Cancer immunotherapy utilizes the immune system to fight cancer and has already moved from the laboratory to clinical application. However, and despite excellent therapeutic outcomes in some hematological and solid cancers, the regular clinical use of cancer immunotherapies reveals major limitations. These include the lack of effective immune therapy options for some cancer types, unresponsiveness to treatment by many patients, evolving therapy resistance, the inaccessible and immunosuppressive nature of the tumor microenvironment (TME), and the risk of potentially life-threatening immune toxicities. Given the potential of nanotechnology to deliver, enhance, and fine-tune cancer immunotherapeutic agents, the combination of cancer immunotherapy with nanotechnology can overcome some of these limitations. In this review, we summarize innovative reports and novel strategies that successfully combine nanotechnology and cancer immunotherapy. We also provide insight into how nanoparticular combination therapies can be used to improve therapy responsiveness, to reduce unwanted toxicity, and to overcome adverse effects of the TME. Cancer immunotherapy can provide powerful and long-lasting anti-cancer responses in patients with advanced or metastasized tumors that are otherwise resistant to conventional therapy . MechaniExtensive preclinical research and first clinical data demonstrate that nanotechnology can overcome some of the challenges that currently limit cancer immunotherapy .However, broadening the clinical applicability of cancer immunotherapy with the help of nanotechnology requires an improved understanding of the mechanisms limiting cancer immune treatment ,7. For eGeneralized immune-boosting therapies improve the overall fitness of immune cells and aim to initiate killing of cancer cells, previously spared by the immune system. In contrast to personalized cancer therapies that target patient-specific oncogenic vulnerabilities, generalized immune-boosting therapies do not require prior knowledge of individual tumor-specific gene mutations or patient-specific immune characteristics such as human leukocyte antigen (HLA) polymorphisms. The first generalized immune-boosting anti-cancer therapy dates back to the year 1891, when William B. Coley started to treat bone and soft-tissue cancer patients with bacterial toxins [Initial modern-day immune-boosting therapies used systemic application of interferon-α (IFN-α) or interleukin-2 (IL-2) and proved that injection of pro-inflammatory cytokines could induce durable anti-tumor immune responses in some cancer types ,10. ThisThe central role of ICIs in cancer immunotherapy was initially demonstrated in melanoma and non-small cell lung cancer patients by the remarkable clinical efficacy of antibodies that target cytotoxic T lymphocyte–associated antigen 4 (CTLA-4) and the programmed cell death protein 1 pathway (PD-1/PD-L1) ,19. WhilN-terminal myristoyl chain coupled to transportan, a cell penetrating peptide, and C-terminal peptides with a TME/TAM homing sequence  dramatically improved treatment responsiveness to anti-CTLA-4, produced similar therapeutic benefits as 10- to 200-fold higher systemic delivery, and generated anti-tumor effects at distant tumor sites [In cases in which tumors are not externally accessible, ICI activity was locally concentrated by producing triblock copolymer-nanoparticles that comprise a central polyethylene glycol (PEG) block flanked by two polypeptide blocks, which contain reactive oxygen species (ROS)-responsive L-methionine (Me) and dextro-1-methyl-tryptophan (D-1MT) and tumor-homing high-affinity peptides derived from placental growth factor–2. The latter bind to fibronectin and collagen type I of the tumor extracellular matrix (ECM) . These nor sites . Similaror sites .Taken together, these pioneering experiments highlight that nanoparticle formulations have the potential to enhance tumor responsiveness to ICI therapy and to reduce unwanted systemic toxicity. Given their potential to fine-tune anti-cancer responses, site-specific nanoparticle delivery and co-packaging with additional immune-boosting agents is likely to advance future ICI-mediated anti-tumor treatments.In contrast to generalized immune-boosting therapies, personalized anti-cancer treatment redirects immune effector cells to patient-specific tumor vulnerabilities. To ensure that the patient is amenable for treatment, upfront knowledge of patient tumor characteristics and information about individual immunological predispositions are required. This means that, for most personalized therapies, critical variables such as individual tumor mutations, expression and presentation of cancer-specific neoantigens, the general immune status, and the nature of HLA alleles have to be tested before therapy can be started.Because T cells are decisive effectors of cancer immunotherapy, treatment strategies that either use patient-derived tumor-reactive T cells, or alternatively, modify the reactive capacity of T cells towards tumor-specific antigens, have been developed. Initial experiments using re-infusion of autologous in vitro expanded tumor-infiltrating T lymphocytes (TILs) demonstrated durable complete responses in some cancer patients ,30. MoreAn excellent alternative to re-infused TILs are GETs. The objective of GETs is to redirect the potent cytotoxic activity of T cells towards tumor-specific antigens. Of note is also that other lymphoid effector cells like natural killer (NK), natural killer T (NKT), and γδ T cells can be modified to recognize tumor-associated antigens and are equally proficient to eliminate cancer cells ,33. A crTo develop a simple and inexpensive method for producing CAR T cells, Smith and collaborators synthesized poly-(β-amino ester) nanoparticles to transfect coding DNA in vivo specifically to T cells . Most imAlthough very effective in destroying tumor cells, CAR T cell applications carry the above-mentioned risk of potentially life-threatening side effects ,41. SuchIn contrast to the very effective therapeutic action of CAR T cells in B cell leukemia, achieving powerful CAR T cell responses to destroy solid tumors remains a challenge. Obvious reasons for this are the poor persistence and tumor infiltration of adoptively transferred CAR T cells, the frequently encountered barriers of an immunosuppressive TME, and the suboptimal in vivo stimulation of CAR T cells by the tumor cells. To overcome these limitations and to be able to enhance CAR T cell activity, one possibility is to attach T cell boosting nanoparticles to their surfaces. Demonstrating the feasibility of such a strategy, the linking of liposomal vesicles loaded with an immune stimulatory A2a adenosine receptor antagonist (SCH-58261) to the surface of CAR T cells markedly improved the anti-cancer CAR T cell responses in mice with leukemia and ovarian cell carcinoma ,44. ThesAn additional major area of research in precision immunotherapy is bi-specific antibodies, also known as bi-specific T cell engagers (BiTEs). In contrast to CARs that contain only one antibody-derived single-chain variable fragment (scFv) for target recognition, BiTEs have two different scFvs. The design of BiTEs allows binding of one scFv to a T cell epitope (often CD3ε) and the other to engage a tumor-associated surface antigen. By interacting with both targets, BiTEs physically link T cells and tumor cells, a bridging that results in T cell activation, cytokine production, and tumor destruction ,49. AccoThe availability of next generation mutanome sequencing (NGMS) in combination with bioinformatics tools that predict the likelihood of a mutated neoantigen to be presented on cancer cells allows the rapid and cost-effective production of customized anticancer vaccines ,56. A fiThe second class of neoantigen-specific antitumor vaccines consist of customized RNA-LPXs loaded with synthetic RNAs expressing multiple patient-specific tumor neoepitopes ,62. BaseThe therapeutic benefit or failure to respond to cancer immunotherapies greatly depends on the TME ,66. TMEsCompared with normal tissue, tumors develop an altered ECM, a tumor-specific neo-vasculature, and increased tissue pressure, all known to hamper homing, proliferation, and activation of immune effector cells ,68. An aSeveral tumor-associated cell types, most notably, regulatory T cells (Tregs), tumor-associated macrophages (TAMs), and myeloid-derived suppressor cells (MDSCs), act as negative regulators for cancer immunotherapy . In the + effector T cells in the B16/B6 melanoma model [Tregs are an important immune suppressive population in the TME and have the potential to inhibit cancer immunotherapy by numerous mechanisms ,86. Thesma model .TAMs represent the prominent innate immune cell type that is paramount for tumor progression and cancer immunotherapy, apart from MDSC and tolerogenic M2-type DCs ,97. DepeThe presence of MDSCs that are recruited from the blood stream into the TME constitutes a major obstacle for immunotherapy . Normall+ T cells . As


We describe a case of suspected COVID-19 pneumonia in a 61-year-old male with known primary central nervous system diffuse large B-cell lymphoma (DLBCL) who underwent restaging PET/CT during the initial peak of infection of COVID-19 pneumonia within the New York region. At the time of his routine PET-CT to assess for disease progression, typical CT imaging features of COVID-19 pneumonia were identified. Upon further investigation, the patient was asymptomatic, and his infection status remained unknown.He was subsequently lost to follow-up with his COVID-19 status pending. Coronavirus disease of 2019 (COVID-19) is a novel coronavirdae virus which causes severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). COVID-19 predominantly manifests as an atypical pneumonia features by nonproductive cough, chest pain, shortness of breath, fever and malaise. The outbreak originated in Wuhan Provence of China, and has now spread to much of the globe, infecting over 16.5 million people worldwide [COVID-19 causes an atypical pneumonia that can be diagnosed by chest radiograph or chest CT imaging. Both modalities illustrate multifocal bibasilar predominant patchy airspace opacities that may progress to confluent consolidations in later stages -3. As thThis diffuse microthrombi burden combined with endothelial destabilization results in a severe multifocal inflammatory response, noted by the histological presence of megakaryocytes, platelets, fibrin, neutrophils and other inflammatory cells . As the It is well known that fluorodeoxyglucose (FDG) positron emission tomography-computed tomography (PET-CT) imaging demonstrates increased uptake across a variety of pathological etiologies including infections, inflammatory processes, and neoplasms. For example, FDG PET-CT imaging has been demonstrated to be useful in localizing foci of infection and inflammation in patients with fever of unknown origin . In caseA 61-year-old male with a history of primary diffuse large B-cell lymphoma (DLBCL) of the CNS was treated by high dose BEAM conditioning chemotherapy followed by autologous hematopoietic stem-cell transplantation. A restaging top of head to mid-thigh FDG PET-CT was performed on March 30th, 2020. The study was performed in an outpatient imaging center within the New York region during a time of high prevalence of COVID-19 infection.As an incidental finding, the lung parenchyma on CT demonstrated interval development of multifocal, bibasilar and peripheral predominant, ground-glass opacities with areas of reticulation within the upper lobes demonstrating prominent FDG uptake Figures -3. The pTypical findings of COVID-19 pneumonia include multilobar, bilateral ground glass opacities with or without consolidation -3. TheseVarious risk factors have been identified in predictors of morbidity and mortality for those with COVID-19 infection including advanced age, obesity, preexisting cardiovascular or cerebrovascular diseases, as well as underlying immunosuppression, including in the setting of malignancy and oncologic therapeutics . Given tFurthermore, according to a recent report by Albano et al., six of 65 (9%) patients undergoing restaging PET/CT for various malignancy demonstrated incidental interstitial pneumonia on CT with corresponding FDG uptake, suggesting SARS-CoV-2 infection may not be infrequent even in asymptomatic patients . In addiTherefore, in regions of high prevalence of COVID-19, several considerations should be made by nuclear medicine staff for oncologic patients undergoing restaging PET/CT scans in order to minimize potential exposure to COVID-19 of this extremely vulnerable patient population . ScreeniWe describe a case of suspected COVID-19 pneumonia in a 61-year-old male with primary DLBCL of the central nervous system who underwent routine restaging PET-CT during the initial peak of COVID-19 infection in the New York region.The patient demonstrated classic imaging findings of early interstitial pneumonia on PET/CT, meeting radiologic diagnostic criteria of SARS-Cov-2 infection on chest imaging. Therefore, taking into consideration the ongoing high prevalence of COVID-19 in the New York region and the patient’s underlying immunocompromised state, early interstitial pneumonia related to underlying SARS-CoV-2 infection was given a high consideration. Although our patient lacked a confirmatory positive PCR result, these typical imaging features should be kept in mind when imaging oncologic patients at high risk for COVID-19 exposure.


