小檗碱对急淋白血病的在体抗肿瘤及与Ara-C的联合作用研究

本实验旨在研究小檗碱(berberine, BBR)对急淋白血病细胞Jurkat的在体抗肿瘤作用及与阿糖胞苷(cytosine, Ara-C)的联合作用。本研究通过体外培养Jurkat细胞株,将细胞注射至裸鼠皮下,建立皮下移植瘤模型,待肿瘤体积长至约100 mm~3时,随机分为两组:对照组和小檗碱处理组,分别口服PBS、200 mg/kg小檗碱,隔天给药一次,隔天记录裸鼠体重及肿瘤体积大小。给药30 d后处死动物,剥离肿瘤组织,称量肿瘤大小,绘制肿瘤生长曲线、体重图及瘤重图,计算抑瘤率;采用免疫组化法检测移植瘤组织中细胞核增殖抗原Ki-67的表达水平。同时,本研究用一线用药阿糖胞苷(cytarabine, Ara-C)与小檗碱联合处理Jurkat细胞,研究两药联用的药效。发现小檗碱处理组移植瘤大小受到明显的抑制,瘤块组织中Ki-67的表达明显降低;Ara-C与小檗碱能协同抑制Jurkat细胞的增殖,联合指数(combination index, CI)1。本研究发现小檗碱能够抑制Jurkat细胞皮下移植瘤的增殖,在体内发挥抗急淋白血病活性,并能与一线用药Ara-C协同产生作用。     

不同方式构建A20鼠B细胞淋巴瘤动物模型

目的探索多种方式构建A20鼠B细胞淋巴瘤动物模型及不同方式造模成瘤的特征。方法鼠源性B细胞淋巴瘤细胞株A20经皮下、尾静脉、脾脏和腹腔接种于同源BALB/c小鼠或先接种裸鼠成瘤后组织块移植BALB/c小鼠,观察动物成瘤时间、成瘤率、成瘤部位;取肿瘤组织和动物脏器行石蜡包埋、病理切片、HE染色观察其组织学特点。结果BALB/c鼠皮下注2×10^6组、2×10^7组和裸鼠瘤组织移植BALB/c小鼠组成瘤率皆为100%,成瘤时间分别为（15.29±3.2）d（、7.0±0.82）d和（6.29±0.49）d。BALB/c小鼠尾静脉注射2×106组、2×107组、脾脏注射组、腹腔注射组成瘤率分别为71.4%、100%、71.4%、14.3%,成瘤时间分别为（76.8±12.0）d、（26.1±7.99）d、（32.6±5.99）d和27 d。尾静脉成瘤部位播及肝脏、脾脏、胰腺、肾脏、食道、胃、肠、肠系膜、脑、淋巴结、骨、子宫、肌肉等多脏器和组织。BALB/c鼠A20成瘤组织学类似人弥漫大B细胞淋巴瘤。结论成功构建A20皮下移植瘤模型、血行播散性模型,为利用有免疫功能动物进行B淋巴瘤相关研究提供了实验平台。     

小鼠Lewis肺癌不同部位皮下移植瘤模型的比较

目的探究不同部位肺癌皮下移植瘤存在的差异,为肺癌研究者提供提供基础依据。方法将稳定表达虫萤光素酶的小鼠Lewis肺腺癌细胞(ll2-luc-m38)分别注射于C57 BL/6小鼠的右腋皮下、右腹股沟皮下、脚垫皮下,定期利用小动物活体成像系统观察小鼠皮下移植瘤成瘤情况及转移情况,观察不同部位荷瘤小鼠的生存时间和死亡率,肿瘤组织取材,采用石蜡包埋、切片、HE染色做出病理学诊断。并将小鼠处死后立即进行肺部组织固定、观察转移灶。对皮下移植瘤行切除术观察小鼠术后生存情况和转移情况。结果腋下组和腹股沟组成瘤时间较早,成瘤率为100%,脚垫组成瘤时间较晚,成瘤率为33%;腹股沟组和腋下组瘤体积增长迅速,其中腹股沟组瘤体积增长最快;接种后第21天腋下组70%的小鼠出现了肺转移,转移灶数目较多;腹股沟组50%小鼠出现肺转移,且转移灶数目较少;脚垫组小鼠未观察到肺转移灶。脚垫组小鼠死亡率最高。腹股沟组和腋下组小鼠可行皮下移植瘤切除术,术后存活率为100%。结论小鼠lewis肺癌皮下移植瘤模型中,腹股沟组和腋下组成瘤率高,可耐受移植瘤切除术,手术死亡风险低,便于监测,操作简单且可重复性高,其中腋下组转移性更好。     

