma pathogenesis and whether there are other key transcriptional and epigenetic programs unique to pediatric IDH-WT glioma that have yet to be identified. It is likely that additional epigenetic or multifactorial drivers exist in pediatric glioma that are distinct from adult tumors. Moreover, larger cohorts are also needed for a deeper understanding of these tumors in general, including for adult gliomas. Namely, the role of secondary mutations in epigenetically driven glioma has only been superficially defined,29 with an unclear role of key oncogenic driver genes such as EGFR, PTEN, RB1, NF1, TERT, and CDKN2A on modulating the tumor microenvironment. In addition, recent evidence suggests IDH-Mut gliomas induce systemic effects on the immune system.72 These effects likely mediate immune suppression and may even have the potential to serve as biomarkers to predict the molecular subtype of the tumor, yet peripheral effects have not been characterized at the single-cell level and represent an important future direction.Along with the addition of new technologies, single-cell multiomic approaches are needed to address a range of unanswered questions. More pediatric glioma atlases, such as the one developed by the Human Tumor Atlas Network,73 Human Biomolecular Atlas Program (HuBMAP),74 and the BRAIN Initiative75 provide an invaluable resource for studying epigenetically driven gliomas. Reference mapping of non-tumor data onto glioma datasets is an important analytical tool for understanding cellular phenotypes and developmental trajectories. Therefore, larger datasets with more extensive profiling of rare cell types will be critical in dissecting the phenotypes of progenitor cells in epigenetically driven gliomas. Equally important is the computational ability to integrate datasets across modalities. Paired multimodal sequencing ameliorates this need in some cases, yet improved computational approaches will enable the expanded analysis of the rich compendium of existing datasets presented in this Review. Lastly, while there is a growing range of sequencing-based integration methods, there is an exciting opportunity for multiomic integration across sequencing and imaging modalities.77As single-cell omics techniques expand, relevant datasets of normal brain and sophisticated computational techniques are required to reconcile data gleaned from such strategies. To this end, the rapidly expanding single-cell omics datasets of non-neoplastic brain generated by consortium projects such as the Human Cell Atlas,78 Alternatively, the COMET system by Lunaphore has demonstrated automated imaging of up to 40 markers in under a day.79 High-throughput approaches will not only optimize standard neuropathology workflows, but allow for the quantitation of higher-dimensional biomarkers which can have prognostic value and inform targeted treatment options. Namely, tissue cellular neighborhoods, which are recurrent functional patterns of spatial organization between cell types, have been shown to be useful variables for identifying and delineating tumor subtypes in other cancer types.81 Moreover, while there is an expanding toolkit of computational approaches to deconvolute spatial motifs and identify important patterns of cellular interaction,82–84 there remains a pressing need for analytical strategies to translate this data into clinically actionable information. In addition to applying multiplexed imaging directly, another avenue of clinical translation involves correlating spatial niches and topological features identified through spatial multiomics with histopathological features on a hematoxylin and eosin-stained slide. Given well-annotated training data, sophisticated machine learning algorithms are well suited to identify useful features that are not readily apparent to the eye of a pathologist and provide meaningful interpretations, and are well poised to greatly expand the scope of clinically relevant information garnered by routine neuropathology.Currently, most high-dimensional multiomic profiling techniques discussed in the present Review are costly and require lengthy protocols and sophisticated laboratory techniques to perform successfully. However, there is a burgeoning opportunity for high-throughput spatial and multiomic technologies to provide direct clinical benefit in the context of digital pathology. For example, the multiplexed immunohistochemistry (IHC) InSituPlex system by Ultivue enables the simultaneous detection of up to 12 protein targets on a single tissue slide with high-throughput capacity on the order of only several hours of imaging time per slide.Single-cell sequencing technologies remain further away from direct clinical application. While they can be used to study the cellular response to therapy as previously noted, whole transcriptome and whole-genome approaches remain too complex and costly to provide routine diagnostic and therapeutic guidance. However, there is strong potential for their future utility in epigenetically driven glioma. For instance, single-cell sequencing may be necessary to identify therapeutic vulnerabilities that are present in rare stem cell-like populations that cannot be detected via deconvolution of bulk RNA-seq. In addition, key discoveries gleaned from multiomic profiling of transcriptional and epigenetic features may motivate the incorporation of bulk epigenetic profiling  into clinical practice. Lastly, while earlier in development, targeted single-cell sequencing approaches, such as those offered by 10x Genomics and BD Rhapsody are more applicable to clinical translation. By capturing a limited panel of genes, these methods greatly reduce cost and increase scalability while preserving the information most critical for clinical diagnostics and treatment planning. In conclusion, epigenetically driven gliomas present an exciting opportunity for high-dimensional multiomic data to uncover complex mechanisms and develop targeted treatments and will undoubtedly provide invaluable data as we seek meaningful life-extending therapies for these horrific brain tumors.


The impacts of the coronavirus disease of 2019 (COVID-19) pandemic on mental health have been relatively severe.This study examined the influence of the COVID-19 especially on depression and suicidal ideation in community-dwelling elderly in Korea.Data were employed from a survey on elderly mental health in Jeollanam-do (southwest province in Korea). A total of 2,423 elderlies were recruited from 22 counties in Jeollanam-do between April and October 2021. We used self-reported questionnaires, including sociodemographic factors, COVID-19 related stress, suicidal ideation, Geriatric Depression Scale-Short Form Korean Version (GDS-SF). Logistic regression was performed to examine the factors on depression and suicidal ideationOf the 2423 subjects, 622 (25.7%) reported depressive symptoms and 518 (21.4%) reported suicidal ideation. The multivariate logistic regression analysis revealed that living alone, poor perceived health status, the worry of COVID-19 infection and restriction of daily activity due to COVID-19 pandemic were significantly associated with depression. Male sex, poor perceived health status, disability in house chores and depressive symptom are risk factors for suicidal ideation.Image:Image 2:Image 3:These findings showed that increased risk factor for depression and suicidal ideation in community dwelling elderly during COVID-19 pandemic. We confirmed that feelings of isolation and negative perception of health were risk factors on depression in community dwelling elderly in the context of the COVID –19 pandemic. Also male, poor self-perceived health status, difficulty of independent living and worry and depression are increased the risk of suicidal ideation among the elderly.None Declared


