Designing a risk prognosis model based on natural killer cell-linked genes to accurately evaluate the prognosis of gastric cancer

Background:This study was aimed at identifying natural killer(NK)cell-related genes to design a risk prognosis model for the accurate evaluation of gastric cancer(GC)prognosis.Methods:We obtained NK cell-related genes from various databases,followed by Cox regression analysis and molecular typing to identify prognostic genes.Various immune algorithms and enrichment analyses were used to investigate the mutations,immune status,and pathway variations among different genotypes.The key prognostic genes were assessed using the least absolute shrinkage and selection operator(Lasso)regression analysis and univariate Cox regression analysis.Thereafter,the risk score(RS)prognosis model was constructed based on the selected important prognostic genes.A Receiver Operating Characteristics(ROC)curve was plotted for analyzing the robustness of the model.Subsequently,the decision and calibration curves were used for assessing the reliability and prediction accuracy of the proposed model.The‘pRRophetic’R software package was utilized for predicting the half-maximal inhibitory concentration(IC50)of immunotherapy and chemotherapy drugs.Results:We screened 21 prognostic genes and three molecular subtypes and found that the C1 subtype had the worst prognosis.Further,the pathways promoting tumor proliferation,such as epithelial-mesenchymal transition were significantly up-regulated.The results also showed that the macrophages in the M2 stage were significantly infiltrated in the C1 subtype,and there was significant overexpression in the C1 subtype,accompanied by a severe inflammatory reaction.The C1 was highly sensitive to drugs like 5-fluorouracil and paclitaxel.The ROC,calibration curve,and decision curve showed that the risk model was robust and strongly reliable.Conclusion:Overall,our proposed NK cell-related RS model can be used as a more accurate prediction index for GC patients,providing a valuable contribution to personalized medicine.

Inositol hexaphosphate-induced enhancement of natural killer cell activity correlates with suppression of colon carcinogenesis in rats

AIM： To investigate the anti-neoplastic effect of inositol hexaphosphate （InsP6 or phytic acid） on dimethylhydrazine （DMH）-induced colon tumor in rats and its effect on blood natural killer （NK） cell activity. METHODS： Healthy Wistar rats, 4 wk old, were divided into control group （fed with common food） and InsP6 group （fed with common food＋2% sodium inositol hexaphosphate in the drinking water）, 15 rats in each group. Both groups were injected with 1,2-dimethylhydrazine subcutaneously （20 mg/kg body weight） once a week for 20 wk. Rats were killed after 21 wk. The whole large intestine was isolated to determine the general condition of tumors and to test blood NK cell activity by lactate-dehydrogenase-release assay. RESULTS： Administration of InsP6 significantly increased blood NK cell activity in DMH-induced colorectal tumor in rats. InsP6 group had a smaller tumor size on average and a smaller number of tumors than the control group. Its mortality was also higher than that in control. However, the variables of body weight and tumor incidence were not significantly different between the two groups. CONCLUSION： InsP6 can increase blood NK cell activity in DMH-induced colon tumor in rats and inhibit tumor growth and metastasis in rats.

Investigation of the Cytotoxicity of CAR-NK-92 Cells Targeting CEA Against Gastric Cancer Cells

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.

Gastric cancer-derived heat shock protein-gp96 peptide complex enhances dendritic cell activation

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.

IMMUNOSUPPRESSIVE STATUS OF THE SPLEEN IN PATIENTS WITH ADVANCED GASTRIC CANCER

Natural Killer (NK) cell activity, T-lymphocyte subsets and circulating im-mune complexes (CIC) were determinated in samples of peripheral blood (PB),splenicvenous blood(SVB) ac scenic blood (SB) from 39 AGC patients undergoing operation(22 with splenectomy,17 without splenectomy), and in samples of PB fromninety-eight healthy adults as controls. The results showed. (1) The NK activity ofperipheral plood lymphocytes (PBAs), splenic venous blood lymphocytes (SVLs) andsplenic cells (SCs) of the patients were lower than that of PBLs controls (highlysignificant): (2) The NK activity of SVAs ed SCs of AGC pothots were thesignihetly lower than that of their own PBLs; (3) Compared with the PBLs of normalcontrols, the SVLs and SCs of the pothots demonstrated a significant fall in CD3+ andCD4+ cells,and a significant increase in CD8+ cells, resulting in an important declinein CD4+/CM8+ cell ratio; (4) Significant declaim in CD4+/CD8+ cell ratio was ofsonoted in SVAs and SCs of AGC pothots when comas with that of their own PBAs,mainly caused by a marked decrease in CD4+ cells, (5) Sera fromPB, SVB and SB of the potients all showed significantly higher CIC valeal taan the serafrom the PB of healthy PB of healthy controls. In conclusion,the spleen appears to exert animmunosuppressive effect in the AGC poticnts, combained splenectomy can thus berightfully included in the resection when indication is present.

