Independent component analysis was performed to identify spatial patterns of coherent variance in KOR binding  across all subjects. Expression of each component was compared between groups and relationships to symptoms were explored using the 17-item Hamilton Depression Rating Scale (HDRS). Three components of variation in KOR availability across ROIs were identified, spatially characterized by [11C]GR103545 VT in (1) bilateral frontal lobe; (2) occipital and parietal cortices, right hippocampus, and putamen; and (3) right anterior cingulate, right superior frontal gyrus and insula, coupled to negative loading in left middle cingulate. In MDD patients, component 3 was negatively associated with symptom severity on the HDRS . There were no group-wise differences in expression of any component between patients and controls. These preliminary findings suggest that KOR signaling in cortical regions relevant to depression, particularly right anterior cingulate, could reflect MDD pathophysiology.Preclinical studies have implicated kappa opioid receptors (KORs) in stress responses and depression-related behaviors, but evidence from human studies is limited. Here we present results of a secondary analysis of data acquired using positron emission tomography (PET) with the KOR radiotracer [ Much preclinical literature implicates the kappa opioid system in maladaptive responses to stress and depression-like behaviors. The endogenous opioid dynorphin is released in response to stress, resulting in preferential activation of the kappa opioid receptor (KOR) . Dynorph11C]GR103545. In that study, we examined a small number of a priori regions of interest (ROIs) based on animal findings: hippocampus, raphe nuclei, amygdala, and ventral striatum GR103545 and a structural magnetic resonance imaging (MRI) scan for image registration and ROI definition. All participants provided informed consent prior to completing any study procedures. Study procedures were approved by the Institutional Review Boards of The New York State Psychiatric Institute and the Yale University School of Medicine.Data from a previous study were re-analyzed . Briefly11C]GR103545, a selective kappa agonist radiotracer UCB-J [11C]GR103545, ICA offers the possibility of characterizing overlapping but distinct spatial distributions of KOR availability based on coherent patterns of variance across subjects. This may be a relevant approach for the KOR/dynorphin system given the local regulation of neuropeptide signaling and of dynorphin in particular. In contrast to neuromodulators such as dopamine and serotonin which are synthesized by well-characterized populations of projection neurons along specific circuits, neuropeptides like dynorphin are typically synthesized and released locally by many unrelated groups of neurons across various brain regions [11C]GR103545 PET data may allowing unmixing of cross-regional patterns of KOR/dynorphin signaling with common regulatory influences or which are similarly implicated in or affected by MDD.ICA is a data-driven method used to identify multiple sources of variance in a dataset, ‘unmixing’ observed signals into maximally independent components and differentiating relevant functional brain signals from variation related to noise . In cont1C]UCB-J . With GR103545 VT were also analyzed using ICA to confirm that similar regional patterns would be captured and allow us to capture relevant variation within larger ROIs. For ROI and voxel-level analyses, regional values or images from all subjects were entered into ICA together. The global mean value was first subtracted from each subject’s data. The number of maximally independent components present in the voxel-level data was determined to be three using a minimum description length criterion approach validated for model order selection in neuroimaging data GR103545 VT is high throughout limbic brain regions including anterior cingulate cortex, insula, hippocampus, and amygdala  in temporal lobes and thalamus. The second  had highest intensity in inferior occipital lobe along with right hippocampus, superior parietal lobe, and putamen. The third  was highly lateralized to the right side, with high positive signal in right anterior cingulate as well as right superior frontal gyrus and insula, and large negative values in left middle cingulate  or in subject loading values for any ICA component .There was not a statistically significant difference between MDD patients and HCs in mean whole-brain r = −0.85, p = 0.0021, Fig. 11C]GR103545 VT values in cingulate subregions directly  or between symptom severity scores and loading for any other component , indicating that similar regional patterns were extracted despite the different level and patterns of variance in the voxel-level data. Accordingly, loading across subjects in the MDD group was also similar between V1 and R3 . V3 including signal in superior frontal cortex with negative signal in ventromedial prefrontal cortex. V3 subject loading was negatively correlated with that of R1 .ICA was repeated using voxel-wise images of GR103545 VT may reflect true biological units of differential KOR/dynorphin regulation, given the regionally specific release and regulation of these peptides. Indeed, post-mortem work suggests regionally specific effects of childhood abuse on KOR binding, with low binding in those with a history of childhood abuse observed in anterior insula but not thalamus or anterior cingulate cortex , mean −0.76 ± 1.71). In some cases, the weighting was in the same direction but stronger on the right than on the left ; in others, the finding was in opposing directions on right vs. left sides . Moreover, the left mid-cingulate was weighted in the opposite direction in the factor as the right anterior cingulate, i.e., KOR signal in left mid-cingulate was positively correlated with depression severity quantified by HDRS, suggesting possible functional differentiation of KOR effects not just by hemisphere but along the anterior-posterior axis of the cingulate. Rostral anterior cingulate has been associated with treatment outcome with a broad range of antidepressant treatment interventions GR103545 radiotracer present a challenge in small, high-binding regions and in conventional voxel-level analyses GR103545 radiotracer, which has slow kinetics contributing to variability in parameter estimates. Future work on this system can take advantage of radioligands with improved kinetic properties [Limitations to this analysis include the small sample size and limitations of the [operties , 68. Givoperties  which maThis study represents the most direct data to date relating quantification of the kappa opioid system in vivo to depression severity. These data highlight regions and networks of KOR/dynorphin binding that merit further study in larger samples. If replicated, these results may suggest that the kappa opioid system can contribute to more severe forms of depression or reflect the consequences of stress due to more severe depression. These networks may be particularly relevant to the somatic and behavioral symptoms of depression captured preferentially by the HDRS. Furthermore, KOR quantification via PET may identify depressed patients with a kappa opioid abnormality, who may be more likely to benefit from kappa-modulating interventions.Supplemental material


With the high spatial resolution and the potential to reach deep brain structures, ultrasound-based brain stimulation techniques offer new opportunities to non-invasively treat neurological and psychiatric disorders. However, little is known about long-term effects of ultrasound-based brain stimulation. Applying a longitudinal design, we comprehensively investigated neuromodulation induced by ultrasound brain stimulation to provide first sham-controlled evidence of long-term effects on the human brain and behavior.Twelve healthy participants received three sham and three verum sessions with transcranial pulse stimulation (TPS) focused on the cortical somatosensory representation of the right hand. One week before and after the sham and verum TPS applications, comprehensive structural and functional resting state MRI investigations and behavioral tests targeting tactile spatial discrimination and sensorimotor dexterity were performed.Compared to sham, global efficiency significantly increased within the cortical sensorimotor network after verum TPS, indicating an upregulation of the stimulated functional brain network. Axial diffusivity in left sensorimotor areas decreased after verum TPS, demonstrating an improved axonal status in the stimulated area.TPS increased the functional and structural coupling within the stimulated left primary somatosensory cortex and adjacent sensorimotor areas up to one week after the last stimulation. These findings suggest that TPS induces neuroplastic changes that go beyond the spatial and temporal stimulation settings encouraging further clinical applications. Non-invasive brain stimulation (NIBS) has been shown to support brain functions in neurological and psychiatric disorders , 2. CommIn recent years, low intensity transcranial focused ultrasound (tFUS) has emerged as a NIBS method that overcomes these drawbacks. With a lateral resolution between 3 and 7 mm, tFUS allows precise brain stimulation of confined regions, for example, the primary somatosensory cortex –8. tFUS Recently, we introduced transcranial pulse stimulation (TPS) –13, a neA first uncontrolled clinical feasibility study in patients with Alzheimer’s disease demonstrated that repeated TPS stimulation of specific cognitive networks improved memory and verbal functions up to three months . These cBesides effectiveness, safety and feasibility, the persistence of favorable effects is certainly