Orthomyxoviridae family. This infectious disease has serious impacts on public health systems and results in considerable mortality and economic costs throughout the world. Based on several experimental studies, massive host immune reaction is associated with the disease severity of influenza. Programmed cell death is typically induced during virus infection as a consequence of host immune reaction to limit virus spread by eliminating niches for virus propagation without causing inflammation. However, in some viral infectious diseases, such as influenza, in the process of immune reaction, aberrant induction of programmed cell death disturbs the maintenance of organ function. Current reports show that there are different types of programmed cell death that vary in terms of molecular mechanisms and/or associations with inflammation. In addition, these novel types of programmed cell death are associated with pathogenesis rather than suppressing virus propagation in the disease course. Here, we review our current understanding of mechanisms of programmed cell death in the pathogenesis of influenza.Influenza is a respiratory disease induced by infection by the influenza virus, which is a member of  The other intracellular PRRs including retinoic acid-inducible gene-I (RIG-I) like molecules, Z-DNA-binding protein 1(ZBP1)/DNA-dependent activator of IFN-regulatory factors (DAI), nucleotide-binding oligomerization domain (NOD)-like molecules, or absent in melanoma 2 (AIM2)-like molecules should recognize the injected and/or de novo vRNP in cytozol to activate intracellular signals promoting several cytokine gene inductions . Among cApoptosis is the most common form of PCD. When cells induce apoptosis, they shrink their cellular shapes and involve their intracellular components  into their cytoplasm to prevent unwanted immune responses.The intracellular mechanism of apoptosis induction is molecularly defined . Most apDuring virus infection, activation of initiator caspases is triggered by several cytokines. These cytokines are called death ligands, which are type-II transmembrane proteins that include tumor necrosis factor (TNF), Fas ligand (FasL) or TNF-related apoptosis-inducing ligand (TRAIL). In the naïve state, death ligands present as homophilic trimers, whereas the specific receptors  present as monomers on cell surfaces. The trimeric complex of death ligands engages monomeric DRs to induce aggregation of the receptors and adaptor proteins that bind to cytoplasmic tail of the receptors. Among the adaptor proteins, Fas-associated protein with death domain (FADD) recruits pro-caspase-8 to its cytoplasmic tail and induces homophilic activation of caspase-8. Activated caspase-8 cleaves and activates its downstream caspases such as effector caspase-3. Caspase-8 also cleaves pro-apoptotic B-cell lymphoma 2 (Bcl2) family proteins, such as BH3 interacting domain death agonist protein (Bid). The cleaved and activated Bid mediates mitochondrial damage and the loss of mitochondrial membrane potential to release cytochrome-c to the cytosol from the interior of mitochondria. The released cytochrome-c binds to apoptotic protease activating factor 1 (Apaf1), an adaptor protein, to form “Apoptosome”, a protein complex that induces auto activation of initiator caspase-9. The activated caspase-9 also cleaves and activates caspase-3 to amplify the apoptotic signal cycle.During influenza A virus infection, functional defects on FasL, a specific ligand of Fas receptor, protect mice. However, deficiency of the FasL gene has almost no effect on virus propagation in mouse lung during lethal influenza virus infection ,32. DuriInduction of TRAIL expression is also linked to influenza pathogenicity. In contrast to FasL, the role of TRAIL is more complex in protecting the host during influenza virus infection. Functional defects in the TRAIL gene in mice cause reduced survival rate during mild influenza virus infection . HoweverNecrosis is often considered as an accidental and unregulated cellular event. However, increasing reports indicate that the phenomenon is also induced by the programmed mechanism in response to several external stimulations such as virus infection. This programmed necrosis is called necroptosis. Necroptosis is an alternative cell death program and is histologically distinguished from apoptosis. When cells induce necroptosis, the cell body bursts and immediately releases intracellular components  that can trigger inflammatory reaction mediated by the innate immune system. The molecular mechanisms that induce necroptosis and their differences compared with those of apoptosis are currently discussed ,40.The induction of necroptosis depends on the formation of necrosomes that consist of at least three kinases: receptor interacting kinase (RIPK)-1 and -3 and mixed lineage kinase domain-like (MLKL) . The intNecroptosis is triggered by several pro-apoptotic stimuli including the death ligands, toll-like receptor agonists or genotoxic stress when caspase activity is blocked . TherefoThe role of necroptosis in host protection against the influenza virus infection is highly controversial. ZBP1/DAI deficiency protects mice during lethal influenza virus infection, although the virus titer is higher in ZBP1/DAI deficient than in wild-type mice . AnotherBased on studies using mice with defects of other necroptosis-related molecules, RIPK3 deficiency leads a high mortality rate in mice infected with influenza virus ,45,46. RTaken together, these studies suggest the importance of necroptosis in regulating influenza virus-mediated cell death in in vitro experiments. DAI-mediated necroptosis also has a crucial role in suppressing virus propagation in vivo.Pyroptosis is a type of cell death that is associated with production of pro-inflammatory cytokines including IL-1β and IL-18 ,48. The Podospora anserina (HET-E) and telomerase-associated protein (TP1) (NACHT), leucine-rich repeats (LRR) and PYD domains-containing protein 3 (NLRP3)) and PRR-adaptor protein to take place. The activated caspase-1 cleaves and converts pro-IL-1β and pro-IL-18 into their mature forms.In cells infected with intracellular microbial pathogens, several microbial components are recognized by cytosolic PRRs, which conserve a caspase activation and recruitment domain (CARD) or a pyrin domain (PYD), such as NOD-like receptor proteins (NLRPs), absent in melanoma (AIM) and pyrin. The CARD-containing PRRs (NLRP1 and NLRC4) directly recruit pro-caspase-1 to their CARDs for aggregation and form inflammasomes to activate caspase-1. Recruitment of apoptosis-associated speck-like protein containing CARD (ASC) is required for formation of inflammasome of PYD-containing PRRs , MHC class II transcription activator (CIITA), incompatibility locus protein from Caspase 1 deficiency reduces the survival rate of mice during non-lethal challenge with influenza virus , whereasActivation of caspase-1 in the hematopoietic but not the stromal compartment recruits several inflammatory leukocytes to the lung during virus infection. These include macrophages, dendritic cells, natural killer cells, neutrophils, and lymphocytes. Recruitment of the cells contributes to the induction of host antiviral immunity  as well In this review, we discuss the molecular mechanisms of the three main types of PCD, and their association with the pathogenicity of the seasonal influenza virus infection in mammals . As descTo control influenza, the first choice for prevention is immunization with influenza vaccines, which induce virus-specific antibodies and cellular responses to eliminate the viruses. These responses depend on antigenicity of the virus, which reduces the effectiveness of this strategy for completely preventing the spread of the virus. This is because the viruses can easily change their antigenicity in response to host antigenic pressures. The genetic diversity of the virus is derived from its RNA-dependent RNA polymerase, which lacks a proofreading system, which DNA polymerase has. This characteristic of the influenza virus (referred to as “antigenic shift”) accumulates point mutations on their genomic sequences and facilitates their escape from host immunological reactions, which consist of antiviral protein-specific antibodies and cytotoxic T lymphocytes. The influenza virus also has another characteristic, which leads its escape from host immune reactions. The viral segment can easily transmit among the different subtypes when they co-infect within the same cell (referred to as “antigenic drift”). This phenomenon can result in rapid and frequent change of the virus serotype .Currently, for therapeutic treatment, several anti-influenza virus reagents such as amantadine have been developed and are used clinically. These reagents are antagonists that selectively block the catalytic pocket of viral proteins, and their specificity depends on the structure of the targeted viral protein . Therefo


Limbic encephalitis associated with antibodies to components of the voltage-gated potassium channel complex (VGKCC-Ab-LE) often leads to hippocampal atrophy and persistent memory impairment. Its long-term impact on regions beyond the hippocampus, and the relationship between brain damage and cognitive outcome, are poorly understood. We investigated the nature of structural and functional brain abnormalities following VGKCC-Ab-LE and its role in residual memory impairment.A cross-sectional group study was conducted. Twenty-four VGKCC-Ab-LE patients  age 63.86 (11.31) years) were recruited post-acutely along with age- and sex-matched healthy controls for neuropsychological assessment, structural MRI and resting-state functional MRI (rs-fMRI). Structural abnormalities were determined using volumetry and voxel-based morphometry; rs-fMRI data were analysed to investigate hippocampal functional connectivity (FC). Associations of memory performance with neuroimaging measures were examined.Patients showed selective memory impairment. Structural analyses revealed focal hippocampal atrophy within the medial temporal lobes, correlative atrophy in the mediodorsal thalamus, and additional volume reduction in the posteromedial cortex. There was no association between regional volumes and memory performance. Instead, patients demonstrated reduced posteromedial cortico-hippocampal and inter-hippocampal FC, which correlated with memory scores . The latter declined as a function of time since the acute illness (r = -0.531).VGKCC-Ab-LE results in persistent isolated memory impairment. Patients have hippocampal atrophy with further reduced mediodorsal thalamic and posteromedial cortical volumes. Crucially, reduced FC of remaining hippocampal tissue correlates more closely with memory function than does regional atrophy. Antibody-mediated limbic encephalitis (LE) is characterised by the subacute onset of amnesia and seizures and is commonly associated with antibodies against components of the voltage-gated potassium channel complex (VGKCC-Ab-LE): leucine-rich glioma-inactivated 1 (LGI1) and contactin-associated protein-like 2 (CASPR2).Nevertheless, the cognitive outcome in LE is difficult to predict and its relationship to brain damage poorly understood. Research so far has focused on the hippocampus, given that this is usually the locus of acute pathology, at least as detected on clinical MRI. Manual delineation of the hippocampus on MRI demonstrates atrophy following VGKCC-Ab-LE.It is less clear whether other areas within broader networks  extra-hippocampal structural damage, (2) reduced FC, and (3) a role of such disruptions in residual memory impairment following VGKCC-Ab-LE.Twenty-four patients