The efficacy of neoantigen vaccine for advanced hepatocellular carcinoma (HCC) is limited largely due to insufficient T cell mobilization and activation. Herein, we develop a spleen-targeted neoantigen mRNA vaccine (STNvac) with highly efficient spleen-selective mRNA transfection. Using a three-dose vaccination regimen, STNvac demonstrates remarkable therapeutic efficacy in orthotopic HCC model with a high likelihood of complete tumor regression and significantly improved survival rates (p < 0.0001). Notably, we identify a distinct ISG15+ CD8+ T cell population as crucial mediators of STNvac-induced immunity with potent antigen-processing and cytotoxic capacities. Intriguingly, STNvac promotes the formation of tertiary lymphoid structures (TLSs) through GZMA-F2R-mediated interactions between ISG15+ CD8+ T cells and antigen-presenting cells (APCs), which is also confirmed in HCC patients. Taken together, our findings demonstrate the potent antitumor efficacy of spleen-targeted mRNA vaccine and reveal its underlying immune cell interactive mechanisms, presenting high potential for clinical translation.

Objective: The aim of this study was to explore the effects of Babao dan (BBD), a traditional Chinese medicine, on gastric cancer (GC) progression in vivo . Materials and Methods: A subcutaneous xenograft mouse model of GC was established using MGC80-3 cells. The terminal deoxynucleotidyl transferase-mediated dUTP: 2’-deoxyuridine 5’-triphosphate -biotin nick-end labeling method was adopted to detect cell apoptosis in vivo . The expression levels of proteins associated with proliferation, apoptosis, and angiogenesis were measured by immunohistochemical staining or western blotting (WB). The activation and protein levels of p-c-Jun N-terminal kinase (JNK), p-p38, p-extracellular-regulated kinase 1/2, p-nuclear factor-κB (NF-κB), and p-STAT3 were examined by Bio-plex and WB. Results: BBD significantly inhibited tumor growth in GC mouse models with no adverse effect on body weight or organ function. It was also found that BBD significantly suppressed the proliferation of GC tumor cells, induced the apoptosis of tumor cells, and inhibited angiogenesis through inactivating with mitogen-activated protein kinase, NF-κB, and STAT3 pathways. Conclusions: BBD exerts suppressive effects on GC tumor growth by regulating multiple pathways in vivo , which may provide a novel treatment option for GC therapy.

An immunosuppressive tumor microenvironment (TME) with inadequate and exhausted tumor-infiltrating cytotoxic lymphocytes and abundant cellular immunosuppressors is the major obstacle responsible for the poor efficacy of PD-1/PD-L1 (programmed cell death 1 and its ligand 1) immune checkpoint blockade (ICB) therapy. Herein, a Janus silica nanoparticle (JSNP)-based immunomodulator is explored to reshape the TME for boosting the therapeutic outcomes of αPD-L1 therapy. The designed JSNP has two distinct domains, namely, an ultra pH-responsive side (UPS), which could encapsulate PI3Kγ inhibitor IPI549 in the pore structure, and a polycation-grafted intra-glutathione (GSH)-sensitive side (IGS), which could absorb CXCL9 cDNA on the surface. The final IPI549@UPS-IGS-PDMAEMA@CXCL9 cDNA (IUIPC) could release IPI549 in weak acid TME to target myeloid-derived suppressor cells (MDSCs) to reverse negative immunoregulation and then release CXCL9 cDNA in tumor cells with abundant GSH for sustained CXCL9 chemokine expression and secretion to improve cytotoxic lymphocyte recruitment signals, thereby jointly restoring tumor sensitivity to PD-1/PD-L1 ICB therapy. As expected, the IUIPC-mediated TME remodeling during αPD-L1 therapy significantly ameliorated TME immunosuppression, as well as induced potent systemic antitumor immune responses, which ultimately achieved a robustly boosted antitumor efficacy proven by remarkable suppression of primary tumor growth, obvious prevention of tumor recurrence, and significant regression of abscopal tumors. Hence, the IUIPC-mediated TME-regulating strategy provides an enormous perspective for the improvement of PD-1/PD-L1 ICB therapy.

