Leukemia is the most common type of childhood malignancy. While the clinical management of pediatric acute leukemia, especially acute lymphoblastic leukemia, underwent a remarkable improvement during the past decades, a subset of the patients still experience relapse and succumb to their disease. Emergence or repositioning of targeted therapies aided by the comprehensive characterization of single leukemic cells using advanced sequencing approaches may provide novel opportunities for therapeutic intervention in these patients. In our review, we summarize the status quo of single-cell-sequencing studies in pediatric leukemia, provide an overview of the current landscape of targeted agents applicable in this disease group, and highlight options for ways single-cell sequencing could further support the decision making related to the clinical management of pediatric leukemia.Single-cell sequencing (SCS) provides high-resolution insight into the genomic, epigenomic, and transcriptomic landscape of oncohematological malignancies including pediatric leukemia, the most common type of childhood cancer. Besides broadening our biological understanding of cellular heterogeneity, sub-clonal architecture, and regulatory network of tumor cell populations, SCS can offer clinically relevant, detailed characterization of distinct compartments affected by leukemia and identify therapeutically exploitable vulnerabilities. In this review, we provide an overview of SCS studies focused on the high-resolution genomic and transcriptomic scrutiny of pediatric leukemia. Our aim is to investigate and summarize how different layers of single-cell omics approaches can expectedly support clinical decision making in the future. Although the clinical management of pediatric leukemia underwent a spectacular improvement during the past decades, resistant disease is a major cause of therapy failure. Currently, only a small proportion of childhood leukemia patients benefit from genomics-driven therapy, as 15–20% of them meet the indication criteria of on-label targeted agents, and their overall response rate falls in a relatively wide range (40–85%). The in-depth scrutiny of various cell populations influencing the development, progression, and treatment resistance of different disease subtypes can potentially uncover a wider range of driver mechanisms for innovative therapeutic interventions. Most cancers are of monoclonal origin and characterized by an unleashed proliferation of tumor cells harboring genomic, epigenomic, and transcriptomic alterations acquired in a stepwise manner or in a single catastrophic event ,2,3,4,5.Next-generation sequencing (NGS) has dramatically revolutionized the research and diagnostics of malignant disorders and massively expanded our knowledge of mechanisms underlying cancer evolution, including transformation, clonal expansion, adaptative selection, and treatment resistance of tumor cells ,12. NoveSingle-cell sequencing (SCS) has been providing an increasingly deep insight into the genetic composition, subclonal architecture, regulatory network, gene expression profile, and even proteome-level phenotype of a wide range of cancers . The amoDue to the easy accessibility of non-adherent, single leukemic cells, oncohematological malignancies have commonly been pivotal in elucidating key biological mechanisms, thus fueling the progress of cancer research and facilitating the development of novel therapeutical modalities . Not surSeveral papers have recently been published on the status quo of single-cell technologies and on their applications in blood cancer research ,74,75,76ETV6-RUNX1-like, KMT2A-like, ZNF384-like, DUX4-rearranged, and MEF2D fusion-harboring subgroups, which have prognostic significance and/or are enriched for specific chromosomal translocations, mutations, tumor-suppressor gene deletions, kinase gene fusions, kinase pathway activation, or dysregulated lymphoid transcription factors, with some of these alterations also providing opportunities for targeted therapeutical intervention , accounting for around 2% of pediatric leukemia, is driven by mutations in the RAS pathway genes, with additional alterations in JAK/STAT signaling and in epigenetic modifier genes contributing to the leukemogenesis. Dissimilar to other types of pediatric leukemia, branching subclonal architecture and the development of relapse from an ancestral clone were not reported to be typical in JMML. Analysis of matching bulk diagnostic and relapse samples suggested that mutations identified at diagnosis persisted till relapse, and all early alterations emerged in a single dominant clone, with acquisition of additional aberrations following a linear fashion. In terms of prognostic significance, the number of mutations instead of their type seem to have prognostic relevance, with higher mutation counts being associated with shorter event-free and overall survival .SCS studies performed in pediatric leukemia to date primarily investigated the subclonal architecture, clonal evolution, developmental state, transcriptional heterogeneity, immunophenotype, and immunomodulatory activity of leukemic blasts. The analyses focused on specific leukemia-propagating cell subsets or complete leukemic cell populations, occasionally also including the interrogation of additional immune cell compartments.IGH) gene rearrangements in single leukemic cells of six patients with B-ALL (5/6 harboring ETV6-RUNX1 gene fusion) using microfluidics-based targeted DNA resequencing [Gawad et al. analyzed the co-segregation of single nucleotide variants (SNVs), deletions, and immunoglobulin heavy chain  in order to scrutinize intrapatient transcriptional heterogeneity at diagnosis . They obWitkowski et al. analyzed diagnostic, remission, and relapse samples of nine patients with B-ALL using droplet-based, single-cell RNA sequencing  and cellular indexing of transcriptomes and epitopes by sequencing , with the latter approach allowing for the simultaneous interrogation of single-cell transcriptome and cell surface protein composition . The autWu et al. re-analyzed the scRNA-seq data generated by Witkowski et al. and integrated it with a large bulk RNA-seq dataset in order to further characterize cell-cell interactions in B-ALL-associated bone marrow microenvironment . They idETV6/RUNX1 B-ALL at diagnosis and during induction therapy (day 15) [ETV6/RUNX1-positive leukemic cell states are most similar to a pro-B state, differ between patients in cell cycle activity, express genes that reprogram the immune microenvironment, and that the induction therapy may modulate leukemic cells towards a pre-B state. Furthermore, the authors demonstrated the feasibility of monitoring the early treatment response using SCS and revealed elevated activity of specific transcription factors, e.g., ETS factors , that could serve as targets for therapeutical intervention.Mehtonen et al. used scRNA-seq for investigating cell states and transcription factor activities during normal B-cell differentiation and compared those to leukemic cell profiles of six patients with (day 15) . Their rNOTCH1 mutations. Fast-cycling, stem-like cells showed high NOTCH activation, while the slow-cycling ones proved to be dependent on PI3K signaling and independent of NOTCH activation, explaining the low success rate that can be achieved by NOTCH inhibition in this entity. The study also demonstrated that cells with PI3K activity already exist at diagnosis, and the PI3K activation is independent of genetic mutations in the PI3K/AKT/PTEN genes, suggesting epigenetic rewiring as an underlying reason. In addition, the study shed light on the mode of immunomodulatory activity of leukemic cells, conferring CD8+ T-cell dysfunction via HAVCR2-LGALS9 interactions that could therapeutically be exploited in the future.Anand et al. investigated patient-derived xenograft (PDX) models generated from diagnostic or relapse samples of pediatric patients with early T-cell progenitor (ETP) ALL or T-ALL using the Smart-Seq2 protocol allowing for full-length scRNA-seq analysis . The autCandelli et al. analyzed a previously identified prednisone-dependent gene expression signature in 15 infants with MLL-rearranged B-ALL using plate-based and droplet-based scRNA-seq methods . They de+CD38− blasts from three patients diagnosed with pediatric AML [Despite the pioneering role of AML in early SCS studies, data generated from pediatric patients are still very limited. Walter et al. performed exome sequencing on five CD34MLL rearrangement and relapsed at month 10 after induction and consolidation therapies. In this patient, mutually exclusive KRAS G13D and FLT3 D835Y mutations were detected, with the latter one gaining selective advantage during disease progression. By investigating pathogenic blasts across all time points, immunophenotypic heterogeneity coupled with common genetic makeup as well as genetic diversity across blasts displaying shared malignant immunophenotype were observed, demonstrating that neither of the two layers of biological information would have been sufficient on their own to comprehensively characterize clinically relevant features in leukemic cells.Demaree et al. simultaneously interrogated the genotype and immunophenotype of single blast cells in longitudinally collected samples of three patients with AML using DNA and Antibody sequencing (DAb-seq), with one patient representing pediatric AML . By analMTOR and SLC2A1. Single-cell genotyping demonstrated that driver aberrations can be backtracked to the hematopoietic stem cell compartment, which showed a high level of clonal dominance and contained a small number of wild-type cells. Emergence of aberrations created linear and branching clonal evolutionary patterns, with RAS-pathway mutations being detected as the earliest alterations. Louka et al. investigated hematopoietic stem/progenitor cells from three patients diagnosed with JMML using scRNA-seq and/or single-cell DNA sequencing (scDNA-seq), with one patient being analyzed by both methods . TranscrThe studies briefly summarized above and listed in a priori extraction of clinically relevant heterogeneity from the biological heterogeneity uncovered by advanced molecular methods.Pharmaceutical research in oncotherapy had to keep pace with the rapid advancement of data generation facilitated by the accelerated development of molecular methods during the past decade. Translation