were recruited into the University of Oxford’s Memory and Amnesia Project (MAP). Patients were identified from neurology clinics within UK NHS Trusts, presented with clinical features typical of LE,Healthy controls were recruited (1) through local advertisement  and (2) through the Oxford Project To Investigate Memory and Ageing . Patients (mean (SD) age 63.86 (11.31) years) and controls (mean (SD) age 62.36 (12.09) years) were matched for age , and were all fluent speakers of English.Clinical assessment included evaluation of medical records for details of presentation onset, subsequent investigations, and response to treatment. Patients were invited for a research-oriented clinical appointment with a consultant neurologist (CRB) for detailed medical history and evaluation of ongoing clinical and cognitive problems.Patient serum samples were tested for VGKCC antibodies from 125I-adendrotoxin labelled rabbit brain extract by routine immunoprecipitation.Neuropsychological tests were administered to assess premorbid intelligence, semantic memory and language, episodic memory, executive function, visuospatial/motor function and mood . The p v10.1136/jnnp-2018-320168.supp1Supplementary dataAn overall composite memory score was derived from memory tests in which patients showed impaired performance at group level as compared with controls (p-corr <0.05), by averaging the age-scaled, standardised scores across these tests. Composite subscores were also formed separately for verbal/visual recall/recognition memory.A consultant neuroradiologist graded signal intensity, volume, and diffusion of left and right hippocampi in the acute T2-weighted clinical MRIs . AbnormaWe acquired 3D T1-weighted structural MRIs and resting-state Blood Oxygenation Level Dependent (BOLD)-weighted fMRI data .http://www.fmrib.ox.ac.uk/fslAutomated segmentation was conducted for the brainstem, thalamus, caudate nucleus, putamen and pallidum, using FSL FIRST  volume discrepancies between the control and patient groups at a whole-brain level, the T1-weighted images were analysed with VBM , and theFunctional MRI data were preprocessed according to a default pipeline  and wereMultivariate pattern analysis: In order to identify seed regions for post-hoc seed-to-voxel FC analyses in an unbiased, data-driven fashion, we used multivariate pattern analysis (MVPA)  involved thresholding of FC maps at a voxel level of p<0.001 and FWE-corrected (p<0.05) at cluster- or peak-level.Region of interest (ROI)-to-ROI FC analysis: In order to investigate the FC of regions identified in the MVPA with MTL structures with enhanced spatial precision, mean time series were extracted from the unsmoothed data in native space for all manually delineated MTL structures. We compared FC between controls and patients , correcting at seed-level (p-corr) for multiple comparisons .Correlation with memory scores: To investigate whether reduced FC was associated with memory performance, correlation analyses between these FC measures and memory scores were conducted across patients and corrected for multiple testing (p-corr). FC measures were residualised against age, sex, and seed ROI volume in order to ensure that memory impairment was associated with genuine dysconnectivity (and not weaker FC due to fewer voxels in the ROI).Key results on autoantibodies and acute MRI findings are presented in Patients were impaired on visual and verbal recall and verbal recognition memory measures; visual recognition memory was intact  0.74 (0.61); patients: mean (SD) −0.54 (0.83)). While patients scored higher than controls on the depression subscale of the Hospital Anxiety and Depression Scale, no participant scored in the severe range. Their performance on all other tests was no different from that of controls . Memory Patients showed bilateral hippocampal atrophy and reduced thalamic volumes. The rest of their volumes did not differ from control volumes . HippocaTotal hippocampal volumes fell below 1.5 SDs from controls for 14/24 patients . HippocaHowever, composite memory scores did not correlate with patients’ hippocampal or thalamic volumes ; S3, andPatients showed bilateral volume reduction in the hippocampal heads and bodies, the mediodorsal thalamus, and the posterior cingulate-precuneus ; table 4Nevertheless, volumes did not correlate with the overall composite memory scores, irrespective of smoothing or FWE-correction levels . CorrelaWe subsequently examined FC abnormalities across the whole brain (MVPA). Patients differed from controls in the FC of the right hippocampus . In ordePatients’ weaker inter-hippocampal FC correlated with their lower overall composite memory scores , with all three composite subscores showing this correlation . Posteromedial cortico-hippocampal FC also correlated with patients’ memory scores  . Inter-hWe conducted a detailed investigation of neuropsychological performance and structural and functional brain imaging following VGKCC-Ab-LE, in order to identify: (1) extra-hippocampal structural damage; (2) reduced hippocampal FC; (3) the role of those in residual cognitive dysfunction following VGKCC-Ab-LE.We identified bilateral hippocampal atrophy that was highly focal within the MTL, along with correlated volume reduction in the mediodorsal thalamus and precuneus-posterior cingulate, which are connected with the hippocampus within the limbic network.Τhe predominant antigenic target was LGI1. The percentage of patients with hippocampal high T2 signal 83%) was in line with previous reports 71%; 78%3. T% was in Capitalising on the strengths of manually segmented MTL structures and whole-brain VBM, we found bilateral  hippocampal atrophy that was highly focal within the MTL. Our findings are consistent with the well-recognised focal T2 signal abnormalities in the hippocampus in the acute phase,The finding of bilateral volume reduction in the thalamus and the precuneus-posterior cingulate cortex in VGKCC-Ab-LE is novel and deserves further investigation. Several facts point towards the interpretation of such reduction as resulting from Wallerian degeneration through an extended limbic circuitet alOur patients demonstrated amnesia with no deficits in other domains, in contrast to other reports (eg, Heine Patients showed striking preservation of visual recognition memory despite profound impairment of verbal and non-verbal recall and verbal recognition memory. There is a long-standing debate about whether recall and recognition memory rely on dissociable neurocognitive processes.Ever since the first description of patient H.M. 60 years ago,Indeed, we found that it was the FC, and not the volume of the hippocampus, that predicted patients’ memory outcome. Our findings do not contradict the well-established role of the hippocampus in memory. Instead, they highlight the significance of an underappreciated aspect of hippocampal damage in explaining hippocampal amnesia, namely, the abnormal FC of residual hippocampal tissue with broader memory networks. Arguably, haemodynamic properties of residual hippocampal tissue may reflect more accurately the extent to which the damaged hippocampus can interact with broader networks in supporting memory processes than hippocampal volumes derived from structural MRI.et al, Buckner et al,In particular, memory impairment was associated with reduced posteromedial cortico-hippocampal and inter-hippocampal FC. The latter declined as a function of time from symptom onset. Inter-hippocampal FC has been linked to memory performance in young adultset al, Finke et alet al, Miller et alWe acknowledge a number of limitations and future directions that should be taken. Sample size was limited by the rarity of the condition, and larger cohorts may reveal more subtle brain-behaviour relationships. Furthermore, our study did not include disease controls, so we are unable to comment on whether our findings generalise to other types of LE. The 3T field strength prevented us from identifying subfield-specific atrophy, as reported elsewhere.


Background: Alternatives in treatment-strategies exist for resectable gastric cancer. Our aims were: (1) to assess the benefit of perioperative, neoadjuvant and adjuvant treatment-strategies and (2) to determine the optimal adjuvant regimen for gastric cancer treated with curative intent. Methods: PubMed, EMBASE, CENTRAL, and ASCO/ESMO conferences were searched up to August 2017 for randomized-controlled-trials on the curative treatment of resectable gastric cancer. We performed two network-meta-analyses (NMA). NMA-1 compared perioperative, neoadjuvant and adjuvant strategies only if there was a direct comparison. NMA-2 compared different adjuvant chemo(radio)therapy regimens, after curative resection. Overall-survival (OS) and disease-free-survival (DFS) were analyzed using random-effects NMA on the hazard ratio (HR)-scale and calculated as combined HRs and 95% credible intervals (95% CrIs). Results: NMA-1 consisted of 9 direct comparisons between strategies for OS . NMA-2 consisted of 16 direct comparisons between adjuvant chemotherapy/chemoradiotherapy regimens for OS  and 14 for DFS . Compared to taxane-based-perioperative-chemotherapy, surgery-alone  and perioperative-chemotherapy regimens without a taxane  were inferior in OS. After curative-resection, the doublet oxaliplatin-fluoropyrimidine (for one-year) was the most efficacious adjuvant regimen in OS . Conclusions: For resectable gastric cancer, (1) taxane-based perioperative-chemotherapy was the most promising treatment strategy; and (2) adjuvant oxaliplatin-fluoropyrimidine was the most promising regimen after curative resection. More research is warranted to confirm or reproach these findings.  Gastric adenocarcinoma is one of the leading causes of cancer related mortality on a global scale . Even afAfter the landmark MAGIC trial, neoadjuvant and perioperative strategies were more frequently applied to improve overall survival . In periWe conducted a systematic review, using NMA, with two primary aims regarding efficacy: (1) compare the clinical benefit of perioperative, neoadjuvant and adjuvant treatment strategies; and (2) to establish the optimal adjuvant regimen after a curative resection for gastric cancer. Our secondary aim was to investigate the safety of different chemo(radio)therapy regimens.n = 15,795 patients) could be included in any of the analyses . For one study, the HR for OS was extracted from a previously conducted meta-analysis  (discovered during treatment or surgery) were omitted and for NMA-2, studies which only included stage III patients were omitted. To account for potential confounding due to differences in surgical techniques between Asia and Western patients, we conducted two sensitivity analyses for the extend of lymph node dissection (D0/D1 versus D2 or higher) and Asian vs. Western patients. Node-split models were created to assess network consistency (between direct and indirect evidence). In case of inconsistency, baseline characteristics were explored for the corresponding studies. Sensitivity analyses were performed, omitting the studies responsible for network inconsistency one by one.Based on currently available data, taxane-containing perioperative chemotherapy is the most promising treatment strategy for resectable gastric adenocarcinoma. If no neoadjuvant treatment has been given, an oxaliplatin-fluoropyrimidine doublet is the most promising adjuvant regimen after a curative resection for resectable gastric adenocarcinoma. The use of adjuvant oxaliplatin has to be further verified in Western gastric cancer patients. Further research is warranted to confirm or reproach our findings.


