While rumors even suggest that it might have killed Napoleon, more recent work suggests ATO as a potential treatment option for acute promyelocytic leukemia [Cellular and Molecular Immunology, Chen et al. address this question, adding yet another chapter to the complex relation between cell death and the immune response + T cells. To elucidate the mechanism of antitumor activity induced by ATO-VAC, the authors analyzed the pattern of ICD factors accompanying ATO-induced cell death. ATO treatment indeed created an exceedingly immunogenic environment through the exposure/release of a myriad of ICD factors. The effects included the release of ATP, cGAMP, and HMGB1 into the extracellular space; translocation of calreticulin to the plasma membrane; enhanced production of IFN-α/β; and transcription of interferon-stimulated genes. Transcriptomic profiling suggested the induction of oxidative stress in ATO-treated tumor cells, which was blocked by a ROS scavenger. To delineate the key pathways involved, western blot analysis of proteins central to autophagy, apoptosis, pyroptosis, ferroptosis, and necroptosis in TC-1 cells was performed. Interestingly, ATO simultaneously or sequentially activated autophagy, apoptosis, ferroptosis, and necroptosis with the possibility of crosstalk among these pathways. ATO-triggered oxidative stress proved indispensable for activating these pathways since the ROS scavenger NAC inhibited ATO-induced cell death, suggesting that oxidative triggers function upstream in this sequence. KO clones lacking each cell death executor were created using CRISPR‒Cas9 technology. Mice receiving Acsl4-/-, Ripk3-/-, or Mlkl-/- whole-cell vaccines displayed a low frequency of adoptively transferred OT-1 cells in the popliteal lymph nodes. The KO clones for apoptotic, necroptotic and ferroptotic factors showed impaired ATO-induced extracellular ATP accumulation and exposure of calreticulin on the plasma membrane. In addition, Acsl4-dependent ferroptosis and Ripk3- and Mlkl-dependent necroptosis seemed to contribute to cGAMP generation and release, suggesting pivotal roles in ATO-induced ICD and the prophylactic efficacy of the vaccine. Notably, ATO-VAC showed substantial therapeutic potential, as the administration of this vaccine to TC-1 cell-bearing mice significantly reduced tumor growth. As expected, ferroptotic and necroptotic pathways played key roles, as evidenced by reduced levels of various immune cells in the tumor microenvironment (TME) and reduced expression of an early activation marker (CD69) and effector cytokines (IFN-γ and TNF-α) by tumor-infiltrating CD8+ and CD4+ T cells in Acsl4-/-, Ripk3-/- and Mlkl-/- mice. ATO-VAC provides a promising agent for creating an immensely immunogenic TME and produces an even stronger antineoplastic effect when combined with immune checkpoint inhibitors, such as anti-PD-1.In their current work, Chen et al. started their investigation by setting up multiparameter drug screening for lethal antineoplastic drugs, including ATO . EvaluatThese results suggest the involvement of various pathways of regulated necrosis. However, a more detailed understanding of the sequential pathway stimulation and the precise triggers and, most importantly, performance of the missing safety assessment for ATO-VAC preclude advancing this approach closer to a clinical test. In addition, not all necrotic-type cell death pathways are immunogenic by nature. Indeed, recent data suggest that ferroptotic cell death can inhibit the antitumor response  and prev


KRAS mutation is the most frequent oncogenic aberration in colorectal cancer (CRC). The molecular mechanism and clinical implications of KRAS mutation in CRC remain unclear and show high heterogeneity within these tumors.Methods: We harnessed the multi-omics data  of KRAS-mutant CRC tumors and performed unsupervised clustering to identify proteomics-based subgroups and molecular characterization.Results: In-depth analysis of the tumor microenvironment by single-cell transcriptomic revealed the cellular landscape of KRAS-mutant CRC tumors and identified the specific cell subsets with KRAS mutation. Integrated multi-omics analyses separated the KRAS-mutant tumors into two distinct molecular subtypes, termed KRAS-M1 (KM1) and KRAS-M2 (KM2). The two subtypes had a similar distribution of mutated residues in KRAS (G12D/V/C etc.) but were characterized by distinct features in terms of prognosis, genetic alterations, microenvironment dysregulation, biological phenotype, and potential therapeutic approaches. Proteogenomic analyses revealed that the EMT, TGF-β and angiogenesis pathways were enriched in the KM2 subtype and that the KM2 subtype was associated with the mesenchymal phenotype-related CMS4 subtype, which indicated stromal invasion and worse prognosis. The KM1 subtype was characterized predominantly by activation of the cell cycle, E2F and RNA transcription and was associated with the chromosomal instability (CIN)-related ProS-E proteomic subtype, which suggested cyclin-dependent features and better survival outcomes. Moreover, drug sensitivity analyses based on three compound databases revealed subgroup-specific agents for KM1 and KM2 tumors.Conclusions: This study clarifies the molecular heterogeneity of KRAS-mutant CRC and reveals new biological subtypes and therapeutic possibilities for these tumors. Colorectal cancer (CRC) ranks third in terms of new cases and represents the second leading cause of cancer-related death worldwide . reported that the KRAS-G12V and KRAS-G12C mutants preferentially activate RTK and RAL signaling and decrease AKT activation, whereas the KRAS-G12D mutant is prone to activating PI3K and MEK signaling Recently, allele-specific covalent inhibitors that can specifically bind to the cysteine residue in the KRAS-G12C mutant have shown promising outcomes in the clinic Previous large-scale omics studies on KRAS-mutant cancers have greatly advanced our understanding of the molecular diversity of KRAS-driven cancer .) data to comprehensively characterize the molecular network and biological heterogeneity and to refine the molecular stratification of KRAS-mutant CRC, which may facilitate the development of combination therapies that are more suitable for KRAS-mutant CRC. Here, we report that KRAS-mutated CRC tumors can be divided into two distinct molecular subtypes based on integrated transcriptomic, proteomic and phosphoproteomic profiling but not on the mutated residues. The two subtypes were characterized by different clinical features, biological pathways, copy number alterations, and phosphorylation cascades associated with KRAS signaling activation. Kinase network analysis and drug sensitivity prediction also revealed potential therapeutic agents that may be helpful to treat the specific subtype. Overall, these proteogenomic analyses present new avenues for biological discoveries and therapeutic development in KRAS-mutant CRC tumors.In this study, we integrated multi-omics  colon cancer database The somatic mutation and copy number alteration (SCNA) segment data from the CPTAC cohort and TCGA-COAD/READ cohort were downloaded and curated for genomic analysis. Tumors with nonsynonymous mutations  in KRAS were considered KRAS-mutant tumors. The significantly mutated genes (SMGs) were curated from previous studies We adopted three types of omics data—mRNA transcriptome data, imputed proteomic data and imputed phosphoproteomic data (the data imputation procedure is described in the supplementary files), to a similarity matrix using the R