To address these challenges, smart drug delivery systems (SDDSs) have been developed to overcome tumor cell resistance to immunomodulators, restore or boost immune cell activity, and magnify immune responses. To combat resistance to small molecules and monoclonal antibodies, SDDSs are used to co-deliver numerous therapeutic agents to tumor cells or immunosuppressive cells, thus increasing the drug concentration at the target site and improving efficacy. Herein, we discuss how SDDSs overcome drug resistance during cancer immunotherapy, with a focus on recent SDDS advances in thwarting drug resistance in immunotherapy by combining immunogenic cell death with immunotherapy and reversing the tumor immunosuppressive microenvironment. SDDSs that modulate the interferon signaling pathway and improve the efficacy of cell therapies are also presented. Finally, we discuss potential future SDDS perspectives in overcoming drug resistance in cancer immunotherapy. We believe that this review will contribute to the rational design of SDDSs and development of novel techniques to overcome immunotherapy resistance.Cancer immunotherapy, a therapeutic approach that inhibits tumors by activating or strengthening anti-tumor immunity, is currently an important clinical strategy for cancer treatment; however, tumors can develop drug resistance to immune surveillance, resulting in poor response rates and low therapeutic efficacy. In addition, changes in genes and signaling pathways in tumor cells prevent susceptibility to immunotherapeutic agents. Furthermore, tumors create an immunosuppressive microenvironment  Cancer immunotherapy is a treatment strategy to keep cancer under control and eliminate cancer by reactivating and maintaining the immune system to identify and eliminate tumor cells. Cancer immunotherapy includes the use of cancer vaccines, cell therapy, immune checkpoint inhibitors, therapeutic antibodies, and small-molecule inhibitors5; however, the clinical responsiveness to immunotherapy varies greatly. Specifically, immune checkpoint blockade (ICB) has a clinical response rate of < 30%6. This finding is primarily a consequence of drug resistance to the immune system. Tumors have a complex genetic network that allows tumors to develop resistance to drugs that are detrimental to growth. For example, tumor cells overexpress programmed cell death protein 1 ligand (PD-L1), which inhibits cytotoxic T lymphocyte (CTL) activity by interacting with programmed cell death protein 1 (PD-1) on the T cell surface7. Indoleamine 2,3-dioxygenase 1 (IDO-1), which is overexpressed in tumor cells, reduces CTL function by increasing the conversion of tryptophan-to-kynurenine8. Mutations in gene expression or epigenetic changes result in decreased drug uptake, a greater efflux by tumor cells, or changes in drug target structure9. Furthermore, tumors have a heterogeneous metabolic regulatory network that maintains a complex heterogeneous immunosuppressive microenvironment by producing abnormal metabolites and recruiting various cells10. It is difficult for monoclonal antibodies and immune cells to overcome this barrier or become inactivated in response to numerous immunosuppressive cells or metabolites in the immunosuppressive microenvironment. These factors render tumor cells resistant to existing immunotherapies. Therefore, developing innovative techniques to overcome drug resistance is critical for enhancing cancer immunotherapy.Cancer immunotherapy has become a major area of research in cancer treatment during the last decade owing to the documented extraordinary success in treating or managing tumors11. SDDSs accurately deliver and release therapeutic agents to disease target sites (precise delivery and intelligent drug release) in response to specific stimuli by altering the structural functionalized components. For example, use of the acid-sensitive linker, maleic acid amide, to construct SDDSs has been shown to cause stimulus-activated disintegration in a tumor acid environment12. Matrix metalloproteinase-2 (MMP-2)- and MMP-9-sensitive materials respond to the tumor microenvironment by overexpressing MMP-2 and MMP-913. Furthermore, reactive oxygen species (ROS)-responsive materials have been synthesized using thione linkers or boronic acid-containing adducts14. Moreover, some materials with external stimuli-sensitive elements, such as light, electricity, magnetic fields, and temperature, have demonstrated improved targeted drug delivery17. Owing to these reports, SDDSs are thought to be a promising approach to overcome tumor drug resistance and enhance cancer immunotherapy18. SDDSs efficiently target tumor cells by passive or active targeting, enriching drug accumulation, triggering tumor cell death, boosting tumor antigen exposure, and improving immune recognition19. In contrast, SDDSs target immune cells by altering the functional phenotype of immune cells, causing immune cells to evolve in an anti-tumor direction, and enhancing immune system function20.With the rapid advances in life sciences and nanotechnology, smart drug delivery systems (SDDSs) are under development for cancer immunotherapy24; however, these reports did not discuss the application of SDDSs in overcoming drug resistance in cancer immunotherapy. Because drug resistance is currently one of the most significant issues in immunotherapy, it is imperative to discuss the use of SDDSs in immunotherapy to overcome drug resistance. Summarizing current SDDS evidence for overcoming drug resistance in cancer immunotherapy will also assist researchers in developing more efficient drug carriers and promote the use in immunotherapy. To this end, we have outlined the current mechanisms of drug resistance in cancer immunotherapy. Then, we focused on the mechanisms underlying SDDSs and applications of SDDSs from the perspective of overcoming drug resistance. Lastly, we discussed the existing obstacles and provide future prospective for the use of SDDSs in overcoming drug resistance in cancer immunotherapy .Several reviews have highlighted the progress of SDDS research for cancer immunotherapy in recent years, thus providing inspiring and prospective insight into the design of these delivery systems for cancer immunotherapy25. Patients who have primary resistance to ICB do not respond to the initial therapy. The most straightforward reason why a tumor does not respond to immune checkpoint therapy or adoptive cell therapy (ACT) is a lack of recognition by T cells due to an absence of tumor antigens. Alternatively, patients develop adaptive resistance if the cancer cells have tumor antigens but develop mechanisms to avoid antigen presentation on the major histocompatibility complex (MHC)-restricted surface due to alterations in the antigen-presenting machinery. Patients who develop acquired resistance initially have an objective response to ICB therapy with anti-CTLA-4 or anti-PD-1 will relapse over time, even despite receiving continued therapy. With the advances in immunology and cell biology, researchers have recognized that drug resistance to cancer immunotherapy, whether primary, adaptive, or acquired, is a continual dynamic evolutionary process that is mediated by tumor cells and the tumor microenvironment (TME)18. Given the intricacy and diversity of tumor immunotherapy resistance mechanisms, we have categorized the mechanisms as follows: (1) tumor cell-intrinsic mechanisms rely on the baseline expression of resistance driver proteins and mutations in tumor cells; and (2) tumor cell-extrinsic mechanisms block cell death pathways and enhance survival behaviors and characteristics via dynamic interactions between the tumor and the TME.There are three common clinical forms of resistance to cancer immunotherapy, as follows: (1) primary resistance, in which the tumor does not respond to immunotherapy from the beginning; (2) adaptive immune resistance, in which the tumor is recognized by the immune system but the tumor is protected by adapting to the immune attack; and (3) acquired resistance, in which the tumor initially responds to immunotherapy but becomes resistant to the drug after a period of treatment and begins to relapse26. Several inherent processes have recently been identified, as follows: (1) low drug uptake and high drug efflux; (2) target mutation; (3) low tumor antigenicity; (4) surface downregulation of surface MHC; (5) dual role of interferon (IFN) signaling; and (6) regulation of oncogenic signaling.Intrinsic cellular resistance mechanisms refer to the expression or inhibition of genes or pathways in tumor cells that limit immune cell infiltration or suppress the immunologic TME. These processes may exist intrinsically in tumor cells or emerge during tumor progression to influence drug uptake and T cell activation, thus preventing tumor death by the immune system27. In this case, a gene encodes a transmembrane protein that prevents drug entry into the cell while excreting the drug. Multidrug resistance protein 1 (MDR1), multidrug resistance-associated protein 1 (MRP1), and breast cancer resistance protein (BCRP) are the three most frequently studied transporter proteins firmly linked to drug resistance in numerous cancers30. All of these transporter proteins have broad basal specificity and excrete foreign chemicals from the cell, thus allowing cancer cells to develop drug resistance. Drug resistance reduces tumor antigen production, which reduces tumor recognition and killing by the immune system, and affects the efficacy of immunotherapy following chemoresistance. The current therapeutic strategy is to use combinations of treatment agents to halt the process of tumor drug resistance, thus prolonging patient survival31. When the drug target changes, the efficacy is reduced, eventually leading to drug resistance