RESUMO
Clinically, a considerable number of non-small cell lung cancer (NSCLC) patients are unable to receive or resist chemotherapy, and the efficacy of non-chemotherapy treatment strategies based on anti-angiogenic agents combined with immune checkpoint blockade is still unsatisfactory. Neoantigen vaccine, based on personalized tumor DNA mutations, could elicit tumor specific T cell infiltration into the tumor site, exerting potent anti-tumor efficacy. Here, we evaluated the feasibility and safety of a new antitumor strategy by adding neoantigen vaccine to the regimen of bevacizumab and anti-PD-1 antibody. Firstly, 7 novel immunogenic neoantigen peptides were identified and developed for neoantigen vaccine (LLCvac), which can elicit strong antitumor immune response in vivo. Then, in orthotopic lung cancer model, LLCvac further combining with bevacizumab and anti-PD-1 antibody exerted a stronger antitumor effect, exhibiting significant decrease of tumor volume without obvious toxicity. Furthermore, tumor immune microenvironment assessment also showed that the proportion of neoantigen-specific T cells in blood could be induced dramatically by the combined therapy. And a large amount of neoantigen-specific Ki67-positive CD8+ T cells were found in tumor tissues, which infiltrated tumor tissues effectively to kill tumor cells expressing identified neoantigens. Overall, these results suggested that this combined therapy could safely induce robust antitumor efficacy, serving as an effective chemotherapy-free strategy for NSCLC treatment.
Assuntos
Vacinas Anticâncer , Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Humanos , Antígenos de Neoplasias , Bevacizumab/uso terapêutico , Vacinas Anticâncer/farmacologia , Vacinas Anticâncer/uso terapêutico , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Linfócitos T CD8-Positivos , Neoplasias Pulmonares/tratamento farmacológico , Microambiente TumoralRESUMO
Water transport through water channels, aquaporins (AQPs), is vital for many physiological processes including epithelial fluid secretion, cell migration and adipocyte metabolism. Water flux through AQPs is driven by the osmotic gradient that results from concentration differences of solutes including ions. Here, we developed a novel optogenetic toolkit that combines the light-gated anion channel GtACR1 either with the light-gated K+ channel HcKCR1 or the new Na+ channelrhodopsin HcNCR1 with high Na+ permeability, to manipulate water transport in Xenopus oocytes non-invasively. Water efflux through AQP was achieved by light-activating K+ and Cl- efflux through HcKCR1 and GtACR1. Contrarily, when GtACR1 was co-expressed with HcNCR1, inward movement of Na+ and Cl- was light-triggered, and the resulting osmotic gradient led to water influx through AQP1. In sum, we demonstrate a novel optogenetic strategy to manipulate water movement into or out of Xenopus oocytes non-invasively. This approach provides a new avenue to interfere with water homeostasis as a means to study related biological phenomena across cell types and organisms.
Assuntos
Aquaporinas , Água , Channelrhodopsins/genética , Channelrhodopsins/metabolismo , Água/metabolismo , Aquaporinas/genética , Aquaporinas/metabolismo , Transporte Biológico , Permeabilidade , Oócitos/metabolismoRESUMO
Our previous studies showed that extremely low frequency magnetic fields (ELF-MFs) inhibited tumor growth and change proportion of splenic regulatory T cells (Treg cells). Here, we focus on the effect of ELF-MFs on lung metastatic melanoma mouse model and the regulatory mechanism of ELF-MFs on the differentiation of Treg cells. Tumor-bearing mice were exposed to sham ELF-MFs and ELF-MFs (0.4 T, 7.5 Hz) 2 h/day for 27 days. Metastatic tumor burden of lung was significantly decreased after ELF-MF treatment. Compared to the control group, expressions of matrix metalloproteinase (MMP2, MMP9) and forkhead box P3 (Foxp3) in lung nodules significantly decreased in the ELF-MF group. Moreover, in vitro, after stimulated with anti-CD3, anti-CD28 antibodies and transforming growth factor-ß (TGF-ß) and treated with ELF-MFs for 2 h, expression of Foxp3 in total T cells was significantly decreased. Differentiation rate of Treg cells was inhibited from 32.0% to 22.1% by ELF-MFs. Furthermore, reactive oxygen species (ROS) was increased and phospho-serine/threonine protein kinase (p-AKT) was inhibited in both T cells and Jurkat cells. ROS scavenger N-acetyl-l-cysteine reversed inhibition of AKT pathway and expression of Foxp3 from 18.6% to 26.6% in T cells. Taken together, our data show that ELF-MF exposure promoted the inhibitory effect of ROS on AKT pathway and decreased Foxp3 expression, which provides an explanation for why ELF-MF exposure can inhibit differentiation of Treg cells and enhance antitumor effect in metastatic melanoma mouse model.
