体内阻断胰腺癌细胞FasL表达抑制肿瘤细胞免疫反击的初步研究

目的:探讨体内抑制胰腺癌细胞表面的FasL表达,能否抑制胰腺癌细胞对免疫系统的反击作用。方法:反义核酸技术扩增C57BL/6小鼠FasL mRNA 35~371位点核苷酸序列互补的cDNA片断,构建pBlast质粒后,转染人Panc-1胰腺癌细胞株,蛋白质印迹观察细胞株FasL蛋白表达的变化,ELISA法检测培养上清可溶性FasL水平。将细胞注射到C57BL/6小鼠胰腺包膜下,观测瘤体的直径变化,免疫组化观测肿瘤组织内浸润淋巴细胞的数量。结果:在稳定转染编码反义FasL cDNA的质粒后,胰腺癌细胞表面的FasL表达明显下调;其FasL蛋白表达下降了80%;FasL表达下调对体外生长的胰腺癌细胞无明显影响,但能减少同系免疫活性鼠体内肿瘤的发展。转染了质检DNA的胰腺癌细胞接种组瘤体的平均直径为1.53 mm,而未转染质检DNA的胰腺癌细胞接种组为2.98 mm,两组比较差异有统计学意义,P<0.01。与未来转染组相比,转染组CD45 TIL细胞数量明显增多,是未转染组的3.23倍,P<0.01。结论:胰腺癌细胞表面FasL表达的下调,能在体内增加免疫系统的抗肿瘤能力,从而为"肿瘤反击"促进肿瘤细胞浸润的学说提供了有力的证据。     

Fas (CD95/APO-1) and Fas ligand expression in normal pancreas and pancreatic tumors. Implications for immune privilege and immune escape

BACKGROUND: Fas (CD95/APO-1) and Fas ligand (FasL) play key roles in immunologic homeostasis and immune privilege and may regulate normal cell turnover. Earlier studies had suggested that FasL-positive pancreatic carcinoma cell lines can induce apoptosis in T cells, thereby evading host immune surveillance. In the current study the authors have characterized the expression of Fas and FasL in the normal pancreas and in pancreatic neoplasia. METHODS: Pancreatic resection specimens with ductal-type adenocarcinoma or intraductal dysplasia (n = 41), nonductal pancreatic neoplasms (n = 5), and chronic pancreatitis (n = 4) were examined for Fas and FasL expression by immunohistochemistry. The results in invasive adenocarcinoma were compared to those for benign ducts and intraductal dysplasia, and correlated with clinicopathologic features of the tumors and with patient survival. RESULTS: Fas was expressed in the normal pancreatic ducts and in intraductal dysplasia in a mixed membrane/cytoplasmic pattern. In all cases of invasive ductal-type adenocarcinoma, membranous Fas could not be detected; cytoplasmic Fas staining was reduced or completely lost. Loss of Fas expression in pancreatic ductal-type adenocarcinomas significantly correlated with poorer differentiation and extrapancreatic spread of the tumors and was associated with a shorter overall survival. FasL expression was present in the normal pancreatic ducts as well as in islet cells and was maintained in all pancreatic tumors. CONCLUSIONS: These results implicate the Fas pathway in the regulation of physiologic cell turnover and immune privilege in the normal pancreas and indicate that loss of Fas expression is correlated with malignant transformation and biologic aggressiveness in pancreatic adenocarcinomas. This may represent a mechanism by which pancreatic tumor cells become resistant to apoptosis and escape immune surveillance in vivo.     