package “CancerSubtypes” https://xcell.ucsf.edu/), with the algorithm run using the xCell signature.We utilized the xCell algorithm Single-cell RNA-seq and metadata were curated from the Samsung Medical Center (SMC cohort) https://portals.broadinstitute.org/ccle/) https://depmap.org/portal/).Available clinical annotations and expression profiles of human colorectal cancer cell lines (N=112) were obtained from the Broad Institute Cancer Cell Line Encyclopedia (CCLE) project .We summarized the KRAS activation signaling pathway and relevant inhibitors of each node Figure A. Once KAccumulating evidence has focused on the genomic mutations that are implicated in tumorigenesis and immune evasion and confer selective advantages during the evolution of cancer. We first compared the tumor mutation load in KRAS-Mut versus KRAS-WT and observed that there was no difference in the TCGA and CPTAC cohorts F-S1G. Im+ T cells, macrophages, pDCs, etc. , SPP1+ macrophages (myeloid cells) and myofibroblasts  were significantly increased in KRAS-Mut tumors  and Metascape analyses in the KM1 and KM2 tumor subsets at the RNA, protein, and phosphoprotein levels. The pathway enrichment at the RNA and protein levels indicated that KM1 was mainly enriched in the peroxisome, MYC targets, and Wnt β-catenin pathways, whereas KM2 was mainly enriched in the EMT, coagulation, angiogenesis, KRAS signaling-up, and myogenesis processes Figure A. PTM anTo explore the potential vulnerability of KRAS-Mut subtypes in CRC, we compared the gene dependencies based on large-scale RNAi screening among the colorectal cancer cell lines in the Cancer Dependency Map (DepMap) dataset. Forty-one KRAS-mutant CRC cell lines were curated and classified into the KM1 or KM2 subset. We identified 31 genes with significantly different dependency scores  between the KM1 and KM2 subsets Figure A. Using To further identify the subset-specific therapeutic agents for KRAS-mutant CRC subtypes, we applied three drug response databases  to investigate the potential therapeutic agents of KM1 and KM2 cell subsets Figure C. Differin vitro and in silico evidence, were considered to hold the most promising therapeutic potential in patients with KM2 CRC. Moreover, floxuridine and CDKi hold the promising therapeutic potential in patients with KM1 CRC. We also explored the potential gene regulators that highly correlated with sensitivity to MEKi, ERKi or AKTi in each KRAS subtype, which may be potential indicators for the response to such inhibitors  dataset to identify compounds for which the gene expression patterns were opposite the KRAS subtype-specific expression patterns . Six compounds for the KM2 subtype and three compounds for the KM1 subtype had a CMap score of less than -90 Figure D. We furhibitors B. Finallhibitors E.KRAS is frequently mutated in CRC and is involved in the occurrence, progression, immune evasion, and treatment resistance of CRC. Due to the broad heterogeneity of KRAS mutations, this group of CRC patients requires more precise and personalized treatment. In this study, genomic, transcriptomic, proteomic and phosphoproteomic analyses were leveraged to explore the clinicopathologic features and molecular networks in KRAS-Mut CRC tumors. Single-cell RNA-seq was also employed to delineate the TME cellular landscape of KRAS mutant CRC tumors. Our integrative analysis intrinsically separated KRAS-mutant tumors into two subtypes, KM1 and KM2, which are characterized by different genomic variations, survival outcomes and biological phenotypes. We further utilized a large-scale drug screening system based on KRAS-mutant CRC cell lines and identified potential therapeutic agents for the specific KRAS-Mut subtypes. Our study confirmed the value of proteogenomic integration in uncovering novel cancer biological implications of oncogenic driver KRAS mutations and further demonstrated the utility of proteogenomics in guiding therapeutic regimens.+ macrophages, myofibroblasts and IgG+ plasma cells) related to KRAS mutation and suggested the roles of genetic alterations in tumor cells that determine the formation of these unique tumor microenvironments in CRC. Giopanou et al. found that lung macrophages and epithelium-secreted SPP1 drove tumor-associated inflammation, while SPP1 promoted early tumorigenesis by promoting the survival of KRAS-mutated cells, especially in KRAS-G12D-driven tumors Previous studies reported that cytotoxic T cells, neutrophils and the interferon gamma pathway were suppressed in KRAS-Mut tumors . confirmed that EMT is a cause of both intrinsic and acquired resistance to KRAS G12C inhibitors in KRAS G12C-mutant non-small-cell lung cancer Considering the inferior clinical outcomes of KRAS-mutant CRC tumors, we performed an integrative clustering analysis to better stratify patients for therapeutic interventions and provide a more precise assessment of phenotypic heterogeneity. The KM2 subtype was associated with the mesenchymal phenotype-related CMS4 subtype and enriched in EMT, TGF-β and angiogenesis pathways, which indicated stromal invasion and worse prognosis. Studies have revealed that EMT-related molecules promote KRAS-driven tumor development and suppress sensitization to MEK inhibitors Interestingly, the two KRAS mutational molecular subtypes exhibited few differences in driver gene mutations. However, the copy number variations in cytobands of chr2p, chr5p, chr12p, etc. indicated significant alteration of amplification or deletion between the two KRAS subtypes. Cytoband amplification in the chr5p15.33 region contained the TERT gene, which is related to the cell cycle and regulation of Wnt-beta catenin signaling. Chr5q22.2 contains the tumor suppressor gene APC, whose deletion or inactivation leads to activation of the Wnt signaling pathway and loss of a tumor-suppressive function. Overexpression of WNT5B and FOXM1, located on chr12p13.33, has been reported to be associated with cancer cell migration and proliferation in vitro and in vivoDespite the breakthroughs in allele-specific inhibitors targeting KRAS-G12C mutant cancer in situ remains to be further validated in large-scale primary samples.The clinical relevance of the identified molecular signatures for subtyping was largely validated at the mRNA level but not at the protein/phosphoprotein level because of the limited clinical proteomics data for KRAS-Mut patients. Therefore, further validation of the molecular subtype classification in a large-scale prospective clinical cohort of patients with CRC is required. Furthermore, potential drug combination intervention strategies in specific KRAS-mutant CRC subsets are expected to be further explored in xenograft models and clinical trials. The representativeness of the signatures and drug sensitivities for CRC In summary, our integrated proteogenomic characterization revealed new molecular subtypes and therapeutic opportunities for targeting signaling proteins, phosphosites, and genomic alterations in KRAS-mutant colorectal cancer. Although validation of these therapeutic hypotheses is beyond the scope of our current study, our characterization of molecular phenotypes and mutation profiles may enable substantial advances in revealing the molecular heterogeneity of KRAS-Mut tumors. We hope that both the specific observations and hypotheses delineated in this manuscript may lead to the development of more promising KRAS-targeted approaches.Supplementary figures and table legends.Click here for additional data file.Supplementary tables.Click here for additional data file.


