Lymph node metastasis (LNM) is a crucial risk factor influencing an unfavorable prognosis in specific cancers. Fundamental research illuminates our understanding of tumor behavior and identifies valuable therapeutic targets. Nevertheless, the exploration of fundamental theories and the validation of clinical therapies hinge on preclinical experiments. Preclinical models, in this context, serve as the conduit connecting fundamental theories to clinical outcomes. In vivo models established in animals offer a valuable platform for comprehensively observing interactions between tumor cells and organisms. Using various experimental animals, including mice, diverse methods, such as carcinogen-induced tumorigenesis, tumor cell line or human tumor transplantation, genetic engineering, and humanization, have been used effectively to construct numerous models for tumor LNM. Carcinogen-induced models simulate the entire process of tumorigenesis and metastasis. Transplantation models, using human tumor cell lines or patient-derived tumors, offer a research platform closely mirroring the histology and clinical behavior of human tumors. Genetically engineered models have been used to delve into the mechanisms of primary tumorigenesis within an intact microenvironment. Humanized models are used to overcome barriers between human and murine immune systems. Beyond mouse models, various other animal models have unique advantages and limitations, all contributing to exploring LNM. This review summarizes existing in vitro and animal preclinical models, identifies current bottlenecks in preclinical research, and offers an outlook on forthcoming preclinical models.
Animals ; Humans ; Mice ; Lymphatic Metastasis/pathology* ; Disease Models, Animal ; Cell Line, Tumor

Objective To explore the differences in clinical characteristics of sudden sensorineural hearing loss(SSNHL)between patients with coronavirus disease 2019(COVID-19)and those without-COVID-19.Methods 31 SSNHL patients with COVID-19 who were hospitalized in a department during the COVID-19 epidemic period(from December 2022 to January 2023)were included as the study group,and 12 SSNHL patients without COVID-19 who received treatment during the non-COVID-19 epidemic period(from December 2021 to January 2022)were co-llected as the control group.Two groups of patients received standardized treatment,and their clinical characteristics and prognosis were compared.Clinical characteristics of SSNHL patients with COVID-19 during the COVID-19 epidemic period were analyzed.Results The time interval from patients developed COVID-19 to the onset of SSNHL in the study group was 3-30 days.The time interval from onset to consultation were 3(1,7)days and 5(4,6)days in the study group and the control group,respectively,with no significant difference(P＞0.05).The average age of patients in the study group was(44.16±13.54)years,which was higher than that of the control group(35.23±9.24)years,and the difference was statistically significant(P＜0.05).The hearing at damaged fre-quency of the study group improved by 6.5(0.5,24.5)dB after therapy,which was lower than that of the control group(36.0[27.0,38.0]dB),with statistically significant difference(P＜0.05).There was a negative correlation of the interval of consultation with the average improvement level of hearing at damaged frequency(r=-0.318,P=0.033).The longer the interval,the less the hearing improvement and the worse the therapeutic effect.Corre-lation analysis was further conducted on the time interval from development of COVID-19 to SSNHL onset and the time interval of consultation in patients in the study group,which showed no correlation(r=-0.337,P=0.059).There was no statistically significant difference in the types and degree of SSNHL between two groups of patients(both P＞0.05).After standardized treatment,the rate of ineffective patients in the study group was 56.25％,which was higher than that in the control group(15.38％),and the difference was statistically significant(P＜0.05).Conclusion After adjusting the COVID-19 prevention and control policies,there are more hospitalized COVID-19 patients with SSNHL,with a higher proportion in patients of older age,with poorer efficacy,and inef-fective treatment.The earlier the treatment for SSNHL patients,the better the effect can achieve.COVID-19 may be a potential inducement and/or etiological factor of SSNHL,and further research is needed.