Human pancreatic cancer cells disable function of Fas receptors at several levels in Fas signal transduction pathway

The aims of this study were to evaluate the functional expression of Fas receptors (Fas) in human pancreatic cancer cell lines; Capan-1, AsPC-1, BxPC-3, PANC-1, and MIA PaCa-2 and to search for the mechanisms of receptor-mediated inhibition of Fas signaling in these cells. Despite the expression of Fas receptors at considerable levels, exposure of these cells to agonistic Fas antibodies (500 ng/ml) induced only minimal apoptosis in 4 cell lines. The mechanisms allowing resistance to Fas-mediated apoptosis are complex. Using RT-PCR, we identified molecules which might counteract the apoptogenic signal at several levels of Fas signal transduction pathway. The most striking findings were the overexpression of Fas decoy receptors (DcR3), Fas associated phosphatase-1 (FAP-1), and FLICE-inhibitory protein (c-FLIP) in the resistant cell lines as well as in pancreatic cancer surgical specimens. In conclusion, pancreatic cancer cells express three molecules that can abrogate Fas function at different levels of Fas signaling cascade, resulting in resistance to Fas-mediated apoptosis, and this may promote the progression of this malignancy.     

Human pancreatic cancer cell lines do not express receptors for somatostatin.

The in vivo administration of somatostatin (SS) or its analogues is capable of suppressing the growth of pancreatic cancer in experimental animals. We examined the effects of SS-14 and its analogue RC-160 on the in vitro growth of two human pancreatic cancer cell lines MiaPaCa-2 and Panc-1 stimulated with epidermal growth factor (EGF) or insulin-like growth factor 1 (IGF-1). Neither SS-14 nor RC-160 inhibited the growth of either cell line. In contrast RC-160 did inhibit the EGF-stimulated growth of a rat pancreatic cancer cell line AR42J. Binding studies with 125I-Tyr11 somatostatin revealed the presence of a single class of high affinity binding sites with a Kd of 0.20 +/- 0.05 nM and a Bmax of 2.1 +/- 0.26 pmoles mg-1 protein on AR42J but not displaceable binding was observed on MiaPaCa-2 or Panc-1. We conclude that lack of receptors accounts for the failure of SS-14 and RC-160 to influence the growth of human pancreatic cancer in vitro. These results, taken together with other findings, lead us to question the therapeutic efficacy of somatostatin and its analogues as mono-therapy in the treatment of human pancreatic cancer.     

肿瘤干细胞免疫逃逸机制及相关免疫疗法的研究进展

肿瘤干细胞（cancer stem cell,CSC）与正常干细胞生物学特征相似,具有自我更新、无限增殖和抗化学毒物损伤的能力, 与肿瘤的发生、治疗、预后、复发和转移关系极为密切。在肿瘤发生初期,虽然机体的免疫监视系统可以有效地对肿瘤细胞进行识 别和清除,但CSC可通过下调抗原加工和提呈机制成分、分泌免疫抑制因子、高表达免疫检查点分子以及激活免疫耐受信号通路等 机制调控免疫细胞功能,或促进抑制性免疫微环境的建立以逃避免疫系统对其的清除,从而使肿瘤得以进展。目前,靶向CSC与免 疫系统相互作用的多种方法正在被积极研究中,一些靶向CSC的新型免疫疗法正处于临床研发阶段。本文综述了近年来CSC相关 免疫逃逸机制及针对免疫逃逸的可能有效的治疗手段（包括DC疫苗、CAR-T细胞、免疫检查点抑制剂等）的研究进展。     

Leukemic dendritic cells: potential for therapy and insights towards immune escape by leukemic blasts.

Dendritic cells (DCs) are a system of potent antigen-presenting cells (APCs) specialized to initiate primary immune responses. DCs are considered important elements in the induction of specific antitumor cytotoxic effectors. At present, because of potential therapeutic implications, the critical role of DCs in cancer patients is under intensive investigation. Interactions between DCs and acute myeloid leukemia cells represent an attractive model for the study of DC physiology. Moreover, DCs can be a valuable therapeutic tool for the adjuvant treatment of leukemic patients. However, DC subsets in vivo may also be affected by leukemogenesis and may contribute to the escape of leukemia from immune control. The aim of this review is to shed further light on this paradoxical picture where the line between immune tolerance and immune defense is narrow.     