MET amplification is rare in treatment-naïve metastatic colorectal cancer (CRC) tumors, but can emerge as a mechanism of resistance to anti-EGFR therapies. Preclinical and clinical data suggest that patients with MET amplified tumors benefit from MET-targeted therapy. Cabozantinib is an inhibitor of multiple tyrosine kinases, included c-MET. Panitumumab is an inhibitor of EGFR. This report describes a patient with KRAS, NRAS, and BRAF wild-type metastatic CRC who experienced disease progression on all standard chemotherapy and anti-EGFR antibody therapy. The patient was enrolled in a clinical trial evaluating the combination of cabozantinib plus panitumumab. After only 6 weeks of treatment, the patient experienced a significant anti-tumor response. Although tumor tissue was negative for MET amplification, molecular profiling of cell-free DNA (cfDNA) revealed MET amplification. This case represents the first report showing the activity of cabozantinib in combination with panitumumab in a patient with metastatic CRC, and suggests that MET amplification in cfDNA may be a biomarker of response. A clinical trial targeting MET amplified metastatic CRC is currently underway. MET proto-oncogene, c-MET is a disulfide-linked glycoprotein consisting of an extracellular α-subunit and a membrane spanning β-subunit , is implicated in tumorigenesis, proliferation, invasiveness, metastasis, and resistance to cancer treatment . Encoded-subunit . Hepatoc-subunit . HGF/c-M-subunit . Aberranfication .MET gene amplification has been observed in multiple tumor types, including colorectal cancer (CRC) (MET amplification is one of the key mechanisms mediating both primary (MET amplification leads to acquired resistance to EGFR tyrosine kinase inhibitors (TKI)s by persistent activation of ERBB3 signaling (MET amplification can be detected with or without the presence of the EGFR T790M “gatekeeper” mutation (MET amplification is low (~3 %) in patients with untreated NSCLC, but increases to 5–22% in patients who develop acquired resistance to EGFR TKI therapy (MET amplification under the selective pressure of anti-EGFR therapy supports the notion that MET amplification is a driver of acquired treatment resistance (er (CRC) , 6, gaster (CRC) , 8, genier (CRC) , head aner (CRC) , non-smaer (CRC) , 12, neuer (CRC) , and ovaer (CRC) , 15. MET primary  and acqu primary  to epideignaling  and MET mutation . The pre therapy , 19, 20.sistance .MET amplification is associated with resistance to anti-EGFR antibodies, including cetuximab and panitumumab. In mice engrafted with MET amplified CRC tumors, treatment with cetuximab is ineffective, suggesting that MET amplification may be responsible for intrinsic resistance to anti-EGFR antibodies  of the tumor tissue biopsies identified MET amplification as one of the most common genomic alterations  of patients with RAS wild-type tumors after exposure to anti-EGFR antibody therapy, but not found at an elevated frequency in anti-EGFR antibody-naïve patients  also supports  therapy . In anotpatients . In addipatients . These fMET amplified patient-derived colorectal cancer xenograft models, MET tyrosine kinase inhibitors (TKIs) reversed resistance to EGFR blockade  for use as monotherapy for metastatic medullary thyroid cancerA 57-year-old male was initially diagnosed with locally advanced rectal cancer T3N1M0) and treated with neoadjuvant chemoradiation followed by surgical resection  biopsy revealed metastatic adenocarcinoma consistent with CRC primary. He received first-line treatment with FOLFIRI plus bevacizumab, but eventually experienced disease progression. He then progressed on a clinical trial combining capecitabine with an investigational therapy, followed by progression on regorafenib.KRAS and NRAS wild-type, he was then treated with anti-EGFR antibody therapy (panitumumab). After 7 months of disease control, imaging revealed a new hypermetabolic LN at the right common iliac chain, and irinotecan was added to panitumumab. This treatment was eventually discontinued due to disease progression. A new biopsy of a mediastinal LN was performed and next generation sequencing (NGS) revealed that the tumor was still KRAS, NRAS, and BRAF wild-type, and there was no evidence of MET amplification  or between different lesions throughout the body , as described previously  in Guardant Health. RN has ownership interest (including patents) in Guardant Health. The remaining 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.


Energy-resolving photon-counting detectors (PCDs) separate photons from a polychromatic X-ray source into a number of separate energy bins. This spectral information from PCDs would allow advancements in X-ray imaging, such as improving image contrast, quantitative imaging, and material identification and characterization. However, aspects like detector spectral distortions and scattered photons from the object can impede these advantages if left unaccounted for. Scattered X-ray photons act as noise in an image and reduce image contrast, thereby significantly hindering PCD utility. In this paper, we explore and outline several important characteristics of spectral X-ray scatter with examples of soft-material imaging (such as cancer imaging in mammography or explosives detection in airport security). Our results showed critical spectral signatures of scattered photons that depend on a few adjustable experimental factors. Additionally, energy bins over a large portion of the spectrum exhibit lower scatter-to-primary ratio in comparison to what would be expected when using a conventional energy-integrating detector. These important findings allow flexible choice of scatter-correction methods and energy-bin utilization when using PCDs. Our findings also propel the development of efficient spectral X-ray scatter correction methods for a wide range of PCD-based applications. Image contrast in transmission X-ray imaging results from attenuation variations of material types within the object. While these attenuation variations are spectrally dependent, conventional energy-integrating detectors (EIDs), widely used in clinical and industrial X-ray imaging, do not capture this important information. Significant developments in the area of energy-resolving photon-counting detectors (PCDs) are happening globally. PCDs developed by CERN Medipix collaboration ,2 are soEnergy-resolving PCDs are direct conversion detectors. Impinging X-ray photons are converted into charge packets in the semiconducting wafer bump-bonded to a virtually pixellated electronic readout. The application-specific integrated circuit (ASIC) consists of an individual readout for each pixel. This includes an electronic discriminator to exclude photons above a given threshold energy value selected by the experimenter. Using efficient energy-calibration methods, the experimenter can accurately link these electronic thresholds to the corresponding photon energy .Detector performance is highly influenced by the semiconductor sensor material. Charge-sharing limitations, such as charge diffusion and X-ray fluorescence, reduce the spectral resolution by falsely attributing a single-incident photon as one or more lower-energy photons. These distortions restrict detector design since influence increases for thicker sensors and smaller pixel sizes. High-Z materials (such as CdTe and CdZnTe ) have deInaccurate spectral-intensity measurements also occur due to photons scattered from the object. The spectral characteristics of scattered X-rays differ from the primary due to the energy transfer and angular deflection of the collision. Maintaining low scatter-to-primary ratios (SPRs) is crucial for spectral imaging where scatter degrades spectral fidelity and hence material identification ,20, inclHere, we report on the spectral characteristics of incoherent X-ray scatter in transmission X-ray imaging using an energy-resolving PCD. We used both Monte Carlo simulations and bench-top experiments to identify key features. As a specific application example, we considered spectral and object parameters relevant to applications such as mammography. Identifying spectral and detector features that vary the SPR can help develop better PCD imaging design and scatter-correction techniques. We use both simulations and experiments to explore these questions.The BEAMnrc software package  was usedIn our study, X-ray photons were incident on a homogeneous PMMA slab with lateral dimensions of 12.4 × 12.4 cmmography . Photoelmography ,27) wereDetection was performed by recording the energy of each X-ray photon that crossed the scoring region. This region was centered on the transmission axis 70 cm from the source with an area of 12.4 × 12.4 cmt is slab thickness. This was compared against the ideal  or knowFinally, an EID detection scheme was approximated by summing photon counts across the spectral distributions with weights corresponding to their respective energies,IEID=∫THkExperiments were conducted on a bench-top X-ray imaging system to validate the simulation results and to gain additional insights. X-rays were generated using a Hamamatsu microfocus X-ray tube unit (L8122-01) consisting of a tungsten anode target and a 200 Contrast loss due to increased scatter is demonstrated with experimentally obtained projections of a breast phantom in As shown in Equation , photonsigations ,29,30,31In comparison to an ideal detector with 100% absorption across the entire spectrum, detectors with Si sensors have poor and significantly decreasing absorption efficiency with increasing photon energies. This results in SPR deviation when using a detector with a thin Si sensor (here, 300 ness see . This isDistinction from the above can be made when considering spectrally resolved imaging with sufficiently narrow energy windows. Referring to Equation , in a spAdditionally, X-ray scatter reduces apparent sample attenuation by increasing photon counts across the detector. The percent error of the estimated attenuation coefficient can be expressed asIt was also observed that attenuation errors due to the presence of scatter see a,b were Increasing the air gap between object and detector is an effective technique to reduce the number of scattered photons from reaching the detector, and to improve the estimated attenuation accuracy (compare top and bottom In conclusion, we illustrated several key spectral characteristics of scattered X-ray photons when using an ideal detector and a silicon detector. These general characteristics can be applied to any PCD. For high-Z sensors like CdTe and CdZnTe, there are additional detector distortions to consider and for which to correct . Our finding that a large number of high-energy bins in PCD measurement exhibit lower SPR than a conventional EID X-ray detector, used clinically and industrially, can offer the means to effectively use these detectors without physical antiscatter grids. The spectral characteristics we reported could also be used for developing design and algorithmic strategies for energy-weighting, and material-decomposition and -characterization methods for which these detectors are being developed.


A study of Mendelian randomization involving 40 studies and 133449 patients hasshown that polymorphism of the gene encoding the IL-6 receptor was related to asignificant reduction in the incidence of coronary events, suggesting a causal role inatherosclerosis.1 seems to be implicated inatherosclerotic plaque instability.4The TNF-alpha, another biomarker of higher expression evidenced in the study by Farsky etal.,5Despite the substantial advance in surgical and percutaneous procedures, as well as inthe clinical therapy involving new antiplatelet, anticoagulant, lipid-lowering,anti-hypertensive and anti-hyperglycemic agents, the residual risk remains elevated andnew anti-inflammatory therapies have been proposed.6 a prospective,randomized, placebo-controlled clinical trial, involving post-myocardial infarctionpatients who maintained elevated high-sensitivity C-reactive protein levels, has shownthat treating inflammation with the monoclonal antibody canakinumab reduced inflammatorymarkers and cardiovascular events during clinical drug treatment.The CANTOS study,1 and suggest that patients receiving baremetal stents might need additional anti-inflammatory therapy. However, new prospectivestudies of efficacy and safety, in addition to lower cost of that therapy, are requiredto its definitive incorporation into clinical practice.7Those data show the relevance of the findings of the study by Farsky et al.