姜黄素和紫杉醇联用对前列腺癌PC3裸鼠移植瘤作用的研究

目的:探讨姜黄素和半量紫杉醇联用对前列腺癌PC3裸鼠移植瘤生长增殖的影响及其机制。方法:构建人雄激素非依赖性前列腺癌细胞系PC3裸小鼠皮下移植瘤模型,随机分为溶酶对照组,姜黄素组,紫杉醇组和姜黄素+紫杉醇(半量)组(n=6)。姜黄素每隔2天腹腔注射100mg/kg,紫杉醇每3天腹腔注射10mg/kg,联合组紫杉醇减半,对照组注射药物溶剂。每6天测量计算移植瘤体积并绘制肿瘤生长曲线。药物注射30天后,取移植瘤组织标本分别进行免疫组化和定量RT-PCR,检测金属基质蛋白酶2(MMP2),增殖细胞核抗原(PCNA)蛋白及mRNA的表达。结果:移植瘤体积在24～30天时,姜黄素组和紫杉醇组肿瘤体积较对照组有不同程度的减小。姜黄素+紫杉醇(半量)组在第30天内肿瘤生长体积和紫杉醇组相近,30天后合用组肿瘤体积小于单纯紫杉醇组(P<0.05)。姜黄素和紫杉醇明显降低PC3移植瘤组织中PCNA和MMP2mRNA的表达(P<0.01)。姜黄素+紫杉醇(半量)组瘤组织中PCNA和MMP2的mRNA表达均低于紫杉醇组(P<0.05)。免疫组化染色显示PCNA和MMP2蛋白表达在治疗组均降低,和瘤组织中mRNA表达变化相一致。结论...     

C57BL/6小鼠Lewis皮下移植瘤肺转移灶的病理及超微结构观察

目的 构建C57BL/6小鼠Lewis皮下移植瘤肺转移模型,观察肺转移灶病理以及超微结构变化。方法 C57BL/6小鼠右前肢腋窝皮下接种Lewis细胞,定期测量肿瘤体积,绘制生存曲线,对存活期35 d以上的小鼠进行解剖学观察,HE染色检测肺组织病理变化,透射电镜观察肺组织的超微结构。结果 接种14 d小鼠右前肢腋窝皮下成瘤率为100%,22 d小鼠开始出现死亡,平均生存周期(33.00±6.98)d;解剖学可见肺表面出现典型转移灶,外观呈圆形半透明状,周围有出血;HE染色显示肺转移灶染色深,外观近似圆形,与周围组织界限明显,肿瘤细胞排列紧密,内部可见丰富的毛细血管;透射电镜下可见肺转移灶内肿瘤细胞具有显著的异型性,出现巨核、双核和奇异型核,胞质内含有大量的糖原颗粒和线粒体数量,肿瘤细胞间可见细胞连接。结论 延长存活时间可使皮下移植瘤小鼠形成典型的肺转移灶,肺转移灶内病理学和超微结构变化符合恶行肿瘤的病变特点。     