in tumor cells32. For example, several anti-cancer medications inhibit topoisomerase II, resulting in DNA damage, inhibition of DNA synthesis, and the end of the mitotic process. Further, cancer cells become resistant to topoisomerase II through a variety of mechanisms33 due to mutations in the topoisomerase II gene. In addition, human epidermal growth factor receptor 2 (HER2), a tyrosine kinase receptor of the EGFR family, is overexpressed in 30% of patients with breast cancer, and resistance can develop following long-term use of inhibitors targeting this receptor34.A drug must reach the target site at an adequate concentration to elicit a therapeutic effect. When a drug fails to attain the concentration required for therapy within a tumor cell, the drug fails to destroy the tumor, which may be due to drug entry failure or drug excretion. One of the most common issues pertaining to immunotherapy, is multidrug resistance gene amplification35. For example, long-stranded non-coding RNA (lncRNA) expression promotes degradation of antigenic peptide loading complexes (PLCs) and intrinsic tumor suppressors (Rb and p53) in triple-negative breast cancer (TNBC), resulting in antigenic downregulation and intrinsic tumor suppression36. Several studies have shown that tumor neoantigens are useful at inducing anti-cancer immune responses and exerting effectiveness in immunosuppressive malignancies38. Specialized T lymphocytes targeting neoantigens have been shown to proliferate and gain anti-cancer activity in response to ICB in a mouse sarcoma model39. Therefore, neoantigen-based cancer vaccines are projected to be an effective method for combating drug-resistant cancers40.Decreasing tumor antigenicity and neoantigen production result in loss or diminished T cell recognition41. IFN is known to stimulate or enhance MHC I antigen presentation42, whereas deficiencies in antigen processing can impair MHC I surface expression. For example, changes in proteasome subunits or transporter proteins (TAPs), deletion of the 2-microglobulin B2M gene, or secondary changes in the structure of human leukocyte antigen (HLA)-1 class molecules all contribute to decreased MHC I production in tumor cells45. Developing targeted therapeutic strategies to address these alterations is expected to maintain MHC expression on the tumor surface, thereby improving the tumor-killing effect of T cells46.Tumor cells escape T cell killing by downregulating the surface MHC I pathway47. IFNs inhibit tumor growth, promote tumor cell apoptosis, and increase immune cell activity48; however, IFNs also induce immune evasion by upregulating multiple immune checkpoints49. For example, T-cell responses against tumor antigens lead to IFN-γ expression in the TME, which activates Janus kinase (JAK)–signal transducer and activator of transcription (STAT) signaling, resulting in PD-L1 expression50. Induction of PD-L1 expression can be prevented by disrupting the tumor cell response to IFNγ signaling, rendering PD-1-PD-L1 blocking ineffective; however, this strategy represents a mechanism of resistance not only to the immune checkpoint, but also to anti-tumor immunity. Stronger anti-tumor effects are expected when ICB is combined with immunotherapy targeting IFNs51.IFNs are pleiotropic cytokines that have a key role in the coordination of tumor-immune system interactions+ dendritic cells (DCs)52. The mitogen-activated protein kinase (MAPK) signaling pathway contributes to cancer immune evasion by increasing expression of immunomodulatory cytokines (IL-6 and IL-10)53. Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) protein inactivation mutations increase tumor cell resistance to cytotoxic T cells54. It is expected that therapies that specifically target these signaling pathways will address resistance in tumor immunotherapy.Oncogenic signaling pathways may be related to drug resistance at many stages of cancer development, including tumor initiation, growth, invasion, and metastasis. For example, activation of the WNT/β-catenin signaling pathway in tumor cells prevents T cells from entering into the TME and reduces the number of CD10356.Cell extrinsic mechanisms include regulatory T cells (Tregs), myeloid-derived suppressor cells (MDSCs), tumor-associated macrophages (TAMs), tumor-associated neutrophils (TANs), aberrant physiologic indicators, and suppressive immune checkpoints, all of which contribute to tumor immunotherapy resistance57. These cells directly block the cytotoxic function of CD8+ T and NK cells, resulting in tumor cells that escape immune system destruction. Tregs, for example, suppress effector T cell responses through the secretion of suppressor cytokines, such as IL-10, IL-35, and TGF-β, as well as direct cellular interaction58. MDSCs regulate the immune system in a variety of ways, including the release of anti-inflammatory and suppressive cytokines , inducible nitric oxide synthase (iNOS) and Arg-1 expression, immune checkpoint expression, and synergy with other immune cells, such as Th17 and Tregs60. TAMs express PD-L1 and release IL-1061. TAMs also promote tumor growth by secreting MMPs, scraping the basement membrane, remodeling epithelial cell motility, releasing VEGF to stimulate angiogenesis, and recruiting Tregs and MDSCs62. Therefore, preventing tumor immune escape by targeting immunosuppressive cells at various stages of cancer growth could be a potential immunotherapeutic technique63.Cancer cells secrete chemokines and cytokines that recruit MDSCs, TAMs, Treg, TANs, and other immunosuppressive cells to primary and secondary tumor sites in primary tumors64. For example, an acidic environment (pH 6.2–6.9), overexpression of numerous enzymes, insufficient oxygen supply, and high glutathione (GSH) and ATP concentrations are some of the associated factors65. Additionally, aberrant metabolites produced by mutant tumor cells have been shown to influence effector T cell activity66. These signals can cause anti-tumor immune cells to stop functioning or increase the development of immunosuppressive receptors  on the surface of effector cells, thus boosting immunotherapy resistance68. Consequently, the development of targeted medicines or TME-responsive drug delivery systems for these abnormal physiologic indicators is predicted to reverse unfavorable TME conditions and boost or restore effector cell activity, thus overcoming immunotherapy resistance.It has been demonstrated that biochemical reactions in the TME cause significant metabolic abnormalities, resulting in disparities between the TME and normal tissuesSDDSs provide precise, targeted drug delivery and release in response to certain stimuli, thus potentially overcoming drug resistance in cancer immunotherapy. Current SDDSs utilize the following strategies to overcome drug resistance, as follows: (1) combining immunogenic cell death (ICD) with cancer immunotherapy; (2) reversing the tumor immunosuppressive microenvironment; (3) regulating IFN and PTEN signaling pathway; and (4) enhancing the efficacy of ACT. The rationale and recent research results for the different strategies are discussed below.69. ICD is recognized for its capacity to increase tumor antigen exposure, induce the release of tumor cell components, stimulate DC maturation, and activate adaptive antitumor immunity by activating T cells70. Currently, ICD inducers have been discovered in chemotherapeutic drugs, such as mitoxantrone and oxaliplatin71. ICD inducers are promising improved therapeutic applications due to anti-tumor cytotoxicity and activation of anti-cancer immunity; however, because of tumor efflux, the drug dose not attain a therapeutic concentration and the success rates of these agents in clinical practice are poor. SDDSs actively or passively target tumor tissues and deliver large amounts of ICD inducers to tumor cells, which increases the intracellular drug concentrations and efficiently induces ICD, resulting in increased DC maturation and CTL activation73. Li et al.74 proposed a dual endoplasmic reticulum (ER)-targeting strategy for photodynamic therapy (PDT) and photothermal therapy (PTT). ER-targeted pardaxin (FAL) peptide-modified indocyanine green (ICG)-conjugated hollow gold nanospheres (FAL-ICG-HAuNS) and hemoglobin (Hb) liposomes were used in this system (FAL-Hb liposomes). Under near-infrared (NIR) light irradiation, this nanosystem generated ICD and calreticulin (CRT) exposure, which served as an “eat-me” signal to enhance antigen presentation by DCs. This outcome triggered a cascade of immunologic responses, including CD8+ T cell growth and the release of cytotoxic cytokines. ER-targeted SDDS selectively regulates ER stress, thereby providing a controlled method for ICD-mediated tumor immunotherapy.ICD is a mechanism by which tumor cells control the anti-tumor