of the enormously growing data, related to acute leukemia (AL) pathobiology, to a certain patient frequently needs data-mining approaches ,125. MaiCREBBP, IKZF1, and NT5C2, were rarely lost if present at diagnosis or in early therapy phase of ALL. Therefore, early detection of these alterations, which requires minimal residual disease (MRD)-level targeted screening, may predict an increased probability of treatment failure and may provide a rationale for therapy intensification or novel drug approaches before further evolution of leukemia towards relapse [BCR-ABL1-fusion-positive ALL, whose numerical sex chromosome aberrations and loss of chromosome Y resulted from late clonal evolution caused difficulties in the interpretation of chimerism analysis at the time of relapse, after sex-mismatched aHSCT [In AL, the predisposing germline genetic material, the repertoire of abundant mutations of de novo disease and that of relapsed leukemia are significantly different; hence, the biological behavior and treatment responsiveness of AL in patients may show substantial changes over time ,85,98. C relapse . Ediriwi relapse . Second, relapse . From a  relapse . Differeed aHSCT . Without− subclone allowing ALL cells to evade CD19-targeted donor chimeric antigen receptor T-cell (CAR-T) therapy in a patient [Targeted immunotherapy is an emerging therapeutic option to achieve remission or constitute a bridge to aHSCT in childhood ALL. Currently, we are not aware of all the factors determining the clinical efficacy of boosting the anti-leukemia immune activity of own or donor immune cells. The thorough cell-level examination of tumor and immune cell clonality by SCS can identify new predictive biomarkers for immunotherapy. Rabilloud et al. used scRNA-seq to identify a relapse-driving CD19 patient . In anot patient . These eETV6-RUNX1 fusion, ETV6-RUNX1-like expression pattern, DUX4 rearrangement, and high hyperdiploid karyotype, while BCR-ABL1 fusion, intrachromosomal amplification of chromosome 21 (iAMP21), IKZF1plus copy number profile, and TCF3-HLF fusion are associated with inferior outcomes [CREBBP aberrations predict corticosteroid resistance [NT5C2 alterations may predispose to poor response to 6-mercaptopurine [NUP98 rearrangement, special activating alteration of FLT3 , and WT1 mutations confer poor prognosis and drive the clinician towards aHSCT. DNMT3A mutations, representing early clonal events in adult AML, are associated with decreased sensitivity to anthracyclines, although they are rare in childhood [Putative genomics-based stratification criteria in childhood AL expand in a rapid manner, in parallel with deeper genomic-transcriptomic characterization of real-world patient cohorts. Recent data on genetic subgroups as well as on prognostic significance of alterations and/or potentially applicable targeted therapies are summarized in outcomes ,132. Cersistance , while stopurine ). In pedhildhood ,133. Co-hildhood ,135.CREBBP, IKZF1, KRAS, NRAS, NT5C2, PTPN11, TP53, and WHSC1, and found no correlation between these aberrations and unfavorable clinical outcome [NT5C2 mutations in a large cohort of relapsed ALL patients and observed that subclonal but not clonal mutations were associated with inferior EFS and with a higher nonresponse rate to relapse treatment. Nonetheless, in the vast majority of cases, these subclonal NT5C2 mutations disappeared by the time of treatment failure or second relapse, indicating that mechanisms driving relapse progression have a previously underappreciated complexity and cannot just simply be attributed to mutations associated with the occurrence of the first relapse [In pediatric leukemia, interrogation of recurrent genomic aberrations at the single-cell level seems especially be crucial in the light of some recent studies that have deeply scrutinized the association of clonal and subclonal alterations with clinical outcome. Jerchel et al. analyzed RAS pathway mutations in B-ALL and found that clonal but not subclonal aberrations are associated with dismal clinical outcome . Antic e outcome . On the  relapse . These eBCR-ABL1+ pediatric ALL treatment. The introduction of TKIs brought a breakthrough in the cure of this patient group, which previously had aHSCT as the only efficient treatment option. Recently, TKI administration has also emerged in other genetic subgroups. A surprisingly rapid and durable response was observed to dasatinib in NUP214-ABL1+ T-cell and NCOR1-LYN-fusion-positive BCR-ABL1-like ALL patients [BCR-ABL1-like ALL may constitute firm druggable entities  approved more small-molecule targeted agents (SMTAs) than altogether in the previous six decades . Developpatients ,147. Subentities , but mordult AML . ConsequAvailability and affordability of genomic profiling conferred by the spread and decreasing cost of NGS facilitated the utilization of genetic data in clinical decision making. Over the past decade, parallel progress in genomics technologies and genomics-driven drug discovery has created an opportunity to test whether a broader knowledge of genetic aberrations present in an individual’s tumor can guide treatment selection and lead to improved outcome of a certain patient. The clinical team responsible for childhood cancer care had to capitalize this opportunity and establish pipelines encompassing all steps from in-depth tumor characterization to personalized treatment suggestions. This ambition founded multidisciplinary molecular tumor boards (MTBs) composed of experts, including members from pediatric oncology/hematology, molecular pathology, cytogenetics, medical genetics, cancer biology, and bioinformatics. MTBs usually hold weekly meetings to discuss all patient related results and suggest treatment modifications or genetic counseling if a cancer-risk gene constellation has been uncovered. As examples, some highly organized pediatric MTBs are listed in ex vivo, circumventing the labor-intensive and time-consuming tasks of primary cell culturing needed to achieve sufficient cell numbers for standard viability tests. Recently, single-cell transcriptomics was also successfully used for examining the effectiveness of prednisolone in eliminating relapse-initiating clones and identifying patients with need for intensified treatment in MLL-rearranged infant ALL [Hopefully, the precision oncology approaches will be further accelerated by SCS, which may drive the clinicians’ choice and widen the indication spectrum of SMTAs. A cell line study by McFarland et al. proposed that long-term effect of an SMTA on tumor cell viability might be predicted more precisely based on short-term (6–24 h post-treatment) transcriptional response monitoring than by multiomic characterization of the initial tumor sample . In thisfant ALL .Studies focusing on the translation of NGS data acquired at single blast level to clinical decision making are in their early infancy. Most clinically oriented SCS experiments provide information on the clonal architecture of leukemia, including very minor subpopulations, and on subclonally co-occurring genetic alterations, which may modify targeted treatment strategies. As mentioned before, special relapse-fated blast subpopulations, so-called diagnosis relapse-initiating (dRI) clones, can be identified prior to chemotherapy initiation in childhood B-cell precursor ALL . These sETV6-RUNX1+ ALL, scRNA-seq revealed elevated activity of B-lineage differentiation transcription factors (TFs), also including the former leukemia genome-wide association study hit ETS domain-containing protein Elk-3 (ELK3) [SCS also aided an advanced understanding of developmental aspects of blast cell biology in childhood ALL . In a st3 (ELK3) . Leukemi3 (ELK3) .IDH1+ AML, SCS demonstrated clonal dominance gain after targeted therapy [JAK2 V617F mutated cell subset  was observed as a response to selection pressure conferred by multiple lines of chemotherapy and isocitrate dehydrogenase 1 (IDH1) inhibitor ivosidenib treatment. Single-cell analysis greatly helped to deconvolute the clonal architecture of the patient’s myeloid malignancy, as it revealed shared JAK2 and IDH1 mutations in the majority of subclones. In such a case, combination therapy of SMTAs might be highly beneficial relative to Janus kinase (JAK) or IDH inhibitor monotherapy, as previously published preclinical data suggest [SCS datasets also identified therapeutically relevant information in JMML hematopoietic stem/progenitor cells (HSPCs), where reduced clonogenicity and induced apoptosis was observed after glucose transporter 1 (GLUT1) inhibitor treatment, which seemed to be synergistic with mitogen-activated extracellular signal-regulated kinase (MEK) inhibition . Even aH therapy . Expansi suggest .These examples demonstrate the potential of SCS to support decision making in a clinical environment, but further translational studies are clearly required for clarifying the robustness of the method and for assessing the feasibility of its implementation in the diagnostic workflow of pediatric leukemia. Strikingly, SCS has started to emerge in currently running clinical trials with an aim to pinpoint the cell of origin of leukemogenic alterations formed in utero or to assess clonal diversity and evolution of pre-leukemic and leukemic populations with a view to correlate these findings with somatic mutation monitoring, in vitro chemotherapy resistance, MRD, and patient outcome ,161.SCS has been expanding our knowledge of the biological background of pediatric leukemia, and several studies reported novel findings with direct or indirect therapeutic relevance, potentially influencing the clinical management of patients in the future. Driven by recent technological advancements, the number of simultaneously analyzable cells and approaches supporting multimodal data generation have been permanently increasing, paving the way towards a comprehensive multiomic data landscape. The current number of papers reporting single-cell studies in pediatric leukemia is still very limited, and the widespread application of SCS methods in general faces multiple challenges posed by data sparsity, quantitative uncertainty, incomplete reference databases, relatively high cost, batch effects, and the rapid emergence of computational tools developed outside the recommended frameworks of standardized benchmarking . The pra