青蒿琥酯对肺癌细胞裸鼠皮下移植瘤生长和放射敏感性的影响研究

目的:探讨青蒿琥酯对肺癌细胞裸鼠皮下移植瘤生长和放射敏感性的影响。方法:将肺癌细胞接种至裸鼠皮下,腹腔注射青蒿琥酯,同时给予放射处理记为联合组,设置青蒿琥酯组(不照射处理)、放射组(腹腔注射生理盐水)和对照组(腹腔注射生理盐水,不放射处理),测量肿瘤体积,取瘤体并称取瘤体重量,脱氧核糖核苷酸末端转移酶介导的缺口末端标记法(TUNEL)法检测组织中细胞凋亡水平,Western blot检测组织中活化的含半胱氨酸的天冬氨酸蛋白水解酶3(Cleaved Caspase-3)、信号转导与转录因子3(STAT3)、磷酸化的STAT3(p-STAT3)、p38丝裂原活化蛋白激酶(p38MAPK)、磷酸化的p38MAPK(p-p38MAPK)水平。结果:青蒿琥酯组、放射组、联合组皮下移植瘤体积和重量均明显低于对照组,联合组肿瘤重量和体积明显低于青蒿琥酯和放射组,差异均具有统计学意义(P0.05)。青蒿琥酯、放射组、联合组肿瘤组织中细胞凋亡率、Cleaved Caspase-3水平和p-p38MAPK/p38MAPK均明显高于对照组,联合组肿瘤组织中细胞凋亡率、Cleaved Caspase-3水平和p-p38MAPK/p38MAPK高于青蒿琥酯和放射组,差异均具有统计学意义(P0.05)。青蒿琥酯、放射组、联合组肿瘤组织中p-STAT3/STAT3水平明显低于对照组,联合组肿瘤组织中p-STAT3/STAT3水平低于蒿琥酯组和放射组,差异均具有统计学意义(P0.05)。结论:青蒿琥酯能够抑制肺癌细胞裸鼠皮下移植瘤生长,促进癌细胞凋亡,增加放疗敏感性,作用机制可能与p38MAPK、STAT3信号通路有关。     

人脐带间充质干细胞对裸鼠的促瘤和致瘤性研究

目的研究人脐带间充质干细胞(hUCMSCs)对动物是否具有促瘤性,以及不同代次是否有致瘤性,为其临床应用提供安全性依据。方法分别用人淋巴瘤细胞Raji接种于CB-17 SCID小鼠皮下、用人结肠癌细胞WiDr接种于BALB/c裸鼠皮下,建立皮下移植瘤模型,WiDr成模动物用完全随机法分为4组(n=8):模型对照组、hUCMSCs低、中、高剂量组;Raji成模动物用完全随机法分为5组(n=7):空白对照组、模型对照组、hUCMSCs低、中、高剂量组。hUCMSCs均静脉注射给药,观察hUCMSCs对皮下移植瘤的增殖是否有影响。另外,观察不同代次的hUCMSCs是否有致瘤性,NOD/SCID小鼠分别皮下接种hUCMSCs的第2、6、10代的细胞,注射后3周,16周对动物剖检,观察hUCMSCs皮下结节的形成及是否有肿瘤转化的倾向。对照组和hUCMSCs组的瘤体积及瘤重采用成组t检验进行统计分析,并计算hUCMSCs组相比对照组的相对肿瘤抑制率T/C%。结果 WiDr肿瘤模型中,第43天hUCMSCs中、高剂量组的瘤体积均显著低于模型对照组,分别为(586.7±274.4)mm~3、(689.5±114.8)mm~3 vs(945.9±234.0)mm~3,P0.05,但T/C%值40%;第51天hUCMSCs低、中高剂量组瘤体积均显著低于模型对照组,分别为(777.8±346.7)mm~3、(793.1±358.6)mm~3、(800.7±116.5)mm~3 vs(1300.0±356.8)mm~3,P均0.05,但T/C%值均40%;表明hUCMSCs在3个剂量下均未对WiDr肿瘤的生长有影响,无显著促进或抑制作用。Raji肿瘤模型中,hUCMSCs治疗组瘤体积与对照组相比在所有时间点差异均无统计学意义(P0.05),显示hUCMSCs在本实验条件下对肿瘤生长没有明显的抑制和促进作用。两个模型的病理检测均未见对肿瘤增殖有明显影响。3个不同代次的hUCMSCs注射到NOD/SCID后16周对动物剖检,也没有观察到肿瘤转化的倾向。结论 hUCMSCs对裸鼠WiDr和Raji移植瘤模型的肿瘤增殖无促进作用,不同代次的hUCMSCs在NOD/SCID裸鼠体内移植也不具有致瘤性。     

