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Objective To analyze the therapeutic effect of anti-programmed death receptor 1(PD-1)/pro-grammed death ligand 1(PD-L1)and the characteristics of intestinal flora in patients with non-small cell lung cancer(NSCLC).Methods A total of 81 NSCLC patients admitted to the People's Hospital of Xinjiang Uygur Autonomous Region from January 2020 to January 2022 were taken as the research object.According to the patients'immunotherapy response,the patients were divided into non-response group and response group.The differences in clinical data and intestinal flora distribution between the two groups were compared,and the correlation between PFS and intestinal flora a diversity index was analyzed by Spearman correlation.Results The proportion of smoking patients in response group was significantly lower than that in non-re-sponse group,and the difference was statistically significant(x2=4.648,P=0.031).Chao1 index,ACE index and shannon wiener index patients in non-response group were lower than those in response group,while Simpson diversity index was higher than that in response group,with statistical significance(P＜0.05).Chao1 index,ACE index and shannon wiener index were positively correlated with PFS(r=0.526,0.579 and 0.539,all P＜0.05),while Simpson diversity index was negatively correlated with PFS(r=-0.867,P＜0.001).The principal coordinate analysis was used to analyze the β diversity structure of intestinal flora.The first principal component contribution rate was 70.36％,and the second principal component contribution rate was 16.63％.Conclusion The diversity and distribution of intestinal flora in NSCLC patients are related to anti-PD-1/PD-L1 therapy.The higher the diversity of intestinal flora,the more sensitive the anti-PD-1/PD-L1 therapy.
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BACKGROUND:Immunotherapy enhances the anti-cancer immune response in many ways,so combined immunotherapy is a better choice.Ultrasound-targeted microbubble destruction technique delivers drugs,genes,antibodies and cytokines directly to the cytoplasm of immune cells and enhances the immune response.However,the application of ultrasound-targeted microbubble destruction technique in the treatment of ovarian cancer with both CXC chemokine receptor 4 antibody and programmed death-ligand 1 antibody has not been reported. OBJECTIVE:To investigate the effect of ultrasound irradiation on the proliferation and migration of ovarian cancer cells with CXC chemokine receptor 4 antibody and programmed death-ligand 1 antibody double targeted nanobubbles. METHODS:IOSE-80 normal ovarian epithelial cells,SKOV3 and CAOV3 ovarian cancer cells were cultured and expanded.Double labeling fluorescence immunoassay was used to co-locate CXC chemokine receptor 4 and programmed death-ligand 1 protein.Western blot assay was used to detect the relative expression of CXC chemokine receptor 4 and programmed death-ligand 1 protein in three kinds of cells and screen out the experimental cells,i.e.,pure nanobubbles,nanobubbles carrying CXC chemokine receptor 4 antibody,nanobubbles carrying CXC chemokine receptor 4 and programmed death-ligand 1 antibody.SKOV3 ovarian cancer cells in the logarithmic growth phase were taken and divided into six groups for treatment.Group A was added with McCoy's 5A medium.Group B was added with McCoy's 5A medium containing stromal cell-derived factor-1.Group C was added with pure nanobubble solution and McCoy's 5A medium containing stromal cell-derived factor-1.Group D was added with nanobubble solution containing CXC chemokine receptor 4 antibody and McCoy's 5A medium containing stromal cell-derived factor-1.Group E was added with nanobubble solution containing CXC chemokine receptor 4 and programmed death-ligand 1 antibody and McCoy's 5A medium containing stromal cell-derived factor-1.Pure nanobubble solution was added in group F.After ultrasonic irradiation for 120 seconds and incubation for 48 hours,the survival rate of cells was measured by CCK-8 assay,and the healing and migration ability of cells in groups B-E were measured by wound healing test. RESULTS AND CONCLUSION:(1)Immunofluorescence staining showed that CXC chemokine receptor 4 and programmed death-ligand 1 protein could be expressed in all three kinds of cells.Western blot assay showed that the expression levels of CXC chemokine receptor 4 and programmed death-ligand 1 in SKOV3 and CAOV3 ovarian cancer cells were significantly higher than those in IOSE-80 normal ovarian epithelial cells(P<0.05).(2)CCK-8 assay results exhibited that the cell survival rate of group B was higher than that of group A(P<0.05).The cell survival rate of group F was lower than that of group A(P<0.05).The cell survival rate of groups B-E decreased gradually,and there were significant differences between the two groups(P<0.05).(3)Wound healing test demonstrated that the cell healing rate of groups B-E decreased gradually,and there were significant differences between the two groups(P<0.05).(4)The results show that the use of CXC chemokine receptor 4 antibody and programmed death-ligand 1 antibody double targeted nanobubbles under ultrasound-targeted microbubble destruction can significantly inhibit the proliferation and migration of ovarian cancer cells.