Clinically, metastatic rectal cancer has been considered a subset of left-sided colon cancer. However, heterogeneity has been proposed to exist between high and middle/low rectal cancers. We aimed to examine the efficacy of anti-epidermal growth factor receptor (EGFR) treatment for middle/low rectal and left-sided colon cancers.This study enrolled 609 patients with metastatic colorectal cancer who were treated with anti-EGFR therapy. They were divided into groups based on primary tumour locations: the right-sided colon, the left-sided colon or the middle/low rectum. The efficacy of first-line and non-first-line anti-EGFR treatment was analysed. Genomic differences in colorectal cancer data from The Cancer Genome Atlas (TCGA) were investigated and visualised with OncoPrint and a clustered heatmap.On first-line anti-EGFR treatment, patients with middle/low rectal tumours had significantly lower progression-free survival, overall survival, and overall response rates  than those with left-sided colon cancer . Similar outcomes were also identified on non-first-line anti-EGFR treatment. In TCGA analysis, rectal tumours displayed genetic heterogeneity and shared features with both left- and right-sided colon cancer.Anti-EGFR treatment has lower efficacy in metastatic middle/low rectal cancer than in left-sided colon cancer. RAS-wild-type (RAS-wt) left-sided mCRC [Colorectal cancer (CRC) originating from the right and left side of the colon differs in embryologic, epidemiologic, genetic and molecular aspects –4. In thded mCRC .In terms of mCRC, the rectum has been commonly categorised as part of the left-sided colon in previous trials and research and treated according to the same treatment principles described in the ESMO and NCCN guidelines , 16. HowThe definition of rectal cancer itself, which could differ surgically, anatomically, and/or biologically, remains inconclusive , 23. AccIn our study, we aimed to compare the efficacy of anti-EGFR therapy in treating metastatic middle/low rectal cancers and left-sided colon cancers. Right-sided colon cancers were also added for a complete analysis of the impact of primary tumour locations. CRC data from The Cancer Genome Atlas (TCGA) were analysed to validate our clinical results at a molecular level.RAS and BRAF mutations were excluded due to their unsuitability for anti-EGFR treatment. Patients were classified based on their primary tumour locations, including the right-sided colon , the left-sided colon , and the middle/low rectum . Patients with different primary tumour locations were treated with chemotherapy plus anti-EGFR (cetuximab or panitumumab) or anti-vascular endothelial growth factor  agents. They were divided into two groups based on their treatment sequences: one (the first-line group) receiving anti-EGFR first and the other (the non-first-line group) receiving anti-EGFR following anti-VEGF. All patients were administered at least two cycles of anti-EGFR regimens. The inclusion flowchart is presented in detail in Supplemental Fig. th Edition stage at first presentation (AJCC stage), metastasectomy , pathology, histological grade, mucinous component, signet cell component, lymphovascular invasion status, perineural invasion status, carcinoembryonic antigen  level and carbohydrate antigen 19-9  level, was collected. All materials and protocols in this study were compliant with the Declaration of Helsinki and Good Clinical Practice guidelines. The study protocols were approved by the ethics committee and institutional review board of the Taipei Veterans General Hospital.This is a retrospective large-scale cohort study. Data were collected between May 2005 and December 2019 at the Taipei Veterans General Hospital, Taiwan. Patients with pathologically confirmed mCRC were eligible for enrolment, while those with Median overall survival (OS) was defined as the time from diagnosis of metastasis to cancer death or loss to follow-up. Median progression-free survival (PFS) represented the time from the beginning of anti-EGFR treatment to disease progression, as confirmed by radiological images  or intolerable toxicity. The overall response rate  and disease control rate  of metastatic tumours were recorded based on the Response Evaluation Criteria in Solid Tumours (RECIST) version 1.1 . PatientIn order to clarify the genomic differences among left-sided colon, right-sided colon and rectal cancers, TCGA colorectal data were utilised as the training set to explore the differential expression of genomic features between left- and right-sided colon cancer. The significantly different expression of genomic features in the training set was compared with the potentially preventive genomic features in rectal cancer as a validation model.RAS or BRAF mutations, non-primary tumour samples or unspecified tumour locations were excluded from the evaluation .Statistical comparisons were based on nonparametric tests. The correlations between clinicopathological variables and treatment responses were analysed using the chi-square test or Fisher’s exact test, where appropriate. PFS and OS curves were calculated through the Kaplan–Meier method. As for univariable and multivariable analyses, Cox proportional hazards regression was used to determine the effects of primary tumour locations and particular factors and to obtain hazard ratios (HRs) with corresponding two-tailed 95% confidence intervals (CIs). Cox regression models were conducted in the subgroup analysis to evaluate the impact of anti-EGFR treatment in primary tumour locations and sequences stratified by baseline variables. A two-sided n = 125), the left-sided colon (n = 313) and the middle/low rectal (n = 171) tumour groups are presented in Table A total of 609 patients were included in this study. The baseline characteristics of patients in the right-sided colon n = 25, the lThe PFS, OS, ORR and DCR results from different groups based on primary tumour location and lines of anti-EGFR treatment are presented in Figs. P = 0.007), OS , and ORR  were significantly higher in the left-sided CRC group than in the middle/low rectal tumour group. Significantly longer PFS  and DCR  were also observed in patients with left-sided tumours compared to middle/low rectal tumours under non-first-line anti-EGFR therapy. Conversely, a comparable OS outcome was observed between left-sided tumours and middle/low rectal tumours  under non-first-line anti-EGFR therapy. The group of patients with right-sided CRC showed the worst PFS, OS, ORR and DCR in both first-line and non-first-line anti-EGFR treatment. The characteristics of treatment responders and non-responders among middle/low rectal cancer patients are summarised in Supplemental Table In terms of the first-line anti-EGFR therapy group, PFS  and DCR  between the left-sided colon and rectum groups under first-line anti-EGFR treatment  revealed no significant differences in PFS  had more SNP mutations than left-sided colon (n = 1944) or rectal (n = 379) tumours. There were more SNP overlaps between rectal and right-sided colon tumours (n = 590) than between rectal and left-sided colon tumours (n = 251). The genomic distribution and types of SNPs based on tumour locations are detailed in Fig. Genetic bioinformation in the mutation annotation format from 161 tumour samples with 1–100% SNP mutations was grouped by primary tumour location and visualised. The Venn diagram Fig.  and UpSeFor the epigenomic DNA methylation analysis, a volcano plot Fig.  and a liPrimary tumour location has been considered a useful biomarker to predict the prognosis and the treatment effects of bioagents in mCRC. Although the rectum is routinely grouped in the left-sided colon in current treatment guidelines, this large-scale retrospective study, nevertheless, demonstrates that anti-EGFR therapy has lower efficacy in metastatic middle/low rectal cancer than in left-sided colon cancer, potentially leading to better clinical practice principles for mCRC management.k-means clustering, k = 3) are presented in Fig. KRAS mutation rates in rectal cancer compared with right- and left-sided colon cancer were reported [In our TCGA investigation, distinctions in a genomic level between the rectal and the left-sided colon tumours are identified. The molecular differences serve some possible explanations to our clinical findings. In Fig. reported , 35. Leereported .RAS-wt mCRC patients and mentioned that the benefit of anti-EGFR agents against rectal cancers may be overestimated [P = 0.028) and ORR  of rectal tumours in the AVF2107g trial [Clinically, the differential efficacy of anti-EGFR therapy between the rectal and the left-sided colon tumours was proposed in the previous literature. The analysis of a Phase III trial of panitumumab (PICCOLO) reported similar PFS outcomes between rectal and right-sided colon cancers in stimated . Loupaki7g trial . Another7g trial . Several7g trial , 26, 28.P = 0.010) and ORR  than left-sided colon tumours, while no significant differences in PFS  and DCR  were seen in the first-line anti-EGFR treatment group [Analyses of left-sided colon and rectal tumours were also performed using the traditional cut-off point  with the aim of testing our hypothesis . Thus, the guiding principles for mCRC treatment selection according to primary tumour location, especially tumours in the middle/low rectum, need further evaluation and confirmation.In several previous studies, anti-EGFR as a first-line treatment has shown higher efficacy and a greater survival benefit than anti-VEGF in treating left-sided, patients . HoweverSubgroup analyses for all patients  remain to be investigated. Specific genomic features affecting the efficacy and resistance rate of anti-EGFR therapy should also be explored to demonstrate their relationship with primary tumour locations.In conclusion, the differential efficacy of anti-EGFR treatment between the middle/low rectal cancers and the left-sided colon cancers has been established. Moreover, TCGA genomic analysis partially supported our findings that rectal cancer is not a homogeneous group and that it shares features with both left-sided colon and right-sided colon cancer. The usefulness of primary tumour location for the basis of anti-EGFR treatment decisions warrants further exploration.Supplemental informationDataset 1