critical for clinical applications. Up to now, little is known about the temporal dynamics and long-term effects of ultrasound-based NIBS. The majority of the tFUS studies evaluated neurophysiological online effects in humans , 18, 19 The major aim of this exploratory study was to probe first sham-controlled long-term effects of TPS and to investigate related neurophysiological mechanisms in healthy human participants. To this end, a randomized sham-controlled cross-over design was used with repeated sessions of verum and sham TPS targeting the cortical somatosensory representation of the right hand. Basic somatosensory processing was targeted, as this represents a well localizable function in the brain with well-established tests to assess perceptional functions  and behavioral implications (sensorimotor abilities). Comprehensive MRI investigations were used to assess anatomical and functional alterations at the stimulation site and in connected sensorimotor networks. As we assume that TPS induces functional neuroplasticity by increasing the functional coupling between the stimulation site and connected brain areas, the primary outcome of this study represents the resting state functional connectivity in primary and secondary somatosensory networks. Ongoing functional up-regulation via NIBS might induce morphological changes, such as enlarged dendritic spines, increased synapse density, and modified interneural connections . Thus, wThis randomized, sham-controlled, and double-blind study was conducted at the Medical University of Vienna. Twelve healthy male participants with 18–35 years of age  that were right-handed and did not suffer from any neurological, psychiatric, or major somatic disease were recruited. For each participant, the study duration was seven weeks including one week pause between the two experimental blocks  comprised MR measurements and behavioral assessments of tactile and sensorimotor functions. In the second week of each block (week 2 and 6) three TPS interventions on three consecutive days were applied, either as real (verum) brain stimulation or as placebo (sham) stimulation. The MR and behavioral measurements were repeated in the third week of each block (week 3 and 7) to assess post-stimulation changes. Each subject received one block with sham and one block with verum TPS using a within-subject crossover design. The order of the experimental conditions was counterbalanced and randomly assigned. The experimenter applying TPS was informed about the actual condition (sham or verum), experimenters conducting MRI, safety evaluations, behavioral assessments, as well as data analysis were blinded.2 and pulse repetition rates of 1–5 Hz  = 10.95). The ISPTA fulfils the DIN EN 61689 norm, and the maximum peak pressure lies well below tissue damaging pressure levels , the ISPTA is marginally higher (ISPTA = 94 mW/cm2) and the MI exceeds respective FDA limits (MI = 1.90)  was computed with fslmaths first. For the ROI-analyses of DTI indices, the Human Sensorimotor Tracts Labels  implemenAfter the MR measurements, behavioral assessments of tactile and sensorimotor functions were performed. The tactile spatial discrimination threshold was measured using a 2-point orientation discrimination task . Here, aThe relations between neurophysiological measures demonstrating a TPS effect and behavioral scores  were examined by correlation analyses. As data were not normally distributed, a non-parametric Spearman’s rank correlation analysis was applied. Previous literature demonstrated an non-linear relation between age and DTI indices . To accoGlobal efficiency (GE), as the capacity for parallel information processing within a network , was sigTBSS analyses of DTI indices  did not show significant effects in the white matter tract skeleton. In contrast, ROI analysis revealed significantly reduced AD within the white matter tracts in the primary somatosensory ROI  and primary motor ROI  after verum TPS compared to the respective baseline  did not show significant effects of TPS, neither on a whole-brain level nor on ROI-level. Safety evaluations of the clinical structural sequences (T2-weighted FLAIR and FLASH images) did not reveal any signs of bleedings, edema, or other morphological alterations after the sham or verum interventions.For the coin rotation task as a measure for manual dexterity and sensorimotor processing, factorial analysis did not show significant effects for the factors  and their interaction (p = 0.195). On a descriptive level, time needed for 20 coin rotations remained at about the same level after the sham intervention , but decreased 0.75 s on average in the verum stimulation .Performance in the 2POD task as an indicator for tactile acuity improved slightly in the post-stimulation test  compared to the respective baseline  for both, verum and sham condition. However, these observations did not reach statistical significance .Correlation analysis between significant neurophysiological variables  and behavioral tests did not reveal significant results. However, a trend between AD in the motor ROI and 2POD performance points to a subtle relation between improved axonal status and tactile acuity , whereby the accuracy improved slightly in the second experimental block  compared to the first block . The subjective certainty of the condition assignment, rated between 1 (not certain) and 10 (very certain), increased steadily from 5.58 ± 2.64 (n = 12) in the first TPS session to 7.33 ± 1.87 (n = 12) in the 6th session. Sensations at the scalp were frequently reported, with tactile impressions  being common in both conditions (Table Using repeated TPS applications in healthy participants, we provide the first sham-controlled evidence of long-term effects of ultrasound-based NIBS on human brain structure and function. One week after the last TPS stimulation of the cortical somatosensory hand representation, global efficiency in the sensorimotor network of the stimulated left hemisphere significantly increased. Further, TPS improved white matter microstructure in the stimulated left sensorimotor regions.For feasible and effective clinical applications of NIBS, persisting changes of brain functions and associated symptom relief are required. Contrary to investigations of online-effects of tFUS , 18, 19,Previous NIBS studies have demonstrated that increased functional connectivity within a network induced by brain stimulation is related to improved behavioral functions or clinical symptoms supported by this network. For example, upregulation of the motor network connectivity by intermittent theta-burst stimulation was correlated with symptom relief in patients with upper limb paresis  and incrIn contrast to our initial hypothesis, functional up-regulation was not found to be significantly correlated with behavioral improvements in this study. This lack of significant enhancement of tactile acuity and manual dexterity after TPS might be caused by statistically underpowered behavioral tests due to the small sample size, learning and ceiling effects regarding sensorimotor functions in healthy participants. Yet, functions corresponding to the stimulation site might be influenced by early, direct effects of ultrasound NIBS . PossiblAfter the verum TPS stimulation, we found decreased AD in the left primary motor and left primary somatosensory ROIs, while it remained unchanged after sham stimulation. Although TPS stimulation was confined to the somatosensory cortex, both ROIs displayed a comparable AD decline after stimulation. Mirroring functional connectivity results, that demonstrate a global efficiency increase in a widespread sensorimotor network, microstructural integrity seems to be promoted in primary motor areas and potentially in other regions that are highly interconnected with the stimulated somatosensory cortex. In the developing brain, axial and radial diffusivity decrease as indicators for axonal and myelin formation, respectively , while Like in the clinical feasibility study in patients with Alzheimer’s disease , repeateRatings directly after the TPS interventions imply that the participants were able to distinguish between sham and verum condition to a certain extent, particularly after repeated applications. This is in contrast with our observations with moving TPS handpiece applications, where AD patients’ differentiation capability was close to chance. Presumably, sensory impressions like knocking, pressure and slight pain at the stimulation site informed the participants about the actual condition. However, a direct effect of the subjective condition assignment on the outcome variables, particularly on resting state functional connectivity and white matter microstructure, seems unlikely. Still, stimulation settings minimizing sensory impressions at the stimulation site should be tested for future studies.As the current investigation focused on long-term TPS effects, immediate effects of the stimulations were not assessed. However, online or short-term effects during or directly after the stimulation could be informative about requirements and extent of further neuroplastic reorganization. Thus, future studies should include longitudinal assessments starting immediately after the stimulation to estimate the temporal dynamics of TPS effects on brain and behavior.Repeated TPS targeting the primary somatosensory cortex increased functional coupling and improved white matter structural integrity in the sensorimotor network up to one week after the stimulation. These findings suggest that TPS induces neuroplastic changes that go beyond the spatial and temporal stimulation settings. This evidence of sham-controlled long-term effectivity, safety and feasibility encourages further clinical applications of TPS.