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ObjectiveTo investigate the possible mechanism of Ruyi Heibai Power （如意黑白散， RHP） in the treatment of vitiligo. MethodsTwenty-four C57BL/6 mice were randomly divided into blank group， model group， high-dose and low-dose RHP groups， with 6 mice in each group. Model group， high- and low-dose RHP groups were all applied hydroquinone to establish vitiligo animal model. After modeling， High- and low-dose RHP groups were given 7.02 g/kg and 2.34 g/kg of RHP by gavage， respectively， while the blank group and model group were intragastrically given 10ml/kg of normal saline， once a day for 36 days. After administration， the skin lesions were observed with naked eye， and HE staining was used to observe the melanin content of the skin lesions. Immunohistochemistry was used to determine the expression of CD3+ and CD8+ T cells in skin tissue. Immunofluorescence staining was used to measure the expression of programmed death receptor 1 （PD-1） in the skin lesion tissue. RT-PCR was used to detect programmed cell death ligand 1 （PD-L1） mRNA expression. ELISA was used to detect serum superoxide dismutase （SOD）， tumor necrosis factor （TNF-α）， and tyrosinase （TYR） levels. ResultsCompared to those in the blank group， the skin of the mice in the model group was pale， and the melanin content was significantly reduced under the microscope after HE staining； the rate of excellent and good skin lesions decreased， and the melanin granules in the cells around the epidermis and hair follicles decreased significantly； the expression of CD3+ and CD8+ T cells in skin tissue increased significantly， and the expressions of PD-L1 mRNA and PD-1 decreased； the content of TYR decreased， while the content of SOD and TNF-α increased （P＜0.05）. Compared to those in the model group， the skin color of high- and low-dose RHP groups were deepened， and the melanin content increased； the rate of excellent and good skin lesions increased， as well as the melanin granules in the spinous cell layer， basal cells and hair follicles； the expression of CD3+ and CD8+ T cells in the skin lesions decreased， while PD-L1 mRNA and PD-1 expression increased； the content of TYR increased， while the content of SOD and TNF-α decreased （P＜0.05）. Compared to the low-dose RHP group， the high-dose group had a larger pigment recovery area in the modeling area， an increased rate of excellent and good skin lesions， an increase in spinous cell layer， basal cells， and hair follicle melanin granules， a decrease in CD3+ and CD8+ T cells expression， an increase in the expression of PD-L1 mRNA and PD-1， an elevated TYR content， and decreased SOD and TNF-α contents （P＜0.05）. ConclusionRHP can increase skin melanin content of vitiligo mice.The mechanism of action may be related to activating the PD-1/PD-L1 signaling pathway， and then reducing the destruction of melanocytes by T cell-mediated autoimmunity.
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Sepsis is a life-threatening organ dysfunction due to dysregulation of the body's response to infection. The immunosuppressive phase of sepsis is also an important cause of high morbidity and mortality in patients with advanced sepsis. Myeloid-derived suppressor cell (MDSC), as a heterogeneous population of Gr1 +CD11b + immature cells, play a pivotal role in the immune process of advanced sepsis together with programmed death-1 (PD-1) and programmed death-ligand 1 (PD-L1) with immunosuppressive properties. This review summarized the research progress of PD-1/PD-L1 pathway-mediated mechanism of MDSC in septic immunosuppression in recent years. The mechanism of action of PD-1/PD-L1 pathway in the immunosuppressive stage, the effects of PD-1/PD-L1 pathway on the "dual-signaling" model of MDSC through the signaling pathway and cytokines, as well as the time course of PD-1/PD-L1 pathway mediation in sepsis were described.
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There have been breakthroughs in the development and clinical application of immuno-therapeutic agents in recent years.As the clinical targets of second-generation immune checkpoint inhibitors(ICIs),programmed death-1(PD-1)and its ligand(programmed death-ligand 1,PD-L1)have become one of the hot spots in drug discovery.The Food and Drug Administration of the USA and National Medical Products Administration in China have approved a variety of PD-1/PD-L1 monoclonal antibody drugs(such as nivolumab,pembrolizumab and atezolizumab),which are marketed for the treatment of small-cell lung cancer,rectal cancer,colon cancer,and melanoma,among other diseases.However,in the clinical application of PD-1/PD-L1 monoclonal antibody drugs,it is found that they can cause different degrees of immune-related adverse reactions in the lung,liver,kidney,gastrointestinal system,endocrine system and skin,and even the emergence of hyperprogressive disease.Effective monitoring and management of clinical applications of PD-1/PD-L1 monoclonal antibody drugs and rational use of some drugs can improve the immunotherapy process and reduce the effects of adverse reactions and hyperprogressive diseases in patients under immunotherapy.
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@#Objective To investigate the effect of programmed death ligand-1(PD-L1)overexpression on epithelial mesenchymal transformation(EMT)in endometrial cancer cells and its possible mechanism.Methods Ishikawa/PD-L1,a PD-L1 stable overexpression cell line constructed in the laboratory and the control Ishikawa/EV were used.The efficiency of PD-L1 overexpression was detected by qPCR and Western blot assay.The cell migration ability and invasion activity were detected by scratch assay and Transwell assay.The EMT-related protein and the phosphorylation level of nuclear transcription factor-κB(NF-κB)were detected by Western blot.Results Compared with the control group,the migration ability and invasion activity of Ishikawa/PD-L1 were significantly enhanced,and the expression of E-cadherin was significantly down-regulated,whereas vimentin,N-cadherin and p-p65 were significantly increased.Conclusion PD-L1 can promote EMT by inducing the activation of NF-κB pathway,and thus enhance the migration and invasion ability of endometrial cancer cells.