Assuntos
Diferenciação Celular , Campos Magnéticos , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Espécies Reativas de Oxigênio/metabolismo , Linfócitos T Reguladores/citologia , Animais , Ativação Enzimática , Feminino , Fatores de Transcrição Forkhead/genética , Regulação da Expressão Gênica , Humanos , Células Jurkat , Neoplasias Pulmonares/prevenção & controle , Neoplasias Pulmonares/secundário , Melanoma/patologia , Camundongos , Fosfoproteínas/antagonistas & inibidores , Fosfoproteínas/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Linfócitos T Reguladores/metabolismoRESUMO
Radiotherapy (RT) has been reported to induce abscopal effect in advanced hepatocellular carcinoma (HCC), but such phenomenon was only observed in sporadic cases. Here, we demonstrated that subcutaneous administration of Toll-like receptor 3 (TLR3) agonist poly(I:C) could strengthen the abscopal effect during RT through activating tumor cell ferroptosis signals in bilateral HCC subcutaneous tumor mouse models, which could be significantly abolished by TLR3 knock-out or ferroptosis inhibitor ferrostatin-1. Moreover, poly(I:C) could promote the presentation of tumor neoantigens by dendritic cells to enhance the recruitment of activated CD8+ T cells into distant tumor tissues for inducing tumor cell ferroptosis during RT treatment. Finally, the safety and feasibility of combining poly(I:C) with RT for treating advanced HCC patients were further verified in a prospective clinical trial. Thus, enhancing TLR3 signaling activation during RT could provide a novel strategy for strengthening abscopal effect to improve the clinical benefits of advanced HCC patients.
Assuntos
Carcinoma Hepatocelular , Ferroptose , Neoplasias Hepáticas , Poli I-C , Receptor 3 Toll-Like , Receptor 3 Toll-Like/metabolismo , Receptor 3 Toll-Like/agonistas , Animais , Carcinoma Hepatocelular/radioterapia , Carcinoma Hepatocelular/patologia , Neoplasias Hepáticas/radioterapia , Neoplasias Hepáticas/patologia , Humanos , Camundongos , Poli I-C/farmacologia , Masculino , Feminino , Linhagem Celular Tumoral , Camundongos Endogâmicos C57BL , Modelos Animais de Doenças , Camundongos Knockout , Pessoa de Meia-IdadeRESUMO
Protein purification is the vital basis to study the function, structure and interaction of proteins. Widely used methods are affinity chromatography-based purifications, which require different chromatography columns and harsh conditions, such as acidic pH and/or adding imidazole or high salt concentration, to elute and collect the purified proteins. Here we established an easy and fast purification method for soluble proteins under mild conditions, based on the light-induced protein dimerization system improved light-induced dimer (iLID), which regulates protein binding and release with light. We utilize the biological membrane, which can be easily separated by centrifugation, as the port to anchor the target proteins. In Xenopus laevis oocyte and Escherichia coli, the blue light-sensitive part of iLID, AsLOV2-SsrA, was targeted to the plasma membrane by different membrane anchors. The other part of iLID, SspB, was fused with the protein of interest (POI) and expressed in the cytosol. The SspB-POI can be captured to the membrane fraction through light-induced binding to AsLOV2-SsrA and then released purely to fresh buffer in the dark after simple centrifugation and washing. This method, named mem-iLID, is very flexible in scale and economic. We demonstrate the quickly obtained yield of two pure and fully functional enzymes: a DNA polymerase and a light-activated adenylyl cyclase. Furthermore, we also designed a new SspB mutant for better dissociation and less interference with the POI, which could potentially facilitate other optogenetic manipulations of protein-protein interaction.