Single-chain polymeric nanoparticles are artificial folded soft nano-objects of ultra-small size which have recently gained prominence in nanoscience and nanotechnology due to their exceptional and sometimes unique properties. This review focuses on the current state of the investigations of click chemistry techniques for highly-efficient single-chain nanoparticle construction. Additionally, recent progress achieved for the use of well-defined single-chain nanoparticles in some promising fields, such as nanomedicine and catalysis, is highlighted. Of the several controlled polymerization methods described in next section, reversible addition fragmentation chain transfer (RAFT) polymerization allows an exquisite control of the molecular weight distribution of the single-chain nanoparticle precursor and is a tolerant technique to monomers having a broad range of functional groups, without involving any metal catalyst. Single-chain nanoparticles (SCNPs) are the smallest unimolecular nano-objects size < 15 nm) that can be prepared starting from a linear polymer chain through 5 nm thatcollapse ,3, enzymcollapse ,5, drug/collapse ,7,8,9, icollapse ,11,12, scollapse , catalytcollapse , and smapolymer functionalization, allowing the decoration of the uniform polymeric precursor chains with appropriate functional groups for the corresponding folding step to collapsed single-chain nanoparticles. Under certain circumstances, the use of this second technique can be avoided through the preparation of well-defined copolymer precursors containing both inert and reactive functional groups distributed along the individual chains . A typical example of polymer functionalization step is the facile and quantitative transformation of the chloromethyl functional groups in poly(4-chloromethyl styrene-co-3-trimethylsilyl-propyn-1-yl methacrylate) copolymers to azidomethyl moieities via azidation with sodium azide [The second technique involved is intrachain folding/collapse being used for the efficient transformation of the precursor coils to folded unimolecular nanoparticles. Obviously, when compared to the long evolution of proteins to their current functional status in living organisms, the field of single polymer folding to functional nanoparticles is still in its infancy. A nice example of highly-efficient folding/collapse method is certainly that based on intrachain click chemistry which has been used for robust, permanent single-chain nanoparticle construction via copper-catalyzed azide alkyne cycloaddition (CuAAC) [The third fundamental technique involved is  (CuAAC) .via CRP in nanoscience and nanotechnology applications [The development of controlled radical polymerization (CRP) processes allowing the facile construction of polymers approaching the polydispersity values of well-defined anionic “living” polymers with controlled architectures  and good tolerance towards the presence of a broad range of monomer functional groups has given rise to an exponential use of specialty polymers synthesized ications . Currentications , atom trications  and nitrications . A commoRecently, ring opening metathesis polymerization (ROMP) using second generation of Grubbs catalysis has gained significant interest as a relatively new controlled polymerization technique . A summavia thiol-ene addition was reported by Serniuk et al. [et al. [et al. [Polymer functionalization aims at the quantitative and selective modification of a given polymer using relatively mild conditions without any side reactions. Polymer functionalization is also known as post-polymerization modification or polymer analogous modification and it has a long history in polymer science . For natk et al.  in 1948. [et al.  on 1963. [et al. ,51,52 invia the above post-polymerization modification reactions is shown in The most efficient and used polymer functionalization reactions are : (1) thiNotably, covalent bonding and non-covalent/dynamic-covalent bonding interactions have been used during controlled folding of linear polymeric precursors to permanent and reversible/stimuli-responsive single-chain nanoparticles as summarized in via click chemistry reactions leading to permanent single-chain nano-objects synthesized by starting from appropriate click chemistry precursors  has evolved into a common coupling technique in organic synthesis, polymer chemistry and materials science. In particular, the click chemistry concept [i.e., applicable under a broad range of conditions with a multitude of starting substrates), (4) modular, (5) to give stable compounds, and (6) to show high yields. Additional requirements for reactions involving one or more polymeric reagents [via click chemistry: (1) intrachain homocoupling, (2) intrachain heterocoupling, and (3) crosslinker-induced collapse.The independent discovery in 2002 by Sharpless et al.  and Meld [et al.  that Hui concept  imposes reagents  to be clet al. [i.e., metal-free C-C click chemistry) involving benzocyclobutene (BCB) functional groups that were activated at very high temperature  and its later removal were demanding procedures that limited their broad use. Further refinements of this technique were performed by Croce et al.  in 2007  [et al.  in 2012 i.e., alkyne homocoupling) by Sanchez-Sanchez et al. [via redox-initiated RAFT polymerization of single-chain nanoparticle precursors containing well-defined amounts of naked acetylenic functional groups available for intrachain metal-catalyzed C-C coupling. Accurate control over the molecular weight, polydispersity and composition of the linear poly(methyl methacrylate-co-propargyl acrylate), poly(MMA-co-PgA), precursor was demonstrated by working up to a maximum polymerization time of 20 h  and a maximum PgA monomer content in the feed of 35 mol%. A very recent breakthrough in intrachain homocoupling for single-chain nanoparticle formation has been the use of Glaser-Hay coupling , was used for the first time for the highly-efficient RT synthesis of bioconjugable poly(methyl methacrylate)-based single-chain nanoparticles by Ruiz de Luzuriaga et al. [Copper-catalyzed azide alkyne cycloaddition, CuAAC  terpolymers synthesized by RAFT polymerization using a one-pot procedure and the continuous addition technique introduced by Harth et al. [et al. [co-4-azidomethyl styrene-co-3-trimethylsilyl-propyn-1-yl methacrylate) terpolymers by a controlled and highly-efficiently azidation step with sodium azide in DMF. By using the intrachain heterocoupling method, bioconjugable polymeric nanoparticles were synthesized from poly single-chain nanoparticles with different intrachain cross-linking degree, showing a broad thermal coil-to-globule phase transition at neutral pH that was shifted to higher temperatures when compared to that of the SCNP precursor.As a result, multifunctional polystyrene-based nanoparticles with diameters in solution of close to 4.2 nm were obtained showing improved rheology behavior (when mixed with elastomeric polymers) and useful fluorescence emission (when aromatic conjugation was maximized). More recently, this method has been employed by Ormategui et al.  to synthvia urea formation from isocyanates and diamines  as schematically depicted in et al. [Facile preparation of single-chain nanoparticles by working under strict absence of water n et al.  in 2009.Nanoparticles with diameters ranging from 8 to 20 nm were synthesized with these materials showing physical properties  fully consistent with a collapsed, three-dimensional structure. More efficient and reproducible collapse was observed when the isocyanate copolymer was added to a solution of the diamine, by maintaing the total chain concentration below 0.1 mM. It was found that only about 75 mol% of the available isocyanate functional groups along the backbone underwent cross-linking with the remaining 25 mol% being available for further reaction with monofunctional amines. A similar volume reduction and cross-linking extend was observed for both styrenic and methacrylate based materials illustrating the efficiency and orthogonality of the urea-based intramolecular cross-linking reaction. Nanoparticle diameter was tuned either by controlling the molecular weight or the mole percentage of isocyanate functional groups.via intrachain CuAAC was demonstrated by Ruiz de Luzuriaga et al. [The high versatility of the cross-linker induced collapse method a et al.  in 2010 N-isopropyl methacrylate) single-chain nanoparticles were synthesized in the 3–20 nm size range by starting with well-defined statistical copolymers with pending Cl- groups along the backbone that were transformed to N3- functional groups by treatment with NaN3 prior to cross-linker induced collapse. The generality of the method was illustrated by synthesizing single-chain nanoparticles of different chemical structure, solubility, functionality, physico-chemical properties and hence potential applications, covering from unconventional polyelectrolytes, melt rheology additives, drug delivery nanocarriers, fluorescent nanoprobes and magnetic resonance image agents [With the assistance of this method, polystyrene, polyacrylate), polymethacrylic acid, poly(sodium styrenesulfonate) and poly were synthesized in a pionnering work by Hamilton et al. [Molecular dendritic transporter nanoparticle vectors based on hydrophilic single-chain nanoparticles post-modified n et al.  in 2009 et al. in 2010 [Facile hydrophobic guest  encapsulation capabilities of biocompatible surface-functionalizable single-chain nanogels showing a diameter of 16 nm was demonstrated by Ryu et al. [Ultra-fine (7.4 nm in size) L-phenylalanine anilide-imprinted polymer single-chain nanoparticles have been synthesized by Njiang et al. [As demonstrated by Tamura et al.  in 2009,3+ ones, enhance water proton relaxivity during image contrast in magnetic resonance imaging, MRI, of body tissues and fluids. Gd3+-containing nanoparticles were reported by Perez-Baena et al. [3+ ions. The relaxivity value of the Gd3+-loaded single-chain nanoparticles on a per Gd basis was 6.78 mM−1 s−1, representing a modest 2-fold increase over a reference commercial low-molecular-weight Gd3+ chelate.Certain stable paramagnetic metal ion complexes, such as Gda et al.  in 2010 et al. [in situ growth of ZnS crystalline nuclei, leading to ZnS quantum dot formation was performed by treating directly Zn2+-containing single-chain nanoparticles with a sodium sulfite solution. The maximum photoluminescence intensity at 362 nm and the corresponding quantum yield were found to increase from 25 to 135 and from 2 to 17%, respectively, upon decreasing the size of the single-chain nanoparticles. Single-chain nanoparticles containing CdS quantum dots were also synthesized by these authors exhibiting bright fluorescence centered at 450 nm with a quantum yield of 45%.Very recently, water soluble single-chain nanoparticles containing ZnS nanocrystals (4.1 nm in size) have been reported by Zhu et al.  by usinget al. [in vivo imaging applications. These single-chain nanoparticles present, after excitation at 350 nm, two maxima located at 391 and 407 nm respectively, in the fluorescence spectrum as well as a small shoulder at 424 nm. Control experiments in which the triazole-benzene-triazole conjugation was disturbed or absent failed to show this fluorescence pattern.As illustrated by Oria et al.  in 2010 et al. [−3 h−1. Recently, chiral nano-objects exhibiting catalytic activity towards carbonyl reductions in water without showing catalyst decomposition or hydrolysis have been reported by Terashima et al. [−1. Following the compartmentalization concept, Huerta et al. [−1 by using only 0.5 mol% of supported L-proline organocatalyst. First report on the use of catalytic single-chain nanoparticles for carbonate hydrolysis was due to Wulff et al. . Solublea et al. . These ca et al.  have repAdditional use of single-chain nanoparticles for different applications has been proposed, such as rheology-improving agents for melts of thermoplastics , elastomDifferent click chemistry methods have been implemented for efficient single-chain nanoparticle construction since the first one developed in 2002 based on Diels-Alder-type reactions taking place at 250 °C . Recent refinement of this click technique allows one to carry out the synthesis at 150 °C that is still far from ideal RT conditions. via intrachain CuAAC  introduced in 2008, and (2) the construction of single-chain nanoparticles at RT under normal oxygen atmosphere via intrachain Glaser-Hay coupling  developed in 2012. The requirement for a severe anhydrous reaction medium clearly limits the scope of the current version of metal-free N-C click chemistry via intrachain bisurea formation for single-chain nanoparticle preparation at RT. Two significant breakthroughs in the field are worth mentioning: (1) the synthesis of single-chain nanoparticles at RT via intrachain click chemistry are expected to occur in the very near future, allowing the use of these versatile, artificial folded nano-objects of ultra-small size in a growing number of emerging applications.Undoubtedly, with the introduction/rediscovery of alternative click chemistry reactions  new advances in single-chain nanoparticle construction


