With the development of ever more powerful and versatile high-throughput sequencing techniques and innovative ways to capture single cells, mapping the multicellular tissues at the single-cell level is becoming routine practice. However, it is still challenging to depict the epigenetic landscape of a single cell, especially the genome-wide chromatin accessibility, histone modifications, and DNA methylation. We summarize the most recent methodologies to profile these epigenetic marks at the single-cell level. We also discuss the development and advancement of several multi-omics sequencing technologies from individual cells. Advantages and limitations of various methods to compare and integrate datasets obtained from different sources are also included with specific practical notes. Understanding the heart tissue at single-cell resolution and multi-modal levels will help to elucidate the cell types and states involved in physiological and pathological events during heart development and disease. The rich information produced from single-cell multi-omics studies will also promote the research of heart regeneration and precision medicine on heart diseases. Nkx2.5 KO, Hand1 KO, etc.)  , in the O, etc.)  or mouseO, etc.) . ResearcThe first single-cell whole-transcriptome profiling was reported by Tang et al., in 2009. Single oocytes were picked by mouth pipette, followed by reverse transcription, polyA tailing, and second-strand synthesis . Later, The cell-type-specific epigenetic landscape is shaped by unique distributions of DNA methylation, chromatin accessibility, key histone marks, and transcription factors (TFs) binding across the genome. Genome-wide profiling of these epigenetic features facilitated the mechanistic study of the developmental process and gene function. The epigenomic profiling technologies include Assay for Transposase Accessible Chromatin with high-throughput sequencing (ATAC-seq) , ChromatTo capture multi-modal information of single cells, samples can be split into multiple parts for multi-omics sequencing separately. However, multi-omics sequence information from the same cells can genuinely reveal the correlation between different layers of epigenetic modification and gene expression . With thThis review will summarize the platforms for single-cell capture and multi-omics sequencing, the recently emerged technologies and analysis tools, and their potential application in cardiovascular research.in vitro transcription by T7 RNA polymerase . These Various platforms are engineered to capture single cells to suit different needs. Based on where the cell barcoding takes place, the single-cell sorting strategy can be classified into three types, cell-per-well (CPW) strategy , dropletCPW strategies  directly put one cell into one well or tube, followed by library construction separately. Droplet-based strategies  use a “co-flow” device for cell encapsulation with barcoded microparticles to capture mRNA. SCI strategies use the intact cell or nucleus as an indexing unit, and each cell will, with high probability, have a unique barcode combination after multiple rounds of split-pool indexing . Here weThe earliest CPW strategy relied on labor-intensive manually picking single cells into each tube . Later, 5 microwells. Each microwell can seed one cell and one barcoded bead. Cells are seeded into microwells by gravity, followed by beads settling. After cell lysis in the plate, RNA transcripts are released into the microwell and captured by beads conjugated with barcoded primers; afterward, the beads are collected, and subsequent reverse transcription is performed in bulk. Compared to microfluidic-based workflows, microwell-based workflows offer higher cell throughput, higher tolerance for cell size variation, and gentle treatment by eliminating shear stress from micro-fluidic channels. Microwell-based methods can interrogate a high volume of samples in a single assay, albeit producing lower sequencing depth. It is an excellent choice for large-scale single-cell profiling. For example, Han et al. depicted the cell atlas of mice (more than 400 thousand cells) and human cells (more than 700 thousand cells) using the microwell platform (Microwell-based methods (agarose-based microwell or BD Rhapsody™ microwell cartridge) offer another solution to improve the throughput and partially overcome the constraints in cell size. . These mplatform .Droplet-based platforms pack single cells and barcoded beads into nanoliter droplets in oil emulsions and utilize these tiny droplets as the reaction chambers. This approach eliminates the need for microfluidic chambers or individual tubes and significantly improves the throughput.3∼104 cells per run) with sufficient gene coverage , but they all require specific instruments for droplet generation and have a limitation on cell size (less than 30 µm in diameter for most pipelines).There are three state-of-art droplet-based scRNA-seq strategies, inDrops , Drop-seThe single-cell combinatorial indexing (SCI) method is a new high-throughput single-cell sequencing paradigm. It takes advantage of the intactness of the cell nucleus during reverse transcription and Tn5 tagmentation. The nuclear membrane is a physical barrier where the indexing reaction occurs inside. Cells are indexed in dozens of wells simultaneously, and multiple rounds of indexing are carried out through pooling and splitting cells into other dozens of wells. After multiple rounds of split-pool indexing, each nucleus will, with high probability, have a unique barcode combination. The SCI method quickly gained popularity because it requires no special instruments other than conventional flow cytometry equipment, and the cost per cell is vastly reduced.C. elegans cells with a median of 575 Unique Molecular Identifiers (UMIs). Other methods employed more complex indexing steps to increase the input or sample multiplexing. For example, in 2018, Alexander B. Rosenberg et al. developed SPLiT-seq that introduced four rounds of barcoding , which yielded over 21 million barcode combinations, vastly decreased the probability of barcode collisions , single-cell Chromatin immunoprecipitation assays with sequencing (scChIP-seq), and single-cell DNA methylome sequencing (scDNAme-seq).Open chromatin regions often harbor more transcription factor (TF) binding sites and regulatory regions, which are of great interest. Several enzymes preferably cut the open regions of genome DNA  and thus have been used to interrogate chromatin accessibility  Schones. DNase-b4 fragments were generated from each cell and mapped to the human genome   . In addier cell) .Modification of histone tails regulates the structure of chromatin and significantly influences gene expression. Several well-known histone modifications are associated with active transcription, including H3K4me1 [active and primed enhancers ], H3K4meChIP-seq has been widely adopted to profile different types of histone modification and define various regulatory elements in cardiovascular development and disease studies . HenikofDNA methylation is a fundamental epigenetic modality closely linked to cell identity and transcriptional regulation. Methylation at the promoter region often marks silenced genes, while methylation in the gene body is a feature of actively transcribed genes . BisulfiBased on bulk WGBS and RRBS methods, scRRBS  and scBSSeveral methods have been developed to profile DNA methylome, transcriptome, and other epigenetic information simultaneously. Most methods physically separate the nucleus and the cytoplasm for bisulfite sequencing and RNA-seq, such as scMT-seq  and scTrIn recent years, it has become feasible to simultaneously profile chromatin accessibility, histone modification, TF binding, DNA methylation, and transcriptome from the same cell, namely single-cell multi-omics sequencing (scMulti-omics-seq). ScMulti-omics-seq avoids the need to match datasets from parallel samples, reducing the technical variations. ScMulti-omics-seq can potentially be used to distinguish fluctuation in gene expression and different cell states due to a typical larger feature set than scRNA-seq, which is limited by the number of genes expressed and sequenced .The first single-cell RNA-ATAC co-assay method is sciCAR-seq, published in 2018 by Jay Shendure and his colleagues, who had adopted the concept of SCI to scMulti-omics-seq by performing reverse transcription and tagmentation consecutively in the same nuclei before the second round of indexing . This re4). Paired-tag adopted three rounds of barcoding through ligation, reaching a theoretically thousand folds higher complexity. Unique reads generated by Paired-tag varied among different histone markers but generally fell within 1-10k, sufficient for analyzing differentially modified regulatory elements. CoTECH generates a dataset with a slightly lower number of unique reads. Zhu et al. profiled ∼70,000 mouse brain cells on H3K4me1, H3K4me3, H3K27ac, H3K27me3, and H3K9me3 genome-wide association and the transcriptome. They successfully partitioned cells into different neuron types using H3K4me1 and H3K27ac modals alone, but H3K4me3, H3K27me3, and H3K9me3 modals failed to resolve the cellular heterogeneity and only clustered cells into broad brain cell types, indicating that it may be unfeasible to link different histone modification modalities directly if matched RNA information is not profiled. Using the transcriptome to bridge with other modalities, they assembled five histone modification datasets to form a comprehensive epigenetic landscape of mouse neurons.Recently published Paired-tag  and CoTEPaired-tag and CoTECH can only profile transcriptome and one single histone modification at a time, so it is not possible to study the relationship between different histone modifications around the same genomic region. To map multiple histone modifications simultaneously in the same cell, multi-CUT&Tag  and MulTNew bioinformatics methods are needed to analyze the vast amount of data generated from scMulti-omics sequencing experiments. Comparative analysis of the same modality, e.g., scATAC-seq, from different experiments turns out to be challenging, as researchers often need to remove batch effects due to different cell capturing strategies, library construction methods, or sequencing platforms. The choice of batch-removal strategy can be significantly determined by the research topic. According to a benchmark study , if cellMulti-omics data analysis needs to consider distinct modalities’ different sizes and unique nature. Chromatin accessibility and DNA methylome datasets contained information across the whole genome, and histone modifications also mark a significant fraction of the genome. These datasets are significantly larger than the transcriptome  and TF binding regions. Dozens of computational strategies have been developed to perform integrative analysis of multi-modal datasets, including matched (multi-omics data from the same cell) or unmatched datasets  . For matGWAS has identified hundreds of thousands of genetic variants associated with the broad spectrum of human traits and diseases, most of which are noncoding . It is cIn recent years, increasing numbers of studies employed scRNA-seq to depict the cell atlas of embryonic, adult, and diseased heart and vasculatures, such as dilated cardiomyopathy , congeniSampling on cardiovascular tissues, especially from the heart, requires special cautions. The size of CM (100–150 μm by 20–35 μm) poses a significant challenge for single-cell analysis. Cui et al. adopted modified STRT-seq  to constSome studies have adopted scRNA-seq to profile human pluripotent stem cell (hPSC) derived CMs. Data mining in the scRNA-seq helped optimize hPSC differentiation protocols . Yang etScATAC-seq, combined with scRNA-seq, provides valuable insights into characteristics underlying developmental plasticity. Information from single-cell chromatin accessibility during the cardiovascular system development can predict the cell-type-specific regulatory TFs through motif enrichment analysis. Furthermore, researchers are more convinced by the more stable epigenetic landscape of the cell for cell-type identification, while RNA expression can be variable and change rapidly .Only a few published research used scATAC-seq to profile embryonic or adult human hearts. Most of them focus on constructing the regulome atlas . Hocker FN1 gene and adjacent to MMP2 and FBLN2 genes, which all showed higher accessibility in the left atrium. These findings may indicate a more activated fibroblast state associated with higher ECM production in the left atrium (The human cardiac CRE atlas derived from the scATAC-seq can be a valuable reference to advance our understanding of gene regulatory mechanisms. For example, for cardiac fibroblasts, there is more differential accessible CREs between right and left ventricles than between atriums and ventricles. Researchers have detected several fibroblast-specific CREs at t atrium .The technology to acquire and analyze multi-omics information from a single cell is rapidly evolving. Single-cell analysis has already provided an unprecedented amount of information about the cellular composition of the heart during development, homeostasis, and diseased conditions. However, there are many pending questions. How does the epigenetic landscape change in different parts of the heart and cell types during embryo development? How do different cardiac cells interact with immune cells during myocardial infarction and heart failure? Are there intermediate cell states during heart regeneration? How do drugs affect different types of cells in the heart? Answering these complex questions requires full resolution of cardiac cell heterogeneity at multiple layers. The multi-modal information at the single-cell level will significantly improve our effort to understand the molecular mechanism regulating cell fate and states in healthy and diseased conditions and find better biomarkers and drug targets to treat cardiovascular diseases.