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Abstract Introduction This study aims to explore Programmed Death Receptor-1 (PD-1) and Programmed Death Ligand-1 (PD-L1) variations in Lung Cancer (LC) tissues and Peripheral Blood (PPB) and their association with immunotherapy efficacy and prognosis. Method 72 patients with LC were included in the LC group and 39 patients with concurrent benign lung disease were included in the benign group. PD-1/PDL-1 was compared in PPB and lung tissue. All LC patients were treated with immunotherapy. The relationship between PD-1/PDL-1 in LC tissue and PPB and immunotherapy efficacy was analyzed. Patients were divided into death and survival groups, and PD-1/PDL-1 in tumor tissues and PPB were compared. Results The authors found that PD-1 and PDL-1 positive expression in lung tissue and PPB in LC patients was elevated. Combined detection of PD-1 and PDL-1 was effective in diagnosing LC and evaluating the prognosis of LC patients. PD-1 and PDL-1 positive expression was reduced after disease remission while elevated in dead patients. The 3-year survival rate of patients with PD-1 positive expression was 45.45 % (25/55), which was lower (82.35 %, 14/17) than those with PD-1 negative expression. The 3-year survival rate of patients with positive and negative expression of PDL-1 was 48.78 % (20/41) and 61.29 % (19/31), respectively. Discussion The present results demonstrated that PD-1 and PDL-1 are abnormal in cancer tissue and PPB of LC patients. The combined detection of PD-1 and PDL-1 has diagnostic value for LC and evaluation value for the efficacy and prognosis of immunotherapy.
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Abstract Objectives The aim of the study was to investigate clinical significance of soluble PD-L1 (sPD-L1) serum level in head and neck cancer and to evaluate its role as a possible prognostic and predictive biomarker. Methods A prospective analysis of sPD-L1 levels in 60 patients diagnosed and treated due to malignant and non-malignant lesions in the region of head and neck was performed in peripheral blood by an ELISA test. Results The range of sPD-L1 in the study group was 0.16-1.63 ng/mL, mean 0.64 ± 0.32. There were no differences in the mean sPD-L1 regarding patients' age, sex, and the localization of the lesion. Statistically significant difference was revealed in the average sPD-L1 level (p = 0.006) depending on the histopathological advancement of the lesions, 0.704 ± 0.349 and 0.512 ± 0.177 respectively in the malignant and benign group. The separate analysis of laryngeal lesions confirmed statistical difference in sPD-L1 (p = 0.002) for the malignant lesions (0.741 ± 0.353) compared with the benign (0.489 ± 0.175). The sPD-L1 level of 0.765 ng/mL or higher, revealed 35% sensitivity and 95.5% specificity for the diagnosis of head and neck malignant lesions (AUC = 0.664, 95% CI 0.529‒0.8, p-value = 0.039). The 1-year DFS was 83.3% in the group of patients with low sPD-L1 levels (< 0.765 ng/mL) and 53.8% in patients with high sPD-L1 (≥0.765 ng/mL). The 2-year OS were 68% and 69.2% respectively in both groups. The log-rank test confirmed statistically significant prognostic value of sPD-L1 level for 1-year DFS (p-value = 0.035). Conclusions sPD-L1 is a promising prognostic and early recurrence predictive biomarker for head and neck cancers, most significantly for laryngeal lesions. Level of evidence 3.
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OBJECTIVE@#To explore the effect of microRNA-424-5p (miR-424-5p) on the drug resistance of diffuse large B-cell lymphoma (DLBCL) cells by regulating the programmed death receptor-1 (PD-1)/programmed death ligand-1 (PD-L1) signaling pathway.@*METHODS@#Human DLBCL cell line CRL2631 cells were induced to construct CRL2631-CHOP resistant cell line. RT-qPCR and Western blot were used to detect the expression levels of MiR-424-5p, PD-L1 mRNA and protein, and multidrug resistance gene-1 (MDR-1) protein in CRL2631 cells and CRL2631-CHOP cells, respectively. The target genes of MiR-424-5p was verified by dual luciferase reporter assay. The miRNA simulation/interference technology and thiazole blue (MTT) method were used to detect the resistance of CRL2631 cells and CRL2631-CHOP cells to CHOP.@*RESULTS@#Compared with CRL2631 cells, the drug resistance of CRL2631-CHOP cells to CHOP and the levels of MDR-1 protein (P<0.05), PD-L1 mRNA and protein in the cells were significantly increased (both P<0.001), while the relative level of MiR-424-5p was significantly reduced (P<0.001). The result of the dual luciferase reporter assay showed that PD-L1 was the direct downstream target gene of MiR-424-5p (P<0.001). After transfection of MiR-424-5p inhibitor, the resistance of CRL2631 cells to CHOP drugs increased, and the expression level of MDR-1 protein (P<0.01), PD-L1 mRNA and protein also increased significantly (both P<0.01). After transfection of MiR-424-5p mimics, the resistance of CRL2631-CHOP cells to CHOP drugs decreased, and the expression level of MDR-1 protein (P<0.001), PD-L1 mRNA and protein also decreased significantly (both P<0.001). Overexpression of PD-L1 could reverse the inhibitory effect of upregulating MiR-424-5p on PD-L1 (P<0.001).@*CONCLUSION@#Down-regulation of MiR-424-5p enhances the drug resistance of DLBCL cells by regulating the PD-1/PD-L1 signaling pathway.