immune response in response to external stimuli75; however, because IDO-1 inhibitors do not have a significant role in tumor immunotherapy, IDO-1 inhibitor use alone frequently fails to achieve therapeutic efficacy, similar to the combination with PD-1 checkpoint inhibitors for melanoma76. This finding could be attributed to limited T cell infiltration in tumor tissues and inhibition of anti-tumor immune activation. Combining IDO-1 inhibitors with ICD inducers using a SDDS may boost the immunotherapeutic efficacy of IDO-1 inhibitors. Feng et al.77 designed a TME-activated prodrug nanoparticle (BCPN) to achieve antitumor immunotherapy by synergistically inducing ICD and IDO-1 inhibition . In both acidic and reducing TMEs, BCPN released oxaliplatin (OXA), while GSH activated the IDO-1 inhibitor prodrug, NLG919. Activated OXA increased CTL aggregation in cancer cells by activating ICD, while NLG919 inhibited IDO-1-mediated immunosuppression. To achieve spatiotemporal control of tumor aggregation and deep penetration of nanoparticles, Feng et al.77 also developed light-induced nanoparticles (LINCs) to control tissue penetration and drug release of these carriers by NIR, while inducing ICD and IDO-1 inhibition to improve tumor immunotherapy78. To more accurately adjust the cascade release of nanodrugs in TME, Hou et al.79 reported boolean logic prodrug nanoparticles (BLPNs) for co-delivery of pheophorbide A (PPa) and NLG919 . As a biocomputational platform, BLPNs are activated cascade-after-cascade in response to various inputs. First, the enzymatic breakdown of the peptide sequence, GALGLPG (GG), in the TME by MMP-2 and MMP-9 overexpression permits tumor-specific accumulation and retention of BLPNs. Second, in the presence of GSH, the disulfide bond dissolves, thus releasing NLG919. Finally, 2-(diisopropylamino) ethyl methacrylate (DPA) decomposes to liberate PPa. The integration of various stimulatory signals into one chain can result in tumor-specific distribution and activation of BLPNs, thereby opening up new avenues for overcoming drug resistance in cancer immunotherapy.IDO-1 overexpression in tumor cells stimulates Tregs, while reducing effector T and NK cell activities. IDO-1 drugs inhibit tryptophan breakdown and reverses tumor cell suppression of T lymphocytes80. Therefore, several SDDS strategies have been created to deliver PD-1/PD-L1 antibodies6. Using SDDSs to co-deliver ICD inducers and PD-1/PD-L1 antibodies not only activates anti-tumor immunity but also prevents immunosuppression, which is a promising technique for tumor immunotherapy.Although ICD inducers activate the immune system, the ICD inducer effectiveness is restricted by tumor resistance, especially due to the existence of immune checkpoints. One of the most important immune checkpoints for CTL activity suppression is the PD-1/PD-L1 pathway. Some PD-1/PD-L1 checkpoint inhibitors are currently available commercially; however, due to tumor heterogeneity and tumor-intrinsic resistance mechanisms, such as altered IFN signaling pathways, increased mutations, defective antigen presentation, and drug-resistant gene expression, the clinical response rate of PD-1/PD-L1 therapies is approximately 30%81 proposed an MMP-2-sensitive anti-PD-L1 antibody (aPD-L1)/ICG-based nanoparticle (S-aPD-L1/ICG@NP) that counteracts drug resistance in cancer immunotherapy by blocking the PD-1/PD-L1 cascade . Through enhanced permeability and retention (EPR), aPD-L1/ICG@NP passively accumulates at the tumor site and is activated to release aPD-L1 in high MMP-2-expressing tumors to block PD-L1 on tumor cells. ICG increases intratumoral CTL infiltration and sensitizes tumors to PD-L1 inhibition. As a potent nanoplatform, this system can be used to deliver immune checkpoint inhibitors to overcome drug resistance during cancer immunotherapy. Zhou et al.82 developed a bispecific nanomodulator (IQS) to trigger ICD and dual blockade of the PD-L1 and IDO-1 pathways, thus enhancing effective antitumor immunity . IQS is self-assembled from ICG, the BRD4 inhibitor, JQ1, and the IDO-1 inhibitor, BMS986205 (BMS). PTT and ICD are effectively induced under NIR irradiation. Surprisingly, IQS reverses PTT-mediated PD-L1 upregulation and IDO-1 activation, resulting in increased CTL invasion. Furthermore, IQS causes immunologic memory effects that significantly suppress tumor growth. In addition to PD-1/PD-L1, the immunologic checkpoint, CD47, which is expressed on numerous malignant tumor cells, operates as a “do not eat me” signal to block DCs from phagocytosing tumor cells83. Monoclonal anti-CD47 antibodies (aCD47) have been tested in clinical trials and have shown great potential84. Zhou et al.85 designed a TME-activated vesicle for co-delivery of the OXA prodrug and PEGylated photosensitizer (PS) to trigger ICD in tumor cells. The platform showed that the combined effect of prodrug vesicle-induced ICD and aCD47-mediated CD47 blockage boosts anti-tumor immunity of the ICD inducer. Given that CD47 is overexpressed in a variety of tumor cells, ICD plus aCD47 may be a potential strategy for overcoming ICD inducer resistance in cancer immunotherapy.Wang et al.86. In addition, the development of immunosuppressive metabolites in the TME impairs cholesterol metabolism in tumor-infiltrating CD8+ T cells87. As a result, inhibiting tumor cell cholesterogenesis is a viable strategy for overcoming immunotherapy resistance. Liu et al.88 used lipid nanovesicles co-encapsulated with the MMP-2 enzyme-responsive proinfiltration peptide, iRGD-modified Ppa, and the cholesterol esterase (ACAT) inhibitor, avasimibe, to modulate cholesterol metabolic pathways and enhance PDT . Nanoparticles passively accumulate at the tumor site, and in response to MMP-2, iRGD is released from the nanoparticles, promoting deep tumor penetration of avasimibe. Cholesterol metabolism is critical for T cell signaling and effects89. Avasimibe has the potential to inhibit cholesterol metabolism in CD8+ T cells and tumor cells, restoring T cell function. Furthermore, PDT-triggered ICD is enhanced by regulation of cholesterol metabolism. This study described a novel approach to improve the efficacy of ICD by modulating cholesterol metabolic pathways.It has been reported that elevated cholesterol levels are a common drug resistance phenomenon, and that elevated cholesterol mediates nuclear translocation of the transcription factor, SP1, which binds directly to the estrogen-related receptor alpha (ERRα) promoter. By activating the EGFR/Src/Erk signaling axis, transcription is allowed to occur in the presence of EGFR-TKIs, thus resulting in acquired resistance to EGFR-TKIs90 created a synthetic high-density lipoprotein (sHDL) to regulate the interactions of intratumoral M2 macrophages, DCs, and MDSCs . Vadimezan, an agonist of the mouse interferon gene stimulator, STING, and/or gemcitabine (Gem), a DNA synthesis inhibitor, were loaded into the sHDLs. Gem selectively kills M2 macrophages in sHDLs, whereas vadimezan promoted DC maturation and monocyte differentiation into antitumor M1 macrophages. These sHDLs effectively reverse M2-mediated immunosuppression, decrease MDSC differentiation, and increase the percentage of M1 macrophages, thereby significantly improving immunotherapeutic efficacy. Furthermore, using a biomimetic delivery system to target immunosuppressive cells can effectively evade immune system clearance, while allowing active targeting of drugs to specific cells for precise delivery. Wang et al.91 created a self-assembled nanorethrocyte system [V(Hb)] for delivery of the chemotherapeutic, adriamycin (DOX), to reprogram TAMs . For effective cell killing, the Hb fraction binds to endogenous plasma haptoglobin (Hp) and specifically targets M2-type TAMs via CD163 surface receptors. TAM targeting effectively reduces levels of immunosuppressive cytokines (IL-10 and TGF-β), while increasing immunostimulatory IFN-γ and CTL anti-tumor killing responses. This endogenous TAM-targeting biomimetic system is a promising tool that can be used in conjunction with ICB to combat drug resistance in cancer immunotherapy.Targeting immunosuppressive cells in the TME, such as MDSCs and TAMs, to restore anti-tumor immunity by inhibiting immunosuppressive cell function is thought to be a promising strategy for overcoming drug resistance in cancer immunotherapy. Antagonism of immunosuppressive cells on T-cell function can be decreased, boosting ICB potency; however, such antagonism can minimize the production of aberrant metabolites in the TME and cause drug inactivation. Wang et al.in situ genetic engineering of immunosuppressive cells in the TME has been developed as a novel strategy for overcoming drug resistance in cancer immunotherapy. This engineering strategy transforms immunosuppressive cells into