Colorectal cancer(CRC)is the third most common cancer and the second leading cause of cancer-related deaths worldwide.Dendritic cells(DCs)constitute a heterogeneous group of antigen-presenting cells that are important...Colorectal cancer(CRC)is the third most common cancer and the second leading cause of cancer-related deaths worldwide.Dendritic cells(DCs)constitute a heterogeneous group of antigen-presenting cells that are important for initiating and regulating both innate and adaptive immune responses.As a crucial component of the immune system,DCs have a pivotal role in the pathogenesis and clinical treatment of CRC.DCs cross-present tumor-related antigens to activate T cells and trigger an antitumor immune response.However,the antitumor immune function of DCs is impaired and immune tolerance is promoted due to the presence of the tumor microenvironment.This review systematically elucidates the specific characteristics and functions of different DC subsets,as well as the role that DCs play in the immune response and tolerance within the CRC microenvironment.Moreover,how DCs contribute to the progression of CRC and potential therapies to enhance antitumor immunity on the basis of existing data are also discussed,which will provide new perspectives and approaches for immunotherapy in patients with CRC.展开更多
MicroRNAs(miRNAs)have received much attention in the past decade as potential key epigenomic regulators of tumors and cancer stem cells(CSCs).The abnormal expression of miRNAs is responsible for different phenotypes o...MicroRNAs(miRNAs)have received much attention in the past decade as potential key epigenomic regulators of tumors and cancer stem cells(CSCs).The abnormal expression of miRNAs is responsible for different phenotypes of gastric cancer stem cells(GCSCs).Some specific miRNAs could be used as promising biomarkers and therapeutic targets for the identification of GCSCs.This review summarizes the coding process and biological functions of miRNAs and demon-strates their role and efficacy in gastric cancer(GC)metastasis,drug resistance,and apoptosis,especially in the regulatory mechanism of GCSCs.It shows that the overexpression of onco-miRNAs and silencing of tumor-suppressor miRNAs can play a role in promoting or inhibiting tumor metastasis,apart from the initial formation of GC.It also discusses the epigenetic regulation and potential clinical applications of miRNAs as well as the role of CSCs in the pathogenesis of GC.We believe that this review may help in designing novel therapeutic approaches for GC.展开更多
Objective:To explore the effect and mechanism of prostaglandins D2(PGD2)on the stemness of gastric cancer stem cells(GCSCs).Methods:7901-GCSCs were enriched by serum-free culture method;then the positivity rate of CD4...Objective:To explore the effect and mechanism of prostaglandins D2(PGD2)on the stemness of gastric cancer stem cells(GCSCs).Methods:7901-GCSCs were enriched by serum-free culture method;then the positivity rate of CD44,a stemness marker,was detected by flow cytometry in serum-free cultured 7901-GCSCs;the sphere-forming ability was detected by the sphere-forming assay after stimulation with different concentrations of PGD2(2.5,5,10)μg/mL,and the expression of stemness-related indicators(OCT4,CD44)and autophagyrelated proteins(LC3,Beclin-1)after PGD2 stimulation was detected by the western blot assay in different concentrations.The expression of stemness-related indexes(OCT4,CD44)and autophagy-related proteins(LC3,Beclin-1)were detected by Western blot assay after stimulation with different concentrations of PGD2.The expression of autophagy-related proteins after stimulation with different concentrations of CQ(2.5,5,10)μM was detected by Western blot experiment.The protein expression of autophagy-related proteins(LC3,Beclin-1)and stemness-related indexes(OCT4,CD44)was detected by Western blot experiment after PGD2 as well as PGD2+CQ treatment.Results:Flow cytometry results showed that the expression of CD44 positivity was increased in serum-free cultured 7901-GCSCs compared with gastric cancer cells SGC-7901(P<0.05),which fulfilled the needs of subsequent experiments.The results of stem cell spheroid formation assay showed that the spheroid formation ability of 7901-GCSCs in the PGD2 group was significantly weakened compared with that of the DMSO group(P<0.05).Western blot results showed that the protein expression of stemness-related indexes(OCT4,CD44)was down-regulated in the 7901-GCSCs in the PGD2 group compared with that of the DMSO group(P<0.05),and the expression of autophagy-related proteins(LC3,Beclin-1)expression increased(P<0.05).Compared with the DMSO group,the expression of autophagy-related proteins(LC3,Beclin-1)was decreased in the CQ group(P<0.05).Western blot results also showed that the expression of cellular autophagy-related proteins and stemness-related indexes in the PGD2+CQ group was not significantly changed compared with that of the DMSO group(ns:the difference was not significant),suggesting that the CQ could block the effect of PGD2 on the expression of stemness markers in 7901-GCSCs.7901-GCSCs stemness inhibition.Conclusion:PGD2 may affect the stemness of 7901-GCSCs by regulating autophagy.展开更多
BACKGROUND Gastric cancer is one of the most common malignant tumors worldwide,and surgical resection is one of the main ways to treat gastric cancer.However,the immune status of postoperative patients is crucial for ...BACKGROUND Gastric cancer is one of the most common malignant tumors worldwide,and surgical resection is one of the main ways to treat gastric cancer.However,the immune status of postoperative patients is crucial for prognosis and survival,and immune cells play an important role in this process.Therefore,it is helpful to understand the immune status of postoperative patients by evaluating the levels of peripheral blood immune cells,especially total T cells(CD3+),helper T cells(CD3+CD4+),and suppressor T cells(CD3+CD8+),and its relationship to sur-vival.AIM To analyzed the immune cells in peripheral blood of patients with gastric cancer after surgery,detect the levels of total T cells,helper T cells and suppressor T cells.METHODS A total of 58 patients with gastric cancer who received surgical treatment were included in the retrospective study.Flow cytometry was used to detect the level of peripheral blood immune cells and analyze the correlation between total T cells,helper T cells and inhibitory T cells.To explore the relationship between these immune markers and patient survival.RESULTS The results showed that the levels of total T cells,helper T cells,and suppressor T cells changed in patients after gastric cancer surgery.There was a significant positive correlation between total T cells,helper T cells and suppressor T cells(r=0.35,P<0.01;r=0.56,P<0.01).However,there was a negative correlation between helper T cells and suppressor T cells(r=-0.63,P<0.01).Follow-up showed that the survival rate of patients in the high-level total T cell group was significantly higher than that in the low-level group(28.87±24.98 months vs 18.42±16.21 months).The survival curve shows that the curve of patients in the high-level group is shifted to the upper right,and that of the low-level group is shifted downward.There was no significant difference between the levels of helper T cells and suppressor T cells and patient survival time.CONCLUSION By detecting peripheral blood immune cells with flow cytometry,we can initially evaluate the immune status of patients after gastric cancer surgery and initially explore its relationship with patient survival.展开更多
Objective:To construct CAR-NK-92 cells targeting carcinoembryonic antigen(CEA)and study their killing effect on gastric cancer cells.Methods:CAR-NK-92 cells targeting CEA were constructed.After co-culturing CAR-NK-92 ...Objective:To construct CAR-NK-92 cells targeting carcinoembryonic antigen(CEA)and study their killing effect on gastric cancer cells.Methods:CAR-NK-92 cells targeting CEA were constructed.After co-culturing CAR-NK-92 cells with MKN-45 gastric cancer cells,the killing effect of CAR-NK-92 cells was detected by a lactate dehydrogenase release assay.The secretion levels of gamma interferon and granulocyte-macrophage colony-stimulating factor were measured using an ELISA assay.Results:The lactate dehydrogenase release assay showed that CAR-NK-92 cells had a significant killing effect on MKN-45 cells compared to CON-NK-92 cells,and the difference was statistically significant(P<0.001).ELISA results indicated that the levels of gamma interferon and granulocyte-macrophage colony-stimulating factor secreted by CAR-NK-92 cells and MKN-45 target cells were significantly increased after co-culture(P<0.001).Conclusion:CAR-NK-92 cells targeting CEA exhibit a significant killing effect on CEA-positive gastric cancer cells.展开更多
AIM： To investigate whether bone marrow-derived denritic cells pulsed with tumor lysates induce immunity against gastric cancer ex vivo. METHODS： c-kit＋ hematopoietic progenitor cells were magnetically isolated wit...AIM： To investigate whether bone marrow-derived denritic cells pulsed with tumor lysates induce immunity against gastric cancer ex vivo. METHODS： c-kit＋ hematopoietic progenitor cells were magnetically isolated with a MiniMACS separator from BALB/c mice bone marrow cells. These cells were cultured with cytokines GM-CSF, IL-4, and TNFα to induce their maturation. They were analysed by morphological observation, phenotype analysis, and mixed lymphocyte reaction （MLR）. Bone marrowderived DCs （BM-DCs） were pulsed with tumor cell lysate obtained by rapid freezing and thawing at a 1：3 DC：tumor cell ratio. Finally, cytotoxic T lymphocyte （CTL） activity and interferon gamma （IFNγ） secretion was evaluated ex vivo. RESULTS： c-kit^＋ hematopoietic progenitor cells from mice bone marrow cells cultured with cytokines for 8 d showed the character of typical mature DCs.Morphologically, observed by light microscope, these cells were large with oval or irregularly shaped nuclei and with many small dendrites. Phenotypically, FACS analysis showed that they expressed.high levels of la, DEC-205, CD11b, CD80 and CD86 antigen, moderate levels of CD40, and negative for F4/80. Functionally, these cells gained the capacity to stimulate allogeneic T cells in MLR assay. However, immature DCs cultured with cytokines for 5 d did not have typical DCs phenotypic markers and could not stimulate allogeneic T cells. Ex vivo primed T cells with SGC-7901 tumor cell lysate-pulsed （TP） DCs were able to induce effective CTL activity against SGC-7901 tumor cells （E：T = 100：1, 69.55% ± 6.05% specific lysis）, but not B16 tumor cells, and produced higher levels of IFNγ, when stimulated with SGC-7901 tumor cells but not when stimulated with B16 tumor cells （1575.31 ± 60.25 pg/mL in SGC-7901 group vs 164.11± 18.52 pg/mL in B16 group, P 〈 0.01）. CONCLUSION： BM-derived DCs pulsed with tumor lysates Can induce anti-tumor immunity specific to gastric cancer ex vivo.展开更多