Human small cell lung cancer cells express functional VEGF receptors, VEGFR-2 and VEGFR-3

Studies have suggested that the vascular endothelial growth factors (VEGFs)/VEGF receptors (VEGF-Rs) system plays an important role in tumour growth and metastasis. We conducted the present study to clarify whether small cell lung cancer (SCLC) cells express functional VEGF-Rs and VEGFs, and their biological significance in the SCLC progression. We examined expression of VEGF and VEGF-C, and their receptors, VEGFR-2 and VEGFR-3, in five SCLC cell lines, NCI-H82, H209, H510, H526 and H660, by Western blotting. We evaluated whether hypoxic conditions up-regulate these protein expressions. We also examined whether VEGF addition and VEGF-D addition cause phosphorylation of the mitogen-activated protein kinase (MAPK) as well as VEGFR-2 and VEGFR-3. Further, we investigated whether VEGF addition and VEGF-D addition induced the proliferation and migration of the SCLC cells. VEGF, VEGF-C, VEGFR-2 and VEGFR-3 were detectable by Western blotting in all five SCLC cell lines,. The VEGF-Rs and VEGFs expression levels were increased by an incubation under hypoxic conditions in NCI-H82. VEGF addition and VEGF-D addition caused phosphorylation of MAPK as well as the VEGF-Rs themselves, and induced proliferation and migration of the SCLC cells. These results suggested potential of VEGF signal-pathway inhibitors as anti-cancer agents in SCLC treatment disturbing growth and migration of the cancer cells.     

Micronuclei levels in peripheral blood lymphocytes as a potential biomarker for pancreatic cancer risk

To find biomarkers for risk prediction of pancreatic cancer (PC), we evaluated the frequency of micronuclei (MN) in peripheral lymphocytes of 346 patients with PC and 449 healthy controls. The levels of baseline MN (mean ± standard error of micronucleated cells per 1000 binucleated cells) were significantly higher in patients (15.3 ± 0.3) than those in controls [9.7 ± 0.5; adjusted for body mass index (BMI), P < 0.001]. Using the median levels found in controls as the cut point, 78.9% of patients and 43.7% of controls had a higher frequency of MN. Logistic regression analysis with adjustment for known risk factors for PC showed that having a higher level of MN was significantly associated with increased risk of PC [odds ratio (OR): 8.32, 95% confidence interval (CI): 5.06-13.67, P < 0.001]; and the risk was much higher in men than in women [OR (95% CI): 14.19 (7.09-28.40) versus 4.19 (1.90-9.27)]. The level of MN was not associated with disease stage or resection status but was related to smoking status in men and to BMI in women among patients. The level of MN was higher in smokers (14.5 ± 0.6) than in nonsmokers (12.1 ± 0.6; P = 0.023) and in obese (25.3 ± 2.8) versus normal weight individuals (17.7 ± 0.8; P = 0.024). These data showed that elevated level of MN in peripheral lymphocytes was associated with increased risk of PC.     

IL-12调控胃癌细胞凋亡及免疫逃逸因子分泌机制探讨

目的 探讨白细胞介素-12(IL-12)调控胃癌细胞凋亡及免疫逃逸因子分泌的途径.方法 培养胃癌SGC7901细胞,随机分为空白对照组、感染阴性对照(NC)腺病毒的NC腺病毒组、感染IL-12腺病毒的IL-12腺病毒组、转染NC质粒的NC质粒组、转染STAT4质粒的STAT4质粒组、转染NC siRNA的si-NC组、...     