不同方法建立的人大细胞肺癌裸鼠移植瘤模型的生物学特点

目的比较三种常用的皮下移植瘤造模方法建立的人大细胞肺癌NCI-H460裸鼠移植瘤模型的不同生物学特点,为不同的研究寻找合适的造模方法提供实验依据。方法分别用NCI-H460细胞,NCI-H460移植瘤组织块和移植瘤匀浆液对于BALB/c-nu/nu裸鼠建立皮下移植瘤模型,运用一般生物学指标观察三种移植瘤的成瘤率、瘤重、倍增时间和组织形态;采用全自动生化分析仪检测其外周血中丙氨酸氨基转氨酶（ALT）、天冬氨酸氨基转移酶（AST）、血糖（G1u）、尿素氮（BUN）和肌酐（CREA）等生化指标;利用血球分析仪检测白细胞总数（WBC）并进行分类,最后体外对其腹腔巨噬细胞吞噬活性和NK细胞活性进行了考察。结果本实验中细胞法和匀浆法的成瘤率及肿瘤生长速率显著高于埋块法且其生长更为均一,差异较小。接种5周后,与正常裸鼠比较,三组荷瘤小鼠血液中ALT、AST显著升高,BUN、CREA显著降低,埋块组的AST和BUN两项指标显著高于其他两荷瘤组。此外,接种2周后,荷瘤裸鼠的GLU显著低于正常裸鼠,匀浆液组的GLU降得最低。白细胞中,三种方法组荷瘤小鼠血液中LYM%、MN%、HGB均有降低,匀浆液组和细胞培养组的荷瘤小鼠血液中WBC、NEUT%、PLT显著高于埋块组。免疫细胞活性方面,两种细胞均呈现出正常细胞组〉匀浆组〉细胞组〉埋块组的趋势。结论细胞培养法接种数量可控,肿瘤生长均匀,适合建立不同实验需求的移植瘤模型,组织块移植法适于建立中药抗肿瘤筛选的动物模型,而匀浆液移植法则不推荐使用。裸鼠的生理生化状态和免疫功能与肿瘤的生长有密切的关系。     

探讨不同浓度Matrigel对小鼠Lewis肺癌异位皮下移植瘤的影响

目的 探讨不同浓度Matrigel对小鼠Lewis肺癌异位皮下移植瘤的影响。方法 将24只C57BL/6J Nifdc小鼠随机分为肺癌组、50%基质胶组、75%基质胶组、100%基质胶组,每组各6只。将Lewis肺癌细胞(LL/2)分别与0%、50%、75%、100%Matrigel按照体积1∶1混合,于小鼠右侧前肢腋下进行皮下注射,每日测量各组小鼠体重、饮食量及饮水量;观察比较各组成瘤时间、成瘤率和肿瘤大小,于肿瘤形成后每天测量肿瘤的长径、短径,计算肿瘤体积变化;于肿瘤接种第15天处死动物,完整剥离肿瘤组织并称重,HE染色检测肿瘤组织病变情况。结果与肺癌组相比,75%基质胶组小鼠自造模第2天起体重显著升高(P<0.05或P<0.01);100%基质胶组小鼠自造模第10天起,体重明显上升(P<0.05或P<0.01)。各组小鼠平均饮食量于造模8 d后有所降低,饮水量总体呈现上升趋势。各组小鼠成瘤率均为100%,添加基质胶组的小鼠相比肺癌组小鼠成瘤时间早,肿瘤生长速度快,且肿瘤较大。其中,75%基质胶组肿瘤体积增长最快,肿瘤重量最大,为(1358.88±388.1...     