Background: Coronary artery disease (CAD) is a significant health concern today that has assumed epidemic proportions worldwide and in India.Objective: The pathobiology of CAD or ischemia with non-obstructive coronary arteries disease (INOCA) in most cases involves coronary microvascular dysfunction (CMD) that often requires personalized pheno-approach based on the risk stratification and diagnostic yield, especially in patients with recurrent angina episodes. Chronic therapy with anti-anginal is often fraught with challenges of unipolar action, tolerance, and oxidative stress that precludes consistent benefits in clinical cases of CMD represented by microvascular angina (MVA). To further elucidate the clinical role and relevance of long-term vasodilator therapy, including nicorandil, a Knowledge, Attitude, and Perception (KAP) survey was planned to be conducted.Methods: Based on responses to the KAP survey questionnaire, Delphi-mediated graded recommendations were developed by a specialist panel as per Agency of Health Care and Quality Systems (AHRQ) criteria.Results: Management of INOCA with MVA requires a multidisciplinary approach involving non- or invasive procedures that may be relevant for persistent or refractory angina before instituting symptomatic and long-term use of nicorandil for beta-blocker intolerant and associated clinical phenotypes.Conclusion: The analyses provide a real-life approach to the management of INOCA and angina non-obstructive coronary artery disease (ANOCA) with underlying CMD that may be relevant in outpatient settings in India. Cardiovascular disease (CVD) related to coronary arteries is a leading cause of morbidity and mortality in India and worldwide.The prevalence of CVD, including coronary artery disease (CAD), has significantly increased in the last three decades in India. The majority is higher in urban areas at 2.5-12.6% compared to 1.4-4.6% in rural areas. The systemic review of Indians with CAD further highlighted the prevalence of risk factors such as smoking (8.9-40.5%), hypertension (13.1-36.9%), and diabetes mellitus (0.2-24.0%). In most cases, the door-to-needle time after myocardial infarction was >300 mins, while the discharge medications were skewed towards anti-platelets, beta-blockers, and renin-angiotensin-aldosterone system (RAAS) inhibitors ATP) channels to alleviate endothelial dysfunction while alleviating further reduction in coronary (macro) and capillary (micro) vascular bed resistance leading that avoiding no-reflow or slow flow phenomenon in ischemia-reperfusion injury models .Anti-anginal drugs, including Nicorandil, might contribute to more favorable acute clinical outcomes due to the inhibition of platelet aggregation and other antithrombotic and anti-inflammatory effects. In addition, nicorandil closes the inner mitochondrial membrane pore to maintain an energy gradient in myocardial ischemic tissues to limit the infarct size or reperfusion injury Figure .The multimodal action of nicorandil as a K+ ATP channel activator offers arterio-venular dilation at therapeutic dosages while correcting microvascular dysfunction, unlike other long-acting nitrates Table .Nicorandil: Clinical evidenceNicorandil as oral or IV/IC in offers differential and unique action as a K+ ATP opener for a potential clinical role in CSA and acute coronary syndrome (ACS) requiring percutaneous coronary intervention (PCI), unlike long-acting nitrates , includiNicorandil is a clinically effective recommended option for managing vasospastic angina ,10.Nicorandil, in addition, may be beneficial in patients with primary CMD, as highlighted by clinical studies in cases of cardiac syndrome X and MVA ,12.Nicorandil, as an intravenous or intracoronary injection followed by an oral strategy, has been shown to improve flow-related CMD as chronic slow flow or no-reflow in CSA or ST-elevation myocardial infarction (STEMI) cases undergoing PCI -17. A meIn the Osaka Acute Coronary Insufficiency Study (OACIS) registry, 1846 consecutive patients with multivessel disease, in most cases with recent AMI undergoing emergency PCI, were evaluated for the clinical impact of nicorandil. Of these patients, 535 were treated with oral nicorandil at discharge, while the remaining 1311 patients did not receive nicorandil at discharge. Nicorandil treatment was associated with a nearly 50% reduction in all-cause mortality following release for AMI  .A nationally representative panel of experts responded and reviewed the literature involving randomized controlled trials for vasodilators and nicorandil before developing consensus on their clinical role and positioning in the management of ASCVD (including INOCA) and ACS requiring PCI. The LoE and LoA were developed after a review of literature and publications featuring in indexed databases of Medline, PubMed, SCOPUS, INDEX MEDICUS, and COCHRANE Systemic reviews using the keywords as Ischemic non-obstructive coronary artery disease, angina with non-obstructive coronary arteries, Microvascular angina, Nicorandil, Chronic coronary syndrome, Type 2 diabetes.The panel evolved a set of clinical recommendations to evaluate the role and relevance of vasodilators and nicorandil in ASCVD, including INOCA that was validated with the strength of evidence with the level of agreement (LoA) reached with each of the members Table .LimitationsThis analysis is limited by the interpretation of the evidence categorized as experimental and real-life observational studies as registries conducted with a heterogenous patient population of CAD and ACS cases requiring PCI with a coronary stent.The analyses provide a real-world approach to the management of INOCA with nicorandil as initial-line therapy. GDMT involving nicorandil in post-PCI settings may be relevant in cases of ASCVD with persistent angina or with demonstrable or suspected instances of chronic slow flow (CSF) during cardiac catheterization. The risk characterization for CSF requiring consistent therapy with nicorandil includes local ethnicity traits of small coronary architecture, T2D, polyvascular disease, and high coronary artery calcium (CAC) scores in most cases of ASCVD.


Pro are conserved among vertebrates, characterized by pleiotropic regulatory functions and have been found to discriminate colorectal tumors from normal colorectal mucosa. In the current study, we investigated the prognostic utility of 5′-tiRNA-ProTGG levels in CRC. For this purpose, total RNA was extracted from 155 malignant colorectal tumors and 74 adjacent non-cancerous tissue specimens, polyadenylated and reverse-transcribed using an oligo-dT adapter as primer. Real-time quantitative PCR (qPCR) was used to assess the levels of 5′-tiRNA-ProTGG. Kaplan-Meier survival analysis demonstrated that high 5′-tiRNA-ProTGG levels predict both poor disease-free survival (DFS) and overall survival (OS) of CRC patients. Of note, high 5′-tiRNA-ProTGG levels retain their unfavorable prognostic value in patients with rectal cancer and/or moderately differentiated CRC (grade II). More importantly, multivariate cox regression analysis highlighted that the overexpression of 5′-tiRNA-ProTGG constitutes an adverse prognostic factor predicting short-term relapse of CRC patients independently of the established prognosticators in CRC. Finally, bioinformatics analysis unveiled a potentially critical role of 5′-tiRNA-ProTGG regarding the maintenance of cellular homeostasis, signaling, cell communication, and cellular motility.Colorectal cancer (CRC) is the second most lethal cause of cancer-related deaths in Europe. Fragments of tRNA According to global cancer statistics, as presented by the Global Cancer Observatory (GCO) in 2018, colorectal cancer (CRC) accounts for approximately 12% of annually diagnosed cancers, being reported as the second most common cause of cancer-related deaths in Europe. The progression of benign colorectal incidentalomas to malignant neoplasms is a lengthy process that is mediated through genomic instability, aberrant DNA methylation and mutations in crucial oncogenes and/or tumor-suppressor genes . HoweverThe tumor-node-metastasis (TNM) classification system for colorectal tumors constitutes the most robust prognosticator to determine the survival of patients. Even in the later revisions of the TNM staging, the extent of the tumor (T), the nodal positivity (N) and the presence or absence of metastatic disease (M) retain their strong prognostic potential regarding the outcome of the disease and guide the clinical management of the patients . As we aTowards that direction, the pleiotropic action of non-coding RNAs (ncRNAs) in the regulation of gene expression makes them as putative diagnostic, prognostic or predictive molecular markers not only in CRC, but also in a wider spectrum of human diseases ,9,10,11.Transfer RNA (tRNA)-derivatives, which are widely known as tRNA-derived small RNAs (tsRNAs), are endogenous single-stranded sncRNAs with an approximate length of 14–40 nt. There are mainly two types of tsRNAs, including tRNA-derived stress-induced RNAs (tiRNAs) and tRNA-derived fragments (tRFs), which differ in the cleavage position of the precursor or mature tRNA transcripts, as well as in their length . In brieRecent scientific evidence has shown that tsRNAs participate in the regulation of the expression of protein-coding genes and as a result they are implicated in a wide range of molecular mechanisms underlying cancer development by possessing several biological roles. More specific, tsRNAs can regulate the expression of cancer-related genes, by forming complexes with Argonaute (AGO) and PIWI family members, imitating the function principles of miRNAs and PIWI-interacting RNAs (piRNAs) ,20. AddiLeu, previously known as miR-1280, has been reported to be deregulated in colorectal tumors, repressing the Notch pathway   from a total of 125 patients who did not present distant metastasis at the time of diagnosis (M0). Using Cox univariate regression analysis  a nearlyTGG-positive compared to 5′-tiRNA-ProTGG-negative tumors . However, when tiRNA-ProTGG expression status was included in a multiparametric Cox regression model along with all the important clinicopathological parameters failed to retain its independent prognostic power in CRC .Concerning the OS of CRC patients, Kaplan-Meier curves demonstrated that 5′-tiRNA-ProTGG expression status, patients were stratified based on important clinicopathological variables, such as tumor location and tumor grade. Initially, the significance of 5′-tiRNA-ProTGG expression status was evaluated regarding tumor location. Patients whose tumor is located in rectum and overexpresses 5′-tiRNA-ProTGG demonstrated a significantly higher probability of tumor recurrence (p = 0.027) (p = 0.047)  A and inf= 0.047) B. Subseqhort DFS C and