Despite several advances in targeted therapies for breast cancer, breast-cancer-associated death remains high in women. This is partially due to the lack of reliable markers predicting metastatic disease or recurrence after initial therapy. Recent research into the clinical validity of circulating cancer-specific biomarkers as a “liquid biopsy” is of growing interest. Of these, exosomal microRNAs (miRNAs) are promising candidate biomarkers for clinical use in breast cancer. In addition to their diagnostic value, exosomal miRNAs play an important role in predicting clinical outcome or treatment response. In this review, it is focused on the findings concerning exosomal miRNAs in relation to disease detection, prognostic impact and therapeutic effect in breast cancer, and discuss their clinical utility. Improvements in the early detection of breast cancer by conventional screening programs or development of novel molecular-targeted therapy may contribute to the reduction of breast cancer-related death. Breast cancer represents a heterogenous disease with varied biological characteristics and clinical outcomes. Based on molecular subtypes, namely, the expression of estrogen receptor (ER), progesterone receptor and human epidermal growth factor receptor-2 (HER2), the disease is classified into four distinct subtypes; luminal A, luminal B, HER2-enriched, and triple negative (TN),4. A better understanding of these subtypes is required to determine the need for appropriate therapy, such as endocrine therapies, chemotherapy, and molecular-targeted therapies. Although these therapies are commonly used for early breast cancer patients, some have experienced resistance following therapy, leading to loco-regional or distant recurrence,6. Many efforts have been made to explore the potential of various biomarkers to predict clinical outcome or treatment response-9, nevertheless, the mechanisms underlying the acquisition of resistance to therapy are still poorly elucidated.Breast cancer is one of the most commonly-diagnosed cancers worldwide and one of the leading causes of cancer death in women,11. To overcome the problem of tumor heterogeneity, the novel diagnostic tool which is referred to as a “liquid biopsy” is an ideal methodology,13. In recent years, there have been a number of advances in the field of molecular profiling to accurately predict clinical outcome and treatment response in early breast cancer. Circulating cancer-specific biomarkers can be evaluated by minimally-invasive techniques and are easily accessible to analyze compared to tissue sampling, as well as providing a real-time assessment. To date, several candidate biomarkers, including RNA, microRNA (miRNA), DNA, and proteins, have been suggested.Currently, clinical determinations of treatment are based on biopsies of tumor tissues, that may not be representative of the entire tumor burden,18. Bioinformatic techniques have revealed that each miRNA has binding affinity to hundreds of target genes. They are known to act functionally as oncogenes or tumor suppressor genes. Besides their intracellular function, there are some evidences of miRNA activity in cell to cell communication. In breast cancer, the expression of miRNAs is frequently deregulated during tumorigenesis as well as various types of cancer. To date, numerous miRNAs are reported to be valuable biomarkers for critical regulators of tumor initiation, metastasis and chemoresistance-24. In particular, circulating miRNAs can be found in several different body fluids and reflect specific disease types. After released into the blood circulation, miRNAs are commonly incorporated into microvesicles, bound to lipoproteins such as HDL, or form complexes with Argonaute-2 (Ago2) proteins. It has been suggested that the majority of miRNAs in circulation are selectively concentrated in exosomes,27 and exosomal miRNAs are considered the main source of circulating miRNAs.MiRNAs are single-stranded and non-coding RNAs with 19 to 22 nucleotides in length, which regulate gene expression at the post-transcriptional level by binding to partially-complementary sites in the 3’-untranslated regions of target messenger RNAs (mRNAs), leading to translational repression or regulation of mRNA degradation. They can be transferred from primary tumor cells to distant organs via the circulation; thus, they reflect the origin of the secreting cells. The cancer-derived exosomes may be important mediators of intercellular communication,29 and the presence of miRNA in exosomes was first reported by Valadi et al.. Exosomal miRNAs are the most abundant and crucial biomolecules which play a pivotal role in implementing the tumor microenvironment and the growth and metastasis of cancer cells. They have been proposed as novel circulating biomarkers for the early detection of several types of cancers-32, based on findings indicating that cancer patients have greater amounts of circulating exosomes than those in healthy controls,34.Exosomes are small membranous vesicles with a diameter of 30-100 nm and consisting of lipid, proteins, DNA, miRNAs and mRNAs. They are actively secreted from various types of cells into the circulationThere have been many reports on circulating miRNAs, whereas limited data are available regarding exosomal miRNAs. In this review, we focus on the role of exosomal miRNAs as markers for possible applications in breast cancer detection, prognosis, and response to treatment. Moreover, we discuss the issue of exosome research in terms of clinical application.-32, based on findings indicating that cancer patients have elevated levels of tumor-derived exosomes in blood, compared with those in healthy controls,34. To date, there have been few studies involving analysis of exosomal miRNAs in the diagnosis of breast cancer . These miRNAs could successfully differentiate breast cancer patients from healthy control subjects. The area under the curve (AUC) was 0.73 to differentiate them when miR-1246 and miR-21 were combined in the analysis, that is considered moderate performance. MiR-1246 suppressed the expression of Cyclin G2, leading to breast cancer progression. A recent study by Li et al. indicated the potential use of an exosomal miR-106a-363 cluster as a novel diagnostic biomarker. Four plasma-derived exosomal miRNAs , and four serum-derived miRNAs  from breast cancer patients were expressed at significantly higher levels than those from healthy controls. Interestingly, two overlapping miRNAs (miR-106a-5p and miR-20b-5p) were consistently upregulated also in breast cancer tissues. The functional mechanisms of the miR-106a-363 cluster in breast cancer have not been fully reported, whereas all but one miRNA (miR-106a-3p) are involved in the “proteoglycans in cancer” pathway according to the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis.Exosomal miRNAs in body fluids have potentials of the novel circulating biomarkers for the detection of several cancers,37. Eichelser et al. analyzed circulating cell-free and exosomal miR-101, miR-372, and miR-373 in preoperative blood serum. In analyses of 50 breast cancer patients and 12 healthy women, the levels of exosomal miR-101 and miR-372 were significantly higher in the serum of patients with breast cancer than in healthy controls. Moreover, the levels of circulating exosomal miR-373 were higher in receptor-negative and TN tumors than in hormone receptor-positive carcinomas or healthy controls. In addition, Stevic et al. also demonstrated different exosomal miRNA signatures in HER2-positive and TN breast cancer patients. The expression levels of exosomal miRNA in plasma of 435 breast cancer patients, consisting of 211 HER2-positive and 224 TN patients, were analyzed. The analysis revealed that the expression of five miRNAs were higher and 13 were lower in HER2-positive than in TN breast cancer patients. Among them, exosomal miR-335, miR-628, and miR-422a were combined into a logistic regression model and these combined scores could discriminate between TN and HER2-positive breast cancer patients with a sensitivity 68% and a specificity of 81%. This is the first study to measure the exosomal miRNAs derived from a large cohort of 435 breast cancer patients, further indicating the different exosomal miRNA patterns among subtypes.Differential expressions of subtype-specific miRNAs have also been reported in several studiesin situ (DCIS),39. It has been demonstrated that the miR-223-3p levels of IDC patients showed the highest fold-change compared with those in DCIS patients and healthy controls. In vitro analysis revealed that transfection of MCF-7 cells with the miR-223-3p gene significantly promoted cell proliferation and cell invasion ability. In another study, exosomal miR-16, miR-30b, and miR-93 were analyzed in 111 IDC patients, 42 DCIS patients, and 39 healthy women. The level of exosomal miR-16 was higher in the plasma of breast cancer and DCIS patients than in healthy women. Meanwhile exosomal miR-93 was higher in DCIS patients than in healthy women, but lower in IDC patients. These findings suggest that the different signatures of the corresponding miRNAs are associated with a particular biology of breast tumors. This may be promising for the identification of biomarkers if specific miRNAs could be used to distinguish DCIS cases that have a higher likelihood of future disease progression. Taken together, these findings suggest that several exosomal miRNAs could be good candidates for early breast cancer detection and differentiation of tumor subtypes.Several studies have compared exosomal miRNAs derived from invasive ductal carcinoma (IDC) and ductal carcinoma et al., described analysis using a PCR-based microarray which contained 47 different miRNAs in exosomes derived from the plasma of 32 breast cancer patients. They observed differences in the levels of six miRNAs between primary and recurrent breast cancer. In particular, the levels of miR-20a and miR-30b were lower in exosomes from recurrent cases than in those from primary breast cancer patients. The functions of the corresponding miRNAs are unclear, but miR-30b may regulate the Cyclin E2 gene (CCNE2),41. Ichikawa et al. demonstrated that trastuzumab induced the expression of miR-30b in breast cancer cells and upregulation of this gene inhibited cell growth by targeting CCNE2. According to the finding, downregulation of miR-30b may be involved in trastuzumab resistance in HER2-positive breast cancer patients. In another study, we have previously demonstrated differential expression of exosomal miRNAs between breast cancer patients with and without recurrence. Of 384 