Autologous Natural Killer Cell/Natural Killer T Cell Immunotherapy of Malignant Diseases

Adoptive immunotherapy, the therapeutic infusion of ex vivo activated cancer-fighting white blood cells that was pioneered by Dr. Steven Rosenberg over 30 years ago, has become more widespread due to outstanding published research documenting the clinical efficacy of this strategy. Based on the well-established in vivo functions of NK and NKT cells, their integral role in the efficacy of certain chemotherapeutic and immunomodulatory agents, and their direct therapeutic action as displayed in clinical trials, the use of autologous natural killer cell infusions is an appropriate and warranted therapeutic option for the treatment of malignant diseases, especially in patients whose disease is refractory to standard treatments such as chemotherapy and radiation.

Breast cancer stem-like cells can promote metastasis by activating platelets and down-regulating antitumor activity of natural killer cells

OBJECTIVE:To investigate whether cancer stem cells(CSCs) more efficiently activating platelets and evading immune surveillance than non-CSCs thus promoting metastasis.METHODS:We enriched and identified sphereforming cells(SFCs) and coincubated washed platelets with several platelet activators including collagen,4T1 and SFCs.Platelet-coating tumor cells,platelet activation and TGF-β1 release were analyzed.Then natural kell cells(NK) were incubated with supernatants of different activated platelet samples what we called sample release(SR).The degranulation assay and NKG2 D expression on NK cells were conducted by flow cytometry.Finally tissue factor(TF) expression of SFCs or 4T1 were evaluated by western blot.RESULTS:Breast cancer cell line 4T1 could form spheres in serum-free medium at low adherence.Sphere-forming cells expressed high levels of the CD24-/lowCD44 + stem cell phenotype.Both sphere-forming cells or 4T1 were coated with abundant platelets while sphere-forming cells induced significantly higher expression of platelet activating receptor CD62 p than 4T1 did(P < 0.01).And sphere-forming cells induced platelets to produce more TGF-β1 than 4T1 did(P < 0.01).Furthermore,sample releases induced by sphere-forming cells caused more vigorous inhibition of NK cells antitumor reactivity(P < 0.05) and reduced NKG2 D expression(P < 0.01).The final results showed that sphere-forming cells expressed higher levels of TF than 4T1(P < 0.05).CONCLUSION:Our findings indicate that CSCs could efficiently activate platelets,induce platelets to secrete more TGF-β1,decrease NKG2 D expression and inhibit antitumor activity of NK cell,compared with 4T1.And higher levels of TF expression of CSCs may account for this correlation of CSCs and platelets.

Lymphomatoidgastropathy mimicking extranodal NK/T cell lymphoma,nasal type:A case report

Extranodal natural killer(NK)/T-cell lymphoma,nasal type,exhibits aggressive tumor behavior and carries a poor prognosis.Recently,lymphomatoid gastropathy with NK/T cell infiltration into gastric mucosa has been recognized as a pseudo-malignant disease which regresses without treatment.Because the conventional immunohistochemical criteria of lymphomatoid gastropathy is similar to that of extranodal NK/T-cell lymphoma nasal type,it is difficult to distinguish between the two conditions by histopathological evaluation only.Here,we report a rare case of lymphomatoid gastropathy in a 57-year-old female.Gastroendoscopy on routine check-up revealed elevated reddish lesions < 1 cm in diameter in the gastric fornix and body.Although repeat endoscopies at 1 and 6 mo later revealed no gastric lesions at any locations without any treatments,at 12 mo later gastric lymphomatoid lesions recurred at gastric fornix and body.Histological examination of endoscopic biopsy specimens at 12 mo showed atypical NK cell infiltration with CD3+,CD4-,CD5-,CD7+,CD8-,CD20-,CD30-,CD56+,CD79a-and T-cell-restricted intracellular antigen-1+ into gastric mucosa.After treatment for Helicobacter pylori(H.pylori) eradication,the lesions disappeared in all locations of the gastric fornix and body over the subsequent 12 mo.Here,we report a case of H.pylori-positive lymphomatoid gastropathy with massive NK-cell proliferation,and also review the literature concerning newly identified lymphomatoid gastropathy based on comparison of extra nodal NK/T-cell lymphoma nasal type.In any case,these lesions are evaluated with biopsy specimens,the possibility of this benign entity should be considered,and excessive treatment should be carefully avoided.Close follow-up for this case of lymphomatoid gastropathy is necessary to exclude any underlying malignancy.