The long road from emerging biotechnologies to commercial “green” biosynthetic routes for chemical production relies in part on efficient microbial use of sustainable and renewable waste biomass feedstocks. One solution is to apply the consolidated bioprocessing approach, whereby microorganisms convert lignocellulose waste into advanced fuels and other chemicals. As lignocellulose is a highly complex network of polymers, enzymatic degradation or “saccharification” requires a range of cellulolytic enzymes acting synergistically to release the abundant sugars contained within. Complications arise from the need for extracellular localisation of cellulolytic enzymes, whether they be free or cell-associated. This review highlights the current progress in the consolidated bioprocessing approach, whereby microbial chassis are engineered to grow on lignocellulose as sole carbon sources whilst generating commercially useful chemicals. Future perspectives in the emerging biofoundry approach with bacterial hosts are discussed, where solutions to existing bottlenecks could potentially be overcome though the application of high throughput and iterative Design-Build-Test-Learn methodologies. These rapid automated pathway building infrastructures could be adapted for addressing the challenges of increasing cellulolytic capabilities of microorganisms to commercially viable levels. Many of the social and technological advances in the last century, from transportation fuels to materials and pharmaceuticals, have been due to an increase in our understanding and utilisation of organic chemistry . Much ofSaccharomyces cerevisiae; an antitumor alkaloid derived naturally from Papever somniferum (opium poppy) . Th. Th43]. Hemicellulose is a heterogenous polysaccharide that is comprised of a diverse array of C5 and C6 sugar monomers. It generally contains a xylan (major component), galacto(gluco)mannan or xyloglucan backbone  with brap-coumaryl-, coniferyl- and sinapyl alcohols (p-phenyl- (H type), guaiacyl- (G type) and syringyl (S type) structural monomers. Lignin is covalently linked to both cellulose and hemicellulose, and provides the plant with structural support and impermeability. It also functions as a resistance against microbial attack and oxidative stress. Given these characteristics, lignocellulose requires pre-treatment to remove lignin to release the cellulose prior to enzymatic saccharification. Lignin is generally not considered to be a target carbon source for microorganism cultivation, but instead is a source of valuable phenolic synthons for the production of high-value compounds . T. Tp-coumompounds ,50. Currently, most commercial and pilot scale processes utilising lignocellulose as a feedstock require physical and/or chemical pre-treatments to remove hemicellulose and lignin, reduce the crystallinity of the cellulose and minimise the release of hemicellulose-derived inhibitory compounds . The resultant amorphous regions of the cellulose then undergo enzymatic hydrolysis by commercial cocktails of cellulolytic enzymes to release glucose for later fermentations . There aA second group of pre-treatments are physio-chemical processes, such as steam explosion and hot liquid water treatment . Steam eOverall, there have been extensive studies on determining the most efficient and cost-effective method for lignocellulose pre-treatment to maximise glucose output for later fermentation ,55,56,57More than 160,000 cellulases have been identified , which sCellulose is enzymatically degraded to glucose units (C6 sugar) by glycoside hydrolases (cellulases) via the hydrolysis of its β-1,4 glycosidic bonds . The comT. aurantiacus generates C1 oxidised polysaccharide oligomers from cellulose with a non-reducing end oxidised species. This enzyme enabled an increase in microcrystalline cellulose degradation by other cellulases in the presence of gallic acid. This new class of enzymes are known as copper-dependent lytic polysaccharide monooxygenases (LPMO) . T. TE. col3.6 g/L) . E. coli has been isolated from bovine rumen that was capable of fermenting corn straw directly to both ethanol and hydrogen gas  homologue/variant, transcriptional/translational control, vector backbone  and DNA De novo robotic workflows and robotic technology platforms are key to biofoundry development as they can rapidly generate libraries of potential pathways and generate comparative performance data to enable the identification of the most suitable parts for target compound production. Machine-learning techniques are an integral part of the “Learn” step, as information from initial datasets can help make more informed decisions in future rounds of pathway design and screening dataset size . The biofoundry approach has been demonstrated using a “pressure test” at the MIT-Broad foundry, where the challenge was to produce novel pathways to 10 compounds in just 90 days . They suA similar rapid prototyping of microbial cell factories challenge by SYNBIOCHEM was performed to benchmark the capabilities of a biomanufacturing pipeline. They successfully produced 17 potential material monomers and key intermediates out of 25 identified compounds within 85 days . This waBeyond proof of principle research, the biofoundry approach is a valuable tool when considering scaled processes. Biofoundaries could be implemented for pathway redesign and/or microbial chassis optimisation to increase chemical titres to be commercially competitive with existing natural and synthetic routes. In the case of CBP technologies, this approach could also be exploited to increase the currently slow rate of glucose release from cellulose, which impacts on the growth rates of the microorganisms. Key “parts” to be optimised could include maximising the synergism between (hemi)cellulolytic homologues, improving cellular excretion by de novo design of secretion tags and varying the relative transcriptional/translational rates necessary to generate the most efficient cellulolytic pathways. This is of particular importance when utilising (Gram-negative) bacterial chassis, where active cellulase secretion is severely limited. Successful implementation of the biofoundry approach for CBP requires efficient and sensitive high throughput screening techniques to ensure a sufficient library size can be sampled to maximise the chance of finding improved variants. Examples include the scaling of the International Union of Pure and Applied Chemistry (IUPAC) standardised methods for the detection of reducing sugars to a microtitre plate scale . Other mThe often unacknowledged or underplayed bottleneck to the commercialisation of a synthetic biology generated microbial biomanufacturing process is the need to replace pure glucose-based carbon sources with cost-effective alternative feedstocks. Lignocellulose appears to be an ideal low-cost abundant waste feedstock, provided the extensive network of polymeric fibres can be degraded to release the abundant sugars contained within. However, the enzymatic degradation of lignocellulose is a complex process requiring thermochemical and/or mechanical pre-processing followed by the synergistic action of a variety of saccharolytic and lignin-degrading enzymes. The incorporation of an efficient cellulolytic system within microbial systems can be hampered by bottlenecks such as cost-effective lignocellulose pre-processing, lack of expression of functional glycosylated fungal cellulases in bacteria and the often-poor secretion of all the required (hemi)cellulases. The light on the horizon is the advent of the biofoundry approach, whereby traditional synthetic biology pathway design is replaced by iterative Design-Build-Test-Learn cycles. Between automated pathway design and synthesis, high throughput screening and machine learning, the process from concept to functionally efficient cellulolytic and high titre chemical producing industrial chassis might be realised in a relatively short time (months rather than years). The future outlook in the CBP field will therefore depend on the continued development of efficient automated and tunable engineering platforms, to accelerate both fundamental and applied biotechnological research to realise commercially successful biomanufacturing platforms.


ICB is a promising form of cancer immunotherapy that aims to boost the anti-tumoral immune response. This response is primarily driven by the presentation of different types of antigens at the cancer cell membrane via the type I major histocompatibility complex (MHC I). Neoantigens are tumor-specific antigens that are small (mostly 9-mers) mutated peptides that generally result from somatic mutations. High tumor mutational burden, which results in the formation of many neoantigens, is currently one of the main biomarkers for ICB response prediction. Because ICB is only effective in a minority of patients, new therapeutic strategies are required to augment this response.A recent study by Lu et al.Cell,1 provide preclinical evidence that pharmacological modulation of the spliceosome results in the generation of a substantial amount of highly immunogenic, splicing-derived neoantigens, augmenting the immune response in mice following ICB treatment. If translated to the clinic, these findings raise the exciting possibility of treating otherwise unresponsive cancers with immunotherapy, previously only effective in cancers with increased mutation burden such as melanoma.The authors of the study, published in B2m knock out tumor cells confirmed that this drug-induced cancer growth impairment was dependent on T cells and MHC I peptide presentation. Since the compounds did not have an effect on cancer cells themselves, it would have been interesting to see if a CRISPR-mediated knockout of, e.g., RBM39 would result in a viable cell line. If so, the RBM39 null cells could be investigated to determine if they displayed inferior growth compared to an unaltered line in vivo because of T cell immunity.The authors explored the effects of two splicing modulating drugs: indisulam and MS-023. The former was previously shown to degrade the accessory splicing factor RBM39, while the latter inhibits type I protein arginine methyltransferase (PRMT) enzymes. They elegantly demonstrate that these drugs have little effect on cancer cell viability in a petri dish, but result in a profound suppression of tumor growth following engraftment into mice. This discrepancy hinted towards an anti-cancer immune response and further experiments using T cell depletion and To determine whether splicing modulation could enhance ICB therapy responses, the authors engrafted mice with syngeneic tumors, followed by pretreatment with splicing inhibitors starting on day 3 and treatment with anti-PD1 on day 7. A significant reduction of tumor growth was detected for simultaneous indisulam (or MS-023) and anti-PD1 therapy, which exceeded the effect of either drug alone. This response was seen for different engrafted tumor cell types . LLC was particularly interesting as this cell type is resistant to anti-PD1 monotherapy, yet combination therapy with splicing modulation significantly reduced tumor growth. The anti-tumoral responses resulted in improved survival as compared to mono treatment, with 50% of the mice engrafted with MC38 and treated with MS-023 and anti PD-1 being alive and tumor-free after 3 months. These mice also responded to an additional challenge with MC38 cells 6 months later, suggesting these mice retain immune memory. No toxicity or inflammatory changes were observed in non-tumor tissues. It is worth noting that MC38 cells are generally sensitive to ICB therapy and the other models did not have as great a response.The authors then explored the molecular mechanisms behind these promising results. As expected, they identified several splicing alterations with intron retention and exon skipping the most common alterations. Using a combination of in silico MHC binding predictions and mass spectrometry, they demonstrate that these alterations result in a substantial amount of MHC I binding peptides. Interestingly, they confirm that a rather large proportion (up to 43%) of these putative neoantigens are immunogenic, resulting in neoantigen-specific CD8+ T cell activation Fig. .Fig. 1Ph2) , splicing-derived peptides are often completely novel to the immune system, resulting in much better immune responses, as illustrated in the study (28–43% of predicted splicing-derived neoantigens were immunogenic versus an estimated amount of 6% for mutation-derived neoantigens2) Fig. .3, safety needs to be carefully monitored in additional species than mice before clinical trials are initiated. Further, clinical effectiveness is uncertain as several immune evasion mechanisms have been described in human tumors that could interfere with (splicing-induced) neoantigen presentation . Relatedly, it is also unclear from the study whether splicing modulation can induce long-lasting immune responses. In this regard, it remains to be established whether memory T-cells are raised against these splicing-derived neoantigens and whether any reaction is still possible after the removal of the splicing inhibitor.It remains to be determined whether the approach will result in direct clinic applications. While the authors did not observe any toxicity in their mice models, inflammatory side effects could be a major clinical concern, especially when combined with ICB. In humans, drug treatments usually continue for several months. Since splicing inhibition could have the risk of causing side-effects such as developmental defects4 The study also hints at a largely underappreciated role of the noncoding genome in neoantigen formation and is in line with the work of Laumont et al.5 who demonstrated that 90% of tumor-specific antigens are derived from noncoding regions.Apart from its direct translational and clinical relevance, the findings provide important insights in cancer biology and tumor evolution. Assuming that splicing inhibition could indeed be a powerful source of highly immunogenic neoantigens, strong negative selective forces are expected against genomic alterations in the genes encoding the targeted proteins. This sheds new light on the interpretation of previously detected negative selection signals in RNA splicing genes.In conclusion, the study of the groups of Abdel-Wahab and Bradley provides important insights in cancer immunobiology with direct clinical relevance. Further translational and clinical research is required to demonstrate whether pharmacologic splicing modulation is a realistic treatment option in a clinical cancer immunotherapy setting.