Although acute inflammatory responses are host-protective and generally self-limited, unresolved and delayed resolution of acute inflammation can lead to further tissue damage and chronic inflammation. The mechanism of pain induction under inflammatory conditions has been studied extensively; however, the mechanism of pain resolution is not fully understood. The resolution of inflammation is a biosynthetically active process, involving specialized pro-resolving mediators (SPMs). In particular, maresins (MaRs) are synthesized from docosahexaenoic acid (DHA) by macrophages and have anti-inflammatory and pro-resolving capacities as well as tissue regenerating and pain-relieving properties. A new class of macrophage-derived molecules—MaR conjugates in tissue regeneration (MCTRs)—has been reported to regulate phagocytosis and the repair and regeneration of damaged tissue. Macrophages not only participate in the biosynthesis of SPMs, but also play an important role in phagocytosis. They exhibit different phenotypes categorized as proinflammatory M1-like phenotypes and anti-inflammatory M2 phenotypes that mediate both harmful and protective functions, respectively. However, the signaling mechanisms underlying macrophage functions and phenotypic changes have not yet been fully established. Recent studies report that MaRs help resolve inflammatory pain by enhancing macrophage phagocytosis and shifting cytokine release to the anti-inflammatory M2 phenotypes. Consequently, this review elucidated the characteristics of MaRs and macrophages, focusing on the potent action of MaRs to enhance the M2 macrophage phenotype profiles that possess the ability to alleviate inflammatory pain. Inflammation is an immune response to harmful stimuli, including pathogens, damaged cells, toxic compounds, surgery, or irradiation . InflammInflammatory pain indicates increased mechanical and thermal sensitivity due to inflammatory reactions . These mIn general, the resolution of acute inflammation is an active rather than a passive process that requires the biosynthesis of SPMs including lipoxins (LXs), resolvins (Rvs), protectins (PDs), and maresins (MaRs), derived from the omega-6 fatty acid arachidonic acid (AA) and the omega-3 polyunsaturated fatty acids docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) . SPMs tuMaRs are believed to act as potent protective mediators of macrophage function ,40 and pThe inflammatory response after tissue damage is an important biological process that is essential for the survival of living organisms . When tiWhen the wound healing reaction is well organized and controlled, the inflammatory response is quickly resolved and normal tissue structure is restored . HoweverSPMs are bioactive autacoids enzymatically produced from the omega-6 fatty acids arachidonic acid (AA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA); they include lipoxins (LXs), resolvins (Rvs), protectins (PDs), and maresins (MaRs) generated via the action of lipoxygenases (LOXs) and cytochrome p450 (CYP450) and cyclooxygenase-2 (COX-2) enzymes usually found in different cell types residing in inflammatory environments ,63. AlthThe mechanisms involved in acute inflammatory conditions are critical for the restoration of tissue homeostasis ,20. The MaRs (from macrophage mediator in resolving inflammation) are a fourth family of DHA-derived SPMs . In macrMacrophages also produce a family of bioactive peptide-conjugated mediators called MCTRs . MCTR co13S, 14S-epoxy-DHA(eMaR) stimulates the conversion of the M1 macrophage phenotype to M2 and blocks LTA4 hydrolase . MaR1 poNaïve macrophages are widely distributed in all tissues via circulation through the bloodstream . These cMacrophages participate in the biosynthesis of SPMs with both anti-inflammatory and pro-resolving properties . SPMs arThe level of this potent leukocyte agonist decreases in the later stages of the self-limited inflammatory response . It is pAlthough IL-10 has anti-inflammatory properties, its contribution to the healing process is not fully established. In a recent report, MaR1 increased the levels of IL-10 postoperatively for 14 days . NotablyMacrophages are involved in the processes of homeostasis, tissue repair, and regeneration . They arMaR1 not only regulates the resolution of inflammation, but also plays a powerful role in preventing hyperalgesia sensitivity in inflammatory- and chemotherapy-induced chronic inflammatory pain ,27. MaR1Escherichia coli infection-mediated delays in tissue regeneration of planaria, in addition to protecting mice from second-organ reflow injury and promoting repair by limiting neutrophil infiltration [E. coli infections by increasing bacterial phagocytosis and limiting neutrophil infiltration [In the periphery, various cytokines contribute to neutrophil recruitment into the tissue and, consequently, an increase in inflammatory processes and pain ,110. Intltration . Furtherltration . Phagocyltration . Increasltration ,93. HoweMaRs belong to the most recently uncovered family of anti-inflammatory lipid mediators with pro-resolving activity in the amelioration of inflammation. The activation of MaRs in macrophages enhances phagocytosis and helps to reverse inflammatory pain by shifting cytokine release to an anti-inflammatory state. However, M1/M2 macrophage imbalance, reduced SPM formation, impaired synthesis of DHA, and aging reduce and generally impair the resolution of inflammation under pathological conditions. MaRs have been shown to alleviate this deficiency by reversing and improving the function of macrophages . However


Chemical reporter, HMGAM-yne, facilitates the detection and identification of lysine HMGylation on histones. Lysine 3-hydroxyl-3-methylglutarylation (HMG-K) is a newly identified PTM that can occur non-enzymatically in mitochondria. However, the substrate scope of this new PTM remains insufficiently explored, which has greatly hindered the progress in interpreting its regulatory mechanisms and cellular functions. Here, we report the development of an alkyne-functionalized chemical reporter (HMGAM-yne), for the detection and identification of cellular HMGylated proteins. HMGAM-yne is cell-permeable and metabolically incorporated into proteins in living cells. Subsequent biorthogonal conjugation enables fluorescence visualization and identification of the protein substrates of HMG-K. Using HMGAM-yne, we also identified Sirt5 as an ‘eraser’ that regulates HMGylation in cells. In addition to the known mitochondrial HMG-K proteins, HMGAM-yne facilitates the discovery of multiple nuclear proteins, including histones, as novel substrates of lysine HMGylation. Posttranslational modifications (PTMs) are involved in regulating a variety of biological processes by affecting their substrate proteins' structure, activity, cellular localization and interactions with other biomolecules. Diverse types of PTMs have been discovered, such as acetylation, methylation, phosphorylation and ubiquitinylation.et al. discovered that negatively charged acyl-CoAs with five carbons in their acyl chains, including HMG-CoA, can effectively acylate protein lysine residues by forming highly reactive anhydride intermediates.Lysine 3-hydroxyl-3-methylglutarylation (HMG-K) is a newly identified PTM that can occur non-enzymatically in mitochondria . HirscheBased on our previous work on the chemical reporter for lysine malonylation,tert-butyldimethylsilyl (TBS) ethers 1 (93% yield). The remaining secondary alcohol was then transformed into ketone 2 by mild Swern oxidation (92% yield). For the synthesis of the homopropargyl alcohol derivative 3via the Grignard reaction, propargylmagnesium bromide was prepared in the presence of mercury chloride at 0 °C to avoid its rearrangement into allenylmagnesium bromide. Subsequent dropwise addition of the obtained Grignard reagent to the solution of ketone 2 led to the formation of 3 (80% yield). After the deprotection of the two TBS groups, the resulting diol 4 was converted to its corresponding dinitrile 6via a tosylate intermediate 5 . The following hydrolysis of the dinitrile 6 afforded the dicarboxylate 7 (30% yield), which was further masked by AM esters to give the desired HMGAM-yne (80% yield).The synthesis of HMGAM-yne followed an eight-step route by using glycerol as a starting material . The twoi)-catalyzed azide–alkyne cycloaddition (CuAAC or ‘click’ chemistry) to conjugate the reporter-labeled proteins to a fluorescent dye (rhodamine). The labeled proteins were then resolved by SDS-PAGE and visualized by in-gel fluorescent scanning. As shown in We first examined whether HMGAM-yne could be metabolically incorporated into cellular proteins. To this end, HeLa S3 cells were treated with HMGAM-yne. After metabolic labeling, the whole-cell lysates were subjected to a Cu has been identified as a de-HMGylase.via fluorescence microscopy. As shown in We finally used HMGAM-yne to investigate the cellular distribution of HMGylated proteins. HeLa cells were metabolically labeled with HMGAM-yne. After fixing, the labeled proteins were conjugated with rhodamine-azide for imaging We speculated that the labeled proteins might be histones. By isolating histones from the cells, we indeed observed robust labeling of all the core histones with HMGAM-yne . In addi6H8O4, mass shift: +144.0423 Da) as a variable modification. We indeed identified histone peptides with a mass shift of +144.0423 Da at six lysine sites in five different histone peptides, including histone H2A Lys 119, H3 Lys9, Lys14, Lys18, Lys27, and H4 Lys59  analysis. The acquired MS/MS spectra were analyzed using Maxquant software to identify potential HMGylated lysine residues by setting HMG (CH4 Lys59 . All theWe have developed a chemical reporter, HMGAM-yne, for the study of protein lysine HMGylation. HMGAM-yne allows for efficient metabolic labeling, robust fluorescence detection, and identification of HMGylated proteins. In addition to known HMGylated proteins in mitochondria, we also identified nuclear proteins as novel substrates, including histones. HMGAM-yne-based fluorescence detection also enabled the analysis of the dynamics of protein lysine HMGylation and identification of de-HMGylase, Sirt5. The current study will provide an important alternative method to study protein lysine HMGylation. When combined with methods to inhibit or activate the functions of HMG-regulating enzymes, our probe could be used to determine cellular substrates of de-HMGylases, such as sirtuins. Further identification of HMGylation sites and functional validation of these substrates will contribute to the comprehensive study of protein lysine HMGylation.There are no conflicts to declare.Supplementary informationClick here for additional data file.


