Osteosarcoma (OS) is the most common bone malignancy and preferably occurs in children and adolescents. Despite significant advances in surgery and chemotherapy for OS over the past few years, overall survival rates of OS have reached a bottleneck. Thus, extensive researches aimed at developing new therapeutic targets for OS are urgently needed. Autophagy, a conserved process which allows cells to recycle altered or unused organelles and cellular components, has been proven to play a critical role in multiple biological processes in OS. In this article, we summarized the association between autophagy and proliferation, metastasis, chemotherapy, radiotherapy, and immunotherapy of OS, revealing that autophagy‐related genes and pathways could serve as potential targets for OS therapy. In this review, we summarized the role of autophagy in OS proliferation, metastasis, chemotherapy, radiotherapy, and immunotherapy. And we think that autophagy‐related genes and pathways could serve as potential targets for OS therapy. This process is manifested by the formation of double‐membrane vesicles  to engulf damaged organelles, unfolded proteins, pathogens, etc., followed by transporting them to lysosomes for breakdown and digestion.A dual role of autophagy is observed in multiple tumor types, as well as in osteosarcoma. On the one hand, OS tumor cells utilize autophagy as a means of therapy resistance, proliferation, and cancer stem cell protection.22.1Autophagy can be divided into three subtypes: chaperone‐mediated autophagy (CMA), microautophagy, and macroautophagy.2.2Under situations of nutrient deprivation, hypoxic conditions, and/or chemotherapeutic treatment, mTORC1 is inhibited or AMPK is activated, which then activates the downstream serine/threonine Unc‐51‐like kinase 1 (ULK1). The activated ULK1 can interact with both the focal adhesion kinase family interacting protein of 200 kDa (FIP200) and the conjugate of autophagy‐related protein 13 (ATG13) and ATG101 to form the ULK1‐FIP200‐ATG13‐ATG101 complex, which is also known as the ULK1 initiation complex.Upon its formation, the ULK1 initiation complex leads to activation of the class III phosphoinositide 3‐kinase (PI3K) complex. The PI3K complex, which is essential for vesicle nucleation, is composed of vacuolar protein sorting 34 (VPS34), ATG14, the activating molecule in BECN1‐regulated autophagy protein 1 (AMBRA1), and the scaffold protein Beclin‐1.This step is characterized by two ubiquitin‐like systems, the first system involves the formation of the ATG5‐ATG12‐ATG16 complex which is conjugated by the ATG7 and ATG10. In the second system, the protease ATG4 converts microtubule‐associated protein 1‐light chain 3 (LC3) into LC3‐I and stimulates the binding of ATG7 to attract ATG3 resulting in the ligation of phosphatidylethanolamine (PE) with LC3‐I to form LC3‐PE complex .LC3‐II further acts in cargo recognition by directly interacting with cargo receptors such as sequestosome 1 (SQSTM1/p62) and neighbor of BRCA1 (NBR1).ATG9 recruits lipid membrane which is derived from mitochondria, plasma membrane, Golgi, or the endoplasmic reticulum to the forming AVs to close the vesicle.In this step, the AVs fuse with lysosomes to generate autophagolysosomes. Rab GTPases, membrane‐tethering complexes , and soluble N‐ethylmaleimide‐sensitive factor attachment protein receptors (SNARE) involve in regulating this process.The membranes and the contents of the AV are degraded by lysosomal hydrolases. The autolysosome is broken down and degradation products, such as amino acids, peptides, and free fatty acids, are released into the cytoplasm to be reused, excreted into the bloodstream. These final products can be used for protein synthesis or can be used as source of energy for cell survival.The process of autophagy involves more than 30 autophagy‐related genes (ATGs) and includes the following seven consecutive steps Figure . And the2.3The regulation of autophagy is closely linked to distinct autophagy‐related signaling pathways, including (i) PI3K/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) signaling pathway; (ii) adenosine monophosphate‐activated protein kinase (AMPK) signaling pathway; and (iii) Beclin‐1/Bcl‐2‐related signaling pathway  is a class of lipid kinase whose main biochemical function is to catalyze phosphatidylinositol (PI) phosphorylation at the D3 position and convert phosphatidylinositol 4,5‐bisphosphate (PIP2) into phosphatidylinositol 3,4,5‐triphosphate (PIP3).2.3.2Another crucial signaling pathway AMPK, which inhibits autophagy through a variety of actions, including its regulatory effect on the MAPK/ERK pathway, the suppression of PI3K‐AKT/mTOR pathway, and the phosphorylation of ULK1 complex in autophagy.2.3.3Another important mechanism found to participate in autophagy regulation and tumorigenesis involves Beclin‐1, originally recognized as a Bcl‐2‐binding protein. It is a part of the PI3K complex, which initiates the formation of the phagophore. Beclin‐1 contains a BH3‐binding domain and has been found bound to Bcl‐2 in the form of a dual protein complex.33.1A growing number of studies have shown that autophagy positively regulates tumor cell proliferation in multiple cancer types. Cell proliferation is normally inhibited through downregulating autophagy‐related genes such as ATG7 in cervical cancer, breast cancer, and osteosarcoma.In the previous paragraph, we explored that autophagy could positively regulate OS cell proliferation. However, a mass of studies has pointed out that activation of autophagy could inhibit proliferation of OS cells.BECN1 was the first tumor suppressor to link autophagy to tumors, and its monoallelic deletions have been found in as many as 40% to 75% of various human tumors, such as prostate, breast, and ovaries.3.2Some important regulatory pathways such as PI3K/AKT/mTORC1 pathway and ERK/MAPK pathway similarly play an important role in activating autophagy and inhibiting proliferation of OS cells. Liu et al found that ginsenoside‐Rg5 (Rg5) reduced the phosphorylation of PI3K, Akt, and mTORC1 activation. Conversely, LC3‐mediated autophagy upregulated significantly. More importantly, Rg5 inhibited proliferation of OS cells potently in a dose‐dependent manner.3.3The 5‐year survival rate in patients with nonmetastatic OS could reach 75%; however, this value is just 20% in metastatic patients.Neoadjuvant chemotherapy regimen consisting of MTX, DOX, and CIS were first proposed in the 1970s as a means to shrink primary lesions, minimize surgical complications, and treat distant micrometastases, significantly improving overall survival in OS patients. Although neoadjuvant chemotherapy has significantly improved survival rate in OS patients, a mass of patients subsequently develops chemoresistance, which is now the major cause of higher mortality.Recently, some researches have shown that chemotherapy‐resistant OS cells are often accompanied by an increased autophagy.Although numerous studies demonstrated that chemotherapy triggered a cytoprotective autophagy, autophagic cell death has received increasing attention. Autophagic cell death is a type of cell death mediated by autophagy which is distinct from apoptosis or necrosis.We conclude from above that mild autophagy could increase chemoresistance because of cytoprotective effect, whereas excessive autophagy reverses chemoresistance through activating cell death Table . Thus, o3.4Local radiotherapy has been found to have some positive effects in OS patients who are inoperable and poorly responsive to chemotherapy.It has been reported that activation of the nuclear factor epithelioid 2‐related factor 2 (NRF2) pathway conferred resistance to radiation therapy in U‐2 OS cell. And this process was regulated by autophagy‐mediated activation of ERK 1/2 kinases.3.5Over the past few years, there has been increasing evidence that the immune system plays a critical role in progression and treatment of tumors. The most studied is the improvement of cancer immunotherapy by blocking PD‐1/PD‐L1 immune checkpoints. In recent years, enhancing the efficacy of antitumor immunotherapy through autophagy has become a promising therapeutic approach. Tumor‐promoting effects of autophagy could be abrogated through immune checkpoint blockers (ICBs) in combination with autophagy inhibitors.+ T cells were activated to inhibit OS growth and metastasis. Meanwhile, Ren et al.Consistently, encouraging results are obtained from immunotherapy in advanced, metastatic, and recurrent OS.4In this review, we summarized the role of autophagy in OS proliferation, metastasis, chemotherapy, radiotherapy, and immunotherapy. Overall, autophagy plays a dual role in different stages of OS. However, there is no specific mechanism that defines this dual role of autophagy in OS. Until now, clinical outcomes of OS have not met expectations. A growing number of studies have confirmed that autophagy plays a dual role in regulating OS chemoresistance through exerting cytoprotective effects or causing autophagic cell death. Hence, either inhibition of cytoprotective autophagy or enhancement of autophagic cell death can improve chemosensitivity of OS. Besides, autophagy inhibition can enhance the efficacy of radiotherapy and immunotherapy in OS. Although autophagy regulator has great potential in the treatment of OS, the practical utility of all kinds of autophagy regulators in clinical practice remains to be investigated. We look forward to more specific autophagy modulators which could improve the efficacy of anti‐OS therapies will be found in the near years.Biao Ning: Conceptualization ; data curation ; formal analysis ; project administration ; resources ; visualization ; writing – original draft (lead); writing – review and editing (lead). Yixin Liu: Data curation ; formal analysis ; investigation ; resources . Tianhe Huang: Formal analysis ; methodology ; project administration ; supervision ; validation . Yongchang Wei: Conceptualization (lead); funding acquisition (lead); methodology (lead); project administration (lead); resources (lead); supervision (lead).There is no funding for this review.All authors acknowledge that there is no conflict of interest in this review.This is a review article and the need for ethics approval and consent was waived.Table S1.Click here for additional data file.


