Following dramatic success in many types of advanced solid tumors, interest in immunotherapy for the treatment of colorectal cancer (CRC) is increasingly growing. Given the compelling long-term durable remission, two programmed cell death 1 (PD-1)-blocking antibodies, pembrolizumab and nivolumab (with or without Ipilimumab), have been approved for the treatment of patients with metastatic colorectal cancer (mCRC) that is mismatch-repair-deficient and microsatellite instability-high (dMMR-MSI-H). Practice-changing results of several randomized controlled trials to move immunotherapy into the first-line treatment for MSI-H metastasis cancer and earlier stage were reported successively in the past 2 years. Besides, new intriguing advances to expand the efficacy of immunotherapy to mCRC that is mismatch-repair-proficient and low microsatellite instability (pMMR-MSI-L) demonstrated the potential benefits for the vast majority of mCRC cases. Great attention is also paid to the advances in cancer vaccines and adoptive cell therapy (ACT). In this review, we summarize the above progresses, and also highlight the current predictive biomarkers of responsiveness in immunotherapy with broad clinical utility. Colorectal cancer (CRC) is the third most common cancer and the second cause of cancer-related death worldwide. It is considered as a global health issue with an urgent unmet need of new therapeutic strategies Immunotherapy is aiming at harnessing the immune system to battle cancer. Immune checkpoint inhibitors (ICIs), modulating the interaction of T cells, antigens-presenting cells (APCs) and tumor cells to help unleash suppressed immune responses, emerged as a very effective therapy for patients with mCRC that is mismatch-repair-deficient (dMMR) or microsatellite instability-high (MSI-H) (termed dMMR/MSI-H mCRC). Owing to the efficacious, stable and durable responses, pembrolizumab and nivolumab (with or without Ipilimumab) were approved by US Food and Drug Administration (FDA) for the treatment of these patients. However, mCRC is characterized by insufficient mutated tumor antigens Mismatch repair (MMR) plays a critical role in maintaining DNA fidelity Nearly 15% of all CRCs are dMMR-MSI-H, and the percentage is associated with tumor stage Pembrolizumab and nivolumab (with or without Ipilimumab) are clinically approved in 2017 as the second-line treatment for patients with dMMR-MSI-H mCRC. KEYNOTE016 (NCT01876511) is a phase II trial using pembrolizumab to treat patients with refractory mCRC Given the cogent efficacy of immunotherapy in dMMR-MSI-H mCRC, the interest in exploring the potential of immunotherapy for the first line treatment of CRC patients, even in earlier-stage, is increasingly growing. The promising data of several randomized clinical trials has elicited tremendous excitement KEYNOTE177 trial has attracted great attention, as an international randomized phase III study to assess pembrolizumab monotherapy verse standard of care in previously untreated stage 4 dMMR-MSI-H CRC patients The efficacy and safety of first-line treatment with combined nivolumab and low-dose ipilimumab in patients of dMMR-MSI-H tumors were evaluated in CheckMate142 Additionally, COMMIT (NCT02997228) is an undergoing randomized phase III trial of mFOLFOX6/bevacizumab combination with or without atezolizumab in 347 previously untreated patients with dMMR-MSI-H mCRC. The primary end point is PFS. The secondary end points are OS, ORR, DCR and incidence of adverse events Adjuvant chemotherapy is indispensable for stage 3 CRC. Aiming to evaluate the potential efficacy of immunotherapy in adjuvant treatment, ATOMIC (NCT02912559), an ongoing phase III randomized controlled trial, enrolled 700 patients with stage 3 dMMR-MSI-H colon cancer. The patients were divided into 2 groups, which received 6 months of FOLFOX or FOLFOX plus atezolizumab for 6 months followed by atezolizumab alone for 6 months, respectively High efficacy of neoadjuvant immunotherapy in early-stage CRC has been proved. NICHE (NCT03026140), a phase II exploratory study, enrolled 40 patients with stage 1-3 colon cancer, including 21 patients with dMMR tumors and 20 with pMMR tumors NRG-GI002 (NCT02921256) is a randomized phase II trial to study the efficacy of veliparib or pembrolizumab when combined with chemotherapy and radiation therapy in patients with locally advanced rectal cancer (LARC) VOLTAGE (NCT02948348) is a phase I b/II open-label single-arm study to investigate the safety and efficacy of sequential use of neoadjuvant immunotherapy after chemoradiotherapy with capecitabine and subsequent surgery in patients with locally advanced resectable rectal cancer Hopefully, neoadjuvant immunotherapy has the potential to become standard therapy of dMMR-MSI-H CRC in the near future.Unlike dMMR-MSI-H CRC, pMMR-MSI-L tumors, which contribute to 95% of all mCRC cases, harbor a much lower mutation burden and poor recruitment of immune cells, leading to an unsatisfactory response to ICIs. With the deepening understanding of tumor mircoenvirnment of CRC, new discoveries and strategies of immune modulation have been explored and tested in pMMR-MSI-L mCRC patients to overcome primary ICI resistance.Combination of PD-1/PD-L1 and CTLA-4-blocking antibodies has the potential to offer synergistic benefits to patients Preclinical data suggested that the anti-tumor activity of ICIs may be enhanced by inhibiting PGE2 synthesis Panitumumab, a monoclonal antibody (mAb) targeting the epidermal growth factor receptor (EGFR), is a standard therapy in KRAS/NRAS/BRAF wild-type mCRC, the resistance of which is associated with increased CTLA-4 and PD-L1 expression Preclinical work highlighted that radiotherapy (RT) could cause immunogenic cell death (ICD), which subsequently cause release of damaged-associated molecular patterns (DAMPs), increase of antigen presentation by APCs, priming of T cells and antitumor effects via the abscopal effect Although interim results of a single arm phase II study (NCT02437071) reported only 1 of 22 patients of pMMR-MSI-L CRC responded to the combination therapy of pembrolizumab and external beam radiation Preclinical data suggested that the inhibition of MEK, a downstream effector of the RAS-MAPK pathway, increased tumor expression of MHC-I and PD-L1, stimulated clonal expansion of peritumoral T cells, and enhanced anti-tumor activity of ICIs Accordingly, the combination strategy using the MEK inhibitor cobimetinib and the PD-L1 inhibitor atezolizumab was tested in a phase I b study (NCT01988896) Preclinical data suggested that anti-angiogenic agents could increase CD8+ T cell infiltration and enhance the anti-tumor activity of CD8+ T cell by upregulating the expression of PD-L1, reducing immunosuppressive cells , and enhancing interactions between APCs and dendritic cells Based on this theory, a phase I b study NCT01633970) showed promising results. Of 14 patients with pMMR/MSI-L rCRC received ICI plus anti-angiogenic agents (atezolizumab + bevacizumab), 1 patient had an objective response and 9 patients had stable disease 3970 showIn LEAP-005 (NCT03797326), a nonrandomized, open-label, phase II study, the efficacy and safety of treatment with combined pembrolizumab and Lenvatinib in patients with previously treated advanced non-MSI-H/pMMR colorectal cancer were preliminarily evaluated Bispecific antibodies are a new class of targeted therapeutics designed to bind two different sites on one antigen or two antigens. By targeting two different antigens,it simultaneously bridges the tumor cell and the T cell, hence enhance the intertumoral T cell infiltration and activation. CEA-CD3 is the first reported bispecific antibody showing significant efficacy in MSS CRC Another 3 emerging bispecific antibodies, TRAILR2-CDH17 (BI 905711) GCC-CD3 PF-07062119) and CD137-PD-L1 (FS222), all showed potent T-cell mediated anti-tumor activity in CRC in preclinical trials 62119 andThere are some alternative approaches of immune modulation worthy of high anticipation. One promising strategy is combining the immunotherapy with cancer vaccines, which may augment the host antitumor immune response Oncolytic viruses (OVs) can cause direct lysis of tumor cells and promote anti-tumor immune response by inducing immunogenic cell death Intestinal bacteria play crucial roles in various fundamental physiopathologic processes Although existing for more than a century, cancer vaccines have barely received response in patients with CRC. Recently, cancer vaccines have elicited renewed interest owing to the convinced efficacy of immunotherapy. Multiple trials aiming to find the right antigenic stimulants are under investigation.PolyPEPI1018 consists of 6 synthetic peptides with 12 unique epitopes derived from 7 conserved cancer testis antigens (CTAs) and optimized to induce long lasting CRC specific T cell responses Guanylyl cyclase C (GCC), a membrane-spanning receptor synthesizing the second messenger cyclic GMP (cGMP), is normally restrictedly expressed by intestinal epithelial cells and a subset of neurons, but universally expressed by metastatic colorectal tumors Autologous vaccine OncoVAX is an active specific immunotherapy, which utilizes the patient's own tumor cells containing all relevant tumor-associated antigens to activate the body's immune system to prevent tumor progression after surgery. A randomized phase IIIb trial is being conducted to evaluate the recurrence status of colon cancer following surgery in 500 patients with stage 2 colon cancer after treatment with OncoVAX (NCT02448173) Another highly anticipated novel treatment to stimulate tumor immunity is adoptive cell therapy (ACT). ACT selects either host cells exhibiting antitumor activity or host cells engineered with chimeric antigens receptors (CARs) or antitumor T cell receptors (TCR) to augment the host antitumor immune response The levels of CEA are low or absent in normal cells, but abundant in CRC The shed natural killer group 2D (NKG2D) ligands from tumor cells may downregulate NKG2D expression on NK and T cells, contributing to tumor immune escape Encouraging results on TILs were shown in a case report Immunotherapy has significantly changed clinical management of CRC. Nevertheless, there is urgent need to explore specific biomarkers to predict responsiveness of immunotherapy. Emerged biomarkers in CRC immunotherapy are classified into 4 main types: tumor mutations, pre-existing immune responses, PD-L1 expression and the microbiota. A limited number of candidate biomarkers in CRC are listed below.Tumor mutational burden (TMB) refers to the total number of somatic mutations per coding area of a tumor genome, which can measure all non-synonymous coding mutations in a tumor exome POLE and POLD1 are crucial for polymerase ε and δ encoding, respectively, which are essential for proofreading and fidelity in DNA replication Tumor infiltrating lymphocytes (TILs) are associated with improved survival in retrospective studies of CRC patients, particularly for cytotoxic CD8+ T cells Based on the concept of Immunoscore, the classification of tumors was redefined as hot, altered and cold, which are routinely used referring to T cell-infiltrated, inflamed but non-infiltrated, and non-inflamed tumors, respectively PD-L1, a co-inhibitory receptor ligand, is recognized as one of the most widely studied biomarkers assayed by immunohistochemical staining, while PD-L1 expression status has not proved to be associated with the efficacy of ICIs in CRC to date. In KEYNOTE016 (NCT01876511), a phase II trial investigating pembrolizumab in patients with refractory mCRC, PFS or OS appeared irrespective of PD-L1 expression level 2AR signaling 2AR signaling could be a promising pathway, through which the gut microbiota shows synergistic effect with immunotherapies. Further studies investigating the mechanisms of the gut microbiota regulating host antitumor immune response are needed.Gut microbiota have large effect on the efficacy of immunotherapies in multiple cancers, and the composition of the intestinal microbiome could be a potential predictor of the efficacy of ICIs Undoubtedly, great progress has been advanced in CRC immunotherapy over the past few years. Recently, FDA granted approval to pembrolizumab and nivolumab (with or without Ipilimumab) for the second-line treatment of patients with dMMR-MSI-H mCRC based on the cogent data from 2 phase II trials. Based on the compelling data of KEYNOTE177, FDA approved pembrolizumab for the first-line treatment of this subgroup in 2020. Multiple trials exploring potential benefit of ICIs as the first-line treatment for patients with dMMR-MSI-H CRC and those with early-stage dMMR-MSI-H CRC are underway and highly anticipated.However, the real critical challenge is to find ways to overcome immunotherapy primary resistance in the vast majority of patients with pMMR-MSI-L mCRC. To modulate immune cells and enhance the therapeutic efficacy, various ICIs-based strategies were tested in this subgroup, such as combinational therapy with antibodies blocking PD-1 and CTLA-4, ICIs combined with radiotherapy, ICIs combined with small molecule TKIs such as MEK inhibition and anti-angiogenic agents, and ICIs combined with bispecific antibodies. Although several early-phase trials presented promising data, further studies are warranted to validate the safety and efficacy. Alternative attempts to stimulate tumor immunity and enhance anti-tumor activity including ACT and cancer vaccines are promising areas of active research.Moreover, biomarkers-based treatment is the inevitable trend of immunotherapy. Selection criteria are indispensable to identify patients who may benefit from these agents. Although some of aforementioned promising biomarkers emerged, discovery and validation of sensitive and specific biomarkers remain as an extremely active area of investigation.With insights gained from additional trials aiming to develop effective therapeutic strategies, novel combination and biomarkers will likely help to guide clinicians towards a more personalized treatment for CRC patients. It is reasonable to believe that immunotherapy may soon change the treatment landscape for CRC.


