Ursodeoxycholic acid (UDCA) is a naturally occurring, low-toxicity, and hydrophilic bile acid (BA) in the human body that is converted by intestinal flora using primary BA. Solute carrier family 7 member 11 (SLC7A11) functions to uptake extracellular cystine in exchange for glutamate, and is highly expressed in a variety of human cancers. Retroperitoneal liposarcoma (RLPS) refers to liposarcoma originating from the retroperitoneal area. Lipidomics analysis revealed that UDCA was one of the most significantly downregulated metabolites in sera of RLPS patients compared with healthy subjects. The augmentation of UDCA concentration (≥25 μg/mL) demonstrated a suppressive effect on the proliferation of liposarcoma cells. [¹⁵N₂]-cystine and [¹³C₅]-glutamine isotope tracing revealed that UDCA impairs cystine uptake and glutathione (GSH) synthesis. Mechanistically, UDCA binds to the cystine transporter SLC7A11 to inhibit cystine uptake and impair GSH de novo synthesis, leading to reactive oxygen species (ROS) accumulation and mitochondrial oxidative damage. Furthermore, UDCA can promote the anti-cancer effects of ferroptosis inducers (Erastin, RSL3), the murine double minute 2 (MDM2) inhibitors (Nutlin 3a, RG7112), cyclin dependent kinase 4 (CDK4) inhibitor (Abemaciclib), and glutaminase inhibitor (CB839). Together, UDCA functions as a cystine exchange factor that binds to SLC7A11 for antitumor activity, and SLC7A11 is not only a new transporter for BA but also a clinically applicable target for UDCA. More importantly, in combination with other antitumor chemotherapy or physiotherapy treatments, UDCA may provide effective and promising treatment strategies for RLPS or other types of tumors in a ROS-dependent manner.

Objective:To investigate the expressions of cystathionine-β-synthase(CBS)and cystathionine-γ-lyase(CSE)and their effects on the malignant biological behaviours of breast cancer cells,and to elucidate their mechanisms.Methods:The breast cancer tissue and paracancerous normal tissue from 15 cases of patients were selected,and RT-qPCR and Western blotting methods were used to detect the mRNA and protein expression levels of CBS and CSE in breast cancer tissue,paracancerous normal tissue,MCF-7 cells,and MDA-MB-231 cells.The MCF-7 cells were divided into siNC group(transfected with siNC)and siCBS group(transfected with siCBS),and the MDA-MB-231 cells were divided into ovNC group(transfected with CSE over-expression empty plasmid)and ovCSE group(transfected with CSE over-expression plasmid).CCK8 assay was used to detect the proliferation activities of breast cancer cells in various groups,Transwell assay was used to detect the numbers of migration and invasion cells in various groups,and Western blotting method was used to detect the protein expression levels of E-cadherin,N-cadherin and Vimentin proteins in the breast cancer cells in various groups.Results:Compared with paracancerous normal tissue,the expression levels of CBS and CSE mRNA and proteins in breast cancer tissue were increased(P＜0.05 or P＜0.01).Compared with MDA-MB-231 cells,the CBS mRNA expression level in the MCF-7 cells was increased(P＜0.05);compared with MCF-7 cells,the expression level of CSE protein in the MDA-MB-231 cells was decreased(P＜0.05).Compared with siNC group,the proliferation activity,the numbers of migration and invasion cells,the expression levels of N-cadherin and Vimentin proteins in the MCF-7 cells in siCBS group were significantly decreased(P＜0.05),and the expression level of E-cadherin protein was increased(P＜0.05).Compared with ovNC group,the proliferation activity,the numbers of migratoin and invasion cells,and the expression levels of N-cadherin and Vimentin proteins in the MDA-MB-231 cells in ovCSE group were increased(P＜0.05),while the expression level of E-cadherin protein was significantly decreased(P＜0.05).Conclusion:The expressions of CBS and CSE are upregulated in breast cancer tissue,and high levels of CBS and CSE promote proliferation,migration,invasion and epithelial-mesenchymal transition(EMT)of breast cancer cells.