Background:Despite the availability of chemotherapy drugs such as 5-fluorouracil(5-FU),the treatment of some cancers such as gastric cancer remains challenging due to drug resistance and side effects.This study aimed t...Background:Despite the availability of chemotherapy drugs such as 5-fluorouracil(5-FU),the treatment of some cancers such as gastric cancer remains challenging due to drug resistance and side effects.This study aimed to investigate the effect of celastrol in combination with the chemotherapy drug 5-FU on proliferation and induction of apoptosis in human gastric cancer cell lines(AGS and EPG85-257).Materials and Methods:In this in vitro study,AGS and EPG85-257 cells were treated with different concentrations of celastrol,5-FU,and their combination.Cell proliferation was assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide(MTT)assay.The synergistic effect of 5-FU and celastrol was studied using Compusyn software.The DNA content at different phases of the cell cycle and apoptosis rate was measured usingflow cytometry.Results:Co-treatment with low concentrations(10%inhibitory concentration(IC10))of celastrol and 5-FU significantly reduced IC50(p<0.05)so that 48 h after treatment,IC50 was calculated at 3.77 and 6.9μM for celastrol,20.7 and 11.6μM for 5-FU,and 5.03 and 4.57μM for their combination for AGS and EPG85-257 cells,respectively.The mean percentage of apoptosis for AGS cells treated with celastrol,5-FU,and their combination was obtained 23.9,41.2,and 61.9,and for EPG85-257 cells 5.65,46.9,and 55.7,respectively.In addition,the 5-FU and celastrol-5-FU combination induced cell cycle arrest in the synthesis phase.Conclusions:Although celastrol could decrease the concentration of 5-fluorouracil that sufficed to suppress gastric cancer cells,additional studies are required to arrive at conclusive evidence on the anticancer effects of celastrol.展开更多
Despite the continuous developments and advancements in the treatment of gastric cancer(GC),which is one of the most prevalent types of cancer in China,the overall survival is still poor for most patients with advance...Despite the continuous developments and advancements in the treatment of gastric cancer(GC),which is one of the most prevalent types of cancer in China,the overall survival is still poor for most patients with advanced GC.In recent years,with the progress in tumor immunology research,attention has shifted toward immunotherapy as a therapeutic approach for GC.Programmed cell death protein 1(PD-1)inhibitors,as novel immunosuppressive medications,have been widely utilized in the treatment of GC.However,many patients are still resistant to PD-1 inhibitors and experience recurrence in the advanced stages of PD-1 immunotherapy.To reduce the occurrence of drug resistance and recurrence in GC patients receiving PD-1 immunotherapy,to maximize the clinical activity of immunosuppressive drugs,and to elicit a lasting immune response,it is essential to research the tumor microenvironment mechanisms leading to PD-1 inhibitor resistance in GC patients.This article reviews the progress in studying the factors influencing the resistance to PD-1 inhibitors in the GC tumor microenvironment,aiming to provide insights and a basis for reducing resistance to PD-1 inhibitors for GC patients in the future.展开更多
To investigate the effects of dendritic cells (DCs) transfected with full length wild type p53 and modified by gastric cancer lysates on immune response, the wild type P53 was transducted to DCs with adenovirus, and t...To investigate the effects of dendritic cells (DCs) transfected with full length wild type p53 and modified by gastric cancer lysates on immune response, the wild type P53 was transducted to DCs with adenovirus, and the DCs were modified by gastric cancer lysates (Lywt-P53DC). The concentration of the surface molecules (B7-1, B7-2, MHC-Ⅰ, MHC-Ⅱ) of all DCs was determined by FACS, and the ability of the DCs to induce efficient and specific immunological response in anti-51Cr-labeled target cells studied. BALB/c mice model infected with DCs and Mk28 was established. CTL response in mice immunized with Lywt-p53DC and the effectiveness of Lywt-p53DC in the treatment of tumor-bearing mice was assayed. FACS revealed that the surface molecules of Lywt-P53 DC had a high expression: for B7-1 86.70 %±0.07 %, B7-2 18.77 %±0.08 %, MHC-Ⅰ 87.20 %±0.05 %, MHC-Ⅱ 56.70 %±0.07 %; The T lymphocytes had a specific CTL lysing ability induced by Lywt-P53DC with the CTL lysis rate being 81 %. The immune protective effect of Lywt-p53DC group was more obvious than any other groups (P<0.05). The tumor diameter in Lywt-p53DC group was 3.10±0.31 mm, 2.73±0.23 mm, 3.70±0.07 mm on the day 13, 16 and 19, smaller than DC, wtp53DC and LyDC groups (P<0.05). On the other hand, the growth rate of tumor in Lywt-p53DC group was slower than any other groups (P<0.05). It was suggested that DCs transfected with wild type P53 and modified by gastric cancer lysates had specific CTL killing capability.展开更多
Objective:To evaluate the effects of ethanol extract from Ardisia gigantifolia leaves on cell proliferation and cancer stem cell(CSC)number in gastric cancer.Methods:The inhibitory effect of Ardisia gigantifolia extra...Objective:To evaluate the effects of ethanol extract from Ardisia gigantifolia leaves on cell proliferation and cancer stem cell(CSC)number in gastric cancer.Methods:The inhibitory effect of Ardisia gigantifolia extract on the proliferation of MKN45 and MKN74 gastric cancer cells was assessed using 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide assay.Non-adherent culture(3D)model was used to evaluate the effect of the extract on tumorsphere size and number.Moreover,the expression of CD44,ALDH,and p21 was determined by immunofluorescence analysis.Flow cytometric analysis was performed to evaluate cell cycle arrest and the expression of gastric CSC markers CD44 and ALDH.Real-time PCR analysis was also carried out to assess the effect of the extract on the expression of cell cycle-regulated genes.Results:Ardisia gigantifolia extract effectively inhibited cell proliferation with an IC_(50)of 55.7μg/m L in MKN45 cells and 123.6μg/m L in MKN74 cells.The extract also arrested cell cycle in the G_(0)/G_(1)phase as well as significantly reduced the size and number of tumorspheres.The markedly increased expression of p21 was observed at both m RNA and protein levels in the extract-treated adherent cells and tumorspheres.In addition,Ardisia gigantifolia extract significantly reduced the number of CD44-and/or ALDH-expressing gastric CSC.Conclusions:The development of gastric CSC can be inhibited by the ethanol extract of Ardisia gigantifolia.展开更多
Schisandrin B(Sch B)is a monomer with anti-cancer and anti-inflammatory effects,which are isolated from the plant Schisandra chinensis(Turcz)Baillon.We investigated the anti-gastric cancer(GC)effects of Sch B and its ...Schisandrin B(Sch B)is a monomer with anti-cancer and anti-inflammatory effects,which are isolated from the plant Schisandra chinensis(Turcz)Baillon.We investigated the anti-gastric cancer(GC)effects of Sch B and its underlying molecular mechanisms.The Cell Counting Kit-8 assay was used to determine the effects of Sch B on the viability of GC and normal cell lines.Hoechst/propidium iodide staining and flow cytometry were used to assess the apoptosis induction of Sch B.Western blotting was used to evaluate the effects of Sch B on downstream apoptotic proteins.The DCFH-DA fluorescent probe was used to assess the regulatory effects of Sch B on reactive oxygen species(ROS)levels and related signaling pathways in GC cells.The results showed that Sch B could regulate the phosphorylation level of mitogen-activated protein kinase(MAPK)by upregulating ROS accumulation in gastric cancer cells,and then reduce the expression of nuclear factor kappa B(NF-κB)and phosphorylated transcription 3(p-STAT3).In addition,Sch B downregulated the cell cycle proteins cyclin-dependent kinase 2/4/6 and cyclin D1/E,and arrested cells in the G0/G1 phase.Moreover,it also inhibited cell migration,which was reversed with Nacetylcysteine pretreatment.In summary,Sch B has killing effects on GC cells by upregulating the production of intracellular ROS and regulating the MAPK/STAT3/NF-κB signaling pathway,leading to the migration arrest and apoptosis of GC cells.展开更多