Due to its tumor-specificity and limited side effects, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has shown great potential in cancer treatments. However, the short half-life of TRAIL protein and the poor death receptor (DR) expression of cancer cells severely compromise the therapeutic outcomes of TRAIL in clinical studies. Herein, a novel ROS-dependent TRAIL-sensitizing nanoplatform, CPT MV, with a Ce6-PLGA core and a TRAIL-modified cell membrane shell was explored to improve the in vivo circulation stability of TRAIL and to amplify TRAIL-induced apoptosis. CPT MV could produce ROS in the targeted cells upon laser irradiation to improve death receptor (DR)-5 expression and trigger Cyt c release from mitochondria. When engaged with TRAIL, the up-regulated DR5 could recruit more Fas-associated death domain (FADD) to transport the extrinsic apoptotic signal to the initiator caspase (caspase 8) and then the executioner caspase (caspase 3), while leaked Cyt c could trigger the intrinsic apoptotic pathway to further strengthen TRAIL-induced apoptosis. Therefore, the designed CPT MV could enhance TRAIL-mediated apoptosis driven by photo-triggered oxidative stress, which provides a very promising approach to clinically overcome tumor resistance to TRAIL therapy.

Despite the huge potential of NK cells in adoptive cell therapy (ACT), formidable physical barriers of the tumor tissue and deficient recognizing signals on tumor cells severely prevent NK cell infiltrating, activating and killing performances. Herein, a novel nano-immunomodulator AuNSP@αCD16 is explored to remodel the tumor microenvironment (TME) for improving the antitumor effects of adoptive NK cells. The as-prepared AuNSP, with a seaurchin-like gold core and a cationic polymer shell, exhibited an excellent gene transfection ability and a stable NIR-II photothermal capacity. The AuNSP could trigger mild photothermal intervention to partly destroy tumors and collapse the dense physical barriers, making a permeable TME for NK cell migration and infiltration. What’s more, the AuNSP could achieve αCD16 gene transfection to modify tumor surface with human CD16 antibody, marking a unique structure on tumor cells for NK recognition and then lead to strong NK activation by CD16-mediated antibody-dependent cellular cytotoxicity (ADCC). The AuNSP@αCD16 induces an immune-favorable TME for NK cell performing killing functions against solid tumors, increasing the release of cytolytic granules and proinflammatory cytokines, which ultimately could achieve a robustly boosted NK cell-based immunotherapy. Hence, the designed AuNSP@αCD16-mediated TME reconstituting strategy provides a substantial perspective for NK-based ACT on solid tumors.

目的:探讨八宝丹(babao dan,BBD)体外抑制淋巴管生成的作用机制.方法:将人淋巴内皮细胞(human lymphatic endothelial cells,HLECs)分为不同浓度的 BBD 组,即0 g·L-1 BBD 组、0.25 g·L-1 BBD 组、0.5 g·L-1 BBD 组及0.75 g·L-1 BBD组.MTT法检测细胞增殖情况;Hoechs33258染色实验观察细胞凋亡情况;细胞划痕实验考察细胞损伤修复能力;Transwell实验考察细胞的迁移能力;管腔形成实验分析细胞淋巴管生成能力;Western Blot检测血管内皮生长因子-C(vascular endothelial growth factor-C,VEGF-C)、血管内皮生长因子受体-3(vascular endothelial growth factor receptor-3,VEGFR-3)、基质金属蛋白酶-2(matrix metalloproteinase-2,MMP-2)和 MMP-9蛋白表达水平.结果:BBD可显著降低HLECs增殖活力及迁移率(P＜0.01);显著升高HLECs凋亡率(P＜0.01);显著抑制HLECs损伤修复能力;显著降低HLECs管腔生成数量及淋巴管生成相关蛋白VEGF-C、VEGFR-3、MMP-2及MMP-9蛋白表达水平(P＜0.05).结论:BBD可促进HLECs凋亡,抑制其增殖、迁移及淋巴管生成,作用可能与下调VEGF-C、VEGFR-3、MMP-2和MMP-9蛋白表达水平有关.