anti-tumor subtypes, resulting in long-lasting tumor killing effects. Chen et al.92 developed a nanoparticle-hydrogel capable of generating glioma stem cell (GSC)-specific chimeric antigen receptor macrophages/microglia (CAR-MΦs) around the lumen of postoperative glioma lesions . After intraluminal delivery, nanoparticles transfer CAR genes into broadly-distributed M2-type macrophages, resulting in CAR-MΦs in a glioma animal model. CAR-MΦs exhibit M1-type macrophage features and are capable of seeking out and engulfing GSCs as well as clearing remaining GSCs in TME by triggering adaptive anti-tumor immune responses. When combined with the aCD47 antibody, the number of positive immune response cells increases.In addition to direct drug delivery to immunosuppressive cells, 94. Treatment techniques targeting the ECM typically involve matrix-degrading enzymes to hydrolyze the excess protein matrix or functional ablation of the ECM with suitable medicines. Tan et al.95 reported a bioinspired lipoprotein (bLP) that induces efficient PTT to remodel the ECM . Multiple stromal cells and ECM components of the TME are extensively disrupted following bLP-mediated PTT, resulting in a 4.27-fold increase in the secondary accumulation of bLP in tumors, deep penetration throughout the tumor block, and a 27.0-fold increase in cancer cell accessibility. Notably, ECM remodeling greatly suppressed tumor development and resulted in a 97.4% inhibition of lung metastasis. Wang et al.96 constructed a pH-responsive dextran (DEX)-hyaluronidase (HAase) polymer (DEX-HAase) nanoparticle . DEX-HAase dissociates within the acidic TME, releasing native HAase, which induces the breakdown of HA and weakens the ECM structure, allowing oxygen and other therapeutic agents to penetrate more effectively. After pretreatment with DEX-HAase, the therapeutic response to PDT combined with aPD-L1 is greatly improved. This study presented a new adjuvant nanodrug that disrupts the ECM to enhance PDT immunotherapy. Although ECM degradation improves the permeability of nanomedicines and immune cells, ECM degradation also facilitates tumor cell extravasation into blood vessels, resulting in tumor metastasis and recurrence93. As a result, how to minimize tumor cell extravasation is critical to consider in ECM-targeted therapy. Liu et al.97 developed a self-assembled liposome that was subsequently conjugated with anti-fibroblast (CAF) antibodies to ensure that prolyl isomerase Pin1 inhibitors are specifically delivered to CAFs. This technique efficiently reduces CAF function while causing less ECM damage and represents an alternative strategy for overcoming the stromal barrier in pancreatic cancer.The aberrant deposition of ECM outside tumor cells increases the pressure inside the solid tumor, thus reducing the ability of nanomedicines and immune cells to penetrate deep into tumors, which leads to drug resistancein situ creation of oxygen by catalyzing excess H2O2 in the TME; and (2) direct supply of oxygen via oxygen carriers.The excessively active growth of tumors creates a number of abnormal metabolic microenvironments, such as abnormal elevation of glycolipid metabolism and accumulation of the glycolytic product, lactate, thus generating an acidic environment (pH = 6.5–6.8), internal hypoxia, upregulation of ROS levels, and overexpression of protease levels. Among these effects, the creation of an acidic and hypoxic TME not only promotes tumor growth, but also significantly inhibits the efficacy of chemotherapeutic drugs and photosensitizers, which may be inactivated by structural alterations in the acidic environment. Current SDDS-based management of an acidic tumor environment is mostly accomplished through the following: (1) depletion of lactate in the TME; and (2) suppression of lactate synthesis in tumors. Modification of the hypoxic environment of the tumor primarily consists of the following: (1) the 98 developed CaCO3 nanoparticles (DNCaNPs) for co-encapsulating DOX and alkylated NLG919 (aNLG919). CaCO3 permits therapeutic medicine to enter deep into a tumor and effectively neutralizes the acidic tumor pH, thus facilitating immunosuppression reversal. Zhang et al.99 used a vesicular nanoparticle (VNP) system to deliver siLdha to tumor sites to knock down lactate dehydrogenase (LDHA), which plays a key role in lactate production, to reduce lactate production, reverse the acidic microenvironment of tumors, restore T cell anti-tumor function, and enhance the therapeutic effect of the immune checkpoint inhibitor, PD-1 antibody . Prasad et al.100 developed a bioinorganic nanoparticle composed of polyelectrolyte albumin complexes and manganese dioxide (MnO2 NPs) to synthetically regulate oxygen depletion, acidity, and ROS in the TME. MnO2 NPs catalyze the synthesis of O2 from H2O2 and neutralize acidic pH, thus improving the oxygen-depleted and acidic environment of tumors and increasing the sensitivity of oxygen-dependent radiotherapy. Hemoglobin in erythrocytes is an ideal oxygen-conducting material because it is a natural in vivo oxygen delivery protein. Tang et al.101 directly attached activated erythrocytes to nanoparticles loaded with photosensitizers to improve the therapeutic effect of photosensitizers using the oxygen supply capacity of erythrocytes, and effectively inhibited the growth of malignant glioblastomas.Zhu et al.25. IFN improves immune function by increasing MHC-I expression102. Therefore, IFN has a role in antigen detection and the interactions between adaptive and innate immune cells. To this end, loss-of-function mutations and genetic changes in the IFN signaling system are linked to clinical drug resistance in immunotherapy104. Recently, STING has been linked to anti-infection, anti-inflammatory, and anti-tumor therapies105. Activation of the STING signaling pathway in APCs causes cellular production of IFN-I, which triggers a T cell-mediated adaptive immunologic response. Natural STING agonists, such as cyclic GMP-AMP (cGAMP), have a weak entrance retention capacity. Furthermore, non-specific activation of STING may result in a wide inflammatory response.105 SDDSs enable the targeted delivery of STING agonists of APCs to attenuate the negative effects, while overcoming immunotherapy resistance in malignancies due to decreased IFN production109. Zhou et al.110 demonstrated an acid-responsive polymer nanovaccine that activates the STING pathway . The nanovaccine contains a STING agonist and a neoantigen that accumulates in lymph nodes and promotes DC uptake. The STING agonist stimulates the STING pathway in DCs, increases IFN-β production, and enhances T cell activation. When combined with an anti-PD-L1 antibody, STING increased antitumor activity in a 4T1 breast tumor model, implying that activation of the STING pathway eliminated drug resistance to ICB therapy. To manipulate STING activation, Li et al.111 created an ultrasonic (US)-guided cancer immunotherapy platform using nanocomplexes made of cGAMP electrostatically coupled to microbubbles targeting APCs . The nanocomplexes are passively deposited in LNs, and the linked microbubbles attached to APCs are used to efficiently transfer cGAMP to the cytoplasm via ultrasound, which triggers the cGAS-STING signaling pathway and effectively initiates an antigen-specific T cell immune response. This process not only provides a perfect platform for STING activation under controlled conditions, but also advances imaging-guided cancer immunotherapy. Furthermore, STING agonists can be targeted and administered to tumor tissues to generate a local immune response. Dane et al.112 used cleavable linkers to attach STING-activated cyclic dinucleotides (CDNs) to PEGylated lipids and incorporated the lipids into disk-shaped nanoparticles (LND-CDNs). LND-CDNs penetrate tumors more effectively than liposomes and expose tumor cells to STING agonists. The uptake of LND-CDNs by tumor cells increases co-localization of CDNs and tumor antigens in DCs, resulting in significant T-cell activation.Tumor cell intrinsic factors that contribute to immunotherapy resistance include the expression or inhibition of genes and pathways in tumor cells that prevent immune cell infiltration or function within the TEM113. In addition, IFN expression causes the activation of several immunologic checkpoints, resulting in immune evasion114. Zhai et al.115 developed an epigenetic nanoinducer (OPEN) loaded with ORY-1001 (a selective lysine-specific histone demethylase 1 inhibitor) and overexpressing PD-1 membrane-modified T cells to overcome these challenges . The OPEN identified PD-L1 on tumor cells and internalized it, upregulating intra-tumor IFN, membrane MHC-I, and membrane PD-L1 expression. MHC-I facilitated neoantigen presentation. In addition, PD-L1 overexpression increased the tumor-specific