实验动物准备对裸鼠移植瘤模型microPET 显像的影响

目的 探讨实验动物准备条件对18 F-FDG microPET 裸鼠移植瘤模型显像的影响,以选择最佳的实验动物准备条件.方法 36 只人表皮样癌细胞A431 裸鼠皮下移植瘤模型.随机分为6 组（6 只/组）;A 组：无禁食、室温（20 ～22）℃、无麻醉（注射18 F-FDG 后60 min 清醒状态）、尾静脉注射18 F-FDG;B 组：禁食（6 ～8）h、加温（30 ～32）℃、麻醉（吸入2%异氟烷麻醉）、尾静脉注射18 F-FDG;C 组：无禁食、加温、麻醉、尾静脉注射18 F-FDG;D组：禁食、室温、麻醉、尾静脉注射18 F-FDG;E 组：禁食、加温、无麻醉、尾静脉注射18 F-FDG;F 组：禁食、加温、麻醉、腹腔注射18 F-FDG.注射18 F-FDG 约1 h 后,行microPET 显像,测量皮下移植瘤、颈部肌肉、棕色脂肪、脑、肝脏、肾脏、心脏、哈氏腺最大每克组织摄取率（%ID/gmax ）.扫描前裸鼠均测血糖.结果 （1）B 组、C 组、F 组裸鼠的血糖水平与肿瘤摄取之间均呈直线负相关.（2）棕色脂肪：A 组摄取最高（8.03 ±1.29）,B 组摄取降低71.98%（P ＝0.000）.颈部肌肉：A 组摄取最高（16.07 ±5.20）,B 组摄取降低最多达81.84%（P ＝0.000）.各组脑、心脏、肝脏、肾脏、哈氏腺摄取差异无统计学意义.（3）A 组皮下移植瘤/组织或器官的摄取率最低.B 组移植瘤/颈部肌肉,移植瘤/肝脏,移植瘤/棕色脂肪的摄取率较A 组分别升高6.50 倍、1.29 倍、4.76 倍（P 均＜0.05）,肿瘤与组织或器官的图像对比度明显改善.（4）第1 次microPET 显像,尾静脉注射与腹腔注射皮下移植瘤摄取值差别无统计学意义（P ＝0.364）.第2 次microPET 显像,腹腔注射腹腔可见不同程度显像剂浓聚,其他正常组织、器官及皮下移植瘤的摄取均减低.腹腔注射方式,两次皮下移植瘤的摄取值差异有统计学意义（P ＝0.025）.结论实验动物准备明显影响18 F-FDG 在裸鼠正常组织的分布及皮下移植瘤的摄取.禁食、加温、麻醉及尾静脉注射方式,可以改善肿瘤对18 F-FDG 的摄取,保证图像有较好的稳定性及可重复性.     

Mannose receptor (MR) engagement by mesothelin GPI anchor polarizes tumor-associated macrophages and is blocked by anti-MR human recombinant antibody

Tumor-infiltrating macrophages respond to microenvironmental signals by developing a tumor-associated phenotype characterized by high expression of mannose receptor (MR, CD206). Antibody cross-linking of CD206 triggers anergy in dendritic cells and CD206 engagement by tumoral mucins activates an immune suppressive phenotype in tumor-associated macrophages (TAMs). Many tumor antigens are heavily glycosylated, such as tumoral mucins, and/or attached to tumor cells by mannose residue-containing glycolipids (GPI anchors), as for example mesothelin and the family of carcinoembryonic antigen (CEA). However, the binding to mannose receptor of soluble tumor antigen GPI anchors via mannose residues has not been systematically studied. To address this question, we analyzed the binding of tumor-released mesothelin to ascites-infiltrating macrophages from ovarian cancer patients. We also modeled functional interactions between macrophages and soluble mesothelin using an in vitro system of co-culture in transwells of healthy donor macrophages with human ovarian cancer cell lines. We found that soluble mesothelin bound to human macrophages and that the binding depended on the presence of GPI anchor and of mannose receptor. We next challenged the system with antibodies directed against the mannose receptor domain 4 (CDR4-MR). We isolated three novel anti-CDR4-MR human recombinant antibodies (scFv) using a yeast-display library of human scFv. Anti-CDR4-MR scFv #G11 could block mesothelin binding to macrophages and prevent tumor-induced phenotype polarization of CD206(low) macrophages towards TAMs. Our findings indicate that tumor-released mesothelin is linked to GPI anchor, engages macrophage mannose receptor, and contributes to macrophage polarization towards TAMs. We propose that compounds able to block tumor antigen GPI anchor/CD206 interactions, such as our novel anti-CRD4-MR scFv, could prevent tumor-induced TAM polarization and have therapeutic potential against ovarian cancer, through polarization control of tumor-infiltrating innate immune cells.     