To assess the long-term (20-year) endocrine therapy benefit in premenopausal patients with breast cancer.Secondary analysis of the Stockholm trial  randomly assigning 924 premenopausal patients to 2 years of goserelin (3.6 mg subcutaneously once every 28 days), tamoxifen , combined goserelin and tamoxifen, or no adjuvant endocrine therapy (control) is performed. Random assignment was stratified by lymph node status; lymph node–positive patients (n = 459) were allocated to standard chemotherapy . Primary tumor immunohistochemistry (n = 731) and gene expression profiling (n = 586) were conducted in 2020. The 70-gene signature identified genomic low-risk and high-risk patients. Kaplan-Meier analysis, multivariable Cox proportional hazard regression, and multivariable time-varying flexible parametric modeling assessed the long-term distant recurrence-free interval (DRFI). Swedish high-quality registries allowed a complete follow-up of 20 years.P = .016). Genomic low-risk patients (n = 305) significantly benefitted from tamoxifen , and genomic high-risk patients (n = 158) from goserelin . Increased risk from the addition of tamoxifen to goserelin was seen in genomic high-risk patients . Moreover, long-lasting 20-year tamoxifen benefit was seen in genomic low-risk patients, whereas genomic high-risk patients had early goserelin benefit.In estrogen receptor–positive patients , goserelin, tamoxifen, and the combination significantly improved long-term distant recurrence-free interval compared with control . Significant goserelin-tamoxifen interaction was observed (This study shows 20-year benefit from 2 years of adjuvant endocrine therapy in estrogen receptor–positive premenopausal patients and suggests differential treatment benefit on the basis of tumor genomic characteristics. Combined goserelin and tamoxifen therapy showed no benefit over single treatment. Long-term follow-up to assess treatment benefit is critical. Standard adjuvant treatment of premenopausal estrogen receptor (ER)–positive breast cancer is tamoxifen for 5 years or more.7 Additional ovarian function suppression (OFS), such as the luteinizing hormone-releasing hormone-agonist goserelin, and/or chemotherapy is recommended in high-risk disease, often defined by standard clinical markers including lymph node involvement, high tumor grade, high proliferation, high genomic risk signature scores, and an age below 40 years.7 Therefore, although clinicians are becoming more inclined to recommend OFS, it is not clear which patients who should be offered the treatment.9Premenopausal patients with breast cancer have an increased risk of fatal disease.Key ObjectiveThe long-term endocrine therapy benefit in premenopausal patients with breast cancer, diagnosed early in life, is important to understand, given the long-term risk of distant recurrence in estrogen receptor (ER)–positive disease. The unique STO-5 trial with a complete follow-up of 20 years randomly assigned premenopausal patients to adjuvant goserelin, tamoxifen, combined goserelin-tamoxifen therapy, or no endocrine therapy (control). To our knowledge, for the first time the long-term benefit of goserelin and tamoxifen in premenopausal patients is assessed.Knowledge GeneratedThis study demonstrates long-term benefit from 2 years of adjuvant endocrine therapy in ER-positive premenopausal patients. Furthermore, it suggests long-lasting benefit from tamoxifen in genomic low-risk patients with long-term risk of distant recurrence, whereas genomic high-risk patients have early risk and benefit from goserelin.RelevancePremenopausal patients with ER-positive breast cancer have long-term benefit of endocrine therapy; however, the heterogenous metastatic potential gives rise to differential treatment benefit and a need for personalized endocrine therapy.11 However, random assignment to OFS only was not included in SOFT, and since patient enrollment was in the early 2000s, random assignment to an untreated group was not possible. Moreover, subanalysis concluded that the main effect from combined tamoxifen and OFS was seen in a group of slightly younger (median age 40 years) clinically high-risk patients who had regained their premenopausal status after prior chemotherapy.11 Contrary to SOFT, the Zoladex in Premenopausal Patients (ZIPP) trial suggested that combined goserelin and tamoxifen therapy is not superior to either modality alone.12 It might seem controversial that a combined treatment is not better than single treatment, but this was also seen in the Arimidex, Tamoxifen Alone or in Combination (ATAC) study in postmenopausal patients, where the combination of aromatase inhibitor and tamoxifen did not improve survival compared with aromatase inhibitor alone.13 A strength of the Stockholm part of the ZIPP trial (STO-5) is that patients were strictly randomly assigned to the four trial arms, and therefore, the STO-5 trial has also been analyzed separately.19 Furthermore, since the premenopausal endocrine therapy benefit was not clearly established at trial start, random assignment to no adjuvant endocrine therapy was included in the STO-5 trial. Thus, this four-arm trial now enables unique assessment of endocrine therapy versus control, in combination with unique long-term follow-up from high-quality Swedish registries.Results from the Suppression of Ovarian Function Trial (SOFT) suggested that addition of OFS to tamoxifen significantly improves survival in premenopausal patients, with a relatively short median follow-up of 8 years.26 Given this, longer follow-up is needed to understand the true endocrine treatment benefit in ER-positive breast cancer. In addition, the long-term benefit of adjuvant OFS therapy remains unknown, as there is a general lack of clinical trials with 10 or more years outcome data on this. The STO-5 trial has now reached a complete follow-up of 20 years; here, we present the long-term endocrine therapy benefit in premenopausal patients with breast cancer randomly assigned to two years of goserelin, tamoxifen, the combination of the two, or no adjuvant endocrine therapy. Moreover, we assess treatment arm–specific endocrine therapy benefit according to genomic risk stratification, using the molecular 70-gene signature risk prediction tool.27 This signature has known prognostic utility among young patients with breast cancer,29 but whether it has endocrine therapy predictive value remains unexplored.Ample evidence shows that patients with ER-positive breast cancer have a steady long-term risk of developing distant metastatic recurrences, with a large proportion of these events occurring beyond 10 years after primary diagnosis.19 Random assignment included four trial arms: 2 years of goserelin (3.6 mg subcutaneously once every 28 days), tamoxifen , combination of goserelin and tamoxifen, or no adjuvant endocrine therapy (control). Random assignment was stratified in three groups on the basis of patients' lymph node status; 0, 1-3, and 4 or more positive lymph nodes  enrolled 924 premenopausal patients diagnosed with invasive operable breast cancer.Informed consent was obtained before random assignment, and the trial was approved by the Karolinska Institutet Regional Ethics Committee. The trial was approved and initiated before the practice of trial registration in Sweden. The trial center was the Stockholm Regional Cancer Center.32Follow-up until December 31, 2016, was from Swedish high-quality national and regional registries of high validity and essentially complete coverage.33 ER-positive and PR-positive status was defined by a threshold of 10% or greater and Ki-67 was categorized as low (< 15%) and intermediate/high (≥ 15%). HER2 positivity was defined as intensity 3+ by IHC. ER status and PR status for patients with missing IHC data but with available immunoassay measurements35 (n = 4) were determined from this approach (Data Supplement). A total of 584 patients had ER-positive tumors  analysis of ER, progesterone receptor (PR), human epidermal growth factor receptor 2 (HER2), and proliferation marker Ki-67 was performed in 2020 in collaboration with the Karolinska University Hospital Pathology Department following standardized clinical protocols; details are given in the Data Supplement. Analysis was conducted for 731 patients with available primary tumor formalin-fixed paraffin-embedded blocks with sufficient invasive tumor cells for analysis. The 731 patient subset had similar distribution of patient and tumor characteristics as the 924 originally enrolled patients (Data Supplement). The percentage of cancer cells positive for each marker was scored by experienced breast cancer pathologists. According to Swedish National Guidelines,36Tumor size (T) was categorized into three groups according to clinical guidelines: T1a/b (≤ 10 mm), T1c (11-20 mm), and T2-T3 (> 20 mm). Tumor grading was performed according to the Nottingham Histologic Score system (Elston grade).38 In total, 586 tumors passed the 70-gene signature quality check, whereof 463 were ER-positive ,41 including distant metastasis or fatal breast cancer  as the event. Adjustments included standard clinical patient and tumor characteristics . Given trial stratification by lymph node status, this simultaneously adjusts for chemotherapy and locoregional radiotherapy. Interaction between goserelin and tamoxifen was tested by including a product term in the Cox proportional hazard regression model. All analyses were based on intention-to-treat. Analyses were performed in R version 3.5.2 and SAS version 9.4. All statistical tests were two-sided, and P values < .05 were considered statistically significant.The long-term endocrine therapy benefit was assessed by univariate Kaplan-Meier analysis and multivariable Cox proportional hazard regression. To assess how risk and treatment effect varied over the 20-year follow-up, multivariable time-varying analysis was conducted by flexible parametric survival modeling;Patient and tumor characteristics of the 584 ER-positive patients in the STO-5 trial were well-balanced between the four trial arms Table . The medP = .026, Fig Survival proportions at 20 years by DRFI were 71.6%, 66.0%, 67.1%, and 59.7% for patients randomly assigned to goserelin, tamoxifen, the