Ursodeoxycholic acid(UDCA)is a naturally occurring,low-toxicity,and hydrophilic bile acid(BA)in the human body that is converted by intestinal flora using primary BA.Solute carrier family 7 member 11(SLC7A11)functions to uptake extracellular cystine in exchange for glutamate,and is highly expressed in a variety of human cancers.Retroperitoneal liposarcoma(RLPS)refers to liposarcoma originating from the retroperitoneal area.Lipidomics analysis revealed that UDCA was one of the most significantly down-regulated metabolites in sera of RIPS patients compared with healthy subjects.The augmentation of UDCA concentration(≥25 μg/mL)demonstrated a suppressive effect on the proliferation of liposarcoma cells.[15N2]-cystine and[13Cs]-glutamine isotope tracing revealed that UDCA impairs cystine uptake and glutathione(GSH)synthesis.Mechanistically,UDCA binds to the cystine transporter SLC7A11 to inhibit cystine uptake and impair GSH de novo synthesis,leading to reactive oxygen species(ROS)accumulation and mitochondrial oxidative damage.Furthermore,UDCA can promote the anti-cancer effects of ferroptosis inducers(Erastin,RSL3),the murine double minute 2(MDM2)inhibitors(Nutlin 3a,RG7112),cyclin dependent kinase 4(CDK4)inhibitor(Abemaciclib),and glutaminase inhibitor(CB839).Together,UDCA functions as a cystine exchange factor that binds to SLC7A11 for antitumor activity,and SLC7A11 is not only a new transporter for BA but also a clinically applicable target for UDCA.More importantly,in combination with other antitumor chemotherapy or physiotherapy treatments,UDCA may provide effective and promising treatment strategies for RLPS or other types of tumors in a ROS-dependent manner.

AIM:To investigate the immunosuppressive effects of M2-like tumor-associated macrophages(M2-TAMs)on CD8+T cells within the tumor microenvironment of esophageal cancer.METHODS:Multiplex fluores-cence immunohistochemistry was used to analyze the distribution of immune cells in esophageal cancer tissues.An in vitro co-culture system was established,and flow cytometry along with Calcein-AM/PI staining was employed to assess the im-pact of M2-TAMs on CD8+T cell function.The GEPIA database was utilized to evaluate the prognostic significance of PDCD1 expression in esophageal cancer patients and to analyze the correlations between gene expressions.Immunohisto-chemistry(IHC)was performed to detect the expression of TGF-β1 in esophageal cancer tissues.Flow cytometry and en-zyme-linked immunosorbent assay(ELISA)were used to measure PD-1,IFN-γ and TNF-α expression in CD8+T cells fol-lowing treatment with a TGF-β1 inhibitor.RESULTS:Compared with early-stage(stage I)esophageal cancer patients,the patients with advanced disease(stages Ⅱ to Ⅳ)exhibited dynamic changes in the infiltration of CD4+T cells,CD8+T cells,Tregs,and M2-TAMs within tumor tissues,with significant correlations observed among these cell populations(P<0.05).The distribution of M2-TAMs and Tregs was positively correlated with poor prognosis(P<0.05),while that of CD8+T cells was negatively correlated(P<0.05).In contrast,CD4+T cell infiltration showed no significant association with clinical outcomes(P>0.05).Co-culture of CD8+T cells with M2-TAMs resulted in significant downregulation of CD107a,granzyme B,IFN-γ and TNF-α expression(P<0.01).Additionally,M2-TAM-treated CD8+T cells co-cultured with esophageal cancer cells led to reduced apoptosis of cancer cells.High expression of PDCD1 was significantly associated with poor prognosis(P<0.05),and significant correlations were observed between CD8A and PDCD1 expression,as well as between TGF-β1 and CD274 gene expression(P<0.01).TGF-β1 was also significantly associated with CD163+macro-phage infiltration and the progression of esophageal cancer(P<0.05).Treatment with a TGF-β1 inhibitor in the M2-TAM and CD8+T cell co-culture system significantly down-regulated PD-1 expression and increased the secretion of IFN-γ and TNF-α(P<0.01).CONCLUSION:The TGF-β1 derived from M2-TAMs inhibits the antitumor activity of CD8+T cells in the esophageal cancer microenvironment,suggesting potential therapeutic targets for overcoming immunosuppression in esophageal cancer.

