Purpose To investigate the role of tumor-associated neutrophils(TANs)in the sternness characteris-tics of breast cancer cells.Methods Flow cytometry was used to identify and validate the successful generation of TANs.Breast cancer cell lines were co-cultured with TANs-conditioned supernatant,and their sphere-forming capacity was assessed.Western blot and reverse transcription-quantitative polymerase chain reaction(RT-qPCR)were used to detect the expression of stem cell-associated markers.In breast cancer clinical specimens,immunohistochemistry(IHC)was performed to quantify TANs infiltration and the expression of sternness-related markers.Correlations be-tween TANs infiltration and sternness marker expression,as well as their associations with clinicopathological features of breast cancer patients,were analyzed.Results Flow cytometry results demonstrated a significantly higher survival rate of TANs compared to normal neutrophils(NEUs)(P＜0.05).In vitro,TANs-derived supernatant significantly en-hanced the sphere-forming capacity of breast cancer cells compared to NEU-derived supernatant(P＜0.05).RT-qPCR analysis revealed that the mRNA expression levels of sternness-related markers such as CD133 and SOX9 in tumor cells was significantly increased after TANs supernatant co-cultured with breast cancer cells(P＜0.05).The results of western blot further confirmed that the protein expression levels of CD133 and SOX9 were markedly increased in the TANs supernatant group relative to the NEU supernatant group(P＜0.01).Immunophenotypic analysis showed that the infiltration density of CD66b+TANs in breast cancer tissues was positively correlated with the expression of CD133(r=0.429,P＜0.001)and SOX9(r=0.561,P＜0.001)in tumor cells.Prognostic and clinicopathological analy-ses indicated that patients in the high CD66b+TANs infiltration group,high CD133 expression group,and high SOX9 expression group had significantly shorter progression-free survival(PFS)than those in the corresponding low-expres-sion/infiltration groups(P＜0.05).Furthermore,the infiltration density of CD66b+TANs was significantly associated with patient age,histological grade,and lymph node metastasis status(all with P＜0.05).CD133 expression was cor-related with patient age,lymph node metastasis,and progesterone receptor(PR)status(all with P＜0.05),while SOX9 expression was associated with patient age and lymph node metastasis status(all with P＜0.05).Conclusion TANs can promote the acquisition and maintenance of sternness characteristics in breast cancer cells.These findings may provide novel insights into the development of neutrophil-targeted immunotherapeutic strategies for breast cancer.

Objective To investigate the clinical characteristics,treatment strategies,and prognostic factors of adenoid cystic carcinoma(ACC)of the external auditory canal(EAC),and to provide evidence for optimizing surgical extent and adjuvant therapy.Methods A retrospective cohort study was conducted on 58 patients with pathologically confirmed ACC of the EAC treated in Department of Otorhinolaryngology Head and Neck Surgery,Beijing Tongren Hospital,Capital Medical University,between January 2001 and December 2021.All patients underwent surgical treatment,with some receiving adjuvant radiotherapy.The primary outcome was local recurrence,while secondary outcomes included overall survival(OS)and local recurrence-free survival(LRFS).Survival analysis was performed by the Kaplan-Meier method,and Cox regression models were used to identify risk factors for recurrence.Results The median follow-up time for the entire cohort was 6.27(3.25,11.30)years.The 1-year,3-year,and 5-year OS rates were 96.55％,91.37％,and 89.66％.43.10％of cases were classified as T4 stage at diagnosis,indicating advanced local progression.Local recurrence occurred in 23 patients(39.66％),and distant metastasis was observed in 28 patients(48.28％),with pulmonary metastasis accounting for 92.86％of cases.Multivariate analysis revealed that the solid histological pattern(HR=2.729,95％CI:1.025-7.226,P=0.044)and perineural invasion(PNI)(HR=9.891,95％CI:3.525-27.752,P＜0.01)were independent risk factors for local recurrence.Conclusion ACC of the EAC is characterized by a high propensity for local recurrence and distant metastasis.The solid histological pattern and perineural invasion are critical prognostic determinants.Multimodal therapy(surgery combined with adjuvant radiotherapy)may improve clinical outcomes,and early diagnosis and intervention are pivotal for enhancing survival rates.