miRNAs, three  were significantly upregulated and eight  were significantly downregulated in patients with recurrence. In contrast to growing evidence of diagnostic potential for exosomal miRNAs, there is limited data concerning their prognostic potentials.Several miRNAs derived from exosomes can be involved in tumor metastasis and recurrence of breast cancer. There are a few articles revealing an association between exosomal miRNAs derived from breast cancer patients and clinical outcome. One of them, by Ni et al. revealed that taking miR-200-rich exosomes from metastatic 4T1 cells and co-culturing them with poorly-metastatic 4T07 cells resulted in transfer of miR-200 and downregulation of ZEB2 which caused the 4T07 cells to revert to an E-cadherin-expressing epithelial phenotype. Moreover, when the 4T07 cells were treated systemically with 4T1 exosomes, the incidence of lung metastasis increased. More recently, a study by Kia et al. showed that poorly-metastatic breast cancer cells (MCF-7) treated with exosomes derived from highly-metastatic breast cancer cells (MDA-MB-231) displayed overexpression of miR-9 and miR-155, which target the tumor suppressor genes, PTEN and DUSP14. They demonstrated that the corresponding miRNAs were enriched in metastatic TN breast cancer and could be transferred into other cells to alter the expression of target genes.Functional studies in cell lines or xenograft models have also revealed that exosome-mediated transfer of several miRNAs has been implicated in promoting tumor growth, cell invasion and preparation of a metastatic niche . MiR-105 is considered to be a potent regulator of migration, acting by targeting the tight junction protein ZO-1, leading to increased vascular permeability in distant organs. Similarly, the role of exosome-associated miR-939 in breast cancer has also been investigated. This gene is a candidate to target VE-cadherin, a component of the adherens junctions involved in vessel permeability. The authors revealed that miR-939 was found to be highly expressed in TN subtypes catalogued in The Cancer Genome Atlas (TCGA) dataset. In the analysis of their cohort of 63 TN breast cancer patients, patients with high expression of miR-939 and nodal involvement showed a 6-fold higher risk of relapse compared to those without. Further, analysis of TN breast cancer cell lines showed that miR-939 directly targets VE-cadherin leading to an increase in permeability of a monolayer of human umbilical vascular endothelial cells.During multiple steps of the metastatic process, cancer cells lose adhesion to the extracellular matrix and migrate into the circulation to reach the pre-metastatic niche. Exosomal miRNAs are implicated in these processes, which include cell migration, invasion, angiogenesis, bone marrow dormancy and reprogrammed metabolism. Exosomal miR-105 is reported to be released by cancer cells, which then destroys tight junctions and the integrity of natural barriers against metastasis. Tominaga et al. demonstrated that injection of brain metastatic cancer cell-derived exosomes into the tail vein of mice resulted in more brain metastasis compared with mice treated with human mammary tumor cell-derived exosomes.The exosome-derived miR-181c promotes destruction of the BBB through the abnormal localization of actin via downregulation of its target gene, 3-phosphoinositide-dependent protein kinase-1 (PDPK1). In analysis of breast cancer patients (n = 56), miR-181c in exosomes collected from brain metastasis patients (n = 10) was significantly higher compared with non-brain metastasis patients (n = 46), suggesting an important role of miR-181c in clinical utility for brain metastatic cancer. In addition, astrocyte-derived exosomal miR-19a has been reported to contribute to brain metastasis. Zhang et al. showed that breast tumors after dissemination to the brain lost PTEN expression, which was regulated by miRNAs from astrocytes. Astrocyte-derived exosomal miR-19a reversibly downregulated PTEN expression in cancer cells, leading to increased secretion of cytokine chemokine (C-C motif) ligand 2 (CCL2) and recruitment of myeloid cells to enhance outgrowth of brain metastatic tumor cells. Their findings signify the dynamic and reciprocal cross-talk between tumor cells and the metastatic niche.MiR-181c is involved in migration of cancer cells through the blood–brain barrier (BBB)et al. demonstrated that miR-10b was highly expressed in metastatic breast cancer MDA-MB-231 cells compared to non-metastatic breast cancer cells. Also, exosomal miR-10b secreted from MDA-MB-231 cells increased the invasive ability of nonmalignant immortalized human mammary epithelial cells. In particular, neutral sphingomyelinase 2 (nSMase2) or ceramide promoted secretion of exosomal miR-10b which then suppressed the protein level of its target genes such as Homeobox D10 (HOXD10) and Kruppel Like Factor 4 (KLF4), indicating functional significance.Another study by Singh . Currently, there is growing research interest focusing on how different microenvironments affect bone marrow dormancy, and exosomes can play a crucial role in this. One study has revealed an association of miR-23b with bone marrow dormancy. Ono et al. generated a bone marrow-metastatic human breast cancer cell line (BM2). Co-culture of BM2 and bone marrow mesenchymal stem cells (BM-MSCs) revealed suppression of proliferation, a decrease in stem cell-like properties, and inhibition of invasion in BM2 cells. MiR-23b derived from BM-MSCs can induce dormancy through the suppression of a target gene, myristoylated alanine-rich C kinase substrate (MARCKS), which encodes a protein that promotes cell cycling and motility. In a cohort of 10 patients, miR-23b expression was increased in metastatic bone lesions compared to matched primary breast tumors. In another study, bone marrow stromal cell-derived miR-127, miR-197, miR-222, and miR-223 led to cancer cell quiescence via down-regulation of CXCR12, a chemokine that interacts with CXR4 and CXR7 receptors. Cancer cells entering quiescence or cell cycle arrest could acquire resistance to chemotherapy. Bliss et al. revealed that breast cancer cells prime mesenchymal stem cells to release exosomes containing miR-222/223, which in turn promotes quiescence in a subset of breast cancer. In an immunodeficient mouse model of dormant breast cancer, therapy with antagomiR-222/223 sensitized breast cancer cells to carboplatin-based therapy and increased host survival.MiR-223 is also regarded as a specific miRNA for tumor-associated macrophages. In a co-culture system, miR-223 released from IL-4-activated macrophages could be transferred to MCF-7 and MDA-MB-231 cells, leading to promotion of invasion of breast cancer cells via disruption of the Mef2c-β-catenin pathway.Breast cancer patients often experience recurrence decades after initial treatment and this seems to involve dormancy in the bone marrow. Donnarumma et al. have demonstrated that breast cancer cells exposed to CAF exosomes containing miR-21, miR-378e, and miR-143, exhibited a significantly increased capacity to promote stemness properties, EMT phenotype, and anchorage-independent cell growth. Thus, the release of CAF exosomes may be responsible for the delivery of miRNAs that promote oncogenic signaling in breast cancer cells. In a large cohort of TCGA database breast cancer patients (n = 744), the patients with lower levels of miR-378 had longer overall survival, suggestive of a prognostic role for this gene.Cancer-associated fibroblasts (CAFs) are vital constituents of the tumor microenvironment and play a major role in cancer initiation, angiogenesis, invasion, and metastasis of breast cancer. Fong et al. showed that cancer cells suppress glucose uptake by non-tumor cells in the pre-metastatic niche, by secreting exosomal miR-122 that downregulates the target genes pyruvate kinase (PKM) and citrate synthase (CS). Both in vitro and in vivo studies have shown that exosomal miR-122 can reprogram the glucose metabolism of lung fibroblasts, brain astrocytes, and neurons that are abundantly present in the pre-metastatic sites of breast cancer.Reprogrammed glucose metabolism is an emerging hallmark of cancer and several miRNAs have been implicated in metabolism and metabolic disorderset al. collected exosomes from MCF-7 and MDA-MB-231 breast cancer cells after treatment with docosahexaenoic acid (DHA). Several miRNAs  were increased by DHA treatment in exosomes from breast cancer cell lines, but not in exosomes from normal breast cells. Exosomes derived from DHA-treated MCF-7 cells increased expression of the above miRNAs in recipient endothelial cells. Furthermore, the transfection of miR-23b and miR-320b into endothelial cells decreased expression of their pro-angiogenic target gene  and significantly inhibited tubular formation. These data suggest that the miRNAs transferred by exosomes mediate the anti-angiogenic action of DHA and support its potential use in cancer therapy. These findings demonstrate the capacity of exosomes derived from mesenchymal stem cells (MSCs) in the tumor microenvironment to exert antitumor effects by down-regulating vascular endothelial growth factor expression in recipient cancer cells.Some miRNAs are involved in modulation of angiogenesis. Hannafon , but there are only limited reports focusing on the exosomal miRNAs in resistance to endocrine therapy; we found only one article focused on this topic. Wei et al. suggested that exosomal miR-221/222 may be responsible for tamoxifen resistance in breast cancer. They generated tamoxifen-resistant MCF-7 (TamR) cells and compared them to tamoxifen-sensitive MCF-7 (wt) cells. They found that exosomes derived from MCF-7 (TamR) cells were able to enter into MCF-7 (wt) cells, enhancing tamoxifen resistance in recipient MCF-7 cells. The elevated miR-221/222 effectively reduced the expression of the target genes P27 and ERα. These findings are supported by another study by Miller et al., revealing that tamoxifen-resistant breast cancer cells display up-regulation of miR-221/222 and significant reductions in p27 levels. Taken together, these findings suggest that anti-miR-221/222 may be a potential therapeutic target to overcome tamoxifen resistance.The current treatment option for breast cancer varies according to tumor subtype or disease stage, which includes chemotherapy, endocrine therapy, molecular-targeted therapy, surgery, and