Fos-related antigen 1(Fra-1)is a nuclear transcription factor that regulates cell growth,differentiation,and apoptosis.It is involved in the proliferation,invasion,apoptosis and epithelial mesenchymal transformation o...Fos-related antigen 1(Fra-1)is a nuclear transcription factor that regulates cell growth,differentiation,and apoptosis.It is involved in the proliferation,invasion,apoptosis and epithelial mesenchymal transformation of malignant tumor cells.Fra-1 is highly expressed in gastric cancer(GC),affects the cycle distribution and apoptosis of GC cells,and participates in GC occurrence and development.However,the detailed mechanism of Fra-1 in GC is unclear,such as the identification of Fra-1-interacting proteins and their role in GC pathogenesis.In this study,we identified tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein eta(YWHAH)as a Fra-1-interacting protein in GC cells using co-immunoprecipitation combined with liquid chromatography-tandem mass spectrometry.Experiments showed that YWHAH positively regulated Fra-1 mRNA and protein expression,and affected GC cell proliferation.Whole proteome analysis showed that Fra-1 affected the activity of the high mobility group AT-hook 1(HMGA1)/phosphatidylinositol-4,5-bisphosphate 3-kinase(PI3K)/protein kinase B(AKT)/mechanistic target of rapamycin(mTOR)signaling pathway in GC cells.Western blotting and flow cytometry confirmed that YWHAH activated HMGA1/PI3K/AKT/mTOR signaling pathway by positively regulating Fra-1 to affect GC cell proliferation.These results will help to discover new molecular targets for the early diagnosis,treatment,and prognosis prediction of GC.展开更多
Objective: Cancer immunotherapy has made remarkable advances in recent years, but its effectiveness in treating gastric cancer is often limited by the complexity of the tumor microenvironment and the lack of effective...Objective: Cancer immunotherapy has made remarkable advances in recent years, but its effectiveness in treating gastric cancer is often limited by the complexity of the tumor microenvironment and the lack of effective biomarkers. This study aimed to identify effective biomarkers for immunotherapy treatment by characterizing the tumor microenvironment.Methods: We retrieved the RNA-seq data from gastric cancer patients treated with the programmed death 1(PD-1) blockade pembrolizumab. Differentially expressed genes associated with clinical outcomes were identified and further analyzed using gene ontology(GO) and Kyoto Encyclopedia of Genes and Genomes(KEGG) pathway analysis. Gene signature scores were calculated by single sample Gene Set Enrichment Analysis(ssGSEA). The infiltration levels of immune cells were quantified using the xCell website. Cell type enrichment analysis was performed to compare treatment response and non-response groups, and regression analysis was used to investigate the relationship between interferon gamma(IFNγ) immune response and immune cell infiltration. Biomarkers were identified using least absolute shrinkage and selection operator(LASSO) analysis.Results: Compared to normal tissues, cytokine activity and interleukin-6 production were highly activated in gastric tumors. Responders to pembrolizumab showed significantly up-regulated expression of IFNγ responserelated genes. Cell type enrichment analysis revealed that Th1 cells were significantly enriched in the tumor microenvironment of responders. Regression analysis indicated that Th1 cells induced IFNγ response more efficiently than other cell types. Using signatures of Th1 cells, stromal cells and IFNγ response, a set of eight genes were identified that effectively predicted the efficacy of immunotherapy treatment and patient prognosis.Conclusions: Th1 cells promote therapeutic efficacy of PD-1 blockade by promoting IFNγ immune response in gastric cancer. The identified biomarkers have the potential to improve the effectiveness of immunotherapy treatment for gastric cancer patients.展开更多
AIM: To prepare a cancer vaccine (H(22)-DC) expressing high levels of costimulatory molecules based on fusions of hepatocarcinoma cells (H(22)) with dendritic cells (DC) of mice and to analyze the biological character...AIM: To prepare a cancer vaccine (H(22)-DC) expressing high levels of costimulatory molecules based on fusions of hepatocarcinoma cells (H(22)) with dendritic cells (DC) of mice and to analyze the biological characteristics and induction of specific CTL activity of H(22)-DC. METHODS: DCs were isolated from murine spleen by metrizamide density gradient centrifugation, purified based on its characteristics of semi-adhesion to culture plates and FcR-,and were cultured in the medium containing GM-CSF and IL-4. A large number of DC were harvested. DCs were then fused with H(22) cells by PEG and the fusion cells were marked with CD11c MicroBeads. The H(22)-DC was sorted with Mimi MACS sorter. The techniques of cell culture, immunocytochemistry and light microscopy were also used to test the characteristics of growth and morphology of H(22)-DC in vitro. As the immunogen, H(22)-DC was inoculated subcutaneously into the right armpit of BALB/C mice, and their tumorigenicity in vivo was observed. MTT was used to test the CTL activity of murine spleen in vivo. RESULTS: DC cells isolated and generated were CD11c+ cells with irregular shape, and highly expressed CD80, CD86 and CD54 molecules. H22 cells were CD11c- cells with spherical shape and bigger volume, and did not express CD80, CD86 and CD54 molecules.H(22)-DC was CD11c+ cells with bigger volume, being spherical, flat or irregular in shape, and highly expressed CD80, CD86 and CD54 molecules, too. H(22)-DC was able to divide and proliferate in vitro, but its activity of proliferation was significantly decreased as compared with H(22) cells and its growth curve was flatter than H(22) cells. After subcutaneous inoculation over 60 days, H(22)-DC showed no tumorigenecity in mice, which was significantly different from control groups (P【0.01). The spleen CTL activity against H(22) cells in mice implanted with fresh H(22)-DC was significantly higher than control groups (P 【 0.01). CONCLUSION: H(22)-DC could significantly stimulate the specific CTL activity of murine spleen, which suggests that the fusion cells have already obtained the function of antigen presenting of parental DC and could present H(22)specific antigen which has not been identified yet, and H(22)-DC could induce antitumor immune response; although simply mixed H(22) cells with DC could stimulate the specific CTL activity which could inhibit the growth of tumor in some degree, it could not prevent the generation of tumor. It shows that the DC vaccine is likely to become a helpful approach in immunotherapy of hepatocarcinoma.展开更多
AIM： To investigate the effect of a vaccine with recombinant adenovirus interleukin-12 （AdVIL-12） transduced dendritic cells （DCs） against colon cancer in mice. METHODS： DCs and AdVIL-12 were incubated together ...AIM： To investigate the effect of a vaccine with recombinant adenovirus interleukin-12 （AdVIL-12） transduced dendritic cells （DCs） against colon cancer in mice. METHODS： DCs and AdVIL-12 were incubated together at different time intervals and at different doses. Supernatant was collected and tested for IL-12 by enzyme-linked immunosorbent assay （ELISA）. In order to determine whether tumor cell lysate-pulsed （TP） AdVIL-12/DCs enhance therapeutic potential in the established tumor model, CT26 colon tumor cells were implanted subcutaneously （s.c.） in the midflank of naive BALB/c mice. Tumor-bearing mice were injected with a vaccination of CT26 TP AdVIL-12/DCs on d 3 and 10. As a protective colon tumor model, naive BALB/c mice were immunized s.c. in their abdomens with CT26 TP AdVIL-12/DCs twice at seven day intervals. After the immunization on d 7, the mice were challenged with a lethal dose of CT26 tumor cells and survival times were evaluated. Subsequently, cytotoxic T lymphocyte （CTL） activity and interferon gamma （IFNy） secretion was evaluated in the immunized mice, and assayed CTL ex vivo. RESULTS： Murine DCs were retrovirally transduced with AdVIL-12 efficiency, and the AdVIL-12 transduced DCs secreted a high level of IL-12 （AdVIL-12/DCs, 615.27 ± 42.3 pg/mL vs DCs, 46.32 ± 7.29 pg/mL, P 〈 0.05）. Vaccination with CT26 TP AdVIL-12/DCs could enhance anti-tumor immunity against CT26 colon tumor in murine therapeutic models （tumor volume on d 19：CT26 TP AdVIL-12/DCs 107 ± 42 mm^3 vs CT26 TP DCs 383± 65 mm^3, P 〈 0.05） and protective models. Moreover, the CT26 TP AdVIL-12/DC vaccination enhances tumor-specific CTL activity, producing high levels of IFN7 in immunized mice. Ex vivo primed T cells with AdVIL-12/DCs were able to induce more effective CTL activity than in primed T cells with CT26 TP/DCs （E：T = 100：1, 69.49% ± 6.11% specific lysis vs 37.44% ＋ 4.32% specific lysis, P 〈 0.05）.CONCLUSION： Vaccination with recombinant AdVIL-12 transduced DC pulsed tumor cell lysate enhance antitumor immunity specific to colon cancer in mice.展开更多
AIM To investigate the role of heat shock protein (HSP)glycoprotein (gp) 96 in dendritic cells (DCs) and lymphocytes induction in gastric cancer (GC). METHODS Human GC cell lines KATOIII, MKN-28 and SGC-7901 were infe...AIM To investigate the role of heat shock protein (HSP)glycoprotein (gp) 96 in dendritic cells (DCs) and lymphocytes induction in gastric cancer (GC). METHODS Human GC cell lines KATOIII, MKN-28 and SGC-7901 were infected with adenovirus gp96 at a multiplicity of infection of 100. gp96-GC antigen peptide complexes were purified. MTT (3-(4,5-dimethylthiazol-2-yl)2,5- diphenyltetrazolium bromide) assay, lactate dehydrogenase (LDH) release assay and enzyme-linked immunosorbent assay were used to determine allo-reactive T cell stimulation, natural killer (NK) cell activity and expression of cytokines (such as interleukin (IL)-10, IL-12, interferon (IFN)-gamma and tumor necrosis factor (TNF)-alpha), respectively. Effect of cytotoxic T lymphocyte (CTL) on DCs incubated with HSP-gp96 was also evaluated by LDH release. All assays were performed in triplicate and the average values were reported. Comparison between groups was conducted using Student's t test. RESULTS T cells incubated with HSP-gp96 exhibited a marked increase in proliferation in a dose-dependent manner (P < 0.05). NK cell activity after gp96-GC peptide complex treatment was significantly higher than that after antigen peptide treatment (P < 0.05). The activity of CTLs incubated with DCs from three GC cells lines was obviously higher than that stimulated by GC antigen at ratios of 50: 1, 25: 1, 10: 1, and 5: 1 (P < 0.05). Furthermore, the secretion of TNF-alpha, IL-10, IL-12 (P70) and IFN-alpha markedly increased after incubation with HSP-gp96 (P < 0.05). CONCLUSION HSP-gp96 promotes T cell response, enhances DC antigen presentation and induces cytokine secretion, as well. HSP-gp96 has potential as immunotherapy for elimination of residual GC cells.展开更多