OS hort DFS D time inTGG in CRC, we performed a functional enrichment and pathway analysis of 5′-tiRNA-ProTGG target genes. A total of 951 genes were predicted by both IntaRNA and RNAhybrid as putative targets of 5′-tiRNA-ProTGG. GO term enrichment analysis revealed that the predicted target genes of 5′-tiRNA-ProTGG , bearing TGG anticodon sequence (5′-tiRNA-ProTGG). Our results indicated decreased expression of 5′-tiRNA-ProTGG in cancerous tissue specimens compared to matched adjacent colonic mucosa. On the other hand, multivariate Cox regression analysis showed that the overexpression of 5′-tiRNA-ProTGG constitutes an adverse prognostic factor predicting short-term relapse of CRC patients independently of the established prognosticators such as patients’ age, tumor size and location, histologic grade of the tumor and TNM stage. Furthermore, univariate Cox regression analysis revealed a significantly increased probability of CRC patients overexpressing 5′-tiRNA-ProTGG to succumb earlier. The bootstrapped analysis used for internal validation and strengthened the statistical significance of these results. All the findings were further confirmed by Kaplan-Meier survival analysis, which indicated that overexpression of 5′-tiRNA-ProTGG was associated with both low DFS and OS of CRC patients.In the current work, we evaluated for the first time the clinical significance of a proline tiRNA in CRC. This molecule derived from a tRNATGG retained their adverse association with both DFS and OS within the subgroups of non-metastatic (M0) patients as well as patients with moderately differentiated colorectal tumors and rectal carcinomas. Histological grade of the disease remains a critical prognosticator of the outcome, nevertheless, intra- and inter-observer variability during tumor grading can often lead to misclassification, especially between well- and moderately differentiated tumors [TGG in moderately differentiated tumors (G2) may be used as an ancillary tool to identify patients at increased risk of relapse, alleviating some of the intrinsic limitations of histological grading. In the same context, overexpression of 5′-tiRNA-ProTGG tumors retained its unfavorable prognostic potential in the subgroup of patients with localized rectal cancer. Although rectal carcinomas represent one-third of total CRC cases, they differ from colon cancer in terms of anatomy, embryological origin and function and for this reason they require a different treatment approach [Moreover, high levels of 5′-tiRNA-Prod tumors . The impapproach .TGG constitutes an unfavorable predictor of both OS and DFS in CRC patients in line with survival data presented by OncotRF database, where 5′-tiRNA-ProTGG expression levels have been correlated with adverse prognostic outcome in a wide spectrum of malignant neoplasms including breast, stomach, prostate, skin as well as head and neck cancers [TGG expression levels between cancerous and non-cancerous tissue samples unveils a slightly overexpression of 5′-tiRNA-ProTGG in non-cancerous tissues . As a result, the deregulation of 5′-tiRNA-ProTGG expression may represents a late event in colorectal carcinogenesis and/or 5′-tiRNA-ProTGG functions as a potential double-edged sword in CRC.Unequivocally our results illustrate that the increased expression of 5′-tiRNA-Pro cancers . HoweverArgCCG targets CLDN1 and inhibits epithelial to mesenchymal transition (EMT) of colorectal cancer cells [Arg derivatives produced in a DICER1-dependent mechanism can also drive progression of colorectal cancer under hypoxic conditions [TGG. Recent studies highlight the pleiotropic functions of tsRNAs derived from tRNAPro in the fine-tuning of protein synthesis by interacting directly with ribosomes as well as the regulation of gene expression by presenting with miRNA- and piRNA-like functions [TGG, including AMPK signaling, autophagy, MAPK signaling and MTOR signaling. The fine-tuning of these key pathways is critical to ensure the maintenance of cellular homeostasis, whereas their deregulation drives the initiation and progression of colorectal tumors [Several studies highlight the implication of tsRNA in colorectal carcinogenesis ,48,49. Aer cells . Paradoxnditions . This innditions ,53. Thesunctions ,55,56. Cl tumors ,58,59.TGG expression levels in CRC, serving as versatile marker for both rectal and colon tumors. The wide spectrum of clinical information that is provided by 5′-tiRNA-ProTGG can be utilized by medical personnel to further ameliorate the therapeutic management of the disease. Moreover, we are aware that our research failed to provide strong evidence regarding the functional role of the described tiRNA. However, in this initial attempt we mainly focused on the evaluation of the prognostic significance of 5′-tiRNA-ProTGG expression levels in CRC. Future studies should intensify their efforts towards the elucidation of the biological role of tiRNAs in CRC. At the same time, additional validation of these results, using a more homogenous patient cohort in terms of CRC type could unveil more valuable information regarding the additional clinical value of 5′-tiRNA-ProTGG in specific CRC forms.This research study revealed, for the first time, a potential prognostic value of 5′-tiRNA-ProTGG expression levels with the prognosis of CRC patients. This tendency was further confirmed in patients with moderately differentiated and non-metastatic (M0) colorectal tumors as well as rectal carcinomas. Our analysis highlights the independent prognostic value of 5′-tiRNA-ProTGG expression levels concerning disease recurrence, when compared to the established prognostic markers in CRC. Moreover, bioinformatics analysis revealed a potential multifaceted biological role for 5′-tiRNA-ProTGG in CRC. A more intense functional study of the tiRNAs is prerequisite to fully uncover the intrinsic networks implicated in cancer development and progression. The clinical usefulness of 5′-tiRNA-ProTGG would be further enhanced if we consider that several tiRNAs can be found in the blood of patients with a variety of human diseases [TGG in biological fluids, such as serum, plasma and stools of CRC patients and evaluate its role as a candidate non-invasive tumor marker. Simultaneously, our study can offer new insights in the field of biomarker research for CRC, by introducing tiRNAs as promising molecular markers, which can be used synergistically in comprehensive prognostic panels.In summary, the present research study unveils an adverse association of the elevated 5′-tiRNA-Prodiseases . It woul


To address this gap, we extended the modeling framework ARCADE (Agent-based Representation of Cells And Dynamic Environments) to include CAR T-cell agents . We conducted in silico experiments to investigate how clinically relevant design choices and inherent tumor features—CAR T-cell dose, CD4+:CD8+ CAR T-cell ratio, CAR-antigen affinity, cancer and healthy cell antigen expression—individually and collectively impact treatment outcomes. Our analysis revealed that tuning CAR affinity modulates IL-2 production by balancing CAR T-cell proliferation and effector function. It also identified a novel multi-feature tuned treatment strategy for balancing selectivity and efficacy and provided insights into how spatial effects can impact relative treatment performance in different contexts. CARCADE facilitates deeper biological understanding of treatment design and could ultimately enable identification of promising treatment strategies to accelerate solid tumor CAR T-cell design-build-test cycles.Chimeric antigen receptor (CAR) T-cell therapy shows promise for treating liquid cancers and increasingly for solid tumors as well. While potential design strategies exist to address translational challenges, including the lack of unique tumor antigens and the presence of an immunosuppressive tumor microenvironment, testing all possible design choices  The six FDA-approved CAR T-cell therapies and many studies expanding CAR designs exclusively target “liquid” cancers that typically derive from CD19+ B-cells (Chimeric antigen receptor (CAR) T-cell therapy combines advances in cellular engineering and personalized medicine for patient-specific, targeted cancer treatment . This th B-cells . CD19 CA B-cells . In cont B-cells .CAR T-cells are currently less effective for treating solid tumors due unique complexities of both the tumor microenvironment (TME) and tumors themselves. First, TME barriers prevent CAR T-cell infiltration . These b+:CD8+ CAR T-cell ratios  models to understand factors influencing CAR T-cell receptor signaling and downstream activation  provide ideal eriments . Past ABeriments , vasculaeriments , immune eriments , and tumeriments . HoweverIn this study, we systematically explore CAR T-cell therapy designs in solid tumor contexts by adding CAR T-cell agents to an established ABM  comprising tissue cell agents  and dynaCARCADE provides a flexible framework for characterizing and exploring hypothesized dynamics of population-level tumor responses to CAR T-cell treatment by defining individual CAR T-cell, cancer, and healthy cell features and rules.dish and tissue, respectively.ARCADE comprises tissue cell agents with individual subcellular metabolism and signaling modules that influence the cell-level decision making rules and drive emergent population- and environment-level dynamics . Tissue + T-cells that primarily provide cytotoxic functions and CD4+ T-cells that primarily provide stimulatory functions (+ only), cytotoxic (CD8+ only), and paused. Undesired states include apoptotic, senescent, exhausted, anergic, and starved. Cells change state according to the rule set and to their current state  on a tissue cell’s decision to proliferate or migrate. CAR T-cell agents use the tissue cell metabolism module with modifications to capture the influence of IL-2 signaling and antigen-induced activation on T-cell metabolism: 1) increased metabolic preference for glycolysis; 2) increased glucose uptake rate; and 3) increased fraction of glucose used to produce cell mass  Frauwir. CAR T-c T-cells  and on g T-cells  . In all simulations, cancer cell and CAR T-cell counts follow experimentally observed trends, including conditions with effector-to-target (E:T) ratios less than one where cancer cell killing occurs over several days . This observation suggests that it is not possible to optimize both efficacy and safety when healthy cells express antigen, and the most useful strategies—typically less aggressive treatments—balance these objectives  cancer cells did not grow beyond the initial number, and 2) no more than 50% of the initial healthy cells were killed off.CAR T-cell therapy has great potential for use in solid tumor contexts, which include a complex tumor microenvironment, vasculature, spatial