Berberine inhibits human tongue squamous carcinoma cancer tumor growth in a murine xenograft model

Our primary studies showed that berberine induced apoptosis in human tongue cancer SCC-4 cells in vitro. But there is no report to show berberine inhibited SCC-4 cancer cells in vivo on a murine xenograft animal model. SCC-4 tumor cells were implanted into mice and groups of mice were treated with vehicle, berberine (10 mg/kg of body weight) and doxorubicin (4 mg/kg of body weight). The tested agents were injected once per four days intraperitoneally (i.p.), with treatment starting 4 weeks prior to cells inoculation. Treatment with 4 mg/kg of doxorubicin or with 10 mg/kg of berberine resulted in a reduction in tumor incidence. Tumor size in xenograft mice treated with 10 mg/kg berberine was significantly smaller than that in the control group. Our findings indicated that berbeirne inhibits tumor growth in a xenograft animal model. Therefore, berberine may represent a tongue cancer preventive agent and can be used in clinic.     

The MRC1/CD68 Ratio Is Positively Associated with Adipose Tissue Lipogenesis and with Muscle Mitochondrial Gene Expression in Humans

Background

Alternative macrophages (M2) express the cluster differentiation (CD) 206 (MCR1) at high levels. Decreased M2 in adipose tissue is known to be associated with obesity and inflammation-related metabolic disturbances. Here we aimed to investigate MCR1 relative to CD68 (total macrophages) gene expression in association with adipogenic and mitochondrial genes, which were measured in human visceral [VWAT, n = 147] and subcutaneous adipose tissue [SWAT, n = 76] and in rectus abdominis muscle (n = 23). The effects of surgery-induced weight loss were also longitudinally evaluated (n = 6).

Results

MCR1 and CD68 gene expression levels were similar in VWAT and SWAT. A higher proportion of CD206 relative to total CD68 was present in subjects with less body fat and lower fasting glucose concentrations. The ratio MCR1/CD68was positively associated with IRS1gene expression and with the expression of lipogenic genes such as ACACA, FASN and THRSP, even after adjusting for BMI. The ratio MCR1/CD68 in SWAT increased significantly after the surgery-induced weight loss (+44.7%; p = 0.005) in parallel to the expression of adipogenic genes. In addition, SWAT MCR1/CD68ratio was significantly associated with muscle mitochondrial gene expression (PPARGC1A, TFAM and MT-CO3). AT CD206 was confirmed by immunohistochemistry to be specific of macrophages, especially abundant in crown-like structures.

Conclusion

A decreased ratio MCR1/CD68 is linked to adipose tissue and muscle mitochondrial dysfunction at least at the level of expression of adipogenic and mitochondrial genes.     

Activation state of stromal inflammatory cells in murine metastatic pancreatic adenocarcinoma