Gastric cancer (GC) is one of the most common gastrointestinal malignancies in the world. Growing evidence emphasizes the critical role of long non-coding RNA (lncRNA) in GC tumorigenesis. The aim of the research was to elucidate the effect and mechanism of Babao Dan (BBD) on lymphangiogenesis of GC in vitro and in vivo via lncRNA-ANRIL/VEGF-C/VEGFR-3 signaling axis. The present study investigated BBD significantly decreased the expression of lncRNA-ANRIL and VEGF-C in GC cells (AGS, BGC823, and MGC80-3) by using real-time quantitative polymerasechain reaction (RT-qPCR) and the secretion and expression of VEGF-C by (enzyme linked immunosorbent assay) ELISA and western blot (WB). BBD significantly inhibited the tumor xenograft of GC growth and the expression of lncRNA-ANRIL, VEGF-C, VEGFR-3 and LYVE-1 in vivo. BBD reduced serum VEGF-C level. In vitro, BBD inhibited the tube formation and decreased the cell viability, proliferation and migration of HLECs by using tube formation, MTT, Hoechst and Transwell assays. In addition, WB assay found that BBD decreased the expression levels of VEGF-C, VEGFR-3, matrix metallopeptidase 2 (MMP-2) and matrix metallopeptidase 9 (MMP-9), and RT-qPCR assay found that the mRNA expression levels of lncRNA-ANRIL, VEGF-C, VEGFR-3, MMP-2, MMP-9, CDK4, Cyclin D1, and Bcl-2 were down-regulated, and the expression of p21 and Bax were increased. Taken together, these results demonstrated that BBD inhibited lymphangiogenesis of GC in vitro and in vivo via the lncRNA-ANRIL/VEGF-C/VEGFR-3 signaling axis.

目的 以低氧诱导因子1(HIF-1)/miR-210回路为切入点,探讨解毒消癥饮(JXY)干预不同肝癌细胞株后葡萄糖代谢的变化以及相关分子生物学作用机制.方法 制备JXY乙酸乙酯提取物并建立体外低氧HepG2、Hep3B、Huh7肝癌细胞模型,通过MTT法和Western blot实验检测低氧、常氧条件下肝癌细胞增殖活性和HIF-1α蛋白表达,以确定低氧细胞模型的成功建立.通过MTT、Hoechst染色检测不同浓度JXY(0、0.05、0.1、0.2 mg/mL)对肝癌细胞存活率和凋亡的作用,以确定最佳干预浓度.将肝癌细胞分为常氧0 mg/mL组、常氧0.1 mg/mL组、低氧0 mg/mL组、低氧0.1 mg/mL组,分别采用细胞爬片染色实验、划痕和迁移实验、葡萄糖含量检测、细胞代谢能量检测、荧光定量PCR及Western blot分别检测肝癌细胞形态学变化、转移侵袭能力和葡萄糖代谢通路中相关因子的变化.结果 与常氧条件下培养的肝癌细胞相比,低氧条件下细胞增殖活性明显增强且随着培养时间的增长而不断升高;HIF-1α蛋白表达也明显上调,且随着培养时间的延长蛋白表达也逐渐升高(P<0.05);与常氧组相比,JXY低氧条件下呈时间和剂量依赖性的抑制肝癌细胞的增殖(P<0.05),在常氧和低氧条件下,与对照组相比,JXY促进肝癌细胞凋亡及抑制迁移及葡萄糖摄取(P<0.05).进一步检测发现JXY下调HIF-1α、葡萄糖转运体蛋白-1(GLUT-1)、己糖激酶(HK)、6-磷酸果糖激酶(PFK)、单羧酸转运蛋白-4(MCT-4)蛋白和miR-210的表达水平(P<0.05).结论 JXY可能通过抑制HIF-1/miR-210的表达,进而抑制肝癌细胞葡萄糖代谢关键酶,从而抑制细胞的糖酵解能力,达到抑制肝癌细胞的增殖、侵袭及转移的目的.