dynamics, and potentially antigen-expressing healthy cells. Predicting how the plicates —to analytissue is initialized with a bed of healthy cells in vascularized tissue that was inoculated with cancer cells and grown for 30 d. At t = 21 d, treatment began by adding a specified total dose of CAR T-cells, each expressing 5 × 104 CARs with the given CAR affinity, and CD4+:CD8+ ratio. CAR T-cells were spawned at locations adjacent to vasculature to mimic intravenous trafficking to the tumor; they were not spawned adjacent to vessels that are too small in diameter for CAR T-cells to pass through. Files used to generate tissue simulations are described in A tissue  sorted from best to worst outcome in terms of difference in healthy and cancer cell counts normalized to start of treatment  are shuffled from their original rankings in dish. One of the worst ranked treatments in dish (11th) jumped to 5th in tissue, while a middle-ranked simulation (7th) fell to 13th in tissue. These data predict that the most effective treatment conditions in dish will perform similarly in tissue assuming perfect CAR T-cell trafficking. Even with perfect trafficking, performance in dish does not exactly correlate with performance in tissue. Certain conditions may outperform in vivo conditions compared to their performance in vitro.Comparing simulation rankings between reatment . Nearly n tissue . The rantissue (+:CD8+ ratio. Most conditions that improve in rank use the relatively higher (though still objectively moderate) CAR affinity and higher CAR T-cell dose, and nearly all conditions that decrease in rank (from dish to tissue) have higher cancer antigen expression level. This finding is surprising given earlier observations that lowest CAR affinity with highest cancer antigen expression level combinations were most effective.Trends in how each feature impacts relative rank reveal which features most strongly dictate performance in tissue . There adish and tissue trends/rank result from differences in spatial dynamics. In dish, both healthy and cancer cells are well-mixed across the simulation, even after treatment, which results in an even spatial distribution of CAR T-cells , we find similar distributions of cells in effector states across dish and tissue simulations , which is known to be an important factor in dictating CAR T-cell efficacy, persistence, and dynamics; rather, we approximate CAR behavior independent of ICD and find that broad trends hold despite not accounting for this factor explicitly. The model could be tuned to capture the effect of different ICD choices on CAR T-cell function. Similarly, the analysis can be tuned to change the definition of effective treatment outcomes to further penalize healthy cell killing . When treating B-cell cancers, off-tumor effects like B-cell aplasia are manageable with treatment, and healthy cell killing is less of a concern. In glioblastomas, EGFR is expressed on cancer cells, healthy brain cells, and other tissues, making healthy cell killing a greater risk of morbidity and mortality . Anotherin vivo CAR T-cell therapy. Additionally, while the current model was designed to facilitate analysis of treatments for solid tumors, particularly through the use of the tissue simulations, the dish simulations could be adapted to investigate liquid cancer treatment strategies. Future expansions could also incorporate trogocytosis, a processes by which CAR T-cells pick up tumor antigens from cancer cells and then experience fratricidal killing by other CAR T-cells, to investigate how this phenomena affects CAR T-cell persistence (Expanding the agents, environment, or subcellular functions included in CARCADE offers opportunities for future model development and use in the field of CAR T-cell engineering. The present model comprises CAR T-cells and cancerous and healthy tissue cells; addition of macrophages, regulatory T-cells, natural killer cells, and other regulatory or supporting cell types or environmental factors could enable investigation of CAR T-cell therapy in a more complete and complex immune environment. In future studies, it may be particularly important to include the cell and environmental factors that contribute to immunosuppressive environments, as this is a common issue faced with sistence .dish and/or tissue contexts. CARCADE is designed to enable interrogation of questions and phenomena that are beyond the scope of the current study. For example, future studies using the current model could include a more granular consideration of CAR T-cell trafficking within the tumor by removing the assumption of perfect trafficking. Tumor immune escape could be investigated by creating multiple tumor subpopulations with variable antigen expression levels or susceptibly to killing. Additionally, inter- and intra-tumor heterogeneity could be integrated by simulating tumors that comprise multiple populations with different parameters and/or differing levels of heterogeneity. Ultimately, integrating CARCADE into the CAR T-cell design process could accelerate the design-build-test cycle, saving resources and time associated with new therapeutic development.Overall, we believe that CARCADE will prove valuable for CAR T-cell designers and enable cross-cutting collaborations to facilitate further model development or tuning to specific contexts and questions of interest. By further refining the model using experimental data, CARCADE could help suggest potential promising strategies for experimental pursuit by testing strategies in in silico experiments where we treated monoculture dish, ideal and realistic co-culture dish, and tissue contexts with CAR T-cells. All model details, including adaptation of tissue cell agents, development of CAR T-cell agents, development and adaptation of subcellular modules, development of dish plating, and all simulation setups and analyses are described in detail in the Supplementary Methods Details section of the Supplementary Material (CARCADE was developed by extending ARCADE, an existing multi-scale, multi-class agent-based model that includes tissue cells and hemodynamic environments. We used CARCADE to generate Material .https://github.com/bagherilab/CARCADE. Scripts used to process and analyze data are available on GitHub at https://github.com/bagherilab/carcade_mapping_design_space.All source code for CARCADE is available on GitHub at


COVID-19 is a respiratory infectious disease caused by the coronavirus SARS-CoV-2. Lung cancer is the leading cause of all cancer-related deaths worldwide. As both SARS-CoV-2 and lung cancer affect the lungs, the aim of this narrative review is to provide a consolidation of lessons learned throughout the pandemic regarding lung cancer and COVID-19. Risk factors found in lung cancer patients, such as advanced cancers, smoking, male, etc., have been associated with severe COVID-19. The cancer treatments hormonal therapy, immunotherapy, and targeted therapy have shown no association with severe COVID-19 disease, but chemotherapy and radiation therapy have shown conflicting results. Logistical changes and modifications in treatment plans were instituted during the pandemic to minimize SARS-CoV-2 exposure while maintaining life-saving cancer care. Finally, medications have been developed to treat early COVID-19, which can be highly beneficial in vulnerable cancer patients, with paxlovid being the most efficacious drug currently available.Cancer patients, specifically lung cancer patients, show heightened vulnerability to severe COVID-19 outcomes. The immunological and inflammatory pathophysiological similarities between lung cancer and COVID-19-related ARDS might explain the predisposition of cancer patients to severe COVID-19, while multiple risk factors in lung cancer patients have been associated with worse COVID-19 outcomes, including smoking status, older age, etc. Recent cancer treatments have also been urgently evaluated during the pandemic as potential risk factors for severe COVID-19, with conflicting findings regarding systemic chemotherapy and radiation therapy, while other therapies were not associated with altered outcomes. Given this vulnerability of lung cancer patients for severe COVID-19, the delivery of cancer care was significantly modified during the pandemic to both proceed with cancer care and minimize SARS-CoV-2 infection risk. However, COVID-19-related delays and patients’ aversion to clinical settings have led to increased diagnosis of more advanced tumors, with an expected increase in cancer mortality. Waning immunity and vaccine breakthroughs related to novel variants of concern threaten to further impede the delivery of cancer services. Cancer patients have a high risk of severe COVID-19, despite being fully vaccinated. Numerous treatments for early COVID-19 have been developed to prevent disease progression and are crucial for infected cancer patients to minimize severe COVID-19 outcomes and resume cancer care. In this literature review, we will explore the lessons learned during the COVID-19 pandemic to specifically mitigate COVID-19 treatment decisions and the clinical management of lung cancer patients. As the Coronavirus-disease-2019 (COVID-19) pandemic enters its third year, the ongoing health crisis has affected every aspect of the healthcare system worldwide. First reported in December 2019 in China, SARS-CoV-2, which stands for “severe acute respiratory syndrome coronavirus 2”, quickly spread to other countries, leading the World Health Organization (WHO) to declare COVID-19 a worldwide pandemic in March 2020. Since then, SARS-CoV-2 infection rates have increased exponentially, on a worldwide scale, with most countries experiencing severe outbreaks in multiple waves. As of 15 July 2022, there have been over 556,000,000 confirmed cases of COVID-19 worldwide, and almost 6,350,000 deaths reported to the WHO, making the COVID-19 pandemic one of the deadliest pandemics in history )) AND ((((COVID-19) OR (SARS-CoV-2)) OR (“COVID-19”[Mesh])) OR (“SARS-CoV-2”[Mesh])))((((neoplasm) OR (cancer) OR  OR (“Neoplasms”[Mesh])) AND ((((COVID-19) OR (SARS-CoV-2)) OR (“COVID-19”[Mesh])) OR (“SARS-CoV-2”[Mesh])))) AND ((outcome) OR (determinant)) AND (cohort).The main online database, PubMed, was searched from inception until March 2022 for studies on cancer and COVID-19 outcomes, and trials evaluating treatments against mild-to-moderate COVID-19. Two different query strings were used for the cancer and COVID-19 outcomes search: The first query was used to search specifically for observational studies pertaining to lung cancer and COVID-19 outcomes, while the second query was broadened to all cancers. Regarding the eligibility criteria, we focused on articles with titles or abstracts that (a) had patients with active cancer or recent