radiotherapy. Approximately 70% of breast cancer tumors express ER and patients with ER-positive tumors are candidates for endocrine therapy. It is a major issue if cancer cells acquire resistance to endocrine therapy. There have been several studies analyzing miRNAs related to treatment resistance, but their efficacy is often limited by the emergence of chemoresistance. The acquisition of chemoresistance requires multiple regulatory changes of the tumor microenvironment, some of which are caused by exosomes. To improve the clinical outcome of cancer patients, accurate biomarkers for early prediction of response or resistance to chemotherapy are needed.Anthracycline and taxane-containing agents are most commonly used as adjuvant chemotherapy for early breast canceret al. on this topic. They analyzed several exosomal miRNAs which were determined based on analysis with a microRNA array card using the patients’ plasma samples before neoadjuvant therapy . The expression of miR-155 and miR-301 in exosomes of HER2-positive and TN breast cancer patients most significantly predicted pathological complete response (pCR) which is considered as a surrogate marker for prognosis, in uni- and multivarite models. MiR-301 regulates the phosphatase and tensin homolog (PTEN)/Akt and nuclear factor-kappa B (NF-κB) signaling pathways,65 and also binds to the ESR 1 gene, leading to estrogen-independent growth of breast cancer.There have been few studies concerning the association between exosomal miRNAs and treatment efficacy in the clinical setting. We found one article by Stevic et al. focused on the exosomal miR-1246, which has been reported to function as a protooncogene in lung and other cancers. In their study, miR-1246 was highly expressed in metastatic breast cancer MDA-MB-231 cells compared to non-metastatic breast cancer cells or non-malignant breast cells. Further, the transferred miR-1246 promoted invasion in non-malignant human mammary epithelial (HMLE) cells in part by targeting Cyclin G2 (CCNG2). The HMLE cells, after treatment with exosomes from MDA-MB-231 cells transfected with miR-1246, gained resistance to docetaxel, epirubicin, and gemcitabine.In contrast, we identified several functional studies using cell lines to investigate the association of exosomal miRNAs with drug response . Basical,69. Chen et al. generated two drug resistance models; MCF-7/adriamycin (Adr) and MCF-7/docetaxel (Doc), to investigate the mechanisms of chemotherapy failure. They demonstrated that following transfer of exosomes derived from MCF-7/Adr and MCF-7/Doc to recipient MCF-7/sensitive cells, the levels of miR-100, miR-222, miR-30a, and miR-17 were all significantly increased in the recipient cells. Further, MCF-7/Adr and MCF-7/Doc displayed a marked reduction of PTEN, which is a target of miR-222, compared to MCF-7/sensitive cells. Zhong et al. established three resistant cell lines by exposing the parental MDA-MB-231 cell line to docetaxel (Doc), epirubicin (Epi), and vinorelbine (Vin), respectively. They found some miRNAs were dysregulated in resistant cell lines relative to sensitive MDA-MB-231 cells; there were 10 consistently up-regulated miRNAs in MDA-MB-231/Doc resistant cells and their exosomes, 11 in MDA-MB-231/Epi resistant cells, and four in MDA-MB-231/Vin resistant cells . Gene and miRNA expression profiling revealed 138 genes and 70 miRNAs were significantly differentially expressed among the corresponding MCF-10A and the untreated MCF-10A cells, affecting mostly the phosphoinositide 3-kinase (PI3K)/AKT, MAPK, and hypoxia inducible factor (HIF)1A pathways. Ten of these miRNAs control some genes APC), hydroxysteroid 17-β dehydrogenase 12 (HSD17B12), MYC, SMAD1 and SMAD3. The relevance of the corresponding genes was also shown by another study. They found miR-155 induction in exosomes isolated from cancer stem cells and resistant cells. When miR-155 exosomes were transfected into recipient sensitive cells, they underwent EMT and gained intermediate resistance to doxorubicin and paclitaxel therapy. It is well-known that miR-155 mediates the loss of CCAAT enhancer-binding protein (C/EBP)-β,75, which in turn, causes loss of transforming growth factor (TGF)-β and leads to EMT in breast cancer cells. They also observed repression of TGF-β, C/EBP-β and forkhead box O (FOXO)3a in cells transfected with miR-155.Ozawa et al. reported the association of miR-134 with drug sensitivity. They identified miR-134 as the most substantially down-regulated miRNA in the aggressive TN breast cancer cells, Hs578Ts(i)8 and their exosomes compared to their parental (Hs578T) counterparts. In analysis of clinical samples, miR-134 was found to be significantly down-regulated in breast cancer when compared to its levels in healthy breast tissue. Further, the transfection of miR-134-enriched exosomes into Hs578Ts(i)8 cells reduced the levels of its target genes, signal transducer and activator of transcription 5B (STAT5B) and heat shock protein 90 (Hsp90), leading to a reduction in migratory and invasion ability. The corresponding cells showed increased sensitivity to an anti-Hsp90 drug. Their functional studies support the potential use of miR-134 as a therapeutic agent in TN breast cancer.O’Brien As described in this section, there is accumulating evidence supporting an association between exosomal miRNA and resistance to anthracycline or taxane-containing agents in breast cancer cells. There are few reports of exosomal miRNAs on the effects of molecular-targeted therapy. It remains a challenge for future research to explore its association with other drugs, such as eribulin or anti-HER2 therapy.. For example, Bovy et al. examined the expression levels of exosomal miR-503 in patients after neoadjuvant chemotherapy. Interestingly, these levels increased after therapy, whereas miR-503 in primary tumors subjected to this treatment did not change. The authors speculated that the increased miR-503 levels in the circulation after chemotherapy did not originate from the tumor, but from the endothelial cells. Exosomes can be secreted by various types of cells. It will be ideal if cancer-specific exosomes or exosomal miRNAs can be more accurately isolated to investigate how donor tumor cells select or sort the packaging of miRNAs into exosomes to regulate their progression. Several efforts to solve this issue have been undertaken, involving surface protein markers on exosomes, such as CD24 or EpCAM, but no standard markers to differentiate cancer-specific exosomes from those of other origin have been identified.Exosomal miRNAs can be attractive future diagnostic markers and therapeutic targets but there remain some unsolved problems which must be overcome before they can be applied in clinical practice. First, exosome research is based on the findings that a higher secretion of exosomes may be a general feature of cancer patients compared to healthy individuals and miRNAs encapsulated in exosomes reflects originating tumor biology. Nevertheless, some studies have suggested that miRNAs derived from exosomes could not parallel those in primary tumors,80. Meanwhile snRNU6 is not detectable in most blood samples. However, these reference genes could not always fit exclusively to individual samples with different populations and characteristics. We therefore need to assess them within each dataset. As another method, most suitable miRNAs can be tested by software such as the Genorm Algorithm. In addition to endogenous normalization, exogenous normalization  to remove the technical and interindividual variability are required. Anyway, currently there are no unified and standard reference genes in exosome research.Second, there is no reliable endogenous gene for normalization of exosomal miRNAs. According to several reports, miR-16-5p, miR-423-3p and miR-191-5p are often recommended as reference miRNAs, since they are constantly expressed through the data,82, which confuses the understanding of their biological mechanisms. To explore the biological functions of identified miRNAs, bioinformatic analysis using programs such as TargetScan, miRbase, miRWalk, DIANA-microT and TarBase are needed.Finally, there are difficulties in interpretation and statistical analysis of obtained data. Recent studies tend to examine comprehensive expression of exosomal miRNAs such as microarray or next-generation sequencing (NGS), which consequently offer us enormous data regarding candidate miRNAs. It is difficult to determine whether all the differentially-expressed miRNAs have a meaningful role in the process of cancer development. In addition, one miRNA has binding affinity to numerous mRNAs and is involved in the downregulation of many factors that participate in different cancer-relevant signal transduction pathways. Some miRNAs, such as miR-155 and miR-335, have the potential to exert both tumor-suppressive and oncogenic effectsInvestigation of the involvement of exosomal miRNAs in breast cancer could provide promising diagnostic and prognostic biomarkers as well as increasing understanding of the biological mechanisms of cancer development. There is accumulating evidence regarding the role of exosomal miRNAs, especially in functional studies, whereas evidence of their role in the clinical setting remains inadequate. We found few overlapping exosomal miRNAs between the studies, so a large clinical cohort may be required to obtain reproducible data., which makes it difficult to understand the comprehensive features of disease conditions and identify unique targets. A panel of specific miRNAs as real-time biomarkers, in combination with other conventional clinical biomarkers, might improve the diagnostic and predictive power. We believe that this review will provide the opportunity for a more critical evaluation of the clinical value of exosomal miRNAs.With regard to experimental studies, it is of interest that not only cancer cells but surrounding cells such as endothelial cells, pericytes, fibroblasts, and immune cells, secrete exosomes to modulate tumor-favorable or -unfavorable conditions. Considering the potential of exosomes as cancer biomarkers, it is becoming necessary to understand the many findings related to the tumor microenvironment. Although individual miRNAs have been abundantly explored as we describe above, the entry of exosomal miRNAs into clinical practice is hampered since not all the identified miRNAs have been fully checked using clinical samples. To expand the research field into clinical studies could help identify those patients who are most likely to experience recurrence or who are most likely to acquire resistance or respond to therapy. Breast cancer tumors are characterized by extensive heterogeneity  and comprise several subtypes with different molecular profiles