OBJECTIVES: To fabricate an injectable composite microsphere hydrogel reinforced with silk fibroin/hyaluronic acid microspheres, achieving synergistic enhance-ment of mechanical robustness and biofunctionality. METHODS: Methacrylated hyaluronic acid (HAMA) and thiolated silk fibroin (TSF) were synthesized. Monodisperse microspheres generated via microfluidics were UV-cured (420 nm) through thiol-ene click reaction. These microspheres were embedded in a TSF/HAMA matrix to form photo-cured composites. The grafting rate of TSF and HAMA was characterized by H1-NMR; particle size distribution of microsphere hydrogels in soybean oil was observed by optical microscopy; gel point of composite microsphere hydrogels was determined by advanced extensional rheometer; microscopic morphology of microsphere hydrogels was observed by scanning electron microscopy; elemental distribution of microsphere hydrogels was detected by X-ray energy dispersive spectroscopy; tunability of composite microsphere hydrogels was observed by inverted confocal microscopy; mechanical properties of composite microsphere hydrogels were tested by compression testing; swelling ratio, degradation rate and water retention rate of composite microsphere hydrogels were measured by gravimetric method. Cytotoxicity of the composite microsphere hydrogels was determined by Calcein-AM/propidium iodide dual staining and CCK-8 assay; cell migration capability was observed by scratch assay. RESULTS: The grafting rates of HAMA and TSF was 48.03% and 17.99%, respectively. Microsphere hydrogels with particle sizes of (43.3±1.2), (78.1±3.0), and (130.8±1.9) μm were prepared. The gel time of the composite microsphere hydrogels was 48-115s. The laser confocal imaging confirmed dynamic regulation characteristics of the composite microsphere hydrogels. The compressive strength of the composite microsphere hydrogels reached 22.7 kPa and maintained structural integrity at 40% strain after 20 compression cycles. The composite microsphere hydrogels exhibited differential deswelling behaviors in simulated physiological environments, and reducing microsphere particle size could significantly enhance its stability under moist conditions. The degradation rate of the composite microsphere hydrogels was (49.1±0.9)% after 200 h, and water retention rate was maintained at 40%-60% after 96 h. Biocompatibility assays confirmed >95% cell viability and unimpaired cell migration abilities. CONCLUSIONS The TSF/HAMA composite microsphere hydrogel developed in this study has characteristics of rapid fabrication, adjustable mechanical properties, enhanced environmental stability and excellent biocom-patibility, thus providing a new material solution for tissue repair and regenerative medicine.
Fibroins/chemistry* ; Hydrogels/chemistry* ; Microspheres ; Hyaluronic Acid/chemistry* ; Humans

Interfering hepatitis B virus (HBV) capsid assembly holds promise as a therapeutic approach for chronic hepatitis B (CHB). Novel anti-HBV agents are urgently needed to overcome drug resistance challenges, with targeted protein degradation (TPD) emerging as a hopeful strategy. Herein, we report the first degradation of HBV core protein (HBC), a multifunctional structural protein, using small-molecule degraders developed by hydrophobic tagging (HyT) technology. Structure-activity relationship (SAR) analysis identified compound HyT-S7, featuring an adamantyl group, exhibiting potent inhibitory activity (EC₅₀ = 0.46 μmol/L, HepAD38 cells) and degradation ability (DC₅₀ = 3.02 ± 0.54 μmol/L) in a dose- and time-dependent manner. Mechanistic studies demonstrated that the autophagy-lysosome pathway was a potential driver of HyT-S7-induced HBC degradation. Remarkably, HyT-S7 effectively degraded 11 drug-resistant mutants, including highly resistant strains P25G and T33N, to Phase III drug GLS4. Furthermore, cellular thermal shift assay, surface plasmon resonance assay, and molecular dynamics simulations revealed the precise mode of HyT-S7 binding to HBC with the adamantyl group potentially mimicking protein misfolding to facilitate HBC degradation. This first proof-of-concept study highlights the potential of HyT-mediated TPD in HBC as a promising avenue for discovering novel HBV and other antiviral agents with favorable drug resistance profiles.

