Inhibiting membrane rupture with NINJ1 antibodies limits tissue injury.

Plasma membrane rupture (PMR) in dying cells undergoing pyroptosis or apoptosis requires the cell-surface protein NINJ11. PMR releases pro-inflammatory cytoplasmic molecules, collectively called damage-associated molecular patterns (DAMPs), that activate immune cells. Therefore, inhibiting NINJ1 and PMR may limit the inflammation that is associated with excessive cell death. Here we describe an anti-NINJ1 monoclonal antibody that specifically targets mouse NINJ1 and blocks oligomerization of NINJ1, preventing PMR. Electron microscopy studies showed that this antibody prevents NINJ1 from forming oligomeric filaments. In mice, inhibition of NINJ1 or Ninj1 deficiency ameliorated hepatocellular PMR induced with TNF plus D-galactosamine, concanavalin A, Jo2 anti-Fas agonist antibody or ischaemia-reperfusion injury. Accordingly, serum levels of lactate dehydrogenase, the liver enzymes alanine aminotransaminase and aspartate aminotransferase, and the DAMPs interleukin 18 and HMGB1 were reduced. Moreover, in the liver ischaemia-reperfusion injury model, there was an attendant reduction in neutrophil infiltration. These data indicate that NINJ1 mediates PMR and inflammation in diseases driven by aberrant hepatocellular death.

Development of an 18F-labeled anti-human CD8 VHH for same-day immunoPET imaging.

PURPOSE: Cancer immunotherapies (CITs) have revolutionized the treatment of certain cancers, but many patients fail to respond or relapse from current therapies, prompting the need for new CIT agents. CD8+ T cells play a central role in the activity of many CITs, and thus, the rapid imaging of CD8+ cells could provide a critical biomarker for new CIT agents. However, existing 89Zr-labeled CD8 PET imaging reagents exhibit a long circulatory half-life and high radiation burden that limit potential applications such as same-day and longitudinal imaging. METHODS: To this end, we discovered and developed a 13-kDa single-domain antibody (VHH5v2) against human CD8 to enable high-quality, same-day imaging with a reduced radiation burden. To enable sensitive and rapid imaging, we employed a site-specific conjugation strategy to introduce an 18F radiolabel to the VHH. RESULTS: The anti-CD8 VHH, VHH5v2, demonstrated binding to a membrane distal epitope of human CD8 with a binding affinity (KD) of 500 pM. Subsequent imaging experiments in several xenografts that express varying levels of CD8 demonstrated rapid tumor uptake and fast clearance from the blood. High-quality images were obtained within 1 h post-injection and could quantitatively differentiate the tumor models based on CD8 expression level. CONCLUSION: Our work reveals the potential of this anti-human CD8 VHH [18F]F-VHH5v2 to enable rapid and specific imaging of CD8+ cells in the clinic.

The Role of MR Assessments of Cardiac Morphology, Function, and Tissue Characteristics on Exercise Capacity in Well-Functioning Older Adults.

BACKGROUND: The relationship between resting cardiac indices and exercise capacity in older adults was still not well understood. New developments in cardiac magnetic resonance imaging (MRI) enable a much fuller assessment of cardiac characteristics. PURPOSE/HYPOTHESIS: To assess the association between exercise capacity and specific aspects of resting cardiac structure, function, and tissue. STUDY TYPE: Cross-sectional study. POPULATION: A total of 112 well-functioning older adults (mean age 69 years, 52 men). FIELD STRENGTH/SEQUENCE: All participants underwent 3.0 T MRI, using scan protocols including balanced steady-state free precession cine sequence, modified look-locker inversion recovery, and T2-prepared single-shot balanced steady-state free precession. ASSESSMENT: Demographic and geriatric characteristics were collected. Blood samples were assayed for lipid and glucose related biomarkers. All participants performed a symptom-limited cardiopulmonary exercise test to achieve peakVO2 . Cardiac MRI parameters were measured with semi-automatic software by S.Y., an 18-year experienced radiologist. STATISTICAL TESTS: Demographic, geriatric characteristics and MR measurements were compared among quartiles of peakVO2, with different methods according to the data type. Spearman's partial correlation and least absolute shrinkage selection operator regression were performed to select significant MR features associated with peakVO2 . Mediation effect analysis was conducted to test any indirect connection between age and peakVO2 . A two-sided P value of <0.05 was defined statistical significance. RESULTS: Epicardial fat volume, left atrial volume indexed to height, right ventricular end-systolic volume indexed to body surface area and global circumferential strain (GCS) were correlated with peakVO2 (regression coefficients were -0.040, -0.093, 0.127, and 0.408, respectively). Mediation analysis showed that the total effect of peakVO2 change was 43.6% from the change of age. The proportion of indirect effect from epicardial fat volume and GCS were 11.8% and 15.1% in total effect, respectively. DATA CONCLUSION: PeakVO2 was associated with epicardial fat volume, left atrial volume, right ventricular volume and GCS of left ventricle. EVIDENCE LEVEL: 4 TECHNICAL EFFICACY: Stage 5.