The Special Issue entitled “New Strategies in Cancer Pharmacotherapy: Development of Hormonal Antineoplastic Drugs, Cytotoxic Drugs and Targeted Therapies” was conceived with the idea of compiling information on the latest advances in the treatment of both hormone-dependent and hormone-independent cancers. The Special Issue includes both reviews and original research manuscripts addressing the efficacy of new compounds in cancer therapy. It is well known that the effectiveness of classical cytotoxic drugs commonly included in cancer treatment protocols is limited, and their use is often accompanied by severe adverse effects, which highlights the importance of the continuous search for “next-generation” compounds and/or delivery strategies. Recent developments have seen hormone analogues or antagonists, inhibitors directed against membrane receptors, monoclonal antibodies raised against cancer biomarkers, inhibitors of immune checkpoint pathways, newly designed derivatives of pre-existing molecules, or even metabolites derived from hormone-like analogues addressed in terms of their effectiveness either alone or in combination with classic cytotoxic drugs.N--4-{thienopyrimidin-4-yl}piperazine-1-carboxamide (compound 6), was found to be more potent than amuvatinib in a cell line-specific manner under glucose starvation, indicating that compound 6 might be a new molecule with potential anti-cancer activity. Interestingly, these compounds synergize with a vascular endothelial growth factor (VEGF) inhibitor in vivo [Kauerová et al. investigated the antitumor effects of recently designed ring-substituted 1-hydroxynaphthalene-2-carboxanilide derivatives, formulated by the extension of salicylanide structure properties. The new halogenated hydroxynaphthalene carboxamides were effective in inhibiting the proliferation of monocytic leukemia (THP-1) and breast adenocarcinoma MCF-7) cell lines, preventing progression through G1/S transition. Moreover, one of the compounds synthesized and tested, compound 10, triggered apoptosis, suggesting that nitro-substituted hydroxynaphthalene carboxamides might represent a moiety model with promising anticancer properties [compound , was fou cell lin in vivo . Melaton in vivo . Wilms’ The review by Tan and Choo explores the application of pluripotent stem cells (PSCs) for the discovery of new biomarkers and generating antibodies against those biomarkers. The monoclonal antibodies generated against PSCs might have applications in cancer-targeted therapy to kill cancer cells, and conversely, monoclonal antibodies already in use in cancer might be repurposed for regenerative medicine, making it safer . GrywalsOverall, the 13 contributions accepted in this Special Issue highlight the promising perspectives for analogues, targeting therapies such as monoclonal antibodies, new-generation derivatives raised from other molecules and new forms, and drug delivery in the future of cancer treatments.


Teaching Point: The typical imaging features of a retroperitoneal solitary fibrous tumor are relatively large-sized, have a well-defined margin, an avidly heterogeneous enhancement on arterial phase, a prolonged enhancement on delayed phase, and serpentine vessels along the periphery of the tumor. A 30-year-old female was admitted to our hospital after an elevated carbohydrate antigen  was discovered on a health check-up. Her past medical history was unremarkable. Contrast-enhanced computed tomography (CT) was performed to check for abdominal malignancy. Axial and coronal reformatted arterial phase CT images Figure , B, C shSFTs are rare spindle-shaped cell tumors that account for less than 2% of all soft-tissue tumors . They arLess than 100 cases of retroperitoneal SFTs have been reported in the English literature, with a mean size of approximately 9 cm [


Usually, miRNAs function post-transcriptionally, by base-pairing with the 3′UTR of target mRNAs, repressing protein synthesis in the cytoplasm. Furthermore, other regions including gene promoters, as well as coding and 5′UTR regions of mRNAs are able to interact with miRNAs. In recent years, miRNAs have emerged as important regulators of both translational and transcriptional programs. The expression of miRNA genes, similar to protein-coding genes, can be epigenetically regulated, in turn miRNA molecules (named epi-miRs) are able to regulate epigenetic enzymatic machinery. The most recent line of evidence indicates that miRNAs can influence physiological processes, such as embryonic development, cell proliferation, differentiation, and apoptosis as well as pathological processes  through epigenetic mechanisms. Some tumor types show repression of tumor-suppressor epi-miRs resulting in cancer progression and metastasis, hence these molecules have become novel therapeutic targets in the last few years. This review provides information about miRNAs involvement in the various levels of transcription and translation regulation, as well as discusses therapeutic potential of tumor-suppressor epi-miRs used in in vitro and in vivo anti-cancer therapy. Although research into RNA biology has been ongoing for more than two decades, almost each year brings new discoveries. Until recently, it was thought that microRNAs (miRNAs) act mainly in the cytoplasm at the post-transcriptional level. Interestingly, miRNAs can exert regulatory effect both in the cell  in which they are produced and in neighboring cells. The latter intracellular transfer of miRNA is mediated by gap junction channels or exosomes . InteresMature miRNAs (mainly guide strands) form a complex with Argonaut (AGO) proteins called miRNA-induced silencing complex (miRISC) which interact with other proteins including DICER, TRBP, PACT and GW182. The miRNA specific region called ‘seed sequence’  base-pairs with miRNA recognition elements (MREs) located on mRNA targets. miRISC complex binding to 3′UTR leads to mRNA cleavage or mRNA decay and finally translation inhibition. For example, full complementarity of the seed region to MRE induces AGO-2 endonuclease activity resulting in mRNA cleavage and destabilization of AGO2-miRNA interaction that ultimately promotes miRNA degradation . In contAHNAK gene contains a miR-222 target sequence within the coding region hence miR-222 can directly down-regulate its expression [In addition, miRNAs can also bind to target sites in the coding region and inhibit translation. Probably, target sequences in the coding regions are used when the 3′UTR are too short or adjust to protein abundance of alternative splice variants . For exapression . Additiopression .Surprisingly, some studies have reported that miRNAs binding to 3′UTR or 5′UTR regions can up-regulate gene expression by increasing translation rates of proteins.Xenopus  [It was demonstrated that translation activation can depend on the cell cycle state and proteins that are bound to the AGO2-miRNA complex within the 3′UTR. Indeed, in serum starved cells Fragile-X-mental retardation related protein 1 (FXR1) was recruited by the AGO2-miRNA complex associated with AU-rich elements (AREs) at the 3′UTR and activated translation . The miRcomplex) .miR-122 binding to RNA of hepatitis C virus protects the single-stranded 5′ end from cytosolic exonuclease activities (increasing RNA stability against Xrn1) and enhances the recruitment of ribosomes as well as possibly providing a scaffold for binding of other essential factors for translation [miR-10a that interacts with the 5′ terminal oligopyrimidine (5′TOP) motif of ribosomal protein mRNAs and enhance their translation during amino acid starvation [miR-346, produced mostly in brain tissues, binds to 5′UTR of the receptor interacting protein 140 (RIP140) mRNA facilitating its association with the polysomes and finally activating translation via an AGO2-independent manner [Furthermore, it was revealed that miRNAs that bind to the 5′UTR can enhance translation through various mechanisms. For example, liver specific nslation ,16,17,18arvation . Moreovet manner .Surprisingly, studies have shown bidirectional transport of the core components of miRNA silencing machinery. It was demonstrated that several proteins including Importin-8, Importin α/β (Impα/Impβ) and Exportin-1 (XPO1) mediate shuttling of key RISC components from the cytoplasm to the nucleus, moreover, XPO1 is able to transport the nuclear RISC (miRNA-Ago-TNRC6A complex) to the cytoplasm ,22.On the one hand, TNRC6A protein, also known as GW182, can be independently transported into the nucleus binding directly to Impβ and interacting with Impα ,24. On tMoreover, it is proposed that miRNA nuclear localization can also be controlled by nuclear localization signal sequences in miRNA molecules or full processing of pre-miRNAs in the nucleus. Several studies show that various motifs, including AGUGUU-motif, 5′-UUGCAUAGU-3′ and 5′-AGGUUGKSUG-3′ motifs (where K is a uridine or a guanine) as well as the consensus ASUS sequence (where S is a cytosine or a guanidine) are presented in many miRNAs and are engaged in the nuclear translocation ,28,29. Ilet-7f are involved in the transcriptional repression of proliferation-promoting genes regulated by the retinoblastoma (Rb)/E2F repressor complex in senescence [let-7f have been localized in the promoters of two E2F-target genes CDC2 and CDCA8. Similarly, nuclear miR-522 suppresses transcription of CYP2E1 gene by interacting with its promoter forming a DNA:RNA hybrid which probably prevents binding of Pol II and transcription factor [let-7i, miR-138, miR-92a and miR-181d bind to the TATA-box motifs and enhance the promoter activities of interleukin-2, insulin, calcitonin or c-Myc, respectively [miR-744-5p and miR-466d-3p leading to transcriptional upregulation [miR-26a-1, miR-339, miR-3179, miR-24-1 