Immunotherapy of Cancer—A Historical Note

We examined the possibility that the anti-estrogens, tamoxifen (TX) and toremifen (TO) interacted?with the immune system. Indeed, both TX and TO stimulated cells mediated cytotoxicity reactions by various killer cells: killer T (TK), natural killer (NK), lymphokine activated killer (LAK) cells. Both TX and TO inhibited the growth of tumors that express estrogen receptors. Thus these antiestrogens inhibited tumor growth and stimulated killer cells for cytotoxicty on such tumors. Therefore these agents were presumed to stimulate tumor immunity. We tested the P815 mouse mastcytoma with TK, LK, and TX or TO. A therapeutic effect was observed in both experiments. The SL2-5 murine lymphoma was tested with NK and TX cells or TO cells and successful immunotherapy was observed.?We digested human breast carcinomas and lung tumors with collagenase. The killer cells were separated from tumor cells on Ficoll gradients. TX and TO enhanced the cytotoxic effect of autologous killer cells on the corresponding tumor cells. This experiment indicates that the results obtained in animals are also valid for human malignant disease.

基于WGCNA筛选胃癌相关自然杀伤细胞基因及潜在中药预测

目的:通过加权基因共表达网络分析(WGCNA)筛选胃癌相关自然杀伤细胞基因,探讨自然杀伤细胞相关基因与胃癌预后相关性,预测潜在治疗的中药。方法:运用WGCNA分别构建TCGA-STAD数据集和GSE84437数据集胃癌自然杀伤细胞相关基因共表达网络,获得自然杀伤细胞相关基因。通过单因素Cox回归分析获得与预后相关基因,对预后相关基因进行生存分析,获得与生存相关的基因,以GSE84433作为验证集。运用CIBERSORT反卷积算法计算生存相关基因在22种免疫细胞中的浸润情况。将预后相关的基因映射到Coremine Medical数据库,获得潜在中药,并对筛选中药进行四气、五味、归经及功效的频数统计。结果:通过WGCNA及差异分析,筛选出98个与自然杀伤细胞相关基因,单因素Cox分析共获得22个与预后相关的基因,通过生存分析,发现5个基因与生存显著相关,且与大部分免疫细胞存在相关性,并在GSE84433数据集得到验证。将预后相关基因映射到Coremine Medical数据库,获得潜在治疗中药186味,其中姜黄、郁金等10味中药出现频数较高,药性以温、寒为主,药味以苦、辛、甘为主,归经以肝经、肺经、胃经、脾经为主,功效以补虚药、清热药为主。结论:IL32、IRF8、INFG、SNX10、BATF2等5个胃癌相关自然杀伤细胞基因与胃癌预后相关,姜黄、郁金等中药可能通过提高自然杀伤细胞的活性和数量改善胃癌的预后。本研究揭示中药可能通过改变肿瘤免疫微环境改善患者生存预后,为后续中药研究提供思路。

KIR不相合的NK细胞对乳腺癌细胞的体外杀伤作用

目的:研究自然杀伤(natural killer,NK)细胞表面杀伤细胞免疫球蛋白样受体(killer immunoglobulin-like recep-tor,KIR)和HLA-Cw配体不相合的异基因NK细胞对乳腺癌细胞的体外杀伤作用;分析杀伤活化受体NKG2D及其MICA配体表达水平与NK细胞对乳腺癌细胞杀伤敏感性之间的关系。方法:取10例健康人及5例乳腺癌患者外周血10ml,采用MACS系统NK细胞免疫磁珠分选试剂盒负向分选高纯度NK细胞。取乳腺癌细胞(MCF-7、MDA-MB-435s和SK-Br3)和NK细胞各1×105个,碱裂解法抽提DNA,PCR-SSP方法分别检测HLA-Cw位点、KIR位点表达。MTT法检测KIR相合与不相合组的NK细胞对乳腺癌细胞的杀伤率。流式细胞术检测NK细胞NKG2D表达水平以及RT-PCR方法检测乳腺癌细胞MICA表达。结果:MACS系统分选出的NK细胞,经流式细胞术检测,其纯度在90%以上;KIR与HLA-Cw相合组的NK细胞对乳腺癌细胞株的杀伤率明显高于不相合组,G1组和G2组NK细胞对MDA-MB-435s(G1组)杀伤率分别为(73.2±14.5)%和(34.2±7.6)%,对SK-Br3(G1组)杀伤率为(67.3±12.5)%和(36.5±7.7)%,而对MCF-7(G2组)杀伤率分别为(36.7±8.5)%和(76.5±11.7)%。结果还显示,3株乳腺癌细胞均表达MICA分子;NK细胞与MCF-7细胞共培养,可上调NK细胞表面NKG2D的表达。结论:NK细胞对乳腺癌细胞的杀伤并非由KIR的不相合机制介导;乳腺癌细胞表面表达的MICA分子可上调NK细胞表面NKG2D的表达,激发NK细胞对乳腺癌细胞的细胞毒效应。