Breast cancer(BC) is the most common malignant neoplasm and the cause of death by cancer among women worldwide. Its development, including malignancy grade and patient prognosis, is influenced by various mutations tha...Breast cancer(BC) is the most common malignant neoplasm and the cause of death by cancer among women worldwide. Its development, including malignancy grade and patient prognosis, is influenced by various mutations that occur in the tumor cell and by the immune system's status, which has a direct influence on the tumor microenvironment and, consequently, on interactions with non-tumor cells involved in the immunological response. Among the immune response cells, dendritic cells(DCs) play a key role in the induction and maintenance of anti-tumor responses owing to their unique abilities for antigen cross-presentation and promotion of the activation of specific lymphocytes that target neoplasic cells. However, the tumor microenvironment can polarize DCs, transforming them into immunosuppressive regulatory DCs, a tolerogenicphenotype which limits the activity of effector T cells and supports tumor growth and progression. Various factors and signaling pathways have been implicated in the immunosuppressive functioning of DCs in cancer, and researchers are working on resolving processes that can circumvent tumor escape and developing viable therapeutic interventions to prevent or reverse the expression of immunosuppressive DCs in the tumor microenvironment. A better understanding of the pattern of DC response in patients with BC is fundamental to the development of specific therapeutic approaches to enable DCs to function properly. Various studies examining DCs immunotherapy have demonstrated its great potential for inducing immune responses to specific antigens and thereby reversing immunosuppression and related to clinical response in patients with BC. DCbased immunotherapy research has led to immense scientific advances, both in our understanding of the antitumor immune response and for the treatment of these patients.展开更多
AIM To investigate the enhanced cytotoxic T lymphocyte responses against pancreatic cancer (PC) in vitro induced by dendritic cells (DCs) engineered to secrete anti-DcR3 monoclonal antibody (mAb). METHODS DCs, T lymph...AIM To investigate the enhanced cytotoxic T lymphocyte responses against pancreatic cancer (PC) in vitro induced by dendritic cells (DCs) engineered to secrete anti-DcR3 monoclonal antibody (mAb). METHODS DCs, T lymphocytes and primary PC cells were obtained from PC patients. DCs were transfected with a designed humanized anti-DcR3 monoclonal antibody heavy and light chain mRNA and/or total tumor RNA (DC-tumor-anti-DcR3 RNA or DC-total tumor RNA) by using electroporation technology. The identification, concentration and function of anti-DcR3 mAb secreted by DC-tumor-anti-DcR3 RNA were determined by western blotting and enzyme-linked immunosorbent assay. After co-culturing of autologous isolated PC cells with target DCs, the effects of secreting anti-DcR3 mAb on RNA-DCs' viability and apoptosis were assessed by MTT assay and flow cytometry. Analysis of enhanced antigen-specific immune response against PC induced by anti-DcR3 mAb secreting DCs was performed using a Cr-51 releasing test. T cell responses induced by RNAloaded DCs were analyzed by measuring cytokine levels, including IFN-gamma, IL-10, IL4, TNF-alpha and IL-12. RESULTS The anti-DcR3 mAb secreted by DCs reacted with recombinant human DcR3 protein and generated a band with 35 kDa molecular weight. The secreting mAb was transient, peaking at 24 h and becoming undetectable after 72 h. After co-incubation with DCtumor- anti-DcR3 RNA for designated times, the DcR3 level in the supernatant of autologous PC cells was significantly down-regulated (P < 0.05). DCs secreting anti-DcR3 mAb could improve cell viability and slow down the apoptosis of RNA-loaded DCs, compared with DC-total tumor RNA (P < 0.01). The anti-DcR3 mAb secreted by DC-tumor-anti-DcR3 RNA could enhance the induction of cytotoxic T lymphocytes (CTLs) activity toward RNA-transfected DCs, primary tumor cells, and PC cell lines, compared with CTLs stimulated by DC-total tumor RNA or control group (P < 0.05). Meanwhile, the antigen-specific CTL responses were MHC class I-restricted. The CD4+ T cells and CD8+ T cells incubated with anti-DcR3 mAb secreting DCs could produce extremely higher level IFN-gamma and lower level IL4 than those incubated with DC-total tumor RNA or controls (P < 0.01). CONCLUSION DCs engineered to secrete anti-DcR3 antibody can augment CTL responses against PC in vitro, and the immune-enhancing effects may be partly due to their capability of down-regulating DC apoptosis and adjusting the Th1/Th2 cytokine network.展开更多
Objective： The aim of the present study was to investigate the effects of 5-fluorouracil（5-Fu） and oxaliplatin on the function and activation pathways of mouse dendritic cells（DCs）, and to clarify whether 5-Fu/ox...Objective： The aim of the present study was to investigate the effects of 5-fluorouracil（5-Fu） and oxaliplatin on the function and activation pathways of mouse dendritic cells（DCs）, and to clarify whether 5-Fu/oxaliplatin combined with the CD1d-MC38/α-galactosylceramide（α-GC） tumor vaccine exhibits synergistic effects on the treatment of colon cancer in mice.Methods： The combination of the Toll like receptor（TLR） ligands and/or 5-Fu/oxaliplatin was added into myeloid-derived DCs in vitro culture. DC phenotypic changes were detected by flow cytometry, and the secretion of DC cytokines was detected by cytometric bead array（CBA）. A MC38 mouse colon cancer model was constructed and the DCs were isolated from the spleen, tumor tissue and lymph nodes following intraperitoneal injection of 5-Fu/oxaliplatin. The cell phenotypes were detected by flow cytometry. The tumor infiltrating leukocytes,splenocytes and lymph node cells were co-cultured with the dead MC38 tumor cells, and the secretion levels of interferon-γ（IFN-γ） were detected. 5-Fu/oxaliplatin combined with our previously developed CD1d-MC38/α-GC tumor vaccine was used to inhibit the growth of MC38 colon cancer in mice, and the tumor growth rate and survival time were recorded.Results： 5-Fu/oxaliplatin exerted no significant effect on the expression of the stimulating phenotypes of DCs in vitro, while it could reduce the expression of programmed death ligand 1/2（PD-L1/L2） and promote interleukin-12（IL-12） secretion by DCs. Furthermore 5-Fu/oxaliplatin was beneficial to the differentiation of T-helper 1（Th1） cells. 5-Fu/oxaliplatin further enhanced the stimulating phenotypic expression of DCs in tumor bearing mice, decreased PD-L1/L2 expression, and specifically activated the lymphocytes. The CD1d-MC38/α-GC tumor vaccine combined with 5-Fu/oxaliplatin could exert a synergistic role that resulted in a significant delay of the tumor growth rate, and an increase in the survival time of tumor bearing mice.Conclusions： 5-Fu/oxaliplatin decreased the expression of the DC inhibitory phenotypes PD-L1/L2, promoted DC phenotypic maturation in tumor bearing mice, activated the lymphocytes of tumor bearing mice, and exerted synergistic effects with the CD1d-MC38/α-GC colon cancer tumor vaccine.展开更多
BACKGROUND: Dendritic cells (DCs) are the most important antigen-presenting cells in the human body, and DCs with different mature status possess different or even opposite functions. This study was designed to explor...BACKGROUND: Dendritic cells (DCs) are the most important antigen-presenting cells in the human body, and DCs with different mature status possess different or even opposite functions. This study was designed to explore the influence of insulin on the functional status of cord blood-derived DCs and on DC-induced cytotoxic T lymphocyte (CTL) activity against pancreatic cancer cell lines. METHODS: Mononuclear cells were isolated from fresh cord blood. Interleukin-4 (IL-4) and granulocytemacrophage colony-stimulating factor (GM-CSF) were used to induce or stimulate the mononuclear cells. Insulin at different concentrations served to modify DCs, and then DC morphology, number, and growth status were assessed. The DC immunophenotype was detected with a flow cytometer. The IL-12 in DC supernatant was determined by ELISA. DC functional status was evaluated by the autologous mixed lymphocyte reaction. T lymphocytes were induced by insulin-modified DCs to become CTLs. The CTL cytotoxicity against pancreatic cancer cell lines was determined. RESULTS: Mononuclear cells from cord blood can be differentiated into DCs by cytokine induction and insulin modification. With the increase in insulin concentration (2.5-25 mg/L), the expression of DC HLA-DR, CD1 alpha, CD80, and CD83 was significantly increased, the DC ability to secrete IL-12 was significantly improved, DC function to activate autologous lymphocytes was significantly enhanced, and the cytotoxicity of CTLs induced by insulin-modified DCs against pancreatic cancer cell lines was significantly strengthened. CONCLUSIONS: Insulin may facilitate DC induction and maturation, and improve the reproductive activity of autologous lymphocytes. The cytotoxicity of CTLs induced by insulin-modified DCs against pancreatic cancer cell lines was significantly enhanced. Insulin may serve as a factor modifying DCs and inducing CTLs in vitro in insulin biotherapy.展开更多
基金This study was supported by grants from the National Natural Science Foundation of China(Grant Nos.82222058,82073197,82273142,and 82173256).