and miR-24-2) are able to induce expression of neighboring genes and function as enhancer (cis-acting DNA elements) regulators [miR-24-1 (located in the enhancer region) increases expression of FBP1 and FANCC genes and triggers direct chromatin state alteration of the FBP1 enhancer that activate transcription. Another notable fact is that transcriptional gene silencing (TGS) and transcriptional gene activation (TGA) can be achieved by miRNA-mediated epigenetic regulation. Indeed, miRNA directs the RNA-induced transcriptional silencing complex (RITS), which consists of chromatin remodeling enzymes  and DNA methyltransferase (DNMT3A), to promoter leading to the transition of active chromatin structure to silent heterochromatin [miR-320 directs to the promoter region AGO-1 that acts as the effector protein for transcriptional silencing of POLR3D gene [POLR3D promoter [miR17-5p and miR20a, encoded within a poly-miRNA cluster miR-17-92, are involved in the acquisition of heterochromatin marks at the promoters through seed-paring manner [miR-223-RISC interaction with the promoter of nuclear factor I-A (NFI-A) results in the recruitment of Polycomb group complex and histone-modifying enzymes that repress transcription of NFI-A, an important step for granulocytic differentiation [miR-10a with AGO-1 and AGO-3 reduces HOX4 expression in human breast cells mediating in de novo DNA methylation and accumulation of repressive chromatin marks  at its promoter [Although the functions of nuclear miRNAs have not been fully elucidated, it is suggested that they can regulate both transcriptional rates and post-transcriptional levels of mRNAs. miRNA-promoter interaction mediated by AGO proteins may either suppress or activate transcription depending on the location of their target region and epigenetic status of the promoter ,30. Genonescence . The putn factor . On the ectively . Also, Cgulation . A recengulators . MoreoveR3D gene . Furtherpromoter . Anotherpromoter .miR-744 and Ccnb1 gene, miRNA-mediated TGA rely on the recruitment of AGO proteins and RNA Pol II enrichment as well as active chromatin marks  at the regulated gene promoters [miR-373 activates transcription of E-cadherin and CSDC2 genes only via enrichment of RNA Pol II at their promoters [miR-205 induces the expression of IL24 and IL32 tumor suppressor genes by targeting specific sites in their promoters as well enrichment of RNA Pol II and active chromatin modifications [In contrast, AGO-miRNA complex may activate the expression of target loci by either disruption of the recruitment of silencing proteins  to lncRNAs (long non-coding RNAs) or recruitment of protein complex containing transcriptional activators  ,41. In tromoters . Moreoveromoters , while mmiR-709 is involved in the post-transcriptional regulation of the pri-miR-15a/miR-16-1, binding to a 19-nt recognition element and preventing processing of primary transcripts, thus, nuclear miRNAs can influence the biogenesis of other miRNAs suggesting hierarchical structures among miRNAs [MALAT1), has two MRE’s which are recognized and bound by miR-9 [miR-675-5p binding site within H19 RNA transcripts has been identified and the overexpression of miR-675-5p significantly downregulated the level of the H19 transcript [XIST), has seed-paring sites for miR-210 which modulates its RNA level [miR-671 directs AGO2-mediated cleavage of a circular antisense transcript of the CDR1 gene and negatively regulates this non-coding antisense transcript [Similar to cytoplasmic miRNAs, nuclear miRNAs can also mediate post-transcriptional gene silencing (PTGS) inducing degradation of target mRNAs. Several studies suggest that miRNAs contribute to the regulation of miRNA precursors and lncRNA transcripts . For insg miRNAs . Furtherg miRNAs . Indeed,by miR-9 . Subsequanscript . So far,NA level . Additioanscript .Pol II elongation, which favors exon inclusion [miRNAs are able to indirectly modulate alternative splicing by regulating translation of various splicing factors. However, mounting evidence suggests that AGO-miRNA complexes can affect the regulation of alternative splicing directly in the nucleus by epigenetic and non-epigenetic mechanisms. A co-immunoprecipitation study has identified multiple AGO-associated splicing factors, moreover, AGO-1, AGO-2 and DICER1 knockdown and overexpression experiments confirmed their involvement in splicing decisions at alternatively spliced exons ,51. Advanclusion . Surprisnclusion .Taken together, the above considerations illustrate the complex regulatory mechanisms of miRNA-mediated gene expression in the cytoplasm and the nucleus. It should be emphasized, that miRNAs are involved in many crucial cellular regulatory processes and may activate or inhibit gene expression at both transcriptional and post-transcriptional level. Thus, deregulation of miRNAs biogenesis and function can disrupt these processes and finally lead to a wide range of human diseases. Hence, miRNAs are valuable as diagnostic and prognostic biomarkers for many diseases, including cancer, diabetes mellitus, cardiovascular pathologies and neurological disorders. Moreover, miRNAs are considered as molecular targets of novel therapies and treatment strategies.Over the past few decades growing evidence has linked epigenetic mechanisms with the regulation of gene expression. Epigenetic markers such as DNA methylation and post-translational modifications of histone tails can rearrange the structure of chromatin leading either to activation or repression of transcription activity . It Recently, a subclass of miRNAs, referred to as epi-miRNAs, that influence the expression of genes encoding epigenetic effector and reader proteins, has been identified . Due to miR-101-1 loci) as well as mutations and chromosomal rearrangements [miR-29 family  act as tumor suppressor miRs in lung cancer and regulate transcript levels of DNMT3A and DNMT3B [miR-29b oligonucleotides, potentiates a hypomethylating effect of DNMT1 inhibitors (decitabine or azacitidine) resulting in better AML response for treatment probably due to the inhibition of other DNMT isoforms that are not efficiently suppressed by these agents [miR-29b mimics inhibit HDAC4 expression in multiple myeloma cell lines, reduce migration potential and increase apoptosis, therefore, this approach could offer a novel targeted therapy [miR-148a combination therapy with either cisplatin or doxorubicin significantly enhanced apoptosis in urothelial cell carcinoma of the bladder cell lines [miR-200b) was more effective than doxorubicin alone, blocking tumor growth and preventing relapse [miR-137 in neuroblastoma tumors [miR-29a and miR-185 in hepatocellular cancer cells [miR-148a in breast cancer cells [To date, several causes have been found that influence the activity of miRNAs, their down-regulation is coupled with epigenetic silencing or genomic abnormalities, such as gene amplification, deletions and microdeletions  or chemically modified poly(nucleic acids), however, cellular uptake of free synthetic miRs are limited because of the ease in which they a degraded in biofluids . In ordese model . Howeverse model ). For exsurvival . DespiteThe discovery of therapeutic epi-miRs potential in cancer therapy makes them attractive candidates for next-generation cancer treatment. It therefore seems likely that profiling of miRs expression and then using appropriate epi-miR-based therapeutics may revolutionize cancer treatment by enabling the reversal of the epigenetic program of tumor cells to a more normal state.miR-34a mimic (drug: MRX34) used in patients with different types of cancer. Double-stranded miR-34a was encapsulated into a liposome-formulated nanoparticle and administered intravenously [miR-16 mimic (drug: MesomiR-1) exhibited hopeful results in patients with pleural mesothelioma and non-small cell lung cancer. Double-stranded miR-16 was delivered by non-living bacterial minicells with a targeting moiety  [However, we realize that turning epi-miR-based therapy into clinical practice faces challenges. Indeed, some clinical trials with miRNA drugs have not always produced satisfactory results. For example, the FDA (Food and Drug Administration) halted phase I clinical trial of venously . Althougr cells) . This isKnowledge in the miRNA field is steadily increasing and recent information about the mechanisms of action, especially their involvement in epigenetic regulation has shed new light on cellular regulatory networks.Interestingly, mature miRNAs are present in both the nucleus and the cytoplasm, therefore they can be involved in the regulation of transcription and translation processes. Nuclear miRNAs can influence gene expression via transcriptional activation or transcriptional gene silencing and shaping alternative splicing, Cytoplasmic miRNAs mainly mediated translation inhibition, however, some miRNAs are capable of activating translation of their target mRNA. A growing body of evidence suggests that miRNAs can act as regulators of the cell epigenome through translation inhibition of proteins engaged in epigenetic control and/or interaction with lncRNA. Considering the pervasive role of miRNAs in numerous biological processes, especially tumorigenesis, better understanding of their role in epigenetic regulation will aid the development of new therapeutic strategies.Currently, miRNA-based treatment approaches for cancer, including tumor-suppressor epi-miRs, are tested in in vitro and in vivo experiments. Although results seem promising further studies will be needed to clarify the safety and effectiveness of epi-miR therapy in clinical practice. We strongly believe that re-introduction of tumor-suppressor epi-miRs will allow for more effective, personalized therapies in the near future.