Objective To explore the differences in clinical characteristics of sudden sensorineural hearing loss(SSNHL)between patients with coronavirus disease 2019(COVID-19)and those without-COVID-19.Methods 31 SSNHL patients with COVID-19 who were hospitalized in a department during the COVID-19 epidemic period(from December 2022 to January 2023)were included as the study group,and 12 SSNHL patients without COVID-19 who received treatment during the non-COVID-19 epidemic period(from December 2021 to January 2022)were co-llected as the control group.Two groups of patients received standardized treatment,and their clinical characteristics and prognosis were compared.Clinical characteristics of SSNHL patients with COVID-19 during the COVID-19 epidemic period were analyzed.Results The time interval from patients developed COVID-19 to the onset of SSNHL in the study group was 3-30 days.The time interval from onset to consultation were 3(1,7)days and 5(4,6)days in the study group and the control group,respectively,with no significant difference(P＞0.05).The average age of patients in the study group was(44.16±13.54)years,which was higher than that of the control group(35.23±9.24)years,and the difference was statistically significant(P＜0.05).The hearing at damaged fre-quency of the study group improved by 6.5(0.5,24.5)dB after therapy,which was lower than that of the control group(36.0[27.0,38.0]dB),with statistically significant difference(P＜0.05).There was a negative correlation of the interval of consultation with the average improvement level of hearing at damaged frequency(r=-0.318,P=0.033).The longer the interval,the less the hearing improvement and the worse the therapeutic effect.Corre-lation analysis was further conducted on the time interval from development of COVID-19 to SSNHL onset and the time interval of consultation in patients in the study group,which showed no correlation(r=-0.337,P=0.059).There was no statistically significant difference in the types and degree of SSNHL between two groups of patients(both P＞0.05).After standardized treatment,the rate of ineffective patients in the study group was 56.25％,which was higher than that in the control group(15.38％),and the difference was statistically significant(P＜0.05).Conclusion After adjusting the COVID-19 prevention and control policies,there are more hospitalized COVID-19 patients with SSNHL,with a higher proportion in patients of older age,with poorer efficacy,and inef-fective treatment.The earlier the treatment for SSNHL patients,the better the effect can achieve.COVID-19 may be a potential inducement and/or etiological factor of SSNHL,and further research is needed.

Objective To explore the differences in brain functions between individuals with good acclimatization to high altitudes and those with poor acclimatization based on microstate and functional connectivity analysis of resting-state electroencephalogram(EEG)activities.Methods A total of 44 residents exposed to the high-altitude environment of 3 650 m above sea level were enrolled.They were divided into two groups according to their findings for the high-altitude acclimatization index(AAI),with 22 in the good acclimatization group and 22 in the poor acclimatization group.The good acclimatization group had 10 males and 12 females of the mean age of(20.48±2.09)years.Among them,10 were Tibetans,and 12 were Han Chinese.The poor acclimatization group consisted of 18 males and 4 females,with a mean age of(18.90±1.25)years.Among them,7 were Tibetans,and 15 were Han Chinese.The K-means clustering algorithm was used to classify EEG microstates into four categories of A,B,C,and D.The average duration,coverage,occurrence frequency,and transition probabilities between microstates were compared.Coherence analysis(COH)was performed to assess the strength of brain network connectivity.Results Compared to the good acclimatization group,the poor acclimatization group exhibited a longer duration,higher occurrence frequency,and coverage of microstate C.The coverage of microstate D was also higher,while the occurrence frequency and coverage of microstate B were lower.The poor acclimatization group showed a decrease in transitions from microstate B to A or D,while transitions from C to B or D,and those from D to A or C increased,showing statistically significant differences between the groups(P＜0.05).In the α,β,δ,and θ frequency bands,the poor acclimatization group had higher COH strength in regions such as the frontal and occipital lobes,showing symmetrical functional connectivity between the left and right hemispheres.Conclusion The good and poor high-altitude acclimatization groups exhibit different neural mechanisms in terms of microstates and functional connectivity.There are significant differences between individuals with good acclimatization and those with poor acclimatization regarding visual information processing,interference resistance,brain network stability,and coherence.

Objective·To explore the correlation between the genetic variations rs7305526 and rs11615756 of nitric oxide synthase 1(NOS1)and the human sleep traits,including insomnia,sleep duration,and clinical quantitative traits related to obstructive sleep apnea(OSA).Methods·The NOS1 gene expression pattern at the whole-brain level using the Allen Human Brain Atlas dataset was analyzed.Subsequently,we performed expression quantitative trait locus(eQTL)analysis to investigate the impact of rs7305526 and rs11615756 on NOS1 gene expression.Regression analysis was conducted to assess the associations between rs7305526 and rs11615756 with insomnia and sleep duration based on the United Kingdom Biobank(UKB)Genome-Wide Association Study(GWAS)dataset.Furthermore,we explored the relationships between rs7305526 and rs11615756 with clinical quantitative traits of OSA,such as respiratory events,oxygen levels,and sleep traits,using clinical monitoring data from the Shanghai Sleep Health Study Cohort(SSHS)based on standard polysomnography(PSG).Results·The NOS1 gene demonstrated elevated levels of expression in various brain regions crucial for regulating sleep,namely the amygdala,basal forebrain,striatum,and thalamus,as well as in the respiratory center,including the mesencephalon and pontine tegmentum.In contrast,the expression level of NOS1 gene was significantly reduced or absent in areas such as the cerebral cortex and cerebellum.Variants rs7305526 and rs11615756 were significantly negatively correlated with the expression levels of NOS1 in the cerebral cortex.Additionally,rs11615756 was also significantly negatively correlated with the expression level of NOS1 in the amygdala.Analysis of the UKB GWAS data revealed that the variant rs7305526 was not significantly associated with either insomnia or sleep duration,while rs11615756 demonstrated a noteworthy negative correlation specifically with sleep duration.Data obtained from the SSHS indicated a significant association between rs7305526 and alterations in clinical quantitative traits of OSA,including lowest pulse blood oxygen saturation(LSpO2),apnea-hypopnea index(AHI),and the ratio of non-rapid eye movement(NREM)stage 2 sleep duration.Although rs11615756 showed a notable negative correlation solely with the quantity of NREM stages 2 and 3,both rs11615756 and rs7305526 showed significant correlations with some respiratory events and oxygen traits after stratification according to the severity of OSA.Conclusion·Genetic variants of NOS1 gene are respectively associated with human sleep duration traits and OSA-related variables,suggesting that NOS1 gene plays a crucial regulatory role in human sleep and clinical quantitative traits of OSA.The regulation of sleep traits by rs7305526(C>A)is independent of its regulation of respiratory events and oxygen traits.