cancer treatment at the time of acquiring COVID-19, either evaluating only lung or thoracic cancer patients or analyzing all cancer types but containing a substantial number of lung cancer patients, and of both sexes; (b) analyzed outcomes of COVID-19 severity or mortality; (c) original studies that were observational cohort studies; and (d) whether anticancer treatments affected COVID-19 outcomes was evaluated.The following query string was used to search for studies pertaining to therapies used to treat mild-to-moderate COVID-19:((“Drug Therapy”[MeSH Terms]) OR (drug therapy)) AND (((((COVID-19) OR (SARS-CoV-2)) OR (“COVID-19”[Mesh])) OR (“SARS-CoV-2”[Mesh]))).The PICOS search tool was used to selecting relevant studies pertaining to mild-to-moderate COVID-19 treatments : (1) popCOVID-19 is caused by the single-stranded RNA virus SARS-CoV-2, which primarily spreads by airborne transmission . COVID-1The second phase, seen only in cases of sCOVID-19 pneumonia, is the result of an overreactive and generalized immune response to a viral infection of the lower respiratory tract, specifically the lungs. ARDS can develop in this phase, leading to acute respiratory failure and death. ARDS is a form of severe lung injury, clinically diagnosed by severe hypoxemia and bilateral radiographic opacities following SARS-CoV-2 infection and is marked by damage in the lung parenchyma, increased pulmonary vascular permeability, and a considerable loss of oxygenated lung tissue. ARDS is further characterized by extensive pulmonary vascular injury, with endothelial dysfunction, including upregulated pro-coagulation factors, inhibited fibrinolysis, and pro-thrombotic coagulopathies ,23. In AGiven the viral S protein–host ACE-2 receptor interaction for initial viral entry, comorbidities that affect host ACE-2 receptor levels may lead to increased rates of SARS-CoV-2 infection and subsequent severe disease due to altered immune statuses. For example, type II diabetes mellitus (T2DM) has been found to be causally related to an increased level of ACE-2 receptors, along with increased furin-like enzymes needed for S protein activation . DiabetiThe impact of other comorbidities on the risk of COVID-19 infection and severity have also been described: hypertension, in which ACE-2 inhibitors as treatment for hypertension are known to upregulate ACE-2 receptor expression, leading to higher susceptibility to COVID-19 infection; COPD, where upregulated ACE-2 receptor expression, lung damage, altered local immunity, and increased mucous production contribute to increased COVID-19 infection and morbidity; and obesity, which is linked to reduced oxygen saturation in the blood due to compromised ventilation at lung bases and the presence of low-grade chronic inflammation, with abnormal levels of cytokines, adipokines, and interferon expression due to an altered immune system, making obese individuals susceptible to COVID-19 . PotentiLC patients, and generally many cancer patients, are older, with multiple co-morbidities, making them highly susceptible to SARS-CoV-2 infection and severe disease. A study by Fowler et al.  determinLC is the second most commonly diagnosed carcinoma worldwide, in both men and women, with an estimated 234,030 new cases occurring in the U.S. alone in 2018 . LC, beiLung cancers are classified based on their histopathological appearances into non-small cell lung cancers (NSCLCs) and small cell lung cancers (SCLCs). NSCLCs occur in 80% of all LCs and are further subdivided into multiple subtypes, including adenocarcinomas, squamous cell carcinomas, and large cell carcinomas, accounting for approximately 40%, 20%, and <3% of all LCs, respectively. SCLCs account for the remaining 20% of LCs and are almost exclusively associated with smoking. They are often highly aggressive with evidence of metastatic disease already present at the time of diagnosis. Patients with SCLCs are highly responsive to treatment, but most relapse within two years following treatment. Microscopically, the sizes of SCLC tumor cells are much smaller versus NSCLCs, with a fine granular chromatin without prominent nucleoli, a high mitotic and apoptotic rate, leading to necrosis, and a scanty cytoplasm. SCLCs are often located centrally, along the major airways .Trends in overall LC mortality rates have decreased recently due to significant advances in clinical management of LCs  . HoweverTo provide more insights into the relationship between SARS-CoV-2 and LC, we will discuss the pathophysiological roles of increased inflammation and immunosuppression in LC.In LC, inflammation is known to play a pivotal role in tumorigenesis and cancer progression. A chronic inflammatory state induced by smoking or chronic infections is consistently associated with LC risk ,47,48,49While COVID-19 ARDS is acute and LC pathogenesis is chronic, aberrant inflammation and immune dysregulation appear to play important roles in LC and sCOVID-19. A heightened inflammatory state marked by elevated IL-6 and CRP are involved in progression and poor outcomes in both diseases. Regarding altered immunity, significantly increased levels of PD-1 are seen in both sCOVID-19 and LC. This increase in T-cell exhaustion markers suggests that suppression of T-cell-mediated immune function occurs through PD-1/PD-1L interactions and plays an important role in both diseases; immune cells cannot effectively clear their targets, resulting in either viral persistence or LC tumor survival. The immunosuppressed and chronic pro-inflammatory TME in LC patients may provide an optimal environment for SARS-CoV-2 replication while simultaneously increasing the risk to develop the aberrant inflammatory response seen in COVID-19 CRS.Age is associated with both LC incidence and sCOVID-19 ,40. SignAdditionally, effective innate immune responses become compromised with aging and can lead to an increase in generalized inflammation, as perhaps a compensatory immune mechanism. Type I interferon (IFN) production, a signaling protein essential for initial antiviral response, appears to be delayed with aging, leading to increased pro-inflammatory cytokine release and a decrease in anti-viral natural killer cell activation. Innate immune cells also have a reduced capacity to respond effectively to DAMPs and PAMPs. Neutrophils migrate less accurately during infection and have reduced phagocytic and intracellular killing capacities, while macrophages that secrete pro-inflammatory cytokines are more easily activated . In the T cells are required to generate a highly effective and specific immune response against invading pathogens. In the context of aging and immunosenescence, the naïve T-lymphocyte population dramatically decreases in older adults due to involution of the thymus, the main site of T-cell production during puberty, and incurs a further decrease due to gradual, continuous age-related hematopoietic stem cell insufficiency . CD8+ T Because of inflamma-aging and immunosenescence, both antiviral and anticancer immune functions weaken in aging adults, with LC patients, who are often older, expected to be particularly vulnerable to sCOVID-19. In COVID-19, a low-grade chronic inflammatory state in aging adults provides favorable conditions for the progression of COVID-19 to CRS and ARDS. Meanwhile, impaired innate and adaptive immune responses lead to viral persistence, which further exacerbates inflammation. In LC, an altered lung TME from inflamma-aging and decreased immunosurveillance are able to elicit tumor progression .Cohort studies measuring COVID-19 outcomes in cancer patients have shown that certain cancer types suffered from higher rates of sCOVID-19 versus the general population. An early Italian study reported that patients with hematological cancer or LC were more vulnerable to sCOVID-19 than other cancers. Of note, LC was associated with a fourfold increased risk of mortality from sCOVID-19 versus non-cancer COVID-19 patients . A seconGiven that early studies reported increased rates of sCOVID-19 and mortality in LC, we reviewed larger cohort studies to clarify the characteristics of general cancer patients and LC patients with COVID-19 to identify the risk factors for sCOVID-19 in this patient group. A summary of the studies discussed below recapitulates the cohort sample size, COVID-19 outcomes, and risk factors associated with sCOVID-19 .Overall, COVID-19 mortality in cancers patients ranges from 13% to 33.7%, rates that appear much higher compared to non-cancer COVID-19 patients . RegardiHospitalization rates for cancer patients ranged as high as 76% and 78.3% in the studies by Provencio et al.  and GaraIn the studies reviewed, the most frequent risk factors for COVID-19 identified in cancer patients were older age, co-morbidities , male sex, and smoking history . CertainCancer patients appear to be at a high risk of mortality overall, with lung cancer being associated with increased COVID-19 mortality in two studies, including the large-scale study by Chavez-MacGregor et al. . FurtherPrevious reports showed, early in the pandemic, concerning data about their administration, systemic anti-cancer therapies as a risk factor for increased COVID-19 morbidity and mortality ,73. RegaCancer therapies have been affected by COVID-19 in ways other than increased risk of sCOVID-19 outcomes. Certain LC therapies might account for complications with clinical presentations similar to COVID-19 pneumonia. While no association was found between immunotherapy and COVID-19 mortality, the administration of immune checkpoint inhibitors (ICIs) as immunotherapy can lead to severe pneumonitis as a complication. Checkpoint-inhibitor-induced pneumonitis (CIP) is caused by inflammation in the lung due to uncontrolled reactivation of T-cell activity and reversal of exhaustion signaling, with an incidence rate of 3–5% in cancer patients undergoing ICI treatment . ClinicaHealthcare settings worldwide have been immensely affected by the high volume of patients due to the pandemic, combined with an increased need for intensive-care beds and iatrogenic transmission of SARS-CoV-2. As a result, hospitals were forced to deliver alternative caregiving models. Otolaryngology (ENT) departments are especially vulnerable to SARS-CoV-2 transmission given their frequent direct contact to saliva and mucous suspensions, ranging from physical examination of the oral cavities to procedures, such as rhinoscopies and laryngoscopies. ENT clinics implemented strict alternative standard operating procedures to minimize contact to SARS-CoV-2 during the pandemic. Individual safety practices in clinics include COVID-19 screening before ENT evaluation, the use of telemedicine in non-urgent evaluations, and the use of PPE . Changes to the physical environment were also implemented, involving