Recent advances in the understating of tumor immunology suggest that cancer immunotherapy is an effective treatment against various types of cancer. In particular, the remarkable successes of immune checkpoint-blocking antibodies in clinical settings have encouraged researchers to focus on developing other various immunologic strategies to combat cancer. However, such immunotherapies still face difficulties in controlling malignancy in many patients due to the heterogeneity of both tumors and individual patients. Here, we discuss how tumor-intrinsic cues, tumor environmental metabolites, and host-derived immune cells might impact the efficacy and resistance often seen during immune checkpoint blockade treatment. Furthermore, we introduce biomarkers identified from human and mouse models that predict clinical benefits for immune checkpoint blockers in cancer. New immunotherapies for cancer known as immune checkpoint blockers (ICBs) may help treat many tumor types. Homeostasis of the immune system is controlled by proteins known as immune checkpoints; cancer cells evade the anti-tumor immune response by displaying ligands of the immune checkpoint molecules, thereby incapacitating tumor-killing immune cells. ICBs disable the checkpoints, making cancer cells vulnerable to the immune response. Although ICBs are effective against many cancers, some patients show resistance or relapse. In a review of ICBs, Young-Jun Park and Yeonseok Chung at Seoul National University, South Korea, report that combining two ICBs can improve efficacy, and discuss new pre-, and post-treatment tests to determine whether a drug will work for a particular patient. They also report that manipulating patients’ intestinal microflora can improve response to ICBs. The findings reported may help to optimize these promising treatments. To reproduce this phenomenon, William Coley attempted to inject inactivated or live Streptococcus pyogenes organisms into a patient with neck and tonsil cancer. Due to bacterial-induced inflammation, the patient developed a high fever; however, the tumor burden regressed. Oncologists at the time assumed that the bystander killing effect of inflammation reduced tumor size but disregarded Coley’s approach due to the lack of exact scientific proof and risks concerning the inoculation of infectious organisms. By the 1990s, however, the development of mouse systems of pure genetic background enabled researchers to revisit the cancer immune-surveillance theory and to elucidate how the tumor environment is sculpted by the immune system to eventually either be eliminated or ignored4.Historical records suggest that scientists have sought for over a century to activate and harness the body’s immune response in order to eradicate cancerous cells. It was first attempted in 1891, based on observations regarding the remissions of tumors after surgery in patients with infections5 has encouraged the placement of immunotherapy at the forefront of cancer treatment. CTLA-4 was first known as a member of the immunoglobulin superfamily induced by activated T cells to transmit self-inhibitory signals6. Subsequently, antibodies blocking the programmed cell death protein 1 (PD-1):PD-L1 pathway, now referred to as an immune checkpoint, along with CTLA-4, have demonstrated promising effects in patients for treating more than ten types of cancers, including non-small-cell lung carcinoma (NSCLC) and renal cell carcinoma (RCC)9. Soon after, additional immune checkpoints were discovered, leading researchers to focus on the development of new-generations of immune checkpoint blockers. However, numerous populations of cancer patients remain uncured by these treatments, necessitating novel therapeutic solutions for non-responders.Despite decades of bench-side research, it has only been several years since immunotherapy was allowed to move into the mainstream of cancer therapeutics in the clinic. Recent approvals by the US Food and Drug Administration (FDA) of ipilimumab, a cytotoxic T lymphocyte-associated protein 4 (CTLA-4) blocking antibody, for the treatment of melanomaIn this review, we summarize the current status of immune checkpoint blockers in clinical settings and discuss the efficacy of applying several immune checkpoint-blocking antibodies in combination. We also introduce comprehensive clinical studies that identify biomarkers for distinguishing responsive from non-responsive or resistant cancers in patients treated with immune checkpoint blockers. We also discuss how regulating tumor-autonomous factors is critical for immuno-resistance and how using agents that control tumor-extrinsic factors influence anti-tumor immunity.10. In the tumor environment, however, tumors hijack these inhibitory mechanisms to avoid anti-tumor immune responses.Immune checkpoints consist of various inhibitory pathways that act as homeostatic regulators of the immune system and are crucial for maintaining central/peripheral tolerance as well as reducing excessive systemic inflammation in the body11. CTLA-4 binds CD80 and CD86 with a far higher affinity than CD28, thereby outcompeting for the same ligands and inhibiting TCR signaling12. As a result, CTLA-4-blocking antibodies augment the binding of CD80/86 to CD28 rather than to CTLA-4 and also deplete Tregs in the tumor environment that consistently express CTLA-4 . Together with TCR-mediated signal 1, CD28 ligation with CD80/86 on antigen-presenting cells (APCs) delivers signal 2 to trigger T-cell survival and expansion by inducing interleukin (IL)-2 productionA-4 Fig. 13,14.Fig16. In 2010, a surprising result came from a phase III clinical study of the GP100 peptide vaccine with an anti-CTLA-4 mAb, ipilimumab . Unexpectedly, only ipilimumab-treated patients showed prolonged survival compared with patients treated with the peptide vaccine alone or with the vaccine combined with ipilimumab17. These clinical outcomes allowed the FDA to approve ipilimumab for the treatment of melanoma in humans and led to additional approvals for the treatment of RCC18. Additionally, phase II and III studies showed that anti-CTLA-4 blockade treatment in advanced-melanoma patients resulted in a 22% 3-year survival rate and durable responses extending beyond 10 years19.Once the CTLA-4 signal was shown to restrict the activity of T cells, agents that shut down this signaling molecule became candidates for combination treatment with existing cancer therapeutics21 -γ during therapy, indicating that PD-L1 blockade might have an advantage over the PD-1-blocking antibody in some cases28.PD-1, another member of the inhibitory receptor family, is also expressed on T cells during TCR stimulation. The expression of its ligands, PD-L1 and PD-L2, is regulated by inflammatory cytokines. While PD-L2 is exclusively induced on APCs, PD-L1 is expressed on tumor cells, epithelial cells, and immune cells. These molecules inhibit TCR downstream signaling when ligated with PD-121 Fig. . Two dif+ (exhausted-like) CD8 T cells in the PBMCs of melanoma and lung cancer patients30. Despite such clinical data, it is still yet unknown whether the expansion of ICB-responsive exhausted-like CD8 T cells is driven by the direct therapeutic engagement of peripheral or tumor-infiltrating populations or what functionally distinguishes the ICB-responsive from the non-responsive exhausted-like CD8 T cells. However, chronic viral infection models suggest that it is possible that expansion of specific tumor-infiltrating PD-1+ CD8 T-cell subsets in response to ICB results from the selective expansion of a distinct progenitor CD8 T-cell population in the secondary lymphoid organs31.As to the novel mechanistic explanation for the immune responses that lead to anti-PD-1 blockade-mediated tumor rejection, recent studies have shown that anti-PD-1 therapy leads to a dynamic expansion and proliferation of PD-133. Agents blocking LAG-3 are being tested in clinical trials against multiple cancers.Recent studies have suggested that several proteins expressed on T cells regulate exhaustion. Lymphocyte-activation gene 3 (LAG-3) induced on activated T cells and Tregs binds to MHC class II or galectin-3, which transduces an inhibitory signal in T cells or enhances the suppressive activity of Tregs34. It was first demonstrated that TIM-3 controls T-cell unresponsiveness in chronic inflammation. Upon interaction with galectin-9 or other undefined ligands, TIM-3-expressing T cells undergo apoptosis and lose effector functions35. Further analysis showed that TIM-3 expression was upregulated in tumor-infiltrating lymphocytes (TILs) in melanoma and NSCLC patients37.T-cell immunoglobulin and mucin domain-containing 3  is another cell surface molecule involved in T-cell exhaustion38, and is a phenotypic marker of inert CD8 T cells or mediates Treg suppression of other effector cells40. As such, blockade of TIGIT might be considered as an attractive target in conjunction with the CTLA-4/PD-1 pathways. The current status of the development and approval of immunotherapeutic agents is listed in Table T-cell immune receptor with immunoglobulin and ITIM domain (TIGIT) is also implicated in the inhibition of T-cell activation. TIGIT expression is tightly regulated in lymphocytes, especially in Tregs and CD8 T cells41. Notably, this study demonstrated that 28% of patients with clinical benefits showed 80% or greater tumor regression24. In a sequential phase II study, 61% of the patients who received the combination therapy exhibited objective responses compared to the 11% of those treated with ipilimumab alone42. Although not statically significant in comparison with the PD-1 single blockade group (nivolumab), the progression-free survival (PFS) rate was clearly prolonged in the combination group compared to that of either monotherapy group 44. Up-to-date clinically tested combination therapies and ICB development time-lines are summarized in Table 47.To improve therapeutic efficacy, a number of preclinical and clinical studies have been conducted to examine if combination treatment of immune checkpoint blockers with conventional treatments, such as chemotherapies, targeted therapies, radiation therapies, and other immunotherapeutic agents, could improve outcomes. So far, the most favorable prognosis has been the combination of CTLA-4 and PD-1-blocking antibodies. In the first clinical study performed to determine the antibody combination dose, objective response rates were exhibited in over 40% of patients across all doses50. This unresponsiveness to ICBs can be identified in two types of patients: patients who did not respond at all (primary resistance), and patients who relapsed after a partial response to ICBs (acquired resistance)51. These non-responder patients endure high treatment costs and toxicities with little benefit from the treatments. To sustain the success that ICBs have achieved in the treatment of various tumors in clinical settings, specific prognostic indicators should be identified to predict whether a patient would be rescued by ICB treatment . Non-responders were treated with pembrolizumab, and biopsies were obtained at early and late stages of treatment. Among those samples, analysis of biopsies collected immediately after CTLA-4 or PD-1 blockade treatment provided highly correlated predictors for response rates. In an immunological assay, the density of CD8+, CD4+, CD3+CD45RO+ PD-1+ or PD-L1+ cells was higher in responders compared to non-responders. The proximity between CD68+ myeloid cells and CD8+ T cells was also higher in responders, though not significantly. In addition, a recent study has demonstrated that peripheral blood mononuclear cell (PBMC) immuno-profiling could predict response rates in melanoma patients treated with nivolumab or pembrolizumab. Using high-dimensional single-cell mass cytometry, the authors searched for differential signatures in PBMCs from responders vs. non-responders54. Among alterations, the increase in classical monocyte (CD14+CD16−CD33+HLA-DR+) frequency was the most prominent in responders, indicating that biomarkers from blood sample collections, rather than those from invasive tumor samples, can be used in clinical practice  in some tumor compartments55. In 2014, whole-exome sequencing analysis on tumor samples from ipilimumab or tremelimumab-treated patients identified that the mutational load was directly correlated with a clinical benefit in advanced-melanoma patients56. Of note, the neoantigen landscape was also elucidated in patients with a strong response, and the peptides derived from these neoantigens activated T cells from patients ex vivo. Furthermore, a similar tendency in the circumstances of PD-1 blockade was identified. NSCLC patients treated with pembrolizumab were analyzed for tumor DNA sequencing57. The patients