accumulation and absorption of subsequent OPEN, which inhibited PD-L1 expression. Complementing intratumoral IFNs by modulating epigenetics and limiting the immunosuppressive function improves CTL recruitment, proliferation, and activity, with significant anticancer efficacy in numerous tumor types.Cells must have functional STING proteins for cGAMP to initiate STING signaling. It has been demonstrated that epigenetic silencing of STING or cyclic guanosine monophosphate (GMP)-adenosine monophosphate (AMP) synthase (cGAS) impairs STING signaling in cancer cells117. Restoration of PTEN expression by plasmid DNA transfection of tumor cells can reverse immunotherapy resistance118; however, systemic instability has hindered the clinical application of plasmid DNA. To overcome these issues, Islam et al.119 used polymer-lipid hybrid nanoparticles for the systemic delivery of modified PTEN mRNA to tumor cells, which effectively overcame several potential challenges in mRNA delivery, including large size, high negative charge, easy degradation, and poor protein translation. These carriers can enter cancer cells, protect mRNA from degradation, and restore natural immune function in vivo. PTEN deficiency promotes resistance to T cell-mediated immunotherapy, suggesting that restoration of PTEN function by PTEN mRNA nanoparticle technology may be useful in the application of immunotherapy and in rescuing drug sensitivity in immunotherapy-resistant tumors120. Liu et al.121 further designed ApoE-modified pH-responsive mRNA nanoparticles (ABNPs@mRNA) coated with red blood cell membranes as a non-invasive brain delivery system for selective delivery of PTEN mRNA across the blood-brain barrier to treat glioblastomas.The collective clinical evidence has demonstrated that the deletion or mutation of tumor suppressor genes may be associated with the TME and poor response or resistance to ICB therapy. Deletion of the PTEN gene, a protein that negatively regulates the PI3K-AKT pathway, has been shown to be associated with decreased T-cell infiltration and immunosuppressive cell proliferationvia the delivery of immune cells generated and expanded in vitro122. In addition to CAR-T, which has been approved by the United States Food and Drug Administration (FDA) for the treatment of hematomas, NK cell-, DC-, and macrophage-based ACT have been developed. Immune cells have a longer half-life and systemic dispersion than small chemicals and biologics, and have the potential to overcome physiologic boundaries and recognize and respond to pathogenic stimuli123. These benefits make immune cells promising cancer-fighting weapons; however, bacause of the immunosuppressive TME in tumor tissues, it is difficult for immune cells to move efficiently to the lesion location124. The few immune cells that reach the tumor are likewise vulnerable to failure and death as a result of local immunosuppressive signals125. This resistance to immune cell therapy due to intrinsic and extrinsic tumor environmental factors has limited the clinical application of ACT. By attaching to diverse biofunctional molecules, SDDSs avoid drug resistance to ACT126. Nanoparticles and antibodies have high structural adaptability and can enable multifunctionalization of immune cells via cell surface conjugation, giving immune cells increased efficacy or the ability to bypass immunosuppressive barriers127. Tang et al.128 reported that activating TCR signaling increases the reduction potential on the surface of T cells and produced a protein nanogel (NG) capable of responding to reduction potential changes . The NGs are comprised of therapeutic protein molecules connected together by a disulfide-containing bis-N-hydroxy succinimide (NHS) linker. Anchoring NGs to CD45 on T cells prevents endocytosis by the cells. Using an IL-15 super agonist combination (IL-15Sa) as the test medication, T cells equipped with NG backpack were able to rapidly grow within the tumor (16 times more than systemic IL-15Sa treatment) but remained quiescent in the peripheral circulation. Hao et al.129 also used hydrophobic forces to bind lipids to the T cell membrane before coupling the complex to avasimibe-encapsulated liposomes via a click reaction on the T cell membrane. Avasimibe was restricted to the T cell surface throughout T cell circulation and extravasation, then released locally to increase the cholesterol concentration in the T cell membrane, promote fast aggregation of T cell receptors, and sustain T cell activation.In contrast to other types of immunotherapies, ACT directly induces a robust antigen-specific immune response 130 created a disk-like particle that adheres to the surface of macrophages and modulates the phenotype . The backpack is comprised of two layers of polylactic acid-hydroxyacetic acid co-polymer (PLGA) with polyvinyl alcohol (PVA) and IFN-γ. Owing to the form effects, macrophages do not endocytose the backpack. Macrophages carrying the IFN backpack remain in M1 for an extended period of time, preserving the phenotype within immunosuppressive tumors and increasing anti-tumor activity. Meng et al.131 constructed an NK cell backpack . Complementary bioorthogonal groups {azide (N3)/bicyclo[6.1.0]nonyl} (BCN) were inserted into NK cells (N3-NK) and tumor cells (BCN-Raji). IL-12-containing nanoparticles (ILNPs) were conjugated to the surfaces of NK cells using aCD45 antibodies. The bioorthogonal method successfully promoted NK cell tumor targeting. Sustained IL-12 release activated NK cells and significantly increased proliferation and activation, hus timproving the therapeutic potential. Furthermore, IL-21 induced the recruitment of numerous immune cells, thereby activating the innate immune system. The fundamental mechanism underlying NK cell-mediated tumor cell death is the creation of an immunosynaptic (IS) detection of cancer cells. In light of this, Im et al.132 developed a smart DOX-releasing NK cell for solid tumor immunotherapy. NK cells were modified with IS-responsive drug-loaded micelles, and upon IS formation, the micelles disintegrated to release DOX with the production of acidic granule contents, effectively increasing NK cell tumor death. These “backpack” techniques successfully improve the function of immune cells, increasing the potential applicability.In addition to T cells, nanoparticles can modify macrophages and NK cells to provide multivalent activities. Shields et al.133 created CAR-T cells in combination with HAase and aPD-L1 antibodies to increase therapeutic efficacy against solid tumors . The modified HAase destroys hyaluronic acid and damages the tumor ECM, allowing CAR-T cells to enter solid tumors. Furthermore, the aPD-L1 antibody significantly suppresses PD-1 on the tumor surface, preventing drug resistance in cancer immunotherapy. This approach is expected to address issues, such as CAR-T solid tumor invasion rates.Moreover, backpacks provide immune cells with the ability to bypass immunosuppressive obstacles. Zhao et al.In this review we have discussed how SDDSs can overcome drug resistance in cancer immunotherapy. Multiple therapeutic compounds can be selectively administered to tumor cells or immune and stromal cells in the TME using SDDSs to combat drug resistance and promote anti-cancer immunotherapy by activating ICD in tumor cells, reversing tumor immunosuppression in the TME, or altering IFN signaling pathways. In addition, SDDSs combined with ACT provide a better therapeutic effect and have the ability to overcome immunosuppressive barriers in immune cells, effectively overcoming drug resistance to ACT.in situ or various forms of factor-induced tumor models, as well as the use of humanized tumor cells or humanized mice that can better replicate the process of tumor treatment in clinical settings. Finally, SDDSs will need to address the obstacle of drug resistance by more than simply encapsulating therapeutic medications in delivery vehicles to be taken up by tumor cells; instead, employing SDDSs to preferentially transport drugs to immune cells appears to be a more viable approach. Although immune cells have an important role against cancer in all stages of cancer formation, progression, and treatment, different drug resistance mechanisms are fundamentally an adaptive development of cancer cells. The use of SDDSs to modify or equip immune cells not only improves immune cell function but also strategically realizes the all-around effect of the immune system against cancer. Overall, future novel SDDSs for overcoming drug resistance in cancer immunotherapy will undoubtedly widen the applicability of immunotherapy in solid tumors.Effective SDDS-based techniques for overcoming drug resistance and improving cancer immunotherapy are emerging; however, significant difficulties remain in achieving good therapeutic effects and practical applications. First, some SDDSs currently being developed have non-biodegradable components that may induce toxicity in the organisms. The intrinsic immunomodulatory potential of certain materials used to produce SDDSs may result in excessive inflammatory responses or drug resistance. Therefore, one of the keys to the clinical translation of SDDSs is the creation of biocompatible and biodegradable materials. Furthermore, the use of diverse bio-derived vesicles as drug delivery carriers has considerably reduced the possible negative effects of SDDSs. Second, while most recent studies have shown SDDSs to be effective in mouse models, there is no certainty that SDDSs would be effective in human cancers owing to species differences. Most contemporary laboratory models are subcutaneous tumor models, and the tumor cells used in the laboratory are cell lines with well-defined phenotypes that differ dramatically from tumors produced in the natural environment. Therefore, future studies are needed to build