OBJECTIVE To evaluate the clinical value of dose-escalated postoperative radiotherapy(PORT)in improving local control and survival outcomes for head and neck adenoid cystic carcinoma(ACC)patients.METHODS This retrospective study analyzed 336 ACC patients treated with surgery plus PORT at Beijing Tongren Hospital from January 2015 to January 2021.Cohort stratification compared high-dose(>60 Gy,n=146)and conventional-dose(≤60 Gy,n=190)regimens.Survival analysis employed Kaplan-Meier estimates with log-rank testing,complemented by multivariate Cox regression for risk adjustment.RESULTS The cohort demonstrated 39.29%(132/336)cumulative local failure rate.The overall survival rates at 1,3,and 5 years after surgery were 98.81%,94.05%,and 90.48%,respectively.Dose-response relationships revealed:1.Significantly reduced local recurrence with high-dose PORT(28.08%vs.47.89%,P<0.001),corresponding to 41.37%lower recurrence risk(a HR=0.59,95%CI=0.38-0.91;P=0.041);2.Superior progression-free survival in the high-dose group(3-year:86.99%vs.76.32%;5-year:82.19%vs.66.32%,all P<0.05);3.Comparable overall survival between groups(median OS:200 vs.160 months,P=0.292).CONCLUSION Dose escalation beyond 60 Gy significantly enhances locoregional control and progression-free survival in head and neck ACC without conferring overall survival advantage,likely reflecting the disease's characteristic indolent metastatic progression.These results establish>60 Gy as an optimal dose threshold for PORT in high-risk ACC management.

Objective To investigate the clinical characteristics,treatment strategies,and prognostic factors of adenoid cystic carcinoma(ACC)of the external auditory canal(EAC),and to provide evidence for optimizing surgical extent and adjuvant therapy.Methods A retrospective cohort study was conducted on 58 patients with pathologically confirmed ACC of the EAC treated in Department of Otorhinolaryngology Head and Neck Surgery,Beijing Tongren Hospital,Capital Medical University,between January 2001 and December 2021.All patients underwent surgical treatment,with some receiving adjuvant radiotherapy.The primary outcome was local recurrence,while secondary outcomes included overall survival(OS)and local recurrence-free survival(LRFS).Survival analysis was performed by the Kaplan-Meier method,and Cox regression models were used to identify risk factors for recurrence.Results The median follow-up time for the entire cohort was 6.27(3.25,11.30)years.The 1-year,3-year,and 5-year OS rates were 96.55％,91.37％,and 89.66％.43.10％of cases were classified as T4 stage at diagnosis,indicating advanced local progression.Local recurrence occurred in 23 patients(39.66％),and distant metastasis was observed in 28 patients(48.28％),with pulmonary metastasis accounting for 92.86％of cases.Multivariate analysis revealed that the solid histological pattern(HR=2.729,95％CI:1.025-7.226,P=0.044)and perineural invasion(PNI)(HR=9.891,95％CI:3.525-27.752,P＜0.01)were independent risk factors for local recurrence.Conclusion ACC of the EAC is characterized by a high propensity for local recurrence and distant metastasis.The solid histological pattern and perineural invasion are critical prognostic determinants.Multimodal therapy(surgery combined with adjuvant radiotherapy)may improve clinical outcomes,and early diagnosis and intervention are pivotal for enhancing survival rates.