with significantly enhanced clinical efficacy were imprinted with an elevated nonsynonymous mutation load. Among them, the signatures related to smoking-induced mutation, neoantigen burdens, and DNA repair mutation were linked to a favorable clinical efficacy.Given that the magnificent efficacy of immunotherapy was demonstrated in human studies, cytotoxic T cells work properly when they recognize antigen loaded onto MHC molecules on the surface of tumor cells. Because T cells are selected to maintain central tolerance in the thymus, these antigens would be the mutated forms of self-antigens, so-called neoantigens, created by tumor-specific chromosomal alterations and viral genomic substances. In this context, the efficacy of ICBs has been markedly outstanding in patients with highly mutated tumor burdens58. This microsatellite instability (MSI) is associated with the response of ICBs. In a phase 2 clinical study for pembrolizumab-treated patients with progressive metastatic colorectal carcinoma, the rate of the objective response was higher in patients with mismatch repair-deficient tumors than in patients with mismatch repair-proficient tumors59. A total of 1782 somatic mutations were detected in tumors with a mismatch repair deficiency, while only 73 mutations were identified in mismatch repair-proficient tumors, and higher mutation loads were associated with longer progression-free survival. Furthermore, a study indicated that not only colorectal cancer but also 12 different types of tumors harboring MMR deficiency were sensitive to PD-1 blockade60. Interestingly, there are several patients with high mutation loads who do not respond to ICBs62. This might be the consequence of intratumor heterogeneity, as multiregional genetic analysis of tumors from metastatic RCC patients revealed that spatially separated tumors exhibited discordant somatic mutation patterns63. In NSCLC and melanoma patients, sensitivity to pembrolizumab and ipilimumab was apparent in patients with high neoantigen loads and low heterogeneity in their tumor64, indicating that tumor clonal variability should be considered as a biomarker for the ICBs-responder prediction  proteins significantly enhanced the error rate in tumor cells, which can result in abnormal DNA microsatellites66, suggesting that these characteristics could be utilized as indicators to predict response rates. In contrast, the expression of a few genes, such as vascular endothelial growth factor (VEGF), was lower in responders in comparison with non-responders, arguing that the resistance mechanism could be a target for combination therapy, as other have suggested68. In a consecutive study, whole-exome sequencing was performed on the biopsies from the same cohort of metastatic melanoma patients treated with sequential ICBs to complement the previous study69. In patients resistant to single or double ICBs, there was a high burden of copy number loss in chromosomes in which various tumor suppressor genes  were located. Although more efforts are needed to expand the cohort size and to broaden these criteria, this study provides an important guide on how to manage the ICB regimen. The inspection of biopsies obtained at each time point provides several mechanisms by which the efficacy of ICBs is restricted  exhibited a favorable response to PD-1 blockade. To clarify the causality between ICBs efficacy and specific commensal species, germ-free mice were recolonized with the bacteria isolated from patient stool using fecal microbiota transplantation (FMS). Mice transplanted with stool from responders exhibited significant regression of tumor growth compared to those receiving stool from non-responders. In addition, microbiota composition in melanoma patients treated with ipilimumab was analyzed to distinguish specific bacteria associated with the efficacy and adverse effects, such as colitis75. This study revealed that the Faecalibacterium genus and other Firmicutes were responsible for the prolonged survival of patients and the occurrence of ICB-mediated-colitis. Although more investigations should be performed to confirm the relation between the microbiota and the clinical response, oncologists may need to consider the proper use of anti- or probiotics before and during ICB treatments to maximize the therapeutic effect . Then, inflammatory stimuli activate DCs to migrate into adjacent lymph nodes in which T cells are educated by DCs presenting tumor antigens. The differentiation of tumor-specific T cells into effector T cells crucially contributes to tumor immunosurveillance. The interaction between CD28 and CD80/86 is indispensable for fully activating T-cell functions, while PD-1 and CTLA-4 blunt their ligation. Activated tumor-specific T cells differentiate into effector cells and then home to the tumor tissues attracted by chemokines and attack tumor cells expressing antigens loaded onto MHC molecules. Though ICBs potentiate T-cell activation, the other steps of immune-induced tumor-killing should be operated precisely to control tumor growth. In the following sections, we introduce the mechanisms of how both tumor-intrinsic and -extrinsic factors regulate immune activation cycles to restrict ICB efficacy.77. IPRES (Innate anti-PD-1 Resistance Signatures) describe a set of genes inherent to the patient that have been attributed to the primary mechanism of resistance to PD-1 blockade66. Comparative transcriptome analysis between melanoma and pancreatic patients who do and do not respond to PD-1 blockade revealed that non-responders showed an enrichment of genes associated with mesenchymal transition, wound healing, and angiogenesis66. In another aspect, other genomic indicators of poor immunogenicity, such as epigenetic downregulation of chemokines (lower T-cell recruitment), upregulation of endothelin receptors , MHC downregulation and low neoantigen load (lower antigen exposure), and impaired DC function (lower antigen presentation), have been described and attributed to what is popularly termed “cold” tumor types79. Therapeutic interventions to improve the responsiveness of these “cold tumors” to ICB are being investigated with various combinational therapies in clinical trials.Approximately 40 to 60% of melanoma patients treated with nivolumab, as well as over 70% of patients treated with ipilimumab, show primary resistance80. Loss of function mutations in Janus kinases JAK1 and JAK2 were observed in patients who exhibited no-response to pembrolizumab81  as well as in inducing MHC expression on tumor cells, whereas chronic IFN-γ exposure induces further mutations in tumor cells and leads to PD-L1 upregulation84.As mentioned above, tumors with higher initial mutational burdens appeared to be positively correlated with therapeutic outcomes. However, the mutations in tumor cells do not always guarantee a favorable response to ICBs, as intratumoral heterogeneity and mutations that might be advantageous for immune-escape and survival can be another manifestation of the high mutational burdenb81 Fig. . IFN-γ r+ DCs in tumor tissues. β-catenin suppressed CCL4 secretion, which is important for the recruitment of T cells/DCs into the tumor bed  tumor suppressor loss has also been attributed to the resistance to PD-1 blockade in a mouse tumor model. The PI3K pathway was activated in the absence of PTEN, promoting immunosuppressive CCL2 and VEGF secretion. Tumor growth significantly regressed in the ICBs and PI3K inhibitor co-treatment group89  to catalyze tryptophan into kynurenine, which induces T-cell dysfunction due to the deficiency of essential amino acid97. Furthermore, IDO enhances MDSCs and Treg cell recruitment into the tumor microenvironment99. IDO inhibitors have been shown to potentiate anti-tumor effects in combination with immune checkpoint blockers100 and are on the brink of being approved in clinical trials. Adenosine, also abundant in the tumor environment, inhibits effector functions of NK cells/CD8 T cells and induces suppressive M2 macrophage differentiation and myeloid-derived suppressor cell (MDSC) accumulation.It is now commonly accepted that cancer cells constitute suppressive networks with surrounding stromal cells and immune cells103. Two ectonucleotidases, CD39 and CD73, have critical roles in generating adenosine. In inflammation, ATP released from cancer or immune cells is converted by CD39 into AMP, which CD73 then metabolizes into adenosine104  of metastatic melanoma patients, MDSCs were higher in non-responders compared with responders during ipilimumab therapy, indicating discriminative biomarkers for the ICB response107. Recently, an attempt to overcome MDSC suppression by targeting the gamma isoform of phosphoinositide 3-kinase (PI3Kγ) has been introduced108. A PI3Kγ inhibitor converted MDSCs into immunogenic myeloid cells, and subsequent tumor-specific CTL responses were enhanced in combination with ICBs. Other myeloid compartments, such as tumor-associated macrophages (TAMs) and neutrophils (TANs), have been shown to contribute to ICB resistance in distinct ways. TAMs have been primarily known to induce angiogenesis and enhance tumor survival via immunosuppressive factors and degradative proteins, thus facilitating therapeutic resistance109. Furthermore, recent in vivo imaging analysis revealed that PD-1- macrophages engulf anti-PD-1 antibodies that bind to PD-1+ CD8+ T cells in a FcγR-dependent manner to disrupt PD-1 efficacy110.Accumulating evidence indicates that MDSC infiltration in tumor tissues is strongly attributed to poor prognosis in ICB-treated patients due to the suppressive activity of the tumor-specific T-cell response111. In a mouse model of KRAS-mutant lung tumor, pro-inflammatory IL-17A cytokine secretion has been shown to recruit neutrophils to the tumor sites, and anti-Ly6G depletion of neutrophils was found to be more effective than PD-1 blockade in treatment of the tumor112. Positive correlation between KRAS mutation and IL-17 levels was also found in lung cancer patients112. In an analysis of 720 advanced-melanoma patients treated with ipilimumab, a higher neutrophil baseline and neutrophil-to-lymphocyte ratio in the blood were found to be significantly associated with lower survival rates113. Taken together, TANs seem to contribute to resistance against ICBs.TANs are known to also release secretory factors, such as MMP2 and hepatocyte growth factor (HGF), to induce ECM remodeling and recruit other immunosuppressive immune cells that aid in cancer immune-escape+ T-cell infiltration into the tumor environment114. Combination of ICBs with TGF-β blockers proved to be synergistic in tumor mouse modelsTGF-β is another powerful negative regulator of effector T cells that is actively exploited by various compartments of the TME. A recent study of patients with metastatic bladder cancer who did not respond to atezolizumab therapy has shown that tumor-associated fibroblast- and collagen-rich extracellular matrices upregulate TGF-β expression, which could inhibit CD8115. In a mouse model, therapeutic sensitivity to PD-1 blockade was restored in combination with a TIM-3-blocking antibody injection  were greater than those observed in gene expression , suggestive of a large rewiring of accessible chromatin networks associated with the exhaustion state. Thus, co-treatment with epigenetic and/or metabolic modulators has been proposed to overcome the inherent limitations of ICBs120.On the other hand, epigenetic studies have revealed that although ICBs do lead to transcriptional reprogramming within the exhausted T-cell pool, they induce minimal development in genes associated with memory T-cell generationThe cancer field in the past few years has witnessed a great advancement in the comprehension of immune-surveillance in the tumor environment. Along the way, researchers have assumed that the regulation of immune checkpoint pathways might have an impact on anti-tumor immunity, thereby leading to the successful administration of CTLA-4/PD-1-blocking antibodies to treat many types of cancers. However, only a minority of patients’ tumors were reduced by this treatment due to tumor-intrinsic or -extrinsic resistance mechanisms, necessitating a clarification of genomic, epigenomic, transcriptomic, as well as cellular features linked with tumor response and resistance. Using novel molecular and diagnostic technologies, the resistance mechanisms should be fully addressed to offer custom-made therapies or optimal therapeutic combination to potentiate clinical responses for patients with a variety of tumors.122, therapeutic vaccines that induce immune responses to tumor-specific antigen124, and agents that remove suppressive myeloid cells 126.In other aspects, several attempts have been made to stimulate anti-tumor activity and/or to eliminate immune-threatening suppressors: the development of agonistic antibodies against costimulatory receptors 71. Considering the conditions of cellular, genomic, and microbial status within individual patients, strategies mentioned above will be integrated optimally to augment the therapeutic effect, which could, in the near future, save the lives of patients suffering from uncontrolled malignancies.Importantly, recent breakthrough discoveries have demonstrated that some species of microbiota could manipulate anti-tumor immune responses to give rise to favorable clinical responses through the induction of DC maturation and a Th1 response, indicating that probiotics or antibiotics-mediated microbial rearrangement perhaps stimulate effective immune responses