Assuntos
Adenilil Ciclases/isolamento & purificação , DNA Polimerase Dirigida por DNA/isolamento & purificação , Proteínas de Escherichia coli/isolamento & purificação , Optogenética , Engenharia de Proteínas , Proteínas de Xenopus/isolamento & purificação , Adenilil Ciclases/genética , Animais , Membrana Celular/enzimologia , Membrana Celular/genética , Análise Custo-Benefício , DNA Polimerase Dirigida por DNA/genética , Escherichia coli/enzimologia , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Luz , Mutação , Optogenética/economia , Ligação Proteica , Engenharia de Proteínas/economia , Multimerização Proteica , Proteínas Recombinantes de Fusão/isolamento & purificação , Fatores de Tempo , Fluxo de Trabalho , Proteínas de Xenopus/genética , Xenopus laevis/genética , Xenopus laevis/metabolismoRESUMO
A hallmark of systemic lupus erythematosus (SLE) is the consistent production of various auto-antibodies by auto-reactive B cells. Interferon-α (IFN-α) signaling is highly activated in SLE B cells and plays a vital role in the antibody response by B cells. Previous studies have shown that CD180-negative B cells, which are dramatically increased in SLE patients, are responsible for the production of auto-antibodies. However, the association between CD180 and IFN-α signaling remains unknown. In the present study, we explored the effect of CD180 on regulating the activation of IFN-α signaling in B cells. We found that the number of CD180-negative B cells was increased in MRL/Mp-Fas(lpr/lpr) lupus-prone mice compared with wild-type mice. Phenotypic analysis showed that CD180-negative B cells comprised CD138+ plasmablast/plasma cells and GL-7+ germinal center (GC) B cells. Notably, ligation of CD180 significantly inhibited the IFN-α-induced phosphorylation of signal transducer and activator of transcription 2 (STAT-2) and expression of IFN-stimulated genes (ISGs) in a Lyn-PI3K-BTK-dependent manner in vitro. Moreover, ligation of CD180 could also inhibit IFN-α-induced ISG expression in B cells in vivo. Furthermore, the Toll-like receptor 7 and Toll-like receptor 9 signaling pathways could significantly downregulate CD180 expression and modulate the inhibitory effect of CD180 signaling on the activation of IFN-α signaling. Collectively, our results highlight the close association between the increased proportion of CD180-negative B cells and the activation of IFN-α signaling in SLE. Our data provide molecular insight into the mechanism of IFN-α signaling activation in SLE B cells and a potential therapeutic approach for SLE treatment.
Assuntos
Antígenos CD/metabolismo , Linfócitos B/metabolismo , Interferon-alfa/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Tirosina Quinases/metabolismo , Transdução de Sinais , Quinases da Família src/metabolismo , Tirosina Quinase da Agamaglobulinemia , Animais , Regulação para Baixo , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Lúpus Eritematoso Sistêmico/imunologia , Lúpus Eritematoso Sistêmico/patologia , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos MRL lpr , Fenótipo , Fosfotirosina/metabolismo , Fator de Transcrição STAT2/metabolismo , Baço/metabolismo , Baço/patologia , Receptor 7 Toll-Like/metabolismo , Receptor Toll-Like 9/metabolismoRESUMO
Mesenchymal stem cells (MSCs) play an important role in the pathology of preeclampsia (PE). Our previous microarray analysis found that microRNA-494 (miR-494) is highly expressed in decidua-derived MSCs (dMSCs) from PE. We hypothesized that aberrant expression of miR-494 in dMSCs is involved in PE development. In the present study, we found that miR-494 arrests G1/S transition in dMSCs by targeting CDK6 and CCND1. We also found that supernatant from miR-494-overexpressing dMSCs reduces HTR-8/SVneo migration and impairs HUVEC capillary formation by suppressing VEGF. Taken together, we report an unrecognized mechanism of miR-494 affecting dMSC proliferation and function in the pathology of PE.