AIM:To investigate the immunosuppressive effects of M2-like tumor-associated macrophages(M2-TAMs)on CD8+T cells within the tumor microenvironment of esophageal cancer.METHODS:Multiplex fluores-cence immunohistochemistry was used to analyze the distribution of immune cells in esophageal cancer tissues.An in vitro co-culture system was established,and flow cytometry along with Calcein-AM/PI staining was employed to assess the im-pact of M2-TAMs on CD8+T cell function.The GEPIA database was utilized to evaluate the prognostic significance of PDCD1 expression in esophageal cancer patients and to analyze the correlations between gene expressions.Immunohisto-chemistry(IHC)was performed to detect the expression of TGF-β1 in esophageal cancer tissues.Flow cytometry and en-zyme-linked immunosorbent assay(ELISA)were used to measure PD-1,IFN-γ and TNF-α expression in CD8+T cells fol-lowing treatment with a TGF-β1 inhibitor.RESULTS:Compared with early-stage(stage I)esophageal cancer patients,the patients with advanced disease(stages Ⅱ to Ⅳ)exhibited dynamic changes in the infiltration of CD4+T cells,CD8+T cells,Tregs,and M2-TAMs within tumor tissues,with significant correlations observed among these cell populations(P<0.05).The distribution of M2-TAMs and Tregs was positively correlated with poor prognosis(P<0.05),while that of CD8+T cells was negatively correlated(P<0.05).In contrast,CD4+T cell infiltration showed no significant association with clinical outcomes(P>0.05).Co-culture of CD8+T cells with M2-TAMs resulted in significant downregulation of CD107a,granzyme B,IFN-γ and TNF-α expression(P<0.01).Additionally,M2-TAM-treated CD8+T cells co-cultured with esophageal cancer cells led to reduced apoptosis of cancer cells.High expression of PDCD1 was significantly associated with poor prognosis(P<0.05),and significant correlations were observed between CD8A and PDCD1 expression,as well as between TGF-β1 and CD274 gene expression(P<0.01).TGF-β1 was also significantly associated with CD163+macro-phage infiltration and the progression of esophageal cancer(P<0.05).Treatment with a TGF-β1 inhibitor in the M2-TAM and CD8+T cell co-culture system significantly down-regulated PD-1 expression and increased the secretion of IFN-γ and TNF-α(P<0.01).CONCLUSION:The TGF-β1 derived from M2-TAMs inhibits the antitumor activity of CD8+T cells in the esophageal cancer microenvironment,suggesting potential therapeutic targets for overcoming immunosuppression in esophageal cancer.

Objective:To investigate the CT imaging features of fat-poor primary retroperitoneal liposarcoma ( PRPLS).Methods:The CT signs of 43 fat-poor PRPLS cases among 128 PRPLS patients confirmed by pathology, including multiple nodules composing or multiple nodules fusing, tumor heterogeneity, were retrospectively evaluated.Results:Of 43 fat-poor PRPLS cases, 28 cases(65%) showed multiple nodules composing or multiple nodules fusing on CT images, 15 cases (35%)demonstrated single mass. Seventeen cases showed tumor heterogeneity on pre-contrast scan, 18 cases showed tumor heterogeneity on contrast scan, 13 cases showed tumor heterogeneity on both pre-contrast and contrast scan, 22 cases (51%)showed tumor heterogeneity on pre-contrast or contrast scan.Conclusion:CT signs of multiple nodules composing or multiple nodules fusing, especially tumor heterogeneity may help establishing diagnosis of fat-poor PRPLS.

Objective:To discuss the effect of sialic acid-binding immunoglobulin-like lectin-15(Siglec15)derived from M2 tumor-associated macrophages(M2-TAMs)on promoting the malignant biological behavior of the esophageal squamous cell carcinoma(ESCC)through bioinformatics analysis,and to validate the findings through cell experiment.Methods:The Tumor Immune Estimation Resource(TIMER)online Database was used to analyze the expression differences and immune infiltration of Siglec15 in pan-cancer and adjacent normal tissues.Real-time fluorescence quantitative PCR(RT-qPCR)method was used to detect the expression levels of Siglec15 mRNA in M2-TAMs and ESCC EC109 and KYSE150 cells.Based on the non-contact co-culture of M2-TAMs and ESCC cells,the following groups were set up,such as EC109/KYSE150 group,EC109/KYSE150+si-NC group(transfected with si-NC sequence),and EC109/KYSE150+si-Siglec15 group(transfected with si-Siglec15#1 and si-Siglec15#2 sequences).CCK-8 method was used to detect the proliferation activities of the cells in various groups;wound healing assay was used to detect the wound healing rates of the cells in various groups;Transwell chamber assay was used to detect the numbers of migration and invasion cells in various groups;flow cytometry was used to detect the apoptotic rates of the cells in various groups.Results:The bioinformatics analysis results showed that compared with adjacent normal tissue,the expression levels of Siglec15 mRNA in pan-cancer tissues such as esophageal cancer,colon cancer,and head and neck squamous cell carcinoma tissues were increased(P＜0.05 or P＜0.01),and the expression level of Siglec15 mRNA in esophageal cancer tissue was significantly positively correlated with the infiltration of the macrophages(P＜0.05).Compared with the EC109 cells and KYSE150 cells,the expression level of Siglec15 mRNA in M2-TAMs was significantly increased(P＜0.01).There was no significant difference in the proliferation rate of the cells among EC109/KYSE150 group,EC109/KYSE150+si-NC group,and EC109/KYSE150+si-Siglec15 group(P＞0.05).Compared with EC109/KYSE150 group,after treated for 24 and 48 h,the wound healing rate of the cells in EC109/KYSE150+si-NC group was increased(P＜0.01),the numbers of migration and invasion cells were increased(P＜0.05),and the apoptotic rate was decreased(P＜0.01).Compared with EC109/KYSE150+si-NC group,the wound healing rates of the cells in EC109/KYSE150+si-Siglec15#1 group and EC109/KYSE150+si-Siglec15#2 group were decreased(P＜0.05),the numbers of migration and invasion cells were decreased(P＜0.05),and the apoptotic rates of the cells had no significant difference(P＞0.05).Conclusion:Siglec15 derived from M2-TAMs may be a key factor in promoting the migration and invasion of the ESCC cells.