Medicines that are based on known molecules and are further developed to address healthcare needs and deliver relevant improvement for patients, healthcare professionals and/or payers are called value-added medicines (VAMs). The evaluation process of VAMs is heterogeneous across countries, and it has been primarily designed for originator pharmaceuticals with confirmatory evidence collected alongside pivotal clinical trials. There is a mismatch between evidence requirements by public decision-makers and evidence generated by manufacturers of VAMs. Our objective was to develop a core evaluation framework for VAMs.Potential benefits offered by VAMs were collected through a systematic literature review and allocated to separate domains in an iterative process. The draft list of domains and their applicability were validated during two consecutive virtual workshops by health policy experts representing countries with different economic statuses, geographical and decision-making contexts.Based on 158 extracted studies, the final consensus on the evaluation framework resulted in 11 value domains in 5 main clusters, including unmet medical needs, health gain , patient-reported outcomes, burden on households, and burden on the health care system.The proposed framework could reduce the heterogeneity in value assessment processes across countries and create incentives for manufacturers to invest in incremental innovation. However, some domains may not be equally relevant or accepted in all countries, therefore the core framework needs thorough adaptation in specific jurisdictions.The online version contains supplementary material available at 10.1186/s12962-021-00311-6. To ensure the sustainability of health care financing, international guidelines recommend the first-line use of off-patent medicines in major diseases , 2. In tdrug repurposing should refer only to developing new indications of established medicines or it should cover all categories of incremental (evolutionary) pharmaceutical innovation . Table S2. Search syntax for the systematic literature review. Table S3. Illustrative benefits of value added medicine test cases according to the domains of the core evaluation framework as reported by the IQVIA report [17]. Table S4. A non-exhaustive list of value propositions in each value domain.


Metabolic pattern reconstruction is an important factor in tumor progression. Metabolism of tumor cells is characterized by abnormal increase in anaerobic glycolysis, regardless of high oxygen concentration, resulting in a significant accumulation of energy from glucose sources. These changes promotes rapid cell proliferation and tumor growth, which is further referenced a process known as the Warburg effect. The current study reconstructed the metabolic pattern in progression of cancer to identify genetic changes specific in cancer cells. A total of 12 common types of solid tumors were included in the current study. Gene set enrichment analysis (GSEA) was performed to analyze 9 glycolysis-related gene sets, which are implicated in the glycolysis process. Univariate and multivariate analyses were used to identify independent prognostic variables for construction of a nomogram based on clinicopathological characteristics and a glycolysis-related gene prognostic index (GRGPI). The prognostic model based on glycolysis genes showed high area under the curve (AUC) in LIHC (Liver hepatocellular carcinoma). The findings of the current study showed that 8 genes  were correlated with overall survival (OS) and recurrence-free survival (RFS). Further analysis showed that the prediction model accurately distinguished between high- and low-risk cancer patients among patients in different clusters in LIHC. A nomogram with a well-fitted calibration curve based on gene expression profiles and clinical characteristics showed good discrimination based on internal and external cohorts. These findings indicate that changes in expression level of metabolic genes implicated in glycolysis can contribute to reconstruction of tumor-related microenvironment. Energy metabolism pattern of tumor cells shows significant differences, compared with oxidative phosphorylation (OXPHOS) in normal cells. Energy metabolism is reprogrammed in tumor cells, in a process known as Warburg effect to maintain survival and meet the high demand for synthesis of biological macromolecules5. Warburg effect represents change in glucose utilization by tumor cells from oxidative phosphorylation to glycolysis, which is now acknowledged as a major feature hallmark of tumors7. This change in energy metabolism is determined by complex factors, including pressure on tumor microenvironment and genetic changes11. Enhanced glycolysis of tumor cells is mainly promoted by increased expression or activity of key glycolysis enzymes12. Previous studies have explored agents that target tumors by inhibiting activity of key enzymes in the tumor glycolysis pathway. In addition, studies report that specific inhibition of glycolysis is associated with significant tumor suppression, and induces cell death. Glycolytic key enzymes such as hexokinase 2 (HK2), phosphofructosidase (PFK), and M2-type acetone kinase (PKM2) are tumor markers, and their expression and activity can affect tumor glycolysis, which in turn affects proliferation of tumor cells17. However, studies have not explored glycolytic-related factors in refining stratification and management of cancer patients. Early diagnosis and personalized treatment can effectively improve survival of cancer patients. Histopathology analysis can be used to predict prognosis and outcome of cancer patients. However, patients with the same pathology present with different prognoses owing to different molecular subtypes thus limiting use of histopathological characteristics19. Advances in high-throughput nucleotide sequencing technology in the recent years enables better understanding of the dynamic changes in tumor cells at the molecular level. A single gene cannot accurately predict the outcome of cancer patients. However, several biomarker combinations can improve sensitivity and specificity of patient outcomes. Multiple biomarkers that are highly correlated with survival and prognosis can identify high-risk patients, ameliorate poor prognosis of cancer patients, and can be used for development of effective intervention therapy.Cells undergo changes in energy metabolism patterns for biosynthesis, depending on cell function and availability of metabolites. In addition to oxidative phosphorylation of glucose, other metabolic pathways, including lipid, nucleotide, and amino acid metabolism can provide energy to meet the biosynthetic requirements for cell growth and proliferationGene set enrichment analysis (GSEA) is used in genomic research to identify potential biological mechanisms implicated in disease. In the current study, a gene signature was developed through GSEA analysis, based on genes implicated in glycolytic metabolic pathways. Tumor glycolysis metabolic patterns of 12 cancer types , were explored through a comprehensive analysis of genome and transcriptome profiles of TCGA dataset. A GRGPI signature was developed for LIHC and multiple risk characteristics that can effectively predict prognosis of patients were determined. The findings showed that glycolysis-related risk characteristics can be used to identify patients with poor outcomes in high-risk group. In addition, Cox multivariate hazard ratio analysis showed that the risk score performed better compared with other clinical variable in evaluating patient prognosis.https://portal.gdc.cancer.gov/), International Cancer Genome Consortium  database, and Gene Expression Omnibus  database. Datasets with insufficient sample size (< 200) or missing clinical information were excluded. The raw counts were transformed into transcripts per kilobase million (TPM) values for subsequent analysis.Transcriptome expression profiles were obtained from multiple data repositories, including The Cancer Genome Atlas Program  and FDR values were used to explore enriched pathways in each phenotype. GSEA was performed to explore differences in glycolysis-related gene sets between tumor tissues and matched normal tissues. P value and FDR value at < 0.05 were set as the threshold.Molecular Signatures Database (MSigDB) was used to identify gene sets and specific biological processes that are significantly differentially expressed in different groups. Analysis using MSigDB resulted in a statistically significant improvement in connectivity between data expression pattern and biological processes, ignoring the clear differential gene threshold21. Multivariable logistic regression analysis was performed after the LASSO (least absolute shrinkage and selection operator) analysis, which simultaneously selects the variables and penalizes the model coefficients for overoptimism22. Multivariate analysis was performed using the Cox proportional hazards (Cox-PH) model to identify independent predictors of survival. Covariates with a P value < 0.05 were used for subsequent risk prediction model construction based on multivariate analyses results. Standardized risk score was calculated using the formula shown below:Univariate Cox regression models were constructed to explore the statistical relationships between mRNA expression levels and RFS or OS. A linear regression model with a stepwise forward method was used to predict significant variations between variables, with the beta value (β) from univariate Cox regression analysis as the weighting factor23. P value less than 0.05 denoted statistical significance.Patients with complete clinicopathological characteristics were divided into a high- and a low-risk group, based on the median value of the risk score. Kaplan–Meier curves wee generated to compare differences in survival probability in low- and high-risk groups. Log-rank test P < 0.05 was conducted to explore significance of survival time differences. All these analyses were performed packages in R version 3.6.1https://www.proteinatlas.org/)24. Immunohistochemical staining results were evaluated by two independent pathologists, based on the integrated index by multiplying the intensity by the proportion of immunopositive cells of interest.Immunohistochemical slides and relative clinical pathology information were retrieved from the Human Protein Atlas database (Ensembl version: 92.38)  was used to construct a scale-free co-expression network. Subsequently, genes with the same expression pattern based on Pearson's coefficient were concentrated into specific gene modules. The top 2 modules that had the strongest association with subgroups were selected for further analysis. GO and KEGG pathway enrichment analyses were performed to explore whether genes from various terms are significantly enriched than expected in the subgroupsThe identities