A wealth of parent-report research shows adaptive functioning difficulties in autistic children, with parent-report influenced by a number of child factors. Adaptive functioning in autistic children is known to vary across settings; however, no research has yet explored factors influencing education professional-report. This study investigated the rate and profile of impairment, and child factors influencing education professional-reported adaptive skills in 248 autistic children. Twelve children were < 3 years (min age for available normative data on the adaptive function measure), so were removed from the analyses. Results replicated parent-literature; adaptive skills were negatively associated with age and informant-reported autism severity, and positively associated with nonverbal ability and expressive language. Adaptive functioning is important for real-world outcomes, e.g. educational attainment, independence, and support needs. Improving our understanding of adaptive functioning in the education context may support opportunities for shared learning and enhance personalised support .The online version of this article (10.1007/s10803-021-04930-z) contains supplementary material, which is available to authorized users. Adaptive functioning describes practical, everyday skills required to meet the demands of the environment. Such difficulties are commonly reported in autistic individuals  would show relative strengths in T-VABS-II Motor Skills. Differences in other domains were explored for the whole sample.CA, nonverbal and language ability, and informant-report of autism severity would show a significant relationship with T-VABS-II ABC and domain scores.In order to address this knowledge gap, we used the baseline data of the Paediatric Autism Communication Trial-Generalised . We used a repeated measures ANOVA to compare T-VABS-II domain scores.Based on findings from the parent VABS literature, we undertook a series of multiple linear regression analyses to investigate whether there were concurrent associations between T-VABS-II ABC/domain scores and child CA, autism severity, nonverbal ability, language ability, and child behaviour, which was supported by correlations between variables in our own data (Supplementary Table 1). All models were examined to ensure that they did not violate the assumptions of linear regression, including multicollinearity (using the VIF). While ADOS-2 CSS did not correlate with T-VABS-II standard scores, we included it to explore prior variable reports regarding direct assessments of autism severity. There were no significant differences between MSEL VR and FM AE scores  so we used a mean score to calculate the MSEL NVDQ . Participants performed significantly better on the ROWT than EOWT ; therefore, we entered these predictors separately, rather than deriving a language quotient.Descriptive statistics (including N) for each measure are presented in Table Regression analyses for T-VABS-II ABC and domain scores are reported in Table As far as the authors are aware, this is the first study to investigate education professional reporting of adaptive functioning in autistic children. We saw high rates of impairment, with at least 50% of children in the ‘low’ range on T-VABS-II ABC/domains (> 2SD below norm). Consistent with our hypotheses, we found relative strengths in Motor Skills, and teacher-report was significantly associated with child factors such as CA, parent-report autism severity, teacher-report behaviour problems, nonverbal and language ability.Our higher relative T-VABS-II Motor Skills performance is consistent with previous parent-report findings . Children with lower adaptive abilities require greater special education provision Below is the link to the electronic supplementary material.


Chirality in biomolecules is ubiquitous in our world, but oral nanomedicines constructed from chiral peptides are extremely rare, principally because of the immature nanofabrication and inadequate bioavailability of chiral nanostructures.Methods: To realize the oral administration of chiral peptides and break through their forbidden zone in intracellular space, a chiral-peptide supramolecular (DPAICP) camouflaging with the membrane from milk-derived extracellular vesicles (ME) was developed herein through an aqueous-based growth method of chiral peptide Au(I) infinite covalent polymer (DPAICP) involving in organothiol D-peptides and Au3+, and a feasible camouflage technology using ME.Results: DPAICP@ME possessed favorable pharmaceutical properties to remain stable during the gastrointestinal absorption and blood circulation, and showed the satisfactory tumor accumulation through oral medication. Expectedly, oral DPAICP@ME played its predetermined role in vivo to restore p53 signaling pathway for cancer therapy in B16F10 homograft malignant melanoma model, LLC Lewis orthotopic transplantation model of lung cancer and patient-derived orthotopic xenograft (PDOX) mice model of colon cancer. Moreover, oral DPAICP@ME augmented the action of immunotherapy by Anti-PD1 through the further T-cell activation.Conclusion: The de novo design of the bionic chiral-peptide supramolecule provides a practicable strategy for the construction of biomimetic chiral peptide-derived nanostructures that can be taken orally, and likely boosts chiral nanomedicine discovery efforts for a wider range of diseases including cancer. Chirality, or handedness, in molecules is ubiquitous in our world, and its major performance is the appearance of left- and right-handed opticity, so-called enantiomers While some success has been achieved in the clinical translation of therapeutics derived from D-enantiomeric peptides, significant challenges remain to develop their oral therapeutic agents, thereby limiting their clinical application. Oral delivery has advantages over the current regime of therapy by injection or infusion, especially for chronic disease such as cancer therapy with long-term dosing requirements, because the oral drug can provide a long-time and continuous exposure of target cells in a relatively lower thus safer concentration with continuing-administration in separated time periods, resulting in the enhanced efficacy and depressed side effects  via the “neonatal” Fc receptor, FcRn Fortunately, the favorable proteolysis resistance endows D-peptides with the capacity to withstand the multi-protease environment in digestive tract in vivo, and CPPs conjugation as well as liposome encapsulation always suffered from the fussy manufacturing process and rapid remove by the liver and spleen Besides, for a long time, intracellular space is the forbidden zone for the chiral peptides towards PPI regulation, because of the ineffective cellular internalization of D-peptides de novo supramolecules assembled from a pro-apoptotic chiral peptide termed MVP. For the well-defined nanostructures of chiral-peptide derived supramolecule, we devised an “one-pot”, two-step and aqueous-based growth method involving organothiol D-peptides and Au3+ to construct a D-peptide Au infinite covalent polymer (DPAICP). Next, the ME membrane was separated with milk proteins through ultracentrifugation, and the medicative and absorbable artificial milk can be obtained after camouflaging the chiral-peptide supramolecular assembly with ME membrane. As expected, DPAICP@ME played its predetermined role in vivo through oral administration to restore p53 signaling pathway for cancer therapy in B16F10 homograft malignant melanoma model, LLC Lewis in situ model of lung cancer and patient-derived orthotopic xenograft (PDOX) mice model of colon carcinoma. Importantly, oral DPAICP@ME augmented the action of immunotherapy by Anti-PD1 through the further T-cell activation. The de novo design of the ME membrane camouflaged chiral-peptide supramolecular assembly provides a practicable strategy for the construction of well-defined chiral peptide-derived nanostructures that can be taken orally, and likely boost chiral nanomedicine discovery efforts for a wider range of diseases including cancer.To realize the oral administration of chiral peptides and break through their forbidden zone in intracellular space, we attempted to turn mike into an oral nanotherapeutic by replacing milk proteins with DMBP) 3+ and D-peptide, an extra mercapto-contained chiral Cys residue and a hydrophilous chiral Arg residue were introduced to the C-terminus of chiral MVP. Notably, for comparative study, natural MVP were also designed, in which DMBP was changed to a levorotatory dodecameric motif binding to MDM2 with the almost identical affinity  chiral peptide oligomerization and 2) nanostructures growth with ME membrane fusion. In the first step, the aqueous Au3+ was reduced by the thiol in chiral MVP and formed a polymeric structure of [MVP-S-Au(I)]n termed p as evidenced by the increased Mark-Houwink molecular weight  on healthy C57/B6 mice. Time-dependent ICP-MS measurements of mice blood yielded metabolic kinetics, supporting the prolonged blood-circulation time after ME coating  effect.5 tumor cells at the right side of the hips, and ICP-MS was employed to quantify the 197Au from tissues of tumor-carrying mice after the DPAICP@ME or DPAICP oral administration at the dosage of 2 mg/Kg. As shown in Figure In theory, the optimized stability and macrophages escape would further promote the tumor accumulation of DPAICP@ME Additionally, we further investigated the acute toxicity of DPAICP@ME after single oral administration towards C57/B6 mice with healthy immune system at a dosage of 20 mg/Kg, which was ten times the therapeutical concentration. As expected, no obvious loss of weight can be found in the one week after administration Figure , nephrotAnother key design of DPAICP@ME is its responsiveness to intracellular reduction environment. The release kinetics of chiral MVP was monitored by High-performance liquid chromatography (HPLC) upon the incubation with or without 10 mM GSH that can simulate the intracellular reduction environment in vivo performance, we firstly explored the pharmacokinetic characteristics and biodistribution of DPAICP@ME both by oral medication (ig) and intravenous injection (iv). As show in 3 (n = 5/group). After a 9-day intragastric administration (ig) of isopyknic normal saline , DPAICP@ME, LPAICP@ME and a drug-free carrier termed NPAICP@ME at the dosage of 2 mg/Kg every other day, immunohistochemical staining analysis showed that DPAICP@ME substantially more active than its L-enantiomer LPAICP@ME in degrading MDM2 and its homologous protein MDMX and reactivating p53, in sharp contrast to the placebo NS and NPAICP@ME  were intravenously inoculated into C57BL/6 mice. After 2-weeks, 18 mice bearing orthotopic LUAD were divided into three group randomly, and administered intragastrically each other day with 2 mg/Kg DPAICP@ME, LPAICP@ME or equivoluminal NS, respectively in a 14-day treatment period commenced three days after tumor challenge. At day 14 post-administration, in comparison to NS and LPAICP@ME, DPAICP@ME remarkably reduced LUAD burden as supported by the number of pulmonary nodule  lower than the DPAICP@ME group  in which LLC cells  model of colon cancer was established by implanting tumor tissues from a colorectal cancer patient to the colon of NOD/SCID mice orthotopically Figure A. To ver+ cell, CD8+ cell and T lymphocyte after the DPAICP@ME treatment  as the content of the engineered exosome, and developed a feasible nanostructures packaging technique using the membrane from cow milk exosomes as the shell of the engineered exosome. In this proof-of-concept research, a D-peptide-derived p53 activator that can specifically degrade MDM2 in cancer cells was used here to construct the DPAICP@ME. Expectedly, DPAICP@ME played its predetermined role in vivo through oral administration to restore p53 signaling pathway for cancer therapy in B16F10 homograft malignant melanoma model, LLC Lewis in situ model of lung cancer and PDOX mice model of colon cancer. Importantly, oral DPAICP@ME augmented the action of immunotherapy by Anti-PD1 through the further T-cell activation. Thus, we can expect that the combination therapy between DPAICP@ME and immunotherapy has the potential to achieve the eradication in early tumors and partially sensitive advanced tumors with systemic metastasis. In short, the de novo design of the ME membrane camouflaged chiral-peptide supramolecular assembly provides a practicable strategy for the construction of biomimetic chiral peptide-derived nanostructures that can be taken orally, and likely boost chiral nanomedicine discovery efforts for a wider range of diseases including cancer. More importantly, while our nanoengineering strategy of biomimetic chiral nanostructures construction helps overcome existing pharmacological obstacles for oral peptide, this chiral chemistry itself is not restricted to nanoengineer peptide per se as it is amenable to construct other biomimetic chiral nanostructures derived from proteins, nucleic acids, and other classes of chiral drug compounds as well.Hereby, to fabricate well-defined chiral peptide-derived nanostructures and realize its oral administration, an extra-small bionic chiral-peptide supramolecular assembly termed DPAICP@ME was designed as oral therapeutic. In details, we devised an “one-pot”, two-step and aqueous-based growth method involving organothiol D-peptides and AuSupplementary experimental section and figures.Click here for additional data file.