Coexisting metabolic syndrome (MetS) and renal artery stenosis (RAS) are linked to poor renal outcomes. Mesenchymal stem/stromal cell- (MSC-) derived extracellular vesicles (EVs) from lean animals show superior ability to repair the experimental MetS+RAS kidney compared to EVs from MetS pig MSCs. We hypothesized that MetS leads to selective packaging in porcine EVs of microRNAs capable of targeting mitochondrial genes, interfering with their capacity to repair the MetS+RAS kidney. n = 7 each) were studied after 16 weeks of diet-induced MetS and RAS (MetS+RAS) and MetS+RAS 4 weeks after a single intrarenal delivery of EVs harvested from allogeneic adipose tissue-derived MSCs isolated from Lean or MetS pigs, and Lean or MetS sham controls. Single-kidney blood flow (RBF) and glomerular filtration rate (GFR) were assessed in vivo with multidetector CT, whereas EV microRNA cargo, renal tubular mitochondrial structure and bioenergetics, and renal injury pathways were assessed ex vivo.  Five groups of pigs ( microRNA sequencing revealed 19 dysregulated microRNAs capable of targeting several mitochondrial genes in MetS-EVs versus Lean-EVs. Lean- and MetS-EVs were detected in the stenotic kidney 4 weeks after administration. However, only MetS-EVs failed to improve renal mitochondrial density, structure, and function or attenuate oxidative stress, tubular injury, and fibrosis. Furthermore, Lean-EVs but not MetS-EVs restored RBF and GFR in MetS+RAS.  MetS alters the cargo of mitochondria-related microRNAs in swine MSC-derived EVs, which might impair their capacity to repair the poststenotic kidney in MetS+RAS. These observations may contribute to develop approaches to improve the efficacy of MSC-EVs for patients with MetS. Renal artery stenosis (RAS) is becoming more frequently identified in patients with chronic kidney disease and may affect almost 7% of adults older than 65  and moreOver the last decade, there has been a growing interest in application of cell-based regenerative therapies for renal disease. In particular, adipose tissue-derived mesenchymal stem/stromal cells (MSCs) have demonstrated important regenerative properties in both experimental , 5 and cA significant proportion of patients with RAS presents with metabolic syndrome (MetS), and their coexistence reduces renal clinical benefits and increases progression to dialysis after renal revascularization . MetS caEmerging experimental evidence suggests that mitochondrial abnormalities and dysfunction may be implicated in the pathogenesis of several forms of renal disease, including MetS , 17, RASn = 7 each, We followed the methods of Eirin et al. 2020 . The MayWe isolated EVs from MSCs, which were previously collected from the omental fat tissue from 14 female domestic pigs at the end of the study. Seven pigs (MetS group) were fed a MetS diet consisting of high fat and high fructose  , whereas6) by ultracentrifugation, as we have described before [We isolated EVs from supernatants of MSCs  and downregulated (foldchange < 0.7 and p ≤ 0.05) in MetS-EVs compared to Lean-EVs. Similarly, miRNAs enriched (foldchange > 1.4 and p ≤ 0.05) in MetS-EVs compared to MetS-MSCs were identified. For validation purposes, we randomly selected 4 miRNAs (2 upregulated and 2 downregulated in MetS-EVs versus Lean-EVs) and measured their expression by quantitative polymerase chain reaction (qPCR) using RNA U6 small nuclear 6 pseudogene (RNU6B) as normalization/internal control, a common reference gene in miRNA expression studies [http://www.genecards.org/), a searchable, integrative database that contains detailed information for all annotated genes.We studied the miRNA content of Lean- and MetS-MSC-derived EVs and their parent MSCs using high-throughput miRNA-seq, as we have previously described . We anal studies . We then studies  to ident studies , a softwp values adjusted [https://www.gsea-msigdb.org/gsea/index.jsp).We explored the gene and protein cargo of Lean- and MetS-MSC-derived EVs with high-throughput mRNA sequencing (mRNA-seq) and liquid chromatography-mass spectrometry (LC-MS/MS) proteomic analysis, as we have previously described , 33. We adjusted . Mitochoadjusted  and clasn = 21). Six weeks after diet initiation, animals were anesthetized using intramuscular tiletamine hydrochloride and zolazepam hydrochloride and xylazine . Ketamine (0.2 mg/kg/min IV) and xylazine (0.03 mg/kg/min) were the drugs of choice to maintained anesthesia. Unilateral RAS was induced in all animals by placing a balloon catheter in the right renal artery with the help of fluoroscopy. This catheter was wrapped with an irritant coil that generated gradual narrowing of the renal artery, as well as hypertension in 1-2 weeks [Additional pigs were placed on a MetS diet for 16 weeks  [n = 7 each). The dose of EVs is based on previous studies from our group in swine MetS+RAS which showed that systemic delivery of EVs was not associated with any side effects or complications and exerted important renoprotective effects [n = 7 each) underwent only sham procedures .Six weeks following RAS induction, pigs were anesthetized in a similar manner with Telazol®. We then performed a renal angiography to confirm that a significant degree of stenosis was achieved in RAS pigs. Then, we treated MetS+RAS pigs with a single infusion of allogeneic Lean- or MetS-EVs (1 × 10protein) , 41, or  effects , 23, 41.Four weeks after treatment with Lean- or MetS-EVs, we collected blood samples from the inferior vena cava to measure total and low-density lipoprotein (LDL) cholesterol, triglyceride, fasting glucose, and insulin levels. To calculate insulin resistance, we utilized the homeostasis model assessment of insulin resistance (HOMA-IR) . Then, rOne or two days after later, we euthanized all pigs with intravenous sodium pentobarbital  . The kidRenal hemodynamics and function in the single kidney were assessed with a Somatom Sensation-128 MDCT scanner , as we have previously described –47. We iTo explore EV retention and localization, we stained stenotic kidney sections with the tubular marker cytokeratin , as previously described , 41. LabImmunofluorescence staining with the mitochondrial outer membrane marker preprotein translocases of the outer membrane (TOM)-20  was performed to assess the renal mitochondrial density. We took 15-20 images from random fields and quantified them using ZEN®. The renal mitochondrial structure was assessed using a digital electron microscopy (Phillips CM10) at the Mayo Clinic Electron Microscopy Core. Stenotic kidney sections were preserved in Trump's fixative, mounted on mesh grids, and stained with aqueous uranyl acetate and lead citrate. Ten representative tubular cells from 10 different tubules were randomly selected for analysis. The tubular cell mitochondrial area and matrix density were measured in 10 representative mitochondria in these cells using the National Institutes of Health software ImageJ (Version 1.5) .202) production, cytochrome-c oxidase (COX)-IV activity, and ATP/ADP levels were calculated by colorimetric methods  [The mitochondrial function was assessed in isolated mitochondria  . Hydrogectively) .Production of superoxide anion was assessed in stenotic kidneys by immunofluorescence microscopy using dihydroethidium (DHE) . Tubulart-test with Tukey's post hoc test) and nonparametric  tests as appropriate. Regressions were calculated by the least-squares fit to compare renal mitochondrial damage and poststenotic injury. All tests were two-tailed, and p ≤ 0.05 was considered statistically significant.Statistical analysis was performed using JMP 14.1 . We used the Shapiro–Wilk test to detect deviations from normality. Results were expressed as mean + SD for normally distributed data, but as median (interquartile range) for nonnormally distributed data. We used parametric . MetS pigs also developed hyperlipidemia, reflected in increased total and LDL cholesterol, and triglyceride levels. Although fasting glucose levels were similar among the groups, fasting insulin and HOMA-IR levels were comparably higher in all MetS groups, indicating early prediabetic MetS [The systemic characteristics of all pigs at 16 weeks are presented in tic MetS .We compared the miRNA content of Lean- and MetS-EVs using miRNA sequencing analysis, which identified 11 miRNAs upregulated and 8 miRNAs downregulated in MetS-EVs versus Lean-EVs . qPCR anWe then performed target analysis of miRNAs differentially expressed in MetS-EVs and found that these miRNAs are capable of targeting a total of 433 mitochondria-related genes Table  primarilIn addition, we compared the gene and protein cargo of Lean- and MetS-EVs using mRNA-seq and LC-MS/MS proteomic analysis. mRNA-seq identified a total of 738 mitochondria-related genes, of which 43 were upregulated and 29 downregulated in MetS-EVs versus Lean-EVs . ProteomTo explore whether MetS impaired the reparative capacity of EVs, we treated MetS+RAS pigs with a single injection of Lean- and MetS-EVs. Four weeks after the intraarterial administration, 2-3% of injected both Lean- and MetS EVs were retained in the stenotic kidney. Immunofluorescence cytokeratin staining similarly identified EV clusters in the tubulointerstitium of Lean-EV and MetS-EV-treated kidneys, some of which colocalized with renal tubular cells .Ex vivo studies of EV-treated kidneys showed that the mitochondrial density that decreased in MetS+RAS versus Lean and MetS and increased in MetS+RAS+Lean-EVs failed to improve in MetS+RAS pigs treated with MetS-EVs Figures . Transmi202 in isolated mitochondria that was elevated in MetS compared to Lean and further increased in MetS+RAS decreased in MetS+RAS animals treated with Lean-EVs, but not in those treated with MetS-EVs  correlated with renal tubulointerstitial injury (injury score and fibrosis), whereas mitochondrial energy production (ATP generation) correlated inversely with renal oxidative stress (DHE staining). Therefore, our observations support the premise that renal mitochondrial injury is implicated in the pathogenesis of MetS+RAS and underscore the potential of Lean-EVs to preserve the poststenotic kidney.Mitochondrial structural damage and dysfunction are often accompanied by increased oxidative stress, mainly due to production of superoxide from complexes I and III , and in e stress . Indeed,202 generation, or increase COX-IV activity and ATP production. Moreover, MetS-EVs did not attenuate renal oxidative stress, tubular injury, or tubulointerstitial fibrosis, or improve RBF or GFR, indicating that MetS limited the capacity of MSC-derived EVs to repair the stenotic kidney.Contrarily, MetS-EVs failed to preserve mitochondria or improve the stenotic kidney function. Unlike Lean-EVs, MetS-EVs did not improve the renal mitochondrial density, area, or matrix density, did not decrease mitochondrial HTo explore mechanisms by which MetS failed to improve the mitochondrial structure and function in MetS-EV-treated pigs, we characterized the EV cargo of miRNA, which are important regulators of the mitochondrial function that modulate the expression of mitochondria-related genes . We idenRemarkably, we found that diet-induced MetS also altered the expression of mitochondria-related genes and proteins in swine MSC-derived EVs, which were involved in important mitochondrial functions, such as organization, transport, and oxidation reduction processes. Therefore, MetS-induced changes in the mRNA, miRNA, and protein cargo of EVs could have interfered with the capacity of EVs to preserve renal mitochondria and repair the poststenotic kidney in MetS+RAS.Our study is limited by the short duration of the disease and the use of relatively young pigs. However, our model recapitulates the synergistic interaction between human RAS and MetS, and 16 weeks of high-fat/fructose diet sufficed to alter the miRNA cargo of MSC-derived EVs. The cargo of cultured MSC-derived EVs might differ from the content of EVs secreted by MSCs in vivo. Isolation and culture conditions may affect the behavior of MSCs and their daughter EVs, making their in vivo activity difficult to predict . Our preIn summary, our study shows that MetS modifies the miRNA cargo of swine MSC-derived EVs. miRNA targets include several mitochondrial genes encoding for proteins implicated in mitochondrial energy pathways. Delivery of MetS-EVs failed to improve the renal mitochondrial or poststenotic kidney structure and function in MetS+RAS. Renal mitochondrial damage correlated with renal injury, implying that MetS-induced posttranscriptional regulation of mitochondria-related genes could have partly contributed to impair the in vivo reparative capacity of MetS-EVs. Additional studies are needed to test the therapeutic efficacy of Lean- and MetS-MSC-derived EVs in patients with MetS and RAS.