Sociodemographic factors associated with the first administration of anti-seizure medication in patients with focal epilepsy in Western China.

BACKGROUND: Epilepsy is a severe chronic neurologic disease with a prevalence of 0.7% worldwide; anti-seizure medications (ASMs) are the mainstay of epilepsy treatment. The effects of sociodemographic factors on the characteristics of initial treatment in patients with newly diagnosed focal epilepsy in Western China are unknown. This study was conducted to explore sociodemographic factors associated with initial treatment characteristics. METHODS: Patients with focal epilepsy on continuous ASM treatment who visited to our epilepsy center at Sichuan Provincial People's Hospital between January 2018 and December 2019 were recruited. Data on initial treatment status and sociodemographic variables were obtained from the patients with a questionnaire designed by our researchers. We examined whether sociodemographic factors were associated with epileptic patients' access to neurologists and prescriptions of individual ASMs. RESULTS: A total of 569 patients completed this study. We found that patients with a higher education level, aged < 16 years, and with a higher household disposable income were more likely to receive treatment from a neurologist than their counterparts. Patients with a lower personal income level and who were treated at a junior hospital were more likely to receive prescriptions for carbamazepine, and those who were younger than 16 years were less likely to receive prescriptions for carbamazepine and oxcarbazepine. Patients with a higher education level, with a higher household disposable income level, who were younger than 16 years, and who were treated at a senior hospital were more likely to receive prescriptions for levetiracetam than their counterparts. Adult, female patients with focal epilepsy treated at a senior hospital were more likely to receive prescriptions for lamotrigine. CONCLUSIONS: This observation suggests that sociodemographic characteristics are associated with access to neurologists and prescriptions of individual antiepileptic drugs. These data may help public health officials establish guidelines for doctors and distribute resources according to the needs of different patient groups.

Monoclonal antibody targeting the ß-barrel assembly machine of Escherichia coli is bactericidal.

The folding and insertion of integral ß-barrel membrane proteins into the outer membrane of Gram-negative bacteria is required for viability and bacterial pathogenesis. Unfortunately, the lack of selective and potent modulators to dissect ß-barrel folding in vivo has hampered our understanding of this fundamental biological process. Here, we characterize a monoclonal antibody that selectively inhibits an essential component of the Escherichia coli ß-barrel assembly machine, BamA. In the absence of complement or other immune factors, the unmodified antibody MAB1 demonstrates bactericidal activity against an E. coli strain with truncated LPS. Direct binding of MAB1 to an extracellular BamA epitope inhibits its ß-barrel folding activity, induces periplasmic stress, disrupts outer membrane integrity, and kills bacteria. Notably, resistance to MAB1-mediated killing reveals a link between outer membrane fluidity and protein folding by BamA in vivo, underscoring the utility of this antibody for studying ß-barrel membrane protein folding within a living cell. Identification of this BamA antagonist highlights the potential for new mechanisms of antibiotics to inhibit Gram-negative bacterial growth by targeting extracellular epitopes.

Effects of valproate, lamotrigine, and levetiracetam monotherapy on bone health in newly diagnosed adult patients with epilepsy.

PURPOSE: The aim of this study was to evaluate the effects of valproate (VPA), lamotrigine (LTG), and levetiracetam (LEV) on bone turnover and bone mineral density (BMD) in newly diagnosed adult patients with epilepsy. METHODS: Eligible adult patients who were newly diagnosed with epilepsy were treated with VPA, LTG, and LEV. The chemical indicators of bone metabolism and BMD were measured before treatment and 2 years after treatment with different antiseizure medication (ASM) monotherapies. Then, the differences in these parameters before and after treatment were analyzed. RESULTS: One hundred twenty-four patients completed the 2 years follow-up; 43 received monotherapy with VPA, 32 received LTG, and 49 received LEV. Serum parathyroid hormone (PTH), bone alkaline phosphatase (B-ALP), and ß-cross-linked C-telopeptide of type I collagen (ß-CTX) levels were elevated in adult patients after 2 years of VPA administration; the serum procollagen I intact N-terminal peptide (PINP) level was noticeably higher in patients after LEV treatment than before treatment. Meanwhile, the BMD of the lumbar spine and femoral neck did not change in patients treated with VPA, LTG, and LEV. CONCLUSIONS: Valproate altered bone turnover in adult patients with epilepsy, while LTG and LEV did not exert harmful effects on bone health in adult patients.