Macrophages and neutrophils are the main components of the innate immune system and play important roles in promoting angiogenesis, extracellular matrix remodeling, cancer cell proliferation, and metastasis in the tumor microenvironment (TME). They can also be harnessed to mediate cytotoxic tumor killing effects and orchestrate effective anti-tumor immune responses with proper stimulation and modification. Therefore, macrophages and neutrophils have strong potential in cancer immunotherapy. In this review, we briefly outlined the applications of macrophages or neutrophils in adoptive cell therapies, and focused on chimeric antigen receptor (CAR)-engineered macrophages (CAR-Ms) and neutrophils (CAR-Ns). We summarized the construction strategies, the preclinical and clinical studies of CAR-Ms and CAR-Ns. In the end, we briefly discussed the limitations and challenges of CAR-Ms and CAR-Ns, as well as future research directions to extend their applications in treating solid tumors. Since tInnate immune cells play important roles in responding to pathological conditions and maintaining immune homeostasis. Among them, macrophages and neutrophils are plastic immune cells with diverse functional phenotypes responsive to different environmental signals . Macroph22.1Given the key roles of macrophages in tissue repair and inflammation, macrophage-based cell therapies have been applied in various diseases. Danon et al. treated human ulcers using macrophages prepared from human peripheral blood monocytes . The useTumor-associated macrophages (TAMs) are the most abundant immune cells in the TME with heterogenous populations, which mostly adopted M2-like phenotypes with immune-suppressive properties . TherefoWith the advantages of high biocompatibility and phagocytic ability, studies have manipulated macrophages as drug carriers, which delivered targeted therapies to diseased tissues with improved drug stability , 27. In Recently, combining the unique properties of macrophages with tumor antigen-induced activation, CAR-Ms have evolved as promising adoptive cell therapies towards solid tumors Figure 12.2As the most abundant immune cells in circulation, neutrophils play central roles in the early immune responses to tissue damage or infection . GranuloActivated neutrophils secreted various pro-inflammatory cytokines and activated T cells by antigen presentation . They alHowever, under the effects of tumor suppressive microenvironment and cytokines, neutrophils in the TME are mostly polarized to acquire N2-like phenotypes, with tumor-promoting functions through driving angiogenesis, extracellular matrix remodeling, metastasis, and immune suppression . Thus, c33.1The basic principles of CAR design in the field of CAR-T cells were applicable to macrophages and neutrophils. Current studies in CAR-Ms and CAR-Ns mostly used classical CAR design consisting of an extracellular antigen recognition domain, a hinge domain, a transmembrane domain, and one or more cytoplasmic signal domains , HER2 6, and PSM3.2Due to the high expression of pattern recognition receptors and non-proliferative nature of macrophages and neutrophils, they are more resistant to gene transfer compared to T cells , 67, 68.3.2.1+ hematopoietic stem cells, which were then differentiated into macrophages (See section 2.3). Retroviruses (excluding the lentivirus subclass) are generally considered ineffective in transfecting non-proliferative or poorly proliferating cells (Lentivirus and retrovirus vectors can mediate the integration of transgenes into host genomes with high efficiencies and are commonly used for stable gene transfer to T cells. Though the primary mouse bone marrow derived macrophages (BMDMs) were infected by human immunodeficiency virus type 1 (HIV-1) lentivirus at high efficiency , 69, stung cells , and theng cells . Introdung cells . Robertsng cells . AlthougAdenovirus (AdV) belongs to the family of double-stranded DNA viruses . AdV vec3.2.2AAVS1 gene locus of human pluripotent stem cells (hPSCs) by CRISPR/Cas9 system through electroporation followed by differentiation into macrophages with M1-like phenotypes . Non-viral methods can deliver larger fragments of transgenes with flexibility, and lower the manufactural time and costs . The pro3.3in-vitro and animal studies. Since primary macrophages in blood is scarce, peripheral blood monocytes represent the main resource for CAR-Ms used in pre-clinical and clinical studies  followed by resting and treatment by LPS and IFN-γ , providi vectors . However vectors , 84.+ hematopoietic stem and progenitor cells (HSPCs) using lentivirus to generate CAR-HSPCs, which were then differentiated into functional CAR-Ms in the presence of M-CSF/GM-CSF/IL-3  production, and neutrophil extracellular traps (NETs) Figure 45Novel strategies utilizing the tumor-homing tendency of macrophages to deliver therapeutic substances  hold the potential to further enhance the anti-tumor effects of CAR-Ms while constraining the cytotoxic effects mainly within the local tumor ecological niche and reducing the toxic effects of systemic administration . For exa2 nanoparticles loaded with the chemo-drug tirapazamine infiltrated to brain tumors and cleared the external tumor cells by phagocytosis  (A recent study reported that CAR-Ns carrying SiOocytosis . As neutem (<1%) .6in vivo programming of myeloid cells using LNP encapsulating mRNA, administered to patients with advanced or metastatic epithelial tumors (NCT05969041). Another study aiming to determine the efficacy of newly developed CAR-Ms therapy using tumor organoids derived from breast cancer patients (NCT05007379) has not yet started.Based on promising results in preclinical studies , Carisma7ex vivo or after infusion since they are terminally differentiated cells   and MyD8Various studies have implemented strategies modulating metabolic programs in T cells to enhance the functions of CAR-T cells . Through7.3Due to the limitations of single therapy in cancer treatment, combinatory therapies have become the long-term trend to increase the efficacies of CAR-based cell therapies. As noted above, CAR-Ms and CAR-Ns delivering drugs had combined anti-tumor effects of cell therapies and cytotoxicity from chemo-drugs , 95. Radin vitro (Combination of different cell therapies can present synergistic anti-tumor effects. Mice treated with CAR-Ms and T cells together showed better anti-tumor responses compared to those treated with either cell therapy alone . This main vitro . The infin vitro .In addition, CAR-Ms combined with antibodies, such as anti-PD-1, anti-CD47, anti-FcγRIIB and anti-Siglec-10 promoted the anti-tumor efficacies compared with CAR-Ms alone, either through enhancing the phagocytosis of macrophages or cytotoxic T cell responses , 113–1198Staphylococcus aureus in mouse models, which presented a potential approach for the prevention and treatment of periprosthetic joint infection (in vivo function and persistency of CAR-Ms and CAR-Ns, with effective manufactural routes to improve the yield of engineered cells and reduce the cost and time. More clinical studies are eagerly awaited to comprehensively evaluate the potency and toxicity of CAR-Ms and CAR-Ns in different diseases including solid cancers.In conclusion, CAR-Ms and CAR-Ns have emerged as promising cell therapies with unique characteristics to combat solid cancers. Beyond cancers, they also provide novel treatment options for other diseases. For example, Li et al. utilized a surficial nanoparticle coating that locoregionally generated super bactericidal CAR-Ms to eradicate nfection . CombinaYL: Writing – original draft, Writing – review and editing. QX: Writing – original draft, Writing – review and editing. QG: Writing – original draft, Writing – review and editing.