Objective·To explore the correlation between the genetic variations rs7305526 and rs11615756 of nitric oxide synthase 1(NOS1)and the human sleep traits,including insomnia,sleep duration,and clinical quantitative traits related to obstructive sleep apnea(OSA).Methods·The NOS1 gene expression pattern at the whole-brain level using the Allen Human Brain Atlas dataset was analyzed.Subsequently,we performed expression quantitative trait locus(eQTL)analysis to investigate the impact of rs7305526 and rs11615756 on NOS1 gene expression.Regression analysis was conducted to assess the associations between rs7305526 and rs11615756 with insomnia and sleep duration based on the United Kingdom Biobank(UKB)Genome-Wide Association Study(GWAS)dataset.Furthermore,we explored the relationships between rs7305526 and rs11615756 with clinical quantitative traits of OSA,such as respiratory events,oxygen levels,and sleep traits,using clinical monitoring data from the Shanghai Sleep Health Study Cohort(SSHS)based on standard polysomnography(PSG).Results·The NOS1 gene demonstrated elevated levels of expression in various brain regions crucial for regulating sleep,namely the amygdala,basal forebrain,striatum,and thalamus,as well as in the respiratory center,including the mesencephalon and pontine tegmentum.In contrast,the expression level of NOS1 gene was significantly reduced or absent in areas such as the cerebral cortex and cerebellum.Variants rs7305526 and rs11615756 were significantly negatively correlated with the expression levels of NOS1 in the cerebral cortex.Additionally,rs11615756 was also significantly negatively correlated with the expression level of NOS1 in the amygdala.Analysis of the UKB GWAS data revealed that the variant rs7305526 was not significantly associated with either insomnia or sleep duration,while rs11615756 demonstrated a noteworthy negative correlation specifically with sleep duration.Data obtained from the SSHS indicated a significant association between rs7305526 and alterations in clinical quantitative traits of OSA,including lowest pulse blood oxygen saturation(LSpO2),apnea-hypopnea index(AHI),and the ratio of non-rapid eye movement(NREM)stage 2 sleep duration.Although rs11615756 showed a notable negative correlation solely with the quantity of NREM stages 2 and 3,both rs11615756 and rs7305526 showed significant correlations with some respiratory events and oxygen traits after stratification according to the severity of OSA.Conclusion·Genetic variants of NOS1 gene are respectively associated with human sleep duration traits and OSA-related variables,suggesting that NOS1 gene plays a crucial regulatory role in human sleep and clinical quantitative traits of OSA.The regulation of sleep traits by rs7305526(C>A)is independent of its regulation of respiratory events and oxygen traits.