Human leukocyte antigen-G is closely associated with tumor immune escape in gastric cancer by increasing local regulatory T cells

Human leukocyte antigen-G (HLA-G) plays an important role in tumor cell escape. We investigated HLA-G expression and regulatory T cells (Tregs) infiltrates in patients with gastric cancer (GC), analyzed their relationship with clinicopathologic features, and characterized their role in tumor immune escape. We also investigated the plasma soluble HLA-G level and its potential in the diagnosis of GC. Effect of HLA-G on Tregs was further assessed by coculture experiments in vitro. Most interestingly, HLA-G positive expression was detected in GC tissues and it was significantly correlated with the presence of tumor-infiltrating Tregs. Patients with HLA-G positive expression or high Tregs had significantly poorer survival at 5 years after operation. Multivariate analysis indicated that HLA-G positive expression was an independent prognostic factor of GC. The coculture experiment showed overexpression of HLA-G in GC cell lines significantly enhanced the frequency of Tregs when GC cells were directly cocultured with human peripheral blood mononuclear cell. However, this effect disappeared when the indirect coculture system was applied. Some cytokines such as interleukin-6, interleukin-10, and tumor necrosis factor-α significantly changed in the coculture system. Moreover, plasma soluble HLA-G level in GC patients was higher than that in normal controls. Taken together, our results indicated that HLA-G expression was closely associated with tumor progression and involved in tumor evasion by increasing the frequency of infiltrating Tregs locally. Thus, HLA-G might be a promising predictor for disease prognosis and a possible novel target for immunotherapy in GC patients.     

Soluble Fas ligand released by colon adenocarcinoma cells induces host lymphocyte apoptosis: an active mode of immune evasion in colon cancer

Expression of membrane-bound Fas ligand (mFasL) on colon cancer cells serves as a potential mechanism to inhibit host immune function by inducing apoptosis of host lymphocytes. Membrane-bound FasL can be cleaved and released as a soluble mediator (sFasL), which may spread the apoptosis induction effect. Our study examined whether colon adenocarcinoma cells release sFasL, and induce apoptosis of host lymphocytes without direct cell-cell contact. In 12 consecutive patients with colon adenocarcinoma mFasL was identified in the tumours, sFasL was measured in the sera and apoptosis identified in tumour-infiltrating and peripheral blood lymphocytes. To analyse the function of sFasL, colon cancer cells were primarily cultured; sFasL was isolated from supernatants, measured, incubated with Fas-bearing Jurkat cells, and the resulting apoptosis was analysed. Serum levels of sFasL were significantly elevated in all colon cancer patients with mFasL expression in tumour tissues (n = 8). In these patients, the number of apoptotic lymphocytes was significantly increased within tumour and peripheral blood. Furthermore, sFasL was present in the corresponding supernatants and induced apoptosis of Jurkat cells in a dose-dependent manner. These findings suggest that mFasL-positive colon cancer cells release sFasL, and thus may induce apoptosis of host lymphocytes as a potential mechanism for immune evasion.     

Correlation between IL-10 gene expression and HPV infection in cervical cancer: a mechanism for immune response escape

The present study was performed to determine IL-10 expression in cervical tissues in Mexican women according to the severity of the malignity and its association with HPV infection. IL-10 expression showed a clear tendency to increase during the different cervical cancer stages: 37% in LGSIL; 62% in HGSIL; and 84% in cancer. However, all the patients that expressed IL-10 were HPV positives; we found an association with HPV 16. These results suggest a clear relationship between IL-10, HPV and the stage of cervical cancer disease; this event could contribute to the immunosuppressive micro-environment in the tumor site.     

