膀胱移行细胞癌来源的exosome诱导体外细胞毒性T细胞杀伤效应

目的 观察膀胱移行细胞癌T24细胞来源的exosome体外诱导细胞毒性特异性T淋巴细胞(CTL)对肿瘤细胞的杀伤效应.方法 采用超滤和蔗糖密度梯度离心法分离T24细胞释放的exosome,电镜、Western blot观察exosome的特征.将exosome和肿瘤细胞负载到人外周血分离培养的树突状细胞(Dc)上,并与T细胞体外共同培养,分为exosome致敏DC组、未致敏DC组和对照组,Alamar blue检测CTL对T24细胞的细胞毒活性.结果 T24细胞分泌的exosome为直径约30～90nm的类圆碟形小囊泡.Western blot证实,exosome表达热休克蛋白70(HSP70)、细胞间黏附分子1(ICAM-1)和人细胞角蛋白20(CK20)分子.与未致敏DC组和对照组比较,exosome致敏DC组活化的T细胞对T24细胞有更强的细胞毒活性(P＜0.01).结论 T24细胞来源的exosome负载了HSP70、ICAM-1等免疫相关蛋白;exosome经DC负载后活化CTL产生抗肿瘤活性.     

IL-18基因修饰增强肿瘤细胞裂解物致敏的树突状细胞的抗肿瘤效应

目的:观察经肿瘤细胞裂解物致敏的白细胞介素18(IL-18)基因修饰的树突状细胞(DC)体内诱导的抗肿瘤免疫应答反应.方法:体外培养的小鼠骨髓树突状细胞经IL-18重组腺病毒感染后(IL18-DC),再经Hepal-6肝癌细胞裂解物冲击致敏后通过皮下注射用于荷瘤小鼠的治疗.用ELISA检测细胞因子,4 h51Cr释放法检测NK细胞活性及CTL杀伤活性.结果:致敏IL18-DC组体内诱导NK细胞活性与未致敏IL18-DC组无明显差别(P＞0.05),但明显高于致敏DC组和DC组(均P＜0.01);致敏IL18-DC组体内诱导特异性CTL杀伤活性明显高于IL18-DC组、致敏DC组和DC组(均P＜0.01);致敏IL18-DC组免疫治疗作用明显优于未致敏IL18-DC组、致敏DC组和DC组(均P＜0.01).结论:肿瘤细胞裂解物致敏的IL-18基因修饰的DC疫苗进行体内免疫治疗,能诱导出显著的抗肿瘤免疫反应,为DC介导的肿瘤基因治疗开辟了新的途径.     

免疫原性细胞死亡相关分子的表达机制及对免疫的调节

研究表明,用部分化学药物和放射线等手段治疗肿瘤,肿瘤发生免疫原性细胞死亡（ICD）,随后细胞表面高表达损伤相关分子模式(DAMPs),如钙网蛋白（CRT）、三磷酸腺苷（ATP）、热休克蛋白(HSP)、高迁移率族蛋白B 1（HMGB1）信号分子,增强肿瘤细胞的免疫原性,招募树突状细胞(DC)到肿瘤床并提高其功能,激活特异性的细胞毒性T淋巴细胞(CTL)对肿瘤的攻击。ICD及其DAMPs为肿瘤治疗提供了新的治疗依据和手段,监测化疗前后肿瘤细胞免疫原性的变化,将化疗和免疫治疗有机结合,可提高肿瘤的治疗效果。本文对ICD相关分子的表达机制及对机体免疫的调节等进行综述。     

Bcap37完全细胞抗原冲击树突状细胞诱导特异性抗乳腺癌免疫实验研究

目的　研究 Bcap37完全细胞抗原冲击树突状细胞 (DC)能否诱导特异性抗肿瘤免疫 ,探索乳腺癌的免疫治疗方法。方法　从人外周血分离单核细胞 ,用 GM- CSF和 IL - 4诱导出 DC,反复冻融制备 Bcap37完全细胞抗原 ,将此抗原冲击的 DC与 T细胞共培养 ,以 7天后分离的 T细胞为效应细胞 ,与肿瘤细胞混合培养 ,MTT法测定对肿瘤细胞的抑制作用。结果　单核细胞经细胞因子诱导 8天 ,形态、表型及功能鉴定为成熟 DC;完全细胞抗原冲击 DC诱导的 T细胞对肿瘤细胞具有明显的抑制作用。结论　 Bcap37完全细胞抗原冲击 DC可有效诱导特异性抗肿瘤免疫     