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Humanos , Antígeno B7-H1/metabolismo , Línea Celular Tumoral , Resistencia a Medicamentos , Luciferasas , Linfoma de Células B Grandes Difuso/patología , MicroARNs/metabolismo , Receptor de Muerte Celular Programada 1 , ARN Mensajero , Transducción de SeñalRESUMEN
@#BACKGROUND: This study aimed to explore the changes of programmed death-ligand 1 (PD-L1) and programmed death-1 (PD-1) expression on antigen-presenting cells (APCs) and evaluate their association with organ failure and mortality during early sepsis. METHODS: In total, 40 healthy controls and 198 patients with sepsis were included in this study. Peripheral blood was collected within the first 24 h after the diagnosis of sepsis. The expression of PD-L1 and PD-1 was determined on APCs, such as B cells, monocytes, and dendritic cells (DCs), by flow cytometry. Cytokines in plasma, such as interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), interleukin-4 (IL-4), IL-6, IL-10, and IL-17A were determined by Luminex assay. RESULTS: PD-1 expression decreased significantly on B cells, monocytes, myeloid DCs (mDCs), and plasmacytoid DCs (pDCs) as the severity of sepsis increased. PD-1 expression was also markedly decreased in non-survivors compared with survivors. In contrast, PD-L1 expression was markedly higher on mDCs, pDCs, and monocytes in patients with sepsis than in healthy controls and in non-survivors than in survivors. The PD-L1 expression on APCs (monocytes and DCs) was weakly related to organ dysfunction and inflammation. The area under the receiver operating characteristic curve (AUC) of the PD-1 percentage of monocytes (monocyte PD-1%)+APACHE II model (0.823) and monocyte PD-1%+SOFA model (0.816) had higher prognostic value than other parameters alone. Monocyte PD-1% was an independent risk factor for 28-day mortality. CONCLUSION: The severity of sepsis was correlated with PD-L1 or PD-1 over-expression on APCs. PD-L1 in monocytes and DCs was weakly correlated with inflammation and organ dysfunction during early sepsis. The combination of SOFA or APACHE II scores with monocyte PD-1% could improve the prediction ability for mortality.
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Objective:To investigate the expression of programmed death 1 (PD-1)/programmed death ligand 1 (PD-L1) pathway in patients with acute myeloid leukemia (AML) and its relationship with clinical features and prognosis, and to examine its effect on PD-1-positive natural killer (NK) cells against AML cells in vitro.Methods:The bone marrow samples of 65 AML patients and the peripheral blood of 32 AML patients diagnosed in Affiliated Cancer Hospital of Zhengzhou University from July 2019 to December 2020 were prospectively collected, and the peripheral blood of 24 healthy people was taken as healthy control. The expression level of PD-L1 in bone marrow tumor cells and expression level of PD-1 in peripheral blood NK cells were detected by flow cytometry. The correlations of PD-1 expression in bone marrow tumor cells and PD-1 expression in NK cells with the clinicopathological features, curative effect and prognosis of patients were analyzed. Flow cytometry was used to detect the expression level of PD-L1 in AML cell line THP-1 (target cells) and the expression level of PD-L1 in NK cell line NKL (effector cells). THP-1 cells treated with and without 25 μmol/L of PD-L1 inhibitor fraxinellone were used as experimental group and control group, and co-cultured with NKL cells at different effector-to-target ratios. The apoptosis of THP-1 cells and the expression of NKG2D in NKL cells were detected by flow cytometry, the cell proliferation status was detected by CCK-8 and the cell proliferation inhibition rate was calculated; the levels of interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α) in the supernatant of co-culture system were detected by enzyme-linked immunosorbent assay (ELISA).Results:The proportion of AML patients with PD-L1-positive expression in bone marrow tumor cells was higher than that in the healthy control group [38.5% (25/65) vs. 8.3% (2/24), P = 0.029]. The proportion of AML patients with PD-1-positive expression in peripheral blood NK cells was higher than that in the healthy control group [40.6% (13/32) vs. 12.5% (3/24), P = 0.035]. There were no statistical differences in sex, age, hemogram, proportion of primordial cells, risk stratification, chromosomal karyotype, gene mutation (except NPM1 gene), fusion gene and French-American-British cooperative group (FAB) typing between patients with PD-L1 positive and negative in bone marrow tumor cells and between patients with PD-1 positive and negative in peripheral blood NK cells (all P > 0.05). In relapsed/refractory patients, the proportion of patients with PD-L1-positive expression in bone marrow tumor cells was higher than that in newly treated patients [58.8% (10/17) vs. 31.2% (15/48), P = 0.045]. There was no significant difference in the proportion of patients with PD-1-positive expression in peripheral blood NK cells between relapsed/refractory patients and newly treated patients [(38.5% (5/13) vs. 42.1% (8/19), P = 0.837]. There was no statistical difference in complete remission (CR) rate between PD-L1 positive and negative patients [69.6% (16/23) vs. 74.3% (26/35), P > 0.05]. There was no statistical difference in CR rate between PD-1 positive and negative patients [66.7% (8/12) vs. 70.6% (12/17), P > 0.05]. There was no statistical difference in recurrence rate after CR between PD-L1 positive and negative patients [12.5% (2/16) vs. 19.2% (5/26), P > 0.05]. There was no statistical difference in recurrence rate after CR between PD-1 positive and negative patients [25.0% (2/8) vs. 16.7% (2/12), P > 0.05]. Flow cytometry showed that the positive rate of PD-1 in NKL cells was (67±6)% and the positive rate of PD-L1 in THP-1 cells was (85±5)%. After co-culture with NKL cells, the apoptotic rate and proliferation inhibition rate of THP-1 cells were higher in the experimental group compared with the control group, the expression of NKG2D on the surface of NKL cells was elevated, and the levels of IFN-γ and TNF-α in the co-culture supernatant were increased. Conclusions:In AML patients, the expression of PD-L1 in bone marrow tumor cells is high, and the expression of PD-1 in peripheral blood NK cells is also high. The expression of PD-L1 in bone marrow tumor cells of relapsed/refractory AML patients is higher than that of newly treated patients. Inhibition of PD-L1 expression in THP-1 cells can enhance the tumor killing activity of NKL cells in vitro. The mechanism may be that inhibition of PD-L1 expression in THP-1 cells up-regulates the expression of NKL cell activated receptor NKG2D and promotes the secretion of IFN- γ and TNF- α.