the use of well-defined segregated areas for structurally isolated ENT outpatient rooms, a filter room with well-defined segregated outpatient rooms, filter areas where patients enter wearing a face mask, and a dirty area for safe removal of PPE with a separate clean area dedicated to PPE wearing. Similar changes were implemented in operation rooms as well . In cancThe use of PPE, specifically face masks, is crucial in protecting healthcare workers from SARS-CoV-2. Long-term and constant use of face masks does have its drawbacks; however, a survey study by Maniaci et al.  describeThe circumstances of the COVID-19 pandemic drastically altered cancer care. Throughout the pandemic, healthcare staff implemented many measures to minimize SARS-CoV-2 exposure and continue cancer care. A strategy to prevent COVID-19 outbreaks was used in an Italian radiation oncology hub designed to pursue regular cancer treatment while minimizing COVID-19 exposure during the initial months of the pandemic. The European Institute of Oncology (IEO) received all regional cancer patients from general hospitals and established a central COVID-19-free hub, using strict protocols effective in protecting cancer patients from COVID-19 infection. COVID-19 diagnosis, isolation, and management were rapid as staff had protocols to identify suspicious symptoms  and wide access to same-day diagnostic tests. Additionally, the center minimized the risk of exposure with telemedicine, social distancing, remote working, requirements to wear surgical masks for everyone and PPE for staff, prohibiting relatives and friends from visiting, and mandatory nasopharyngeal swabs for incoming patients . SimilarCertain cancer treatments cannot be delayed, even during a pandemic, due to the rapidly progressive nature of lung neoplasms. Anticancer regimens have been altered to maintain cancer care while minimizing the risks of iatrogenic SARS-CoV-2 exposure and infection. Durvalumab, a PD-1-PD-1L inhibitor, is used as adjuvant immunotherapy in stage III NSCLC. While durvalumab was normally given bi-weekly at 10 mg/kg, the regimen was altered to 20 mg/kg every 4 weeks to reduce hospital visits and minimize contact with SARS-CoV-2, which was complemented with telemedicine for safety monitoring of patients. Despite the doubling of dosage per visit, a study by Joshi et al.  showed aThe use of shortened high-dose hypofractionated RT regimens (HFRT) was suggested as an alternative treatment modality to conventionally fractionated RT (CFRT) during the pandemic ,97. IndeSurgical services are especially vulnerable to delays due to the high risk of SARS-CoV-2 exposure during surgery-related hospitalization and the redirection of surgical resources  for COVID-19 patients during outbreaks. Resectable LCs , thus, require alternative treatments when timely surgery cannot be performed. Stereotactic body radiotherapy (SBRT) for resectable stage I/II NSCLC has been suggested as a viable alternative . SBRT reDespite the current pandemic adaptations from cancer centers, initial delays in cancer diagnoses and treatment were paramount to minimize infection given the risk of sCOVID-19 in LC patients. The early consensus in the CHEST panel report was to delay all screenings until further notice, delay surveillance procedures and evaluations of suspicious nodules for three to six months, and only treat confirmed stage I NSCLC without any delay . These dp < 0.04) [As COVID-19 continued with further outbreaks and waves, it became clearer that delaying cancer treatment was associated with high risks of cancer progression and mortality. Due to the delays that often exceeded three months due to high local prevalence and a general fear of visiting hospitals during the COVID-19 pandemic, a significant decrease in cancer diagnoses was detected during the first wave of the pandemic compared to before the pandemic . An Ohio < 0.04) . These r < 0.04) ,112. Can < 0.04) ,113. Sim < 0.04) . In the  < 0.04) ,116.Worryingly, the decreases in screening, diagnoses, and treatment of cancers seen during the COVID-19 pandemic can potentially lead to a surge in cancer progression. The impacts of delays, interruptions, and cancellations of cancer care services at the initial stages of the pandemic have already been observed by oncological centers worldwide. A majority of the European radiation oncology centers (71.0%), surveyed one year following the onset of the COVID-19 pandemic, reported patients presenting with more advanced diseases . In the As SARS-CoV-2 evolves into novel variants of concern (VOC) over time, it is important to review how the ease of transmission of the virus, its infectivity, antigenicity, viral disease severity, and virulence, are altered in VOCs, with the B.1.617.2 (Delta) and B.1.1.529 (Omicron) VOCs being examples. Mutations in the S protein are the principal mechanism of increased virulence, transmissibility, and immune evasion among the novel VOCs. In Delta, 17 mutations were discovered in the S protein, with notable L452R and T478K mutations on the RBD of the S protein. These substitutions increase binding affinity to ACE2 and decrease immune recognition, enhancing replication and immune evasion . AnotherTo minimize delays of cancer diagnosis and treatment, COVID-19 infection and complications must be minimized by reducing exposure, vaccinations, and early treatment in case of infection. Despite efforts to minimize COVID-19 exposure, risk of SARS-CoV-2 transmission can be difficult to control in clinical settings, especially during outbreaks of novel VOCs. While COVID-19 vaccines have initially shown high efficacy in preventing symptomatic infection, waning immunity against COVID-19 from vaccination has become an increasing challenge. A rapid and significant decrease in humoral response following two doses of the BNT162b2 vaccine has been observed over a six-month period . A similWith the novel VOCs able to escape immunity in healthy individuals, cancer patients appear to have an even higher risk of developing symptomatic infection that can progress to sCOVID-19, despite being fully vaccinated. An observational study that included cases between November 2020 and May 2021 demonstrated that most cancer patients with breakthrough COVID-19 were hospitalized (65%). Severe disease was observed in these hospitalized patients, with 16% ICU admission and 13% mortality rates. These findings were comparable to unvaccinated cancer patients, as the study found no significant differences in mortality between vaccinated and unvaccinated cancer patients . A lack of significant difference was equally found in ICU and hospitalization rates. Patients with active and progressive cancers, hematological malignancies, or undergoing systemic sCT were overrepresented among the vaccinated group with sCOVID-19, suggesting increased vulnerability in these groups. The study further identified lymphopenia as a significant risk factor for mortality in the vaccinated cancer group . As thisDirect-acting antivirals (DAAs) specifically target and inhibit the viral replication mechanism to decrease the viral load. The novel oral COVID-19 antivirals molnupiravir and combination nirmatrelvir–ritonavir (or paxlovid) have both been approved to treat mCOVID-19.4-hydroxycytidine triphosphate, is a nucleoside analog of cytidine or uridine. Viral RNA-dependent RNA polymerase (RdRP) erroneously incorporates NHC triphosphate, leading to mutations, genomic instability, and a reduction in viral replication [Molnupiravir, in its active form β-D-Nlication . The phalication . AnotherIn paxlovid, the nirmatrelvir component is an inhibitor of coronavirus 3C-like protease, an enzyme required to lyse and release viral proteins essential for viral RNA replication and proliferation . Co-admiThe DAA remdesivir, in addition to its current use for hospitalized COVID-19 patients, has also been evaluated to treat mCOVID-19 in outpatients with at least one risk factor for sCOVID-19. Remdesivir is a prodrug that is metabolized into its alanine active form once inside the body to become a nucleotide analogue. Active remdesivir is then incorporated into viral RNA by viral RdRP to promote early chain termination during RNA synthesis, inhibiting replication . A phaseRegarding medications that have been repurposed for COVID-19, fluvoxamine, as a selective serotonin reuptake inhibitor (SSRI), has been evaluated as a potential treatment for non-hospitalized mCOVID-19. Fluvoxamine is known as an inhibitor of acid sphingomyelase (ASM), a host enzyme essential for the generation of ceramide, which is known to facilitate viral entry . Sigma-1Most other SSRIs have shown antiviral mechanisms similar to fluvoxamine as they possess S1R agonism and ASM antagonism . SSRIs, Hydroxychloroquine was originally an antimalarial medication that was repurposed into a disease-modifying antirheumatic drug. Hydroxychloroquine has anti-inflammatory properties, attenuating production of pro-inflammatory cytokines and inflammation, which may protect against the CRS seen in sCOVID-19. Additionally, in vitro studies have shown that hydroxychloroquine could disrupt communication between SARS-CoV-2 S proteins and cell membranes, inhibiting viral entry . Azithrop = 0.005) [Convalescent plasma is an old therapy that has been historically used against multiple infectious diseases, such as MERS-CoV, H1N1, H5N1, etc. . Convale= 0.005) .Neutralizing monoclonal antibodies (nMAb) are also used to treat high-risk mCOVID-19 patients. nMAb are antibodies isolated from convalescent plasma from COVID-19 patients that can inhibit viral replication by specifically targeting and binding on epitopes on the RBD of the S protein, preventing ACE2 attachment and cell entry . CocktaiAdditionally, given that nMAbs are a form of passive immunization, as they involve the use of SARS-CoV-2 specific antibodies, there has been great interest in nMAbs as a form of prophylaxis in high-risk populations for SARS-CoV-2 infection. A recent phase III trial studied casirivimab/imdevimab as PoEP in healthy unvaccinated participants in close contact with an individual confirmed with SARS-CoV-2 infection. An 81.4% rRR was shown in participants exposed to SARS-CoV-2 administered with casirivimab/imdevimab. Phase 3 clinical trials for other nMAb cocktails, such as AZD7442 (tixagevimab/cilgavimab) and sotrovimab, are underway for both PrEP and PoEP in individuals at high risk of sCOVID-19 . A recent paper published on the trial of AZD7442 as COVID-19 PrEP showed promising results in preventing infection for individuals at high risk for exposure or inadequate response .Interferons (IFN) have been well characterized as a key factor in initial immune response to viral infections, including SARS-CoV-2. Type I IFNs have an important role in restricting