combination, or control, respectively. Univariate Kaplan-Meier analysis showed significantly improved long-term DRFI from goserelin (log-rank P = .016).Multivariable analyses also assessed the effect from goserelin in patients treated with or without tamoxifen and vice versa. No significant long-term benefit from the combination of goserelin and tamoxifen was seen, compared with tamoxifen only or goserelin only Table . FurtherThe long-term endocrine therapy benefit was further assessed in the 463 ER-positive patient tumor samples with available 70-gene signature risk classification , but not in genomic low-risk patients (P = .080).Compared with either therapy alone, further multivariable analyses showed no significant long-term benefit from the combination in genomic low-risk or high-risk patients Table . HoweverTime-varying multivariable analysis was conducted to assess how risk for distant recurrence and treatment benefit varies over time. This analysis focused on tamoxifen in genomic low-risk patients and goserelin in genomic high-risk patients, after the significant and potentially time-varying effects observed in the previous analyses.Genomic low-risk patients had a steady long-term risk of distant recurrence. The estimated hazard rates were slightly increased from year 5 to year 10 and remained steady throughout the 20-year follow-up (Data Supplement). Moreover, a long-lasting benefit from tamoxifen was observed from year 4 to year 20 compared with control (Data Supplement) with significantly estimated HRs at years 5, 10, 15, and 20 showing a reduced risk . Significantly improved DRFI from goserelin was seen in the first 8 years compared with control (Data Supplement) with a statistically significant estimated HR of 0.26  at year 5 Table .This study of premenopausal patients with ER-positive breast cancer in a unique controlled randomization study demonstrates a significant 20-year benefit of 2 years of goserelin, tamoxifen, and the combination, compared with no adjuvant endocrine therapy. Furthermore, this study suggests that genomic low-risk patients have significant long-lasting benefit from tamoxifen, whereas genomic high-risk patients have early benefit from goserelin. The combination of goserelin and tamoxifen showed no long-term benefit over single treatment. Advantages of the STO-5 trial include long-term high-quality follow-up and inclusion of a control group of patients randomly assigned to no endocrine therapy.25 and others.23 Thus, long-term follow-up in these patients is essential to understand the true treatment benefit. The mechanisms influencing late recurrences remain undefined, but potentially involve dormant low-proliferative tumor cells whose metastatic growth becomes awakened by undetermined microenvironmental or systemic stimuli, leading to the proliferation-apoptosis equilibrium later shifting in favor of metastatic proliferation.20The risk of distant recurrence and fatal metastatic disease in ER-positive patients remains steady decades after primary diagnosis, as observed by usGenomic low-risk patients in the STO-5 trial have a steady long-term risk of distant recurrence, whereas genomic high-risk patients have high early risk, which demonstrates the heterogenous metastatic potential within premenopausal ER-positive patients. The observed differential treatment benefit suggests that 2 years of the ER agonist/antagonist tamoxifen more effectively prevents late recurrences in genomic low-risk patients, whereas 2 years of goserelin, inducing rapid systemic estrogen depletion, most effectively reduces the early risk in the more aggressive genomic high-risk tumors. These findings emphasize the clinical importance of biologic heterogeneity in ER-positive breast cancer and demonstrate the need to personalize premenopausal adjuvant endocrine therapy on the basis of prognostic and predictive tumor characteristics.6 However, determining patients' risk is challenging and differs between studies. For instance, SOFT used a continuous composite risk measured from a Cox model including different tumor characteristics,42 whereas risk classification was performed by the combination of different tumor characteristics in the MINDACT trial.43 Here, we assessed patients' genomic risk using the 70-gene signature. As expected, high genomic risk is to a greater extent associated with more aggressive tumor characteristics, such as larger tumor size, higher grade, and HER2-positive status, but there is also clear interpatient heterogeneity. Notably, approximately 40% of genomic high-risk patients had lymph node–negative disease or low-proliferative tumors. Furthermore, according to the MINDACT definition,43 22% of the STO-5 genomic high-risk patients were of low clinical risk. Clearly, a better understanding of risk is needed, especially the long-term risk (beyond 5-10 years) is largely unexplored,44 and risk definition will most likely differ between pre- and postmenopausal patients.Today, the addition of OFS to tamoxifen is recommended to clinical high-risk patients, often defined by lymph node involvement, high grade, high proliferation, high genomic risk scores, or an age below 40 years.11 To note, random assignment to OFS only was not included in SOFT, and therefore, an interaction between OFS and tamoxifen could not be assessed. Also, the benefit from the combined therapy was mainly observed in younger clinically high-risk patients with regained premenopausal status after prior chemotherapy. Extended follow-up in SOFT will demonstrate whether there is significant long-term benefit of the combination versus tamoxifen only. Similar to our study, the addition of OFS to tamoxifen was not seen to improve survival in clinically defined low-risk patients in SOFT.11 Moreover, in a meta-analysis from 2007 and in a follow-up study of the ZIPP trials,45 no significant reduction in the risk of recurrence by the addition of goserelin to tamoxifen was observed.46The STO-5 trial observed no benefit of the combination of goserelin and tamoxifen compared with single treatment and also suggests significant interaction between goserelin and tamoxifen. By contrast, SOFT showed improved disease-free and overall survival from the combination of OFS and tamoxifen compared with tamoxifen only, but not in the rate of distant recurrences.48 On the other hand, tamoxifen acts as a mixed agonist/antagonist that when bound to the breast cancer ER alters its transcriptional activity and expression of proliferative-associated genes. In some tissues, including normal endometrium, tamoxifen acts primarily as an ER agonist, accounting for its association with endometrial cancer.50 Given the differences in mechanisms of action, we speculate that when tamoxifen and goserelin are given in combination, the agonistic effects of tamoxifen may counteract the estrogen-depleting effects of goserelin. Consistent with this, the ATAC trial suggested that tamoxifen's ER agonistic properties may counteract the simultaneous withdrawal of estrogen levels by anastrozole in postmenopausal patients.13How goserelin and tamoxifen work on a mechanistic level, alone or in combination, is not fully understood. In brief, the initial response to goserelin is increased gonadotrophin-releasing hormone release in the hypothalamus. However, within a few weeks, desensitization occurs, and the ovarian estrogen production is inhibited, resulting in postmenopausal levels of circulating estrogens.18 However, further investigation is needed to understand potential differences between 2 and 5 years of therapy. Regarding tamoxifen, direct comparison between 2 years of 40 mg once daily as given in the STO-5 trial with today's at least 5 years of 20 mg once daily is difficult. The optimal dose and duration of endocrine therapy in premenopausal patients have not been established and may differ from that for postmenopausal patients.Patients in the STO-5 trial received 2 years of adjuvant endocrine therapy, but standard treatment nowadays is 5 years or more. The observed effect of 2 years of goserelin might be clinically important for patients who are unable to accept or endure 5 years of therapy, given the strong but reversible side effects associated with goserelin.There are limitations to this study. In the ER-positive subset of the STO-5 trial, sample size is a limitation and caution should be taken in the interpretation. Because of sample size, further subanalysis, for instance, on age was not statistically justifiable. Comparable with today's treatment approach, the STO-5 trial design allocated chemotherapy to lymph node–positive patients. Because of trial design, the additional effect from chemotherapy cannot be explored in the STO-5 trial. However, to note, 60% and 47% of genomic high-risk and low-risk patients, respectively, were lymph node–positive and received chemotherapy. Given the unique trial design and long-term follow-up by high-qualitative Swedish registries, results from this study are important to consider along with results from other trials. Importantly, no other studies on adjuvant endocrine therapy including OFS have reached more than a complete follow-up of 10 years or included a control group of patients randomly assigned to no endocrine therapy or random assignment to goserelin only.In conclusion, results from the STO-5 trial with a 20-year follow-up suggest a long-term benefit from 2 years of adjuvant endocrine therapy in ER-positive premenopausal patients. Furthermore, long-lasting benefit from tamoxifen in genomic low-risk patients with steady long-term risk of distant recurrence is observed, whereas genomic high-risk patients with early risk benefit from goserelin. For patients unable to endure 5 years of endocrine therapy, the significant benefit from 2 years of treatment as seen in this study could be helpful for both patients and clinicians. However, further studies are needed to understand the optimal treatment duration. Moreover, no long-term benefit from combined goserelin-tamoxifen therapy over single treatment was observed in the STO-5 trial. This controlled randomization study has limited sample size, but allows unique assessment of the long-term effects of endocrine therapy in premenopausal patients.