This review discusses potential predictors of efficacy of PSMA-targeted radionuclide therapies (TRT) segregated in three major categories: imaging, clinical and molecular.Theranostics of prostate cancer (PC) represents a growing area of development of imaging agents and targeted radionuclide therapeutics against a major target, prostate specific membrane antigen (PSMA). In view of the encouraging efficacy from the use of  One exception to this could be the use of NSE as a potential laboratory indicator for [s needed .177Lu-PSMA-617 demonstrated that a normal pre-treatment Hb level was predictive of ≥50% PSA decline 4 weeks after receiving the third PSMA-RLT dose as well as of OS compared to patients with reduced baseline Hb [Anemia is common among patients with mCRPC and has prognostic relevance, with several factors impacting on its magnitude, including the disease itself, particularly in presence of bone marrow infiltration and systemic therapies ,36. A reeline Hb . Likewiseline Hb .177Lu-PSMA, a platelet count of more than 300 had an independent negative effect on therapeutic response in these patients, regardless of other clinical parameters including age, γ-glutamyl transferase, LDH, hemoglobin, Gleason score, C-reactive protein, and regular need for pain medication [There are scarce data regarding the clinical utility of PLT count in patients treated with PSMA-TRT. In a comprehensive analysis of various clinical predictors of response in 40 progressive mCRPC patients receiving dication .The value of ALP in predicting OS is well established in mCRPC patients undergoing first-line chemotherapy . With re223Ra was able to improve OS in all mCRPC patients with symptomatic bone metastases enrolled in the phase 3 ALSYMPCA trial, regardless of opioid use [177Lu-PSMA revealed that the regular need for analgesics had a negative impact on PSA response (>50% or any PSA decline) [The regular use of opiate analgesics in patients with mCRPC is a surrogate indicator of poor OS . The α-eioid use . The prodecline) . Accordidecline)  and was decline) .177Lu-PSMA-617 [The presence of visceral metastases is a harbinger of poor OS in patients with mCRPC undergoing systemic chemotherapy . It appePSMA-617 ,41. FurtPSMA-617 . InteresPSMA-617 ,43. ThisPSMA-617 .max of the “hottest lesion” in pre-therapeutic PET in a composite predictive score [177Lu-PSMA. The composite score separated two distinct groups of patients: one with ≤2 predictive factors that demonstrated only a 19% risk of progression, and another with three predictive factors, resulting in 90% chance of progressive disease [Given the presence of multiple imaging and clinical factors that can influence therapeutic benefit and OS after PSMA-TRT, an effort was made to retrospectively assess the predictive value of key markers such as ALP (cut-off 135 U/L), PSA (cut-off 200 ng/mL) and SUVve score . This ex disease . ProspecRetrospective tumor microarray studies of PSMA expression in primary prostate tumor tissues supported its prognostic utility as a potential indicator of lethal disease, correlating with a higher Gleason score and PSA at diagnosis . High ex225Ac-labeled PSMA ligands, those with high baseline immunohistochemical PSMA expression H-score of ≥200, defined semiquantitatively on a scale of 0−300 as a composite of the percentage of immunopositive tumor cells (0−100%) and the staining intensity , tended to have longer OS compared to those with H-score < 200  [The question of whether PSMA overexpression in mCRPC patients undergoing treatment with PSMA-TRT could represent a prognostic or predictive tool has yet to be fully elucidated. In a small cohort of 13 patients who received targeted alpha radiation therapy with ctively) .An issue with semiquantitative immunohistochemical assessment of membranous PSMA is the marked tumor heterogeneity among patients and among different metastases within the same patient . For exa177Lu-PSMA-617 RLT, according to a small cohort of 19 men with mCRPC [177Lu-PSMA-617 [During progression to the metastatic, castration-resistant state, the median PSA PFS and OS are significantly shorter in patients with PSMA-positive CTCs compared to those without PSMA mRNA expression . Additioth mCRPC . AlthougPSMA-617 .A main cause of tumor heterogeneity resulting in target heterogeneity (PSMA), is the presence of genomic instability. Genomic instability is often associated with a defective DNA repair machinery. On the other hand, DNA repair defects may shape the response of tumors to DNA damaging agents, including ionizing radiation. Therefore, assessment of DDR gene alterations could be instrumental in predicting response or resistance to PSMA-TRT. This hypothesis was tested by a few studies. Using targeted next-generation sequencing analysis of tumor biopsies and germline DNA samples from mCRPC patients one study found that presence of deleterious DDR mutations in BRCA2, ATM, mismatch repair (MMR), and CDK12 genes was more frequent in patients with high tumor membranous PSMA expression . Transcr25Ac-PSMA-617 [177Lu-PSMA-617 and concomitant enzalutamide is in line with a potential role of DDR defects in sensitizing to PSMA-TRT [225Ac-PSMA-617 [The presence of pathogenic BRCA1 mutations was associated with longer OS (16.1 vs. 7.6 months) after treatment with PSMA-617 . AnecdotPSMA-TRT . DDR altPSMA-617 . HoweverPSMA-617 ,57.In the largest to-date single-institution cohorts of mCRPC patients treated with beta- or/and alpha-emitting PSMA-TRT, deleterious somatic BRCA1 or BRCA2 alterations (copy number variations and point somatic mutations) were found to correlate with longer PFS and PFS/OS respectively, whereas TP53 alterations predicted shorter PFS . The pre68Ga-DOTA-NT-20.3 or 18F-DEG-VS-NT could represent viable alternative options for selecting therapies targeting NTSR1 in mCRPC patients with limited PSMA expression levels [A particular challenge for the efficacy of PSMA-TRT, discussed earlier, is attenuated expression of PSMA during development of resistance or as a manifestation of tumor heterogeneity. To counteract this limitation from an otherwise good but imperfect biomarker (PSMA), additional molecular targets for patient screening, detection of metastatic disease and treatment monitoring are under investigation. One such target is neurotensin receptor1 (NTSR1), which exhibits high or moderate expression in 92% of PC tumors, including all PSMA-negative tissues, suggesting a potential complementary role in targeted imaging or/and therapy . Althougn levels ,61.In a world where precision medicine is becoming the standard approach in oncology, PSMA PET has the potential of being the best available imaging method for mCRPC therapy response evaluation. Along with it, other key indicators at the clinical or/and molecular level, may be able to guide treatment decisions regarding PSMA-TRT .177Lu-PSMA-617 to standard of care in terms of radiographic PFS (8.7 vs. 3.4 months) and OS (15.3 vs. 11.3 months) in men with mCRPC [Since the majority of biomarker signals are derived from retrospective studies, it will be critical to conduct prospective PSMA-TRT trials with a biomarker-embedded design rather than as exploratory variables in order to validate the true significance of each one. Additionally, because different cut-off values have been reported for several of the studied markers, harmonization of data is required for future dichotomous analyses. The phase III VISION study was recently reported as a positive trial, demonstrating the superiority of addition of The predictive and prognostic utility of DDR defects could assist with patient selection and could further be exploited to optimize the activity of PSMA-TRT by adding PARP inhibitors, platinum agents, immune checkpoint inhibitors or other pharmacologic agents targeting DNA repair.One limitation in establishing surrogates of response to PSMA-TRT is that there are specific effects of other systemic therapies, particularly androgen deprivation therapy on PSMA expression by PC cells, leading to potential pitfalls and dynamic changes in the actual target (PSMA) ,63,64.Consequently, it is less likely that one single biomarker would be sufficient to accurately predict responses and outcomes after treatment with PSMA-TRT. Instead, a combination of biomarkers with strong biological rationale each being able to provide complementary predictive and prognostic value would be more likely to accompany the future integration of PSMA-TRT in the armamentarium of systemic therapies for mCRPC.