文摘Colorectal cancer(CRC)is the third most common cancer and the second leading cause of cancer-related deaths worldwide.Dendritic cells(DCs)constitute a heterogeneous group of antigen-presenting cells that are important for initiating and regulating both innate and adaptive immune responses.As a crucial component of the immune system,DCs have a pivotal role in the pathogenesis and clinical treatment of CRC.DCs cross-present tumor-related antigens to activate T cells and trigger an antitumor immune response.However,the antitumor immune function of DCs is impaired and immune tolerance is promoted due to the presence of the tumor microenvironment.This review systematically elucidates the specific characteristics and functions of different DC subsets,as well as the role that DCs play in the immune response and tolerance within the CRC microenvironment.Moreover,how DCs contribute to the progression of CRC and potential therapies to enhance antitumor immunity on the basis of existing data are also discussed,which will provide new perspectives and approaches for immunotherapy in patients with CRC.
基金the National Natural Science Foundation of China,No.82074402the Science and Technology Innovation Project of China Academy of Chinese Medical Sciences,No.CI2021A01802.
文摘MicroRNAs(miRNAs)have received much attention in the past decade as potential key epigenomic regulators of tumors and cancer stem cells(CSCs).The abnormal expression of miRNAs is responsible for different phenotypes of gastric cancer stem cells(GCSCs).Some specific miRNAs could be used as promising biomarkers and therapeutic targets for the identification of GCSCs.This review summarizes the coding process and biological functions of miRNAs and demon-strates their role and efficacy in gastric cancer(GC)metastasis,drug resistance,and apoptosis,especially in the regulatory mechanism of GCSCs.It shows that the overexpression of onco-miRNAs and silencing of tumor-suppressor miRNAs can play a role in promoting or inhibiting tumor metastasis,apart from the initial formation of GC.It also discusses the epigenetic regulation and potential clinical applications of miRNAs as well as the role of CSCs in the pathogenesis of GC.We believe that this review may help in designing novel therapeutic approaches for GC.
基金Natural Science Foundation of Anhui Province(No.1908085MH258)Scientific Research and Innovation Project of Bengbu Medical College(No.Byycxz21004)。
文摘Objective:To explore the effect and mechanism of prostaglandins D2(PGD2)on the stemness of gastric cancer stem cells(GCSCs).Methods:7901-GCSCs were enriched by serum-free culture method;then the positivity rate of CD44,a stemness marker,was detected by flow cytometry in serum-free cultured 7901-GCSCs;the sphere-forming ability was detected by the sphere-forming assay after stimulation with different concentrations of PGD2(2.5,5,10)μg/mL,and the expression of stemness-related indicators(OCT4,CD44)and autophagyrelated proteins(LC3,Beclin-1)after PGD2 stimulation was detected by the western blot assay in different concentrations.The expression of stemness-related indexes(OCT4,CD44)and autophagy-related proteins(LC3,Beclin-1)were detected by Western blot assay after stimulation with different concentrations of PGD2.The expression of autophagy-related proteins after stimulation with different concentrations of CQ(2.5,5,10)μM was detected by Western blot experiment.The protein expression of autophagy-related proteins(LC3,Beclin-1)and stemness-related indexes(OCT4,CD44)was detected by Western blot experiment after PGD2 as well as PGD2+CQ treatment.Results:Flow cytometry results showed that the expression of CD44 positivity was increased in serum-free cultured 7901-GCSCs compared with gastric cancer cells SGC-7901(P<0.05),which fulfilled the needs of subsequent experiments.The results of stem cell spheroid formation assay showed that the spheroid formation ability of 7901-GCSCs in the PGD2 group was significantly weakened compared with that of the DMSO group(P<0.05).Western blot results showed that the protein expression of stemness-related indexes(OCT4,CD44)was down-regulated in the 7901-GCSCs in the PGD2 group compared with that of the DMSO group(P<0.05),and the expression of autophagy-related proteins(LC3,Beclin-1)expression increased(P<0.05).Compared with the DMSO group,the expression of autophagy-related proteins(LC3,Beclin-1)was decreased in the CQ group(P<0.05).Western blot results also showed that the expression of cellular autophagy-related proteins and stemness-related indexes in the PGD2+CQ group was not significantly changed compared with that of the DMSO group(ns:the difference was not significant),suggesting that the CQ could block the effect of PGD2 on the expression of stemness markers in 7901-GCSCs.7901-GCSCs stemness inhibition.Conclusion:PGD2 may affect the stemness of 7901-GCSCs by regulating autophagy.
文摘BACKGROUND Gastric cancer is one of the most common malignant tumors worldwide,and surgical resection is one of the main ways to treat gastric cancer.However,the immune status of postoperative patients is crucial for prognosis and survival,and immune cells play an important role in this process.Therefore,it is helpful to understand the immune status of postoperative patients by evaluating the levels of peripheral blood immune cells,especially total T cells(CD3+),helper T cells(CD3+CD4+),and suppressor T cells(CD3+CD8+),and its relationship to sur-vival.AIM To analyzed the immune cells in peripheral blood of patients with gastric cancer after surgery,detect the levels of total T cells,helper T cells and suppressor T cells.METHODS A total of 58 patients with gastric cancer who received surgical treatment were included in the retrospective study.Flow cytometry was used to detect the level of peripheral blood immune cells and analyze the correlation between total T cells,helper T cells and inhibitory T cells.To explore the relationship between these immune markers and patient survival.RESULTS The results showed that the levels of total T cells,helper T cells,and suppressor T cells changed in patients after gastric cancer surgery.There was a significant positive correlation between total T cells,helper T cells and suppressor T cells(r=0.35,P<0.01;r=0.56,P<0.01).However,there was a negative correlation between helper T cells and suppressor T cells(r=-0.63,P<0.01).Follow-up showed that the survival rate of patients in the high-level total T cell group was significantly higher than that in the low-level group(28.87±24.98 months vs 18.42±16.21 months).The survival curve shows that the curve of patients in the high-level group is shifted to the upper right,and that of the low-level group is shifted downward.There was no significant difference between the levels of helper T cells and suppressor T cells and patient survival time.CONCLUSION By detecting peripheral blood immune cells with flow cytometry,we can initially evaluate the immune status of patients after gastric cancer surgery and initially explore its relationship with patient survival.
基金Henan Provincial Health Commission's 2020 National Health Commission Science Research Fund Henan Provincial Medical Science and Technology Tackling Plan Provincial-Ministerial Joint Project and Soft Science Project"Clinical Study of CAR-NK Cells Targeting Carcinoembryonic Antigen on Gastric Cancer Cells"(Grant No.SBGJ202002093)Henan Province 2022 Science and Technology Development Plan"Study on Pyroglutamate Targeting DJ-1 to Trigger ROS-Induced Cell Death and Protective Autophagy in Pancreatic Cancer"(Grant No.222102310725)。
文摘Objective:To construct CAR-NK-92 cells targeting carcinoembryonic antigen(CEA)and study their killing effect on gastric cancer cells.Methods:CAR-NK-92 cells targeting CEA were constructed.After co-culturing CAR-NK-92 cells with MKN-45 gastric cancer cells,the killing effect of CAR-NK-92 cells was detected by a lactate dehydrogenase release assay.The secretion levels of gamma interferon and granulocyte-macrophage colony-stimulating factor were measured using an ELISA assay.Results:The lactate dehydrogenase release assay showed that CAR-NK-92 cells had a significant killing effect on MKN-45 cells compared to CON-NK-92 cells,and the difference was statistically significant(P<0.001).ELISA results indicated that the levels of gamma interferon and granulocyte-macrophage colony-stimulating factor secreted by CAR-NK-92 cells and MKN-45 target cells were significantly increased after co-culture(P<0.001).Conclusion:CAR-NK-92 cells targeting CEA exhibit a significant killing effect on CEA-positive gastric cancer cells.