Background: To further elucidate the anti-angiogenesis effect of Babao Dan (BBD) in vitro, gastric cancer (GC) cells and human umbilical vein endothelial cells (HUVECs) were used to evaluate the regulation role of BBD by vascular endothelial growth factor A (VEGFA)/vascular endothelial growth factor receptor 2 (VEGFR2) signaling pathway. Methods: After induced by VEGFA, GC cells (AGS, MGC80-3 and BGC823) were treated by different concentrations of BBD and then were detected cell viability, migration and VEGFA level. And the anti-angiogenesis effect of BBD was evaluated with HUVECs. To furtherly mimic the tumor microenvironment of angiogenesis, VEGFA as an inducer (10 ng/mL) was used to trigger a cascade of angiogenesis of HUVECs in vitro. Results: The viability and migration of GC cells with VEGFA-induced or non-induced and VEGFA levels in GC cells were significantly inhibited by BBD with concentration-dependent manner (P<0.01). BBD significantly inhibited the HUVECs viability with concentration-dependent manner (P<0.01), which was consistent with the inhibitory action on augmentation of cell viability induced by VEGFA ( P<0.01). BBD exhibited the similar inhibitory trend on cyto behavioral variability such as wound repairing (P<0.05), migration (P<0.01) and tube formation (P<0.01) and activation effect on cell apoptosis rate (P<0.01) with VEGFA-induced or non-induced. Moreover, BBD notably regulated the levels of VEGFA, VEGFR2, matrix metalloprotein 2 (MMP2) and matrix metalloprotein 9 (MMP9) of HUVECs on present or absent of VEGFA with dose-dependent manner. Conclusions: BBD inhibited GC growth against VEGFA-induced angiogenesis of HUVECs by VEGFA/VEGFR2 signaling pathway in vitro.

SCOPE:To investigate the effect of Qingjie Fuzheng Granule (QFG) on lymphangiogenesis and lymphatic metastasis in colorectal cancer.METHODS:The effects of QFG on the expression and secretion of vascular endothelial growth factor-C (VEGF-C) in HCT-116 cells were investigated both in vitro and in vivo. HCT-116 cells were treated with different concentrations (0.2, 0.5, and 1.0 mg/mL) of QFG. The VEGF-C expression level was determined using RT-qPCR and western blotting, and the VEGF-C concentration in supernatant was measured by ELISA. Tumor xenograft models of HCT-116 cells were generated using BALB/c nude mice, and the mice were randomly divided into a control group (gavaged with normal saline) and QFG group (gavaged with 2 g/kg QFG). The effect of QFG on tumor growth was evaluated by comparing the volume and weight of tumors between two groups. Immunohistochemistry (IHC) and RT-qPCR were performed to detect the expression levels of VEGF-C, vascular endothelial growth factor receptor 3 (VEGFR-3), and LYVE-1 (lymphatic vessel endothelial hyaluronan receptor 1). ELISA was performed to measure the concentration of serum VEGF-C. TMT proteomics technology and Reactome pathway analysis were used to explore the mechanism of QFG inhibiting lymphangiogenesis in tumor. The VEGF-C (5 ng/mL)-stimulated human lymphatic endothelial cell (HLEC) model was conducted to evaluate the effect of QFG on lymphangiogenesis in vitro. The model cells were treated with different concentrations (0.2, 0.5, and 1.0 mg/mL) of QFG. Cell viability was then determined using an MTT assay. The cell migration, invasion, and tube-formation ability were analyzed using transwell migration, matrigel invasion and tube formation assays, respectively. The underlying mechanism was uncovered, the levels of VEGFR-3, matrix metalloproteinase 2 (MMP-2), matrix metalloproteinase 9 (MMP-9), p-PI3K/PI3K, p-AKT/AKT and p-mTOR/ mTOR were detected using western blotting.RESULTS:QFG significantly reduced VEGF-C expression and secretion in HCT-116 cells. QFG evidently suppressed in vivo tumor growth and the expression of VEGF-C, VEGFR-3, and LYVE-1. The serum VEGF-C level was also reduced by QFG. Moreover, TMT proteomics technology and Reactome pathway analysis identified 95 differentially expressed protein and multiple enriched pathway about matrix metalloproteinase and extracellular matrix, which is direct associate with lymphangiogenesis. In vitro experiment, QFG inhibited the viability, migration, invasion and tube formation of HLECs. Additionally, QFG reduced the VEGFR-3, MMP-2, MMP-9 expression levels, and the p-PI3K/PI3K, p-AKT/AKT, p-mTOR/ mTOR ratios.CONCLUSION:QFG can exert its effect on both tumor cells and HLECs, exhibiting ani- lymphangiogenesis in colorectal cancer via the VEGF-C/VEGFR-3 dependent PI3K/AKT pathway pathway.