of patients are not provided in the TCGA and GEO databases, therefore, no approval and informed consent was required from the institutional review board.The study process and analysis were presented as a panoramic flow chart Fig. . A totalTo further explore whether these core genes participated in the glycolysis process, GO and KEGG pathway analyses were performed using ClusterProfiler R package. The findings showed these genes are enriched in several pathways implicated in glucose metabolisms, such as pyruvate metabolic process, pyruvate biosynthetic process, a glycolytic process Figure b–d, and 2 score to avoid overfitting and unnecessary complexity. Selected features were incorporated into a least absolute shrinkage and selection operator (LASSO) regression model to penalize for model complexity overfitting. A total of 8 genes  with nonzero LASSO coefficients  were identified in BLCA, BRAC, HNSC, LIHC, LUAD, and LUSC Table . These fnts Fig. b,c. Multnts Fig. a,d. Notants Fig. e,f. The Risk scores were calculated and used to develop predictive models for prediction of OS and RFS. To verify the assignments of sub-categories, t-SNE was performed to constraint the dimensionality of features. T-SEN analysis showed that the two risk subgroups were scattered in two discrete directions Fig. a–d. TimeThe explanation for many clinical situations one can identify some standard variables that have previously been demonstrated to have prognostic value and are generally measured for most patients having the particular diagnosis. Further, tumor‐related clinicopathological variables associated with the classifier in the current study was explored based on TCGA Fig. , GEO Fi, and ICGFurther stratified analysis was performed to explore independence of the model within the same subgroups of clinicopathological features. Taking advantage of the clinicopathological parameters, TCGA discovery cohort was divided into subgroups based on clinical-pathological features, such as gender , age (≤ 65/ > 65), grade (G1–2/G3–4), stage (Stage I–II/III–IV), T classification (T1–2/3–4), tumor status (Tumor Free/With Tumor). After stratification, the 8-mRNA signature accurately divided cohort into low- and high-risk patients Fig. . SimilarA multiple ROC curve analysis was performed to determine the sensitivity and specificity of the OS/RFS prognostic model and other clinical pathology variables. The 8-mRNA model was used for analysis of the TCGA discovery cohort, TCGA training cohort, GEO, and ICGC cohort, and the prediction quality was compared by evaluating the area under the ROC curve to determine its performance. Multivariate Cox regression and AUC analyses showed that the prognostic model was an independent prognostic indicator with high accuracy  is a highly malignant tumor that accounts for approximately 90% of the total primary liver cancer cases44. In addition to the effects in the cancer environment, AURKA actively promotes DNA repair and acts as a transcription factor to promote cell migration and invasion45. It is located in the mitochondrial membrane where it regulates mitochondrial dynamics and ATP production47. AURKA is an effective prognostic indicator that probably integrates multiple oncogenic events in progression of tumors49. CDK1 (Cyclin-dependent kinase 1) is a serine/threonine-like protein kinase that plays an essential role in controlling cell proliferation at the G2/M point of the cell cycle. Studies report that high CDK1 expression level is an independent predictor for tumor recurrence in one and five years, and compounds that target CDK1 can be novel antitumor reagents52. CENPA is overexpressed in several cancers, and plays an auxiliary but important role in cancer pathogenesis, progression, distant metastases and invasion angiogenesis, etc54. Previous studies report that CENPA is significantly overexpressed in hepatocellular carcinoma (HCC) tumor tissue. CENPA is associated with overall survival (OS), disease-free survival (DFS), relapse-free survival (RFS), and progression-free survival (PFS) of HCC57. Key functions and potential regulatory pathways of DEP domain-containing protein 1 (DEPDC1), a newly discovered gene related to cancer and cell cycle, have been reported in bladder cancer, and other human cancers, such as breast cancer and prostate cancer. Previous studies report overexpression of DEPDC1 in several tumors and reported that it drives tumor pathogenesis through multiple potential mechanisms60. HMMR and KIF20A are important regulators of mitosis and exhibit oncogenic properties in various cancers through multiple mechanisms64. The Warburg pathway enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 4 (PFKFB4) is implicated in regulation of diverse biological processes and plays an important role in regulating glucose metabolism and guiding macromolecule biosynthesis to promote proliferation of cancer cells. Several studies screened and identified PFKFB4 as a poor prognostic factor for multiple tumors through high-throughput analysis67. STMN1 is an oncogene and its aberrant upregulation is closely related to different kinds of tumors69. STMN1 is an independent predictive factor of poor outcome, it is upregulated in hepatocellular carcinoma and promotes migration, invasion, and EMT by activating PI3K/AKT pathway69.Several studies report that some gene signatures are derived from glucose metabolism, including several genes that are critical for glycolysis and are overexpressed in glycolytic cancer cells. Aurora kinase A (AURKA) is an important regulatory protein involved in regulation of chromosome congression/alignment, regulation of chromosome segregation, and regulation of spindle dynamics70. Metabolic mode switching from aerobic oxidation to anaerobic glycolysis is an important characteristic of hepatocellular carcinoma. The Warburg effect results in accumulation of the lactic acid, as the final product of glycolysis. The acidic microenvironment thus mediates immune escape. The acidic environment is formed by continuous accumulation of highly acidic substances such as lactic acid and ketone bodies72. Studies report past decade have found that aerobic glycolysis and the resulting acidification of tumor microenvironment (TME) exert specific inhibitory effects on antitumor immune response mediated by T cells and the activity of tumor-infiltrating myeloid cells. Therefore, targeting sugar metabolism and/or lactic acid production and secretion is an attractive anticancer treatment strategy74. Moreover, the current study explored inflammatory infiltration landscape in HCC tissues based on 22 immune cells using the CIBERSORT tool75. T cells CD8 are important immunomodulatory cytokines, that play a critical role at the interface between innate and adaptive immunity, mainly in antitumor immune response82. Macrophages M2 promote tumor progression and poor prognosis, mainly promoting metastases in target organs84. Thee findings showed that enhanced glycolytic activity contributes to the highly acidic environment in the TME. Tumor-reactive T cells are suppressed resulting in loss-of-function in the acidic TME induced by glycolytic activity, resulting in a critical barrier for efficacy of cancer immunotherapy87. Most immunotherapies target the immune system but not cancer, therefore, immunotherapies are promising foundation for development of treatment regimens for several tumor types. However, complexity of the metabolic regulation of immune cell subsets and effect of the TME may have significant implications for efficacy of these therapies. In the current study, 8 genes implicated in prognosis of HCC were identified. These genes are important regulators in glucose metabolism and energy production, mainly in glycolysis process. These findings indicate that glycolysis pathway is required for proliferation of most cancer cells and for energy production in reprogramming of tumor microenvironment characteristics. Therefore, therapeutic agents can be developed that target glycolysis pathway, thus controlling tumor progression and improving patients prognosis. These findings have remarkable prognostic and therapeutic implications for HCC patients.Dysregulation of glycolytic pathway is a hallmark of oncogenic potential in tumor biology. Reprogrammed glycolytic and mitochondrial pathways are hallmarks of altered energy generation system of malignant cells, and lead to abnormal survival and proliferation of tumor cellsNotably, the current study has some limitations. First, univariate Cox regression analysis and the LASSO method were used to filter glycolytic genes associated with clinical outcomes of HCC and a prognostic model was built through multivariate Cox-PH regression analysis. In the linear regression model, adjustments were made stepwise in major groups, to reveal which variables contributed the most to confounding, and some important components with similar contributions may be ignored. Second, a prognostic risk model was developed and validated based on public databases, which was not verified by prospective clinical trials. Further studies should consider some traditionally recognized clinical factors, which have a significant effect on tumor progression and prognosis of HCC patients. Factors related to a clinical interaction may be missed, such as tumor volume, TP53 mutation, CTNNB1 mutation, lifestyle, patient follow-up time, and relevant therapeutic information. These factors have an effect on the accuracy of the prediction of the model. Therefore, the predictive performance of predictive models based on glycolysis-related gene signatures should be explored further in subsequent studies. In addition, well-designed, prospective, multicenter collaborative trials should explore if clinical decision-making based on these approaches leads to improved clinical risk stratification. To circumvent these limitations, more in vivo and in vitro studies should be conducted to verify the findings of the current study and explore more complex and in-depth biological mechanisms.The current study developed and optimized a novel 8-gene signature for identifying outcomes and recurrence in HCC patients. This predictive model improves accuracy of predicting patient prognosis. Moreover, the 8-gene signature serves as an independent prognostic factor and was superior compared with other clinicopathological features. A nomogram was established based on the GRGPI signatures and clinicopathological characteristics, which significantly improved prognosis in terms of discrimination and effectiveness of clinical decision-making. The findings of the current study provide a basis for prognostic stratification for designing prospective trials of risk-adapted therapies and surveillance strategies. In addition, the findings have clinical implications, and can be used to ensure that more patients benefit from additional systemic treatment.Supplementary Information.