Urothelial bladder cancer (UBC) is a global challenge of public health with limited therapeutic options. Although the emergence of cancer immunotherapy, most notably immune checkpoint inhibitors, represents a major breakthrough in the past decade, many patients still suffer from unsatisfactory clinical outcome. A thorough understanding of the fundamental cellular and molecular mechanisms responsible for antitumor immunity may lead to optimized treatment guidelines and new immunotherapeutic strategies. With technological developments and protocol refinements, single-cell approaches have become powerful tools that provide unprecedented insights into the kaleidoscopic tumor microenvironment and intricate cell-cell communications. In this review, we summarize recent applications of single-cell analysis in characterizing the UBC multicellular ecosystem, and discuss how to leverage the high-resolution information for more effective immune-based therapies. Urothelial bladder cancer (UBC) accounts for more than half a million new diagnoses and 212,536 deaths annually . Muscle Extensive studies have been focused on dissecting the cellular and molecular mechanisms underlying the immune response of UBC, in order to identify clinical biomarkers to predict ICI treatment efficacy, and to design novel single or combination trials of more effective regimens –17. AccuIn this review, we update the current progress of cancer immunotherapy in UBC, summarize the applications of cutting-edge single-cell analysis in decoding the tumor multicellular ecosystem, and discuss future prospects for using these high-dimensional multi-faceted data to guide more effective immune checkpoint therapies.in situ), and are usually managed with TURBT followed by a single dose of intravesical chemotherapy to kill free-floating tumor cells. After the initial TURBT, patients with intermediate or high likelihood of recurrence will receive adjuvant intravesical BCG as maintenance therapy to reduce the risk of progression . Based on these results, pembrolizumab was approved as a second-line treatment for those whose disease progressed during or after platinum-based chemotherapy. In addition, avelumab (JAVELIN Solid Tumor)   and nivod Tumor)  also gaivs 10.6 months; HR, 0.87 ; P = .41)   , atezoliP = .41) . LikewisP = .30) . As a rePembrolizumab and atezolizumab were given accelerated approval for the first-line treatment of cisplatin-ineligible advanced or metastatic UBC, following KEYNOTE-052  and IMviIn contrast, three recent trials with cisplatin-eligible patients consistently showed that first-line ICI monotherapy was not superior to chemotherapy in unresectable locally advanced or metastatic UBC. All these large randomized phase III trials, i.e., IMvigor130, KEYNOTE-361, and DANUBE, observed similar performance of three ICI drugs and platinum-containing chemotherapy in the front-line setting –40. Evenvs 14.3 months; HR, 0.69 ; P = .001). The evident improvement of patient outcomes has led to the FDA approval of avelumab as maintenance therapy in this disease setting   did not meet its primary endpoint of improved disease-free survival in the atezolizumab group over observation (19.4 P = .24) . On the P = .24) . AdditioClinical trials of perioperative immunotherapy are ongoing in patients with advanced urothelial carcinoma. In PURE-01 phase II study, 42% of patients treated with pembrolizumab achieved pathologic complete response (pCR) and up to 54% downstaged to pT1 or lower disease . The ABAAs a reasonable alternative to radical cystectomy, trimodal therapy (TMT) combines maximal TURBT with concomitant radiosensitizing chemotherapy and external-beam radiotherapy to devise bladder-sparing strategies in well-selected patients. Given that ICIs may further augment the immune response triggered by radiotherapy-induced tumor cell death , severalFor patients having BCG-refractory NMIBC with CIS who are unable or unwilling to undergo cystectomy, pembrolizumab was recently approved on the basis of results from KEYNOTE-057 phase II study , 57. TheTo fulfill a robust and durable clinical benefit of tumor immunotherapy, immense efforts have been taken to comprehensively understand the mechanism of action for PD-1/PD-L1 checkpoint blockade . Under pGenerally, FFPE  or snap-frozen clinical samples, though readily available, can only be used for single-nucleus sequencing . The metTo date, single-cell RNA sequencing (scRNA-seq) is the most mature single-cell genomic approach and has a wide spectrum of novel analytic tools to facilitate data interpretation –83. The + T cells in bladder cancer exhibit multiple distinct tumor-specific states of regulatory T cells and cytotoxic CD4+ T cells, which were clonally expanded  mainly includes whole-genome scDNA-seq, whole-exome scDNA-seq, and panel scDNA-seq detecting a few genes of interest. Whole-genome or whole-exome scDNA-seq covers large genomic regions but is limited by sequencing depth, while panel scDNA-seq focuses on a narrow list of target genes but can achieve higher throughput and sequencing depth . Despiteem cells .via CyTOF and RNA-seq analyses, uncovered higher CD8+ T cell populations in murine bladder cancer upon DDR2 depletion and anti-PD-1 treatment, implying that DDR2 inhibition might fuel tumor response to ICIs (Cytometry by time of flight (CyTOF) adopts the single-cell format of flow cytometry technique for multiparameter detection of protein expression using the precision of mass spectrometry . By empl to ICIs .As an evolving field, numerous novel single-cell technologies are in rapid development to extract additional layers of biological information. For example, surface protein levels can also be measured in single cells by oligonucleotide-barcoded antibodies, as illustrated by various methods including CITE-seq and REAP-seq , 93. AnoIt is increasingly evident that various cell populations residing at neoplastic lesions and the interplay of these cellular compartments strongly affect cancer progression and response to immunotherapeutics. Recently, single-cell studies have provided in-depth insights into the composition and architecture of tumor multicellular ecosystem in UBC. By profiling the transcriptome of 52,721 single cells from bladder urothelial carcinoma or peritumor mucosa samples, Chen et al. discovered seven annotated cell types including epithelial cells, endothelial cells, fibroblasts, B cells, myeloid cells, T cells, and mast cells . Despite+ and CD8+ T cell populations , immunotherapy and targeted small molecules  or antibody-drug conjugates  –127. At Ultimately, the multiparametric data derived from single-cell technologies ought to assist UBC patient care and inform treatment recommendations. Achieving the ambitious goal will need joint efforts to develop standard operating procedures for benchmarking and implementing single-cell workflows that meet ethical, regulatory, and temporal requirements. With all foreseeable challenges, this venture would be imperative to transform bladder cancer management and necessitate very close collaboration among physicians, basic researchers and translational scientists. Recently launched large-scale initiatives, including the Human Tumor Atlas Network (HTAN) and the Tumor Profiler (TuPro) study, are poised to accelerate the standardization of key protocols, best-practice guidelines, quality control solutions, metadata schemata, and analytic pipelines , 129. ThThe recent decade has witnessed unprecedented advances in the clinical management of urothelial carcinoma with the advent of various ICIs. The ever-expanding applicable range of ICI therapies in UBC highlights the significant potential of immune-targeted agents and advocates a more thorough interrogation of their mechanistic underpinnings. Despite remaining questions, a number of studies using high-resolution single-cell techniques begin to reveal the identity and state of multiple cell types, the variety and uniqueness of tumor-infiltrating T lymphocytes, as well as the heterogeneity and plasticity of bladder cancer cells. This wealth of information has allowed a better understanding of dysfunctional antitumor immunity in UBC and variable responses to immunotherapy across patients Figure 3JZ and KY contributed equally to the literature search, figure visualization, and manuscript drafting. YF and RZ helped with data curation. HC and GZ conceptualized and supervised the project. All authors contributed to the article and approved the submitted version.This work was supported by the National Natural Science Foundation of China , Shanghai Natural Science Foundation , Ren Ji Hospital Research Funding Projects , Shanghai Jiao Tong University School of Medicine Research Funding Projects (TM201708), and Foundation of Shanghai Hospital Development Center (SHDC12015125).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