The adverse effects of the COVID-19 vaccine have been discovered as the rapid application of the vaccines continues. Neurological complications such as transverse myelitis raise concerns as cases were observed in clinical trials. Transverse myelitis is a rare immune-mediated disease with spinal cord neural injury, resulting in neurological deficits in the motor, sensory, and autonomic system. Vaccine-related transverse myelitis is even rarer. We present a case of acute transverse myelitis after vaccination against COVID-19 with the ChAdOx1 nCOV-19 vaccine (AZD1222), which was the first case reported in Taiwan. Although it rarely occurs, post-vaccination neurological complications should not be ignored. As the pandemic of SARS-CoV-2 continues to spread and concern about vaccination efficacy and safety rises, heterologous vaccination were implemented in health public policy in several countries. A literature review of several clinical trials shows promising effects of mix-and-match vaccination. Further study on different combinations of vaccines can be expected. Since the rapid administration of COVID-19 vaccines, specialists are facing questions regarding potential neurological complications. Acute transverse myelitis (ATM) post-vaccination raises concerns as three cases were observed during ChAdOx1 nCOV-19 vaccine AZD1222) clinical trials among 11,636 participants [22 clinicA 41-year-old man with diabetes under well medical control who works as an emergency physician received his first dose of AZD1222 2 weeks prior to the onset of symptoms. He first presented with left peripheral facial palsy, which was resolved after oral prednisolone. In the following week, a tingling sensation over T4 dermatome was experienced, followed by progressive paresthesia below T4, along with lower-limb weakness and clumsiness, which developed in the 6th week after vaccination. Neurologic examination revealed the loss of pinprick sensation below T4 bilaterally; decreased lower-limb muscle power, which was more severe over the left side; loss of joint position; and vibration over both lower limbs as well as increased bilateral knee reflex.The patient was admitted to the neurology ward for workups 7 weeks post-vaccination. Laboratory analysis showed unremarkable findings in the complete blood cell count, complete metabolic profile, and negative SARS-CoV-2 PCR test. Brain and whole spine magnetic resonance imaging (MRI) were performed. The contrast-enhanced MRI of the spine revealed an intramedullary-enhancing lesion over the spinal cord at the T1 to T6 vertebral levels . The MRIAcute transverse myelitis is a rare clinical syndrome in which immune-mediated processes cause spinal cord neural injury, resulting in motor, sensory, and autonomic dysfunction . Since tAccording to the VAERS of the Taiwan Centers for Disease Control, since the beginning of vaccination administration on 22 March 2021 to 28 July 2021, there were 9,987,157 doses of vaccines administered in Taiwan and 5620 adverse effect cases reported. Among the 2548 cases of severe adverse events, neurological complications, including 208 strokes, 42 facial palsy, seven Guillain–Barre Syndrome reports, two acute disseminated encephalomyelitis, and two transverse myelitis, were recognized . This isThe pathogenesis of transverse myelitis is thought to be immune-mediated from infection, para-infection, autoimmune disease, and paraneoplastic . RelatedDue to the concerns regarding vaccine adverse effects and the persisting pandemic of COVID-19, heterologous vaccine regimens raise discussions. Some countries are now performing heterologous prime-boost vaccine schedules . CurrentAlthough they rarely occur, the association of the COVID-19 vaccine and the disease, along with other neurological complications, should not be ignored. As mix-and-match COVID-19 vaccines are under greater discussion in Taiwan, studies examining it are also on the way. As I  am the patient described above, I recommend that clinicians keep vaccine-related neurological complications in mind. Delayed diagnosis and treatment may cause sequela. I also propose that my experience of the heterologous vaccination could open up discussion regarding the control of the COVID-19 pandemic.


We aimed to understand the role of the transcriptional co-factor Yes-associated protein (Yap) in the molecular pathway underpinning the pathogenic transformation of synovial fibroblasts (SF) in rheumatoid arthritis (RA) to become invasive and cause joint destruction.Pdgfrα-CreER and Gdf5-Cre mice, crossed with fluorescent reporters for cell tracing and Yap-flox mice for conditional Yap ablation. Fibroblast phenotypes were analysed by flow cytometry, and arthritis severity was assessed by histology. Yap activation was detected using Yap–Tead reporter cells and Yap–Snail interaction by proximity ligation assay. SF invasiveness was analysed using matrigel-coated transwells.Synovium from patients with RA and mice with antigen-induced arthritis (AIA) was analysed by immunostaining and qRT-PCR. SF were targeted using Pdgfrα-expressing SF during AIA, with predominant expansion of the Gdf5-lineage SF subpopulation descending from the embryonic joint interzone. Gdf5-lineage SF showed increased expression of Yap and adopted an erosive phenotype (podoplanin+Thy-1 cell surface antigen−), invading cartilage and bone. Conditional ablation of Yap in Gdf5-lineage cells or Pdgfrα-expressing fibroblasts ameliorated AIA. Interleukin (IL)-6, but not tumour necrosis factor alpha (TNF-α) or IL-1β, Jak-dependently activated Yap and induced Yap–Snail interaction. SF invasiveness induced by IL-6 stimulation or Snail overexpression was prevented by Yap knockdown, showing a critical role for Yap in SF transformation in RA.Yap, its binding partner Snail and downstream target connective tissue growth factor were upregulated in hyperplastic human RA and in mouse AIA synovium, with Yap detected in SF but not macrophages. Lineage tracing showed polyclonal expansion of Our findings uncover the IL-6–Yap–Snail signalling axis in pathogenic SF in inflammatory arthritis. Synovial fibroblasts (SF) are key drivers of rheumatoid arthritis (RA) pathogenesis; however, the SF lineages involved and the molecular mechanisms underpinning their pathogenic transformation are incompletely understood.Gdf5-lineage SF, defined by their ontogenic derivation from the joint interzone, are the key pathogenic SF in inflammatory arthritis. Mechanistically, interleukin (IL)-6 signals via Jak to activate Yes-associated protein (Yap), which forms a complex with Snail to drive the invasiveness of SF, and selective targeting of Yap in Gdf5-lineage SF ameliorates inflammatory arthritis.This study shows that This study supports the targeting of IL-6 and Jak in RA, not only for immunosuppression but also to directly control the Yap–Snail-mediated pathogenic behaviour of SF.Rheumatoid arthritis (RA) is a common immune-mediated chronic inflammatory disease causing joint damage and deformities. Current treatment consists of synthetic and biological disease-modifying antirheumatic drugs aimed at systemic immunosuppression. Nonetheless, some patients fail to respond to treatments, and joint damage progression can still occur despite clinical remission.A hallmark of RA pathology is synovitis, causing the synovium to thicken and form a pannus that invades cartilage and bone, driven by pathogenic transformation and proliferation of fibroblasts with infiltration of inflammatory/immune cells. A recent study reported two distinct subsets of synovial fibroblasts (SF), immune effector fibroblasts expressing podoplanin (Pdpn) and Thy-1 cell surface antigen (Thy1) found in the sub-lining synovial tissue and promoting inflammation, and destructive Pdpn+Thy1− fibroblasts in the lining layer mediating cartilage and bone damage.Gdf5-lineage cells, descend from the Gdf5-expressing joint interzone cells in the embryo.Gdf5-lineage cells include the SF in the lining as well as a subset of SF in the sublining.Cell lineage tracing studies have revealed that the adult synovium consists of ontogenetically diverse fibroblast subsets. One subset, the Gdf5-lineage cells underpins synovial lining hyperplasia following acute joint surface injury in mice.The synovial pannus is a tumour-like structure resulting in part from uncontrolled fibroblast expansion. Increased activity of the transcriptional cofactor Yes-associated protein (Yap) is known to cause tissue overgrowth in multiple tissues and organs through stimulating cell proliferation.The role of Yap in immune-mediated inflammatory arthritis and the signalling mechanisms that link Yap activity to the pathogenic transformation of SF await clarification. A reduced severity in the K/BxN serum-transfer arthritis model in mice was reported after treatment with the small molecule verteporfin,Here, we show that Yap is highly expressed by SF in both human RA and mouse AIA and that conditional genetic ablation of Yap in SF ameliorates AIA. Mechanistically, Yap is Jak-dependently activated by interleukin (IL)-6, a key inflammatory cytokine in RA, and forms a complex with the transcription factor Snail to drive the SF invasive phenotype. Our findings identify the IL-6–Yap–Snail signalling axis as a fibroblast-specific therapeutic target in RA synovitis.Materials and methods are available in the 10.1136/annrheumdis-2021-220875.supp1Supplementary dataYAP mRNA expression levels correlated non-significantly with the expression of SNAIL (p=0.083) and significantly with the expression of YAP downstream targets CTGF (p=0.024) and GP130  were mostly quiescent and located predominantly in the synovial lining, while other SF  were found throughout the synovium  phenotype during AIA, as compared with control knees  target cells. Immunostaining of synovium showed a significant decrease in Yap expression in the cKO mice , which moreover revealed a significantly higher expression of Yap in the Gdf5-lineage SF compared with other SF after 7 days of AIA  (Gdf5-lineage cells in synovium during AIA was observed in both Yap WT (p<0.001) and Yap cKO mice , immune infiltrates in synovium (p=0.026) and erosions at the joint margins (p=0.002), and a non-significant trend towards decreased cellular exudate, resulting in an overall arthritis score of 4.9  in the cKO mice, compared with 6.5  in p=0.002; . Despitep=0.023) , extensip<0.001, . Accordinduction . These dYap ablation on the expression of key catabolic and inflammatory mediators by qRT-PCR analysis of Tom+ Gdf5-lineage SF purified from AIA mice. Compared with Gdf5-lineage SF of Yap WT mice, the Gdf5-lineage SF from Yap cKO mice displayed reduced expression levels of critical enzymes mediating SF invasiveness, including Mmp9 (p=0.048), Mmp13 (p=0.044) and Mmp14 (p=0.012),Next, we investigated the effects of conditional Yap cKO mice compared with Yap WT controls after AIA  KO model in which Yap ciKO , althoug Yap cKO . Nonethe WT mice .Together, these findings indicate that Yap has crucial functions in pathogenic SF to promote inflammation and joint destruction, and demonstrate amelioration following its selective targeting in SF in inflammatory arthritis.Yap mRNA expression at least after 24 hours (To assess the ability of key inflammatory cytokines to activate Yap, we used a Yap–Tead reporter construct,24 hours , indicat24 hours , showing24 hours  and relySince the transcription factor Snail, previously reported to be involved in TNF-α-mediated SF activation in RA,Yap in SF ameliorates immune-mediated inflammatory arthritis in vivo and show that IL-6 increases Yap activity through Jak signalling and promotes interaction of Yap and Snail to drive the pathogenic behaviour of SF.SF are key cells in RA that sustain inflammation and induce tissue damage, but the molecular mechanisms underlying these functional characteristics remain to be fully elucidated. Here, we report that ablation of Yap in SF using two different mouse models, a specific role for Yap in driving the pathological behaviour of SF in inflammatory arthritis. The