文摘AIM： To investigate whether bone marrow-derived denritic cells pulsed with tumor lysates induce immunity against gastric cancer ex vivo. METHODS： c-kit＋ hematopoietic progenitor cells were magnetically isolated with a MiniMACS separator from BALB/c mice bone marrow cells. These cells were cultured with cytokines GM-CSF, IL-4, and TNFα to induce their maturation. They were analysed by morphological observation, phenotype analysis, and mixed lymphocyte reaction （MLR）. Bone marrowderived DCs （BM-DCs） were pulsed with tumor cell lysate obtained by rapid freezing and thawing at a 1：3 DC：tumor cell ratio. Finally, cytotoxic T lymphocyte （CTL） activity and interferon gamma （IFNγ） secretion was evaluated ex vivo. RESULTS： c-kit^＋ hematopoietic progenitor cells from mice bone marrow cells cultured with cytokines for 8 d showed the character of typical mature DCs.Morphologically, observed by light microscope, these cells were large with oval or irregularly shaped nuclei and with many small dendrites. Phenotypically, FACS analysis showed that they expressed.high levels of la, DEC-205, CD11b, CD80 and CD86 antigen, moderate levels of CD40, and negative for F4/80. Functionally, these cells gained the capacity to stimulate allogeneic T cells in MLR assay. However, immature DCs cultured with cytokines for 5 d did not have typical DCs phenotypic markers and could not stimulate allogeneic T cells. Ex vivo primed T cells with SGC-7901 tumor cell lysate-pulsed （TP） DCs were able to induce effective CTL activity against SGC-7901 tumor cells （E：T = 100：1, 69.55% ± 6.05% specific lysis）, but not B16 tumor cells, and produced higher levels of IFNγ, when stimulated with SGC-7901 tumor cells but not when stimulated with B16 tumor cells （1575.31 ± 60.25 pg/mL in SGC-7901 group vs 164.11± 18.52 pg/mL in B16 group, P 〈 0.01）. CONCLUSION： BM-derived DCs pulsed with tumor lysates Can induce anti-tumor immunity specific to gastric cancer ex vivo.
基金supported by Shahrekord University of Medical Sciences,Shahrekord,Iran(Ethics Code:IR.SKUMS.REC.1397.119,Grant No.3696 and Ethics Code:IR.SKUMS.REC.1401.197,Grant No.6651).
文摘Background:Despite the availability of chemotherapy drugs such as 5-fluorouracil(5-FU),the treatment of some cancers such as gastric cancer remains challenging due to drug resistance and side effects.This study aimed to investigate the effect of celastrol in combination with the chemotherapy drug 5-FU on proliferation and induction of apoptosis in human gastric cancer cell lines(AGS and EPG85-257).Materials and Methods:In this in vitro study,AGS and EPG85-257 cells were treated with different concentrations of celastrol,5-FU,and their combination.Cell proliferation was assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide(MTT)assay.The synergistic effect of 5-FU and celastrol was studied using Compusyn software.The DNA content at different phases of the cell cycle and apoptosis rate was measured usingflow cytometry.Results:Co-treatment with low concentrations(10%inhibitory concentration(IC10))of celastrol and 5-FU significantly reduced IC50(p<0.05)so that 48 h after treatment,IC50 was calculated at 3.77 and 6.9μM for celastrol,20.7 and 11.6μM for 5-FU,and 5.03 and 4.57μM for their combination for AGS and EPG85-257 cells,respectively.The mean percentage of apoptosis for AGS cells treated with celastrol,5-FU,and their combination was obtained 23.9,41.2,and 61.9,and for EPG85-257 cells 5.65,46.9,and 55.7,respectively.In addition,the 5-FU and celastrol-5-FU combination induced cell cycle arrest in the synthesis phase.Conclusions:Although celastrol could decrease the concentration of 5-fluorouracil that sufficed to suppress gastric cancer cells,additional studies are required to arrive at conclusive evidence on the anticancer effects of celastrol.
基金Natural Science Foundation of Gansu Province,No.21JR1RA186and the Health Industry Research Program of Gansu Province,No.GSWSKY2021-043.
文摘Despite the continuous developments and advancements in the treatment of gastric cancer(GC),which is one of the most prevalent types of cancer in China,the overall survival is still poor for most patients with advanced GC.In recent years,with the progress in tumor immunology research,attention has shifted toward immunotherapy as a therapeutic approach for GC.Programmed cell death protein 1(PD-1)inhibitors,as novel immunosuppressive medications,have been widely utilized in the treatment of GC.However,many patients are still resistant to PD-1 inhibitors and experience recurrence in the advanced stages of PD-1 immunotherapy.To reduce the occurrence of drug resistance and recurrence in GC patients receiving PD-1 immunotherapy,to maximize the clinical activity of immunosuppressive drugs,and to elicit a lasting immune response,it is essential to research the tumor microenvironment mechanisms leading to PD-1 inhibitor resistance in GC patients.This article reviews the progress in studying the factors influencing the resistance to PD-1 inhibitors in the GC tumor microenvironment,aiming to provide insights and a basis for reducing resistance to PD-1 inhibitors for GC patients in the future.
文摘To investigate the effects of dendritic cells (DCs) transfected with full length wild type p53 and modified by gastric cancer lysates on immune response, the wild type P53 was transducted to DCs with adenovirus, and the DCs were modified by gastric cancer lysates (Lywt-P53DC). The concentration of the surface molecules (B7-1, B7-2, MHC-Ⅰ, MHC-Ⅱ) of all DCs was determined by FACS, and the ability of the DCs to induce efficient and specific immunological response in anti-51Cr-labeled target cells studied. BALB/c mice model infected with DCs and Mk28 was established. CTL response in mice immunized with Lywt-p53DC and the effectiveness of Lywt-p53DC in the treatment of tumor-bearing mice was assayed. FACS revealed that the surface molecules of Lywt-P53 DC had a high expression: for B7-1 86.70 %±0.07 %, B7-2 18.77 %±0.08 %, MHC-Ⅰ 87.20 %±0.05 %, MHC-Ⅱ 56.70 %±0.07 %; The T lymphocytes had a specific CTL lysing ability induced by Lywt-P53DC with the CTL lysis rate being 81 %. The immune protective effect of Lywt-p53DC group was more obvious than any other groups (P<0.05). The tumor diameter in Lywt-p53DC group was 3.10±0.31 mm, 2.73±0.23 mm, 3.70±0.07 mm on the day 13, 16 and 19, smaller than DC, wtp53DC and LyDC groups (P<0.05). On the other hand, the growth rate of tumor in Lywt-p53DC group was slower than any other groups (P<0.05). It was suggested that DCs transfected with wild type P53 and modified by gastric cancer lysates had specific CTL killing capability.
基金funded by Vietnam National Foundation for Science and Technology Development(NAFOSTED)under grant number 108.05-2017.331。
文摘Objective:To evaluate the effects of ethanol extract from Ardisia gigantifolia leaves on cell proliferation and cancer stem cell(CSC)number in gastric cancer.Methods:The inhibitory effect of Ardisia gigantifolia extract on the proliferation of MKN45 and MKN74 gastric cancer cells was assessed using 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide assay.Non-adherent culture(3D)model was used to evaluate the effect of the extract on tumorsphere size and number.Moreover,the expression of CD44,ALDH,and p21 was determined by immunofluorescence analysis.Flow cytometric analysis was performed to evaluate cell cycle arrest and the expression of gastric CSC markers CD44 and ALDH.Real-time PCR analysis was also carried out to assess the effect of the extract on the expression of cell cycle-regulated genes.Results:Ardisia gigantifolia extract effectively inhibited cell proliferation with an IC_(50)of 55.7μg/m L in MKN45 cells and 123.6μg/m L in MKN74 cells.The extract also arrested cell cycle in the G_(0)/G_(1)phase as well as significantly reduced the size and number of tumorspheres.The markedly increased expression of p21 was observed at both m RNA and protein levels in the extract-treated adherent cells and tumorspheres.In addition,Ardisia gigantifolia extract significantly reduced the number of CD44-and/or ALDH-expressing gastric CSC.Conclusions:The development of gastric CSC can be inhibited by the ethanol extract of Ardisia gigantifolia.
基金This work was supported by the National Natural Science Foundation of China[Grant No.82060118]the Research Program of Science and Technology at Universities of Inner Mongolia Autonomous Region[Grant No.NJZY20203]+3 种基金the Program for Young Talents of Chifeng University[Grant No.CFXYYT2202]the Central Government Supports Local College Reform and Development Fund Talent Training Projects[Grant No.2020GSP16]the Heilongjiang Touyan Innovation Team Program[Grant No.2019HTY078]the Project for Heilongjiang Bayi Agricultural University[Grant No.XDB202012].
文摘Schisandrin B(Sch B)is a monomer with anti-cancer and anti-inflammatory effects,which are isolated from the plant Schisandra chinensis(Turcz)Baillon.We investigated the anti-gastric cancer(GC)effects of Sch B and its underlying molecular mechanisms.The Cell Counting Kit-8 assay was used to determine the effects of Sch B on the viability of GC and normal cell lines.Hoechst/propidium iodide staining and flow cytometry were used to assess the apoptosis induction of Sch B.Western blotting was used to evaluate the effects of Sch B on downstream apoptotic proteins.The DCFH-DA fluorescent probe was used to assess the regulatory effects of Sch B on reactive oxygen species(ROS)levels and related signaling pathways in GC cells.The results showed that Sch B could regulate the phosphorylation level of mitogen-activated protein kinase(MAPK)by upregulating ROS accumulation in gastric cancer cells,and then reduce the expression of nuclear factor kappa B(NF-κB)and phosphorylated transcription 3(p-STAT3).In addition,Sch B downregulated the cell cycle proteins cyclin-dependent kinase 2/4/6 and cyclin D1/E,and arrested cells in the G0/G1 phase.Moreover,it also inhibited cell migration,which was reversed with Nacetylcysteine pretreatment.In summary,Sch B has killing effects on GC cells by upregulating the production of intracellular ROS and regulating the MAPK/STAT3/NF-κB signaling pathway,leading to the migration arrest and apoptosis of GC cells.