Objective:To investigate the effect of Lamins B2 (LMNB2) on the migration of human retroperitoneal liposarcoma (RPLS) cells SW872.Methods:Immunohistochemistry was used to analyze the the differential expression levels of LMNB2 in 33 RPLS tissue samples . The correlation between LMNB2 expression and clinical prognosis and clinicopathological features was analyzed. siRNA was used to lower the expression level of LMNB2 in tumor cells, and the effect of LMNB2 on the scratch healing ability and migration ability of SW872 cells was examined by using wound-healing assay and transwell migration assay. The expression levels of p-AKT and AKT in each group cells were detected by Western blot.Results:Patients with high LMNB2 expression had a lower recurrence-free survival and overall survival compared to those with low LMNB2 expression, and were more likely to experience recurrence, ( χ2=4.872, P=0.027; χ2=4.180, P=0.041; χ2=7.127, P=0.008). The migration ability of cells was significantly reduced following the silencing of LMNB2 expression ( t=11.240, P<0.01; t=7.445, P<0.01). The expression level of p-AKT in the silencing group was significantly lower than that in the control group, while there was no significant difference in the expression level of AKT between the two groups ( t=9.784, P<0.01). Conclusion:LMNB2 may promote the migration of human retroperitoneal liposarcoma cells SW872 by regulating AKT signaling pathway.

Background::Heterogeneity of tumor cells and the tumor microenvironment (TME) is significantly associated with clinical outcomes and treatment responses in patients with urothelial carcinoma (UC). Comprehensive profiling of the cellular diversity and interactions between malignant cells and TME may clarify the mechanisms underlying UC progression and guide the development of novel therapies. This study aimed to extend our understanding of intra-tumoral heterogeneity and the immunosuppressive TME in UC and provide basic support for the development of novel UC therapies.Methods::Seven patients with UC were included who underwent curative surgery at our hospital between July 2020 and October 2020. We performed single-cell RNA sequencing (scRNA-seq) analysis in seven tumors with six matched adjacent normal tissues and integrated the results with two public scRNA-seq datasets. The functional properties and intercellular interactions between single cells were characterized, and the results were validated using multiplex immunofluorescence staining, flow cytometry, and bulk transcriptomic datasets. All statistical analyses were performed using the R package with two-sided tests. Wilcoxon-rank test, log-rank test, one-way analysis of variance test, and Pearson correlation analysis were used properly.Results::Unsupervised t-distributed stochastic neighbor embedding clustering analysis identified ten main cellular subclusters in urothelial tissues. Of them, seven urothelial subtypes were noted, and malignant urothelial cells were characterized with enhanced cellular proliferation and reduced immunogenicity. CD8 + T cell subclusters exhibited enhanced cellular cytotoxicity activities along with increased exhaustion signature in UC tissues, and the recruitment of CD4 + T regulatory cells was also increased in tumor tissues. Regarding myeloid cells, coordinated reprogramming of infiltrated neutrophils, M2-type polarized macrophages, and LAMP3 + dendritic cells contribute to immunosuppressive TME in UC tissues. Tumor tissues demonstrated enhanced angiogenesis mediated by KDR + endothelial cells and RGS5 +/ACTA2 + pericytes. Through deconvolution analysis, we identified multiple cellular subtypes may influence the programmed death-ligand 1 (PD-L1) immunotherapy response in patients with UC. Conclusion::Our scRNA-seq analysis clarified intra-tumoral heterogeneity and delineated the pro-tumoral and immunosuppressive microenvironment in UC tissues, which may provide novel therapeutic targets.

Humans ; Retroperitoneal Neoplasms ; Liposarcoma ; Calcium-Binding Proteins ; Mixed Function Oxygenases ; Membrane Proteins ; Muscle Proteins