recent demonstration of additional roles for Yap in promoting synovial angiogenesis further supports the possible therapeutic benefits of targeting Yap in RA synovitis.In a previous study using the K/BxN serum-transfer mouse model of inflammatory arthritis and mice engrafted with cartilage and RA-SF, treatment with the non-specific Yap inhibitor verteporfin was shown to reduce arthritis severity and cartilage invasion by RA-SF, respectively.Gdf5-lineage SF drives synovial lining hyperplasia in a traumatic joint surface injury model in mice.Yap in Gdf5-lineage SF reduced not only synovial lining hyperplasia but also immune infiltration and erosive damage in inflammatory arthritis. Decreased synovial lining hyperplasia in AIA was also observed when Yap was knocked out in fibroblasts in adult mice, showing this is not a result of developmental defects resulting from Yap KO.We previously reported that increased Yap activity in Although the upstream regulators of Yap activity are likely to be context-dependent, we uncover a hitherto unappreciated molecular link between Jak-mediated IL-6 signalling, pivotal in RA pathogenesis, and the Yap-mediated invasive SF phenotype. While IL-6 treatment of SF enhanced invasive behaviour, Yap silencing completely prevented the IL-6-induced SF invasion. JAK inhibition with baricitinib was shown to abrogate cytokine-induced invasive behaviour of SF.YAP and GP130 expression in human RA synovium, in accordance with evidence that YAP upregulates GP130 expression in an autoregulatory loop,SF, which do not express the IL-6R, undergo pro-inflammatory sIL6R-mediated trans-signalling, facilitated by cell surface expression of GP130.Snail was previously reported to be highly expressed in RA synovium and SF, and its lentiviral vector-mediated silencing in the joint ameliorated collagen-induced arthritis in rats.Gdf5-lineage SF were largely confined to the synovial lining, as previously reported,Gdf5-lineage SF underwent extensive proliferation and expansion throughout the synovium, indicating a derangement of the anatomical segregation of fibroblast lineages during synovitis. Our findings show the Gdf5-lineage SF to become erosive and support a role for Yap in the destructive SF, possibly through modulation of MMP expression.Yap cKO mice with AIA suggests that the Gdf5-lineage also includes immune effector SF and that Yap may be needed in these cells to sustain inflammation. Indeed, Gdf5-lineage SF from Yap cKO AIA mice displayed reduced expression levels of IL-34, an inflammatory cytokine produced by RA-SF able to support osteoclastogenesisA recent study reported functionally distinct fibroblast subsets in RA synovium, with destructive fibroblasts restricted to the synovial lining layer and immune effector fibroblasts located in the synovial sub-lining.In summary, we report a novel IL-6-Jak-Yap–Snail signalling axis linking immune-mediated inflammation with pathogenic SF in RA. Our findings position Yap in RA-SF at the crossroads between the activated immune system and their destructive phenotype, and suggest Yap for SF-targeted therapy in RA.


Antibody glycosylation has received considerable attention in coronavirus disease 2019 (COVID-19) infections and recently also in vaccination. Antibody glycosylation and in particular immunoglobulin G1 fucosylation levels influence effector functions and are therefore key parameters for assessing the efficacy and safety of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) directed immune responses. This review article summarizes and interprets recent research into antibody glycosylation in COVID-19. Experimental approaches for analyzing the glycosylation of SARS-CoV-2-directed antibody responses are evaluated. The pronounced dynamics, effector functions, clinical utility, and regulation of antibody glycosylation in COVID-19 are assessed. Future research on the role of antibody glycosylation in COVID may cover the glycosylation of other antibody classes beyond immunoglobulin G, the regulation of antibody glycosylation, and the role of non-canonical antibody receptors in determining effector functions. N-glycan is an important structural component that fine-tunes effector functions [N-glycan may elicit qualitatively different immune responses by varying their potential to activate complement and by altering their binding to Fc receptors present on a range of immune cells [Antibodies are abundant soluble glycoproteins in the circulation, various biofluids and mucosal layers playing essential roles in the adaptive immune response . Beyond unctions . Notablyne cells .During homeostasis hardly any intra-individual variation is observed in the composition of the plasma-derived total (or bulk) IgG glycome –7. With Plasmodium falciparium antigens on red blood cells [Substantial alterations of IgG glycosylation are concomitant with various infectious diseases and vaccinations against those , 9–11. Tod cells . Antibodod cells , 22.Recent studies have pointed towards associations between IgG1 glycosylation – especially fucosylation – and coronavirus disease 2019 (COVID-19) severity, but study results are not directly comparable due to differences in cohorts, disease phases and methodologies. This encouraged us to concisely review the available evidence in order to identify commonalities and address discrepancies in methodologies and patient cohorts. Eventually, we provide a broader outlook on IgG glycosylation patterns in SARS-CoV-2 messenger ribonucleic acid (mRNA) vaccination and provide perspectives on the utility of antigen-specific IgG glycosylation analysis as a factor in assessing efficacy and safety of both pathogen- and vaccine-induced immune responses.SARS-CoV-2 infections show largely diverse disease courses, and it became evident during the ongoing pandemic, that an evoked robust anti-SARS-CoV-2 immune response, commonly considered as protective, can in fact lead to aggravated immunopathologies , 24. DisN-glycans of IgG, covering both Fc and Fab glycans alike of all IgG subclasses. This method may be considered the gold standard for antibody glycosylation analysis and features a particularly high precision. Petrovic et al. focused on total IgG glycosylation analysis, and no analysis of SARS-CoV-2 spike protein (S) specific antibodies or anti-SARS-CoV-2 receptor binding domain (RBD) antibodies was pursued [An early study by Petrovic et al. applied ultrahigh-performance liquid chromatography with fluorescence detection (UPLC-FLD) for analyzing total  pursued .In the studies of Larsen et al., Hoepel et al., Bye et al. and Pongracz et al., a common, liquid chromatography – mass spectrometry (LC–MS)-based method was employed to characterize total, anti-S, and to a limited extent anti-SARS-CoV-2 nucleocapsid (N) IgG glycosylation following their affinity purification , 27, 31.Similarly to the afore approach, Chakraborty et al. used LC–MS for the bottom-up analysis of IgG Fc glycosylation, albeit using multiple-reaction monitoring  for the target detection and quantification of pre-selected glycopeptides . FarkashAn alternative MS-based method potentially useful for the assessment of IgG Fc glycosylation in COVID-19 is middle-up antibody analysis, as presented for anti-RBD IgG in a study by Melani et al. Even though this method is promising, the observed high complexity due to multiple different amino acid sequences and glycan structural heterogeneity complicates Fc glycosylation analysis using this approach .Aleuria aurantia to α1,6-fucose linked to N-acetylglucosamine, which has been exploited to quantify corresponding fucosylation levels in an ELISA-setting [Conversely to MS-based methods, Ankerhold et al. used a lectin-based assay to characterize anti-S and anti-N IgG fucosylation. The principle of this method lies in the preferential binding of the lectin -setting .The comparability of the results of the various assays has for a big part not been established. In particular, it would be of interest to see how the results of the MRM- based and PRM-based MS methods relate to established UPLC-FLD and LC–MS high-throughput glycomics approaches. For the latter two methods, very good comparability has been demonstrated for applications in a biomedical setting, providing a basis for the integration of data obtained with these methods .High-throughput technological advances allowed to routinely analyze the IgG glycomes in large clinical cohorts, which contributed to our understanding on how IgG glycosylation signatures associated with changes in health and disease , 37. RecIgG1 Fc fucosylation is in the spotlight of most these studies, and high levels of afucosylated anti-S or anti-RBD IgG1 have been construed as a motif associated with exacerbated immunopathologies in COVID-19 , 31, 35.Of note, anti-S IgG1 fucosylation could not further discriminate between the various hospitalized groups , which iNext to fucosylation, also galactosylation, sialylation and bisection of anti-S IgG1 showed pronounced dynamics in COVID-19, with initially high levels of galactosylation and sialylation as well as low levels of bisection . After hBisection of IgG was found to be low in severe COVID-19, as shown both for total IgG  and antiin vitro functional assays [Some of the above studies found remarkably dynamic glycosylation patterns as exemplified for one patient in Fig. l assays , providiIgG glycosylation is influenced by demographic factors such as age and sex . FurtherIn SARS-CoV-2, it has been hypothesized that the antigen context gives rise to IgG afucosylation, with host membrane-associated antigen presentation as a pre-requisite for the induction of low-fucose responses. This led to the postulation of a signal at the viral protein-displaying host plasma membrane that would trigger afucosylated IgG responses in B cells, yet the nature of this signal remains elusive . FurtherMycobacterium tuberculosis infection showed largely agalactosylated glycosylation patterns, unlike in latent tuberculosis [in vitro and triggered platelet reduction in vivo. These afucosylated IgG1 were postulated to contribute to the immunopathology via antibody-dependent enhancement (ADE) [Altered pathogen-specific antibody glycosylation has been reported to impact their inflammatory potential and functionality , 11, 41.rculosis . IgG siarculosis , with thrculosis , 22. Fornt (ADE) .in vitro cytokine release assay to demonstrate that glycoengineered monoclonal anti-S IgG1 carrying afucosylated glycans – when incorporated in immune complexes with recombinantly expressed spike protein – induced elevated interleukin (IL)-6 release in monocyte-derived macrophages (expressing FcγRIIIa), as compared to its normally fucosylated counterpart. However, it is worth to note that fucosylation levels of the used glycoengineered anti-S IgG1 were way below those observed in patients. Accordingly, the onset of low-fucosylated anti-S IgG1 in critically ill patients was accompanied by a rise in IL-6 (together with C-reactive protein and D-dimer) upon longitudinal sampling [Larsen et al. used an sampling .Consistent with these observations, Chakraborty et al. showed that the affinity and dissociation constant of patient sera, and of both isolated patient-derived and glycoengineered anti-RBD IgG1 for recombinant FcγRIIIa was proportional to the degree of IgG1 afucosylation. Additionally, the same study used in vitro stimulation assays to quantify NK cell degranulation as well as cytokine production by primary monocytes. Using both of these assays employing immune complexes formed with patient-derived IgG1 or glycoengineered anti-RBD IgG1 immune complexes, they found that low-fucosylated anti-RBD IgG1 indeed impacted ADCC as exemplified by increased IL-6, tumor necrosis factor and IL-1β production, when compared to normally fucosylated alternatives .in vitro by blocking ADCC-associated, which is of therapeutic interest in COVID-19 [Hoepel et al. identified the combination of high titers and low fucosylation of anti-S IgG1 as the potential predisposing factor of severe COVID-19. Using