viral replication and spread as a key regulator of the innate and adaptive immune systems . It is sp = 0.001). This study suggests that high-risk mCOVID-19 patients may benefit from IFNs with several trials currently underway for IFN λ as a potential treatment option for mCOVID-19 . There has also been interest in using intranasal IFNs as a prophylaxis strategy, with the prophylactic potential of IFN α nasal drops observed in an open label study on hospital workers with high-risk exposure to SARS-CoV-2 [With the importance of early IFN induction during the initial stages of infection, administration of IFN may provide better benefits in the early mCOVID-19 versus during late sCOVID-19. Multiple clinical trials are underway in evaluating IFNs as a treatment for outpatients exhibiting mCOVID-19 symptoms. A phase II study found efficacy in subcutaneous pegylated type III IFN λ at accelerating viral decline in individuals with asymptomatic COVID-19 . A higheRS-CoV-2 . AnotherAs the COVID-19 pandemic continues into its third year, recurrent outbreaks have affected every aspect of healthcare, including the management of cancer screening, clinical investigations, and treatment.The early consensus from cohort studies identified cancer patients as a particularly vulnerable population for sCOVID-19, with hematological malignancies and metastatic disease being identified as independent risk factors for poor COVID-19 outcomes. While studies have found conflicting results regarding LC as an independent risk factor for sCOVID-19, LC patients often harbor multiple major risk factors shared with sCOVID-19 outcomes. Additionally, immunosuppressed, and pro-inflammatory properties of the TME in LC provide the optimal environment for SARS-CoV-2 replication and spread, which can lead to diffuse cell injury, ARDS, and CRS. Several studies further characterized the following risk factors for sCOVID-19: a smoking history, older age, the male sex, multiple co-morbidities, immunosuppression, and lymphopenia. Many of these risk factors possess similar pathological mechanisms to a pro-inflammatory state and immunosuppression. Regarding the male sex as a risk factor, men with COVID-19 appear to elicit a higher inflammatory response with more pro-inflammatory cytokines and monocytes compared to women. Furthermore, CD8+ T-cell response was shown to be lower in male compared to female patients, with a negative correlation observed between older age and T-cell function observed only in men . One stuAdditionally, a majority of metastatic lung cancer is under more intensive immunosuppressive sCT, leaving them at further increased risk of COVID-19 mortality ,167. RegClinical presentations of treatment-related toxicity and COVID-19 pneumonitis have also become a challenge in oncological care. ICI-related and RT-related pneumonitis have clinical and radiological findings that are highly similar to COVID-19 pneumonia . Risk asDuring the initial waves of the pandemic, oncological care adapted to the best of its abilities to the rapidly changing circumstances of COVID-19. Oncological hubs were created as COVID-19-free centers that received cancer patients from regional hospitals overwhelmed by COVID-19 and safely continued regular cancer care, regardless of local COVID-19 prevalence. These centers were able to prevent COVID-19 outbreaks in their patient populations by following strict safety guidelines  and implementing protocols in the event of suspected COVID-19. Cancer treatment regimens have also been altered to minimize hospital visits and COVID-19 exposure. For stage III NSCLC, durvalumab and RT dosing schedules were modified. An altered dose regimen, with a short fractionation regimen for RT and long interval periods for durvalumab, reduced patient exposure. In cases where certain treatments, such as surgery, were not available due to the pandemic, alternatives, such as SBRT, were proposed. While SBRT is not the standard of care for operable early-stage NSCLC compared to surgery, timely treatment of these patients with SBRT is paramount for favorable outcomes and close follow-ups should be given to patients following SBRT. Additionally, when surgical services resume, surgical resection has been proposed to patients who received SBRT if response to treatment was suboptimal following a thoracic CT scan or if patients seek further treatment .Despite the efforts to adapt cancer care during the COVID-19 pandemic, high volumes of cancellations and delays in cancer screening and treatment have been observed worldwide. Routine cancer services and non-urgent surgeries were shut down for several months due to high COVID-19 infection risk in clinical settings and due to resources being redirected to combat the pandemic. Thus, healthcare providers noticed cancer-stage progression and more advanced and metastatic cancers when care services restarted, with a large backlog expected to last for more than a year. COVID-19-pandemic-related excess cancer mortality, especially from LC, is expected to significantly increase for the next few years ,117,118.Overall, patients receiving systemic cancer treatments are often immunosuppressed and may be more vulnerable to breakthrough infections . A focusMultiple novel promising treatments have emerged to treat mCOVID-19 in patients at high risk for severe disease. Of all the treatments discussed, the novel DAAs paxlovid and molnupiravir, with their effectiveness in preventing sCOVID-19, favorable safety profiles, and oral route of administration, are expected to become the gold standard for treating mCOVID-19 in high-risk outpatients. Paxlovid is the preferred option due to its apparent highest efficacy. Cancer patients with a recent COVID-19 diagnosis could be prescribed with the novel DAA and be safely treated at home. Given the quicker time to recovery with the treatment of COVID-19, patients could resume life-saving anticancer treatment with minimal delays. The other treatments discussed may be good alternatives in case paxlovid is unavailable. If shown to be effective in further large-scale trials, the oral fluvoxamine, along with its accessibility and affordability, could potentially be a highly desirable alternative. Convalescent plasma may also be useful in resource-limited settings where access to antivirals is severely limited or in outbreaks of novel SARS-CoV-2 VOCs that resist most available therapies. COVID-19 prophylaxis for vulnerable cancer patients could be a potentially life-saving method for preventing sCOVID-19 outcomes and safely continue anticancer treatments, especially since they are often exposed to high-risk environments in clinics or hospitals. For all treatments discussed, there is a lack of overall data on the efficacy of these approaches against mCOVID-19, specifically in cancer patients. Most clinical trials reviewed were not able to recruit more cancer patients, where underrepresentation was a major problem. Further specific clinical trials evaluating these treatments in cancer populations are required to better investigate their efficacy.The worldwide spread of Delta and Omicron has shown how rapidly VOCs can mutate its S proteins to increase infectivity and immune escape. These VOC mutations could decrease the efficacy of therapies that target the S protein, while further mutations might arise due to the selective pressure caused by these treatments. As currently available vaccines all generate humoral immunity targeting the S protein, vaccine breakthrough has become an increasingly difficult problem. The challenge of vaccine breakthrough is expected to increase with Omicron and future VOCs. Lymphopenia has been determined as a significant risk factor for sCOVID-19 in the studies reviewed and could have a significant role in breakthrough infections ,79,130.


This is a highly interesting case because, despite the advanced metastatic spread at initial presentation as showed by [68Ga]Ga-PSMA-PET/CT, the primary Pca was detected by none of the diagnostic techniques . However, [68Ga]Ga-PSMA-PET/CT showed a suspicious axillary lesion suitable for biopsy, which finally resulted as Pca metastasis. This case report is therefore a brilliant example of how [68Ga]Ga-PSMA-PET/CT optimized patient’s management.This is a case of [ Definitive diagnosis of Pca relies on histopathology; according to EAU guidelines  and enlarged pelvic and retroperitoneal lymphadenopathies at ultrasound, despite prostate gland negativity at both mpMRI and 12-core saturation biopsy. Nevertheless, [68Ga]Ga-PSMA-11 PET/CT was performed due to its good sensitivity and specificity in staging Pca patients (77% and 97% respectively for lymph node involvement (We present the [olvement ).268Ga]Ga-PSMA-11 PET/CT was then performed as it allows studying prostate gland, lymph nodes and bones at the same time; it was acquired approximately 60 minutes after the injection of 2 Mbq/Kg of [68Ga]Ga-PSMA-11 , and a saturation biopsy was performed; however, none of the 12 samples were positive for Pca. The transrectal ultrasound (TRUS) showed diffuse inhomogeneity of the left lobe, while only 1 out of the 12 saturation biopsy’s random samples indicated acute prostatitis (in the left lobe), still not justifying the major rise in PSA levels. Due to further increase of the PSA biomarker up to 77 ng/mL, a mpMRI was performed resulting in low probability of clinically significant Pca (PI-RADS 2)  but persidelines  and EANM368Ga]Ga-PSMA-11 PET/CT did not show any significant area of focal uptake within the prostate gland  orange arrow), neither at a 90 minutes-delayed scan; that was in accordance with the previous mpMRI (C) showing PI-RADS 2 prostate gland  purple circle and (D) purple arrow, showing an avid right external iliac lymph-node; (E) showing multiple lumbar-aortic lymph-nodes). Further findings were also detected: a single, intense focal bone lesion at L3 vertebral body with osteoblastic pattern  yellow circle, (E) yellow arrow) and a single focal uptake in a left axillary lymph-node  blue circle; (F) blue arrow). The axillary lymph-node was homolateral to the injection site of a recent anti-SARS-Cov2 vaccination and morphologically not clearly malignant; however, due to its high PSMA expression, it was therefore biopsied with a mini-invasive ultrasound-guided approach and finally diagnosed as Pca metastasis. Immunohistochemistry was also performed . Written informed consent was obtained from the individual for the publication of any potentially identifiable images or data included in this article.LV, GC, RM, and LZ contributed to the manuscript conception and design. The first draft of the manuscript was written by LV and GC, and all authors commented on previous versions of the manuscript. All authors contributed to the article and approved the submitted version.