BXD纯系小鼠树突状细胞抗肿瘤免疫功能的基因定位分析

目的:探讨DC的抗肿瘤免疫功能是否与其基因位点有关.方法:选择19株基因型明确的BXD纯系小鼠的树突状细胞(DC),MTT法和ELISA分析对DC吞噬肿瘤细胞的作用;肿瘤细胞诱导DC产生IL-12的能力;经肿瘤细胞刺激后的DC对T细胞增殖和IFN-γ分泌的影响进行测定.用流式细胞仪检测DC表面抗原标志CD80和CD54.有关数据用MapManagerQTX and WebQTL软件对DC抗肿瘤免疫的基因位点进行分析.结果:DC刺激T细胞增殖的作用很大程度取决于BXD各系的遗传差异.DC吞噬肿瘤细胞及诱导IL-12产生的能力都与DC刺激T细胞的增殖相一致(P＜0.01).基因位点定量分析(QTL)分析在第6和第13号染色体上有2个位点可能与DC的肿瘤抗原提呈加工功能和T细胞毒作用有关.结论:不同系的BXD小鼠,其DC的抗原提呈、加工能力不同.DC对肿瘤细胞生长的抑制与IL-12的诱导产生和DC表面共刺激分子的表达呈平行.其有关基因位点分别于第6和第13号染色体上.     

树突状细胞与细胞因子诱导杀伤细胞治疗肿瘤研究进展

树突状细胞(dendritic cells,DC)是已知的功能最强的专职抗原提呈细胞,在抗原加工提呈、抗原识别及T细胞激活中发挥重要作用.而细胞因子诱导的杀伤细胞(cytokineinduced killer cells,CIK)是由多种细胞因子诱导而成,对多种肿瘤细胞具有杀伤作用的细胞毒性T细胞.以DC为基础的肿瘤免疫治疗日益受到人们的重视.文章就近年DC与CIK免疫治疗肿瘤的研究进展作一综述.     

树突状细胞与肿瘤细胞融合瘤苗研究进展

树突状细胞(DC)与肿瘤细胞融合所获得的杂交瘤细胞,既可内源性表达肿瘤抗原,又具DC抗原递呈能力,能够诱导特异抗肿瘤细胞毒性T淋巴细胞(CTL),有望成为安全高效的肿瘤疫苗应用于临床.现将其研究进展作一综述.     

非小细胞肺癌中血管内皮生长因子与树突细胞的关系

血管内皮生长因子(VEGF)是重要的肿瘤细胞生长因子之一,其可以抑制树突细胞(DC)的分化成熟,诱导成熟DC功能障碍,而DC亦可通过自分泌VEGF加重自身的功能障碍,从而介导肿瘤细胞逃逸免疫监视.因此VEGF和DC的相互关系与非小细胞肺癌(NSCLC)的分化程度、淋巴结转移、临床分期密切相关.二者关系可为临床NSCLC的治疗提供相应理论依据.     

树突状细胞联合细胞因子诱导的杀伤细胞对大肠癌细胞株LOVO体外杀伤作用的研究

目的研究树突状细胞(DC)联合细胞因子诱导的杀伤细胞(CIK)对大肠癌细胞株LOVO的杀伤及诱导凋亡作用。方法 从健康供者外周血中提取出单个核细胞, 用不同的细胞因子诱导出DC及CIK细胞, 待DC成熟后将DC与CIK混合培养。细胞计数法测定细胞的增殖、MTT法测定细胞杀伤活性、透射电镜观察肿瘤细胞的凋亡、Western Blot法检测效应细胞表面FasL表达。结果 DC细胞对CIK细胞具有很强的促增殖作用, 且DC-CIK细胞共培养组在各效靶比的杀伤活性均明显高于单独CIK细胞培养组(P＜0.01)。DC可以促进CIK诱导肿瘤细胞发生凋亡, 且DC-CIK、CIK细胞均大量表达FasL。结论 DC细胞可以显著提高CIK细胞的增殖活性和细胞毒活性, 其共培养的作用机制之一可能是通过Fas/FasL途径而实现。     