Traumatic brain injury (TBI) is a leading cause of long‐term disability in young adults and induces complex neuropathological processes. Cellular autonomous and intercellular changes during the subacute phase contribute substantially to the neuropathology of TBI. However, the underlying mechanisms remain elusive. In this study, we explored the dysregulated cellular signaling during the subacute phase of TBI.Single‐cell RNA‐sequencing data (GSE160763) of TBI were analyzed to explore the cell–cell communication in the subacute phase of TBI. Upregulated neurotrophic factor signaling was validated in a mouse model of TBI. Primary cell cultures and cell lines were used as in vitro models to examine the potential mechanisms affecting signaling.Single‐cell RNA‐sequencing analysis revealed that microglia and astrocytes were the most affected cells during the subacute phase of TBI. Cell–cell communication analysis demonstrated that signaling mediated by the non‐canonical neurotrophic factors midkine (MDK), pleiotrophin (PTN), and prosaposin (PSAP) in the microglia/astrocytes was upregulated in the subacute phase of TBI. Time‐course profiling showed that MDK, PTN, and PSAP expression was primarily upregulated in the subacute phase of TBI, and astrocytes were the major source of MDK and PTN after TBI. In vitro studies revealed that the expression of MDK, PTN, and PSAP in astrocytes was enhanced by activated microglia. Moreover, MDK and PTN promoted the proliferation of neural progenitors derived from human‐induced pluripotent stem cells (iPSCs) and neurite growth in iPSC‐derived neurons, whereas PSAP exclusively stimulated neurite growth.The non‐canonical neurotrophic factors MDK, PTN, and PSAP were upregulated in the subacute phase of TBI and played a crucial role in neuroregeneration. Cell–cell communication analysis reveals that the canonical neurotrophic signaling  are upregulated rather than the canonical neurotrophic signaling in the subacute phase of TBI. Activated microglia promotes astrocyte‐secreting MDK, PTN, and PSAP, which enhances neural stem cell proliferation and neurite growth, and may induce spontaneous neuroregeneration. TBI disrupts the normal structure of the brain, leading to temporary or permanent brain impairment, and in severe cases, it may even cause death. Approximately 69 million individuals worldwide suffer from TBI annually.Traumatic brain injury is classified into two categories according to the mechanism of injury: primary and secondary injury. Primary brain injury occurs immediately after a mechanical force is applied directly to the head, leading to acute neural cell death, hemorrhage, hematoma, and brain edema. Secondary injury is initiated after the primary insult and lasts for hours to even years.Previous studies have revealed changes in the genomic profile after TBI by utilizing heterogeneous mixtures of cell populations.Unbiased high‐throughput single‐cell RNA‐sequencing provides a novel perspective for exploring the molecular mechanisms of TBI. A recent study revealed changes in the cell type‐specific genes and pathways in the hippocampus during the acute phase (within 24 h) of concussive brain injury using single‐cell RNA‐sequencing.Using high‐throughput single‐cell RNA‐sequencing technology, we sought to identify the cell populations involved and changes in the signaling pathways in the subacute phase of TBI at the single‐cell level. In this study, we found that cell–cell interactions were significantly increased in astrocytes, microglia, and oligodendrocytes. We found that the signaling of three non‐canonical neurotrophic factors, midkine (MDK), pleiotrophin (PTN), and prosaposin (PSAP), was significantly upregulated, especially in astrocytes. These findings were validated in a time‐course study using a rodent model of TBI. We then investigated the possible roles of MDK, PTN, and PSAP in the pathogenesis of TBI and found that they played a limited role in inflammatory regulation but significantly increased neurite growth and neural progenitor cell (NPC) proliferation. Our findings provide novel insights into the pathology of TBI, paving the way for the development of potential treatments for TBI.22.1http://xteam.xbio.top/CellMarker/).The single‐cell RNA‐sequencing data of TBI (fluid percussion injury model) were collected from Gene Expression Omnibus .2.2Three‐month‐old male mice (C57BL/6) were anesthetized with Avertin . The mice were fixed on a stereotaxic frame, and a 4‐mm‐diameter cranial window centered at 2 mm lateral to the Bregma was performed on the right side of the skull. TBI was induced using a weight drop model. A steel block weighing 45 g with a 3‐mm‐diameter flat tip was dropped onto the exposed cortex from a height of 30 cm with an impact depth of 2 mm. The mice in the sham group were handled in the same manner but without craniotomy and impact. After surgery, mice were placed on a homoeothermic blanket (37°C) until complete recovery from the anesthesia. An analgesic  was subcutaneously injected for postoperative pain control.2.3The mice were sacrificed at 2 days (TBI‐2d), 7 days (TBI‐7d), 14 days (TBI‐14d), and 30 days (TBI‐30d) after TBI. Mice were anesthetized with a lethal dose of Avertin, and then transcardially perfused with 0.01 M phosphate‐buffered saline (PBS) containing 10 U/mL of heparin, followed by 4% paraformaldehyde (PFA) solution. Brains were collected and post‐fixed in 4% paraformaldehyde solution overnight at 4°C and then soaked into 30% sucrose in PBS for dehydration. Brains were sectioned using a cryostat (Leica Biosystems) with 20‐μm thickness when mice brains sunk in the 30% sucrose solution. Brain sections were stored in cryoprotectant  at −30°C until use.Two to three brain sections from Bregma +1 mm to −0.5 mm were chosen for free‐floating immunofluorescence staining. Brain sections were rinsed with PBS and blocked in 10% normal goat/donkey serum in 0.3% Triton X‐100 PBS for 1 h at room temperature (RT) before incubation with primary antibody Table  overnighTo quantify MDK, PTN, and PSAP expression in the peri‐injured site of the cortex, the integrated density of the positive area was analyzed using Fiji software and then normalized by the sham group. The percentage of GAP43 positive was also analyzed using Fiji with a fixed threshold. To evaluate the number of GAP43 positive puncta, a Fiji plugin of “Find Maxima” with 15 of “prominence” was used after subtracting the background of images with 50 pixels of rolling ball radius.To quantify the MDK, PTN, and PSAP expression in different cell types at 7 days after TBI, brain sections were co‐immunostained with NeuN , IBA1 , or GFAP . The percentage of the co‐stained area (per field) was analyzed using Fiji software. The co‐stained area was also normalized by the corresponding positive area of the neural cell markers.2.4The astrocyte was cultured from 1‐day postnatal mice.2.5To detect the effect of MDK, PTN, and PSAP on inflammatory‐related genes expression, BV2 cells were treated with MDK , PTN , and TX14(A) , or co‐stimulated with LPS (500 ng/mL) for 24 h. Total RNA was isolated using TRIzol. The complementary DNA (cDNA) was synthesized via reverse transcription using PrimeScript™ RT reagent Kit (TaKaRa). The inflammatory‐related gene expression was evaluated using real‐time quantitative polymerase chain reaction (qPCR) using a 2× Universal SYBR Green Fast qPCR Mix kit  according to the instruction manual. The primers are shown in Table 2.6To induce human‐derived induced pluripotent stem cells (iPSCs) to differentiate into neural progenitor cells (NPCs) and neurons, feeder‐free iPSCs were maintained in mTeSR™ Plus medium (STEMCELL) and then changed to neural induce medium  for 9 days (daily medium change). Then, cells were dissociated with TrypLE (Thermo Fisher Scientific) and plated on the low growth factor Matrigel Matrix‐coated dishes in NPCs medium  with ROCK inhibitor Y27632  at the first day. The NPC medium was changed daily until neural rosettes formed to acquire NPCs. The NPCs were dissociated with Accutase (STEMCELL) and cryopreserved in liquid nitrogen until use.To evaluate the role of MDK, PTN, and PSAP on the proliferation of human iPSCs‐derived NPCs, the NPCs were seeded on low growth factor Matrigel Matrix‐coated 24‐plate wells in NPCs medium with ROCK inhibitor Y27632. The next day, the medium was changed into fresh NPCs with/without MDK (50 ng/mL), PTN (50 ng/mL), and TX14(A) (500 nM) for 16 h, and then EdU (10 μM) was added for another 8 h. The cells were fixed in 4% PFA. The Edu‐labeled cells were detected using the BeyoClick™ EdU‐594 cell proliferation kit according to the manual instruction. The EdU‐positive cells were counted manually using Fiji software, and the percentage of EdU‐positive cells was analyzed.iPSCs‐derived neurons were used to evaluate the effects of MDK, PTN, and PSAP on neurite growth. A 24‐well plate was coated with 0.1% polyethyleneimine (Sigma‐Aldrich) in 0.15 M borate buffer  for 1 h. After rinsing with distilled water, the plate was coated with low growth factor Matrigel Matrix (Corning) for 1 h. iPSCs‐derived NPCs were plated on the treated 24‐well plate in neuron culture medium  with ROCK inhibitor