Pancreatic ductal adenocarcinoma (PDAC), accounting for 90% of all pancreatic tumors, is a highly devastating disease with poor prognosis and rising incidence. The lack of available specific diagnostics tests and the limited treatment opportunities contribute to this pejorative issue. Over the last 10 years, a growing interest pointing towards mesothelin (MSLN) as a promising PDAC-associated antigen has emerged. The limited expression of MSLN in normal tissues  and its overexpression in 80 to 90% of PDAC make it an attractive candidate for therapeutic management of PDAC patients. Moreover, its role in malignant progression related to its involvement in tumor cell proliferation and resistance to chemotherapy has highlighted the relevance of its targeting. Hence, several clinical trials are investigating anti-MSLN efficacy in PDAC. In this review, we provide a general overview of the different roles sustained by MSLN during PDAC progression. Finally, we also summarize the different MSLN-targeted therapies that are currently tested in the clinic. BRCA1, BRCA2, TP53 or CDKN2A genes also represent risk factors for around 5% of PDAC patients [Pancreatic ductal adenocarcinoma (PDAC) is the most prevalent neoplastic disease of the pancreas representing 90% of pancreatic malignancies . PDAC ispatients ,11. Desppatients . The lacpatients . Early-spatients , but unfpatients . Novel spatients ,17,18.Among the relevant targets, accumulating evidences reveal mesothelin (MSLN) as a potential and promising biomarker of PDAC aggressiveness ,20. Impomesothelin gene encodes a 71-kDa precursor protein, processed into a 31-kDa shed form  and a 40-kDa glycosylphosphatidylinositol (GPI)-anchored membrane protein, MSLN. MSLN can also be processed by splicing or cleavage, to generate a truncated soluble form, the Serum Mesothelin Relative Peptide (SMRP) [MSLN gene inactivation did not reveal any developmental, anatomical nor histological abnormalities—no detectable phenotype is thus mirrored by the absence of mesothelin gene [MSLN was identified by Ira Pastan and Mark Willingham thirty years ago ,24. The e (SMRP) ,26. Three (SMRP) . Althouglin gene .MSLN can bind to mucin MUC16 and this interaction mediates cellular adhesion ,30. MUC1MSLN expression has been evidenced by immunohistochemistry or microarray analyses in nearly 40% of solid tumors . InitialLi M et al. were the first to demonstrate that MSLN expression in PDAC was correlated with an increase of tumor volumes, thus suggesting an intense capacity of these tumorigenic cells to proliferate . ThroughMSLN signaling is also involved in the resistance to TNF-α-induced apoptosis through the activation of the Akt/PI3K/NFκB axis in PDAC . In PDACOverexpression of MSLN in 86% of metastatic tissues derived from ovarian cancers suggests its implication in the metastatic process . As aforMSLN is also involved in the metastatic process through its role in epithelial-mesenchymal transition (EMT). EMT is defined by the acquisition of characteristics of invasive mesenchymal cells by epithelial cells . EMT is Finally, MSLN-expressing tumors were also evidenced to be associated with a significant increase of microvascular density in newly formed pancreatic metastatic tissues—this would suggest a new potential role held by MSLN in promoting angiogenesis . HoweverA tight association between resistance to chemotherapy and MSLN expression has been previously established ,59. InhiA MSLN promoter called Canscript, located between −65 to −45 bp before the initiation transcription site of MSLN, has been identified in MSLN-expressing solid tumors . This prThe association between MSLN expression and clinical outcomes of ovarian, lung, breast cancers and PDAC patients has been extensively studied. MSLN and MUC16 co-expression, assessed by immunohistochemistry, was reported to be associated with increased tumor cell invasiveness and metastasis, and poor clinical outcomes of PDAC patients. Indeed, MUC16 high/MSLN high expression group not only displayed significantly reduced OS when compared to the other group, but also decreased disease-free survival, respectively 11.9 versus 22.8 months and 6.7 versus 10.9 months . Of note®) and validated for the detection and management of mesothelioma in clinical trials [The identification of soluble markers and the development of tests to reliably detect them represent major challenges in oncology. Such markers will be useful: (1) to enable early diagnosis, and (2) to evaluate the therapeutic efficacy of anti-cancer agents. Two soluble forms of MSLN have previously been identified: the MPF (32-kDa) and the 40-kDa form SMRP . The dial trials . Unfortul trials .111In)-radiolabeled monoclonal antibody (mAb) in experimental models of ovarian cancer [64Cu- and 89Zr- radiolabeled mAbs [111In- and 89Zr- radiolabeled mAbs for phenotypic imaging of MSLN-expressing PDAC [Some MSLN-targeting radiotracers serve as a companion marker of anti-MSLN therapies that are currently under clinical development. The different anti-MSLN therapies will be detailed in n cancer . Interesled mAbs ,69,70. Cing PDAC ,72. Neveing PDAC ,73.As discussed above, the limited expression of MSLN in normal tissues as well as its overexpression in a broad range of tumors makes MSLN an attractive and promising target for therapy. Hence, antibody-based drugs, vaccines or also CAR-T cells have been developed to target MSLN-expressing tumors . Also, mPseudomonas exotoxin (PE38) resulted in the recombinant immunotoxin (RIT) called SS1P. SS1P is internalized into MSLN-expressing cells and induces apoptosis by inactivation of the elongation factor 2 [SS1 is an anti-MSLN Fragment antigen binding (Fab). The fusion of SS1 to a factor 2 . The antfactor 2 ,77. Basefactor 2 . Among tfactor 2 . In chemfactor 2 . TherefoIn order to limit the formation of neutralizing antibodies, a second strategy relying on the modification of SS1P sequences has also been evaluated. LMB-100/RG7787, a re-engineered version of SS1P combining SS1 Fab with the truncated and deimmunized PE24 moiety, was shown to restrict tumorigenicity both in vitro and in vivo using preclinical models of PDAC . SimilarAmatuximab (or MORAb-009) is a chimeric high-affinity mAb targeting MSLN inhibiting MUC16/MSLN interaction. Amatuximab eradicates tumor cell through antibody-dependent cellular cytotoxicity , and exeBased on this favorable safety profile, a phase II clinical trial investigating the effect of amatuximab in combination with pemetrexed and cisplatin has been conducted in 89 mesothelioma patients. A slight increase of the OS was then observed in comparison to controls . MoreoveSeveral antibody-drug conjugates (ADC) targeting MSLN have been developed. ADC relies on the specificity of an antibody with the cytotoxic potential of a chemotherapeutic agent. For example, anetumab ravtansine or BAY94-9343 is an anti-MSLN antibody coupled to the maytansinoid tubulin inhibitor DM4 via a disulfide-containing linker. Hence, anetumab ravtansine was reported to display promising anti-tumor activity in preclinical models of PDAC by inducing bystander effects . MoreoveSeveral phase I clinical studies investigating the safety of anetumab ravtansine in combination with pembrolizumab (anti-programmed cell death (PD-1)) or atezolizumab, (anti-programmed cell death ligand 1 (PDL-1)) antibodies, are ongoing . In advanced PDAC, a phase II clinical trial with anetumab ravtansine is currently ongoing in the United States (NCT03023722).DMOT4039A is a humanized anti-MSLN mAb fused to monomethyl auristatin (MMAE), an anti-mitotic agent that blocks the polymerization of tubulin. A single-dose of DMOT4039A prevented tumor growth in a dose-dependent manner in murine models of ovarian cancer, PDAC and mesothelioma, as well as in mice bearing patient-derived tumor xenografts . Also, tBMS-986148 is a mAb conjugated to the alkylating agent duocarmycin, leading to the death of MSLN-expressing cells after internalization, and it is currently undergoing a phase I clinical trial . The eva227Th) conjugates thus represent a new and promising class of therapeutics for targeted alpha therapy. A 227Th-radiolabeled mAb directed against MSLN, the BAY2287411, has recently been assessed in combination with DNA damage response inhibitors using experimental ovarian cancers—this combination revealed a synergistic anti-tumor effect [Targeted alpha therapy is a type of targeted radionucleide therapy that employs high-energy emission of short-range alpha particles to favor and induce cell death. The short-range emission of these particles confines its cytotoxic effect in tumor cells that are specifically targeted, thereby limiting damages to the surrounding healthy tissues . Targeter effect . MoreoveListeria monocytogenes bacterial strain ANZ-100 engineered to express MSLN, has been developed. CRS-207 was evaluated in a dose-escalation study in a total of 17 patients including 7 PDAC and revealed that despite its safety profile and ability to induce immune activation, none of the patients displayed a PR [Cancer vaccines are designed to induce tumor-specific immune response. Elizabeth Jaffee’s team was the first to identify MSLN as immunogenic protein, using GVAX (GM-CSF gene-transfected tumor cell vaccine), which originates from irradiated PDAC cells genetically engineered to secrete the granulocyte-macrophage colony-stimulating factor (GM-CSF). GVAX induced a T-cell response against several antigens including MSLN. Enhanced MSLN-specific CD8 T-cell responses were detected in GVAX treated PDAC patients and associated with longer OS . Subsequyed a PR . Interesyed a PR . UnfortuOther MSLN-targeted vaccines are actually under preclinical development such as the Meso-VAX. Meso-VAX enhanced MSLN-specific T cell populations and anti-MSLN antibodies production when administrated in combination with the adeno-associated virus expressing IL-12 . RecentlAlthough cancer vaccines induce an effective anti-tumor T cell response, their use alone is insufficient to inhibit tumor growth. The up-regulation of PD-L1 has been reported in PDAC and the PD1/PD-L1 signaling pathway was shown to hamper the efficiency of anti-tumor immune response . ImportaTM, tisagenlecleucel) has been approved by the Food and Drug administration and the European Medicine Agencies for the management of patients with acute lymphoblastic leukemia [Among immunotherapies, chimeric antigen receptor T cells (CAR-T) therapy has been described as being one of the most promising novel approaches in order to treat cancer. CAR-T cells are engineered T cells that produce an artificial T-receptor targeting a specific protein. The CARs are composed of an extracellular antigen binding domain, a transmembrane domain and an intracellular domain transmitting T cell activation signals. The first-generation of CARs consisted of only one intracellular signaling domain, a CD3z chain . Despiteleukemia . Howeverleukemia .Preclinical studies performed on mouse models of metastatic PDAC demonstrated that MSLN-directed CAR-T cells induced tumor cytotoxicity and could eradicate lung metastases ,111. RegCurrently, the majority of clinical trials that target MSLN-expressing tumors relies on CAR-T cell therapy. Several phase I/II clinical trials are currently ongoing in PDAC patients, aimed at determining the safety, the anti-tumoral effects and the maximum tolerated dose. In a phase I clinical study, 6 patients with metastatic PDAC were intravenously injected with anti-MSLN CAR-T cells twice per week during three weeks, and no side effects or dose-limiting toxicities were reported . MoreoveWhile CAR-T cell approaches appear to be an appealing strategy, serious side effects such as cytokine release syndrome or anaphylactic shock may however decrease their efficiency. To avoid these complications, a pro-apoptotic suicide gene (iCaspase9) has recently been included—such strategy is actually considered as a possible approach to increase anti-MSLN CAR-T cell tolerance . FinallyThe limited physiological expression of MSLN and its overexpression in a broad range of tumors make it an attractive target for therapy in several cancers including PDAC. Despite high membrane-associated MSLN expression in PDAC, soluble MSLN assay failed in validating their diagnostic potential for PDAC. Nuclear imaging agents currently in development will serve as companion markers for MSLN-targeted therapies. While MSLN-targeted therapies displayed acceptable safety profile, their anti-tumoral efficacy is often limited. However, MSLN, in spite of an incompletely resolved mode of action, remains a very attractive target and several clinical trials are currently ongoing and the medical community awaits their final results with much interest. To take this reasoning further, it is increasingly evident that the benefit of single-agent therapies becomes limited and future directions will necessarily involve combined therapies especially with immune checkpoint inhibitors, immune-stimulation as well as radiotherapy or radio-sensitizing agents.