肺癌gp96-多肽复合物/DC疫苗体外诱导CTL反应的实验研究

目的 研究热休克蛋白gp96－多肽复合物负载树突状细胞（dendritic cell,DC）后，能否诱导出gp96－多肽复合物特异性的细胞毒性T细胞（cytotoxic T lymphocyte,CTL）反应。方法 从一例肺癌患者肿瘤组织中提取gp96-多肽复合物和自体肿瘤细胞溶解物，分别负载从该患者骨髓血中培养的DC。以不同形式的抗原／DC疫苗分别刺由患者外周血中分离的淋巴细胞。采用ELISA法检测淋巴细胞所释放的IFN－γ量作为CTL反应的指标；以Cr^51释放实验分析致敏后的淋巴细胞对不同靶细胞的裂解和杀伤作用。结果 所有肿瘤抗原致敏淋巴细胞后均可以诱导产生CTL反应，其中以gp96－多肽复合物／DC疫苗诱导释放的IFN－γ量最高。肿瘤抗原致敏淋巴细胞后对原代培养的肿瘤细胞的杀伤作用高于PG细胞和K562细胞。结论 自体肿瘤组织中提取的gp96－多肽复合物能诱导出肿瘤特异性CTL反应，而负载DC后能激发起更强的CTL。     

Effect of DC therapy combined with chemotherapy in advanced cancer cases

Dendritic cells (DC) are powerful antigen-presenting cells, and have attracted attention in recent years from the viewpoint of DC vaccine therapy against cancer. However, the existence of an immunosuppressive state in cancer individuals leads to anergy and immunotolerance, which has been reported to be caused by T cell and DC immunosuppressive subsets or cytokines such as Th2, Tc2, CD4+CD25+, DC2 and IL-10 against Th1, Tc1, DC1 and IL-12. Therefore, DC therapy could be incompatible with severe chemotherapy. Conversely, there are some reports that indicate tumor specific cytotoxicity in DC therapy could be augmentedly un exposure to tumor antigen caused by apoptosis in combination radiation or chemotherapy. In this study we examined the usefulness of DC therapy combined with chemotherapy and BRM (PSK) administration by analyzing the immunocyte subsets and cytokines as well as the combination effect. The results indicate this method can be useful in advanced cancer patients.     

非小细胞肺癌患者体外诱导的肿瘤细胞老化与化疗客观疗效关系的研究

Objective： To investigate the relationship between the objective response to combination chemotherapy of taxanes plus cisplatin in non-small cell lung cancer （NSCLC） and docetaxel plus cisplatin （DC regime） induced senescence of tumor cells in vitro. And its relation to mutant P53 protein （m-P53） was also to be evaluated. Methods： Sixty-seven specimen obtained from NSCLC patients from January 1, 2003 to June 30, 2006. The patients consisted of 48 males and 19 females, ranging in age from 54 to 82 years （mean, 67.5 years）, 41 cases were diagnosed as pathological stage Ⅲb, 26 cases were diagnosed as stage Ⅳ. Thirty-nine tumors were confirmed to be adenocarcinomas, 28 were confirmed to be squamous cell carcinomas. All patients accepted 2-6 cycles combination chemotherapy of Taxanes （docetaxel 40 mg/m^2, d1; d8, or paclitaxel 175 mg/m^2, d1） plus cisplatin （CDDP, 25 mg/m^2, d2-4）. Patients were divided into chemoresponsive （CR ＋ PR） and chemoresistant （SD ＋ PD） groups according to objective response status which was evaluated by RECIST system. Tumor cells from specimens of bronchoscopic, surgical biopsy and pleural effusion cell collection had been cultured and treated with DC in vitro. The m-P53 of culture supernatant was measured by ABC-ELISA kit before DC treatment. The telomerase activity was determined by the telomeric repeat amplification protocol （TRAP） based PCR-ELISA kit and apoptosis was determined by TdT-mediated d-UTP-X nick-end labeling （TUNEL） assay. Data represent as both actual detected and positive value. The senescence of tumor cells defined as that, apoptosis rate increased more than 50% to control, and telomerase activity decreased less than 50% to control. Results： There was no significant difference between clinical treatment response and sex, pathological type, specimen origin, or m-P53 status in cultured cell supernatant. Telomerase activity and apoptosis rate was positive in 61.1% （41/67） and 25.4%（17/67） of all samples respectively. A significant difference of senescence of tumor cells treated by DC, was existed between chemoresponsive and chemoresistant patients groups （P 〈 0.05）. Multinomial logistic regression analyses shew that telomerase activity decreased less than 50% in vitro may be an indicator of clinical response for taxanes plus cisplatin chemotherapy. Odds ratio was 4.226, P 〈 0.05. Conclusion： For NSCLC, DC induce lung cancer tumor cells senesce in vitro may be a promising predicator for clinical response, but the relationship between objective response by chemotherapy and detectable m-P53 or DC induced apoptosis is still obscure.     