曲马多对胃肠道肿瘤患者术中免疫功能的影响

目的观察曲马多对胃肠道肿瘤患者术中T淋巴细胞亚群和NK细胞数量的影响。方法 30例胃肠道肿瘤行根治手术的患者随机均分为观察组和对照组。观察组麻醉前肌注曲马多1mg/kg,对照组不使用。于麻醉前、手术1h和术毕抽取外周静脉血,用流式细胞仪检测T淋巴细胞亚群(CD3+、CD3+CD4+、CD3+CD8+)、活化T细胞(CD3+HLA-DR+)和自然杀伤(NK)细胞(CD3-CD16+CD56+)数量的变化。结果两组手术1hCD3+、CD3+CD4+、CD3+CD4+/CD3+CD8+、CD3+HLA-DR+和NK细胞数量均较麻醉前明显下降,对照组明显低于观察组(P<0.05);术毕两组各指标有所回升,但对照组仍明显低于麻醉前水平和观察组(P<0.05)。结论曲马多可减轻胃肠道肿瘤患者术中T淋巴亚群和NK细胞下降的程度,明显改善机体细胞免疫功能的抑制。

胃癌围手术期T细胞亚群和NK细胞活性检测的临床意义

目的 探讨胃癌围手术期细胞免疫功能的变化及与肿瘤分期的关系。方法 采用流式细胞仪测定40例胃癌患者围手术期外周血T淋巴细胞亚群和NK细胞活性。结果 胃癌患者术前外周血CD_3^+细胞、CD_4^+细胞、CD_4^+/CD_8^+比值和NK细胞活性明显低于正常对照组,CD_8^+细胞显著升高,并与病理分期相关,分期越晚改变越大。手术后以上指标可显著改善。结论 胃癌患者细胞免疫功能低下,病理分期越晚细胞免疫功能越低,肿瘤负荷是其主要原因。

自然杀伤细胞活性与血清癌胚抗原关系的研究

作者应用ＬＤＨ释放法测定ＮＫ细胞活性、放射免疫法测定血清癌胚抗原（ＣＥＡ），平行检测２０例正常健康者，４０例胃癌病人的ＮＫ细胞活性及血清ＣＥＡ，结果表明：胃癌病人术前ＮＫ细胞活性明显降低，血清ＣＥＡ明显增高，ＮＫ细胞活性与血清ＣＥＡ呈显著负相关关系。作者推测血清ＣＥＡ可能对ＮＫ细胞活性有抑制作用。

自然杀伤细胞在胃癌组织中的表达及临床意义

目的分析自然杀伤(NK)细胞在胃癌组织中的表达及其与胃癌临床病理特征和预后的关系。方法采用免疫组织化学染色法检测107例胃癌患者手术标本中NK细胞的表达,采用SAS 9.12统计软件包进行资料分析。结果在胃癌、胃炎及胃息肉组织中均可见NK细胞的表达,但胃癌组NK细胞的表达[(31.74±15.93)个/HPF]明显高于慢性胃炎组[(5.02±2.55)个/HPF]和胃息肉组[(4.84±1.55)个/HPF],差异有统计学意义(P<0.01)。NK细胞的表达与胃癌患者的性别、年龄、肿瘤部位、肿瘤大小、淋巴结转移、复发、病理分级、组织学类型和浸润深度均无统计学意义(P>0.05),但TNM分期越高,NK细胞表达水平有明显下降的趋势,差异有统计学意义(P<0.05)。与NK细胞低表达组相比,NK细胞高表达组胃癌患者中位生存时间延长14个月,两组生存曲线差异无统计学意义(χ2=2.888,P>0.05)。多因素COX模型分析显示,与NK细胞低表达组相比,高表达组有降低胃癌死亡风险的趋势(RR=0.68,95%CI=0.41～1.23)。结论 NK细胞的高表达与胃癌患者的生存时间呈正相关,可作为预测胃癌生存期的指标。