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Sepsis is currently the leading cause of death in the intensive care unit, and its survivors also experience long-term immunosuppression and high rates of recurrent infections. At present, the clinical treatment of sepsis is still based on antibiotics, intravenous rehydration, and vasopressors, and there is no targeted drug treatment. However, as the rate of antibiotic resistance continues to increase, immunotherapy is highly anticipated as a new treatment. Patients with sepsis are often accompanied by acute leukocyte immune dysfunction and immunosuppression, which may be an important risk factor for the increasing morbidity and mortality of patients. Targeted inhibition of specific cell surface inhibitory immune checkpoint receptors and ligands, such as programmed death receptor-1 (PD-1), programmed death-ligand 1 (PD-L1), and other targets, can improve the host’s resistance to infection. In this paper, the research progress of PD-1 and PD-L1 in the immune response to sepsis was summarized to provide a theoretical basis for their further application in the treatment of sepsis in the future.
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Objective:To investigate the regulatory effects of ring finger protein 43 (RNF43) on CD8 + T cell-mediated anti-tumor immune reaction in melanoma. Methods:RNF43 gene was over-expressed and knockdown in mouse melanoma cells line B16-OVA by lentivirus infection; In vivo proliferation of mouse melanoma cells line B16-OVA in the Lv-Ctrl-OE, Lv-RNF43-OE, Lv-Ctrl-KD and Lv-RNF43-KD groups was detected by subcutaneous tumorigenesis assay in mice, and the expression levels of CD8 + T cells perforin and interferon γ (IFN-γ) in tumor immune microenvironment of melanoma were detected by flow cytometry; The expression levels of β-catenin and programmed death-ligand 1 (PD-L1) mRNA in cells were detected by quantitative real-time PCR assay; The effect of RNF43 on the transcriptional regulation of PD-L1 was detected by dual-luciferase reporter gene assay. Results:Stable RNF43 over-expressing and RNF43 knockdown mouse melanoma cells lines Lv-RNF43-OE and Lv-RNF43-KD were successfully constructed. The results of subcutaneous tumorigenesis experiment in mice showed that the tumor mass of the Lv-RNF43-OE group was (0.08±0.06) g, which was significantly smaller than that of the Lv-Ctrl-OE group [ (1.04±0.52) g], with a statistically significant difference ( t=3.71, P=0.032) ; The tumor mass of Lv-RNF43-KD group was (1.94±0.29) g, with no statistically significant difference ( t=-1.70, P=0.164) compared with that of the Lv-Ctrl-KD group (1.15±0.74) g. The flow cytometry results showed that the fluorescence intensity of CD8 + T cell perforin in the Lv-RNF43-OE group was 9 034 ± 2 628, which was significantly higher than that in the Lv-Ctrl-OE group (3 847 ±1 637), with a statistically significant difference ( t=-3.35, P=0.015) ; The fluorescence intensity of CD8 + T cell perforin in the Lv-RNF43-KD group was 966±247, which was significantly lower than that in the Lv-Ctrl-KD group (2 226±646), with a statistically significant difference ( t=3.16, P=0.034) ; The fluorescence intensity of IFN-γ of CD8 + T cell in the Lv-RNF43-OE group was 2 422±429, which was significantly higher than that of 1 688±324 in the Lv-Ctrl-OE group, with a statistically significant difference ( t=-2.73, P=0.034) ; The fluorescence intensity of IFN-γ of CD8 + T cell in the Lv-RNF43-KD group was 614 (454, 863), with a statistically significant difference ( Z=-1.96, P=0.050) compared with 1 159 (1 152, 2 068) in the Lv-Ctrl-KD group. The results of quantitative real-time PCR showed that the relative expression level of β-catenin mRNA in the Lv-RNF43-OE group was 0.67±0.16, which was significantly lower than that of 1.00±0.11 in the Lv-Ctrl-OE group, with a statistically significant difference ( t=2.98, P=0.041) ; The relative expression level of PD-L1 mRNA in the Lv-RNF43-OE group was 0.32±0.09, which was significantly lower than that of 1.00±0.09 in the Lv-Ctrl-OE group, with a statistically significant difference ( t=9.13, P=0.001). The results of the dual-luciferase reporter gene assay showed that the PD-L1 promoter luciferase activity in the pCMV6-NC, RNF43, RNF43+β-catenin and β-catenin groups were 1.00±0.00, 0.84±0.00, 1.49±0.00 and 1.57±0.03 ( F=2 218.33, P<0.001). Further pairwise comparison showed that compared with the pCMV6-NC group, PD-L1 promoter luciferase activity was significantly lower in the RNF43 group ( P<0.001) and significantly higher in the RNF43+β-catenin and β-catenin groups ( P<0.001; P=0.003) ; compared with the RNF43 group, PD-L1 promoter luciferase activity was significantly higher in the RNF43+β-catenin group ( P<0.001) . Conclusion:RNF43 may reduce the expression of PD-L1 mRNA in melanoma by inhibiting the expression of β-catenin and promote CD8 + T cell-mediated anti-tumor immune reaction.