The ERBB4/HER4 intracellular domain 4ICD is a BH3-only protein promoting apoptosis of breast cancer cells

ERBB4/HER4 (referred to here as ERBB4) is a unique member of the epidermal growth factor receptor (EGFR) family of receptor tyrosine kinases. In contrast to the other three members of the EGFR family (i.e., EGFR, ERBB2/HER2/NEU, and ERBB3), which are associated with aggressive forms of human cancers, ERBB4 expression seems to be selectively lost in tumors with aggressive phenotypes. Consistent with this observation, we show that ERBB4 induces apoptosis when reintroduced into breast cancer cell lines or when endogenous ERBB4 is activated by a ligand. We further show that ligand activation and subsequent proteolytic processing of endogenous ERBB4 results in mitochondrial accumulation of the ERBB4 intracellular domain (4ICD) and cytochrome c efflux, the essential and committed step of mitochondrial regulated apoptosis. Our results indicate that 4ICD is functionally similar to BH3-only proteins, proapoptotic members of the BCL-2 family required for initiation of mitochondrial dysfunction through activation of the proapoptotic multi-BH domain proteins BAX/BAK. Similar to other BH3-only proteins, 4ICD cell-killing activity requires an intact BH3 domain and 4ICD interaction with the antiapoptotic protein BCL-2, suppressed 4ICD-induced apoptosis. Unique among BH3-only proteins, however, is the essential requirement of BAK but not BAX to transmit the 4ICD apoptotic signal. Clinically, cytosolic but not membrane ERBB4/4ICD expression in primary human breast tumors was associated with tumor apoptosis, providing a mechanistic explanation for the loss of ERBB4 expression during tumor progression. Thus, we propose that ligand-induced mitochondrial accumulation of 4ICD represents a unique mechanism of action for transmembrane receptors, directly coupling a cell surface signal to the tumor cell mitochondrial apoptotic pathway.     

Chemotherapy‐induced immunogenic modulation of tumor cells enhances killing by cytotoxic T lymphocytes and is distinct from immunogenic cell death