Y27632. The next day, the medium was changed to fresh neuron culture medium with/without MDK (50 ng/mL), PTN (50 ng/mL), and TX14(A) (500 nM) and kept in culture for another 72 h. The neurons were fixed with 4% PFA and immunostained with mouse anti‐Tuj1  to visualize neurites. The total length of neurite per cell was analyzed using the Macro tool of “Measure_Skeleton_Length_tool” via Fiji software.2.7t‐test was employed for data with normal distribution, while the Mann–Whitney test was used when data were not normally distributed. For multiple group comparisons, the one‐way ANOVA test was conducted followed by either Tukey's Honest Significant Difference Test (HSD) (for between‐group comparisons) or Dunnett's tests (for comparisons to a control group) if the data passed variance equality and normality tests. For data without variance equality and normality, the Kruskal–Wallis test followed by Dunnett's test was performed for multiple comparisons. Two‐tailed statistical significance tests were used throughout. A p‐value less than 0.05 was considered statistically significant.Data were presented as mean ± SEM or median with interquartile range (in box plots). The assumption of equal variances was tested using the Brown–Forsythe test. And normal distribution was tested using the Shapiro–Wilk test when the sample size was sufficient. For two group comparisons, the student's 33.1Aif1, P2ry12), astrocyte , oligodendrocyte , neuron , endothelial cell , pericyte , border‐associated macrophage , T cells , and ependymal cells , we analyzed 14,676 individual cells in the control group and 10,745 cells in the TBI group from a previous study .3.2Signaling crosstalk via ligand–receptor pairs is critical for cellular physiological and pathological processes. Therefore, we analyzed alterations in cell–cell communications in the subacute phase of TBI using the CellChat R package. The number of inferred cell–cell communications decreased slightly after TBI Figure . HoweverSeveral signaling pathways were significantly enhanced after TBI, including fibroblast growth factor (FGF), CCL, KIT, and non‐canonical neurotrophic factors  signaling Figure . FGF sigWe investigated the cell types in which the cell–cell communication was affected in the subacute phase of TBI. We found that both incoming and outgoing signals from astrocyte1, astrocyte2, astrocyte3, microglia2, and oligodendrocytes were strongly enhanced after TBI Figure . Notably3.3To validate the upregulation of MDK, PTN, and PSAP signaling after TBI, we measured the expression of MDK, PTN, and PSAP at various time points. After TBI, MDK was significantly upregulated on Days 2, 7, and 14, and then decreased on Day 30 Figure . The upr+NeuN+ colocalization area did not change, whereas the PTN+NeuN+ and PSAP+NeuN+ areas were significantly reduced in TBI‐7d mice  increased, whereas PTN and PSAP expression remained unchanged  and 14 patients with TBI (10 with mild TBI and four with moderate/severe TBI treated with decompressive craniectomy), and MDK, PTN, and PSAP expression was detected using dot blotting. Notably, correlation analysis revealed positive correlations among the expression of MDK, PTN, and PSAP Figure , especia3.4We examined the expression of MDK, PTN, and PSAP in microglia and astrocytes under inflammatory conditions (LPS stimulation) in vitro. Our results revealed that LPS stimulation did not affect MDK and PTN expression in BV2 microglia Figure  or in pr3.5Neurotrophic factors are implicated in neural regeneration following brain injury. To test whether MDK, PTN, and PSAP regulate the proliferation of NPCs, we treated human iPSC‐derived NPCs with recombinant MDK, PTN, and the PSAP‐derived peptide TX14(A). The EdU cell proliferation assay indicated that NPC proliferation was significantly increased by MDK and PTN treatment Figure . We alsop = 0.0536 for the GAP43‐positive area) when compared to the results at 2 and 7 days post‐TBI  immunostaining. Both the percentage of GAP43‐positive area and the number of GAP43‐positive puncta significantly decreased after TBI compared with that in the sham group, and this trend persisted even 30 days post‐TBI Figure . It is wI Figure . In comb3.6Il1b, Il4, Il6, Il10, Il13, Ccl2, Tnfa, Tgfb, Nos2, Cd86, Cd206, and Arg1. The results showed that PTN and TX14(A) upregulated Il1b expression in BV2 cells n Figure . We inves Figure . Howevers Figure . These f4High‐throughput single‐cell RNA‐sequencing enables us to uncover novel cell subtypes, cell–cell interactions, and signaling pathways under pathological conditions, providing new insights for developing therapeutic strategies. In the present study, using high‐throughput single‐cell RNA‐sequencing analysis, we unveiled damage‐associated cell–cell communication changes in the subacute phase of TBI. Our results demonstrated that the canonical neurotrophic factor signaling (NT and NPY) was significantly downregulated during the subacute phase of TBI. However, the signaling of the non‐canonical neurotrophic factors MDK, PTN, and PSAP was enhanced in the subacute phase of TBI. The in vivo results showed that the expression of MDK, PTN, and PSAP after TBI was time‐dependent. An in vitro study revealed that MDK, PTN, and PSAP expression was upregulated in astrocytes after inflammatory stimulation, which may play a role in neural regeneration rather than in inflammation regulation.Single‐cell RNA‐sequencing enables the inference of cell–cell communication by analyzing the expression of genes encoding the corresponding ligands, receptors, and intermediary proteins across the interacting cell populations. In this study, we chose the datasets of the control and TBI groups from the reported single‐cell RNA‐sequencing data to explore the common pathological changes in the subacute phase of TBI. We found that the number of cell–cell interactions decreased, while the strength of cell–cell interactions increased, indicating that TBI disrupts neural homeostasis. Information flow analysis, which quantified the total communication probability within the inferred network, revealed that FGF (FGF9‐FGFR1/2/3 interactions) and CCL signaling were upregulated after TBI. These two signaling pathways were mainly derived from the cell–cell interactions among glial cells and inflammatory cells Figure , which iMDK and PTN, the heparin‐binding neurite outgrowth‐promoting factors, have similar structures and functions and have been verified to belong to the neurotrophic factor family.PSAP is a nonenzymic glycoprotein that is targeted at lysosomes.The roles of MDK, PTN, and PSAP in TBI are not fully understood. Both MDK and PTN play an anti‐apoptotic role by inhibiting caspase‐3 expression.MDK, PTN, and PSAP can also mediate inflammation under pathological conditions.Single‐cell RNA‐sequencing analysis revealed the comprehensive cell–cell interactions in the subacute phase of TBI. Taking advantage of the cell–cell communication analysis, we discovered that the signaling of the non‐canonical neurotrophic factors MDK, PTN, and PSAP was enhanced compared with the signaling of the canonical neurotrophic factors BDNF and NPY, after TBI. The upregulation of MDK, PTN, and PSAP expression after TBI may play a role in spontaneous neurogenesis and axonal regeneration after TBI. These new insights can help understand the complex pathological processes of TBI and provide therapeutic targets for TBI. Notably, the TBI model used in the present study was a weight loss‐induced TBI model, which is a more severe model than the fluid percussion injury TBI model used in single‐cell RNA‐sequencing studies. Consistent results from the two different TBI models suggest that enhanced signaling of MDK, PTN, and PSAP is a common pathological feature of both mild and severe TBI. However, the potential of exogenous MDK, PTN, and PSAP to promote brain repair and the underlying mechanisms behind it remain unclear, and further studies in an experimental model of TBI are required.5In summary, we demonstrated that the non‐canonical neurotrophic factors MDK, PTN, and PSAP were upregulated in the subacute phase of TBI and revealed their roles in promoting neuroregeneration. Our findings provide new insights into TBI pathology and offer potential therapeutic targets for TBI intervention.X.Q. carried out experiments, analyzed data, and prepared the manuscript. Y.G. performed the immunofluorescence staining and statistical analysis. M.F.L. calculated a Glasgow Coma Scale (GCS) score of TBI participants and collected blood samples. B.Z. assisted in the qPCR study. J.L. and J.F.W. contributed to the design of in vitro study and the manuscript revision process. M.L. and X.Q. conceived the study. M.L. supervised the experiment and revised the manuscript.The authors declare no conflict of interest.Not applicable.Table S1.Click here for additional data file.Table S2.Click here for additional data file.Table S3.Click here for additional data file.Data S1.Click here for additional data file.Figure S1.Click here for additional data file.Figure S2.Click here for additional data file.Figure S3.Click here for additional data file.Figure S4.Click here for additional data file.Figure S5.Click here for additional data file.