Metastatic colorectal cancers (mCRCs) assigned to the transit-amplifying (TA) CRCAssigner subtype are more sensitive to anti–epidermal growth factor receptor (EGFR) therapy. We evaluated the association between the intratumoral presence of TA signature  and outcomes in CRCs treated with anti-EGFR therapy.The TA-ness classes were defined in a discovery cohort (n = 84) and independently validated in a clinical trial  and other samples using an established NanoString-based gene expression assay. Progression-free survival (PFS), overall survival (OS), and disease control rate (DCR) according to TA-ness classification were assessed by univariate and multivariate analyses.P < .001; validation HR, 0.65; 95% CI, 0.45 to 0.93; P = .018), longer OS , and higher DCR . TA-ness classification and its association with anti-EGFR therapy outcomes were further confirmed using publicly available data (n = 80) from metastatic samples (PFS P < .001) and patient-derived xenografts (P = .042). In an exploratory analysis of 55 patients with RAS/BRAF wild-type and left-sided tumors, TA-high class was significantly associated with longer PFS and trend toward higher response rate .The TA-ness was measured in 772 samples from 712 patients. Patients (treated with anti-EGFR therapy) with TA-high tumors had significantly longer PFS (discovery hazard ratio [HR], 0.40; 95% CI, 0.25 to 0.64; RAS/BRAF wild-type tumors.TA-ness classification is associated with prognosis in patients with mCRC treated with anti-EGFR therapy and may further help understanding the value of sidedness in patients with  Nevertheless, 30%-60% of eligible patients do not benefit from these expensive drugs.4 As a shift from the traditional paradigm of negative molecular selection, we previously demonstrated that the transit-amplifying (TA) CRCAssigner (CRCA) subtype was enriched for cetuximab-responsive tumors,5 a finding independently validated in a clinical study,6 in a panel of CRC xenografts5 and cell lines.7 However, responses were also seen in other groups, such as the poorly differentiated stem-like subtype,7 albeit at a lower frequency. This suggested a scope for refining a previously validated gene-expression–based classifier to assess anti-EGFR therapy response in CRC.Epidermal growth factor receptor (EGFR)-targeting antibodies cetuximab and panitumumab are available treatment options for approximately 40% of patients with metastatic colorectal cancer (mCRC).Key ObjectiveTo evaluate whether the presence of the transit-amplifying (TA) subtype gene signature (dubbed as TA-ness classification) representing the intratumoral transcriptome heterogeneity is associated with anti–epidermal growth factor receptor (EGFR) therapy outcomes.Knowledge GeneratedRAS/BRAF wild-type left-sided tumors (enriched for TA-high) and anti-EGFR therapy benefit.The TA-ness classification is an easily detectable biomarker of intratumoral transcriptome heterogeneity, which was retrospectively evaluated in 712 patient samples, including those from a clinical (CO.20) trial, which showed prognostic significance in patients treated with anti-EGFR therapy. This biomarker provides additional biologic insights for the association between RelevanceRAS/BRAF wild-type left-sided metastatic colorectal cancer who are most likely to benefit from anti-EGFR therapy.With further validation, TA-ness may represent a positive selection biomarker for patients with 5 After asymmetric division, stem cells generate rapidly proliferating TA cells characterized by increased EGFR expression that eventually differentiate into goblet cells and enterocytes.9 We evaluated a hypothesis that tumors with increased TA gene signature expression (irrespective of TA or other subtypes) may be associated with anti-EGFR therapy outcomes. This may capture intratumoral transcriptomic heterogeneity in CRCs with more than one subtype signature coexisting in the same tumor and improve assessment of prognosis and its association with RAS/BRAF wild-type statuses and tumor sidedness in patients with mCRC treated with anti-EGFR therapy.10TA subtype tumors are characterized by gene signatures similar to normal TA cells of the colonic crypt, that is, those in transit between stem cells in the crypt base and differentiated cells at the top of the crypt.NCT02112357) or within the context of a case-control study in Italian institutions . All patients signed an informed consent for translational research and received at least one cycle of anti-EGFR therapy. Nineteen and 12 patients from the RMH cohort were treated before the implementation of KRAS testing (August 2009) and extended RAS testing (December 2011), respectively.12 All patient samples from the PRESSING study had extended RAS/BRAF wild-type tumors.Four independent cohorts of patients with CRC treated with anti-EGFR therapy (n = 315) were examined: one discovery and three validation  cohorts . The disKRAS exon 2 wild-type tumors who had received single-agent cetuximab within the control arm of the CO.20 phase III randomized clinical trial .13 This correlative analysis was approved by the Joint Canadian Cancer Trial Group and Australasian Gastrointestinal Trial Group (CCTG/AGITG) Correlative Sciences and Tumor Biology Committee.One of the clinical validation cohorts included 121 patients with 15 Only samples with known KRAS wild-type status were selected.Two additional public gene expression datasets  of primary CRC samples  and liver mCRC lesions  were evaluated.Formalin-fixed paraffin-embedded (FFPE) tissues were evaluated by a trained pathologist; areas with at least 30% of tumor content were marked on hematoxylin and eosin slides and macrodissected in unstained slides (7- to 10-μm thickness). After deparaffinization, total RNA and DNA were simultaneously isolated using the Ambion RecoverAll kit (discovery) or QIAamp nucleic acid FFPE tissue kit  and quantified with NanoDrop 2000 Spectrophotometer  according to the manufacturer’s instructions. The DNA quantification  was performed using a PICO plate reader and the Qubit dsDNA HS kit (ThermoFisher), with an 8-point reference curve.16 Based on the correlation coefficient values after Pearson correlation analysis between five published CRCA-38 centroids (expression summary of each gene in each subtype)16 and gene expression, each sample was assigned either to TA-high (increased expression of TA signature genes) or TA-low (reduced expression) TA-ness classes. When gene expression profiles were compared with the five CRCA-38 centroids, five correlation coefficients (one for each subtype-centroid) were calculated for each sample. The coefficients were then ranked from highest to lowest; TA-high samples were those with a correlation coefficient value for the TA centroid ranking within the first three highest values; TA-low samples were those with a correlation coefficient for the TA centroid, which is second to last or the lowest. Therefore, the TA-ness classification represents a measure of transcriptome-based intratumoral heterogeneity in mCRC, based on the idea that each sample can contain more than one subtype. This best cut-off for TA-ness classification was established based on the highest accuracy in defining disease control, measured as area under the curve (AUC) of a receiver operating characteristic (ROC) curve (Appendix Thirty-eight published CRCAssigner subtype-specific genes (CRCA-38) were assessed using the NanoString platform  according to a previously validated custom CRC subtype-based gene expression analysis assay.Appendix .P < .05 was used to assess the association between TA-ness classes and percentage of tumor shrinkage (using RECIST) criteria in a subgroup of the discovery cohort. Multivariate analyses were performed for the discovery and the validation cohorts, using Cox proportional hazard regression models with 95% CIs. An ROC curve was built to evaluate the accuracy of TA-ness signature and sidedness in defining anti-EGFR clinical benefit. Although the statistical analysis of discovery cohort was performed by the Institute of Cancer Research statistician, the validation cohort was independently analyzed by CCTG/AGITG investigators blinded to the biomarker cut-off analysis. Additional methods are available in the Data Supplement.Progression-free survival (PFS) was the primary endpoint. Overall survival (OS), disease control rate (DCR), and response rate were secondary endpoints. Kaplan-Meier survival function was used to estimate survival curves followed by log-rank test to analyze differences in survival time. Fisher’s exact test was used to compare categorical variables, and Wilcoxon signed rank test with 16 Moreover, an experiment cohort from 30 patients along with 30 patient-derived xenografts  that were treated with anti-EGFR therapy or vehicle (control) were subjected to the same CRCA gene expression assay. In addition, publicly available gene expression microarray data for 80 patients with mCRC (treated with anti-EGFR therapy) was included as an additional clinical validation cohort.17 This publicly available cohort also served to validate TA-ness classification using a different platform  cohorts after clinical review and quality control of the tumor blocks and tumor-derived RNA . Eighty-Patient characteristics for discovery and validation CO.20 cohorts are shown in the Data Supplement. With the exception of sex, there were no significant differences in patients’ characteristics between the CO.20 subgroup included in this analysis and the overall CO.20 clinical trial cohort (Data Supplement).P = .1; Appendix P < .001) and validation  cohorts ; postprogression treatment information was not available.Using conventional subtyping, 15 of 84 samples belonged to the TA subtype in the discovery cohort. The TA subtype showed a trend toward a longer PFS compared with the other subtypes  and were found in the left side of the colon . The validation CO.20 cohort was completely selected for KRAS wild-type tumors (Data Supplement).In the discovery cohort, TA-high tumors  were predominantly P < .001; 19 in which 30 RAS/BRAF wild-type liver metastases were classified into TA-high (n = 16) and TA-low (n = 14) classes. The percentage of cetuximab-induced tumor volume change in the PDX-based mouse-propagated patient metastatic tumors was significantly associated (P < .042) with the TA-ness signature , the depth of response was associated with the TA-ness classification  in the Khambata-Ford data . To further confirm that the association was independent of the diagnostic sample of origin and to further validate the results, we examined the Khambata-Ford publicly available (microarray) datasetord data . To furtnstrated .RAS/BRAF mutational status, sidedness is a recognized selection factor for anti-EGFR therapy benefit: patients with left-sided tumors benefit more than patients with right-sided tumors.4 However, the biology behind this association remains unclear. First, we further confirmed significant association (P < .001) between TA-ness classification and sidedness in KRAS wild-type primary tumors , high-sensitivity next-generation sequencing RAS/BRAF mutational analysis was available for 118 patients: 71 were classified as RAS/BRAF wild-type, of which 53 were assigned to TA-high (75%) class. The accuracy of the classification (measured as AUC) appeared higher than the accuracy of the sidedness in defining DCR , which warrants additional validation.Beyond RAS/BRAF wild-type and left-sided tumors , the median PFS of TA-high left-sided tumors was significantly longer than that of TA-low left-sided tumors  classification: TA-high and TA-low. This classification has the advantage of providing a qualitative assessment in all the samples, including the non-TA subtypes, overcoming the limitations posed by intratumoral heterogeneity when using the conventional molecular subtyping classification as a potential tool to assess benefit from anti-EGFR therapy. TA-high tumors were significantly and primarily associated with prognosis  in patients treated with anti-EGFR–based therapy in our discovery cohort; this was validated in a KRAS exon 2 wild-type trial cohort of cetuximab-only–treated patients,13 which has the advantage of properly assessing prognostic value in a homogeneously treated population and in the absence of the confounding effect of chemotherapy. The significant prognostic role of TA-high was retained in the RAS/BRAF wild-type and left-sided subgroup. Moreover, TA-low assignment was enriched for RAS/BRAF-mutant tumors, providing a potential alternative method to estimate prognosis and may be a treatment benefit from anti-EGFR therapy when the mutational status is missing. This signature and its association with anti-EGFR treatment outcomes were also confirmed in the publicly available samples from patients with mCRC17 and the preclinical PDX models treated with cetuximab.19 Finally, the TA-ness classification retained prognostic significance when assessed in either archival primary tumors or metastatic samples in multiple cohorts. This is highly clinically relevant, because it means that the classification can be assessed in metastatic lesions when the primary tumor sample is not available or of poor quality; however, intrapatient concordance was not assessed; therefore, additional validation is required.In this study, we explored, for the first time (to our knowledge), a proof-of-concept intratumoral heterogeneity-based transcriptome biomarker of prognosis and potential response in patients treated with anti-EGFR agents along with the clinically established criteria of EREG/AREG, HER2, HER3, EPHA2, or mir-31-3p) and responses to anti-EGFR therapy.20 In contrast, we evaluated a refined form of our previously published gene expression signature (with multiple genes) to identify biologically different CRC subtypes with distinct cellular phenotypes.16 The subtypes summarize a complex network of pathways potentially associated with therapeutic responses, simplifying multiple levels of information derived from heterogeneous samples. Hence, the deployment of subtypes and their signatures, instead of single genes, has the advantage of reducing the dimension of complexity without losing biologic information. Although the CRCA and Consensus Molecular Subtype (CMS) classifications are highly concordant,21 CMS classification was not assessed here because it is technically challenging to dichotomize samples into two groups based on the current CMS classifier (with multiple centroids).Several studies have now evaluated the association between single genes or microRNAs (RAS/BRAF wild-type patient samples. The identification of such patients in the context of clinical trials is challenging; in fact, the negative predictive value of RAS/BRAF mutations was retrospectively demonstrated in multiple clinical trials, and to our knowledge, none of them was designed with an up-front prospective inclusion of extended RAS/BRAF wild-type tumors. Last, this was a proof-of-concept study and was retrospectively designed on preexisting tissue collections in the absence of a control group, limiting the assessment of a TA-ness biomarker as prognostic rather than predictive. In current clinical practice, anti-EGFR therapy is more frequently used in the first-line rather than the chemorefractory setting. Hence, the assessment of the TA-ness in more contemporary first-line trials, including a control arm and with balanced mutational status between arms, is warranted in the future.This study has some limitations. First, the discovery cohort was from two different sources. However, the outcomes were evaluated together as a merged cohort given that these were all patients treated with anti-EGFR therapy within standard practice. Second, there was only a small number of 4 Whether the TA-ness classification can be used as a biomarker to improve patient selection for anti-EGFR therapy benefit in mCRC warrants additional validations in the future.In conclusion, we demonstrated that the detection of the TA-ness classification in primary CRC or mCRC samples shows prognostic significance in patients treated with anti-EGFR therapy and provides an additional biologic explanation for left-sided versus right-sided tumors, which is currently used for the differential anti-EGFR therapy benefit in patients.