an array of techniques, they likewise found increased cytokine release, a pro-inflammatory ribonucleic acid sequencing profile as well as disrupted endothelial barrier function and platelet adhesion associating with anti-S IgG1 afucosylation. Using the same glycoengineered anti-S IgG1 as Larsen et al., they found the proinflammatory cytokine production by macrophages (including IL-6) was blocked by FcγRIIIa-blocking antibodies . In addition, this was also inhibited by small molecule drug fostamatinib to counteract afucosylated anti-S IgG1-induced inflammatory responses COVID-19 .in vitro models and highlighted the role of a platelet-specific Fc receptor FcγRIIa [Bye et al. reported on the association of anti-S IgG1 afucosylation with prothrombotic platelet activation using  FcγRIIa . This fi FcγRIIa , and molUsing a cell-based reporter system, Ankerhold et al. showed that serum pools originating from COVID-19 patients right upon hospitalization were potent in FcγRIIIa activation, after normalizing to antigen-specific IgG titers, although the findings were independent of clinical manifestation and could not be used for the differentiation between severe and critical cases .in vitro functional evidence associating anti-S IgG1 afucosylation with enhanced immune cell activation [in vitro assays [Together, these findings consistently revealed a marked, proinflammatory fucosylation signature on plasma-derived anti-S IgG1 originating from severely ill, SARS-CoV-2 infected inpatients , 31, 35.tivation , 28, 35 tivation . Interesin vivo Fc glycosylation and dynamics thereof can be followed in a relatively well controlled model, given that for example the time between a primer and a booster shot is largely comparable between vaccinees [By vaccination with foreign antigens representing parts or even whole attenuated or killed infectious agents, immune responses are evoked, including induction of pathogen-specific neutralizing antibody and long-lived memory B cell production, that are jointly capable of alleviating and/or eliminating the infections during a later encounter. Vaccination provides a highly interesting setting where accinees . Hithertaccinees , 45, eveaccinees , 41.A recent study by Farkash et al. investigated anti-RBD antibody glycosylation patterns longitudinally, as elicited by the mRNA vaccine BNT162b2. Dynamic Fc compositions and immune receptor engagement were found, different from those in the setting of a natural infection or in convalescents. These antibodies were characterized by high fucosylation and low bisection . While tin vitro, other glycosylation features including bisection, galactosylation and sialylation show promising associations with disease severity pointing towards their clinical biomarker potential. The regulation of antibody glycosylation is poorly understood, and further research is needed to provide a mechanistic understanding of antibody glycosylation at the cellular and systemic level, to design intervention strategies targeting antibody glycosylation in COVID-19 as well as other diseases.Initiation of the adaptive immune response against SARS-CoV-2 is indispensable to fight the infection. It appears that IgG antibodies are key components in protection against COVID-19 with an important role for glycosylation and resulting Fc-mediated effector functions. Recent studies have collectively indicated that a distinct, pro-inflammatory, low-fucosylation glycosylation phenotype marks circulatory IgG produced against the SARS-CoV-2 spike protein in hospitalized patients. This response has been suggested to characterize IgG responses against host-membrane embedded antigens, albeit the underlying mechanisms revealing this await further elucidation. While anti-S IgG1 afucosylation marks high COVID-19 disease severity and associates with numerous inflammatory markers While the role of IgG1 afucosylation in steering effector functions via FcγRIII interaction is receiving due attention, the role of other glycosylation features such as galactosylation, sialylation and bisection is poorly understood. For anti-SARS-CoV-2 antibody galactosylation and sialylation, further research is needed on its role in effector functions and complement activation  and possIn view of the observed glycosylation signatures in COVID-19, we believe that pathogen-specific antibody glycosylation is an important determinant of effector functions, that could be a promising severity marker for infection induced as well as efficacy marker for vaccine induced antibodies in the future.


A variety of innate immune cells such as macrophages, dendritic cells, myeloid-derived suppressor cells, natural killer cells, and neutrophils in the tumor microenvironments, contribute to tumor progression. However, while several recent reports have studied the use of immune checkpoint-based cancer immunotherapy, little work has focused on modulating the innate immune cells. This review focuses on the recent studies and challenges of using nanoparticles to target innate immune cells. In particular, we also examine the immunosuppressive properties of certain innate immune cells that limit clinical benefits. Understanding the cross-talk between tumors and innate immune cells could contribute to the development of strategies for manipulating the nanoparticles targeting tumor microenvironments. Based on the important roles of the various components of tumor microenvironment (TME) involved in tumor progression, strategies to therapeutically modulate the TME have recently emerged as a promising approach for the cancer therapy ,2,3,4. TIn particular, the recent accumulating studies have focused on the relationship between innate immune cells and tumors. The innate immune system originally serves as an initial border of defense against foreign invaders and alerts the adaptive immune system of impending attacks . In TME,Many works were made to target adaptive immunity, such as the use of antibodies against immune checkpoints, such as PD-1, PD-L1 and CTLA4 , or chimeric antigen receptor-T (CAR-T) cell therapy ,15,16. DThe specific and efficient delivery of modulators to innate immune cells can improve the efficacy of cancer therapy. Nanoparticles (NPs), a type of material with a diameter of approximately 10 to 400 nm, can be decorated with functional moieties  or be used to encapsulate therapeutic agents . The repThe general advantages of using NPs to target immune cells are relatively consistent with those of NPs as delivery molecules. In case of targeting DCs with NPs, NPs can be employed for delivering antigen-associated molecules  and adjuvants. This is able to promote long-term immune response against cancers via DC-based adaptive immunity . In TME,Macrophages are major innate immune cells which play critical roles as an immune regulator. In particular, macrophages can participate in anti-tumor immune responses via phagocytosis, the production of inflammatory cytokines, and the attraction of other immune cells. Therefore, macrophages could be a key promising target for a cancer immunotherapy.With featuring their phenotypes and remarkable plasticity, macrophages display two different subtypes that are a double-edged sword: M1-like phenotype and M2-like phenotype macrophages in the TME ,29. AccoHere we introduce several NP-based strategies that target TAMs for suppressing tumor progression: (1) TAMs depletion; (2) inhibiting monocyte recruitment; (3) TAM reprogramming; and (4) blocking CD47- SIRPα signaling , Table 2Depletion of TAM eliminating pro-tumorigenic M2-like macrophages is a very potential strategy to treat a variety of cancer types. There are several studies that have effectively developed NPs as cancer treatments. Synthetic NPs (inorganic NPs or polymer-based NPs) are also considered as the most promising types for TAM depletion due to their characteristics that are easy to be engineered. Recent studies showed that the utilization of dendrimer NPs carrying the chemotherapeutic methotrexate that specifically recognize the folate receptor-2 (FOLR2) increases therapeutic efficacy by depleting TAMs. Since TAMs overexpress FOLR2, it can target macrophages to inhibit angiogenesis and improve anti-tumor effects. A dendrimer nanoparticle combined with the chemotherapeutic methotrexate (G5-MTX Nps) could improve TAMs targeting and alleviate the cancer development . Tian etThe second strategy of TAM targeting is to inhibit the recruitment of macrophages derived from blood monocytes. This approach is to block specific signaling and tumor-derived chemotactic signals . The colTAM depletion and inhibition of macrophage recruitment have the disadvantage of losing powerful immune modulators called antigen presenting cells (APCs). Therefore, recently, reprogramming TAM from M2-like macrophages to M1-like macrophages has attracted attention as a strategy for cancer therapy. One study has shown that M0 and M2 macrophage were converted to M1 macrophage associated with anti-tumor effects using drug-free mannose decorative liposomes . MannosyRecently, extracellular vesicles which have a significant impact on tumor suppression have gained much interest. Particularly, extracellular vesicles, such as exosomes naturally derived from cells, exhibit targeting ability and, therefore, suitability as biomolecular carriers. For example, the use of exosome-mimetic nanocarriers derived from M1 macrophages could improve anti-tumor efficacy by reprogramming macrophages . The othMany recent studies have demonstrated that the inhibition of the CD47-signal regulatory protein-α (Sirpα) signaling is a promising target for activating macrophage phagocytosis. Representing a “don’t eat me” signal, CD47 is overexpressed on the surface of cancer cells in many tumor types . When SiDCs, the most professional APCs in the immune system, are able to bridge the gap between innate and adaptive immune response ,59. OnceGiven the aforementioned roles of DCs in the immune response, it facilitates assuming that DCs act as the “key-player” of cancer immunotherapy by capturing tumor-associated antigens (TAA) and interacting with T cells.Considering the central role of DCs that induce priming and activating antigen-specific T cells in the immune system, DC-based cancer vaccines resulting in a potent anti-cancer immunity by DC maturation were studied over the past few decades ,62. AlthIn the field of drug delivery, NPs are considered as potent materials for delivering various molecules such as small molecules, nucleic acids, and peptides ,68,69. NLiposomes are self-assembling lipid-based NPs composed of amphiphilic phospholipids. Because liposomes have a hollow sphere structure that encompass hydrophilic central space with hydrophobic lipid bilayer, they can deliver both hydrophilic/lipophilic payloads simultaneously ,89,90,91Varypataki and colleagues demonstrated that the synthetic model antigen (OVA24)-engineered and poly (inosinic-polycytidylic acid) (poly(I:C))-adjuvanted liposomes significantly induced DC maturation followed by OVA24-specific CD8+ T-cell responses . AccordiLipid nanoparticles (LNPs) are another subset of lipid-based NPs with structural differences from conventional liposomes. Whereas traditional liposomes are composed of one or more lipid bilayers encompassing a hydrophilic inner space, LNPs form multiple micelle-like structures within their particle core depending on formulating methods . In geneAccording to the experimental results from Blankschtein’s group, it was found that a single immunization with LNPs containing OVA mRNA induced a strong cytotoxic CD8 T-cell activation. Moreover, significant tumor growth inhibition was also confirmed after treatment of LNPs possessing mRNA encoding glycoprotein 100 (gp100) and tyrosinase-related protein 2 (TRP2) in the B16F10 melanoma implanted mice model .As antigen/adjuvants carrier for DC-mediated anti-cancer immunity, inorganic NPs have some advantageous physicochemical properties such as customizing of size, shape, surface functionalization, and structural conformation . Among tIndeed, Lee