The framework integrates cellular-level epigenomic profiles from peak accessibility, motif deviation score, and pseudo-gene activity and learns a shared manifold using the multimodal input, followed by clustering and/or trajectory inference. We apply Destin2 to real scATAC-seq datasets with both discretized cell types and transient cell states and carry out benchmarking studies against existing methods based on unimodal analyses. Using cell-type labels transferred with high confidence from unmatched single-cell RNA sequencing data, we adopt four performance assessment metrics and demonstrate how Destin2 corroborates and improves upon existing methods. Using single-cell RNA and ATAC multiomic data, we further exemplify how Destin2’s cross-modality integrative analyses preserve true cell-cell similarities using the matched cell pairs as ground truths. Destin2 is compiled as a freely available R package available at  Recent advances in single-cell assay of transposase-accessible chromatin followed by sequencing (scATAC-seq) technologies  offer unThe first set of methods call ATAC peaks or segment the genome into bins and take the cell by peak (or cell by bin) matrix as input. Among these methods, Signac , scOpen Notably, most, if not all, of the aforementioned methods carry out “unimodal” analysis with a single type of feature input . One of the earliest methods, SCRAT , proposeTo our best knowledge, no integrative methods are available for a cross-modality analysis of scATAC-seq data, yet it has been shown that the peaks, genes, and motifs all contain signals to separate the different cell types/states. Here, we propose Destin2, a successor to our previous unimodal method Destin , for crocis-regulatory regions from region-topic distribution by cisTopic  and latent Dirichlet allocation (LDA), for the peak matrix. LSI normalizes reads within peaks using the term frequency-inverse document frequency transformation (TF-IDF), followed by a PCA-based dimension reduction . LDA is cisTopic . For they Seurat , followey Seurat . The numy Seurat , which rWith the pre-processed and normalized unimodal data input, Destin2 offers three options for cross-modality integration: consensus PCA (CPCA), generalized/multiple CCA (MultiCCA), and weighted nearest neighbor (WNN). Denote the feature input across (I) CPCA , algebraMultiCCA , on the  package  in R.  from 10x Genomics, adult mouse cortex cells from 10x Genomics, human bone marrow mononuclear cells (BMMCs) , and humWe apply unimodal analysis methods  and Destin2 to these datasets, with UMAP visualizations shown in Across the four scATAC-seq datasets, our results suggest that the multimodal analysis methods proposed by Destin2 improve clustering accuracy compared to conventional unimodal analysis methods using ARI and AMI as assessment metrics . For cLIFor downstream analysis, we first demonstrate how to determine the clustering resolution using the clustree method . SpecifiWe further applied Destin2 to three single-cell RNA and ATAC multiomic datasets of human PBMCs from 10x Genomics, adult mouse cortex cells from 10x Genomics, and mouse skin data from SHARE-seq . See SupSpecifically, we first obtained the low-dimensional embedding using the ATAC data alone by the various unimodal methods and Destin2’s multimodal methods. We then reconstructed the low-dimensional embedding using the RNA data using Seurat’s scRNA-seq processing pipeline . We thenFor the three multiomic datasets, our results suggest that the multimodal analysis methods offered by Destin2 exhibit top or near-top performance. For FOSCTTNN, Destin2’s cross-modality integration results are either top-performing or negligibly different from the top performer ; for agrformance . Interesformance , while Lformance . In realWe propose Destin2 to integrate multimodal peak accessibility, motif deviation, and pseudo-gene activity measures derived from scATAC-seq data. Destin2’s cross-modality integration can finish within a few minutes on a local computer; the computational bottleneck comes from the preprocessing step, which can also finish reasonably fast within an hour across tens of thousands of cells . For peaetc.] have not been thoroughly explored. For CCA, its variants and extensions, such as sparse CCA (For cross-modality joint modeling, Destin2 utilizes three statistically rigorous and computationally efficient methods—CPCA, MultiCCA, and WNN—and shows its outperformance and robustness. Additional methods that fall in the realm of multiomic integration  harse CCA  and decoarse CCA , can pot


With a growing number of patients entering the recovery phase following infection with SARS-CoV-2, understanding the long-term neurological consequences of the disease is important to their care. The neurological complications of post-acute sequelae of SARS-CoV-2 infection (NC-PASC) represent a myriad of symptoms including headaches, brain fog, numbness/tingling, and other neurological symptoms that many people report long after their acute infection has resolved. Emerging reports are being published concerning COVID-19 and its chronic effects, yet limited knowledge of disease mechanisms has challenged therapeutic efforts. To address these issues, we review broadly the literature spanning 2020–2022 concerning the proposed mechanisms underlying NC-PASC, outline the long-term neurological sequelae associated with COVID-19, and discuss potential clinical interventions. COVID-19 is a disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). As of 25 January 2023, there have been over 600 million confirmed cases of COVID-19 and over 6 million deaths worldwide , “post CThe symptomatology of PASC is highly variable as the condition can present with heterogeneous sequelae across multiple organ systems, and it can affect people independently of age and severity of acute infection ,9. Some The WHO defines the “post COVID-19 condition” as an illness that typically arises within three months of infection onset and causes symptoms and effects that last at least two months . HoweverNeurological symptoms are common in people experiencing PASC . A meta-The acute symptomotology of COVID-19, while not a focus of this review, warrants discussion as it can pertain to NC-PASC development. During an active infection, COVID-19 can cause encephalitis ,19, meniThere are currently no diagnostic criteria for NC-PASC . This maExamining the etiology of NC-PASC may assist clinicians in enacting prophylactic measures to slow or prevent the development of PASC. Unfortunately, the presentation of NC-PASC is extremely heterogeneous, and the factors that influence their development are not clearly known . ConsideWith respect to NC-PASC, lingering damage and physiological perturbations caused by acute COVID-19 infection are recurring themes in proposed mechanisms of sequelae development. For instance, Doykov et al. observe a persistent inflammatory response 40–60 days post-infection in asymptomatic and mildly affected  patients ; this suUnderstanding the pathophysiological mechanisms underlying NC-PASC could yield more effective approaches to treatment. However, discordance exists within the literature as to how SARS-CoV-2 infection causes lingering neurological symptoms; namely, do symptoms arise from direct effects of SARS-CoV-2 neuroinvasion and injury in the CNS, or indirect effects causing secondary downstream injury or neurodysfunction? We will consider these possibilities in turn. A graphical summary of the hypothesized pathophysiology of NC-PASC is provided in One broad pathophysiological mechanism supported by a few studies and case reports is neuroinvasion (neurotropism), in which the virus invades the CNS ,41,42,433) in medulla, frontal lobes, and olfactory nerves, although viral proteins were not detected by immunohistochemical labeling . Th. Th40].  