Immune-mediated necrotizing myopathy (IMNM) is a rare form of idiopathic immune myopathy (IIM) that requires immunotherapies, including immunosuppressive medications, if severe. There is a paucity of data regarding outcomes of patients with immune-mediated polymyositis who continue immunosuppressive medications during the COVID-19 pandemic. This is the first reported case of COVID-19 in a patient with IMNM. Despite being on two immunotherapies, having risk factors, and having radiographic abnormalities on chest X-ray, the patient had an unremarkable COVID-19 course. He was discharged from the emergency department with a 7-day course of azithromycin and quickly resumed his immunotherapies, but he experienced a flare in his myositis. The 14-week follow-up computed tomography (CT) was negative for residual pneumonitis or fibrosis. More data are needed regarding management and prognosis of patients with connective tissue diseases who become infected with SARS-CoV-2. Immune-mediated necrotizing myopathy (IMNM) is a recently recognized inflammatory myositis characterized by proximal muscle weakness and rare extra-muscular involvement. It comprises 20 percent of the 2–7 per million yearly cases of idiopathic inflammatory myopathies . IMNM caA 54-year-old white man with a history of immune-mediated necrotizing myopathy and obesity (body mass index (BMI) of 35) presented to the emergency department (ED) with five days of fevers (102–104 degrees Fahrenheit), chills, myalgia, and dry cough. His IMNM was diagnosed 1.5 years prior to this ED presentation. The diagnosis was based on rapidly progressive symmetrical proximal muscle weakness; laboratory tests demonstrated an elevated aldolase  and creatine kinase (CK) , a low titer anti-mitochondrial antibody (1 : 80), an anti-SSA 52 Kd of 24 (reference range <20 units), and a muscle biopsy showing pauci-immune myositis. His biopsy demonstrated an upregulation of MHC1, arguing against IMNM; however, desmin, C5b9, TDP43, Cd3/SMA, and CD163/8 immunostaining confirmed scattered muscle fiber necrosis, myophagocytosis, and degenerating-regenerating fibers consistent with IMNM. CD45 and CD68 immunostaining was not performed. The patient's anti-SRP and anti-HMG-CoA reductase antibodies were notably negative, and he had no prior exposure to a statin. His IMNM had been successfully treated with mycophenolate mofetil  and intravenous immunoglobulins .When we were notified of the high fever, we instructed the patient to stop taking MMF . InitialThe ED physician monitored him and, due to the patient's clinical stability, discharged him home with a seven-day course of azithromycin (500 mg day 1 and then 250 mg daily for 6 more days) and CDC instructions for self-quarantine. The rest of his COVID-19 course was unremarkable. He did not have a recorded fever after initiation of azithromycin and his cough and myalgia resolved 3 days afterward. The patient resumed MMF 6 weeks since first symptoms and monthly infusions of IVIG 8 weeks since first symptoms without recurrence of respiratory symptoms. The patient did not resume IVIG immediately due to his reluctance to return to the infusion center since he thought he may have been infected with SARS-CoV-2 during his last IVIG infusion.Unfortunately, his IMNM disease flared  due to wTo our knowledge, this is the first reported case of COVID-19 in IMNM. Despite a relatively benign course, it is notable that he had multiple risk factors for progression to severe COVID: male sex, obesity, immunosuppressive medication, and an abnormal chest X-ray . His cheSince the beginning of the pandemic, multiple studies established that COVID-19 is a truly systemic disorder : pulmonaWe then searched the current literature for any reports of patients with pre-existing IIM and COVID-19. A case series of 4 patients with rheumatological disorders from Malaysia described an 80-year-old female with polymyositis on azathioprine as having a benign course . No addiOngoing discussion regarding the potential of COVID-19 pneumonitis to evolve into fibrotic lung disease  exists, β and IL-6) levels predict progression to lung fibrosis and death. Many survivors have significantly impaired quality of life, exercise capacity, and even CT abnormalities following ARDS [One of the most important clinical manifestations of COVID-19 pneumonitis is acute respiratory distress syndrome (ARDS); although many patients who develop ARDS survive, elevated interleukin (IL-1ing ARDS , 16. TheThe American College of Rheumatology COVID-19 guidelines suggest restarting DMARDs within 7–14 days from resolution of symptoms, or 10–17 days from a positive SARS-CoV-2 RT-PCR test [in vitro and in vivo activity against several viruses [The management of patients' immunotherapies in connective tissue disease during the COVID-19 era is still being investigated. Our patient was on two immunotherapy medications for suppression of his severe IMNM. MMF is a broad immunosuppressant with activity against T- and B-cells making individuals more susceptible to bacterial and viral infections, but it interestingly demonstrates some  viruses . The blo viruses . Not onlA middle-aged obese male with a history of immune-mediated necrotizing myopathy on mycophenolate mofetil and IVIG had a complete recovery from COVID-19 pneumonia despite receiving immunosuppressive medications and risk factors for progression to severe disease. He completed a course of azithromycin and resumed his immunosuppressive medications without COVID-19 symptom recurrence or lung damage. His underlying IMNM flared  due to t


Epigallocatechin-3-gallate (EGCG) is a primary bioactive phytochemical in green tea. Its therapeutic potential in metabolic diseases has been reported; however, the molecular mechanisms of the anti-obesity effect of EGCG have not been fully elucidated. In this study, we examined the effects of EGCG on lipid metabolism and autophagy in adipose tissue. After 8 weeks of high-fat diet feeding, mice were treated with EGCG (20 mg/kg/day) for 2 weeks to test in vivo anti-obesity effects of EGCG. EGCG treatment improved glucose tolerance and caused body weight loss. Interestingly, reduced adipose tissue mass was more prominent in visceral compared to subcutaneous white adipose tissue. Mechanistically, EGCG treatment increased autophagic flux in white adipose tissue through the AMP-activated protein kinase-mediated signaling pathway. Adipocyte-specific knockout of Beclin1 mitigated the effects of EGCG on visceral adipose tissue mass and glucose tolerance, indicating that the anti-obesity effect of EGCG requires Beclin1-dependent autophagy. Collectively, our data demonstrated that EGCG has anti-obesity effects through the upregulation of Beclin1-dependent autophagy and lipid catabolism in white adipose tissue (WAT). Obesity, a worldwide health problem, is defined as abnormal fat accumulation and excess adiposity, and is generally caused by an imbalance between calorie intake and energy expenditure ,2. NotabGreen tea is consumed throughout the world, and its various health benefits have been reported . ResearcAdipose tissue accumulates excessive energy in adipocytes as triglycerides in lipid droplets (LDs) . NegativHere, we looked into the anti-obesity effects of EGCG using a high-fat diet (HFD)-induced obesity mouse model and demonstrated that EGCG treatment increased autophagy in visceral white adipose tissues (WATs). The induction of autophagy by EGCG was accompanied by the AMPK pathway activation. Further, the anti-obesity effects of EGCG treatment required Beclin1-dependent autophagy.Becn1-floxed mice, as previously described [Male C57BL/6 mice were utilized in experiments following approved protocols by the Institutional Animal Care and Use Committees of Yonsei University (IACUC-201803-704-01) and Seoul National University (SNU-200406-1-1). Mice were raised on a 12-h light/12-h dark cycle and given free access to normal chow diet (NCD) and water at 22 ± 1 °C. Mice were acquired from Central Lab Animal Inc. . Adipoq-CreER (B6.129-Tg(Adipoq-cre/Esr1*)1Evdr/J: stock# 024671) mice were obtained from the Jackson Laboratory . Adipocyte-specific Beclin1 knockout (KO) mice were produced by crossing Adipoq-CreER mice and escribed . Cre recHigh-fat diet (HFD)  were fed for 8 weeks. EGCG  dissolved in distilled water were treated for 2 weeks by oral gavage ,18. The ® reagent . The High-capacity cDNA Reverse Transcription kit  was applied for cDNA synthesis, and qPCR was detected using iQ SYBR Green Supermix . Primers for qPCR are described in Quantitative PCR (qPCR) analysis was conducted through a method described previously . BrieflyImmunoblot analysis was conducted through a method described previously . ProteinHematoxylin/eosin (H&E) staining was conducted using paraffin sections, as described previously . For thet-test was used between two groups to measure statistical significance.For statistical analysis, we used Prism 5 software . Data were shown as mean ± SEM. An unpaired An HFD-induced obesity mouse model was applied to validate the anti-obesity effect of EGCG. After feeding of HFD for 8 weeks, EGCG was administrated to C57BL/6 mice under continuous HFD feeding for 2 weeks. EGCG was detected in the mouse plasma samples . As indiBecn1, Atg7 and Atg12 in gWAT were increased by EGCG administration  by the pro-oxidant action of EGCG in various cell types including adipocytes , hepatocA critical finding of our study is that EGCG induced lipid catabolism in a visceral WAT-specific manner. It is well accepted that the accumulation of visceral WAT belongs to the pathogenesis of insulin resistance and diabetes, while subcutaneous WAT is beneficial for insulin sensitivity and to maintain energy homeostasis. In this regard, the waist-to-hip ratio has been used to determine abdominal obesity as a potential indicator of obesity-related serious health conditions. Although we did not investigate molecular mechanisms of the visceral adipose tissue-specific effect of EGCG, knowledge of this mechanism will help in developing strategies to target visceral fat for metabolic health.Modifying effects of EGCG on gut microbiota has been proposed as one of the potential mechanisms of anti-obesity activity of EGCG . MoreoveIn addition, a reduction in postprandial glucose levels by EGCG is associated with inhibition of alpha-amylase . AlthougIn summary, our results showed that EGCG reduced visceral adiposity in a diet-induced obesity mouse model by activating autophagy and lipolysis in WAT through an AMPK-mediated mechanism. Furthermore, the adipocyte-specific Beclin1 KO mouse model demonstrated that the induction of Beclin1-dependent autophagy was required for the anti-obesity effects of EGCG. In this sense, EGCG is a potential candidate for preventing or treating obesity; however, further investigations will be necessary to determinethe molecular mechanisms of EGCG in targeting visceral WAT.