Heat stroke is a life‐threatening disease with high mortality and complications. Endothelial glycocalyx (EGCX) is essential for maintaining endothelial cell structure and function as well as preventing the adhesion of inflammatory cells. Potential relationship that underlies the imbalance in inflammation and coagulation remains elusive. Moreover, the role of EGCX in heat stroke‐induced organ injury remained unclear. Therefore, the current study aimed to illustrate if EGCX aggravates apoptosis, inflammation, and oxidative damage in human pulmonary microvascular endothelial cells (HPMEC). Heat stress and lipopolysaccharide (LPS) were employed to construct in vitro models to study the changes of glycocalyx structure and function, as well as levels of heparansulfate proteoglycan (HSPG), syndecan‐1 (SDC‐1), heparansulfate (HS), tumor necrosis factor‐α (TNF‐α), interleukin (IL)‐6, Von Willebrand factor (vWF), endothelin‐1 (ET‐1), occludin, E‐selectin, vascular cell adhesion molecule‐1 (VCAM‐1), and reactive oxygen species (ROS). Here, we showed that heat stress and LPS devastated EGCX structure, activated EGCX degradation, and triggered oxidative damage and apoptosis in HPMEC. Stimulation of heat stress and LPS decreased expression of HSPG, increased levels of SDC‐1 and HS in culture supernatant, promoted the production and release of proinflammation cytokines  and coagulative factors (vWF and ET‐1) in HPMEC. Furthermore, Expressions of E‐selection, VCAM‐1, and ROS were upregulated, while that of occludin was downregulated. These changes could be deteriorated by heparanase, whereas they meliorated by unfractionated heparin. This study indicated that EGCX may contribute to apoptosis and heat stroke‐induced coagulopathy, and these effects may have been due to the decrease in the shedding of EGCX. The features of vascular endothelial glycocalyx under stimulation of heat stress and lipopolysaccharide corroborated vascular inflammatory injury and coagulation disorder. For the first time, the study showed that heat stroke triggered endothelial glycocalyx degradation and further induced oxidative damage and apoptosis of human pulmonary microvascular endothelial cells, which impaired its ability to resist inflammatory injury and maintaining vascular permeability. Dislocation of endotoxin was observed secondary to the injury of intestinal mucasa, which increased the level of endotoxin in the blood.vitro models.Collectively, the present study aims to confirm the effects of degradation of EGCX in heat stroke‐induced coagulopathy. Human pulmonary microvascular endothelial cell (HPMEC) was explored to observe the injury characteristic of vascular EGCX under heat stress combined with LPS double hits in 22.12. All experiments were carried out during the logarithmic phase of cell growth. The account of cells was adjusted to 1 × 106/mL by serum‐free medium before culture, inoculating into six‐well plates, and incubated at 5% CO2 and 37°C.HPMEC was purchased from Guangzhou Dewei Biological Technology Co. Ltd, China. HPMEC was cultured in the endothelial cell medium with 10% fetal bovine serum (ScienCell) at 37°C in a humidified incubator with 5% CO2.22.2.1Heat stress and lipopolysaccharide (HS + LPS) were employed to construct the in vitro models of heat stroke in our previous study.2.2.22, 37°C incubators, stimulated with LPS at a concentration of 500 ng/mL for 12 h.Briefly, except for the CON group, HPMEC in HS, HPSE, and UFH groups were subjected to heat stress at 43°C for 30 min, and then moved to 5% CO2.32.3.1To investigate HPMEC apoptosis and heparansulfate proteoglycan (HSPG) concentration, flow cytometry was performed in each group according to our previous methods.2.3.2To examine the intracellular ROS levels, a ROS assay kit that sets dichloro‐dihydro‐fluorescein diacetate (DCFH‐DA) as the probe was used. HPMEC supernatant was withdrawn after modeling from each group, and DCFH‐DA was diluted to 10 μmol/L by 1:1000 in a serum‐free blank medium. Cells were stained with DCFH‐DA (Nanjing Jiancheng) in a 37°C cell incubator for 20 min. The cells were washed three times with a blank medium, observed and photographed by Olympus positive fluorescence system microscope (Olympus Corporation), and analyzed by ImageJ software.2.3.3Cells from each group were washed with PBS for three times (1 min each), washed in PBS for 5 min each. Cells were then permeabilized in precooled 0.25% Triton X‐100 solution for 10 min on ice, blocked in 1% bovine serum albumin  for 1 h. Incubation with anti‐HSPG at 4°C for 2 h (Affinity Biosciences) was followed by three washes (5 min each) with PBS and incubation with Goat Anti‐rabbit complex at 1:20 dilution in the dark at room temperature for 1 h. Finally, the cells were incubated with 4′,6‐diamidino‐2‐phenylindole in the dark for 5 min and then were observed in a fluorescence microscope.2.3.4The level of major degraded products of EGCX, including syndecan‐1 (SDC‐1) and heparin sulfate (HS), was measured by ELISA based on an established procedure. Moreover, ELISA was also performed to detect the concentrations of tumor necrosis factor‐α (TNF‐α), interleukin‐6 (IL‐6), Von Willebrand factor (vWF), and endothelin‐1 (ET‐1).2.3.5The expression levels of E‐selectin, VCAM‐1, and Occludin in HPMEC were investigated via western blot. Cells were collected and lysed in radioimmunoprecipitation assay lysis buffer (LEAGENE), which added with phenylmethylsulfonyl fluoride for 30 min on ice. The protein samples were determined using BCA Protein Assay Kit , separated on 12% sulfate‐polyacrylamide gel electrophoresis, and were then transferred to polyvinylidene difluoride membranes (BIO‐RAD). The membranes were further blocked with 5% nonfat dry milk with 100 mL Tris‐buffered saline with 0.05% Tween 20 (TBST) at room temperature and incubated with rabbit anti‐E‐selectin (SAB), rabbit anti‐Occludin (SAB), rabbit anti‐VCAM1 (SAB) primary antibodies at 4°C overnight. TBST was used for rinsing for 10 min and shaking at room temperature. Peroxidase‐conjugated Affinipure goat anti‐rabbit IgG/FITC and goat anti‐mouse IgG/PE were used for incubating in the dark at room temperature for 1 h. The immunoblots were investigated by enhanced chemiluminescence (ECL), and the protein expression levels were analyzed by ImageJ software.2.42.4.1The ultrastructure of HPMEC was assessed using TEM analysis. HPMEC were cultured on filter membranes were fixed in 2.5% glutaraldehyde in cacodylate buffer (PH 7.4) for 2 h, then fixed in 1% OsO4  for 2 h, dehydrated in a series of ethanol washes, and embedded in Epon 812 Resin according to the standard procedure. Ultrathin section (60–80 nm) was prepared, stained with both uranyl acetate and lead citrate and photographed using a Hitachi 7700 electron microscope.2.5p < .05 was considered to be statistically significant.All statistical analysis of the data was analyzed by SPSS 27.0 and GraphPad Prism 9. All the data were expressed as mean ± standard deviation (mean ± SD). If the variance is uniform, single‐factor descriptive analysis of variance (ANOVA) and least significant difference (LSD) methods were used to analyze comparisons among multiple groups. If not, the Welch test was used. 33.13.1.1p < .05)   Figure , as indi) Figure . These r3.1.2p < .05) . These results revealed that heat stress and LPS may induce EGCX degradation in HPMEC, which showed a positive correlation with the degree of glycocalyx damage.Considering that SDC‐1 and heparin sulfate were identified as the classical degraded products of EGCX.) Figure . UFH mig3.23.2.1The ultrastructure of HPMEC was observed to estimate the effects of heat stress and LPS on HPMEC apoptosis Figure . Normal p < .05)   Figure . This in) Figure , suggest3.3p < .05) . These results indicated that heat stress and LPS directly affect glycocalyx degradation as a potential mechanism to induce a clotting state in HPMEC.To further investigate the effect of heat stroke on the function of coagulation factors, the expression of vWF and ET‐1 was assessed under stimulation of heat stress and LPS. Exposure to stimulation of heat stress and LPS resulted in a statistically significant increase in the expression of vWF and ET‐1  Figure . HPSE trp < .05)  Figure . Compare3.4p < .05)   Figure . These rp < .05)  Figure . These r4In this study, the features of vascular EGCX under stimulation of heat stress combined with LPS confirmed vascular inflammatory injury and coagulation disorder. This study showed that heat stress and LPS triggered EGCX degradation and further induced oxidative damage and apoptosis of HPMEC, which impaired its ability to resist inflammatory injury and protecting vascular permeability.Increase in rectal core temperature induced by heat stress worsened intestinal lesions in mice, leading to a dislocation of endotoxin, and increasing in the level of endotoxin.HSPG was a major element of glycocalyx, which played important roles in endothelial cell homeostatic signals based on their unique structures and interactions with both the intra‐ and extracellular environments. Decreased HSPG prompted degradation of EGCX and vascular disease.High concentrations of serum HS and SDC‐1 were used as principal markers of EGCX shedding, as demonstrated in previous studies.Earlier studies demonstrated that heat stress contributed to injury and apoptosis of endothelial cells by activation of inflammation, which played an important role in organ injury secondary to heat stroke.Inflammation and coagulation played a critical role in the pathophysiological basis of tissues/organs damage secondary to heat stroke.TNF‐α was known to promote inflammation via upregulating the expression of adhesion factors on the surface of the endothelium, such as VCAM‐1 and ICAM‐1.E‐selectin and VCAM‐1 were the important symbols to reflect injury of EGCX and endothelium, which also played critical roles in the spread of inflammation via mediating the adhesion between leukocyte and inflammatory cells.Decreased occludin may be associated with widening gaps between endothelial cells, which aggravated the vascular leakage. Our data indicated that heat stroke‐induced glycocalyx damage enhanced adhesion protein expression, and further led to pathophysiological changes, including increased vascular permeability and leukocyte adhesion.ROS was also associated with the degradation of EGCX, widely involved in signaling transduction and the life process of cells. Excessive ROS may lead to diseases by inducing oxidative stress in mitochondria.Release of ROS may trigger EGCX degradation when exposed to generation of TNF‐α and endotoxin, as well as inflammatory states, including sepsis.As the most abundant molecular chaperone proteins, heat shock proteins (HSPs) expressed under physiological conditions, and increased in expression when cells were exposed to high temperature or hypoxia.There was a limitation in our study. The mechanism of EGCX contributing to inflammatory and oxidative responses related to heat stroke was unclear. Future studies are needed to explore the potential signal pathways and mechanism of dysfunction of vascular endothelium induced by EGCX degradation under heat stroke.5In conclusion, our study provides evidence that degradation of endothelial glycocalyx induced by heat stress and LPS causes endothelium cell apoptosis in HPMEC, which leads to increased generation of inflammatory cytokines and oxidative response, thereby contributing to aggravating inflammation and destroying the vascular permeability.Jiadi Chen: Data curation; formal analysis; project administration; writing—original draft; writing—review & editing. Chengjia Ding: Data curation; formal analysis; software. Jingjing Cao: Data curation; formal analysis; software. Huasheng Tong: Funding acquisition; methodology; project administration; writing—review & editing. Yi Chen: Conceptualization; funding acquisition; methodology; project administration; writing—review & editing.The authors declare no conflict of interest.