Droplet-based microfluidics has been widely applied in enzyme directed evolution (DE), in either cell or cell-free system, due to its low cost and high throughput. As the isolation principles are based on the labeled or label-free characteristics in the droplets, sorting method contributes mostly to the efficiency of the whole system. Fluorescence-activated droplet sorting (FADS) is the mostly applied labeled method but faces challenges of target enzyme scope. Label-free sorting methods show potential to greatly broaden the microfluidic application range. Here, we review the developments of droplet sorting methods through a comprehensive literature survey, including labeled detections [FADS and absorbance-activated droplet sorting (AADS)] and label-free detections . We highlight recent cases in the last 5 years in which novel enzymes or highly efficient variants are generated by microfluidic DE. In addition, the advantages and challenges of different sorting methods are briefly discussed to provide an outlook for future applications in enzyme DE. The variants are expressed in vivo/in vitro or in cell-free system and then selected for improved properties. An effective assay requires tight linkage of genotype and phenotype, so that promising variants could be subjected to further cycles of optimization . The whole process starts from a mutant library of one existing enzyme, or 4 variants with manual operation or ~106 variants with robots per day  is commonly defined as screening no <100,000 samples per day  usually drags droplets with active enzymes inside toward a sorting channel by high-voltage electric pulses. The sorting methods determine the selection threshold for DEP at the junction. Many researches have been done in droplet generation step in the last decade, including w/o droplets  detection. However, there have emerged other novel sorting methods for on-chip droplet HTS sorting recently and shows the trend of label-free sorting. In this work, both traditional labeled sorting methods and new label-free approaches in the past 5 years have been reviewed.Labeled sorting is defined as sorting methods based on characteristics that do not result directly from the enzymatic reaction itself. Chemicals or tagged groups are added into the reaction so that labeled sorting methods could work on them or their derivatives. Fluorescence-activated droplet sorting (FADS) and absorbance-activated droplet sorting (AADS) are two commonly applied labeled sorting methods and also regarded as optical sorting. FADS relies on the fluorophore yield or fluorescent tagging in the droplet. AADS is based on changes in UV or visible light absorption. Its absorption changes are always caused by absorbing reagent yield in the enzymatic reaction or in its coupled assays.FADS is based on a very similar principle to fluorescence-activated cell sorting (FACS). FACS has been considered as the gold standard for single cell sorting  to fluorophore DiFMU by xylanase to evolve xylanase-producing Pichia pastoris and screened out a 1.3-fold mutant. Fenneteau et al. , which is detected by the downstream oxidation of a fluorogenic dye Amplex by horseradish peroxidase. This coupled-enzyme assay is applicable to other oxidases or relative enzymes, like l-asparaginase   and the reduction of the water-soluble tetrazolium salt 2-(4-iodophenyl)-3-(4-nitrophenyl)-5--2H-tetrazolium (WST-1) to give the absorbing dye WST-1 formazan. The sorting frequency could achieve ~1 million droplets per hour. Theoretically, AADS could have much more applications since most small molecules exhibit absorption in the UV and visible regions of electromagnetic spectrum.AADS helps extend the application of microfluidics, by breaking the exclusive boundary of fluorescent readouts. Gielen et al.  firstly AADS has the inert disadvantage of reduced optical pathlength together; its sensitivity is three to four magnitude lower than that of FADS . To addrE. coli cells will be in one 100-pl droplet after single-cell cultivation overnight. Through increasing enzyme molecules, the reaction rate of phenylalanine dehydrogenases (PheDH) improved 12-fold as detected in absorbance assay of droplets. The same strategy might also be applied for other less sensitive sorting methods.Instead of improving the sensitivity of AADS itself, Zurek et al.  recentlyApart from the two common optical (labeled) sorting approaches, a trend of developing label-free detections emerged, which uses intrinsic physical or chemical biomarkers to separate and sort cells. Several major label-free sorting methods are like electrochemical detection, mass spectrometry (MS), and Raman and nuclear magnetic resonance (NMR) coupling with a microfluidic chip. Different from easy coupling of optical sorting set up with microfluidic devices, the following label-free sorting methods often need a specific design or coupling techniques.Electrochemical detection is label free and can be applied to complex samples without interference from suspension, autofluorescence, or staining. Due to the limitation of detection electrode surface size, which should not be larger than the droplet, the size of droplets applied is normally limited to nanoliter. The detection device on a microfluidic chip could also be coupled with a DEP sorting device, by converting electrochemical signal into digital signal and then into alternating current (AC) signal to turn on DEP sorter.Streptococcus mutants was screened  method was developed even without extra electrodes to differentiate droplets of various interfacial tension due to different pH values in droplets   and high accuracy (~98%) were achieved.Unlike MS, RADS is non-invasive but also label-free. RADS could sort cells at a rate of hundreds of cells per minute  were discovered by applying this RADS setup  substrates to improve the sensitivity. Sobota et al.  applied 0 homogeneity.NMR could give information from all states of matter in a wide range of temperatures. Unlike MS, NMR offers an option of a completely non-invasive metabolomic readout. Theoretically, NMR is obviously disadvantageous in its low intrinsic mass sensitivity, which means normal concentrations below 1 mM are hard to observe ,” and “sorting” steps on a chip. For the first three steps, there have been many technological advances in the last decade, and there have already been commercial droplet generation toolkits and droplet manipulation devices like a picoinjector  would be a good choice if richer information is required apart from the enzymatic reaction itself  (Frenzel and Merten, There are also some attempts to extend traditional DEP sorter to multiple channels, so that the whole system could reach a higher efficiency. Frenzel and Merten  designedde novo synthetic enzyme screening. Sorting methods, as the main step in DE, determine the efficiency of the whole system. Sorting devices are developing toward standardized modules compatible with different instruments and microfluidic chips. Combinations of different sorting methods could help gain multiplex perspectives into enzyme and boost a wide range of application.DE of enzymes toward high specificity and efficiency is significant to both scientific researches and industrial applications. Droplet-based microfluidics paves a cheap and convenient way for enzyme DE with ultra-high throughput, and meanwhile, it is becoming a useful tool for XF and FM conceived the concept, conducted literature survey, and drafted and revised the manuscript. All the authors organized the figures and approved them for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Colorectal cancer that has become metastatic continues to have a poor outlook. Many patients will undergo intensive chemotherapy, often with limited effects but many side effects. In other types of cancer, immunotherapy has shown to be effective by targeting the immune cells of the patient and restoring their function to fight off cancer. This review explores the up-to-date evidence for immunotherapy in metastatic colorectal cancer. We have discussed in which patients this treatment is effective, but also why this has not been effective in a large number of patients. By summarizing the key components of the immune cells within the proximity to the tumour and its areas of spread, we discuss how these components can be targeted. Areas of future research are also highlighted, which includes combining immunotherapy with current treatments, such as surgery, radiotherapy and chemotherapy as well as some recent advances from basic and translational studies that promises to improve outcomes in these patients.Metastatic colorectal cancer carries poor prognosis, and current therapeutic regimes convey limited improvements in survival and high rates of detrimental side effects in patients that may not stand to benefit. Immunotherapy has revolutionised cancer treatment by restoring antitumoural mechanisms. However, the efficacy in metastatic colorectal cancer, is limited. A literature search was performed using Pubmed (Medline), Web of Knowledge, and Embase. Search terms included combinations of immunotherapy and metastatic colorectal cancer, primarily focusing on clinical trials in humans. Analysis of these studies included status of MMR/MSS, presence of combination strategies, and disease control rate and median overall survival. Evidence shows that immune checkpoint inhibitors, such as anti-PD1 and anti-PD-L1, show efficacy in less than 10% of patients with microsatellite stable, MMR proficient colorectal cancer. In the small subset of patients with microsatellite unstable, MMR deficient cancers, response rates were 40–50%. Combination strategies with immunotherapy are under investigation but have not yet restored antitumoural mechanisms to permit durable disease regression. Immunotherapy provides the potential to offer additional strategies to established chemotherapeutic regimes in metastatic colorectal cancer. Further research needs to establish which adjuncts to immune checkpoint inhibition can unpick resistance, and better predict which patients are likely to respond to individualised therapies to not just improve response rates but to temper unwarranted side effects. Colorectal cancer (CRC) is the second greatest cancer threat to life in the Western World . Very feBRAFV600E mutant patients sought to address the role of EGRF pathway reactivation in resistance to therapy. Using a combination of standard chemotherapy, encorafenib (BRAF inhibitor) and MEK inhibitor, binimetinib, significantly improved survival in this dismal prognosis disease [However, hope exists for patients with colorectal cancer that targeted therapies can start to be applied to specific subgroups of patients. The recently coordinated BEACON study on the failure