Statins are widely prescribed medications to prevent cardiovascular events such as myocardial infarction and stroke. Both myalgia and myopathy are well-known potential side effects of statins. However, a rare and severe form of statin-induced necrotizing autoimmune myopathy (SINAM) has recently been described and can lead to debilitating weakness, often requiring immunosuppressive therapy. We report a case of a 73-year-old male who made a complete recovery from SINAM following a three-month course of prednisone monotherapy. Myalgia related to statin administration is generally a self-limited side effect. Nevertheless, it is now recognized that a more severe autoimmune myopathy characterized by muscle necrosis can develop in approximately 0.002% of patients treated with statins . As thisA 73-year-old male with a past medical history of complete heart block status post dual-chamber pacemaker insertion and hypertension presented to his primary care physician due to progressive generalized weakness of two-months duration. Physical exam was significant for 3/5 symmetric proximal strength, and initial laboratory workup demonstrated a creatine kinase (CK) level of 8,915 IntUnit/L with an associated mild transaminitis. As the patient had been on atorvastatin 40 mg daily for several years, a preliminary diagnosis of rhabdomyolysis from statin-associated myopathy was presumed, and inpatient admission for intravenous fluid administration was recommended. Following a mild decrease in the CK level, the patient was discharged off statin therapy with recommended outpatient follow up. Due to persistent objective weakness despite the cessation of statin therapy, CK and aldolase levels were rechecked two months later and found to be 1,600 IntUnit/L and 52.2, respectively. After a myositis panel came back benign, an anti-3-hydroxy-3-methyl-glutaryl-coenzyme A (anti-HMG-CoA) reductase antibody was ordered and returned positive at 118 units . As seen in Figure While the primary mechanism behind SINAM remains unknown, statin exposure does lead to increased expression of HMG-CoA reductase. Moreover, binding of statin to 3-hydroxy-3-methyl-glutaryl-coenzyme A (HMG-CoA) reductase can change the confirmation of the protein, which in theory could affect immune system tolerance. Once tolerance is broken, high HMG-CoA reductase levels in regenerating muscle could continue to drive autoimmunity even after statin therapy is discontinued .As the development of an autoimmune myopathy secondary to statin exposure is exceedingly rare, no randomized trials or large enough case series are available on which to base definitive treatment recommendations. However, in a recently published meta-analysis, Nazir et al. found that 84% of reported SINAM cases required two or more immunosuppressants . AlternaAlthough rare, SINAM should be considered in patients with exposure to a statin, presenting with a persistent myopathy, particularly if the offending agent has already been discontinued. Unlike statin-induced myopathy, individuals with SINAM should never be rechallenged with a statin. While this patient made a complete recovery without adding steroid-sparing agents or a second immunosuppressant, large randomized controlled trials comparing different therapeutic regimens are needed to help guide clinical management.


With a lipid-rich environment and elevated oxygen consumption, the central nervous system (CNS) is subject to intricate regulation by lipid-derived electrophiles (LDEs). Investigations into oxidative damage and chronic LDE generation in neural disorders have spurred the development of tools that can detect and catalog the gamut of LDE-adducted proteins. Despite these advances, deconstructing the precise consequences of individual protein-specific LDE modifications remained largely impossible until recently. In this perspective, we first overview emerging toolsets that can decode electrophile-signaling events in a protein/context-specific manner, and how the accumulating mechanistic insights brought about by these tools have begun to offer new means to modulate pathways relevant to multiple sclerosis (MS). By surveying the latest data surrounding the blockbuster MS drug dimethyl fumarate that functions through LDE-signaling-like mechanisms, we further provide a vision for how chemical biology tools probing electrophile signaling may be leveraged toward novel interventions in CNS disease. The abundance of polyunsaturated fatty acids (PUFAs) in neuronal membranes, together with heightened reactive oxygen species (ROS) generation caused by high oxygen consumption, drives LDE production , intracellular LDE dosage is controlled. Control of HaloTag-expression locale and light-exposure time offers spatiotemporal resolution. REX approaches do rely on HaloTag overexpression (with or without POI fusion) and UV-light exposure (1–5 min at 5 mW/cm2); however, potential artifacts are controlled for by a suite of technical controls and RES-sensing-defunct-but-otherwise-functional mutant POIs/constructs  was introduced in mid-2013  is one of the most promising. Its rapid approval followed two large-scale phase-III trials demonstrating the benefit of DMF treatment in relapsing MS patients  in whole blood samples from MS patients at 12 weeks post-DMF treatment MF administration wherein multiple proteins are modified, questions about the validity of these results have been raised. A study using an EAE model designed to mimic the most inflammatory phase of the disease found that Overall, although Nrf2 is clearly activated by DMF, this activation fails to fully account for the therapeutic effects. Ultimately, invoking Nrf2 activation  is somewhat contradictory to the clinically relevant effects of DMF, i.e., immune cell apoptosis -expressing cells, albeit to a lesser extent for the mutant (approximately 5-fold suppression vs. ~12-fold for WT). Therefore, PKCθ is insufficient to fully account for DMF-induced output. Because the study was performed in isolated T cells, the contribution of PKCθ to the MOA of DMF  in vivo remains unsettled.Recent efforts have utilized ABPP to mine the proteome for novel targets of DMF in activated primary human and mouse T cells , leading to enzyme inhibition MF modification of GAPDH to immune modulation  is a G protein-coupled membrane receptor. HCA2 activation downregulates lipolysis , a well-characterized agonist  inhibits RSK2 in a dose-dependent manner . Typical second-order target-adduction rates for approved covalent drugs , has recently been approved by the FDA (US Food and Drug Administration, Functionally sufficient molecular targets and genetically necessary downstream players in DMF’s MOA altogether remain murky. General issues that persist in the LDE signaling field (Parvez et al., All datasets generated for this study are included in the article/supplementary material.Both authors wrote and critically revised the manuscript.US patent application has been filed for the G-REX method. 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.


The number of upregulated genes was higher in the CTC lines obtained after treatment, indicating that they acquired properties to escape treatment pressure. Among these upregulated genes, some are involved in the mTOR and PI3K/AKT signaling pathways. Moreover, cytidine deaminase expression was significantly increased in the CTC lines obtained after failure of the first- and second-line 5-fluorouracile-based treatments, suggesting that these CTCs can eliminate this specific drug and resist to therapy. Several enzymes involved in xenobiotic metabolism also were upregulated after treatment, suggesting the activation of detoxification mechanisms in response to chemotherapy. Finally, the significant higher expression of aldolase B in four of the six CTC lines obtained after treatment withdrawal and cancer progression indicated that these clones originated from liver metastases. In conclusion, these CTC lines generated at different time points during treatment of metastatic colon cancer in a single patient are characterized by the deregulation of different genes that promote (i) drug resistance, (ii) xenobiotic and energy metabolism, and (iii) stem cell properties and plasticity.The characterization of circulating tumor cells (CTCs) holds promises for precision medicine because these cells are an important clinical indicator of treatment efficacy. We established the first and still only nine permanent colon CTC lines from peripheral blood samples of a patient with metastatic colon cancer collected at different time points during treatment and cancer progression. The study objectives were (The online version contains supplementary material available at 10.1186/s12943-021-01326-6. Liquid biopsy-based strategies may transform oncology in the near future . Many stWe established the first nine permanent CTC lines from a 57-year-old patient with metastatic colon cancer (MCC). At diagnosis, he had unresectable widespread MCC with abdominal and mediastinal lymph node invasion and liver metastases. The patient was first treated with the 5-fluorouracile (5-FU)-irinotecan (FOLFIRI) combination and bevacizumab (5 cycles). The first biological progression was observed after the fifth FOLFIRI cycle, and second-line treatment was initiated with 5-FU-oxaliplatin (FOLXFOX) and bevacizumab, but was stopped after the third cycle because of disease progression . The patient died about 6 months after diagnosis due to cancer progression. We derived these CTC lines from blood samples collected before treatment initiation (CTC-MCC-41)  group  ALDOB was strongly expressed in normal liver compared with normal colon, (ii) ALDOB expression was comparable in primary colon adenocarcinoma and normal colon samples, and (iii) ALDOB was upregulated in colon cancer liver metastases compared with primary tumors   drug resistance, with the upregulation of molecules and pathways implicated in drug detoxification (e.g. CDA and the irinotecan/SN38 pathway), (ii) metabolism changes, including upregulation of genes implicated in the xenobiotic metabolism, linked to therapy resistance, and energy metabolism , and the ALDOB gene linked to liver metabolism, and (iii) stemness and plasticity, highlighting their metastasis-competent potential  showed the progressive deregulation of genes involved in cancer aggressiveness hallmarks:  in the CTC-MCC-41, CTC-MCC-41.4, and CTC-MCC-41.5 [ABFG] and [CDE] cell lines. Abbreviations: CTC-BT, CTC line derived before treatment initiation; CTC-AT, CTC lines derived after treatment initiation. Figure S2. RT-qPCR validation of the microarray data. Abbreviations: CTC-BT, CTC line derived before treatment initiation; CTC-AT, CTC lines derived after treatment initiation.