What transpires soon after inhaling Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the respiratory virus causing Coronavirus Disease 2019 (COVID-19)? Where does infection begin? What are the features of subsequent virus spread? How might host responses quickly contain infection? Two recently published manuscripts have evaluated infection in primary cultures of well-differentiated cells to address these questions and bring more light on the proviral and antiviral components operating during the initial days after SARS-CoV-2 exposure. Well before Coronavirus Disease 2019 (COVID-19) advanced to pandemic levels, fundamental questions concerning Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection in the human respiratory system were under consideration. Answers to these questions were necessary to form a general understanding of viral pathogenesis and to frame COVID-19 prevention and treatment strategies. Obtaining answers continues to involve great effort, in large part because the human respiratory tract is complex, encompassing nasal passages, large and small airways, bronchioles, and alveoli, each distinctly organized for specialized functions and each comprising several variable cell types. Complex relationships between initial infection and subsequent coordinated antiviral responses take place in the respiratory epithelium, the details of which determine disease outcomes. Despite the challenges, answers have come forward at a remarkable pace. Sensitive assays detecting viral RNA and protein in COVID-19 patient samples revealed sino-nasopharyngeal, airway, and alveolar cell infection. Animal models revealed the accumulation and distribution of SARS-CoV-2 throughout the pulmonary system [Now, Ravindra and colleagues  and FiegPreferential virus attachment to ciliated cells may be explained by the prominent display of angiotensin converting enzyme 2 (ACE2), the SARS-CoV-2 receptor, on distal cilia extensions . This plAs infection progresses, viruses may spread beyond ciliated cells. Of note, Ravindra and colleagues identified infected basal and club cells as infection continued beyond 1 day. Although Fiege and colleagues did not recognize basal cell infection, and also did not identify TMPRSS2 transcripts in basal cells, the potential for expansive infection deserves further consideration. In the proximal conducting airways, the basal cell is an important progenitor for all the surface epithelial cell types . How theThe Wilen and Langlois teams moved forward from questions of virus tropism to dissect the coordinated innate immune responses to virus infection. The 2 groups identified expected relationships between SARS-CoV-2 RNA accumulation and antiviral interferon transcription, with single-cell resolution revealing that only a small proportion of robustly infected ciliated cells expressed interferons. These few responding cells did, however, produce enough type I and III interferons to elicit pan-ALI expression of interferon-stimulated genes (ISGs). Patterns of ISG expression varied between epithelial cell types, yet overall, ISG products protected bystander cells from infection. Interferons, if sufficiently abundant and present early after infection, will reduce disease in animal models of SARS-CoV  and SARSIn addition to interferons, airway cells release chemokines such as CXCL10 and cytokines including interleukin (IL)-6 and IL-8, which contribute to the early recruitment of adaptive immune cells and onset of inflammation. Hyper-inflammatory responses are a central feature of late-stage COVID-19, with exuberant IL-6 secretion figuring prominently [The reports of Ravindra and colleagues and Fiege and colleagues demonstrate the broad utility of ex vivo infection models. The bronchial epithelial cultures they used were valuable in characterizing cell types infected by SARS-CoV-2, cell susceptibility determinants, cytokine induction and signaling, and antiviral therapeutics. Along with complementary models reflecting diffuse alveolar damage and inflammation, the works help to clarify features of lung injury in COVID-19. Longitudinal studies in more sophisticated coculture and animal models will further help unravel the complexities of immune involvement and injury-repair responses that lead to homeostasis or disease chronicity. Taken in the broad context of the thousands of reports on SARS-CoV-2 and COVID-19, the findings in Ravindra and colleagues and Fiege and colleagues add insights into the initial waves of infection and local airway responses. They feed into meta-analyses of single-cell omics data  and in d


Dynamic communication within adipose tissue depends on highly vascularized structural characteristics to maintain systemic metabolic homoeostasis. Recently, it has been noted that adipose endothelial cells (AdECs) act as essential bridges for biological information transmission between adipose-resident cells. Hence, paracrine regulators that mediate crosstalk between AdECs and adipose stromal cells were summarized. We also highlight the importance of AdECs to maintain adipocytes metabolic homoeostasis by regulating insulin sensitivity, lipid turnover and plasticity. The differential regulation of AdECs in adipose plasticity often depends on vascular density and metabolic states. Although choosing pro-angiogenic or anti-angiogenic therapies for obesity is still a matter of debate in clinical settings, the growing numbers of drugs have been confirmed to play an anti-obesity effect by affecting vascularization. Pharmacologic angiogenesis intervention has great potential as therapeutic strategies for obesity.  The traHerein, we summarize and discuss the dramatically underexplored evidence in favour of the causal role of AdECs in systemic metabolic control. This review provides an overview of the molecular mechanisms of communication between AdECs-adipocyte and AdECs-stromal cells in the control of AT metabolic homoeostasis.2.LYVE1 [LYVE1 as a classic marker of LECs is enriched in AdECs, implicating that AdECs may present LECs-like metabolic and structural features. LECs have been reported to control lipid transport and mediate AT inflammation via regulation of intestine-adipose axis [Endothelial cells in ATs can be divided into arterial endothelial cells (AECs), venous endothelial cells (VECs), capillary endothelial cells (CECs) and lymphatic endothelial cells (LECs) based on anatomy locations . AECs arLYVE1 . LYVE1 aose axis . This obose axis . Althoug3.AdECs can secrete signalling molecules to target neuron cells, immune cells, and progenitors, altering their metabolic pathways and reshaping the AT microenvironment. In this study, we will comprehensively summarize the current studies on molecular signalling linking crosstalk between AdECs-immune cells, AdECs-neuron cells, and AdECs-progenitor cells .Figure 3.1A variety of AdEC-derived factors may target adipose-resident immune cells to regulate adipose metabolism and inflammatory responses . Adipose3.2The close connection between AdECs and sympathetic stimuli might be essential for AT remodelling during overfeeding or cold-induced thermogenesis . AdECs-d3.3+), could be triggered and differentiated into beige adipocytes by retinoic acid (RA), a metabolite of vitamin A [+ progenitor cells requires the upregulation of the VEGF signalling pathway to bind to the promoter of PRDM16, which is a transcription factor that determines the fate of brown and beige fat [AT is a cellular heterogeneous endocrine organ. In addition to adipocytes, adipose progenitor cells (APCs), which can create mature adipocytes, actively participate in metabolic homoeostasis . APCs reitamin A . The proeige fat . Hence, 4.Abnormal communication originating from AdECs or adipocytes leads to obesity and associated complications . Pellegr4.1In recent years, our understanding of the signalling molecules controlling insulin and glucose transport across the AdECs has unremitting increased. Examples of AdECs-derived protective factors include miR-181 and IgG receptor FcgRIIB, which serve as powerful regulators of insulin sensitivity and are implicated in the maintenance of metabolic homoeostasis in AT . MiR-1814.2The functional relevance of adipocyte-AdEC circuitry for adipose lipid turnover was also demonstrated in studies designed to explore the function of AdECs in the maintenance of adipocyte lipid homoeostasis. AdECs have a tight cross-talk with adipocytes for fatty acid (FA) turnover as they secrete VEGF-B and apelin, which promote endothelial FA absorption by modulating FATP4 or CD36 and transport lipids into adipocytes . This li4.3AdECs are the most important cells for AT vasculature involved in self-renewal and construction of the lumen and basement membrane . Most stBeige-like or brown-like AT remodelling is an energy-consuming process that relies on the growth of microvessels to match the energy demands . The angIn addition to paracrine factors derived from AdECs that can regulate vascularization and the browning process, the fibroblast growth factor-21 (FGF21) and murine double minute 2 (Mdm-2) derived from the liver and skeletal muscles can change the phenotype of adipocytes by regulating AdEC-dependent angiogenesis ,57. ReceAGO1, have been found to resist obesity [Obesity causes excess energy to be deposited in the white adipocytes, and its progression leads to a high demand for oxygen and nutrients . Hence,  obesity , whereas obesity .Certain rodent literature held the opposite views that promoting angiogenesis can effectively improve local hypoxia, fibrosis and inflammation induced by the rapid expansion of fat cells. Conversely, targeted delivery of pro-angiogenic factors including VEGFs, PDGF-B and PDGF-CC into AT enhances microvessel growth and can effectively reverse adiposity, macrophage infiltration, and oxidative stress . Hence, In the previous chapter, we have introduced the pros and cons of angiogenesis on fat metabolism in animal experiments. Limited AT vascularization and blood flow were shown to be correlated with hypoxia and insulin resistance, whereas hyperactived angiogenesis may result in unhealthy expansion of WAT . Thus, aExenatide, a glucagon-like peptide 1 receptor (GLP-1 R) agonist, is a first-line clinical drug with dual effects of anti-obesity and insulin resistance . HypoxiaTNP-470 is a synthetic analogue of fumagillin, which selectively inhibits endothelial cell growth and angiogenesis. The angiostatic mechanism of TNP-470 involves suppression of VEGF production in endothelial cells . PreviouPPARγ is a key transcriptional factor for promoting adipocyte differentiation, but enhancing insulin sensitivity. PPARγ induces vascularization to increase adipocyte numbers . RosigliNifedipine is a well-recognized dihydropyridine calcium-channel blocker that is widely used for the treatment of hypertension . ExperimMetformin is used as a first-line treatment in newly diagnosed T2DM patients and ameliorates hyperglycaemia and improves systematic metabolism . AlthougSeveral plant extracts have been reported to exhibit anti-angiogenic activity by suppression of VEGF-mediated proliferation in ECs . For exa4.4Since prohibitin and ANX2 were identified as surface markers of AdECs useful for its targeting with a peptide precision therapy ,99, accu5.Evidence indicating the interaction between adipose-resident cells and AdECs has mined the novel perspective of AT homoeostasis maintenance. We posit that AdECs, as major initiators, effectors, and regulators of metabolic stress, might be a central determinant of unhealthy AT growth. Some metabolites, proteins, miRNAs serve as messengers in the communication between adipocytes and AdECs . Recentl