and colleagues reported that model antigen (red fluorescent protein (RFP)) and adjuvants (CpG ODN)-engineered AuNPs can convert naïve T cells to active ones, as well as promote antigen-specific T-cell proliferation after LN accumulation. In addition, Gulla et al. showed that AuNP-based nanoplex functionalized with a thiolate ligand (SGSH) and caused a long-lasting (for 180 days) immune response against the murine melanoma by delivering a melanoma antigen (MART1)-encoding DNA vaccine .Another type of inorganic NP for improving the effect of DC-based cancer vaccination is the mesoporous silica nanoparticles (MSNs). Although MSNs possess negatively charged and hydrophilic silanol groups (Si-OH) that allow them to target LNs, MSNs showed low migration efficiency to LNs due to their relatively large size (>100 nm). To tackle this, the Sun group fabricated a smaller size of MSNs (around 80 nm) with different pore sizes, showing enhanced MSNs accumulation in LNs and anti-cancer effects .A previous study from Song and colleagues suggested that polysaccharide-functionalized selenium NPs could be a novel therapeutic tool for lung cancer by modulating dysfunctional immune cells in malignant pleural effusion (MPE) . The sel3O4-OVA) and confirmed its DC-stimulatory ability and anti-tumor effects via in vitro/vivo studies. After incubation with Fe3O4-OVA, a significantly enhanced secretion level of Th-1 biased cytokines from DC2.4 cells  was detected compared to free Fe3O4 NPs and soluble OVA only groups. Moreover, it was confirmed that immunization with Fe3O4-OVA facilitated tumor growth inhibition in tumor-bearing mice n induced surface expression level of co-stimulatory  and MHC (HLA-ABC and HLA-DR) molecules [n demonstrated converted chemokine responsiveness pattern from specific for CCL5 to CCL19 [According to the recent study from the Zhang group, fullerene derivatives, a type of carbon nanomaterial, are suggested as a potent stimulator for maturation of DCs. Similarly to the aforementioned NPs used for the cancer vaccine, gadolinium atom-entrapping fullerene derivatives (Gd@C82(OH)x) also have advantageous properties, such as biocompatible, water-soluble, and small size (approximately 25 nm average) ,122. Aftolecules . It is kolecules ,123. Indto CCL19 .The accommodation of MDSCs at tumor tissues is correlated with suppression of T-cell proliferation or promoting the differentiation of regulatory T cells (Tregs). MDSCs are heterogeneous cell types of immature myeloid cells inducing immunosuppressive effects against cancers . The repLiu et al. designed liposomes that functionalized with DNA thioaptamer (T1) for TME targeting. The results revealed that intravenously administered T1 aptamer accumulated in the tumor site, especially to PMN-MDSCs (CD11b+Ly6G+). In in vivo efficacy test using the orthotopic breast cancer mouse model, it was found that T1-functionalized and doxorubicin-encapsulating liposomes decreased PMN-MDSCs and induced infiltration of CD8+ T cells in tumor sites . Thus, aIn tumor-associated pathological conditions, various immune response-related molecules in TME such as IL-1β ,136, IL-NK cells were identified in 1975 as innate lymphoid cells, which play an important role as modulator within the TME and directly kill cancer cells without any activating procedures such as complements or antibodies . ParticuOn the other hand, innate-like T lymphocytes, natural killer T (NKT) cells perceive glycolipid antigens presented by CD1d, MHC class I-like protein. Activation of NKT cells triggers rapid pro-inflammatory cytokine for immune modulation and leads to chemokine responses. NKT cells control downstream innate and adaptive immune responses against cancers by interacting with APCs. Many studies recently focused on the activation of NKT cells, owing to its properties as mediator between innate and adaptive immunities . AccordiNeutrophils , the most plentiful leukocytes, play a critical role in the regulation of invading infections and involved in innate immune responses. They respond to chemotaxis and can rapidly transmigrate into damaged or infected tissues such as TME.Here we introduce several studies utilizing neutrophils for improving cancer therapeutic efficacy. One study developed albumin-based NPs that hitchhike activated neutrophils and promote immune responses in tumor sites . SimilarBased on the knowledge and understanding of the immune cell dependent effects of traditional cancer therapy (chemo- and radiotherapy), cancer immunotherapy has rapidly emerged as a standard tool for comprehensive cancer care. Cancer immunotherapy is a promising anti-cancer treatment that attacks specific cancer cells by strengthening the immune system in our body, unlike well-established conventional cancer therapies targeting directly the malignant cells . In partNote that the failure of current immunotherapies is closely related with the activity of innate immune cells within TMEs. Moreover, recent evidence underscores the importance of cross-talk between innate immune cells and cancer cells in controlling cancer progression, metastasis, and response to various forms of immunotherapy. Therefore, altering TMEs, specifically, targeting innate immune cells to selectively enhance cytotoxic T-cell activation and reduce MDSC/M2 migration, together with limiting the suppressive effects induced in TMEs, is likely to increase the current response rate of ICBs and other immunotherapies. Therefore, innate immunity is now highlighted as a potential combination-target for immune checkpoint therapy.Here, we review the contribution of advanced nanotechnology for the modulation of innate immune cells in TMEs, their significance on current cancer therapies and the potentials for developing novel therapeutic strategies. Despite of recent pioneering research which has ushered in a new era of the nanomaterial-based modulation of innate immune cells in TME, several issues should be addressed before such applications can be translated into the clinical. First of all, given that drug-loaded NPs can evoke immune toxicity through interaction with immune cells, in-depth toxicity studies must be carefully examined. In addition, NPs that modulate innate immune cells should be designed to target only specific cells with fewer off-target effects and to provide a sustained anti-tumor effect to patients. Accumulating data on ligands that differentiate immune cells of tumors and normal tissues. For example, mannose/galactose receptors are present on both M1-type macrophages and M2-type TAMs. Therefore, to maximize the targeting ability of nanomaterials to TME-specific immune cells, additional research is required to discover more specific ligands for innate immune cells. Importantly, a precise understanding of the contribution of the innate immune systems to the anti-cancer immune responses will be a key feature to consider for the development of nanomaterial-based clinical strategies for cancer therapy.


Human cardiac organoids (hCOs) are complex multicellular aggregates that faithfully recapitulate the cardiac tissue’s transcriptional, functional, and morphological features. In recent years, significant advances in the field have dramatically improved the robustness and efficiency of hCOs derivation and have promoted the application of hCOs for drug screening and heart disease modeling. This review surveys the current differentiation protocols, focusing on the most advanced 3D methods for deriving hCOs from hPSCs. Furthermore, we describe the potential applications of hCOs in the pharmaceutical and tissue bioengineering fields, including their usage to investigate the consequences of Severe Acute Respiratory Syndrome CoronaVirus 2 (SARS-CoV2) infection in the heart.Human pluripotent stem cells (hPSCs) constitute a valuable model to study the complexity of early human cardiac development and investigate the molecular mechanisms involved in heart diseases. The differentiation of hPSCs into cardiac lineages  Consequently, the use of human pluripotent stem cells (hPSCs) as an Here, we emphasize the latest breakthroughs in the fields of cell biology and tissue bioengineering that open the road for the generation of 3D human cardiac organoids (hCOs).MESP-1), together with the surface markers Kinase Insert Domain Receptor (KDR) and Platelet-Derived Growth Factor Receptor (PDGFR) and migrate bilaterally forming the cardiac crescent at the midline, the future heart’s site , and BMP4 from hypoblast and anterior endoderm drives the cardiac induction in chick embryos (Xenopus (β (GSK-3β) inhibitor that activates the canonical Wnt/β-catenin pathway. Mesodermal precursors are then differentiated into cardiac mesodermal progenitors by Wnt inhibition  . Indeed,primordium . Overall, this methodology combines hPSC derived-cardiac cells and ECM substrates in casting molds to generate hydrogels . These cβ-adrenergic stimulation. Furthermore, chronic catecholamine-treated EHTs resemble a heart failure model, including contractile dysfunction, CM hypertrophy, and Natriuretic Peptide release , decreased cell cycle activity, and induced a metabolic switch from glycolysis to fatty acid oxidation, which increased CMs oxygen consumption rate and mitochondrial content , developed by Mills et al. in light of the minimal required tissue handling, has been constantly used in recent years. This method relies on a 96-well plate screening platform to identify favorable conditions for hCO growth, maturation, and functions . This ap content .in vitro  caused a robust diastolic dysfunction together with bromodomain-containing protein 4 (BRD4) activation. Significantly, treatment with bromodomain 2 (BD2)-selective BET inhibitors reduced the cytokine storm-induced diastolic dysfunction in hCOs and SARS-CoV-2 infected mice, and has the potential to reduce SARS-CoV-2 infection of cardiac cells (The Heart-dyno system has been recently used to investigate the bilateral ventricular diastolic dysfunctions developed in SARS-CoV-2 patients . To idenac cells .in vivo heart development. Importantly, each model displays unique characteristics that suit specific purposes, but all show some limitations.Here, we reviewed some advanced techniques for the production of cardiac 3D organoids starting from hPSCs. The approaches described above generate morphological and functional models that partially resemble the in vitro models to recapitulate the early stages of heart development and the dynamic cell-to-cell interactions occurring during cardiomyogenesis. This method relies on the intrinsic capacity of hPSCs to self-organize, implying a stochastic arrangement in the three-dimensional space. Thus, this variability hampers the reproducibility of the studies (in vivo organs, such as the four cardiac chambers, valves, and inflow/outflow tract, cannot be faithfully recreated with these models. Furthermore, self-assembly-derived hCOs resemble the characteristics of fetal-like organs and cannot be employed to model heart diseases occurring in later stages of development. In this regard, the directed assembly approach offers a promising alternative based on casting platforms, such as the EHTs, that provide an excellent cellular system to optimize CM maturation while more closely mimicking the in vivo physiology. However, EHT-derived CMs fail to fully recapitulate the adult heart tissue anatomy. Interestingly, the recent application of the Heart-dyno model in SARS-CoV-2 research showed its tremendous potential. Innovative approaches could be based on the formation of multicellular “assembloids,” as recently reported for the brain tissue (ULA-derived self-assembled hCOs represent helpful  studies . On the  studies  and subs studies . Unfortun tissue . Assembln tissue . This apIn conclusion, we reviewed the latest 3D methodologies to derive hCOs from hPSCs, which promise to further advance translational medicine applications of hPSCs. The use of these models could provide novel insights into understanding human cardiogenesis, injury processes, and developmental drug toxicities.