The purpose of the study was to investigate the ability of new parameters in distinguishing high‐risk patients of recurrence from isthmic papillary thyroid carcinomas (iPTCs).One hundred sixteen iPTC patients who underwent total thyroidectomy were identified from 3461 PTC patients from 2014 to 2019. Tumor margin to trachea midline distance (TTD), maximum tumor size (TS), and transverse diameter of trachea (TD) were measured on CT images. Cox proportional hazard models served to identify risk factors associated with recurrence‐free survival (RFS). The iPTC prognostic formula (IPF = TD/(TTD − TS) − TD/TTD) was evaluated to assess the prognosis. RFS was conducted between the different groups using the Kaplan–Meier analysis. The receiver operating characteristic (ROC) curve of each parameter was drawn to predict recurrence.p = 0.001) and PLNM  (p = 0.010) in cPTC  and non‐cPTC (iPTC patients enrolled in this study excluding cPTC) groups differed significantly. A cut‐off point of tumor size >1.1 cm and IPF ≤5.57 were established to have significant differences in prognosis . Multivariate analysis showed that IPF ≤5.57 was independent prognostic factor for RFS .Central lymph node metastasis (CLNM) and extrathyroidal invasion in iPTC were 58.6% and 31.0%, respectively. Regional recurrence occurred in 16 (13.8%) patients, and no patient died or had distant metastasis. The 3‐ and 5‐year RFS of iPTC were 87.5% and 84.5%, respectively. Gender (This study indicated the association between IPF and RFS in iPTC patients and established new models to assess risk factors for recurrence pre‐operation. IPF ≤5.57 was significantly associated with poor RFS and might be promising parameters for predicting prognosis and surgical decision‐making pre‐operation. This study indicated that the previously used classification of iPTC showed no significant differences in prognosis. This is the first study to integrate the isthmic tumor position, trachea width, and tumor size to demonstrate the association between IPF and the prognosis of iPTC. IPF ≤5.57 was significantly associated with recurrence and may be promising parameters that could be used for the prognostic differentiation and preoperative surgical decision‐ making. Most of the studies did not have definite selection criteria for iPTC. Several studies used the hypothesis by drawing two imaginary lines perpendicular to the surface of the skin from the most lateral points of the trachea as the lateral margin of the isthmus. The center of the thyroid cancer is located between these two lines defined as iPTC.In this study, we retrospectively analyzed the clinicopathologic characteristics of iPTC. By measuring the distance from the tumor margin to the midline of the trachea, the maximum diameter of the tumor, and width of the trachea, we analyzed the influence of different locations and sizes of iPTC on the clinicopathologic characteristics and tumor recurrences, sought to find a possible model to facilitate the assessment the characteristics and prognosis of the iPTC pre‐operation.2This retrospective study was approved by the clinical research ethics committee of The First Affiliated Hospital, School of Medicine, Zhejiang University. Written informed consent was obtained from all patients before the study.2.1A total of 3461 PTC patients underwent total thyroidectomy and bilateral cervical central lymph nodes dissection between August 2014 and October 2019 in the department of thyroid surgery of The First Affiliated Hospital, School of Medicine, Zhejiang University were analyzed retrospectively. One hundred sixteen patients (3.4%) had a dominant PTC located in the isthmus. The clinicopathological data of iPTC patients were analyzed.Before surgery, each patient underwent ultrasound‐guided fine‐needle aspiration biopsy (FNAB) and enhanced cervical high‐resolution computed tomography (CT) imaging with a thickness of 1.25 mm. Two experienced surgeons performed all surgical operations (Dr. Wu & Dr. Xie). Thin slice CT images were exported for measurement. CT image data were collected and measured by two technical staff and reviewed by a radiologist to ensure accuracy.The patients in the study met the following inclusion criteria: the center of PTC was in the fan‐shaped area, which the cervical vertebrae as the apex and 45° outside the midline of the trachea (MT). Patients who underwent unilateral lobectomy or isthmectomy, multifocal carcinoma, isthmic microcarcinoma less than 3 mm, secondary surgery, preoperative neck US suspected cervical lymph node metastasis (cN1), and other types of thyroid cancer were excluded.To detect the serum levels of thyroid stimulating hormone (TSH), thyroid peroxidase antibody (TPOAb), and thyroglobulin (Tg), electrochemiluminescence assay  was performed. The reference ranges for TSH, TPOAb, and Tg were 0.350–4.940 mIU/L, 0.00–5.61 IU/mL, and 0.00–55.00 ng/mL, respectively.2.2One hundred sixteen iPTC patients' cervical CT images were collected and measured. CT image of the maximum tumor diameter of the iPTC of each patient was exported. The angle of the CT images will be adjusted if the neck deflection occurs during scanning so that the midline of the trachea and cervical vertebra coincide to avoid measurement deviations. CT images were measured independently by two technicians, and the average value was calculated. A re‐measure will be taken if the results differ significantly.y axis, IPF = TD/RTTD + TD/LTTD). The coordinate system takes the MT as the y‐axis, and the tangent line perpendicular to the y‐axis and passing through the posterior edge of the trachea as the x‐axis. At the same time, assume that the trachea is a circle.TTD (RTTD: TTD on the right side & LTTD: TTD on the left side), TS, and TD were measured simultaneously on the CT image when the iPTC's diameter was largest Figure . To inve2.3All patients received thyroid‐stimulating hormone‐suppressive therapy after surgery. Postsurgical physical examinations were performed every 3–6 months. Cervical CT and FNAB were performed during a follow‐up to evaluate suspected recurrences. Reoperation was performed on patients' suspected recurrence, and postoperative pathology was confirmed.2.4t‐test. Recurrence‐free survival (RFS) was analyzed between the different groups with Kaplan–Meier analysis with log‐rank test. Multivariate recurrence analysis was conducted using the cox proportional hazards regression to identify independent prognostic factors. Results were reported as hazard ratios (HR) and 95% confidence intervals (95% CI). X‐tile software (Version 3.6.1) a bio‐informatics tool produced by Robert et al.,p values <0.05 were defined as statistically significant.Statistical analyses were performed using SPSS version 22.0 (SPSS). The results were expressed as mean ± SD. Differences between categorical variables were assessed using the Chi‐square test or Fisher exact test and continuous variables using Student's 3Among the 3461 patients, 116 patients (3.4%) had PTC located in the isthmus. The clinicopathologic features of iPTC patients were listed in Table p = 0.846, p = 0.361, p = 0.439, p = 0.566, p = 0.230, and p = 0.621, respectively). The mean tumor size was 1.13 ± 0.68 cm in the cPTC group and 1.11 ± 0.57 cm in the non‐cPTC group (p = 0.859). The rate of female patients in the two groups was 89.5% and 63.3%, respectively (p = 0.001). The rate of PLNM  was significantly lower in the cPTC group than in the non‐cPTC group. However, there were no statistically significant differences in recurrence between two groups (p = 0.120).The clinicopathologic features of the cPTC and non‐cPTC groups were compared Table . There wp = 0.502 and p = 0.691)    and lower IPF value (p = 0.029) (Table (p = 0.0009), which indicated that the IPF could accurately predict recurrence in iPTCs. According to Youden's index, the best cutoff value was 5.57. The sensitivity and specificity were 81.25% and 56.00%. IPF >5.57 and IPF ≤5.57 have significant differences in recurrence (p = 0.005)  patients developed recurrence. The median time to recurrence after surgery was 48.5 months . No patients died or had distant metastasis. Kaplan–Meier survival analysis with log‐rank test was performed to predict prognosis. The entire cohort's 3‐ and 5‐year RFS were 87.5% and 84.5%. There were no statistical differences for recurrence in terms of extrathyroidal invasion and age (≤ 55 years)  Figure . There w) Figure . Tumor s) Figure . Reopera.8% patie) Figure .p = 0.034) , tumor size (>1.1 cm) and IPF (≤5.57) were significantly associated with poor RFS. Multivariate cox regression analysis found that IPF (≤5.57) was independent prognostic factor for recurrence . These findings indicated that the classification of iPTC by the lateral margin of the trachea might narrow the scope of patient inclusion. Meanwhile, differentiating the location of iPTC by MT alone may not fully reveal the risk factor of prognosis. The purpose of this study was to analyze a possible convenient model to differentiate iPTC and to evaluate the prognosis of iPTC.There were no studies on the definition of iPTC. Therefore, the definition of 45° fan‐shaped area on both sides of the MT was used for the selection criterion of iPTC in this study. Which accounts for a larger number of patients. The membranous structure of the “C”‐shaped tracheal cartilage may influence the precision of phase measurement. We try to take the intersection of the MT and cervical spine as the vertex of the 45‐degree sector. The cPTC and non‐cPTC groups were established to compare the difference of clinicopathologic features and prognosis. The rate of PLNM was significantly lower in the cPTC group than in the non‐cPTC group (10.5% vs. 23.3%). In the study of Li et al., PLNM was significantly correlated with sex, age, tumor size, and extrathyroidal invasion, and the male ratio in the PLNM (+) group was higher as compared to that in the PLNM (−) group (46.89% vs. 18.56).y‐axis, and a line perpendicular to the y‐axis tangent to the posterior edge of the trachea as the x‐axis to establish a coordinate system. The IPF represents the difference between the slopes of the two straight lines from the two endpoints of the tumor transverse diameter to the origin of the coordinate system (IPF = TD/(TTD − TS) − TD/TTD). The univariate cox regression analysis indicated CLNM (+), tumor size (>1.1 cm) and IPF (≤5.57) were significantly associated with poor RFS. Multivariate analysis indicated that IPF ≤ 5.57 was independent prognostic factor for recurrence. It may be a convenient model for predicting the recurrence and evaluating the boundaries of surgery.Our results indicated that the previously used classification of iPTC showed no significant differences in prognosis. Since iPTC is a tumor type defined by location, a stable coordinate system could be established with the relatively fixed bone structure around the thyroid. Using this coordinate system to measure parameters such as tumor location and the transverse diameter of trachea, we want to find a way to redefine isthmic cancer. Therefore, we have established predictive models to assess risk factors for recurrence. We assume that the trachea is a circle, with the midline of the trachea as the The present study had several limitations. First, the sample size of the iPTC was relatively small. Meanwhile, although carefully measured, the measurement bias should not be ignored. Studies with larger sample sizes and more accurate results are needed to confirm our findings. Second, the inclusion criterion in this study expands the scope of iPTC, which may affect the results. Finally, because of the complexity of iPTC location, the IPF model cannot fully explain all iPTCs. For example, when the iPTC was located on the side of the MT but infinitely close to the MT, regardless of the tumor size, the IPF >5.57, which was not consistent with the actual situation. Thus, further investigation should be explored in the future.5Our results indicated that the previously used classification of iPTC showed no significant differences in prognosis. This is the first study to integrate the isthmic tumor position, trachea width, and tumor size to demonstrate the association between IPF and the prognosis of iPTC. IPF ≤5.57 was significantly associated with recurrence and may be promising parameters that could be used for the prognostic differentiation and preoperative surgical decision‐making.Feng Zhu: Conceptualization (lead); data curation (lead); formal analysis (lead); investigation (lead); software (lead); writing – original draft (lead). FuQiang Li: Data curation ; investigation ; methodology ; resources ; writing – original draft . XiaoJun Xie: Data curation ; formal analysis ; investigation ; methodology ; software ; writing – original draft . YiJun Wu: Conceptualization (lead); data curation ; funding acquisition ; investigation ; methodology ; project administration (lead); supervision (lead); validation (lead); writing – review and editing (lead). Weilin Wang: Conceptualization (lead); funding acquisition (lead); investigation (lead); methodology (lead); project administration (lead); supervision (lead); validation (lead); writing – review and editing (lead).This work was supported by Zhejiang medical science and technology projects [2022KY786].No competing financial interests exist.