基金This work was supported by the Hunan Provincial Natural Science Foundation(2021JJ30915).
文摘Fos-related antigen 1(Fra-1)is a nuclear transcription factor that regulates cell growth,differentiation,and apoptosis.It is involved in the proliferation,invasion,apoptosis and epithelial mesenchymal transformation of malignant tumor cells.Fra-1 is highly expressed in gastric cancer(GC),affects the cycle distribution and apoptosis of GC cells,and participates in GC occurrence and development.However,the detailed mechanism of Fra-1 in GC is unclear,such as the identification of Fra-1-interacting proteins and their role in GC pathogenesis.In this study,we identified tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein eta(YWHAH)as a Fra-1-interacting protein in GC cells using co-immunoprecipitation combined with liquid chromatography-tandem mass spectrometry.Experiments showed that YWHAH positively regulated Fra-1 mRNA and protein expression,and affected GC cell proliferation.Whole proteome analysis showed that Fra-1 affected the activity of the high mobility group AT-hook 1(HMGA1)/phosphatidylinositol-4,5-bisphosphate 3-kinase(PI3K)/protein kinase B(AKT)/mechanistic target of rapamycin(mTOR)signaling pathway in GC cells.Western blotting and flow cytometry confirmed that YWHAH activated HMGA1/PI3K/AKT/mTOR signaling pathway by positively regulating Fra-1 to affect GC cell proliferation.These results will help to discover new molecular targets for the early diagnosis,treatment,and prognosis prediction of GC.
基金financially supported by the National Natural Science Foundation of China (No. 82030079, 82341005, 81972656 and 82173035)the National Science and Technology Major Project of China (No. 2022YFC3400 901)Sino-Russian Math Center in PKU。
文摘Objective: Cancer immunotherapy has made remarkable advances in recent years, but its effectiveness in treating gastric cancer is often limited by the complexity of the tumor microenvironment and the lack of effective biomarkers. This study aimed to identify effective biomarkers for immunotherapy treatment by characterizing the tumor microenvironment.Methods: We retrieved the RNA-seq data from gastric cancer patients treated with the programmed death 1(PD-1) blockade pembrolizumab. Differentially expressed genes associated with clinical outcomes were identified and further analyzed using gene ontology(GO) and Kyoto Encyclopedia of Genes and Genomes(KEGG) pathway analysis. Gene signature scores were calculated by single sample Gene Set Enrichment Analysis(ssGSEA). The infiltration levels of immune cells were quantified using the xCell website. Cell type enrichment analysis was performed to compare treatment response and non-response groups, and regression analysis was used to investigate the relationship between interferon gamma(IFNγ) immune response and immune cell infiltration. Biomarkers were identified using least absolute shrinkage and selection operator(LASSO) analysis.Results: Compared to normal tissues, cytokine activity and interleukin-6 production were highly activated in gastric tumors. Responders to pembrolizumab showed significantly up-regulated expression of IFNγ responserelated genes. Cell type enrichment analysis revealed that Th1 cells were significantly enriched in the tumor microenvironment of responders. Regression analysis indicated that Th1 cells induced IFNγ response more efficiently than other cell types. Using signatures of Th1 cells, stromal cells and IFNγ response, a set of eight genes were identified that effectively predicted the efficacy of immunotherapy treatment and patient prognosis.Conclusions: Th1 cells promote therapeutic efficacy of PD-1 blockade by promoting IFNγ immune response in gastric cancer. The identified biomarkers have the potential to improve the effectiveness of immunotherapy treatment for gastric cancer patients.
基金Supported jby the Natural Science Foundation of Guangdong Province China,No.980180
文摘AIM: To prepare a cancer vaccine (H(22)-DC) expressing high levels of costimulatory molecules based on fusions of hepatocarcinoma cells (H(22)) with dendritic cells (DC) of mice and to analyze the biological characteristics and induction of specific CTL activity of H(22)-DC. METHODS: DCs were isolated from murine spleen by metrizamide density gradient centrifugation, purified based on its characteristics of semi-adhesion to culture plates and FcR-,and were cultured in the medium containing GM-CSF and IL-4. A large number of DC were harvested. DCs were then fused with H(22) cells by PEG and the fusion cells were marked with CD11c MicroBeads. The H(22)-DC was sorted with Mimi MACS sorter. The techniques of cell culture, immunocytochemistry and light microscopy were also used to test the characteristics of growth and morphology of H(22)-DC in vitro. As the immunogen, H(22)-DC was inoculated subcutaneously into the right armpit of BALB/C mice, and their tumorigenicity in vivo was observed. MTT was used to test the CTL activity of murine spleen in vivo. RESULTS: DC cells isolated and generated were CD11c+ cells with irregular shape, and highly expressed CD80, CD86 and CD54 molecules. H22 cells were CD11c- cells with spherical shape and bigger volume, and did not express CD80, CD86 and CD54 molecules.H(22)-DC was CD11c+ cells with bigger volume, being spherical, flat or irregular in shape, and highly expressed CD80, CD86 and CD54 molecules, too. H(22)-DC was able to divide and proliferate in vitro, but its activity of proliferation was significantly decreased as compared with H(22) cells and its growth curve was flatter than H(22) cells. After subcutaneous inoculation over 60 days, H(22)-DC showed no tumorigenecity in mice, which was significantly different from control groups (P【0.01). The spleen CTL activity against H(22) cells in mice implanted with fresh H(22)-DC was significantly higher than control groups (P 【 0.01). CONCLUSION: H(22)-DC could significantly stimulate the specific CTL activity of murine spleen, which suggests that the fusion cells have already obtained the function of antigen presenting of parental DC and could present H(22)specific antigen which has not been identified yet, and H(22)-DC could induce antitumor immune response; although simply mixed H(22) cells with DC could stimulate the specific CTL activity which could inhibit the growth of tumor in some degree, it could not prevent the generation of tumor. It shows that the DC vaccine is likely to become a helpful approach in immunotherapy of hepatocarcinoma.
文摘AIM： To investigate the effect of a vaccine with recombinant adenovirus interleukin-12 （AdVIL-12） transduced dendritic cells （DCs） against colon cancer in mice. METHODS： DCs and AdVIL-12 were incubated together at different time intervals and at different doses. Supernatant was collected and tested for IL-12 by enzyme-linked immunosorbent assay （ELISA）. In order to determine whether tumor cell lysate-pulsed （TP） AdVIL-12/DCs enhance therapeutic potential in the established tumor model, CT26 colon tumor cells were implanted subcutaneously （s.c.） in the midflank of naive BALB/c mice. Tumor-bearing mice were injected with a vaccination of CT26 TP AdVIL-12/DCs on d 3 and 10. As a protective colon tumor model, naive BALB/c mice were immunized s.c. in their abdomens with CT26 TP AdVIL-12/DCs twice at seven day intervals. After the immunization on d 7, the mice were challenged with a lethal dose of CT26 tumor cells and survival times were evaluated. Subsequently, cytotoxic T lymphocyte （CTL） activity and interferon gamma （IFNy） secretion was evaluated in the immunized mice, and assayed CTL ex vivo. RESULTS： Murine DCs were retrovirally transduced with AdVIL-12 efficiency, and the AdVIL-12 transduced DCs secreted a high level of IL-12 （AdVIL-12/DCs, 615.27 ± 42.3 pg/mL vs DCs, 46.32 ± 7.29 pg/mL, P 〈 0.05）. Vaccination with CT26 TP AdVIL-12/DCs could enhance anti-tumor immunity against CT26 colon tumor in murine therapeutic models （tumor volume on d 19：CT26 TP AdVIL-12/DCs 107 ± 42 mm^3 vs CT26 TP DCs 383± 65 mm^3, P 〈 0.05） and protective models. Moreover, the CT26 TP AdVIL-12/DC vaccination enhances tumor-specific CTL activity, producing high levels of IFN7 in immunized mice. Ex vivo primed T cells with AdVIL-12/DCs were able to induce more effective CTL activity than in primed T cells with CT26 TP/DCs （E：T = 100：1, 69.49% ± 6.11% specific lysis vs 37.44% ＋ 4.32% specific lysis, P 〈 0.05）.CONCLUSION： Vaccination with recombinant AdVIL-12 transduced DC pulsed tumor cell lysate enhance antitumor immunity specific to colon cancer in mice.