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Biliary tract cancer is a highly lethal disease composed of diverse epithelial tumors, of which incidence is increasing. Nearly two-thirds of patients with biliary tract cancer are in local advanced stage or metastasized at diagnosis. Systemic therapies are the primary treat-ment options, but the prognosis is poor. In recent years, the emergence of immune checkpoint inhibitors has changed the treatment prospects for advanced solid tumors, and multiple clinical trials and related studies have been conducted worldwide. Based on clinical experiences and pertinent researches in the field, the authors expound upon the research progress in immune checkpoint inhibitors treatment of advanced biliary tract cancer.
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Objective:To investigate the expression and significance of programmed death ligand 1 (PD-L1) and programmed death 1 (PD-1) in colorectal cancer complicated by schistosomiasis.Methods:A total of 134 patients with colorectal cancer who received treatment in Xuancheng People's Hospital during 2014-2021 were included in this study. These patients consisted of 74 patients with colorectal cancer combined with schistosomiasis (patient group) and 60 patients with only colorectal cancer (control group). The expression of PD-L1 and PD-1 in colorectal cancer tissue was detected by an immunohistochemical method. The differences in PD-L1 and PD-1 expression were compared between the two groups. The relationships between PD-L1 and PD-1 expression and clinical pathological characteristics were determined.Results:The positive expression rates of PD-L1 and PD-1 in cancer cells and interstitial lymphocytes were 55.4% and 60.8% respectively in the patient group and they were 35.0% and 40.0% respectively in the control group. The positive expression rates of PD-L1 and PD-1 were significantly higher in the patient group than the control group ( χ2 = 5.55, 5.74, both P < 0.05). The expressions of PD-L1 and PD-1 in the patient group were correlated with lymph node metastasis and high tumor-node-metastasis stage ( P < 0.05). Conclusion:PD-L1 and PD-1 are highly expressed in colorectal cancer complicated by schistosomiasis and are related to their invasive behavior. PD-1/PD-L1 singaling pathway may be involved in the molecular mechanism underlying the occurrence and development of colorectal cancer complicated by schistosomiasis. Blocking PD-1/PD-L1 signaling pathway may be a new strategy for immunotherapy of colorectal cancer complicated by schistosomiasis.
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Objective:To investigate the distribution of M2 tumor-associated macrophage (TAM) in hepatocellular carcinoma (HCC) and their correlation with clinicopathological features, and the significance of programmed death-ligand 1 (PD-L1) expression.Methods:From January 1, 2012 to December 31, 2020, a total of 320 HCC patients who underwent surgical resection at the Third People′s Hospital of Nantong were included. The distribution of CD163 labeled and PD-L1 CD163 double-labeled M2 TAM in HCC tissues was detected by immunohistochemistry, and the cell density was calculated. The cell density> the average cell density (112/mm 2) was judged as high-density, the cell density≤ the average cell density was judged as low-density. The correlation between CD163 positive and PD-L1 CD163 double positive M2 TAM density and the clinical pathological characteristics of HCC and its impact on prognosis were analyzed. Chi-square test was used to analyze the correlation between M2 TAM expression and the clinical pathological characteristics of HCC. Kaplan-Meier method was used to draw survival curves, and log-rank test was used for inter group comparison. Univariate and multivariate Cox proportional hazards regression analysis was used to indentify the relevant factors affecting the prognosis of HCC. Results:TAM were mainly distributed in the tumor edge stroma and tumor sinusoids, CD163 positive M2 TAM were the main macrophage subtype. PD-L1 expression was observed in CD163 positive M2 TAM in HCC tissues, and PD-L1 positive M2 TAM were mainly distributed in the tumor edge stroma. The rate of high-density CD163 positive M2 TAM in HCC tissues was 44.4% (142/320). High-density CD163 positive M2 TAM was correlated with histological grade, TNM stage, and PD-L1 expression on tumor infiltrating immune cells in HCC tissues ( χ2=4.65, 6.72 and 42.19, P=0.031, =0.011 and <0.001). High-density PD-L1 and CD163 double positive M2 TAM in HCC tissues was correlated with microvascular invasion and TNM stage ( χ2=11.96 and 8.74, P=0.001 and 0.004). The median disease-free survival (DFS) time and overall survival (OS) time of patients with high-density CD163 positive M2 TAM were 21 and 36 months, respectively, which were lower than those of patients with low-density CD163 positive M2 TAM (50 and 103 months, respectively); the median DFS time and OS time of patients with high-density PD-L1 CD163 double-positive M2 TAM were 12 and 15 months, respectively, which were lower than those of patients with low-density PD-L1 CD163 double-positive M2 TAM (28 and 45 months, respectively), and the differences were statistically significant (all log-rank tests, all P<0.001). The results of multivariate Cox proportional hazards regression analysis showed that high-density CD163 positive M2 TAM, microvascular invasion and high TNM stage were independent risk factors for evaluating DFS and OS of patients with HCC (DFS time: HR=2.408 (95% confidence interval (95% CI) 1.778 to 3.261), 2.603 (95% CI 1.860 to 3.641), 4.032 (95% CI 2.833 to 5.747), all P<0.001. OS time: HR=2.007 (95% CI 1.457 to 2.764), 4.144 (95% CI 2.881 to 5.960), 4.292 (95% CI 2.915 to 6.329), all P<0.001). Conclusions:High-density of CD163 positive M2 TAM in HCC tissues indicates high malignancy and poor prognosis, and it is an independent prognostic risk factor. The expression of PD-L1 in M2 TAM suggests stronger tumor aggressiveness and worse prognosis in HCC.