Identification of an imidazopyridine scaffold to generate potent and selective TYK2 inhibitors that demonstrate activity in an in vivo psoriasis model.

Herein we report identification of an imidazopyridine class of potent and selective TYK2 inhibitors, exemplified by prototype 6, through constraint of the rotatable amide bond connecting the pyridine and aryl rings of compound 1. Further optimization led to generation of compound 30 that potently inhibits the TYK2 enzyme and the IL-23 pathway in cells, exhibits selectivity against cellular JAK2 activity, and has good pharmacokinetic properties. In mice, compound 30 demonstrated dose-dependent reduction of IL-17 production in a PK/PD model as well as in an imiquimod-induced psoriasis model. In this efficacy model, the IL-17 decrease was accompanied by a reduction of ear thickness indicating the potential of TYK2 inhibition as a therapeutic approach for psoriasis patients.

Conditional Deletion of NF-κB-Inducing Kinase (NIK) in Adult Mice Disrupts Mature B Cell Survival and Activation.

NF-κB-inducing kinase (NIK) is a primary regulator of the noncanonical NF-κB signaling pathway, which plays a vital role downstream of BAFF, CD40L, lymphotoxin, and other inflammatory mediators. Germline deletion or inactivation of NIK in mice results in the defective development of B cells and secondary lymphoid organs, but the role of NIK in adult animals has not been studied. To address this, we generated mice containing a conditional allele of NIK. Deletion of NIK in adult mice results in decreases in B cell populations in lymph nodes and spleen, similar to what is observed upon blockade of BAFF. Consistent with this, B cells from mice in which NIK is acutely deleted fail to respond to BAFF stimulation in vitro and in vivo. In addition, mice with induced NIK deletion exhibit a significant decrease in germinal center B cells and serum IgA, which is indicative of roles for NIK in additional pathways beyond BAFF signaling. Our conditional NIK-knockout mice may be broadly useful for assessing the postdevelopmental and cell-specific roles of NIK and the noncanonical NF-κB pathway in mice.

PILRα negatively regulates mouse inflammatory arthritis.

Paired Ig-like type 2 receptor (PILR)α inhibitory receptor and its counterpart PILRß activating receptor are coexpressed on myeloid cells. In this article, we report that PILRα, but not PILRß, is elevated in human rheumatoid arthritis synovial tissue and correlates with inflammatory cell infiltration. Pilrα(-/-) mice produce more pathogenic cytokines during inflammation and are prone to enhanced autoimmune arthritis. Correspondingly, engaging PILRα with anti-PILRα mAb ameliorates inflammation in mouse arthritis models and suppresses the production of proinflammatory cytokines. Our studies suggest that PILRα mediates an important inhibitory pathway that can dampen inflammatory responses.

Polyclonal hyper-IgE mouse model reveals mechanistic insights into antibody class switch recombination.

Preceding antibody constant regions are switch (S) regions varying in length and repeat density that are targets of activation-induced cytidine deaminase. We asked how participating S regions influence each other to orchestrate rearrangements at the IgH locus by engineering mice in which the weakest S region, Sε, is replaced with prominent recombination hotspot Sµ. These mice produce copious polyclonal IgE upon challenge, providing a platform to study IgE biology and therapeutic interventions. The insertion enhances ε germ-line transcript levels, shows a preference for direct vs. sequential switching, and reduces intraswitch recombination events at native Sµ. These results suggest that the sufficiency of Sµ to mediate IgH rearrangements may be influenced by context-dependent cues.

[Role of SREBP-1c in risk of liver disease associated with the triacylglycerol lipase PNPLA3 I148M variant].