In this study, we utilized gonadotropin-releasing hormone analogue-modified indocyanine green (GnRHa-ICG) to improve the accuracy of intraoperative recognition and resection of endometriotic lesions.  Gonadotropin-releasing hormone receptor (GnRHR) expression was detected in endometriosis tissues and cell lines via immunohistochemistry and western blotting. The in vitro binding capacities of GnRHa, GnRHa-ICG, and ICG were determined using fluorescence microscopy and flow cytometry. In vivo imaging was performed in mouse models of endometriosis using a near-infrared fluorescence (NIRF) imaging system and fluorescence navigation system. The ex vivo binding capacity was determined using confocal fluorescence microscopy.  GnRHa-ICG exhibited a significantly stronger binding capacity to endometriotic cells and tissues than ICG. In mice with endometriosis, GnRHa-ICG specifically imaged endometriotic tissues (EMTs) after intraperitoneal administration, whereas ICG exhibited signals in the intestine. GnRHa-ICG showed the highest fluorescence signals in the EMTs at 2 h and a good signal-to-noise ratio at 48 h postadministration. Compared with traditional surgery under white light, targeted NIRF imaging-guided surgery completely resected endometriotic lesions with a sensitivity of 97.3% and specificity of 77.8%. No obvious toxicity was observed in routine blood tests, serum biochemicals, or histopathology in mice.  GnRHa-ICG specifically recognized and localized endometriotic lesions and guided complete resection of lesions with high accuracy. Endometriosis is a common benign gynecological disease that seriously affects the physical and mental health of women of childbearing age. Laparoscopic surgery is currently the dominant approach for relieving symptoms and promoting fertility in patients with endometriosis . HoweverTraditional imaging approaches such as ultrasound –10 and MIndocyanine green (ICG), the only clinically approved near-infrared (NIR) fluorophore, has been used in intraoperative imaging for endometriosis, but it cannot improve the detection of endometriotic lesions compared to traditional white light imaging owing to its nonspecificity . Specifi111In-labeled GnRH peptides effectively detected human prostate cancer /[μW/cm2]) using Living Image software. Fluorescence intensity was measured by drawing a region of interest (ROI) around the area. The signal-to-noise ratio (SNR) was calculated as the average fluorescence intensity of the tumor divided by that of skeletal muscle or intestine.The synthesis and characterization of GnRHa-ICG are described in the study by Liu et al. . The endn = 8 per group). The number and maximum diameter of the foci confirmed to be endometriotic by histopathology were recorded. To evaluate the accuracy of the probe, ten endometriotic model mice were intraperitoneally injected with GnRHa-ICG, followed by GFP and NIRF imaging. The GFP-positive tissues were resected under the guidance of NIRF imaging, and the sensitivity and specificity of the probe were calculated with the histopathology examination as a reference.The targeted probe GnRHa-ICG (1 mg/kg) was injected intraperitoneally into model mice at the indicated times, and the abdominal cavities were exposed after sacrifice. NIRF image- and white light-guided surgeries were performed using a clinically used fluorescence navigation system . Endometriotic foci were resected under white light and NIRF guidance (n = 3). Two groups received intraperitoneal injections of 1.0 mg/kg GnRHa-ICG and were followed for 2 and 120 h. The control group received the vehicle alone. Blood was drawn to assess alanine transaminase (ALT), aspartate transaminase (AST), blood urea nitrogen (BUN), creatinine (CREA), white blood cells (WBC), and red blood cells (RBC). Heart, lung, liver, spleen, and kidney tissues were harvested for HE staining.GnRHa-ICG toxicity was determined in BALB/c mice .Student's GnRHR expression was observed in 100% (6/6) of endometria (EM), 88.9% (40/45) of ovarian endometrioma (OE), and 88.4% (15/17) of deep endometriosis (DIE) samples by IHC analysis . GnRHR wTo evaluate whether GnRHa peptide binds to endometriotic cell lines, we labeled GnRHa peptide with FITC and incubated GnRHa-FITC with immortalized human endometriotic epithelial 12Z cells. Strong fluorescence signals of GnRHa-FITC were detected in 12Z cells via fluorescence microscopy and flow cytometry Figures .To evaluate the binding capacity of GnRHa-ICG to endometriotic cells, we incubated GnRHa-ICG with the 12Z and ESC cell lines. A significant difference in fluorescence intensity was detected between GnRHR-positive 12Z cells and GnRHR-negative ESC . The meaThe fluorescence intensities of EMT and background tissues (muscle and intestine) per dose group are shown in Figure Ex vivo NIRF images of the EMT foci and other organs demonstrated that the fluorescence signals were mainly concentrated in the EMT foci and liver after intraperitoneal injection of GnRHa-ICG, whereas they were predominantly concentrated in the intestine, liver, uterus, and kidney in the ICG group . The flun = 37) was significantly smaller than that in the white light-guided group   . Under t n = 28) . GnRHa-ITo evaluate the biodistribution of the GnRHa-ICG probes, ex vivo EMTs and organs were collected 2 h postadministration. Confocal imaging confirmed the high accumulation of GnRHa-ICG in the EMT and liver, while ICG showed strong fluorescence signals in the intestine and liver and a weak signal in the EMT . These rThe dynamics of GnRHa-ICG and ICG in mice with endometriosis were evaluated. Mice were intraperitoneally injected with GnRHa-ICG or ICG and monitored for 48–120 h. As shown in Figures To address concerns of possible side effects caused by GnRHa-ICG, we assessed the viability of GnRHR-positive 12Z cells after dose-gradient incubation with GnRHa-ICG. Exposure to a high concentration of GnRHa-ICG did not decrease the cell viability Figure . In miceIn our study, we first reported intraoperative imaging and imaging-guided surgery using the targeted near-infrared fluorescent probe GnRHa-ICG in an endometriotic mouse model, which suggested a strong potential in clinical intraoperative imaging for endometriosis.Over the past few decades, imaging approaches have been reported for the intraoperative detection of endometriosis; however, their diagnostic performance has been limited. Methylene blue, a widely used thiazide dye in surgical imaging, exhibits blue staining of peritoneal endometriotic lesions, but the colored peritoneal areas were not histopathologically confirmed , 26. 5-AAutofluorescence, the natural emission of light by biological structures, such as mitochondria and lysosomes , has bee99mTc to develop a radioactive imaging probe 99mTc-GAP-EDL, which was first reported to target endometriotic lesions in a rabbit model of endometriosis [99mTc-labeled monoclonal anti-VEGF antibody (bevacizumab-99mTc) was used as a radiopharmaceutical for endometriosis imaging in an endometriotic rat model in 2015 [Targeted imaging of endometriosis can be performed by conjugating imaging agents with endometriotic-specific ligands  , 37. In etriosis . Consideetriosis –42, 99mT in 2015 . Moses e in 2015 . HoweverGonadotropin-releasing hormone receptor, which was mentioned in our study as GnRHR-I, is mainly expressed in the anterior pituitary and reproductive tissues, such as the breast, ovary, and prostate –47. HighNIRF imaging is an emerging real-time imaging technology that has been widely used in fluorescence-guided surgery because of its high penetration depth and low autofluorescence background . ICG, thThe use of NIRF imaging for molecularly guided surgical resection of cancers could reduce residual tumor burden and surgical morbidity associated with excising sufficient tissues to avoid positive surgical margins . Based oTo further evaluate the target recognition capability of GnRHa-ICG for endometriosis, white light and NIRF-guided surgeries were performed. GnRHa-ICG could recognize tiny endometriotic lesions that could not be identified under white light, and the minimum detectable lesions were up to 1 mm in diameter. GnRHa-ICG has the potential to help localize tiny residual lesions that the naked eye cannot identify during surgery. Moreover, the signals of ICG decreased exponentially in mice after intraperitoneal administration, whereas GnRHa-ICG reached the highest concentration in the EMTs at 2 h and still had a good SNR within 48 h of administration. This period provides flexible administration time for clinical intraoperative imaging.The toxicity of GnRHa-ICG was assessed, and no hematotoxicity and hepatotoxicity were observed in the study. The short-term administration of GnRHa-ICG is safe in vivo.In summary, GnRHa-ICG specifically recognized and localized endometriotic lesions and showed high fluorescence signals from 2 to 48 h postadministration. Targeted NIRF imaging-guided surgery completely resected the endometriotic lesions with high accuracy. Future clinical applications might help surgeons accurately resect lesions to reduce the postoperative recurrence of endometriosis.