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@#Abstract: Objective The method of "molecular docking - molecular dynamics - in vitro pharmacology" was used to screen and optimize small molecule polypeptide compounds that can block PD-1/PD-L1 binding and have high anti-tumor activity. Methods Using the 5-peptide compound database constructed by our team and the three-dimensional structure data of the PD-L1 protein downloaded from the protein database (PDB), the peptide compounds obtained by flexible molecular docking using Molecular Operating Environment software. The molecular dynamics calculation and analysis of the compounds with the highest GWVI/WSA (generalized Born volume integral/weighted surface area) free energy change value (ΔG) were carried out, including the root mean square deviation (RMSD) of the position change of the weight atom and the interaction energy (the sum of Lennard-Jones potential and Coulombic energy). The blocking effect of ligand compounds on PD-1/PD-L1 binding was analyzed by the homogeneous time-resolved fluorescence (HTRF) technique. The co-culture system of Jurkat T lymphocytes and melanoma B16-F10 cells was established to explore the effect of compound ligands on the killing effect of T cells on tumors and the effect on the secretion level of IL-2 in the co-culture supernatant. Results The selected polypeptide compounds were analyzed by molecular dynamics, and the binding of RGGHA to RGGHH and PD-L1 was stable. There are many kinds of interactions between RGGHA and PD-L1，which can compete with PD-1 ligands to bind Asp122, Try123 and Lys124 sites of the PD-L1 protein and block PD-1/PD-L1 signal transduction. HTRF experiments showed that the binding inhibition rate of RGGHA to PD-1 and PD-L1 was 58.38%, and that of RGGHH was 42.73%. In addition, we established a co-culture system of Jurkat T lymphocytes and melanoma B16-F10 cells to explore the immunostimulatory effect and mechanism of peptides. The results show that RGGHA and RGGHH can significantly increase the secretion level of IL-2, improve the killing ability of T cells against tumor cells and activate the tumor immune microenvironment. Conclusions The study found that the polypeptide compounds RGGHA and RGGHH can effectively block the PD-1/PD-L1 interaction and reactivate the anti-cancer immune response, which can be used as lead compounds for new drug development.
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Objective: To investigate the expression of programmed death protein-ligand 1 (PD-L1) in liver cancer stem-like cells (LCSLC) and its effect on the characteristics of tumor stem cells and tumor biological function, to explore the upstream signaling pathway regulating PD-L1 expression in LCSLC and the downstream molecular mechanism of PD-L1 regulating stem cell characteristics, also tumor biological functions. Methods: HepG2 was cultured by sphere-formating method to obtain LCSLC. The expressions of CD133 and other stemness markers were detected by flow cytometry, western blot and real-time quantitative polymerase chain reaction (RT-qPCR) were used to detect the expressions of stemness markers and PD-L1. The biological functions of the LCSLC were tested by cell function assays, to confirm that the LCSLC has the characteristics of tumor stem cells. LCSLC was treated with cell signaling pathway inhibitors to identify relevant upstream signaling pathways mediating PD-L1 expression changes. The expression of PD-L1 in LCSLC was down regulated by small interfering RNA (siRNA), the expression of stem cell markers, tumor biological functions of LCSLC, and the changes of cell signaling pathways were detected. Results: Compared with HepG2 cells, the expression rate of CD133 in LCSLC was upregulated [(92.78±6.91)% and (1.40±1.77)%, P<0.001], the expressions of CD133, Nanog, Oct4A and Snail in LCSLC were also higher than those in HepG2 cells (P<0.05), the number of sphere-formating cells increased on day 7 [(395.30±54.05) and (124.70±19.30), P=0.001], cell migration rate increased [(35.41±6.78)% and (10.89±4.34)%, P=0.006], the number of transmembrane cells increased [(75.77±10.85) and (20.00±7.94), P=0.002], the number of cloned cells increased [(120.00±29.51) and (62.67±16.77), P=0.043]. Cell cycle experiments showed that LCSLC had significantly more cells in the G(0)/G(1) phase than those in HepG2 [(54.89±3.27) and (32.36±1.50), P<0.001]. The tumor formation experiment of mice showed that the weight of transplanted tumor in LCSLC group was (1.32±0.17)g, the volume is (1 779.0±200.2) mm(3), were higher than those of HepG2 cell [(0.31±0.06)g and (645.6±154.9)mm(3), P<0.001]. The expression level of PD-L1 protein in LCSLC was 1.88±0.52 and mRNA expression level was 2.53±0.62, both of which were higher than those of HepG2 cells (P<0.05). The expression levels of phosphorylation signal transduction and transcription activation factor 3 (p-STAT3) and p-Akt in LCSLC were higher than those in HepG2 cells (P<0.05). After the expression of p-STAT3 and p-Akt was down-regulated by inhibitor treatment, the expression of PD-L1 was also down-regulated (P<0.05). In contrast, the expression level of phosphorylated extracellular signal-regulated protein kinase 1/2 (p-ERK1/2) in LCSLC was lower than that in HepG2 cells (P<0.01), there was no significant change in PD-L1 expression after down-regulated by inhibitor treatment (P>0.05). After the expression of PD-L1 was knockdown by siRNA, the expressions of CD133, Nanog, Oct4A and Snail in LCSLC were decreased compared with those of siRNA-negative control (NC) group (P<0.05). The number of sphere-formating cells decreased [(45.33±12.01) and (282.00±29.21), P<0.001], the cell migration rate was lower than that in siRNA-NC group [(20.86±2.74)% and (46.73±15.43)%, P=0.046], the number of transmembrane cells decreased [(39.67±1.53) and (102.70±11.59), P=0.001], the number of cloned cells decreased [(57.67±14.57) and (120.70±15.04), P=0.007], the number of cells in G(0)/G(1) phase decreased [(37.68±2.51) and (57.27±0.92), P<0.001], the number of cells in S phase was more than that in siRNA-NC group [(30.78±0.52) and (15.52±0.83), P<0.001]. Tumor formation in mice showed that the tumor weight of shRNA-PD-L1 group was (0.47±0.12)g, the volume is (761.3±221.4)mm(3), were lower than those of shRNA-NC group [(1.57±0.45)g and (1 829.0±218.3)mm(3), P<0.001]. Meanwhile, the expression levels of p-STAT3 and p-Akt in siRNA-PD-L1 group were decreased (P<0.05), while the expression levels of p-ERK1/2 and β-catenin did not change significantly (P>0.05). Conclusion: Elevated PD-L1 expression in CD133(+) LCSLC is crucial to maintain stemness and promotes the tumor biological function of LCSLC.