Assuntos
Células-Tronco Mesenquimais/fisiologia , MicroRNAs/fisiologia , Neovascularização Fisiológica , Apoptose , Sequência de Bases , Sítios de Ligação , Movimento Celular , Proliferação de Células , Células Cultivadas , Decídua/patologia , Feminino , Pontos de Checagem da Fase G1 do Ciclo Celular , Expressão Gênica , Células Endoteliais da Veia Umbilical Humana/fisiologia , Humanos , Pré-Eclâmpsia/metabolismo , Pré-Eclâmpsia/fisiopatologia , Gravidez , Interferência de RNA , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
INTRODUCTION: Previous studies have produced controversial results regarding whether mesenchymal stem cells (MSCs) promote or inhibit tumor development. Given the dual role of MSCs in inflammation and cancer, in this study the colitis-associated colorectal cancer (CAC) model was used to examine whether umbilical cord tissue-derived MSCs could prevent neoplasm by inhibiting chronic inflammation. METHODS: MSCs were obtained and identified using flow cytometry. Colitis-associated colorectal cancer model was induced using azoxymethane (AOM) and dextran sulfate sodium (DSS) and MSCs were injected intravenously twice. Levels of immune cells in mesenteric lymph node including regulatory T (Treg) cells were detected using flow cytometry. Naïve T cells and Jurkat cells were co-cultured with MSCs and the effect of MSCs on Treg cells differentiation was evaluated. RESULTS: After injection through tail vein, MSCs could migrate to colon and suppress colitis-related neoplasm. This tumor suppressive effect was characterized by longer colon length, decreased tumor numbers and decreased expression of Ki-67. Moreover, MSCs alleviated the pathology of inflammation in the colitis stage of CAC model and inhibited inflammation cytokines both in colon and serum. Furthermore, Treg cells were accumulated in mesenteric lymph node of MSCs-treated mice while the percentage of T helper cells 2 (Th2) and Th17 were not changed. Of note, MSCs secreted transforming growth factor-ß (TGF-ß) enhanced the induction of Treg cells from naïve T cells. The conditioned medium of MSCs also activated Smad2 signaling, which has been reported to regulate Treg cells. CONCLUSIONS: These results proved that MSCs could migrate to colon tissues and induce the differentiation of Treg cells via Smad2 as so to inhibit the colitis and suppress the development of CAC.
Assuntos
Colite/patologia , Neoplasias Colorretais/prevenção & controle , Células-Tronco Mesenquimais/metabolismo , Proteína Smad2/metabolismo , Linfócitos T Reguladores/imunologia , Animais , Azoximetano , Contagem de Linfócito CD4 , Diferenciação Celular , Linhagem Celular , Movimento Celular , Terapia Baseada em Transplante de Células e Tecidos/métodos , Colite/induzido quimicamente , Colite/imunologia , Colo/citologia , Colo/imunologia , Colo/patologia , Neoplasias Colorretais/patologia , Citocinas/sangue , Sulfato de Dextrana , Modelos Animais de Doenças , Humanos , Inflamação/imunologia , Inflamação/patologia , Inflamação/terapia , Células Jurkat , Ativação Linfocitária/imunologia , Contagem de Linfócitos , Masculino , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/citologia , Camundongos , Camundongos Endogâmicos C57BL , Transdução de Sinais , Linfócitos T Reguladores/citologia , Células Th17/imunologia , Células Th2/imunologia , Cordão Umbilical/citologiaRESUMO
Estrogen is involved in promoting lung cancer cell division and metastasis. MICA and MICB function as ligands for NKG2D, an important immunoreceptor expressed on natural killer (NK) cells. However, whether estrogen regulates MICA/B expression and affects tumor immune escape remains unknown. In this study, we measured the mRNA levels of MICA, MICB and ADAM17in non-small cell lung cancer (NSCLC) cell lines treated with estrogen. Surface expression of MICA/B on LTEP-a2 and A549 was detected using flow cytometry. We demonstrate that both mRNA and secretory protein levels of MICA/B in lung adenocarcinoma cell lines were upregulated by estradiol. Estradiol enhanced the expression of ADAM17, which was associated with the secretion of MICA/B. This secretion of MICA/B downregulated the NKG2D receptor on the surface of NK92 cells and impaired the cytotoxic activity of NK cells. Estradiol enhanced the expression of ADAM17, which was associated with the secretion of MICA/B. Furthermore, a significant correlation between the concentration of estradiol and the expression of MICA was found in tumor tissues of NSCLC patients. Therefore, we conclude that estrogen can regulate the expression and secretion of MICA/B through ADAM17, which helps lung cancer cells escape NKG2D-mediated immune surveillance.