Objective:To determine the expression of Brahma-related gene 1 (BRG1) in cutaneous squamous cell carcinoma (cSCC) tissues and cells, and to investigate molecular mechanisms underlying the regulatory effect of its interaction with activating transcription factor 2 (ATF2) on the proliferation, migration and invasion of cSCC cells.Methods:From 2015 to 2021, 66 paraffin-embedded actinic keratosis (AK) tissue samples and 80 paraffin-embedded cSCC (including squamous cell carcinoma in situ) tissue samples were collected from the Department of Dermatology, Affiliated Hospital 2 of Nantong University, and the diagnoses of all the cases were confirmed histopathologically; at the same time, 35 paraffin-embedded normal skin tissue samples obtained by cosmetic surgery served as normal control group. Immunohistochemical staining was performed to determine the BRG1 expression in cSCC, AK, and normal skin tissues, and correlations between BRG1 expression and clinicopathological parameters of cSCC patients were analyzed. Fresh tissue samples were collected from 12 cSCC patients and 12 healthy controls, and cSCC cell lines A431 and Scl-1 and a human immortalized keratinocyte cell line HaCaT were routinely cultured; real-time fluorescence-based quantitative PCR (qRT-PCR) was performed to determine the mRNA expression of BRG1 in tissues and cells, and co-immunoprecipitation assay and cellular immunofluorescence staining were conducted to analyze the interaction between BRG1 and ATF2. The expression of BRG1 (BRG1 siRNA1 - 5 groups) and ATF2 (ATF2-shRNA group) in A431 and Scl-1 cells was knocked down by RNA interference, and cells transfected with negative control siRNA or shNC served as controls (control siRNA group and shNC group, respectively), cell counting kit-8 (CCK8) assay, colony formation assay, cell scratch assay, and Transwell assay were conducted to evaluate effects of knocking down BRG1 and ATF2 on the proliferation, migration, and invasion of cSCC cells. Comparisons of measurement data among multiple groups were conducted using one-way analysis of variance, and multiple comparisons were conducted using Dunnett- t test. Results:Immunohistochemical staining showed that the expression intensity of BRG1 protein was significantly lower in the cSCC and AK tissues than in the normal skin tissues ( χ2 = 44.40, P < 0.001). qRT-PCR showed that the mRNA expression level of BRG1 was significantly lower in the cSCC tissues (1.345 ± 0.956) than in the normal skin tissues (2.499 ± 1.501, t = 2.25, P = 0.035), and also significantly lower in A431 and Scl-1 cells (0.041 ± 0.002, 0.026 ± 0.003, respectively) than in HaCaT cells (0.135 ± 0.033, t = 4.95, 5.73, P = 0.008, 0.005, respectively). The low expression of BRG1 was associated with tumors at sun-exposed sites ( P = 0.041), low tumor differentiation ( P = 0.001), and high Broder′s grade ( P < 0.001) in the cSCC patients. In both A431 cells and Scl-1 cells, the BRG1 siRNA1 group and BRG1 siRNA2 group showed significantly increased numbers of cell colonies, migratory cells and invasive cells, as well as cell migration rates compared with the control siRNA group (all P < 0.05). Co-immunoprecipitation assay showed that BRG1 protein could bind to ATF2 protein in A431 and Scl-1 cells, and immunofluorescence staining showed that the two proteins were co-localized; compared with the control siRNA group, the BRG1 siRNA1 group (both A431 and Scl-1 cells) and BRG1 siRNA2 group (A431 cells) both showed increased phosphorylation and activation of ATF2 (all P < 0.05) ; in both A431 cells and Scl-1 cells, the shATF2 group showed significantly decreased numbers of cell colonies (both P = 0.001), cellular proliferative activity at 24 - 96 hours (all P < 0.001), and numbers of migratory cells and invasive cells compared with the shNC group (all P ≤ 0.001) . Conclusion:BRG1 was lowly expressed in the cSCC and AK tissues, and could inhibit the proliferation, migration, and invasion of cSCC cells; ATF2 could promote the proliferation, migration, and invasion of cSCC cells; BRG1 may exert an anti-tumor effect by interacting with ATF2 protein and inhibiting phosphorylation-dependent activation of ATF2.

Objective: To explore assessment value of change of brain natriuretic peptide (BNP) level for cardiac function and prognosis judgment in patients with acute heart failure (AHF).Methods: After standard medicinal treatment, according to whether BNP level after treatment reduced <30% or rose compared with at hospitalization, a total of 91 AHF inpatients were divided into BNP reduction ≥30% group (n=67) and BNP reduction <30% or rise group (n=24).Left ventricular ejection fraction (LVEF), left ventricular end-diastolic dimension (LVEDd) and 6min walking distance (6MWD) at discharge were compared between two groups.All patients were followed up for 12 months.Multi-factor Logistic regression analysis was used to analyze risk factors for cardiogenic death.and adverse cardiac events were recorded.Results: Compared with BNP reduction ≥30% group, there were significant reductions in LVEF [(46.00±5.46)% vs.(34.54±5.32)%] and 6MWD [(392.64±153.02)m vs.(136.75±56.25)m], and significant rise in LVEDd [(56.33±4.40)mm vs.(65.96±6.13)mm] in BNP reduction <30% group, P<0.01 all.Multi-factor Logistic regression analysis indicated BNP reduction <30% or rise was the only one independent risk factor for cardiogenic death (OR=2.714, P=0.039).Compared with BNP reduction ≥30% group, there was significant rise in incidence rate of adverse cardiac events (40.3% vs.62.5%) in BNP<30% group, and the Log-rank value was 30.264 (P=0.001).Conclusion: Change of plasma BNP level can be used as an important reference index for the evaluation of cardiac function and prognosis in patients with acute heart failure.