of BRAF directed inhibition alone in Despite these recent advances, surgery provides the potential for cure in patients with metastatic disease. However, sadly most patients are not candidates for resection due to the burden or distribution of disease. Colorectal cancer oncological therapies must look to capitalise on the successes achieved in other cancers with immunotherapies to enhance patient survival, combining these in the future where possible with surgical resection to benefit patients with lower volume oligometastatic disease. An opportunity exists to bring forward treatments that are being used in the most advanced disease to provide opportunities to achieve long-term survival in more patients with borderline resectable/resectable metastatic disease. This review will focus on the mutational and transcriptomic landscape of colorectal cancer and current understanding of drug positioning in disease subtypes. We will illustrate previous and current trials of immune oncological agents in mCRC and explore avenues for combinatorial immune oncological approaches in this disease.Patients presenting with colorectal cancer can be classified by stage of disease. Patients are increasingly presenting early through screening with pre-cancerous polyps or localised disease (stages 0–III). However, there are still a significant proportion of patients that present with stage IV disease, which is frequently metastatic and not surgically operable. Whilst local excision, major colorectal resection and metastasectomy have an important role to play, it is the patients with metastatic colorectal cancer (mCRC) in whom systemic therapeutic strategies offer poor response rates and limited potential for disease control. Increasing understanding of major genotypic and phenotypic variation in colorectal cancer permits a tailored strategy to help predict likely responses to a given agent, which maximises the benefit whilst minimising the suffering from side effects and toxicity in patients who do not stand to benefit.The most common mutations that are observed in colorectal tumours include APC (40%), TP53 (65%), KRAS (50%) and SMAD4 (12%), amongst many others ,8. The mB-type RAF (BRAF) mutations occur in 5–15% of mCRC and are associated with right-sided colon cancer . Patient6 DNA bases) which offers a high number of neoantigens which are recognised as foreign by the local patrolling immune cells [Mismatch repair (MMR) deficiency occurs through mutations or promoter hypermethylation in MLH1, MSH2, MSH6 or PMS2 . Due to ne cells ,21. As ane cells ,23. Thisne cells .Approximately 15–30% of colorectal cancers derive from the serrated pathway in contrast with the conventional adenoma–carcinoma sequence . BRAF anWith the increasing understanding of the heterogeneity of colorectal cancer, the CRC consensus molecular subtype (CMS) classification has been agreed upon through the study of patients at multiple centres and whole transcriptomic analysis of tumour tissue . CMS1  after engagement with antigen-presenting cells (APC) to destroy tumour cells through perforin and granzyme granules. CD4 cells, T-helper-1 cells, support the response through cytokine production, such as IL-2 and IFN gamma, to promote T-cell maturation and activation of macrophages. Galon and colleagues showed that increased CD3 and CD8 T-cells at the tumour core and the invasive front, measurably by the Immunoscore, were associated with better prognosis . Indeed,T-regulatory (Treg) cells infiltrate the tumour microenvironment, which suppresses T-cell activation and the antitumoural response. Tregs produce suppressive cytokines, such as TGF beta, and express checkpoint proteins, such as PD-L1, LAG3 and CTLA4. Whilst increased numbers of CD3 and CD8 T-cells to the invasive front are associated with better outcomes in CRC, increased numbers of Tregs are associated with worse outcomes . AdditioNatural Killer (NK) cells are a critical part of the innate immune response, which function to rapidly cause programmed cell death in cancerous cells lacking MHC I. Increased numbers of tumour-infiltrating NK cells, as well as T cells, in the specimens of resected colorectal liver metastases after neoadjuvant chemotherapy are predictive of overall survival .Dendritic cells (DCs) are resident, specialised APCs which coordinate the innate and adaptive responses. They act to promote tumour rejection through priming effect T-cells and phagocytose tumour antigens. DCs have been a target for vaccines against tumour antigens .Following the extravasation of circulating monocytes, these cells become macrophages. They are part of the innate response and are the centrepiece to cancer-associated inflammation ,36. ActiMyeloid-derived suppressor cells (MDSCs) represent an immature population of bone marrow-derived cells which are broadly granulocytic (G-MDSC) or monocytic (M-MDSC). Following activation, immunosuppressive functions are acquired through upregulation of Arg1 and inducible nitrox oxide synthase  within the TME promoting T cell apoptosis, inhibiting T cell function and suppressing T cell infiltration . As a reNeutrophils are classically the first immune cell recruited to injury or inflammation. Tumour-associated neutrophils (TANs) demonstrate a similar phenotype in the early stage of cancer. These N1 TANs help educate T cells to reject tumours, promote tumour cell apoptosis through TRAIL and secrete reactive oxygen species (ROS). However, N2 TANs which are associated with later-stage cancer, driven by TGF beta, can promote angiogenesis, release MMP8 to degrade the ECM and secrete leukotrienes which expand tumour cells and worsen survival .Thus, the effect of colorectal cancer-associated inflammation leads to dysregulation of multiple signalling pathways and stimulations, a chronic inflammatory process that influences the local immune cell populations and their function and phenotype. Failed antitumoural immunity and an immunosuppressive tumour microenvironment lead to the progress of the primary tumour and create a systemic pre-metastatic niche to promote the spread to local and distant sites. This is summarised in Immune checkpoint inhibition has been the most successful form of immunotherapy for solid tumours. There has been strong evidence leading to a new era of therapy in malignant melanoma and lung cancer with the use of popular immune checkpoint inhibitors, which are being applied to colorectal cancer. The most promising results have been demonstrated in patients with deficient MMR status and MSI-H which is discussed in the next section. The most common immune checkpoint inhibitors in use and their molecular target are summarised in Immune checkpoint inhibition to target metastatic colorectal cancer was investigated by Le et al. in the Keynote-016 study. This phase I study of chemorefractory patients included those with both deficient and proficient MMR status who were treated with pembrolizumab . In patiFurther PD-1 inhibition using nivolumab was utilised in the Checkmate-142 study. Anti-PD-1 monotherapy was used in 74 patients with deficient MMR status . An objeA further cohort of the Checkmate-142 study investigated 119 patients undergoing combination therapy with anti-PD-1 and anti-CTLA-4 immunotherapy . In this group of MMR deficient patients, response rates of 55% with disease control rates (DCR) of 80% were observed. Progression-free survival of 71% at 1 year was achieved, with overall survival (OS) at 85% at 1 year. The combination was more effective compared with nivolumab monotherapy . However, 32% of patients experienced grade 3 or grade 4 side effects [Further evidence for checkpoint inhibition in MMR deficient, microsatellite instability (MSI)-high patients was reported in the Keynote-164 study in 2020 . One hunThe Keynote-177 study was recently published in NEJM . This phA Canadian study recently reported in JAMA Oncology investigated combination immunotherapy with tremelimumab and durvalumab compared with best supportive care in patients with metastatic refractory CRC . In thisThus, there is a rationale for upfront PD1 directed immunotherapy in MMR deficient patients. However, late-stage patients who are refractory to other chemotherapies fail to benefit.The efficacy of immune checkpoint inhibitors has been far less successful in microsatellite stable (MSS) colorectal cancers. The Keynote-016 study discussed in the earlier section had included 18 patients with chemorefractory metastatic colorectal cancer and proficient MMR status were treated with pembrolizumab and found no responses in these patients .Shahda and colleagues presented preliminary results of a phase II study combining pembrolizumab alongside chemotherapy (mFOLFOX6) irrespective of MMR status . They foDue to the poor results seen in immune checkpoint inhibition, this has led to a number of studies using various combination strategies aiming to improve the efficacy of immunotherapy in MMR proficient metastatic colorectal cancer . CombinaIn patients with microsatellite instable-MMR deficient metastatic colorectal cancer, the high tumour mutational burden with high neoantigen rates creates increased MHC I expression and increased numbers of infiltrating CD8 T-cells. The concept of combining immunotherapy with an additional modality attempts to synergistically stimulate the tumour microenvironment to increase interferon-gamma and other cytokines and promote immune cell recruitment.In order to assess the role of atezolizumab alongside traditional chemotherapy, the MODUL trial used a biomarker-driven approach . FollowiA MEK inhibitor (cobimetanib) was used in combination with atezolizumab in the IMblaze 370 trial . This inThe combination of cobimetanib and atezolizumab was reported by Bendell and colleagues in a phase 1b trial in 2016 . This suTargeting carcinogenic embryonic antigen on tumour cells and CD3 T cells is under investigation which aims to promote T cell infiltration and upregulate PD-L1 . A phaseThe immunosuppressive enzyme idoleamine 2,3-dioxygenase (IDO) has been a target of inhibition in combination with nivolumab in the ECHO-204 study . This inAs immune checkpoint inhibitors have low efficacy alone in patients with proficient MMR metastatic colorectal cancer, investigators are studying the potential combination of radiotherapy (RT) and immunotherapy. Segal and colleagues studied 26 patients with unresectable or recurrent metastatic colorectal cancer who have already failed at least two standard therapies . They reThe Checkmate-142 study, which was discussed earlier in this review, also included additional treatment arms utilising combinations of immunotherapy. This included nivolumab and anti-LAG3 antibodies (another co-inhibitory protein), and combination with nivolumab, ipilimumab