Assuntos
Proteínas ADAM/genética , Estradiol/farmacologia , Regulação Neoplásica da Expressão Gênica , Antígenos de Histocompatibilidade Classe I/genética , Neoplasias Pulmonares/genética , Proteínas ADAM/antagonistas & inibidores , Proteínas ADAM/imunologia , Proteína ADAM17 , Adenocarcinoma/genética , Adenocarcinoma/imunologia , Adenocarcinoma/patologia , Adenocarcinoma de Pulmão , Adulto , Idoso , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/imunologia , Carcinoma de Células Escamosas/patologia , Linhagem Celular Tumoral , Feminino , Antígenos de Histocompatibilidade Classe I/imunologia , Humanos , Vigilância Imunológica , Células Matadoras Naturais , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/patologia , Masculino , Pessoa de Meia-Idade , Subfamília K de Receptores Semelhantes a Lectina de Células NK/genética , Subfamília K de Receptores Semelhantes a Lectina de Células NK/imunologia , Estadiamento de Neoplasias , RNA Mensageiro/genética , RNA Mensageiro/imunologia , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Evasão TumoralRESUMO
MicroRNAs (miRNAs) are small regulatory RNAs that bind directly to complementary sequences on target messenger RNA (mRNA) transcripts, resulting in translational repression and/or target mRNA degradation. MiRNAs have been proven to play critical roles on the development, differentiation, and function of immune cells. Regulatory T cells (Tregs) are of importance in maintaining immune homeostasis and self-tolerance. Although many transcription factors and cytokines are known to regulate Tregs, scientists began to focus on the role of noncoding RNA on the regulation of Treg cells. This review provides an overview of the entire microRNA network and specific miRNAs in the development, differentiation, and function of Tregs.
Assuntos
MicroRNAs/genética , MicroRNAs/metabolismo , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismo , Imunidade Adaptativa/genética , Animais , Autoimunidade/genética , Diferenciação Celular/genética , Citocinas/metabolismo , Humanos , Imunidade Inata/genética , Camundongos , Modelos Biológicos , Ribonuclease III/metabolismo , Linfócitos T Reguladores/citologiaRESUMO
Patients with non-small cell lung cancer (NSCLC) with malignant pleural effusion (MPE) have a short median survival time and increased regulatory T cells (Treg). However, it is unclear whether some specific factors in MPE are involved in Treg recruitment in the progression of NSCLC. Here, we found that Treg population was increased in MPE and inversely correlated with patient survival (P < 0.001). Increased level of CXCL1 in MPE was associated with recruitment of Tregs (P < 0.01). Moreover, miR141 regulated expression of CXCL1 in lung cancer cells, whereas the luciferase test confirmed that CXCL1 is a target of miR141. Chemotaxis assay showed that the miR141-CXCL1-CXCR2 pathway regulates migration of Tregs into MPE. Furthermore, miR141 significantly inhibited tumor growth and metastasis in an immune-competent mouse model. This suppressive function was mediated by the CXCL1-CXCR2 pathway and recruitment of Tregs. Our study uncovered a causative link between microRNA and development of MPE. Mechanistically, decreased expressions of miR141, associated with the survival of patients with NSCLC with MPE, resulted in the increased production of CXCL1 and recruitment of Tregs to promote immune escape of tumor.