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Humanos , Animales , Ratones , Proteínas Proto-Oncogénicas c-akt/metabolismo , Antígeno B7-H1/metabolismo , Ligandos , Neoplasias Hepáticas/patología , ARN Interferente Pequeño/metabolismo , Células Madre Neoplásicas/fisiología , Línea Celular Tumoral , Proliferación CelularRESUMEN
Objective: To evaluate the safety and antitumor activity of envafolimab monotherapy in Chinese patients with advanced solid tumors. Methods: This open-label, multicenter phase I trial included dose escalation and dose expansion phases. In the dose escalation phase, patients received subcutaneous 0.1, 0.3, 1.0, 2.5, 5.0 or 10.0 mg/kg envafolimab once weekly (QW) following a modified "3+ 3" design. The dose expansion phase was performed in the 2.5 mg/kg and 5.0 mg/kg (QW) dose cohorts. Results: At November 25, 2019, a total of 287 patients received envafolimab treatment. During the dose escalation phase, no dose-limiting toxicities (DLT) was observed. In all dose cohorts, drug-related treatment-emergent adverse events (TEAEs) for all grades occurred in 75.3% of patients, and grade 3 or 4 occurred in 20.6% of patients. The incidence of immune-related adverse reactions (irAE) was 24.0% for all grades, the most common irAEs (≥2%) included hypothyroidism, hyperthyroidism, immune-associated hepatitis and rash. The incidence of injection site reactions was low (3.8%), all of which were grades 1-2. Among the 216 efficacy evaluable patients, the objective response rate (ORR) and disease control rate (DCR) were 11.6% and 43.1%, respectively. Median duration of response was 49.1 weeks (95% CI: 24.0, 49.3). Pharmacokinetic (PK) exposure to envafolimab is proportional to dose and median time to maximum plasma concentration is 72-120 hours based on the PK results from the dose escalation phase of the study. Conclusion: Subcutaneous envafolimab has a favorable safety and promising preliminary anti-tumor activity in Chinese patients with advanced solid tumors.
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Humanos , Pueblos del Este de Asia , Neoplasias/patología , Anticuerpos Monoclonales Humanizados/uso terapéuticoRESUMEN
Objective: To evaluate the safety and antitumor activity of envafolimab monotherapy in Chinese patients with advanced solid tumors. Methods: This open-label, multicenter phase I trial included dose escalation and dose expansion phases. In the dose escalation phase, patients received subcutaneous 0.1, 0.3, 1.0, 2.5, 5.0 or 10.0 mg/kg envafolimab once weekly (QW) following a modified "3+ 3" design. The dose expansion phase was performed in the 2.5 mg/kg and 5.0 mg/kg (QW) dose cohorts. Results: At November 25, 2019, a total of 287 patients received envafolimab treatment. During the dose escalation phase, no dose-limiting toxicities (DLT) was observed. In all dose cohorts, drug-related treatment-emergent adverse events (TEAEs) for all grades occurred in 75.3% of patients, and grade 3 or 4 occurred in 20.6% of patients. The incidence of immune-related adverse reactions (irAE) was 24.0% for all grades, the most common irAEs (≥2%) included hypothyroidism, hyperthyroidism, immune-associated hepatitis and rash. The incidence of injection site reactions was low (3.8%), all of which were grades 1-2. Among the 216 efficacy evaluable patients, the objective response rate (ORR) and disease control rate (DCR) were 11.6% and 43.1%, respectively. Median duration of response was 49.1 weeks (95% CI: 24.0, 49.3). Pharmacokinetic (PK) exposure to envafolimab is proportional to dose and median time to maximum plasma concentration is 72-120 hours based on the PK results from the dose escalation phase of the study. Conclusion: Subcutaneous envafolimab has a favorable safety and promising preliminary anti-tumor activity in Chinese patients with advanced solid tumors.