Purpose To investigate the role of tumor-associated neutrophils(TANs)in the sternness characteris-tics of breast cancer cells.Methods Flow cytometry was used to identify and validate the successful generation of TANs.Breast cancer cell lines were co-cultured with TANs-conditioned supernatant,and their sphere-forming capacity was assessed.Western blot and reverse transcription-quantitative polymerase chain reaction(RT-qPCR)were used to detect the expression of stem cell-associated markers.In breast cancer clinical specimens,immunohistochemistry(IHC)was performed to quantify TANs infiltration and the expression of sternness-related markers.Correlations be-tween TANs infiltration and sternness marker expression,as well as their associations with clinicopathological features of breast cancer patients,were analyzed.Results Flow cytometry results demonstrated a significantly higher survival rate of TANs compared to normal neutrophils(NEUs)(P＜0.05).In vitro,TANs-derived supernatant significantly en-hanced the sphere-forming capacity of breast cancer cells compared to NEU-derived supernatant(P＜0.05).RT-qPCR analysis revealed that the mRNA expression levels of sternness-related markers such as CD133 and SOX9 in tumor cells was significantly increased after TANs supernatant co-cultured with breast cancer cells(P＜0.05).The results of western blot further confirmed that the protein expression levels of CD133 and SOX9 were markedly increased in the TANs supernatant group relative to the NEU supernatant group(P＜0.01).Immunophenotypic analysis showed that the infiltration density of CD66b+TANs in breast cancer tissues was positively correlated with the expression of CD133(r=0.429,P＜0.001)and SOX9(r=0.561,P＜0.001)in tumor cells.Prognostic and clinicopathological analy-ses indicated that patients in the high CD66b+TANs infiltration group,high CD133 expression group,and high SOX9 expression group had significantly shorter progression-free survival(PFS)than those in the corresponding low-expres-sion/infiltration groups(P＜0.05).Furthermore,the infiltration density of CD66b+TANs was significantly associated with patient age,histological grade,and lymph node metastasis status(all with P＜0.05).CD133 expression was cor-related with patient age,lymph node metastasis,and progesterone receptor(PR)status(all with P＜0.05),while SOX9 expression was associated with patient age and lymph node metastasis status(all with P＜0.05).Conclusion TANs can promote the acquisition and maintenance of sternness characteristics in breast cancer cells.These findings may provide novel insights into the development of neutrophil-targeted immunotherapeutic strategies for breast cancer.

Purpose To investigate the expression of Versican(VCAN)and thrombospondin 2(THBS2)and their relationship with cancer-associated fibroblast(CAFs)and clinicopathological significance in papillary thyroid car-cinoma(PTC).Methods Bioinformatics analyses were performed using PTC single-cell sequencing data from the GEO and TCGA database.Weighted correlation network analysis identified CAFs-related genes,and enrichment analy-sis highlighted pivotal genes associated with CAFs;the expression of VCAN,THBS2,and α-SMA in 130 PTC tissue samples were detected by immunohistochemistry EnVision method.Masson staining evaluated tumor stromal fibrosis.Relationships between these markers,clinicopathological parameters,and CAF proliferation were analyzed.Results Bioinformatics analysis identified VCAN and THBS2 as core genes significantly associated with CAFs,and extracellular matrix-related pathways.The proliferation rate of CAFs in PTC was 83.1％(108/130),with positivity rate of 96.9％(126/130)for VCAN and 75.4％(98/130)for THBS2.The median mesenchymal fibrosis index was 32.4(inter-quartile range:22.7-50.0).High CAF proliferation correlated positively with lymph node metastasis(P＜0.001),higher TNM stage(P＜0.05),and specific histologic subtypes of PTC.Similarly,VCAN expression,THBS2 expres-sion,and the degree of PTC stromal fibrosis were positively correlated with lymph node metastasis(P＜0.001,P＜0.05,and P＜0.001,respectively).Both THBS2 expression and the degree of PTC stromal fibrosis correlated with the histologic subtype of PTC.The percentage of tumor mesenchymal α-SMA-positive cells strongly correlated with the im-mune response score(IRS)of VCAN and THBS2(rs=0.713,P＜0.001;rs=0.646,P＜0.001).Additionally,the percentage of Masson-stained area was positively correlated with the percentage of tumor mesenchymal α-SMA-posi-tive cells,and the IRS of VCAN and THBS2(rs=0.892,P＜0.001;rs=0.729,P＜0.001;rs=0.616,P＜0.001).Conclusion VCAN and THBS2 serve as potential markers for assessing invasiveness and lymph node metas-tasis of PTC.Their strong association with CAFs provides a basis for further investigation of the malignant biological be-havior of PTC.