NC-PASC . The SAROverall, these studies support the hypothesis that SARS-CoV-2 is neuroinvasive and that invasion into the CNS occurs, and that these observations are most consistent with hematogenous spread given the simultaneous appearance of SARS-CoV-2 RNA in anatomically distinct and distant brain regions. In contrast, trans-neuronal viral spread into the CNS would be supported by detection of SARS-CoV-2 RNA in contiguous and anatomically connected brain regions over time. Thus, although SARS-CoV-2 is neuroinvasive in some severely ill patients during acute infection, there is little, if any, evidence for a functional SARS-CoV-2 reservoir within the CNS after resolution of the acute infection. Therefore, the genesis of NC-PASC symptoms likely depends on secondary effects of acute infection.There is some evidence to suggest that NC-PASC could arise from microcirculatory damage and/or immune system dysfunction, primarily by means of hypoxia, cerebrovascular disease, and cytokine-mediated neuroinflammation . In the Many acutely ill COVID-19 patients present with some degree of hypoxia, even if they do not appear to be under respiratory stress . AutopsyA cross-sectional study of COVID-19 patients conducted by Dondaine et al. observed that those people who received oxygen therapy to manage pneumonia also had more severe memory impairment, attentional disorders, and psychomotor slowing months after initial recovery than people who did not require supplemental oxygen therapy for their acute COVID-19 infection . ImportaEndothelial damage resulting from hypoxia can contribute to cell death and cytokine signaling, upregulating an inflammatory response which potentially underlies the cognitive impairment reported by some patients . HoweverCerebral microbleeds are small hemorrhages in the brain associated with structurally abnormal vasculature . They arAlthough an association between COVID-19 and the development of cerebral microbleeds has been established, the underlying pathogenic mechanism is still under investigation. A retrospective case series conducted by Dixon et al. suggests that microbleed patterns in COVID-19 patients are similar to those seen in critically-ill non-COVID-19 patients and in patients with severe hypoxia from other causes . Thus, tChronic systemic inflammation as caused by a dysregulated immune system has been proposed as a pathway for the development and persistence of NC-PASC. Acutely, COVID-19 can cause severe inflammation via “cytokine storm” ,77 and sLymphopenia in acute COVID-19 infection has been associated with disease severity and poor outcomes ,82,83. PSeveral reports also suggest late complications can develop by means of autoimmune pathways. Two potential mechanisms include SARS-CoV-2 as a direct trigger of autoimmune events  or as an indirect trigger via systemic inflammation and immune dysregulation ,88. A stViruses other than SARS-CoV-2 are also known to trigger the development of post-viral syndromes. For instance, myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a complex, multi-systemic, chronic condition characterized by profound neurological and immunological dysfunction following acute viral infection ,92,93. TThe SARS-CoV-1 virus operates analogously to SARS-CoV-2 in that they both target and bind to the mammalian ACE2 receptor using spike proteins . SARS-CoOther viruses contribute to long-term symptoms by directly infecting neurons (neuronotropic viruses) and causing cellular damage. Although SARS-CoV-2 is likely not neuronotropic (not capable of infecting neurons in vivo), understanding the post-acute course of neuronotropic viruses could help guide diagnoses and better elucidate the relationship between neuronal dysfunction (NC-PASC) and direct neuronal infection. Alpha-herpesviruses such as varicella-zoster virus (VZV), herpes simplex virus 1 (HSV-1), and herpes simplex virus 2 (HSV-2) can establish latent neuronal infection in sensory ganglia following primary infection and cause neurological symptoms upon reactivation ,117. HSVAlthough CNS infections caused by arboviruses (arthropod-borne viruses) are generally associated with acute CNS symptoms, some infections are also associated with long-term neurocognitive sequelae . Among tNC-PASC development may in part be driven by the secondary dysregulation of the immune system and response. However, other non-neuronotropic viruses more directly target immune system components, leading to lasting neurological effects. HIV-1 is a neuroinvasive/neurotropic retrovirus that infects CD4 positive immune cells (but not neurons), ultimately leading to systemic immune deficiency when left untreated . PersistEBV primarily infects B lymphocytes, but it can also infect nasopharyngeal epithelial cells and neurons . FollowiSome non-neuronotropic viruses primarily target non-immune cellular components. For instance, chikungunya virus (CHIKV), a non-neuronotropic mosquito-borne virus, can infect a wide range of different cells and tissue types including stromal CNS cells ,113. CHIResearch efforts are a crucial part of improving how the neurological sequelae of COVID-19 infection are managed. Here, we identify broad gaps in knowledge as suggested by previous work in the field and discuss the current state of research with regards to treating NC-PASC.Additional experimentation is required to substantiate the proposed pathways of NC-PASC development and answer outstanding questions. First, the specific mechanisms that link acute hypoxia from COVID-19-associated pneumonia to NC-PASC, such as cognitive deficits, remains unclear. Rahman et al. suggest that a direct effect of the virus on the nervous system (among other systems including the HIF-1 pathway) may play a role in the development of a persistent “silent hypoxia” that can lead to severe tissue damage . NeuroimDysregulation of the immune system is a central feature of poor outcomes of COVID-19 infection. Future studies could further investigate the effect of the virus on BBB integrity and characterize how a pro-inflammatory state disrupts this barrier on the cellular level . CertainHeterogeneity in studies of NC-PASC can impede meta-analyses and may present as differences in methods of symptom assessment and data collection . To imprAs the long-term effects of COVID-19 infection become increasingly prevalent in the coming years, longitudinal prospective studies could be key to furthering our understanding of NC-PASC. Such studies could follow cohorts of patients and note exposures that may increase the risk of developing certain NC-PASC such as fatigue or cognitive impairment. Neuroimaging studies could also be leveraged to identify significant relationships between structural damage to brain tissue and vasculature and chronic symptomatology.Finally, there are no criteria for the diagnosis of NC-PASC excepting a proposed rubric by Moghimi et al. . This isAt present, no drug has been shown to specifically and effectively reduce symptoms of PASC, including neurological complications ,53,125. Hyperbaric oxygen therapy (HBOT) has been reported to improve multiple neurological symptoms including fatigue, executive dysfunction, and inattention in patients experiencing NC-PASC . A randoNC-PASC may be prevented altogether by the proper management of acute COVID-19 infection. Adhering to current standards of care in patients with severe COVID-19 infection and resultant acute respiratory distress syndrome (ARDS) is important for reducing neurological damage that could have long-lasting effects . FurtherSphingosine has been shown to prevent the interaction of the SARS-CoV-2 spike protein with ACE2 , and thuclinicaltrials.gov appear heterogeneous in design and intervention and thus show promise for tackling NC-PASC management from a number of different angles. Although studies aimed at characterizing the broad spectrum of involvements and neurological manifestations associated with NC-PASC are foundational, there is a clear and emerging need for trials of novel treatments as the burden of NC-PASC in the global population continues to increase.As of 25 January 2023, clinical trials related to NC-PASC include observational studies aimed at characterizing the natural history of neurological sequelae ,137 and The management of the neurological complications of post-acute sequelae of COVID-19 infection continues to be a concern in clinics worldwide. Post-mortem studies of brain tissue and observational studies identify pathways associated with hypoxia and dysregulation of the immune system as the main drivers of NC-PASC. However, due to a lack of experimental controls, the precise cellular mechanisms underlying these pathways remain unclear. Future research should focus on elucidating these mechanisms and leveraging this knowledge to advance novel therapeutics.