Antibiotic resistance has posed a great threat to human health. The emergence of antibiotic resistance has always outpaced the development of new antibiotics, and the investment in the development of new antibiotics is diminishing. Supramolecular self-assembly of the conventional antibacterial agents has been proved to be a promising and versatile strategy to tackle the serious problem of antibiotic resistance. In this review, the recent development of antibacterial agents based on supramolecular self-assembly strategies will be introduced. Antibiotic resistance is a growing problem that causes 700,000 deaths per year worldwide, and a recent prediction indicates that bacterial infections will cause 10 million deaths by 2050 ,4,5,6. TResearchers in relevant fields are making great efforts to discover new drugs and develop new antibacterial strategies and technologies to address the serious issues caused by antibiotic resistance ,24,25,26In this review, we briefly highlight and discuss the recent development of antibacterial agents based on the supramolecular self-assembly strategies, which involve the above-mentioned noncovalent interactions. The typical and novel supramolecular self-assembly strategies, which could improve the therapeutic performance of antibacterial agents and overcome their shortcomings, will be focused on in this review. The review is divided into six sections according to the type of conventional antibacterial agents constituting the supramolecular self-assembly, including antibiotics, antimicrobial peptides, cationic surfactants and polymers, antibacterial photodynamic therapy (aPDT) agents, antibacterial photothermal therapy (aPTT) agents, and macrophages.Since their introduction in the 1940s, antibiotics have been heavily applied to treat infectious diseases. After the “Golden Age” of antibiotics, semisynthetic and chemically modified antibiotics have been deemed as the bright way to develop new antibiotics ,35,36,37The host–guest complexations between antibiotics and macrocyclic hosts are highly potential approaches to enhance antibiotics activity. Sinisterra et al. reported that the complexation of doxycycline (Dox) and β-cyclodextrin (β-CD) showed greater antimicrobial activity than free doxycycline  39]. Is. Is39]. Kumari et al. also fabricated the host–guest complexation between resorcin[4]arene and gatifloxacin to improve antibacterial activity . These hEscherichia coli (E. coli) biofilm ) and cationic surfactants  . The antWang et al. also fabricated the host–guest complexation of biocompatible CDs and cationic surfactant (tri(dodecyldimethylammonioacetoxy)-diethyltriamine trichloride, DTAD) to improve the mildness of cationic surfactant . The outTang and coworkers fabricated cationic nanoparticles through the co-assembly of cationic surfactant cetyltrimethylammonium bromide (CTAB) and conjugated polymers (PFVBT), which could enhance the antibacterial effect and lower the cytotoxicity of CTAB . When miThe commonly used antibacterial quaternary ammonium surfactants (QAS) generally lack long-lasting performances. The frequent usage of these agent cause accumulation in the environment, which would exert a huge burden on the ecosystem and selective pressure on bacteria, triggering the emergence of antimicrobial resistance. Supramolecular self-assembly is a potential strategy to fabricate long-lasting antimicrobial agents. For example, Wang et al. fabricated long-lasting antimicrobial aggregate by co-assembling QAS and biocompatible gallic acid through multiple synergistic noncovalent interactions, including electrostatic interaction, hydrogen bonding, hydrophobic interaction, and π-π stacking . Three QTraditional antibiotic abuse has caused a lot of health problems, including antimicrobial resistance. In order to tackle these problems, Lei and coworkers investigated two self-assembling modes between berberine (BBR) and flavonoid glycosides and compared their antibacterial potency . Based oA precise pesticide delivery nanoplatform was fabricated by iron mineralization after electrostatic self-assembly between sodium lignosulphonate (SL) and dodecyl dimethyl benzyl ammonium chloride (DDBAC). The nanoplatform was a core–shell structure, which was excellently stabilized by iron mineralization. The outer layer of the shell was constructed by in situ mineralization with iron ions, while the endothecium of the shell was formed by the electrostatic self-assembly of SL and DDBAC. The nanoplatform was suitable for encapsulating agrochemicals with hydrophobicity and low volatility. The nanoplatform showed a specific response to alkalescency and laccase in the alimentary tract of insects and also improved the photostability and environmental security of agricultural chemicals .Shen et al. designed an electrochemical redox-controlled bacterial inhibition agent [(12-ferrocenyl) benzalkonium bromide (FBZK)] containing ferrocene quaternary ammonium salt based on supramolecular self-assembly and disassembly . They deYang et al. exploited the supramolecular self-assemblies of cationic polyaspartamides and anionic carboxylatopillar[5]arene (CP[5]A) to target Gram-positive bacteria and mitigate antimicrobial resistance  78]. Th. Th78]. co-butyl methacrylate (BMA)), could self-assemble into core–shell nanoparticles with a non-fouling dextran shell and a cationic core. The nanoparticles could diffuse through Gram-positive biofilm and attach to bacterial surfaces. The dextran corona enhanced the bacterial/nanoparticle interactions to promote bacterial detachment from the biofilm, which reduced biofilm biomass. The bacterial debridement mechanism is orthogonal to antibiotic resistance so that it removes biofilms of drug-resistant strains as effectively as those of drug-sensitive strains.Chan-Park and coworkers also developed block copolymer nanoparticles to remove biofilms of drug-resistant Gram-positive bacteria using nanoscale bacterial debridement . The bloAntibacterial Photodynamic therapy (aPDT) is a promising way to eliminate bacteria and biofilm infections. In the aPDT process, the reactive oxygen species (ROS) destroy biomolecules in bacterial cells, leading to cell death ,81,82. T1O2 generation was also enhanced. Thus, the bacterial inhibition ability was efficiently enhanced by promoting ROS generation. Furthermore, metal ions, including Zn(II) and Pd(II), were introduced into the porphyrin core to enhance its ability to produce ROS and improve the efficiency of PDT uril (CB[7]) and porphyrin derivatives (TPOR) to enhance the antibacterial efficiency . As the y of PDT .N,N,N-triethylammonium-1′-propyloxy)-4-methyl-2,5-thiophene chloride) (PMNT) (Supramolecular assembly strategy is applied to manipulate the absorbance wavelength of conjugated polythiophene to enhance its aPDT effect under red light irradiation by Xing and coworkers. They fabricated hybrid hydrogels by co-assembling of tri(ethylene glycol)-functionalized polyisocyanide (PIC) and conformation-sensitive conjugated polythiophene, poly(3-(3′-) (PMNT) . The PICN-(4-boronobenzyl)-N,N-dimethylammonium} phenoxy-substituted zinc(II) phthalocyanine (PcN4-BA) generated the novel photosensitizer, and the boronic acid functionalized on the PCN4-BA endow the self-assembly with bacteria target capacity  and the cationic pedants of poly(phenylene vinylene) derivative (PPV) . When thTang and coworkers also regulated the antibacterial photodynamic effect of oligo(phenylene ethynylene) (OPE) through the supramolecular self-assembly of OPE and DNA . DNA-OPEA supramolecular self-assembly strategy was also applied to improve the biocompatibility and endow the photosensitizer with acid responsiveness. Recently, Zhang and coworkers fabricated an acid-triggered supramolecular porphyrin photosensitizer to combat bacteria and biofilms. The host–guest complexation of carboxylatopillar[5]arene (CP5) and quaternary ammonium-functionalized tetrafluorophenyl porphyrin (TFPP-QA) formed nanoparticles in aqueous solutions, resulting in the supramolecular photosensitizer . The quaN-methyl-4-pyridyl) porphyrin (AgTPyP) and carboxylatopillar[5]arene (P[5]) [Zhang and coworkers also constructed a pH-responsive supramolecular antibacterial photosensitizer AgTPyP@P[5] by the host–guest complexation between a water-soluble photosensitizer silver tetra(e (P[5]) . The hosPseudomonas aeruginosa  and polycationic cyclodextrin (CD-QAS) to enhance the specific intercalation between negatively charged bacteria membrane and the aPDT agent .Photothermal therapy (PTT) is an antibacterial method dependent on the light-induced heat from the photothermal agents. The locally increased temperature (≥41 ℃) inactivates the microbial cell by inducing protein denaturation and aggregation, disruption of cell membrane integrity, and DNA cross-linking ,100,101.E. coli), the supramolecular radical anions were in situ generated, which further generated hyperthermia to induce bacterial cell death under near-infrared light irradiation.Supramolecular radical anions were fabricated to serve as an in situ generated antibacterial near-infrared photothermal agents. The host–guest complexation of PPDI and CB[7] was firstly constructed , which cE. coli induced in situ supramolecular polymer was constructed by Xu and coworkers . T. T106]. E. coli and enhance the PTT of the graphene. The supramolecular carbohydrate-functionalized two-dimensional (2D) surface was constructed by host–guest complexation between hepta mannosylated β-CD (ManCD) and the adamant group on thermally reduced graphene oxide. The host–guest complexation on the surface of thermally reduced graphene oxide not only increased the intrinsic water solubility but also endowed the surface with effective E. coli capture capacity through multivalent interactions. Upon IR laser irradiation, the captured E. coli was killed effectively by the photo-induced heat from the graphene oxide [Regulating the interaction between PTT agents and bacteria through supramolecular self-assembly is an effective way to enhance the therapeutic performance of PTT. A supramolecular carbohydrate-functionalized two-dimensional (2D) surface was fabricated by Haag and coworkers to selectively capture ne oxide .E. coli was decorating with adamantyl (ADA) groups by the mannose-ADA via specific mannose–FimH interactions. Based on the strong and multipoint host–guest interactions between CB[7] on SAR-Macrophage and ADA on the E. coli, the SAR-Macrophage could significantly recognize E. coli and catch and internalize more pathogens. The promoted recognition and capture of E. coli induced the M1 polarization of macrophages to generate ROS and effectively killed the intracellular bacteria. This work provides a supramolecular cell-engineering approach for potential antibacterial applications [The phagocytosis of bacteria by macrophages acts as the front line to protect the human body from bacterial pathogens. The pathogens’ clearance efficiency is always low due to the relatively slow encounter and insufficient capture of bacteria by macrophages. Wang and coworkers developed a supramolecular artificial receptor-modified macrophage (SAR-Macrophage) to enhance the recognition and capture of bacteria in the systemic circulation. SAR-Macrophage was fabricated by decorating CB[7] on the macrophage surface via inserting DSPE-PEG-CB[7] -CB[7]) into the surface membrane of the cells . E. coliications .Antibiotic resistance has caught the attention of researchers from different fields, and plentiful multidisciplinary studies for combating antibiotic resistance have been reported in recent years. Supramolecular assembly is tunable, modular, and responsive, as the formation of assembly is based on noncovalent interactions. Supramolecular self-assembly provides a promising and versatile strategy to construct new antibacterial agents to combat antibiotic-resistant infections. In this review, we hope we have shown the recent development of antibacterial agents based on the supramolecular self-assembly strategies, which include the supramolecular self-assembly of antibiotics, antibacterial peptides, cationic surfactants and polymers, antibacterial photodynamic therapy agents, antibacterial photothermal therapy agents, as well as supramolecular assembly strategy engineered macrophage-bacteria interaction for promoting phagocytosis. These new antibacterial agents showed enhanced antibacterial activity, or the intrinsic shortcomings of the motifs were improved after supramolecular self-assembly.The current state of the art for antibacterial agents based on supramolecular self-assembly strategy exhibits strong potential to treat drug-resistant bacterial infections in the near future. However, there are still some issues that require to be added to promote the clinical translation of the novel antibacterial agents. First, the new antibacterial agents need to possess excellent biocompatibility. Biocompatibility is a basic requirement for constructing the new antibacterial agents, and the supramolecular self-assembly strategy also could be considered for the cytotoxicity of the potential reported antibacterial agents. Second, the stability of the new antibacterial agents needs to be systematically investigated. The new antibacterial agents exert their enhanced activity through the positive effects induced by supramolecular self-assembly, but the supramolecular self-assembly may show instability in the complex biological media. The revelation of the mechanism of the new antibacterial in the complex biological media will help in developing effective strategies for future applications. Third, the generation of resistance needs to be carefully evaluated during the research stage. As the resistance could be developed rapidly, the evaluation of resistance generation promotes feasibility for clinical tests. Last but not least, more new antibacterial agents based on supramolecular self-assembly need to be fabricated.