基金Supported by Science and Technology Department of Zhejiang Province,No.2008C33064
文摘AIM To investigate the role of heat shock protein (HSP)glycoprotein (gp) 96 in dendritic cells (DCs) and lymphocytes induction in gastric cancer (GC). METHODS Human GC cell lines KATOIII, MKN-28 and SGC-7901 were infected with adenovirus gp96 at a multiplicity of infection of 100. gp96-GC antigen peptide complexes were purified. MTT (3-(4,5-dimethylthiazol-2-yl)2,5- diphenyltetrazolium bromide) assay, lactate dehydrogenase (LDH) release assay and enzyme-linked immunosorbent assay were used to determine allo-reactive T cell stimulation, natural killer (NK) cell activity and expression of cytokines (such as interleukin (IL)-10, IL-12, interferon (IFN)-gamma and tumor necrosis factor (TNF)-alpha), respectively. Effect of cytotoxic T lymphocyte (CTL) on DCs incubated with HSP-gp96 was also evaluated by LDH release. All assays were performed in triplicate and the average values were reported. Comparison between groups was conducted using Student's t test. RESULTS T cells incubated with HSP-gp96 exhibited a marked increase in proliferation in a dose-dependent manner (P < 0.05). NK cell activity after gp96-GC peptide complex treatment was significantly higher than that after antigen peptide treatment (P < 0.05). The activity of CTLs incubated with DCs from three GC cells lines was obviously higher than that stimulated by GC antigen at ratios of 50: 1, 25: 1, 10: 1, and 5: 1 (P < 0.05). Furthermore, the secretion of TNF-alpha, IL-10, IL-12 (P70) and IFN-alpha markedly increased after incubation with HSP-gp96 (P < 0.05). CONCLUSION HSP-gp96 promotes T cell response, enhances DC antigen presentation and induces cytokine secretion, as well. HSP-gp96 has potential as immunotherapy for elimination of residual GC cells.
文摘Breast cancer(BC) is the most common malignant neoplasm and the cause of death by cancer among women worldwide. Its development, including malignancy grade and patient prognosis, is influenced by various mutations that occur in the tumor cell and by the immune system's status, which has a direct influence on the tumor microenvironment and, consequently, on interactions with non-tumor cells involved in the immunological response. Among the immune response cells, dendritic cells(DCs) play a key role in the induction and maintenance of anti-tumor responses owing to their unique abilities for antigen cross-presentation and promotion of the activation of specific lymphocytes that target neoplasic cells. However, the tumor microenvironment can polarize DCs, transforming them into immunosuppressive regulatory DCs, a tolerogenicphenotype which limits the activity of effector T cells and supports tumor growth and progression. Various factors and signaling pathways have been implicated in the immunosuppressive functioning of DCs in cancer, and researchers are working on resolving processes that can circumvent tumor escape and developing viable therapeutic interventions to prevent or reverse the expression of immunosuppressive DCs in the tumor microenvironment. A better understanding of the pattern of DC response in patients with BC is fundamental to the development of specific therapeutic approaches to enable DCs to function properly. Various studies examining DCs immunotherapy have demonstrated its great potential for inducing immune responses to specific antigens and thereby reversing immunosuppression and related to clinical response in patients with BC. DCbased immunotherapy research has led to immense scientific advances, both in our understanding of the antitumor immune response and for the treatment of these patients.
基金Supported by National Natural Science Foundation of China,No.81071982
文摘AIM To investigate the enhanced cytotoxic T lymphocyte responses against pancreatic cancer (PC) in vitro induced by dendritic cells (DCs) engineered to secrete anti-DcR3 monoclonal antibody (mAb). METHODS DCs, T lymphocytes and primary PC cells were obtained from PC patients. DCs were transfected with a designed humanized anti-DcR3 monoclonal antibody heavy and light chain mRNA and/or total tumor RNA (DC-tumor-anti-DcR3 RNA or DC-total tumor RNA) by using electroporation technology. The identification, concentration and function of anti-DcR3 mAb secreted by DC-tumor-anti-DcR3 RNA were determined by western blotting and enzyme-linked immunosorbent assay. After co-culturing of autologous isolated PC cells with target DCs, the effects of secreting anti-DcR3 mAb on RNA-DCs' viability and apoptosis were assessed by MTT assay and flow cytometry. Analysis of enhanced antigen-specific immune response against PC induced by anti-DcR3 mAb secreting DCs was performed using a Cr-51 releasing test. T cell responses induced by RNAloaded DCs were analyzed by measuring cytokine levels, including IFN-gamma, IL-10, IL4, TNF-alpha and IL-12. RESULTS The anti-DcR3 mAb secreted by DCs reacted with recombinant human DcR3 protein and generated a band with 35 kDa molecular weight. The secreting mAb was transient, peaking at 24 h and becoming undetectable after 72 h. After co-incubation with DCtumor- anti-DcR3 RNA for designated times, the DcR3 level in the supernatant of autologous PC cells was significantly down-regulated (P < 0.05). DCs secreting anti-DcR3 mAb could improve cell viability and slow down the apoptosis of RNA-loaded DCs, compared with DC-total tumor RNA (P < 0.01). The anti-DcR3 mAb secreted by DC-tumor-anti-DcR3 RNA could enhance the induction of cytotoxic T lymphocytes (CTLs) activity toward RNA-transfected DCs, primary tumor cells, and PC cell lines, compared with CTLs stimulated by DC-total tumor RNA or control group (P < 0.05). Meanwhile, the antigen-specific CTL responses were MHC class I-restricted. The CD4+ T cells and CD8+ T cells incubated with anti-DcR3 mAb secreting DCs could produce extremely higher level IFN-gamma and lower level IL4 than those incubated with DC-total tumor RNA or controls (P < 0.01). CONCLUSION DCs engineered to secrete anti-DcR3 antibody can augment CTL responses against PC in vitro, and the immune-enhancing effects may be partly due to their capability of down-regulating DC apoptosis and adjusting the Th1/Th2 cytokine network.
文摘Objective： The aim of the present study was to investigate the effects of 5-fluorouracil（5-Fu） and oxaliplatin on the function and activation pathways of mouse dendritic cells（DCs）, and to clarify whether 5-Fu/oxaliplatin combined with the CD1d-MC38/α-galactosylceramide（α-GC） tumor vaccine exhibits synergistic effects on the treatment of colon cancer in mice.Methods： The combination of the Toll like receptor（TLR） ligands and/or 5-Fu/oxaliplatin was added into myeloid-derived DCs in vitro culture. DC phenotypic changes were detected by flow cytometry, and the secretion of DC cytokines was detected by cytometric bead array（CBA）. A MC38 mouse colon cancer model was constructed and the DCs were isolated from the spleen, tumor tissue and lymph nodes following intraperitoneal injection of 5-Fu/oxaliplatin. The cell phenotypes were detected by flow cytometry. The tumor infiltrating leukocytes,splenocytes and lymph node cells were co-cultured with the dead MC38 tumor cells, and the secretion levels of interferon-γ（IFN-γ） were detected. 5-Fu/oxaliplatin combined with our previously developed CD1d-MC38/α-GC tumor vaccine was used to inhibit the growth of MC38 colon cancer in mice, and the tumor growth rate and survival time were recorded.Results： 5-Fu/oxaliplatin exerted no significant effect on the expression of the stimulating phenotypes of DCs in vitro, while it could reduce the expression of programmed death ligand 1/2（PD-L1/L2） and promote interleukin-12（IL-12） secretion by DCs. Furthermore 5-Fu/oxaliplatin was beneficial to the differentiation of T-helper 1（Th1） cells. 5-Fu/oxaliplatin further enhanced the stimulating phenotypic expression of DCs in tumor bearing mice, decreased PD-L1/L2 expression, and specifically activated the lymphocytes. The CD1d-MC38/α-GC tumor vaccine combined with 5-Fu/oxaliplatin could exert a synergistic role that resulted in a significant delay of the tumor growth rate, and an increase in the survival time of tumor bearing mice.Conclusions： 5-Fu/oxaliplatin decreased the expression of the DC inhibitory phenotypes PD-L1/L2, promoted DC phenotypic maturation in tumor bearing mice, activated the lymphocytes of tumor bearing mice, and exerted synergistic effects with the CD1d-MC38/α-GC colon cancer tumor vaccine.
基金supported by a grant from the 2008Henan Tackling Key Problems in Science and Technology(No.082102310036)
文摘BACKGROUND: Dendritic cells (DCs) are the most important antigen-presenting cells in the human body, and DCs with different mature status possess different or even opposite functions. This study was designed to explore the influence of insulin on the functional status of cord blood-derived DCs and on DC-induced cytotoxic T lymphocyte (CTL) activity against pancreatic cancer cell lines. METHODS: Mononuclear cells were isolated from fresh cord blood. Interleukin-4 (IL-4) and granulocytemacrophage colony-stimulating factor (GM-CSF) were used to induce or stimulate the mononuclear cells. Insulin at different concentrations served to modify DCs, and then DC morphology, number, and growth status were assessed. The DC immunophenotype was detected with a flow cytometer. The IL-12 in DC supernatant was determined by ELISA. DC functional status was evaluated by the autologous mixed lymphocyte reaction. T lymphocytes were induced by insulin-modified DCs to become CTLs. The CTL cytotoxicity against pancreatic cancer cell lines was determined. RESULTS: Mononuclear cells from cord blood can be differentiated into DCs by cytokine induction and insulin modification. With the increase in insulin concentration (2.5-25 mg/L), the expression of DC HLA-DR, CD1 alpha, CD80, and CD83 was significantly increased, the DC ability to secrete IL-12 was significantly improved, DC function to activate autologous lymphocytes was significantly enhanced, and the cytotoxicity of CTLs induced by insulin-modified DCs against pancreatic cancer cell lines was significantly strengthened. CONCLUSIONS: Insulin may facilitate DC induction and maturation, and improve the reproductive activity of autologous lymphocytes. The cytotoxicity of CTLs induced by insulin-modified DCs against pancreatic cancer cell lines was significantly enhanced. Insulin may serve as a factor modifying DCs and inducing CTLs in vitro in insulin biotherapy.