The immune system plays a vital role in maintaining tissue integrity and organismal homeostasis. The sudden stress caused by myocardial infarction (MI) poses a significant challenge for the immune system: it must quickly substitute dead myocardial with fibrotic tissue while controlling overt inflammatory responses. In this review, we will discuss the central role of myocardial regulatory T-cells (Tregs) in orchestrating tissue repair processes and controlling local inflammation in the context of MI. We herein compile recent advances enabled by the use of transgenic mouse models with defined cardiac antigen specificity, explore whole-heart imaging techniques, outline clinical studies and summarize deep-phenotyping conducted by independent labs using single-cell transcriptomics and T-cell repertoire analysis. Furthermore, we point to multiple mechanisms and cell types targeted by Tregs in the infarcted heart, ranging from pro-fibrotic responses in mesenchymal cells to local immune modulation in myeloid and lymphoid lineages. We also discuss how both cardiac-specific and polyclonal Tregs participate in MI repair. In addition, we consider intriguing novel evidence on how the myocardial milieu takes control of potentially auto-aggressive local immune reactions by shaping myosin-specific T-cell development towards a regulatory phenotype. Finally, we examine the potential use of Treg manipulating drugs in the clinic after MI. Heart and immune system development are closely intertwined, as leukocytes permeate the cardiac tissue during the embryonic stage and remain there throughout life. In addition to their housekeeping functions and recently discovered new roles , 2, dive+ T-cells can significantly affect myocardial repair after MI  that are involved in immunosuppression and play a vital role in maintaining immunological tolerance and overall homeostasis (A growing body of evidence indicates that adaptive immune responses orchestrated by CD4after MI . While ld models . Regulatd models –8. A sereostasis –15.via amphiregulin and interleukin-33 (IL-33) pathways  knockout mice, both of which lack functional CD4+T-cell responses. In all experimental settings, mice without CD4+ T-cell responses showed impaired myocardial leukocyte migration, reduced collagen deposition and unexpectedly decreased survival after experimental MI  of function approaches in an experimental MI model  that drives CD4+ T-cell responses after MI in Balb/C mice  Tregs infiltrated the myocardium via the IL-33 interleukin 1 receptor-like 1(-ST2) axis and favored collagen deposition and infarct maturation through mechanisms that depend on expression of Secreted Protein Acidic And Cysteine Rich (Sparc), a gene involved in collagen calcification and extracellular matrix synthesis , suggesting they might exert suppressive and anti-inflammatory function in this context too  and regulatory (CD25+) TCR-M cells we showed that the myocardium attracts conventional TCR-M cells, which in turn gain FOXP3 expression, demonstrating that the infarcted heart favors in situ Treg conversion , there are currently no cardiac antigens mapped in the widely used C57BL/6 mouse strain (MHC-II haplotype I-Ab) or in humans. This current lack of tools to track heart-specific Tregs in C57BL/6 mice is an important limitation in the field and a major roadblock to translation. The genetic background can critically impact antigen presentation, T-cell responses, autoimmunity predisposition, myocardial function, amongst several other factors , yet the heart seems to tame T-cell responses and direct them toward a regulatory phenotype. Unlike tissues such as skin and liver, the adult heart possesses negligible regenerative capacity, and transient functional impairment can be life-threatening. Interestingly, the myocardium is equipped with several immune inhibitory receptors that can keep T-cell responses at bay under baseline conditions , 54. As function , 56.Areg), effector markers (Tnfrsf9) and collagen synthesis-related genes (Sparc). Further investigation revealed that the IL-33 axis and Sparc were necessary for the Treg-mediated improvement in cardiac function post MI. Martini et al.  and myosin-specific (TCR-M cells) T-cell responses at the single-cell level in the heart and MedLNs. Intriguingly, TCR-Ms clustered separately from bona fide Tregs and showed a transcriptome suggesting an induced Treg signature   and another subset expressing high levels of immune checkpoint inhibitor transcripts . However, further investigation is needed to validate these phenotypic states and eventually resolve the mechanisms underlying their differentiation and function  technology, which allows for unbiased, simultaneous characterization of cellular information across thousands of individual cells. Moreover, all this information can be tracked to TCRα/β sequences at the single-cell level, enabling researchers to analyze the transcriptome response, evaluate clonal expansion and eventually address expanded TCR specificity in unprecedented detail . Considei et al.  used ScRIzumo1r) . More defunction   have a short regeneration window that lasts until shortly after birth , 59. Treneration . Besidesignaling . These o healing , 19, 64. healing . While Socardium , 27, 37.State-of-the-art therapy for infarcted patients has significantly reduced mortality and morbidity over the years, but understanding local immune responses in the MI context may reveal immunological markers of progression toward HF and further improve patient recovery. For instance, the CANTOS (Canakinumab Anti-inflammatory Thrombosis Outcome Study) trial demonstrated that targeting pro-inflammatory cytokines in patients with previous MI lowers the rate of recurrent cardiovascular events .Currently, little is known about T-cell biology in infarcted human hearts. Analyzing human cardiac autopsies showed increased Treg infiltration during the proliferative phase of MI repair. In addition, PET/CT imaging using a CXCR4 probe, as a readout for T-cell activity, revealed elevated signal in the heart draining lymph nodes of infarcted patients compared to control subjects, suggesting the existence of a similar MedLN-Heart T-cell axis following MI , 67. A pWith regard to the immune system, MI substantially differs from surface/mucosal infection; MI results in abrupt release of auto-antigens and DAMPs in a sterile environment in a vital post-mitotic organ with low disease tolerance . Thus, oEW, GR, and MD wrote the manuscript and designed the figures. All authors contributed to the article and approved the submitted version.This work was supported by the Interdisciplinary Centre for Clinical Research Würzburg [E-354 to GR], the European Research Area Network—Cardiovascular Diseases  the German Research Foundation [DFG grants 411619907 and 453989101 to GR. EW received a scholarship from the Graduate School of Life Sciences – Würzburg. GR leads projects integrated in the Collaborative Research Centre ‘Cardio-Immune interfaces’, funded by the German Research foundation (SFB1525 grant number 453989101).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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Data collected demonstrated greater detrimental effects in vitro with cinnamon (9 articles), strawberry (5 articles), and menthol (10 articles), flavors than other flavors. The most reported effects among these investigations were perturbations of pro-inflammatory biomarkers and enhanced cytotoxicity. There is sufficient evidence to support the toxicological impacts of diacetyl- and cinnamaldehyde-containing e-liquids following human inhalation; however, safety profiles on other flavors are elusive. The latter may result from inconsistencies between experimental approaches and uncertainties due to the contributions from other e-liquid constituents. Further, the relevance of the concentration ranges to human exposure levels is uncertain. Evidence indicates that an adequately controlled and consistent, systematic toxicological investigation of a broad spectrum of e-liquid flavors may be required at biologically relevant concentrations to better inform public health authorities on the risk assessment following exposure to EC flavor ingredients.Electronic cigarettes (ECs) are purported to be tobacco harm-reduction products whose degree of harm has been highly debated. EC use is considered less hazardous than smoking but is not expected to be harmless. Following the banning of e-liquid flavors in countries such as the US, Finland, Ukraine, and