Certain chemotherapeutic regimens trigger cancer cell death while inducing dendritic cell maturation and subsequent immune responses. However, chemotherapy‐induced immunogenic cell death (ICD) has thus far been restricted to select agents. In contrast, several chemotherapeutic drugs modulate antitumor immune responses, despite not inducing classic ICD. In addition, in many cases tumor cells do not die after treatment. Here, using docetaxel, one of the most widely used cancer chemotherapeutic agents, as a model, we examined phenotypic and functional consequences of tumor cells that do not die from ICD. Docetaxel treatment of tumor cells did not induce ATP or high‐mobility group box 1 (HMGB1) secretion, or cell death. However, calreticulin (CRT) exposure was observed in all cell lines examined after chemotherapy treatment. Killing by carcinoembryonic antigen (CEA), MUC‐1, or PSA‐specific CD8⁺ CTLs was significantly enhanced after docetaxel treatment. This killing was associated with increases in components of antigen‐processing machinery, and mediated largely by CRT membrane translocation, as determined by functional knockdown of CRT, PERK, or CRT‐blocking peptide. A docetaxel‐resistant cell line was selected (MDR‐1⁺, CD133⁺) by continuous exposure to docetaxel. These cells, while resistant to direct cytostatic effects of docetaxel, were not resistant to the chemomodulatory effects that resulted in enhancement of CTL killing. Here, we provide an operational definition of “immunogenic modulation,” where exposure of tumor cells to nonlethal/sublethal doses of chemotherapy alters tumor phenotype to render the tumor more sensitive to CTL killing. These observations are distinct and complementary to ICD and highlight a mechanism whereby chemotherapy can be used in combination with immunotherapy.     

Effects of an antibody to vascular endothelial growth factor receptor-2 on survival, tumor vascularity, and apoptosis in a murine model of colon carcinomatosis

Vascular endothelial growth factor (VEGF) is the predominant regulator of colon cancer angiogenesis and is associated with a poor prognosis and the development of metastases. We hypothesized that DC101, an antibody against the VEGF receptor-2 (flk-1), may be efficacious in the therapy of colon cancer peritoneal carcinomatosis in a murine model. BALB/c mice underwent intraperitoneal injection of CT-26 colon cancer cells to generate peritoneal metastases. Mice received control solvent or DC101 for up to 60 days. In parallel studies, mice were sacrificed at sequential time points to determine the effect of DC101 on tumor angiogenesis, tumor cell proliferation and apoptosis, and endothelial cell apoptosis. Mice treated with DC101 demonstrated a 30% increase in mean survival. In addition, DC101 also led to a significant decrease in tumor vascularity, growth and tumor cell proliferation. In sequential studies, anti-VEGF-R therapy led to a progressive increase in endothelial cell apoptosis followed by an increase in tumor cell apoptosis. These findings suggest that anti-flk-1 therapy may prolong survival in patients with colon cancer carcinomatosis. The temporal studies demonstrating that anti-flk-1 therapy lead to an increase in endothelial cell apoptosis that in turn lead to an increase in tumor cell apoptosis confirms the role of VEGF as an endothelial cell survival factor.     

Enhanced cancer therapy with the combination of EGFR and VEGFR-2 targeting in an orthotopic glioblastoma model

Using an orthotopic glioblastoma model, we investigated the activity of the combination of monoclonal antibody DC101 against vascular endothelial growth factor receptor-2 (VEGFR-2) and monoclonal antibody C225 against epidermal growth factor receptor (EGFR). Nude mice bearing intracerebral glioblastoma xenografts were administered either DC101 or C225, or the combination via intraperitoneal (i.p.) injection. Histopathological analysis of solid tumor volume, microvessel density, tumor cell proliferation and apoptosis were performed. In the DC101-treated group, solid tumor volume and microvessel density were reduced by 59.7% and 64%, respectively. The tumor cell proliferation level was reduced by 53.2% and tumor cell apoptosis was increased by 66.7% but there was enhanced tumor cell invasiveness. C225 alone reduced the invasiveness of tumor tissue, but had no effect on solid tumor growth, microvessel density, tumor cell proliferation or apoptosis. The combination cancer therapy with C225 and DC101 enhanced tumor treatment with reduced tumor volume, microvessel density, tumor cell proliferation level, and increased cancer cell apoptosis, while decreasing tumor cell invasiveness.     