乳腺癌患者手术前后免疫状况的测定

对３０例乳腺癌病人分别于术前、术后两周检测了静脉末梢血自然杀伤细胞（Ｎａｔｕｒａｌｋｉｌｌｅｒ．ＮＫ）活性和Ｔ淋巴细胞亚群状况，其中２０例于术后６周重复检测。结果显示，乳腺癌病人术前ＮＫ、ＯＫＴ４、ＯＫＴ４／ＯＫＴ８活性均降低，并随病情的进展呈减低趋势，在晚期病人中ＯＫＴ８增高尤其明显。术后２周与术前比较无显著性差异。术后６周时ＮＫ、ＯＫＴ４、ＯＫＴ４／ＯＫＴ８均明显回升，而ＯＫＴ８显著降低。由此可见手术切除癌灶对病人的免疫恢复起积极作用。相关分析表明ＮＫ活性与ＯＫＴ４、ＯＫＴ４／ＯＫＴ８呈正相关关系，说明机体免疫系统具有协同抗癌能力。

肝癌与肝硬化患者外周血NK活性及血清免疫球蛋白、补体C_3的研究

应用^(125)I—UdR释放法检测30例原发性肝癌与30例肝硬化代偿期患者外周血自然杀伤细胞(NK)活性,同时测定患者血清免疫球蛋白及补体C_3含量。结果表明肝癌及肝硬化患者外周血NK活性明显低于正常对照者,且肝癌亦较肝硬化患者NK活性为低,肝癌及肝硬化患者血清IgG含量均较正常人高,补体C_3含量在肝硬化组减低,肝癌组增加。本文提示肝癌及肝硬化患者均存在免疫功能低下,且两者的免疫功能紊乱具有一定的相似性。

Tim-3分子在胃癌患者外周血NK细胞上的表达及临床意义

目的:检测Tim-3分子在胃癌荷瘤个体NK细胞上的表达,探讨Tim-3在胃癌发生发展中的作用。方法:收集2012年8月-2013年1月在苏州大学附属第一医院初治的原发性胃癌患者32例,健康体检者28例为正常对照组。流式细胞仪检测胃癌患者及正常对照组外周血NK细胞上Tim-3的表达,分析其与胃癌患者临床病理特征的关系。结果:胃癌患者外周血CD56+NK细胞上Tim-3的表达量为(49.27±21.06)%,显著高于健康对照组(37.00±17.16)%(P<0.05)。有淋巴结转移者Tim-3+CD56+NK细胞高表达率(55%)高于无淋巴结转移患者(0%);有远处转移患者Tim-3+CD56+NK细胞高表达率(100%)显著高于无远处转移者(40.7%),差异均有统计学意义(P<0.05)。Ⅲ-Ⅳ期胃癌患者Tim-3+CD56+NK细胞高表达率较Ⅰ-Ⅱ期患者高(73.7%vs 15.4%),差异具有显著性(P<0.01)。结论:Tim-3在胃癌的发生发展中发挥着一定作用,是潜在的用于评估胃癌预后的指标。

消化性溃疡及胃癌患者外周血IL-2活性水平及其与NK活性的关系

本文观察了40例健康人,28例胃癌,26例球部溃疡,19例胃溃疡及9例胃溃疡伴不典型增生病人的IL—2活性及NK细胞活性。其中胃癌病人IL—2及NK活性最低,与健康对照组比较,P<0.0005;胃溃疡伴不典型增生病人的IL—2活性及NK活性,虽然高于胃癌病人,但却显著低于健康人,统计学有意义。将正常人和病人的PBMC分别用IL—2处理后,观察IL—2对NK细胞的调节作用,IL—2对正常人及病人的NK细胞活性均有促进作用,但未能使NK活性显著低下的胃癌病人的NK活性恢复至正常人水平。

胃病患者自然杀伤细胞活性的研究

应用^(125)IUdR 释放试验,同时测定胃癌与良性胃病患者外周血自然杀伤细胞活性,发现胃癌病人的NK活性显著低于正常人,慢性萎缩性胃炎病人NK活性虽然高于胃癌病人,但却显著低于正常人。本文还进一步研究了正常人、胃癌切除术后病人及进展期胃癌病人血浆对NK细胞功能的影响。发现进展期胃癌病人血浆对正常人NK活性有明显的抑制作用,提示胃癌病人血浆具有抑制NK细胞活性的物质。