and cobimetanib (MEK inhibitor) and nivolumab with daratumumab (anti-CD38 antibody)—a protein previously associated with tumour cells of myeloma.New targets for therapy continue to be identified via ongoing translational research to identify underpinning molecular mechanisms of immunopathology. Macrophages within the tumour microenvironment have been identified as key players in tumour progression, where an M2 phenotype creates resistance to anti-PD-1/PD-L1 agents . In precOther emerging therapies include antibodies targeting OX40 and CD73 immunotherapy. OX40 (CD134) is a member of the TNF receptor superfamily with a role in co-stimulatory immune responses, which are expressed on T-cells. This interacts with OX40 ligand on antigen-presenting cells resulting in activation of NFKB and NFAT. Given the high levels expressed on Tregs and increases in the generation of those and similar immunosuppressive T cell subsets, OX40 inhibition aims to counteract tumour-infiltrating Tregs and thus aims to improve immune checkpoint efficacy . CD73 isA difficulty remains in defining which patients will respond best to immunotherapy. Consistently across different tumour types, tumour mutational burden is a strong predictor, particularly in melanoma and non-small cell lung cancer . The morMany basic science and clinical researchers are exploring different avenues in order to make stepwise gains in the advancement of care for patients with metastatic colorectal cancer. New technologies allow for better characterisation of the genomic and molecular mechanisms underpinning colorectal cancer and the pathological responses of the local and systemic immune components. More realistic preclinical models using autochthonous models, organoids and patient-derived xenografts in animals permit the study of novel agents which target not just inflammatory pathways but also have potential to reprogramme pro-tumourigenic pathways regulated by cancer-associated lymphocytes, macrophages, neutrophils and cancer-associated fibroblasts.Tauriello and colleagues adopted a model of murine metastatic colorectal cancer with APC, p53, KRAS and TGF-β mutations . This prThe influence of gut microbiota on the tumour microenvironment and, therefore, responses to immunotherapy are wide-ranging, varied, and yet to be fully explored. The microbiome in the gut is predominated by anaerobes, different species of whom have been shown to have influences on different immune cells within tumours, in addition to interacting directly with immune checkpoints. These interactions are thoroughly described in previous review material . PersonaPatients with metastatic colorectal disease range from those with low burden oligometastatic hepatic disease to overwhelming, aggressive widespread metastases. Given the range of colorectal cancer subtypes based on the location of primary and CMS phenotypes, then it is likely that it will take different strategies to improve survival for stratified patient groups . For exaMetastatic colorectal cancer continues to have a dismal prognosis and is a major cause of death in the Western world. Established regimes for chemotherapy have relatively poor rates of response yet cause significant side effects which impair quality of life in end-stage disease. Immunotherapy represents a new dawn of therapeutics, which could not only improve the balance of benefit and side effect profile but also may offer better responses in certain patient groups. We have discussed the goals of the clinical and scientific community in discovering how to translate the benefits and efficacy of immune checkpoint inhibitors and other novel agents to the right groups of patients.


The generation of high affinity antibodies is a crucial aspect of immunity induced by vaccination or infection. Investigation into the B cells that produce these antibodies grants key insights into the effectiveness of novel immunogens to induce a lasting protective response against endemic or pandemic pathogens, such as influenza viruses, human immunodeficiency virus, or severe acute respiratory syndrome coronavirus-2. However, humoral immunity has largely been studied at the serological level, limiting our knowledge on the specificity and function of B cells recruited to respond to pathogens. In this review, we cover a number of recent innovations in the field that have increased our ability to connect B cell function to the B cell repertoire and antigen specificity. Moreover, we will highlight recent advances in the development of both ex vivo and in vivo models to study human B cell responses. Together, the technologies highlighted in this review can be used to help design and validate new vaccine designs and platforms. The humoral arm of the immune system plays a key role in vaccine-mediated immunity through the production of protective high-affinity neutralizing and non-neutralizing antibodies against diverse pathogens . The antRecent developments in B cell technologies have allowed for investigation into novel dimensions of B cell biology. Innovative new techniques have allowed for the connection of the B cell repertoire to serum antibody responses ,7, or thPlasmodium falciparum [Although much work has been done to investigate the antigen specificities of B cells and B cell differentiation, little is known about the relationship between the two or how they may differ in the context of heterogenous B cell populations in distinct tissues. The relationship between antigen specificity and B cell identity can have significant clinical ramifications, such as the discovery of antigen-specific atypical memory B cells (AtM) in certain cases of chronic pathogen exposure such as the malaria-causing parasite lciparum  or HIV [lciparum  that exhlciparum . The rollciparum . There ilciparum ,19,20, blciparum . The lacThe advent of cytometry by time of flight CyTOF, A has beeCellular indexing of transcriptomes and epitopes by sequencing  imaging, that have great potential to reveal migration patterns of maturing B cells within lymphatic tissues. The rapidly evolving field of spatial transcriptomics can reveal single-cell transcriptomic levels of distinct mRNAs in distinct tissue niches ,34,35,36Electron microscopy polyclonal epitope mapping (EMPEM) is a technique that may be used to characterize the epitope targeting of polyclonal sera in response to vaccination or infection. Since its development and initial application to characterizing polyclonal antibody (pAb) responses to HIV-1 BG505 Envelope trimer immunizations , it has Rag1, Rag2, and Prkdc that lead to limited, functionally impaired B and T cell populations and inhibit natural killer (NK) cell development [Il2rg mutation [Rag–/– Il2rg–/– SirpaNOD (BRGS) mice, referred to as BRGST mice [Il2rg gene, the induced TSLP expression rescues SLT development in BRGST mice. As a result, BRGST mice successfully develop physiologically organized SLT, including a larger thymus, as well as mount an improved adaptive immune response to vaccination relative to non-TSLP-expressing mice [Human immune system (HIS) mice may be used to explore the behavior of human immune cells and immune tissues in vivo. HIS mice are generated by xenografting human hematopoietic stem cells and other human tissues into mice with severe genetic immunodeficiencies, inducing tolerance to the xenografted tissues while simultaneously eliminating murine immune cell contributions to experimental observations. Various immunodeficient mice exist for such experiments and typically use mutations in the interleukin-2 (IL-2) receptor γ-chain locus and in the receptor rearrangement proteins elopment . Howevermutation . BecauseGST mice . Becauseing mice . This noBCR knock-in (BCR-KI) mice are an established experimental model, of interest both for understanding antigen-specific B cell development and function in vivo and to test immunogens that can engage particular BCRs. Immunogens able to engage the germline precursors of known broadly neutralizing antibodies (bnAbs) are of great interest for vaccine design. Recently, BCR-KI models have been used to validate novel immunogens that are meant to recruit bnAb precursor B cells against HIV-1 . NotablyBCR-KI mice have been traditionally generated via gene modification in embryonic stem (ES) cells. However, these methods require significant technical skill and equipment as well as long periods of time and planning. The CRISPR/Cas9 knock-in approach facilitates the generation of BCR-KI mice because of its speed and simplicity ,50. UsinAn alternative model for the interrogation of the human immune system outside of humanized mice is ex vivo organoids, namely tonsils. By using tonsil organoids as an experimental system, it is possible to effectively study key aspects of the immune response to vaccination in humans, such as affinity maturation, plasmablast differentiation, class-switch recombination, and memory induction within germinal center reactions . As vaccThe bone marrow is another site of great interest in responses to vaccination because it is where long-lived plasma cells (LLPCs) predominantly reside ,56. LLPCMost investigation into B cell biology in the modern era has been done by isolating B cells from the blood using single cell sorting techniques when probing questions about somatic hypermutation or the antigen specificities of monoclonal antibodies. These types of experiments have granted and continue to grant many novel discoveries, but do not provide insight into the in vivo behavior of B cells, including the entry and participation of B cells into germinal centers. These sites represent key locations for affinity maturation to generate a highly protective antibody response and are thus of great interest for vaccine development. Germinal centers may form in ex vivo models of the human immune system, such as the previously mentioned tonsil organoids. However, most vaccines do not stimulate cells in the tonsils, but rather cells in the draining lymph nodes from the site of vaccination . As suchUsing ultrasound-guided fine-needle aspirates of the draining lymph nodes, it is now possible to safely and minimally invasively probe germinal centers in vivo during the course of the immune response to vaccination in humans ,62. FineLastly, techniques to investigate the spatial organization of single-cells within tissue have continued to yield increasingly high-resolution insight into the transcriptomic profiles of microanatomical structures ,65. ThisIn summary, the novel techniques and innovations discussed in this review have great potential to deepen our understanding of B cell biology at an unprecedented pace. With relative ease, one could sew together a number of these techniques into a striking, multifaceted series of experiments  to compr