Autophagy facilitates the release of immunogenic signals following chemotherapy in 3D models of mesothelioma

We have previously shown that nearly half of mesothelioma patients have tumors with low autophagy and that these patients have a significantly worse outcome than those with high autophagy. We hypothesized that autophagy may be beneficial by facilitating immunogenic cell death (ICD) of tumor cells following chemotherapy. An important hallmark of ICD is that death of tumor cells is preceded or accompanied by the release of damage‐associated molecular pattern molecules (DAMPs), which then can stimulate an antitumor immune response. Therefore, we measured how autophagy affected the release of three major DAMPs: high mobility group box 1 (HMGB1), ATP, and calreticulin following chemotherapy. We found that autophagy in three‐dimensional (3D) models with low autophagy at baseline could be upregulated with the cell‐permeant Tat‐BECN1 peptide and confirmed that autophagy in 3D models with high autophagy at baseline could be inhibited with MRT 68921 or ATG7 RNAi, as we have previously shown. In in vitro 3D spheroids, we found that, when autophagy was high or upregulated, DAMPs were released following chemotherapy; however, when autophagy was low or inhibited, DAMPs release was significantly impaired. Similarly, in ex vivo tumors, when autophagy was high or upregulated, HMGB1 was released following chemotherapy but, when autophagy was low, HMGB1 release was not seen. We conclude that autophagy can be upregulated in at least some tumors with low autophagy and that upregulation of autophagy can restore the release of DAMPs following chemotherapy. Autophagy may be necessary for ICD in this tumor.     

树突状细胞体外诱导抗肿瘤免疫通过诱导肿瘤细胞凋亡抑制裸鼠移植瘤生长

研究树突状细胞（ＤＣ）体外诱导的细胞免疫抑制裸鼠移植瘤生长作用及其机理。方法：联合应用粒／巨噬细胞集落刺激因子ＫＭ－ＣＳＦ）及白介素－４（ＩＬ－４）直接从肝癌患者外周血中培养出ＤＣ,以人肝癌细胞系ＨｅｐＧ２肿瘤细胞的肿瘤抗原粗提物刺激ＤＣ,ＤＣ激活同源的Ｔ淋巴细胞,建立裸鼠人肝癌细胞系ＨｅｐＧ２移植瘤模型,以被激活的Ｔ淋巴细胞治疗裸鼠ＨｅｐＧ２移植瘤并观察治疗效果,检测移植瘤标本肿瘤细胞凋亡情况。结果：ＤＣ诱导的Ｔ细胞免疫能够诱导裸鼠人肝癌细胞系ＨｅｐＧ２移植瘤肿瘤细胞大量凋亡从而抑制裸鼠ＨｅｐＧ２移植瘤生长。结论：经肿瘤抗原激活的ＤＣ作为一新概念上的抗肿瘤疫苗有可能在肿瘤的治疗中发挥重要作用。     

The HER4/4ICD estrogen receptor coactivator and BH3-only protein is an effector of tamoxifen-induced apoptosis

Greater than 40% of breast cancer patients treated with tamoxifen exhibit de novo or acquired tumor resistance. Recent clinical evidence indicates that loss of expression of HER4 is an independent marker for tamoxifen resistance. In direct corroboration with clinical observations, suppression of HER4 expression in the tamoxifen-sensitive MCF-7 and T47D breast tumor cell lines resulted in resistance to tamoxifen-induced apoptosis. Furthermore, HER4 expression was lost in three independent MCF-7 models of acquired tamoxifen resistance. The HER4 intracellular domain (4ICD) is an independently signaling nuclear protein that functions as a potent ERalpha coactivator. In addition, mitochondrial 4ICD functions as a proapoptotic BH3-only protein. Tamoxifen disrupts an estrogen-driven interaction between ERalpha and 4ICD while promoting mitochondrial accumulation of the 4ICD BH3-only protein. BCL-2 inhibition of tamoxifen-induced apoptosis and tamoxifen activation of BAK, independent of BAX, further supports a role for 4ICD during tamoxifen-induced apoptosis. Finally, reintroduction of HER4, but not HER4 with a mutated BH3 domain, restores tamoxifen sensitivity to tamoxifen-resistant TamR cells in a xenograft model. Clinically, breast cancer patients with tumor expression of nuclear 4ICD responded to tamoxifen therapy with no clinical failures reported after 14 years of follow-up, whereas 20% of patients lacking nuclear 4ICD expression succumbed to their disease within 10 years of diagnosis. Our identification of the HER4/4ICD BH3-only protein as a critical mediator of tamoxifen action provides a clinically important role for 4ICD in human cancer and reveals a potential tumor marker to predict patient response to tamoxifen therapy.