Death ligands such as tumor necrosis factor-related apoptosis-inducing ligand  and their corresponding death receptors  not only initiate apoptosis through activation of the extrinsic apoptotic pathway but also exert non-apoptotic biological functions such as regulation of inflammation and cancer metastasis. The involvement of the TRAIL/death receptor signaling pathway in the regulation of cancer invasion and metastasis is complex as both positive and negative roles have been reported. The underlying molecular mechanisms are even more complicated. This review will focus on discussing current knowledge in our understanding of the involvement of TRAIL/death receptor-mediated signaling in the regulation of cancer cell invasion and metastasis. Metastasis is a key cause of cancer death. To metastasize to distant organ sites, cancer cells must detach from the primary tumor sites , translocate into the circulatory system, survive in the circulation and arrest at a distant organ site (extravasation), and finally adapt to the new microenvironment of distant tissues  . TherefoApoptosis represents a major form of cell death. Thus, the acquisition of apoptosis resistance is a key hallmark of cancer and occurs early during tumorigenesis . AccordiThere are two TRAIL death receptors in humans: death receptor 5  and death receptor 4 . Both DR5 and DR4 share redundant functions in triggering apoptosis but have some distinct biological functions, such as regulation of cancer cell metastasis, as discussed below. In contrast, there is only one TRAIL death receptor named murine TRAIL death receptor (mDR) in mice ,10 or moS) and long (FLIPL) splice forms, has two death effector domains and can allow the recruitment of caspase-8. The C-terminal region of FLIPL contains a caspase homolog that lacks caspase activity. Formation of the pro-caspase-8/FLIPL heterodimer complex can limit caspase-8 activation. In FLIPS, there is no such homolog of caspase; instead, there is a short region of 20 amino acids responsible for its ubiquitylation and proteasomal degradation. Both forms of FLIP can compete for caspase-8 homodimer complex assembly, and so, when FLIP is heterodimerized with caspase-8, it inhibits caspase-8-dependent cell death [The cellular FLICE-like inhibitory protein (c-FLIP) is a truncated and dominant-negative homolog of caspase-8 and functions to inhibit death receptor-mediated apoptosis by competing with caspase-8 for binding to FADD ,15. FLIPll death ,17.Epithelial cells normally undergo detachment-induced apoptosis, termed anoikis, once losing anchorage to the extracellular matrix or when adhesion to the correct substrate is disrupted, primarily by triggering the death receptor-mediated extrinsic apoptotic pathway. In contrast, malignant epithelial cells with metastatic potential resist anoikis and can survive in an anchorage-independent fashion ,19.TRAIL is expressed in various tissues, particularly immune cells , and plaBased on this rationale, therapeutic strategies against metastatic cancer cells have been explored. In a breast cancer xenograft model, injection of TRAIL-expressing CD34⁺ cells, CD34-TRAIL(+) cells, did not lead to tumor growth inhibition; however, lungs in the tumor-bearing mice were completely free of metastases at 12 days after the last injection of CD34-TRAIL(+) cells, whereas metastases were present in all control mouse lungs . In a reIt has been shown that tumor cell lines derived from metastatic lymph nodes displayed a greater TRAIL resistance than cell lines derived from the respective primary oral tumors . ConsistMYC-driven lymphomagenesis [Early studies using a mouse knockout model suggest that mDR plays a suppressive role in cancer metastasis without affecting primary tumor growth since mDR knockout significantly increased lymph node metastasis of carcinogen-induced skin carcinomas  and metaagenesis . In agreagenesis . Moreoveagenesis . Similaragenesis . These pReduced DR5 expression in human melanoma tumor samples was reported to be associated with metastatic lesions . Our ownIn the early stage of HNCs with no lymph node metastasis, we found that higher expression of caspase-8 alone or in combination with higher DR5 expression significantly correlated with better disease-free survival and overall survival , suggestDespite the suppressive function of TRAIL/death receptor in the regulation of cancer cell invasion and metastasis as discussed above, TRAIL has also been reported to strongly induce the expression of pro-inflammatory cytokines interleukin-8 and monocyte chemoattractant protein 1 (MCP1), to enhance the invasion of apoptosis-resistant pancreatic ductal adenocarcinoma (PDAC) cells in vitro by upregulation of the urokinase-type plasminogen activator expression, and to strongly increase the distant metastatic spread of pancreatic tumors in vivo . SimilarKRAS mutation in AC accompanied by a higher incidence of metastasis and increased amplification of the TRAIL gene in SCC. Bioinformatic analyses of an additional lung cancer patient database also showed that the risk of disease progression was significantly increased with high TRAIL expression in AC (461 samples) [Bioinformatic analyses of patient data from 839 adenocarcinoma (AC) and 356 squamous cell carcinoma (SCC) of lung cancer cases by cBioPortal  showed that TRAIL expression leads to differential outcomes of disease-free survival in AC and SCC. Oncomine datamining  revealed that TRAIL is upregulated in 167 SCC as compared to 350 AC patients from six data sets. Genomic analyses using cBioPortal revealed high rates of samples) . Patientsamples) .KRAS-driven models of non-small cell lung cancer (NSCLC) and PDAC was shown to reduce tumor growth, blunt metastasis, and prolong survival by inhibiting cancer cell-autonomous migration, proliferation, and invasion. Moreover, this study detected DR5 expression in human PDAC and colorectal cancer tissues and found that high DR5 expression correlated with invasion of human PDAC into lymph vessels and with shortened metastasis-free survival of KRAS-mutated colorectal cancer patients [There was a study demonstrating that mDR and human DR5 promote K-Ras-driven cancer progression, invasion, and metastasis ; these rpatients . Mechanipatients .In a different study using an osteotropic variant of MDA-MB-231 breast cancer cells, knockdown of DR5 increased the levels of E-cadherin and decreased migration with strongly impaired ability to form bone metastases in vivo after intracardiac injection. Evaluating possible underlying mechanisms revealed a strong downregulation of CXCR4, the receptor for the chemokine SDF-1 important for homing of cancer cells to the bone. In accordance, cell migration toward SDF-1 was significantly impaired by DR5 knockdown. Conversely, overexpression of DR5 upregulated CXCR4 levels and enhanced SDF-1-directed migration . This stFADD and caspase-8 are key proteins in transmitting death receptor-mediated apoptotic signaling, whereas c-FLIP is the key inhibitor of this death signaling. However, there are critical roles of these proteins in the regulation of cancer cell invasion and metastasis; some may be largely independent of death receptors ,60,61.High FADD expression was associated with an increased rate of lymph node metastasis of HNCs and with a shorter distant metastasis-free interval when lymph node metastases were present . ConsistAlthough caspase-8 was reported to be associated with FAK and other proteins, resulting in enhancement of cleavage of focal adhesion substrates and subsequent promotion of cancer cell migration and metastasis under apoptosis-compromised conditions , caspaseIn human cancer samples, a high abundance of c-FLIP was found to be significantly associated with lymph node metastasis in both gastric adenocarcinoma  and endoIt is apparent that TRAIL/death receptor signaling is involved in both negative and positive regulation of cancer invasion and metastasis. Under apoptosis-compromised conditions, TRAIL/death receptor has a high likelihood to exert non-apoptotic functions, including invasion/metastasis-promoting activity.It is now known that non-canonical TRAIL/death receptor signaling exists and results in the subsequent activation of various kinases including RIPK1, IκB/NF-κB, mitogen-activated protein kinases (MAPK) p38, JNK, ERK1/2, MAP3K/TAK1, PKC, PI3K/Akt, and Src in cancer cells, particularly under apoptosis-resistant conditions . WheIn addition to the primary DISC formation, TRAIL also induces the formation of a secondary signaling complex that retains the DISC components FADD and caspase-8 but recruits several other factors involved in kinase activation such as RIPK1, TRAF2, and NEMO/IKKγ. This secondary complex formation requires FADD, as well as caspase-8 activity, and activates NF-κB, MAPKs, JNK, and p38 pathways . TherefoAlthough inflammation alone is insufficient for causing cancer or metastasis, the inflammatory response can greatly increase the risk of metastasis through the generation of pro-inflammatory biomolecules , sphingosine-1-phosphate (S1P), matrix metalloproteinases (MMPs), and angiogenic factors  at the site of inflammation ,86,87. WTRAF2 is present in the DISC complex and negatively regulates TRAIL-induced caspase-8 activity . MoreoveIn addition to the positive regulation of cancer cell invasion and metastasis, TRAIL/death receptor signaling is largely involved in the negative regulation of cancer cell migration, invasion, and metastasis, as discussed above. Beyond its apoptosis or anoikis-inducing activity that helps to eliminate circulating metastatic cancer cells to prevent dissemination of cancer cells, activation of the pathway may also exert non-apoptotic functions in the suppression of cancer metastasis. Based on our findings ,96, we sMonocyte chemotactic protein-induced protein-1  encoded by the ZC3H12A gene critically regulates inflammatory responses and immune homeostasis primarily . As its KRAS-mutated cancer cells [HRAS mutant animal models [RAS- or RAF-mutated cells with elevated DR5 expression barely survive in circulation and will be eliminated  if they are sensitive to TRAIL/DR5-mediated apoptosis. A subset of RAS- or RAF-mutated cells with resistance to TRAIL/DR5-mediated killing may survive in the circulation and eventually be able to metastasize to distal organs. Under such a scenario, elevated DR5 may favor the formation of metastasis-related signaling complexes such as the FADDosome as illustrated in Oncogenic mutations of Ras and B-Raf frequently occur in many cancer types and are critical for cell transformation and tumorigenesis. Interestingly, DR5 has been shown to positively regulate cell invasion and metastasis in er cells  while sul models , albeit l models . It is pRAS mutations [TRAIL/death receptor signaling has long been considered an apoptosis-inducing signaling cascade. However, it can also initiate other signaling pathways, unveiling non-apoptosis-related functions, including stimulation of inflammatory responses and cancer metastasis under certain circumstances. Hence, death receptor-mediated signaling complex proteins can serve as an important platform to regulate cancer apoptosis, inflammation, and metastasis depending on cell types or contexts. Under TRAIL-resistant or apoptosis-compromising conditions, activation of TRAIL/death receptor signaling may likely favor the positive regulation of cancer cell invasion and metastasis. Oncogenic Ras proteins such as K-Ras and H-Ras, in general, have shared redundant biological activities . Howeverutations ,56. The utations ,10 and tTRAIL/death receptor-targeted therapeutics including recombinant proteins and agonistic antibodies have been developed for cancer therapy and are currently being tested in the clinic ,13,101. Since resistance to TRAIL/death receptor-mediated apoptosis is a key factor associated with enhanced invasion and metastasis of cancer cells, it is critical to consider strategies aiming to target these cancer cells with resistance to TRAIL/death receptor-initiated apoptosis. Fortunately, many types of resistant cancer cells can be sensitized to TRAIL or death receptor activation-induced apoptosis by various means, particularly some small molecules that are able to increase death receptor expression and/or decrease the levels of c-FLIP and/or other anti-apoptotic proteins such as Mcl-1 ,103. Con