T‐cell engaging bispecific antibodies (BsAbs) have substantial activity in heavily pretreated patients with multiple myeloma (MM). The overall response rate obtained with B‐cell maturation antigen (BCMA)‐targeting BsAbs is approximately 60%–70%, including a high proportion of patients achieving very good partial response or complete response. Comparable efficacy is seen with BsAbs targeting GPRC5D or FcRH5. Cytokine release syndrome is frequently observed with BsAb treatment, but mostly during the step‐up doses and the first full dose. Early intervention with IL‐6 receptor blocking antibodies  prevents escalation to severe manifestations. Infections are also common during treatment and related to the extent of exposure to immune suppressive anti‐MM agents, as well as development of hypogammaglobulinemia due to elimination of normal plasma cells, and probably because of T‐cell exhaustion resulting from continuous BsAb‐mediated T‐cell activation. Adequate monitoring for infections and institution of infectious prophylaxis are essential. Patients treated with GPRC5D‐targteing BsAbs often develop skin and nail disorders and loss of taste, which is likely related to GPRC5D expression in cells that produce hard keratin. Currently ongoing studies are aiming at further improving these results by evaluating BsAbs in combination with other drugs, such as immunomodulatory agents and anti‐CD38 antibodies, as well as the application of BsAbs in earlier lines of therapy, including patients with newly diagnosed disease. We expect that the outcomes of patients with MM will further improve by the introduction of this novel type of T‐cell immunotherapy. At that time his disease was refractory to three immunomodulatory drugs , one CelMOD , two proteasome inhibitors , one CD38‐targeting antibody (daratumumab), as well as alkylating drugs  and one checkpoint inhibitor . Cytogenetic analysis was repeated and revealed the presence of a new chromosome 17p deletion, next to the preexisting hyperdiploidy.2At the time of his 11th relapse, our patient was heavily pretreated and refractory to three important drug classes . These triple‐class refractory MM patients have a very poor prognosis . Patient3Triple‐class refractory patients should always be considered for participation in a clinical trial, which allows early access to novel agents with new mechanisms of action Figure . UnfortuOther approved options include BCMA‐targeting CAR T‐cell therapies (ide‐cel and cilta‐cel). Ide‐cel received European Medicines Agency (EMA) and U.S. Food and Drug Administration (FDA) approval based on an overall response rate of 73% and median PFS of 8.8 months in patients with a median of 6 prior lines of therapy . Cilta‐c4◦C) in the absence of signs of infection. His blood pressure was normal, but his heart rate was increased (100 beats per minute). He was not hypoxic. The diagnosis of grade 1 CRS was made, and he was treated with one intravenous (IV) infusion of tocilizumab. The fever rapidly resolved, and we could continue treatment with teclistamab as planned. As infectious prophylaxis he received valacyclovir, cotrimoxazole, and we started IVIG treatment when he developed hypogammaglobulinemia. Although heavily pretreated he rapidly achieved a deep remission with as best response a stringent complete response (CR).In March 2019, we enrolled our triple‐class refractory patient into the phase 1, dose‐escalation trial with teclistamab (MajesTEC‐1). At that time he received only a fraction of the recommended phase 2 dose (RP2D), which was identified later. Prior to the priming dose and first full dose he received premedication  to prevent severe cytokine release syndrome (CRS). Nonetheless, 1 h following the administration of the first priming dose, he developed a fever  on the tumor cell surface Figure . This reBsAbs come in many different formats but can generally be classified into two groups; IgG‐like BsAbs containing a fragment crystallizable (Fc) domain and those without an Fc domain (e.g. BiTEs and DARTs) Figure . The Fc Trispecific antibodies are also under investigation in both preclinical studies and in clinical trials. Next to binding CD3, these antibodies either target two antigens on the myeloma cell in order to prevent antigen escape, or they have the ability to activate T‐cell co‐stimulatory molecules to prevent T‐cell anergy , 17.66.1B cell maturation antigen (BCMA) is a type III transmembrane glycoprotein that belongs to the tumor necrosis family receptors. It is expressed on healthy and malignant plasma cells and a subset of mature B cells . It prom6.2GPRC5D is an orphan G‐protein coupled receptor. It is a 7‐segment transmembrane protein for which the endogenous ligand(s) and signaling mechanisms remain unknown , 26. GPR6.3FcRH5 is a membrane surface protein that is related to the group of receptors homologous to FcγRI , 31]. I. I31]. I7Proof‐of‐concept that T‐cell engaging bispecific antibodies work in heavily pretreated MM patients was delivered by the clinical study that evaluated AMG 420. This BiTE molecule binds to both BCMA on MM cells and CD3 on T‐cells. AMG 420 has a short half‐life and therefore needs to be administered via continuous IV infusion (every 4 weeks of a 6‐week cycle) . At the In the first‐in‐human, dose‐escalation study, the recommended phase 2 dose (RP2D) of teclistamab was established as a subcutaneous (SC) dose of 1.5 mg/kg with 2 step‐up doses to mitigate CRS (0.06 mg/kg and 0.3 mg/kg) . SC dosiSeveral other IgG‐like BCMA‐targeting antibodies are in development for the treatment of MM, including elranatamab Table . ElranatOther IgG‐like BCMA‐targeting BsAbs in clinical development are alnuctamab (CC‐93269) , HPN217 8Not only BsAbs targeting BCMA have shown great promise, but also BsAbs targeting other surface antigens on MM cells have substantial anti‐MM activity Table . Importa9Talquetamab is the first‐in‐class GPRC5D‐targteing BsAb, which was tested in a first‐in‐man, dose‐escalation study in heavily pretreated MM patients (median of six prior lines of therapy), 48]. T. T48]. TForimtamig (RG6234) is another GPRC5D T‐cell engaging BsAb with a 2:1 (GPRC5D:CD3) configuration, which increased the potency compared to the 1:1 configuration . Forimta10Cevostamab is a BsAb that targets the membrane‐proximal region of FcRH5 on the MM cell surface. The first‐in‐human dose‐escalation study with cevostamab has shown promising activity in heavily pretreated patients . At high1111.1Cytokine‐release syndrome is a frequent manifestation following initiation of BsAb treatment, and mostly observed during the step‐up doses or the first full dose Figure . BsAb‐meDevelopment of severe CRS can be prevented by premedication including steroids . Also the strategy of starting with a low dose of the BsAb, allows for the safe subsequent administration of a higher dose. Some BsAbs use two‐step doses, as a 2‐step‐up dosing regimen appeared to further mitigate severity of CRS, compared to only one step‐up dose , 51]. I. I51]. IWhen CRS occurs it can be effectively treated by using tocilizumab, or steroids. We typically administer tocilizumab directly when a patient develops grade 1 CRS in order to prevent further escalation to more severe manifestations. Other supportive care measures include acetaminophen in case of fever, IV fluids in case of hypotension, and O2 supplementation in case of hypoxia. Intensive care treatment with vasopressor support may be needed in patients who develop high‐grade CRS.11.2For a protein to be an appropriate target for BsAbs, the protein must be uniformly expressed on the cell surface of the tumor cells, but not on critical normal tissues to prevent severe “on target, off tumor” toxicities Figure .Because BCMA, GPRC5D and FcRH5 are also expressed on normal plasma cells, these antibody‐producing cells are also eliminated by BsAbs. This results in hypogammaglobulinemia, which increases risk for development of infections . InteresGPRC5D is also expressed on cells that produce keratin, which may explain the development of skin and nail disorders in patients treated with GPRC5D‐targeting BsAbs. These adverse events are typically of low grade , 56, 57.11.3Especially during the first 1 or 2 cycles patients may develop neutropenia, thrombocytopenia and anemia Figure . This is11.4Patients treated with BsAbs have a high frequency of infections which may be related to (1) the high cumulative exposure to immunosuppressive drugs during previous lines of therapy, (2) development of hypogammaglobulinemia due to eradication of normal plasma cells, 3) occurrence of neutropenia, and (4) the development of T‐cell exhaustion during long‐term BsAb treatment , or toxoplasma gondii. Reactivation of viral infections may also occur during treatment with a BsAb, including cytomegalovirus, herpes simplex virus, herpes zoster virus, BK virus, or hepatitis B virus.In our practice, we give patients treated with a BsAb infectious prophylaxis consisting of valacyclovir to prevent herpes simplex virus and varicella zoster virus‐related infections, as well as PJP prophylaxis with cotrimoxazol or another drug in case of allergy . Patients with increased risk for infections may also benefit from levofloxacin to prevent bacterial infections  . We star12Approximately 4.0 years after start of teclistamab, he is still treated with this agent (once montly), and he remains in a stringent complete remission. He has in fact achieved the deepest and most durable remission, since he was diagnosed with MM. We continued cotrimoxazol and valacyclovir as well as immunoglobulin replacement therapy, because his IgG levels did not recover during treatment. With this supportive care cocktail, he did not develop severe infections, or other complications.13Our patient has a very durable remission, but not all patients respond (primary resistance) and patients may develop disease progression after having achieved a remission (acquired resistance). Both T‐cell‐related features, tumor characteristics, and components of the bone marrow microenvironment may contribute to resistance to T‐cell immunotherapies . Several1414.1Although BsAbs have shown promising activity in heavily pretreated patients, a subset of patients does not respond and eventually many responding patients relapse. Aiming at further improving depth and duration of response, several combination strategies are being investigated. This includes combinations with agents that improve T‐cell function, such as IMiDs , CelMODs , checkpoint inhibitors , as well as CD38‐targeting antibodies , 69], , .