Bcl-2基因在人膀胱癌细胞中表达对CTL凋亡诱导作用的影响

目的:探讨Bcl-2基因在人膀胱癌细胞中表达对细胞毒性T淋巴细胞(CTL)凋亡诱导作用的影响。方法:采用基因重组技术构建真核表达载体pcDNA3.1(+)/Bcl-2;应用脂质体介导的基因转染技术将pcDNA3.1(+)/Bcl-2导入膀胱癌BIU-87细胞,RT-PCR检测Bcl-2基因表达水平;不同浓度抗Fas单克隆抗体(Anti-Fasmab)模拟CTL的Fas-配体(Fas-L)处理转染与未转染Bcl-2基因的膀胱癌细胞,采用四甲基偶氮唑蓝(MTT)比色法、吖啶橙(AO)荧光染色法和流式细胞检测仪(FCM)分析细胞存活和凋亡情况。结果:酶切和核酸测序证明真核表达载体pcDNA3.1(+)/Bcl-2构建成功。将重组质粒转染膀胱癌细胞后,RT-PCR分析表明,转染Bcl-2基因的BIU-87/Bcl-2细胞的Bcl-2基因表达水平较BIU-87细胞和转染空载体的BIU-87/neo细胞显著增高,P<0.01。An-ti-Fasmab作用后,BIU87/Bcl-2细胞的存活率显著高于BIU-87和BIU-87/neo细胞,P<0.05或P<0.01。3种细胞虽均发生凋亡的形态学改变,但BIU87和BIU87/neo细胞改变更为明显,两者的凋亡率分别为(25.33±3.00)%和(27.05±1.75)%,而BIU-87/Bcl-2细胞的凋亡率为(18.06±1.41)%,显著低于前两者,P<0.01。结论:Bcl-2基因高表达通过阻断CTL杀伤膀胱癌细胞的Fas/Fas-L途径,使膀胱癌细胞能够抵御免疫系统中CTL的攻击。     

Enrichment of high proliferation potential colony forming cells from mouse marrow by selecting low-density cells expressing receptors for wheat germ agglutinin

Suspensions enriched for high proliferation potential colony forming cells (HPP-CFC's) were prepared from mouse marrow by selecting low-density cells stained with fluoresceinated wheat germ agglutinin on a fluorescence activated cell sorter. HPP-CFC's were 12-fold more concentrated in the final suspensions and co-enriched with a population of primitive CFU_s detected in the spleens of irradiated mice reconstituted 13 days earlier with marrow. This is consistent with previous observations suggesting that these populations are closely related. The degree of enrichment for other haemopoietic progenitors was in the order HPP-CFC > day 8 CFU_s > BFU_e > GM-CFC > CFU_e.“Extra” colonies developed when human placental conditioned medium (HPCM) was added to cultures of enriched suspensions already containing pregnant mouse uterus extract (PMUE). HPP-CFC's probably formed most of these and we discuss why counting these extra colonies may be more reliable than the conventional “size” assay for HPP-CFC's.     

PDIA6 promotes pancreatic cancer progression and immune escape through CSN5-mediated deubiquitination of β-catenin and PD-L1

Protein Disulfide Isomerase Family A Member 6 (PDIA6) is an endoplasmic reticulum protein that is capable of catalyzing protein folding and disulfide bond formation. Abnormally elevated expression of PDIA6 has been reported to predict poor outcomes in various cancers. Herein, gain-of- and loss-of-function experiments were performed to investigate how PDIA6 participated in the carcinogenesis of pancreatic cancer (PC). By analyzing the protein expression of PDIA6 in 28 paired PC and para carcinoma specimens, we first found that PDIA6 expression was higher in PC samples. Both the overall survival and disease-free survival rates of PC patients with higher PDIA6 expression were poorer than those with lower PDIA6 (n = 178). Furthermore, knockdown of PDIA6 impaired the malignancies of PC cells — suppressed cell proliferation, invasion, migration, cisplatin resistance, and xenografted tumor growth. PDIA6-silenced PC cells were more sensitive to cytotoxic natural killer (NK) cells. Overexpression of PDIA6 had opposite effects on PC cells. Interestingly, COP9 signalosome subunit 5 (CSN5), a regulator of E3 ubiquitin ligases known to promote deubiquitination of its downstream targets, was demonstrated to interact with PDIA6, and its expression was increased in PC cells overexpressing PDIA6. Additionally, PDIA6 overexpression promoted deubiquitination of β-catenin and PD-L1 and subsequently upregulated their expression in PC cells. These alterations were partly reversed by CSN5 shRNA. Collectively, the above results demonstrate that PDIA6 contributes to PC progression, which may be associated with CSN5-regulated deubiquitination of β-catenin and PD-L1. Our findings suggest PDIA6 as a potential target for the treatment of PC.Keyword: Pancreatic cancer, PDIA6, CSN5, β-catenin signaling pathway