Purpose To investigate the expression of Versican(VCAN)and thrombospondin 2(THBS2)and their relationship with cancer-associated fibroblast(CAFs)and clinicopathological significance in papillary thyroid car-cinoma(PTC).Methods Bioinformatics analyses were performed using PTC single-cell sequencing data from the GEO and TCGA database.Weighted correlation network analysis identified CAFs-related genes,and enrichment analy-sis highlighted pivotal genes associated with CAFs;the expression of VCAN,THBS2,and α-SMA in 130 PTC tissue samples were detected by immunohistochemistry EnVision method.Masson staining evaluated tumor stromal fibrosis.Relationships between these markers,clinicopathological parameters,and CAF proliferation were analyzed.Results Bioinformatics analysis identified VCAN and THBS2 as core genes significantly associated with CAFs,and extracellular matrix-related pathways.The proliferation rate of CAFs in PTC was 83.1％(108/130),with positivity rate of 96.9％(126/130)for VCAN and 75.4％(98/130)for THBS2.The median mesenchymal fibrosis index was 32.4(inter-quartile range:22.7-50.0).High CAF proliferation correlated positively with lymph node metastasis(P＜0.001),higher TNM stage(P＜0.05),and specific histologic subtypes of PTC.Similarly,VCAN expression,THBS2 expres-sion,and the degree of PTC stromal fibrosis were positively correlated with lymph node metastasis(P＜0.001,P＜0.05,and P＜0.001,respectively).Both THBS2 expression and the degree of PTC stromal fibrosis correlated with the histologic subtype of PTC.The percentage of tumor mesenchymal α-SMA-positive cells strongly correlated with the im-mune response score(IRS)of VCAN and THBS2(rs=0.713,P＜0.001;rs=0.646,P＜0.001).Additionally,the percentage of Masson-stained area was positively correlated with the percentage of tumor mesenchymal α-SMA-posi-tive cells,and the IRS of VCAN and THBS2(rs=0.892,P＜0.001;rs=0.729,P＜0.001;rs=0.616,P＜0.001).Conclusion VCAN and THBS2 serve as potential markers for assessing invasiveness and lymph node metas-tasis of PTC.Their strong association with CAFs provides a basis for further investigation of the malignant biological be-havior of PTC.

Objective To explore the pain catastrophizing (PC) level of pain in patients with multiple fractures and its influencing factors. Methods A convenience sampling method was used to investigate 156 patients with multiple fractures in the orthopedic trauma department of the First Affiliated Hospital of Nanjing Medical University. The questionnaire included a general information survey, a Digital Pain Rating Scale, PC scale, Positive and Negative Emotion Scale, and Social Rating Scale. Results The average PC score of patients with multiple fractures was (23.22±12.05), with 27 patients (17.20%) reaching the PC level. The average score of the Digital Pain Rating Scale was (6.30±1.49), the score of the Positive Emotion Scale was (27.92±6.06), the score of the Negative Emotion Scale was (23.18±7.00), and the total score of the Social Rating Scale was (27.90±4.61). The results of multiple linear regression analysis showed that pain score, negative emotion level, and social support level had predictive effects in PC among patients with multiple fractures. Conclusion The incidence of PC among patients with multiple fractures is at a moderate to high level. Patients with high pain scores, high negative emotion scores, and low social support are more likely to develop PC.