Hungary, there are growing concerns regarding the safety profile of e-liquid flavors used in ECs. While these are employed extensively in the food industry and are generally regarded as safe (GRAS) when ingested, GRAS status after inhalation is unclear. The aim of this review was to assess evidence from 38 reports on the adverse effects of flavored e-liquids on the respiratory system in both  Tobacco smoking remains the leading cause of preventable deaths globally  or dual users (30.5%) and relatively few never-smokers (4.9%) were EC consumers in Great Britain ASH . A revieAlthough ECs are considered less hazardous than cigarettes, it is less clear how harmful EC use as evidenced by oncogenicity  for ingestion and employed extensively in the food industry. However, there are concerns regarding EC flavoring toxicity following inhalation using EC as evidence for safety following has not been extensively investigated .Flavor additives in food may undergo enzymatic metabolism, which might produce fewer toxic metabolites due to phase I and II metabolism  AND (flavor OR flavor OR flavoring OR flavoring OR flavoring agents OR Flavoring agents OR Flavored OR Flavored)) AND ).title, abstract, key. Whereas the search stream below was used for our Scopus search – ((TITLE-ABS-KEY (“electronic cigarette”)) OR (TITLE-ABS-KEY (“electronic nicotine delivery system”)) OR (TITLE-ABS-KEY (“electronic (non) nicotine delivery system”)) OR (TITLE-ABS-KEY (“e cigarette”)) OR (TITLE-ABS-KEY (“e-cigarette”)) OR (TITLE-ABS-KEY (“e-cig”)) OR (TITLE-ABS-KEY (“e cig”))) AND ((TITLE-ABS-KEY (flavor)) OR (TITLE-ABS-KEY (flavor)) OR (TITLE-ABS-KEY (flavoring)) OR (TITLE-ABS-KEY (flavoring)) OR (TITLE-ABS-KEY (“Flavouring agents”)) OR (TITLE-ABS-KEY (“Flavoring agents”)) OR (TITLE-ABS-KEY (“Flavoured”)) OR (TITLE-ABS-KEY (“Flavored”)) AND (TITLE-ABS-KEY (lung)) OR )) AND ((TITLE-ABS-KEY (lung)) OR ) OR ) OR (TITLE-ABS-KEY (pulmonary))) AND ) AND ))))).A literature search was completed in November 2020, which was updated in November 2021, using Scopus and healthcare database advanced search (HDAS)  following Preferred Reporting Items for Systematic Reviews (PRISMA) guidelines. Only primary articles published in English were included. After a manual screening of titles and abstracts within the 225 and 74 records from HDAS and Scopus, respectively, 38 primary articles (one paper outside the search string) met the inclusion criteria for this systematic review .Figure in vitro studies where human cell lines were exposed to flavored e-liquids, 5 articles were e-liquid chemical composition studies, and 5 reported studies where rodents (mice and rats) were exposed to flavored e-liquids. Analysis of the 38 included reports and their reported adverse effects of flavors are summarized in The search stream included 28 in vitro investigations on cells were carried out on at least two different cell lines belonging to one of the three classes of the cell line: primary (5 articles), immortalized normal (15 articles), and cancerous pulmonary cells (13 articles). Therefore, we have subdivided the reporting of these papers based on the cell lines used in Most in vivo , MUC5AC  production, and oxidative stress markers. There were no human studies where the effects of flavors on the respiratory system were investigated.Similarly, cinnacide (cinnamon-contained), black licorice, banana pudding, vanilla, and tobacco-induced differential toxicity in vivo . Toxic eThis systematic review screened literature to determine which e-liquid flavors and flavoring agents (if any) elicit adverse effects in the airways. The studies identified several flavor categories demonstrating toxicity , suggestThe evidence presented in this review suggests that flavors in e-liquids are not non-hazardous. Evidence regarding the risk of e-liquids might be biased or affected due to the limited number of appropriate toxicological studies. The most frequently reported adverse effects are reduced cell count and viability, altered pro-inflammatory biomarkers, cytokine release, and increased oxidative stress  Leslie .Immortalized human cell lines are normal cells artificially manipulated to proliferate indefinitely. In contrast, cancer cell lines are transformed cells with mutations in oncogenes and tumor suppressor genes. There is evidence to suggest that cancer cells have increased levels of antioxidant enzymes such as superoxide dismutase and catalases compared to healthy cells, potentially as a defense mechanism against oxidative stress or to promote DNA damage and cancer progression  in a gender-dependent manner. In addition, Glynos et al.  cell-only for submerged exposures and air-only controls for air-liquid interface (ALI) exposures, (2) PG/VG/vehicle alone controls, and (3) PG/VG + nicotine controls to better inform on the contributions of the flavor compounds. In addition, justifications for e-liquid dosages might improve the interpretation of the relevance of the findings for health of individuals that vaper. These would lead to a better understanding of the relative contributions of flavor compounds, vehicles, and nicotine to the observed adverse effects of e-liquids and aid the ongoing debate over the safety and banning of flavored-e-liquids.in vitro or in vivo.This review note the following: 1) lack of appropriate controls in many studies to enable reliable interpretation of data for identification of hazards and risks due to flavor components, 2) ambiguity over physiologically relevant doses of e-liquid/flavor used, and 3) lack of assessments of multiple dosing to establish the presence of dose-dependent relationships. Therefore, this section proposes an approach to reduce discrepancies among EC investigations, whether In vitro submerged culture models are extensively employed in EC-induced toxicity screening. Evidence suggests that cytotoxicity of e-liquids in submerged models accurately predicts aerosol cytotoxicity 74% of the time  systems with standardized puff profiles suggested by CORESTA , and the apical surface may be exposed directly to EC vapor. It is probably a better model than adding e-liquid in submerged basal cell culture. Such ALI models focus on anatomical regions of the lung or molecular pathways and aim to model relevant processes in vivo. Pairing this with analytical approaches to identify potential chemical compositions and concentrations  for further investigation may eventually lead to limits on specific flavor chemical concentrations or outright bans from e-liquids. In addition, studies assessing the usefulness of ECs in risk and harm reduction needs to consider comparing e-cigarette data against conventional cigarettes before concluding the relative safety of these devices. This could be a gold-standard protocol to screen e-liquids toxicity that minimizes uncertainties.In contrast to the submerged models, ALI lung cell models that have been validated for adequate transepithelial membrane resistance (TEER) measurements, expression of differential markers for goblet cells (MUC5AC), ciliated cells (FOXJ1), Clara cells (SCGB1A1), and basal cells (KrT5) may be used to investigate physiological and pathophysiological responses of the respiratory tract, molecular events, modes of action and interactions of different cell types. Potentially, these cell models mimic the in vitro studies where the experimental model used to assess the safety of EC flavors closely recapitulates the microenvironments of the 3D human lung  or in vivo animal testing may help discuss the potential extrapolation of EC toxicity to humans, taking into consideration dosage and exposure times required to mimic human exposures  not all flavors produce toxicity, and 3) more evidence-based risk assessments need to be used for those flavors that induced some toxicity to set concentration limits to reduce the potential for causing harm in humans. Although no apparent human studies data accounted for potential adverse pulmonary effects of flavored e-liquids, in vitro and in vivo studies reported toxicity attributed to cinnamon, strawberry, menthol, and other flavors. Therefore, public health authorities need to extrapolate data from these studies to predict risks posed by flavors to human health.Some flavors in e-liquids may elicit pulmonary toxicity. However, these flavors are generally believed to attract long-time tobacco cigarette smokers to switch to EC use. As such, imposing restrictions on the use of flavors based upon reported evidence may be counterproductive. This is because 1) several