目的:探讨解毒消癥饮(JXY)对血管生成因子A(VEGFA)诱导的人脐静脉内皮细胞(HUVECs)凋亡、迁移以及血管生成的体外影响.方法:HUVECs分为对照组、诱导组(VEGFA 10ng/mL)、JXY低剂量组(VEGFA10ng/L+JXY 0.05mg/mL)、JXY中剂量组(VEGFA 10ng/mL+JXY 0.1mg/mL)、JXY高剂量组(VEGFA10ng/mL+JXY 0.2mg/mL),通过MTT、Hoechst、划痕实验,管腔形成实验、Transwell以及Western blot分别检测细胞活力、凋亡、修复能力、血管生成、细胞迁移和VEGFR2、MMP2、MMP9蛋白表达.结果:与诱导组比较,JXY以剂量依赖性地抑制VEGFA诱导的细胞增殖、修复能力,迁移以及体外管腔的形成能力(P＜0.01),促进凋亡(P＜0.05,P＜0.01).进一步研究表明JXY剂量依赖性降低VEGFR2、MMP2、MMP9蛋白表达量.结论:JXY显著抑制VEGFA诱导的HUVECs细胞体外血管生成,与其降低VEGFR2、MMP2、MMP9表达直接相关.

目的 探讨清解扶正颗粒(Qingjie Fuzheng Granules,QFG)对血管生成因子A(vascular endothelial growth factor,VEGF-A)诱导的肿瘤血管生成的抑制作用及其作用机制.方法 体外培养肠癌细胞HCT-8、肝癌细胞Huh7和胃癌细胞MGC80-3,经不同剂量(0、0.25、0.5、1、1.5、2 mg/mL)QFG干预处理后,用MTF法检测细胞活力,Western blot实验检测VEGF-A蛋白表达;体外培养脐静脉血管内皮细胞(HUVECs),HUVECs分为对照组、诱导组(VEGF-A 10 ng/mL)、VEGF-A 10 ng/mL+QFG 0.5 mg/mL组、VEGF-A 10ng/mL+QFG 1 mg/m L组和VEGF-A 10 ng/mL+QFG 2 mg/mL组,通过MTT、DAPI染色、划痕实验、Transwell实验,管腔形成及Western blot实验分别检测细胞活力、凋亡、迁移、血管生成和VEGFR-2蛋白表达.结果 QFG可以抑制肠癌细胞HCT-8、肝癌细胞Huh7和胃癌细胞MGC80-3的细胞活力以及蛋白VEGF-A的表达,且具有统计学差异;QFG能够抑制VEGF-A诱导HUVECs的增殖,促进细胞凋亡,抑制其损伤修复、迁移及管腔生成能力,也可以下调蛋白VEGFR-2的表达水平,具有统计学差异.结论 QFG可靶向VEGF-A抑制肿瘤血管生成.

目的:探讨清解扶正颗粒对体外淋巴管内皮细胞新生淋巴管能力的影响.方法:将体外培养淋巴管内皮细胞分为对照组、实验组(清解扶正颗粒浓度:0.5、1.0、2.0 mg/ml),采用MTT、DAPI染色,划痕损伤修复实验,迁移实验和管腔形成实验分别检测清解扶正颗粒对细胞活力、细胞凋亡、细胞迁移及淋巴管生成能力的影响.结果:清解扶正颗粒可抑制淋巴管内皮细胞增殖,促进细胞凋亡,抑制划痕损伤修复、细胞迁移及淋巴管腔的生成.结论:清解扶正颗粒对体外淋巴管内皮细胞新生淋巴管能力有显著的抑制作用.