Checkpoint inhibitor therapy has introduced a revolution in contemporary anticancer therapy. It has led to dramatic improvements in patient outcomes and has spawned tremendous research into novel immunomodulatory agents and combination therapy that has changed the trajectory of cancer care. However, clinical benefit in patients with colorectal cancer has been generally limited to tumors with loss of mismatch repair function and those with specific germline mutations in the DNA polymerase gene. Unfortunately, tumors with these specific mutator phenotypes are in the minority. Recent pre-clinical and clinical studies have begun to reveal encouraging results suggesting that checkpoint inhibitor therapy can be expanded to an increasing number of colorectal tumors with microsatellite stability and the absence of traditional predictive biomarkers of checkpoint inhibitor response. These studies generally rely on combinations of checkpoint inhibitors with chemotherapy, molecular targeted therapy, radiation therapy, or other novel immunomodulatory agents. This article will review the most current data in microsatellite stable colorectal cancer. The dynamic interplay between tumor cells and the host immune system is instrumental in the evolution, progression and therapeutic susceptibility of colorectal cancer (CRC). Supporting the fundamental role of the immune system in the pathogenesis and course of this disease, colonic neoplastic progression is strongly associated with intestinal inflammatory mediators  and tumoTumor immune evasion is a fundamental hallmark of cancer cells , facilitFor patients with hyper-mutated subtypes of CRC—specifically those with microsatellite instability-high (MSI-H)/mismatch repair-deficient (dMMR) CRC or those with hereditary mutations in the exonuclease domain of the polymerase epsilon, catalytic subunit (POLE)—ICIs have shown remarkable efficacy with a more favorable toxicity profile than traditional chemotherapy . The notHowever, tumors with MSI constitute a small fraction of all colorectal neoplasms in both the metastatic and non-metastatic setting, and patients with the more prevalent MSS CRC have, on the whole, not benefited from single-agent ICI therapy. The exception is limited to roughly 3% of MSS CRC with a high tumor mutational burden and a more MSI-like phenotype . Going fIn an attempt to foster a more immunogenic tumor microenvironment in MSS CRC, chemotherapy has been shown in vitro and in vivo to have favorable synergistic immunomodulatory effects that can potentiate ICI efficacy. As known predictive biomarkers critical to ICI effectiveness, TILs, tumor neoantigens, and tumor PD-L1 expression have been shown to increase after 5-flourouracil plus oxaliplatin (FOLFOX) treatment in murine models of CRC and human tumor sample correlates . LikewisP < 0.001) in patients receiving the PD-1 inhibitor pembrolizumab in combination with platinum-based chemotherapy first-line, irrespective of PD-L1 status [Substantiated by these preclinical findings, results from a phase 3 trial of 559 patients with untreated, metastatic squamous non-small cell lung cancer (Keynote 407) showed an improved median overall survival  of 53% at 24 weeks median follow-up with a disease control rate (DCR) of 100% at 8 weeks . DespiteValidated as robust therapeutic targets in CRC, both the vascular endothelial growth factor (VEGF) and the epidermal growth factor receptor (EGFR) are well-established mediators of tumor growth and proliferation. Targeted agents directed against EGFR, such as cetuximab and panitumumab, and those directed against VEGF, such as bevacizumab, have been shown to facilitate a more immunogenic tumor profile in preclinical models and, as such, are reasonable potential adjuncts to ICIs in MSS CRC. In vitro and in vivo preclinical studies describe increased tumor necrosis receptor CD137 expression on NK and T-cells, decreased immunosuppressive cell populations  and improved T-cell cytotoxicity and growth after EGFR inhibition . SimilarDriven by the preclinical data, an ongoing phase Ib/II study (NCT02713373) is evaluating the combination of cetuximab and pembrolizumab in patients with metastatic, RAS wild-type CRC with at least one prior line of treatment. In preliminary results of nine patients, the combination was well-tolerated despite the increased proportion of hypomagnesemia and led to durable (>16 weeks) disease control in six of the nine patients evaluated . While mAdditional clinical strategies include adding the PD-L1 inhibitor atezolizumab to a backbone regimen of FOLFOX and bevacizumab. In the first-line metastatic CRC setting in 23 patients, these agents together safely yielded an ORR of 52% and a median progression-free survival (PFS) of 14.1 months with a median duration of response (MDR) of 11.4 months in a phase 1b study . SupportSimilar to chemotherapy and anti-angiogenic therapy, preclinical data support the use of radiotherapy as an effective means to augment ICI efficacy by directly contributing to the process of immunogenic cell death. Mechanistically validated in vivo and in vitro, radiotherapy can elicit a pro-inflammatory tumor state through direct tumor destruction, release of tumor antigens, up-regulation of inflammatory cytokines and cell surface molecules , and recruitment of immune cells into the tumor microenvironment . The repThough early clinical efforts combining radiotherapy and ICI in other tumor types are limited, there are small trials showing potential benefit in the treatment of MSS CRC. Early data from a phase II, non-randomized study evaluating pembrolizumab 200 mg every 3 weeks in addition to either radiotherapy or ablation of tumor metastases in patients with advanced, refractory pMMR CRC reported 1 partial response in 11 patients (ORR 9%) who had received radiotherapy after a median of 4 doses of pembrolizumab . No respAs a key intermediate signaling molecule in the mitogen-activated protein kinase (MAPK) pathway, mitogen-activated protein kinase kinase (MEK) regulates cell cycle progression and is frequently aberrantly expressed in cancer cells . ApproxiClinical studies of ICIs and MEK inhibitors in CRC have been, on the whole, unremarkable to date. The 3-arm phase III randomized study IMblaze370 NCT02788279) compared the combination of cobimetinib and atezolizumab with atezolizumab or regorafenib in 363 patients with metastatic, chemo-refractory, >90% MSS, CRC . Neither88279 comNew actionable strategies to circumvent inherent MSS CRC immune resistance necessitate careful dissection of immune pathways and elements of the tumor microenvironment that pose barriers to checkpoint blockade. Unique pharmacologic approaches being studied in early-phase clinical trial are discussed below.In vivo and in vitro experiments have demonstrated that growth suppression of tumor-associated macrophages (TAMs) through inhibition of colony-stimulating factor 1 receptor (CSF1R) can improve the anti-tumor, pro-inflammatory function of macrophages and enhance tumor immunogenicity for optimal ICI synergy . While CLike CSFR1, the rate-limiting, cytosolic enzyme indoleamine 2,3-dioxygenase-1 (IDO1) functions in the process of tumor-mediated immune evasion. Inhibition of this enzyme, which has an essential role in regulating tryptophan catabolism, has been shown to improve tryptophan-dependent T-cell function, decrease the immunosuppressive product kynurenine, and reduce immunosuppressive tumoral cell populations . FollowiTherapeutic cancer vaccines represent an alternative strategy for immune-sensitization that may be rationally combined with ICIs to augment anti-tumor immunity. In particular, the GVAX CRC vaccine, which consists of irradiated CRC cells modified to express granulocyte-macrophage colony-stimulating factor (GM-CSF), is currently being investigated in combination with cyclophosphamide and pembrolizumab in patients with pMMR advanced CRC (NCT02981524). Likewise, the adenovirus carcinoembryonic antigen (ad-CEA) vaccine, designed to procure a CEA-specific immune response, is also being evaluated in combination with PD-L1 inhibition in patients with metastatic or unresectable CRC (NCT03050814). An alternative personalized peptide vaccine, which combines individually selected, human leukocyte antigen (HLA)-matched peptides derived from a panel of tumor-associated antigens, is also being trialed with PD-1 inhibition (NCT02600949). Similarly, a personalized neoantigen vaccine GRT-C901/GRT-R902 is being evaluated in combination with nivolumab and ipilimumab in patients with MSS CRC (NCT03639714). In contrast, oncolytic viral therapy mediates tumor regression through preferentially replicating and destroying tumor cells, leading to a proliferative immune cascade. Talimogene Laherparepvec (T-vec) was the first oncolytic virus approved by the US Food and Drug Administration in 2015. It is a modified herpes simplex virus (HSV) type 1 with an inserted GM-CSF gene delivered via intralesional injection that selectively replicates in and lyses melanoma cells. In a phase III trial, T-vec improved durable response rate and ORRs compared to subcutaneous GM-CSF, although the improvement in OS only trended to statistical significance . The sucThe use of bioengineered T-cell bispecific antibodies (TCBs) with the ability to simultaneously target and connect both a tumor associated antigen and a CD3 T-cell receptor epitope directly enables a localized, antigen-specific T-cell response. Early pre-clinical and clinical data suggest that TCBs against the carcinoembryonic antigen (CEA), which are a known CRC biomarker, can lead to up-regulated expression of PD-L1, increase intra-tumoral lymphocytes and augment anti-tumor activity . The comAdenosine monophosphate is dephosphorylated to adenosine by the extracellular ectonucleotidase CD73, which is highly expressed on the plasma membrane of cancer and immune cells  AdenosinThe gut microbiome has an important role in regulating the innate and adaptive immune response in the presence of foreign pathogens. Specific subpopulations of intestinal microbes have been implicated in the efficacy of ICIs . AlterinPolyinosinic-polycytidylic acid-poly-l-lysine carboxymethylcellulose (poly-ICLC) is a ligand for toll-like receptor 3 and has a critical role in stimulating the systemic innate immune response . ThroughPlease see Therapeutics targeting well-delineated molecular pathways with known roles in tumor immunity offer alternative agents for ICI combination therapy in MSS CRC. The WNT/ β-catenin signaling pathway—frequently mutated in MSI-H tumors and associated with reduced TIL density in CRCs —can be iICIs that target either PD-1 or PD-L1 can have remarkable efficacy in any MSI-H malignancy, including MSI-H CRCs. No clinical differences in efficacy have been detected between these two antibody targets, although PD-1 blockade has been utilized in the majority of CRC studies. With the clinical success of ICIs in the MSI-H population, significant effort is now being deployed to harness the benefits of immune therapy in the MSS CRCs. Combination strategies have shown promise with the administration of ICIs with a variety of additional agents, including cytotoxic chemotherapy, small molecule inhibitors, targeted agents against the MAPK pathway, and radiotherapy techniques. Dual checkpoint inhibition of PD-1 plus CTLA-4 has been approved in MSI-H CRCs but there no reported clinical trials in MSS patients. Additional adjunctive agents under investigation include CSFR1 inhibitors, IDO1 inhibitors, TCBs and a host of other agents. As these novel agents progress through the phases of clinical development, novel predictive markers specific to these agents may become important to optimally select patients most likely to benefit from them. Going forward, more data are needed to address efficacy and tolerability as well as drug sequencing, dosing, and timing to optimize patient benefit. Careful consideration will need to be given to the cost of these medications in the overall paradigm of CRC treatment.