OBJECTIVE: To investigate the relationship between SREBP-1c and the risk of liver disease associated with the triacylglyceride lipase PNPLA3 I148M variant using a human hepatoma cell line model transfected with recombinant lentiviruses. METHODS: Huh7 cells were transfected with control lentivirus or lentivirus containing the PNPLA3 I148M variant (variant). The two cell groups were compared to assess differences in triglyceride content (using oil red O staining), levels of triglyceride and cholesterol (using automated biochemical analyzer), expression of SREBP-lc mRNA (using fluorescence quantitative PCR), and expression of SREBP-1c protein (using western blot. RESULTS: Cells expressing the PNPLA3 I148M variant showed higher triglyceride content (0.54+/-0.03 mmol/L vs. control cells: 0.23+/-0.02 mmol/L; t=22.58, P<0.001), cholesterol level (0.28+/-0.03 mmol/L vs. control cells: 0.13+/-0.02 mmol/L; t =11.83, P<0.001), SREBP-1cmRNA expression (13.59+/-0.60 vs. 11.81+/-0.82; [The abstract and text in the paper say variant increases, but the data shown says the higher value is in the control cells. Please correct to properly express the data.] P=0.001), and SREBP-1c protein expression. The level of SREBP-1c was positively correlated with serum triglyceride in the cells expressing the PNPLA3 I148M variant (r=0.912, P<0.01). CONCLUSION: The risk of liver disease associated with the PNPLA3 I148M variant, which increases lipogenesis, may involve SREBP-1c and a pathway that increases triglycerides.

[Association between the PNPLA3 I148M polymorphism and chronic hepatitis B in a Qingdao Han Chinese population].

OBJECTIVE: To investigate the association between the PNPLA3 rs738409 polymorphism and chronic hepatitis B (CH[B) in a Han Chinese population residing in Qingdao. METHODS: Peripheral blood samples were collected from 185 CHB patients and 164 healthy controls and subjected to polymerase chain reaction (PCR) and DNA sequencing to determine the PNPLA3 genotypes. The relative risk of the rs738409 polymorphism for CHB was estimated by calculating the odds ratio (OR) and 95% confidence interval. RESULTS: The rs738409 G allele frequency was significantly different between the CHB and control groups (31.9% vs.21.9% respectively, P less than 0.05). Compared to he rs738409 C allele, the G allele was associated with an increased risk of developing CHB (OR =1.67, 95% CI:1.18-2.34, P =0.003). Logistic regression model analysis, with adjustment for confounding factors, indicated that carriers of the PNPLA3 rs738409 GG + GC genotype had increased risk of CHB than carriers of the CC genotype (OR =1.76 ,95% CI:1.14-2.71, P =0.011). CONCLUSION: Qingdao Han Chinese who are carriers of the rs738409 G allele are at increased risk of CHB.

[Association between APOC3 promoter region polymorphisms and non-alcoholic fatty liver disease].

OBJECTIVE: To investigate the association between two polymorphisms of the APOC3 gene (T-455C and C-482T) and hereditary risk of non-alcoholic fatty liver disease (NAFLD). METHODS: A total of 287 patients with NAFLD and 310 control subjects were genotyped by PCR and direct sequencing. Serum lipid profiles were also detected by standard biochemical METHODS: One-hundred-and-eighty of the study participants were used to measure the APOC3 content by enzyme-linked immunosorbent assay. Inter-group differences and associations were assessed statistically using Chi square and t tests and logistic and linear regression analyses. RESULTS: The frequencies of neither the genotypes or alleles were significantly different between the NAFLD cases and the controls. Compared with the most common genotypes-455TT or-482CC, none of the variants showed a significant increase in risk of NAFLD or for the clinical and biochemical parameters. The adjusted odds ratios (with 95% confidence intervals) of NAFLD were 1.25 (0.79-1.96) and 1.20 (0.76-1.89) for carriers of the APOC3-455C and-482 T variants respectively (P more than 0.05). CONCLUSION: The T-455C and C-482T polymorphisms of the APOC3 gene are not associated with risk of NAFLD, pathogenic changes in lipid profiles, or insulin resistance in Han Chinese.

Epileptic brain network mechanisms and neuroimaging techniques for the brain network.