+ regulatory T‐cells leading to improved T‐cell numbers and killing capacity . A. A+ regucapacity . Among 5capacity . With bocapacity , 47], [, [+ regucapacity . The inccapacity . For exacapacity , 76, 77,In an attempt to prevent antigen escape, there is also an ongoing study assessing the combination of two BsAbs targeting two different MM‐associated antigens . Based on preclinical data showing that gamma‐secretase inhibitors are capable of increasing BCMA expression on the tumor cell surface and concomitantly reducing soluble BCMA levels, which resulted in enhanced activity of BCMA‐targeting BsAbs , BCMA‐ta14.2Fixed treatment duration and less frequent administration are also explored as novel strategies to prevent BsAb‐mediated T‐cell exhaustion and thereby reduce the frequency of infections and/or improve the durability of response. Recently preclinical studies showed that treatment‐free intervals may also be beneficial to preserve T‐cell fitness and improve anti‐tumor activity .14.3Patients with newly diagnosed disease or early relapsed/refractory disease have superior T‐cell function and numbers when compared to end‐stage patients who received immunosuppressive anti‐MM drugs over a period of many years . Therefo15In conclusion, T‐cell immunotherapy with BsAbs has demonstrated substantial activity in heavily pretreated, triple‐class refractory MM patients. CRS and neurotoxicity are typically less severe with BsAbs than with CAR T‐cell therapy, indicating that also elderly patients can be effectively treated with this new class of agents. Ongoing efforts are exploring combination strategies and earlier use of BsAbs, including its evaluation in newly diagnosed disease. Altogether these efforts will lead to a further improvement of the survival of MM patients.Chloe O'Neill and Niels W.C.J. van de Donk contributed to the writing of the manuscript and literature search.C.O.N. has no conflict of interest. N.W.C.J.v.d.D. has received research support from Janssen Pharmaceuticals, AMGEN, Celgene, Novartis, Cellectis, and BMS and serves in advisory boards for Janssen Pharmaceuticals, AMGEN, Celgene, BMS, Takeda, Roche, Novartis, Bayer, Adaptive, and Servier


Folate was involved in oxidative stress, hepatic lipid metabolism and chronic hepatic inflammation. However, evidence about the association between serum folate level and non-alcoholic fatty liver disease (NAFLD) in general population is scarce. This study aimed to explore the relationship between serum folate level and NAFLD among adults.7,146 adult participants aged 20 years and over who have complete data of serum folate level and liver function biomarkers in NHANES 2011–2018 were included. Serum folate level was measured by isotope-dilution high-performance liquid chromatography coupled to tandem mass spectrometry (LC–MS/MS). And suspected NAFLD was defined according to the United States fatty liver index (USFLI). Logistic regression and the restricted cubic spline models were performed.p for trend<0.001). The non-linear and L-shaped relationship was found between serum folate level and the presence of NAFLD in the restricted cubic spline regression (p for non-linearity <0.01). Consistent with serum total folate, serum 5-Methyltetrahydrofolate level was also inversely associated with the presence of NAFLD.Serum folate level was inversely associated with the presence of NAFLD. When comparing the second, third and fourth quartiles of serum folate level to the lowest quartile, the adjusted ORs of the presence of NAFLD were 0.62 (0.49–0.78), 0.65 (0.51–0.84), and 0.43 (0.32–0.56) respectively (Higher serum folate level may be negatively associated with NAFLD. Non-alcoholic fatty liver disease (NAFLD) is a major chronic liver disease with the estimated prevalence of approximately 25% in the general population worldwide . NAFLD iFolate as a water-soluble B9 vitamin is a unique catalytic substrate for one-carbon transfer reactions , 8. FolaRecently, one noninvasive and accurate index-the United States fatty liver index (USFLI) developed using NHANES III data was used to define NAFLD . The USFNHANES is a national representative survey of the cross-sectional design which include non-institutionalized US civilians. The National Center for Health Statistics of the Centers for Disease Control  conducted the study. The National Center for Health Statistics’ Ethics Review Board approved the protocols of NHANES. Participants were selected through a stratified, multi-stage, sampling design in NHANES. All participants provide written informed consent.n = 143), (2) they have significant alcohol consumption (>30 g/day for men and > 20 g/day for women) , (3) women who were pregnant (n = 82), and (4) their data of serum folate level are missing (n = 766). Ultimately this study included 7,146 participants, comprising 3,310 males and 3,836 females  were used. This study initially included 9,203 participants aged 20 years and over who were scheduled for a fasting blood draw and had blood tested for glucose, insulin and gamma glutamyl transferase (GGT). Participants were also excluded if (1) they are with the hepatitis B surface antigen or hepatitis C antibodies  + 0.0249 × waist circumference + 1.1792 × ln (insulin) + 0.8242 × ln (glucose) -14.7812) / (1 + e −0.8073 × non-Hispanic black + 0.3458 × Mexican American + 0.0093 × age + 0.6151 × ln (gamma glutamyl transpeptidase) + 0.0249 × waist circumference + 1.1792 × ln (insulin) + 0.8242 × ln (glucose) -14.7812) × 100 (Suspected NAFLD was defined according to USFLI which was developed using NHANES data . The for2) × 100 . Accordi2) × 100 , 17, NAFIn NHANES, five folate forms  were measured by LC–MS/MS . The assSerum total folate was calculated by adding the above five folate forms. 5-Methyltetrahydrofolate is the main bioactive form of serum total folate. Therefore, in the analyses we included serum total folate and 5-Methyltetrahydrofolate levels.2 . Diabetes was defined according to one of the following four criteria: self-reported prior diagnosis of diabetes, taking any antidiabetic medication, glycosylated hemoglobin measurement ≥6.5%, or fasting plasma glucose level ≥ 126 mg/dL. Participants with hypertension were defined as taking hypertension medication, self-reported prior diagnosis of hypertension, systolic blood pressure ≥ 130 mmHg, or diastolic blood pressure ≥ 80 mmHg  were calculated and the lowest quartile of serum total folate and 5-Methyltetrahydrofolate level was taken as the reference group. To evaluate potential nonlinear associations between serum total folate and 5-Methyltetrahydrofolate and the risk of having NAFLD, restricted cubic spline analyses were conducted , which were measured in a fasting subsample of participants in NHANES.Descriptive statistics were conducted to characterize the individuals. The continuity variables were reported as the mean ± standard deviation (SD), and the categorical variables were reported as number and percentages. Differences between the group with NAFLD and the group without NAFLD were determined by chi-square tests for categorical variables and by onducted . Three kp ≤ 0.05.According to analytical guidelines of NHANES, appropriate sampling weights should be used considering the stratified, multistage probability sample design. And the new weights were created for the combined NHANES (2011–2018) cycles. All statistical data were analyzed using Stata version 12.0 . Statistical significance was defined as two-sided The characteristics of the study population grouped by the presence or absence of NAFLD are shown in p for trend<0.001) (p for non-linearity <0.01)  . The nony <0.01) . Similary <0.01) . Consisty <0.01) . The resy <0.01) .In this large study of 7,146 adult participants from NHANES 2011–2018, serum total folate and 5-Methyltetrahydrofolate levels were inversely associated with the presence of NAFLD determined by USFLI in adults. Meanwhile, the dose–response relationship between serum total folate and 5-Methyltetrahydrofolate levels and NAFLD was non-linear.2) participants, lower serum folate level was found in patients with NAFLD compared to those participants with normal liver or minimal alterations  . OxidatiThe primary strength of this study was utilization of a large sample size from a nationally representative sample of NHANES. In NHANES there were detailed data on lifestyle, diet, and medical comorbidities, and we adjusted for a large number of covariates that made the results of this study more stable and credible. Moreover, the non-linear dose–response relationship between serum folate level and NAFLD was clearly elucidated using the restricted cubic splines in this study. However, there are a few of limitations that need to be considered. First, reverse causation may be the concern due to the cross-sectional nature of NHANES. Second, in this study noninvasive index (USFLI) but not a liver biopsy was used to identify NAFLD, which might result in the misclassification of NAFLD. However, The USFLI was developed using NHANES data, and the previous studies have demonstrated that the USFLI had a high accuracy .Our results suggest that serum folate level was inversely associated with the prevalence of NAFLD in the general population. Since this study is the first attempt to investigate the association between serum folate level and NAFLD defined by USFLI, more future longitudinal and intervention studies are needed.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.NHANES was approved by the National Center for Health Statistics Research Ethics Review Board.BY performed the statistical analyses, and wrote the manuscript. XL and LX contributed to interpretation of the data and the manuscript draft. YJ conceived the study and reviewed the manuscript. All authors contributed to the article and approved the submitted version.This work was supported by the National Natural Science Foundation of China (no. 81403237), Three-year Action Plan of Shanghai to Accelerate the Development of Traditional Chinese Medicine (no. ZY (2018–2020)-RCPY-2005), and Research project of Shanghai University of Traditional Chinese Medicine for COVID-19 infection (no. 2022YJ-18).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