Objective The objective was to further investigate apoptosis induction by Babao Dan (BBD), which supports its anti-tumor mechanisms, using two human gastric cancer cell lines (AGS and MGC80-3). Methods After treatment with various BBD concentrations, cell viability and cytotoxic effects were investigated using methyl thiazolyl tetrazolium (MTT) and lactate dehydrogenase (LDH) assays, respectively. The following indicators of cell apoptosis were evaluated: Annexin V-APC staining, caspase-3/-8/-9 activation, and mitochondrial membrane potential loss. Apoptosis-related protein levels (including Bcl-2-associated X protein [Bax], B-cell CLL/lymphoma 2 [Bcl-2], factor associated suicide [Fas], and Fas ligand [FasL]) were determined by western blot. The following multi-pathway factors were also assessed: p-ERK1/2, p-JNK, p-p38, and p-NF-κB. Results The MTT and LDH assays both demonstrated increased BBD cytotoxicity. BBD induced cell apoptosis by stimulating caspase-3/-8/-9 activity and destroying the mitochondrial membrane potential. BBD also regulated key factor expression levels including Bcl-2, Bax, Fas, and FasL and down-regulated protein phosphorylation via the MAPK and NF-κB pathway. Conclusions The possible anti-tumor mechanism is that BBD induces apoptosis via the MAPK and NF-κB signaling pathways.

目的:解毒消癥饮(JXY)是应用于围手术期消化道肿瘤的临床验方.前期工作表明,其可抑制肝癌血管的生成,可能与下调缺氧诱导因子-1(HIF-1)及血管内皮生长因子(VEGF)的表达有关.但该方如何下调HIF-1及VEGF的表达进而抗肿瘤血管生成的机制尚不清楚.为探讨JXY抑制肝癌血管新生的作用机制,本文从表观遗传学角度出发,考察JXY对组蛋白乙酰基转移酶p300和CBP的表达情况,从分子水平研究该方调控VEGF表达的作用机制.方法:制备JXY乙酸乙酯提取物,体外建立常氧和乏氧条件下HepG2肝癌细胞的培养,MTT法检测不同乏氧时间下HepG2肝癌细胞的存活率,RT-PCR法检测HepG2肝癌细胞VEGF和HIF-1αmRNA水平,用以确定乏氧模型构建成功.MTT法检测不同浓度JXY对乏氧条件下HepG2肝癌细胞存活率的影响,RT-PCR法检测不同浓度JXY对乏氧条件下HepG2肝癌细胞VEGF和HIF-1αmRNA表达水平的影响,Western blot法检测不同浓度JXY对乏氧条件下HepG2肝癌细胞VEGF、HIF-1α、CBP和P300蛋白表达的影响.在体建立HepG2肝癌细胞裸小鼠皮下移植瘤动物模型,随机分为模型组和JXY组,模型组给予生理盐水,JXY组给予0.06 g/kg/d JXY.Western blot法和免疫组织化学法检测瘤体VEGF、VEGFR、HIF-1α蛋白表达的影响.结果:与常氧组比较,随着乏氧时间的延长,HepG2肝癌细胞存活率先降低后升高,在乏氧7h时达到波谷(P<0.01).此时间点HepG2肝癌细胞VEGF和HIF-1αmRNA的表达水平也明显升高.随着JXY浓度的升高,乏氧条件下HepG2肝癌细胞存活率逐渐降低,VEGF和HIF-1αmRNA和蛋白表达水平也受到抑制,呈现剂量依赖性(P<0.05或P<0.01).在乏氧条件下HepG2肝癌细胞p300和CBP的蛋白表达水平明显高于常氧组,给予不同浓度JXY后,CBP、P300蛋白的表达量逐渐减弱,有剂量依赖性(P<0.05或P<0.01).在体实验也观察到JXY对裸小鼠皮下移植瘤VEGF、VEGFR和HIF-1α蛋白表达水平呈现明显的抑制作用.结论:JXY可能通过抑制组蛋白乙酰基转移酶p300和CBP的表达水平,抑制其与HIF-1α结合形成HIF-1α/p300(CBP)低氧诱导复合物,进而抑制VEGF的转录,最终抑制肿瘤新生血管的生成.

目的 观察八宝丹(BBD)对人脐静脉内皮细胞(HUVEC)增殖和迁移能力的影响. 方法 体外培养HUVEC,使用不同浓度BBD进行干预,MTT法检测HUVEC增殖情况,倒置显微镜观察细胞形态、划痕损伤,Transwell实验观察细胞损伤修复及迁移能力. 结果 BBD能显著降低HUVEC的活力,抑制HUVEC增殖,抑制HUVEC的损伤修复和迁移能力,并呈现剂量依赖性. 结论 BBD具有抑制HUVEC增殖扣迁移的作用.