Epilepsy can be defined as a dysfunction of the brain network, and each type of epilepsy involves different brain-network changes that are implicated differently in the control and propagation of interictal or ictal discharges. Gaining more detailed information on brain network alterations can help us to further understand the mechanisms of epilepsy and pave the way for brain network-based precise therapeutic approaches in clinical practice. An increasing number of advanced neuroimaging techniques and electrophysiological techniques such as diffusion tensor imaging-based fiber tractography, diffusion kurtosis imaging-based fiber tractography, fiber ball imaging-based tractography, electroencephalography, functional magnetic resonance imaging, magnetoencephalography, positron emission tomography, molecular imaging, and functional ultrasound imaging have been extensively used to delineate epileptic networks. In this review, we summarize the relevant neuroimaging and neuroelectrophysiological techniques for assessing structural and functional brain networks in patients with epilepsy, and extensively analyze the imaging mechanisms, advantages, limitations, and clinical application ranges of each technique. A greater focus on emerging advanced technologies, new data analysis software, a combination of multiple techniques, and the construction of personalized virtual epilepsy models can provide a theoretical basis to better understand the brain network mechanisms of epilepsy and make surgical decisions.

The relationship between event-related potential components and suicide risk in major depressive disorder.

BACKGROUND: Suicide is a serious global issue, with major depressive disorder (MDD) being a significant risk factor for suicidal thoughts and behaviors. There is an urgent need to determine whether event-related potential components (ERPs) could be used as an indicator to assess suicidal risk. METHODS: From 2020 to 2023, 258 participants in total were recruited into the study. All participants were divided into four groups: MDD patients at high (n = 66), moderate (n = 66), and low risk (n = 56) of suicide, and healthy controls (HCs)(n = 70). Each participant provided socio-demographic information and underwent evaluations using clinical psychological scales such as 7-item Generalized Anxiety Disorder (GAD-7), Health Questionnaire-9 items (PHQ-9), and Nurses' Global Assessment of Suicide Risk (NGASR). The auditory brainstem response test and ERP examination were performed for all subjects. RESULTS: Our study found that the amplitude of P2-P3 and N2-P3 was significantly reduced in MDD patients at moderate and high risk of suicide, and these were negatively correlated with NGASR total score (all P < 0.05). Point B latency was positively correlated with NGASR total score (P < 0.05). Patients with MDD patients at low risk for suicide had a lower A-B amplitude compared to HCs (P < 0.05). No differences were found in MMN or P50 components between the four groups (all P > 0.05). CONCLUSIONS: MDD patients at higher risk of suicide exhibited severe impairment of cognitive function. ERP indices, such as the amplitude of P2-P3 and N2-P3, could be associated with the risk of suicide in MDD patients.

The role of the "gut microbiota-mitochondria" crosstalk in the pathogenesis of multiple sclerosis.

Multiple Sclerosis (MS) is a neurologic autoimmune disease whose exact pathophysiologic mechanisms remain to be elucidated. Recent studies have shown that the onset and progression of MS are associated with dysbiosis of the gut microbiota. Similarly, a large body of evidence suggests that mitochondrial dysfunction may also have a significant impact on the development of MS. Endosymbiotic theory has found that human mitochondria are microbial in origin and share similar biological characteristics with the gut microbiota. Therefore, gut microbiota and mitochondrial function crosstalk are relevant in the development of MS. However, the relationship between gut microbiota and mitochondrial function in the development of MS is not fully understood. Therefore, by synthesizing previous relevant literature, this paper focuses on the changes in gut microbiota and metabolite composition in the development of MS and the possible mechanisms of the crosstalk between gut microbiota and mitochondrial function in the progression of MS, to provide new therapeutic approaches for the prevention or reduction of MS based on this crosstalk.

Molecular dynamics simulation of PNPLA3 I148M polymorphism reveals reduced substrate access to the catalytic cavity.

A missense mutation I148M in PNPLA3 (patatin-like phospholipase domain-containing 3 protein) is significantly correlated with nonalcoholic fatty liver disease (NAFLD). To glean insights into mutation's effect on enzymatic activity, we performed molecular dynamics simulation and flexible docking studies. Our data show that the size of the substrate-access entry site is significantly reduced in mutants, which limits the access of palmitic acid to the catalytic dyad. Besides, the binding free energy calculations suggest low affinity for substrate to mutant enzyme. The substrate-bound system simulations reveal that the spatial arrangement of palmitic acid is distinct in wild-type from that in mutant. The substrate recognition specificity is lost due to the loop where the I148M mutation was located. Our results provide strong evidence for the mechanism by which I148M affects the enzyme activity and suggest that mediating the dynamics may offer a potential avenue for NAFLD.

[Polymorphism rs738409 in PNPLA3 is associated with inherited susceptibility to non-alcoholic fatty liver disease].