To determine whether serum gamma‐glutamyl transpeptidase (GGT) level is associated with pancreatic cancer risk in a large prospective cohort.The study analyzed serum GGT concentration at baseline of 421,032 participants recruited in the UK Biobank since 2006 through 2010. Information on incidence of pancreatic cancer was obtained from cancer and death registers, updated until 2015 in Scotland or 2016 in England and Wales. Adjusted Cox proportional hazards models were used to measure the association between serum GGT and pancreatic cancer risk.p = 0.025). In the total population, the HR for the highest GGT group was 1.68 (95%CI: 1.22–2.30) versus the lowest GGT group. The HR for the highest GGT group in men (≥50.2 U/L) was 1.72 (95%CI: 1.14–2.61) and that in women (≥31.6 U/L) was 1.75 (95%CI: 1.06–2.88) versus the lowest GGT group.The study identified 586 cases of pancreatic cancer over a median follow‐up period of 7.16 years. In the multivariable‐adjusted Cox model, serum GGT level was associated with 14% higher pancreatic cancer risk (hazard ratio (HR) per one standard deviation increment of log2 GGT level = 1.14, 95% confidence interval (CI) 1.02–1.28, Our findings suggested a positive association of serum GGT in pancreatic cancer etiology, implying the potential of monitoring GGT level for identifying at‐risk individuals for pancreatic cancer. The study analyzed serum GGT concentration at baseline of 421,032 participants recruited in the UK Biobank since 2006 through 2010. Elevated serum GGT level predicted a higher risk of pancreatic cancer after fully adjusting for multiple confoundings. Therefore, serum biomarker GGT may have utility to early detect the high‐risk population with pancreatic cancer. Finally, our phenotypic analysis included 421,032 participants, which were then followed up until the date of cancer diagnosis, death, or the last follow‐up, whichever came first.   by linking records to the national cancer and death registers.2.5Self‐completed questionnaires were used to collect sociodemographic data  and characteristics related to smoking, alcohol consumption, and physical activity. Participants were assigned a Townsend deprivation index that represents their socioeconomic status according to their postcode. The assessment of physical activity was conducted using the International Physical Activity Questionnaire, which was summed over walking, moderate, and vigorous activity as metabolic equivalents (MET min/week). Trained nurses collected data on height, body weight, waist circumference during the initial assessment center visit. All participants were given a touchscreen food frequency questionnaire at recruitment about diet information.2.6The hazard ratios (HRs) and 95% confidence intervals (CIs) of serum GGT level for incident pancreatic cancer were assessed by Cox proportional hazards regression models. GGT was modeled on a continuous scale and also classified into four circulating levels based on sex‐specific quartiles. Log2 transformation of the nonnormally distributed continuous GGT scale was applied to reduce skewness. Z score for GGT standardization was analyzed for a standard deviation (SD) increase in the HR. A series of baseline characteristics were taken into account when fitting the multivariable model (model 1), namely gender, age at recruitment, UK Biobank Assessment Centre, Townsend deprivation index at recruitment, qualifications. In model 2, we additionally adjusted BMI, waist circumference, total physical activity per week, smoking status, alcohol consumption status, and levels of glycated hemoglobin (HbA1c). Furthermore, we adjusted for markers of inflammation and that possibly interrelate/cross talk with the GGT pathway including C‐reactive protein, total bilirubin, alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase, cholesterol, triglycerides, and low‐density lipoprotein direct in model 3. P for trend was also calculated using the median value of each category of GGT concentration as a continuous variable in the models.Furthermore, we examined the possibility of nonlinearity of log2 transformation of serum GGT level as continuous variables to predict pancreatic cancer using cubic spline models.We conducted sensitivity analyses to assess the impact of reverse causality after exclusions of the first year of follow‐up, participants with new‐onset diabetes before the cancer diagnosis within three years, inflammatory bowel disease (IBD), liver or bile duct disease and liver‐toxicity medication use . Furthermore, we corrected the additional confounding by controlling for dietary lifestyle characteristics, such as raw and cooked vegetable intake, fresh and dried fruit intake, frequency of poultry consumption, and first‐degree family history of digestive cancer.p < 0.05 was considered statistically significant. Analyses were conducted using R 3.6.3.Statistical tests were all two‐sided and a value of 33.1A total of 421,032 participants  were included in the study. Among the participants, the mean age was 56.23 (SD 8.10) years. In Table 3.2p‐trend = 0.003). The similar associations were observed for men  and women .Over a median follow‐up time of 7.16 years, a total of 586 incident pancreatic cancer cases were identified. Among them, 93 (0.09%) were in the bottom GGT quartile group and 184 (0.17%) in the top GGT quartile group Table . The posAssociations of log2 transformation of serum GGT level with incident pancreatic cancer appeared to be generally linear , and without new‐onset diabetes within 3 years before the cancer diagnosis . GGT's impact on pancreatic carcinogenesis was not influenced by age, smoking status , alcohol consumption status , or BMI in either subgroup analysis  , never/current smokers , current alcohol drinkers , those with low BMI <25 kg/m3.4After considering  1‐year lag period, we still detected a positive correlation between serum GGT level and pancreatic cancer risk (Q4 vs. Q1: HR 1.66 95%CI 1.20–2.29). When removing participants with new‐onset diabetes before the cancer diagnosis within 3 years, or participants with IBD, or participants with liver or bile duct‐related disease and liver‐toxicity medication use, the results remained essentially unchanged.  After additionally adjusting dietary lifestyle factors or first‐degree family history of digestive cancer, the impact of serum GGT on incident pancreatic cancer held solid.  The identical associations were found in gender stratification. .Xiao et al. reported that metastatic pancreatic cancer patients with higher GGT level had poorer overall survival outcomes. After controlling for possible confounding variables, serum GGT > 48 U/L led to a HR of 1.53 (95%CI: 1.19–1.97) for mortality risk. However, some potential confounders were unavailable with no adjustments made for smoking history, alcohol consumption, or tumor location, etc.11 However, in the latter study, they did not fully consider the sex disparities in serum GGT level, ignoring that men have higher GGT activity than women.To the best of our knowledge, few studies have examined gender disparities in serum GGT levels on the risk of pancreatic cancer. In this study, it was not found that GGT level was correlated with pancreatic cancer incidence in a sex‐aligned fashion. The observed multivariate HR was 1.72  in men and 1.75  in women. In Japanese population, when stratified the gender, the multivariate HR was 1.16 (95%CI: 0.90–1.50) in men and 1.22 (95%CI: 0.92–1.62) in women, supporting the notion that GGT level and cancer incidence are not sex‐dependent.2) and without new‐onset diabetes before the cancer diagnosis within 3 years, although other subgroup analyses with smaller sample size could not show sufficient evidence. There is no interaction among the subgroups including age, smoking status, alcohol drinking status, or BMI. Similarly, in National Health Insurance Service (NHIS) with Korean Cancer Prevention Study (KCPS), associations between serum GGT and site‐specific cancers were diminished in the high BMI (≥25 kg/m2) and the younger group.The subgroup analysis found an association between serum GGT and pancreatic cancer incidence in the population aged ≥60 years or never/current smokers, current alcohol drinkers, with low BMI  are formed,Since in clinical practice, serum GGT measurements are commonly carried out as a quick and inexpensive routine test, when individuals detected with high GGT level, timely intervention could potentially prevent the pancreatic cancer onset. It is worth noting that, the beneficial effect of adhering to a healthy lifestyle such as the consumption of fruit, vegetables,A strength of our study is relied on a nationwide prospective cohort, which gathered extensive data on a broad spectrum of covariates. In this way, we maintained adequate control of potential confounding factors for the associations of interest. Additionally, we provided comprehensive follow‐ups and robust sensitivity analyses. Some limitations of the study should also be noted. No repeated measurements for GGT were available. Besides, pancreatic cancer is relatively rare in Europe. This study was dependent on genetic data of European descent, so generalizability of our findings to other ethnicity is a concern. Further studies are required to investigate whether it hold valid in population with different genetic backgrounds. Finally, other cohorts will be expected to be applied to verify our results and provide more confidence.5Serum GGT level was a positive indicator of pancreatic cancer occurrence. Individuals with high risk of pancreatic cancer might benefit from close surveillance with serum GGT measurements. Further studies need to explore the potential causality of serum GGT level on incident pancreatic cancer.Weiting Liao: Conceptualization (lead); investigation (lead); methodology (lead); visualization (lead); writing – original draft (lead). Yu Yang: Conceptualization ; writing – original draft ; writing – review and editing . Huazhen Yang: Data curation ; investigation ; resources . Yuanyuan Qu: Project administration ; resources ; writing – review and editing . Huan Song: Conceptualization (lead); methodology (lead); resources (lead); supervision (lead); visualization (lead); writing – review and editing (lead). Qiu Li: Conceptualization (lead); funding acquisition (lead); project administration (lead); resources (lead); supervision (lead); validation (lead); writing – review and editing (lead).The work was financially supported by National Science and Technology Major Projects of China (No. 2017ZX09304023), the 1.3.5 project for disciplines of excellence, West China Hospital, Sichuan University (No. ZYJC18010). The funding had no role in study design, data collection, analysis or interpretation, or in writing the report.The authors declare no potential conflicts of interest.The UK Biobank obtained written informed consent from each participant, and the study has been ethically approved by the NHS National Research Ethics Service (16/NW/0274). The biomedical research ethics committee of West China Hospital has also approved this study (2019–1171).