OBJECTIVE: To study the relationship between the patatin-like phospholipase domain-containing protein 3 (PNPLA3) gene and hereditary susceptibility to non-alcoholic fatty liver disease (NAFLD) by detecting single nucleotide polymorphisms (SNPs). METHODS: Peripheral blood DNA from 315 patients diagnosed with NAFLD (including the spectrum of simple steatosis (SS) and non-alcoholic steatosis (NASH)) and 336 control subjects was used to determine the PNPLA3 genotype by polymerase chain reaction (PCR) and direct sequencing. The relationship of SNPs and NAFLD-related markers of liver function were assessed by correlation analysis. RESULTS: The SNP rs738409 was identified in more of the NAFLD patients (allele variant frequencies: NAFLD, 65.40%; NASH: 71.87%; SS, 56.47%) than in the controls (33.18%). Case-control analysis revealed that carriers of the 148GG genotype were at 3.81-fold (95% CI: 3.03 ~ 4.79) higher risk of developing NAFLD and at 1.97-fold (95% CI: 1.41 ~ 2.75) higher risk of progressing from SS to NASH, compared with non-carriers. rs738409 was also found to be associated with serum levels of alanine aminotransferase (ALT) and y-glutamyltransferase (y-GT) (both P less than 0.05). Carriers of the 148GG genotype had significantly higher body mass index, ALT, and fasting insulin than carriers of the 148CC genotype (all P less than 0.05), and significantly higher level of serum HDL than carriers of either the 148CC genotype or the 148GC genotype (both P less than 0.05). CONCLUSION: Polymorphisms in the PNPLA3 gene may play an important role in mediating susceptibility to developing NAFLD in the Chinese population. The rs738409 polymorphism, in particular, is related to development and progression of NAFLD and may play a role in the contribution of PNPLA3 to NAFLD pathogenesis.

The differences of event-related potential components in patients with comorbid depression and anxiety, depression, or anxiety alone.

BACKGROUND: There was a high comorbidity rate of major depressive disorder (MDD) and generalized anxiety disorder (GAD), showing a poor prognosis and significant detrimental impact on functioning. The study aimed to find whether patients with comorbid GAD and MDD had some differences in cognitive functions from patients with MDD or GAD alone. METHODS: 360 adult patients were enrolled from inpatient department of psychiatry from 2020 to 2022. They were divided into three groups with 120 patients for each group: MDD, GAD, and MDD + GAD. All the patients completed psychological evaluation scales including patient health questionnaire-9 (PHQ-9) and 7-item generalized anxiety disorder (GAD-7). All the patients underwent examinations of auditory brainstem response and event-related potentials (ERPs). RESULTS: In MDD + GAD group, P3b latency was significantly longer than patients with MDD alone, and P300 reaction time was positively correlated with total score of GAD-7 and PHQ-9, and PHQ-9 total score was also significantly positively correlated with P2-P3b amplitude (all p < 0.05). In addition, MDD patients had significantly longer P300 reaction time and lower P2-P3b amplitude than the GAD group (p < 0.05). LIMITATIONS: It was a single-center and cross-sectional study, and we used self-report scales as assessment tools. CONCLUSIONS: Patients with MDD and GAD comorbidity might have a worse cognitive function than MDD patients, and the severity of cognitive impairments was positively correlated with the severity of anxiety and depression symptoms.

Research of restricted migration evaluation of MDA-MB-231 cells in 2D and 3D co-culture models.

The restricted migration evaluation is conducive to more complex tumor migration research because of the conformity with in vivo tumors. However, the differences between restricted and unrestricted cell migration and the distinction between different evaluation methods have not been systematically studied, hindering related research. In this study, by constructing the restricted environments on chips, the influence of co-culture conditions on the cancer cell migration capacity was studied. The results showed that the restricted channels can discriminate the influence of weak tumor environmental factors on complex tumor migration behaviors by limiting the free growth instinct of tumor cells. Through the comparison of 2D and 3D restricted migration methods, the extracellular matrix (ECM) restriction was also helpful in distinguishing the influence of the weak tumor environmental factor. However, the 3D ECM can better reflect the tortuosity of the cell migration process and the cooperative behavior among cancer cells. In the anticancer drug evaluation, 3D ECM can more accurately reflect the cytotoxicity of drugs and is more consistent with the drug resistance in the human body. In conclusion, the research will help to distinguish different evaluation methods of cancer cell migration, help researchers select appropriate evaluation models, and promote the research of tumor metastasis.