The histologic presence of macrophages (tumor-associated macrophages, TAMs) and neutrophils (tumor-associated neutrophils, TANs) has been linked to poor clinical outcomes for solid tumors. The exact mechanism for this association with worsened prognosis is unclear. It has been theorized that TAMs are immunomodulated to an alternatively activated state and promote tumor progression. Similarly, TANs have been shown to promote angiogenesis and tumor detachment. TAMs and TANs were characterized for activation state and production of prometastatic mediators in an immunocompetent murine model of pancreatic adenocarcinoma. Specimens from liver metastases were evaluated by immunofluorescence and immunoblotting. TAMS have upregulated expression of CD206 and CD163 markers of alternative activation, (4.14 ± 0.55-fold and 7.36 ± 1.13-fold over control, respectively, P < 0.001) but do not have increased expression of classically activated macrophage markers CCR2 and CCR5. TAMs also express oncostatin M (OSM). We found that TANs, not TAMs, predominantly produce matrix metalloproteinase-9 (MMP-9) in this metastatic tumor microenvironment, while MMP-2 production is pan-tumoral. Moreover, increased expression of VEGF colocalized with TAMs as opposed to TANs. TAMs and TANs may act as distinct effector cells, with TAMs phenotypically exhibiting alternative activation and releasing OSM and VEGF. TANs are localized at the invasive front of the metastasis, where they colocalize with MMP-9. Improved understanding of these interactions may lead to targeted therapies for pancreas adenocarcinoma.     

Clinical Significance of sIL-2R Levels in B-Cell Lymphomas

Elevated soluble interleukin-2 receptor (sIL-2R) in sera is observed in patients with malignant lymphoma (ML). Therefore, sIL-2R is commonly used as a diagnostic and prognostic marker for ML, but the mechanisms responsible for the increase in sIL-2R levels in patients with B-cell lymphomas have not yet been elucidated. We first hypothesized that lymphoma cells expressing IL-2R and some proteinases such as matrix metalloproteinases (MMPs) in the tumor microenvironment can give rise to increased sIL-2R in sera. However, flow cytometric studies revealed that few lymphoma cells expressed IL-2R α chain (CD25) in diffuse large B-cell lymphoma (DLBCL) and follicular lymphoma (FL), and most CD25-expressing cells in the tumor were T-cells. Distinct correlations between CD25 expression on B-lymphoma cells and sIL-2R levels were not observed. We then confirmed that MMP-9 plays an important role in producing sIL-2R in functional studies. Immunohistochemical (IHC) analysis also revealed that MMP-9 is mainly derived from tumor-associated macrophages (TAMs). We therefore evaluated the number of CD68 and CD163 positive macrophages in the tumor microenvironment using IHC analysis. A positive correlation between the levels of sIL-2R in sera and the numbers of CD68 positive macrophages in the tumor microenvironment was confirmed in FL and extranodal DLBCL. These results may be useful in understanding the pathophysiology of B-cell lymphomas.     

The density of macrophages in colorectal cancer is inversely correlated to TGF-β1 expression and patients’ survival

The role of macrophages in colorectal cancer tumorogenesis is complex because they can both prevent and promote tumor development. We investigated CD68-positive cell infiltration in tumor tissue and its correlations with proteins of TGF-β1 signaling pathway and survival of the patients after surgical therapy. A non-selected panel of 210 primary tumors of colorectal origin was investigated immunohistochemically with antibodies against CD68, TGF-β1, TGFβRII and Smad4. Lower CD68 infiltration in tumor stroma was associated with expression of TGF-β1 (p = 0.002) and SMAD4 (p = 0.090) in tumor cell cytoplasm and with TGFβRII expression (p = 0.017) in tumor cells membranes. The absence of SMAD4 immune deposits in tumor cell nuclei was more often seen in biopsies with low number of CD68 in the invasive front (p = 0.044). The low number of CD68-positive cells was significantly associated with several adverse clinical and histological tumor characteristics as the presence of metastases in local lymph nodes (p = 0.047), distant metastases (p = 0.0003), advanced tumor stage (p = 0.006), tumor cell invasion of blood, lymph vessels or perineural invasion (p = 0.004), higher histological types (p = 0.0002) and lower grade of inflammatory infiltration in the invasive front (p = 0.002). Moreover, the low grade of CD68 appeared to be significant unfavorable factors of prognosis of the patients with colorectal cancer. The results of our study confirm the prognostic significance of low level of tumor-associated macrophage infiltration in colorectal cancer as unfavorable marker for survival of the patients.     

Integration of OV6 expression and CD68+ tumor-associated macrophages with clinical features better predicts the prognosis of patients with hepatocellular carcinoma

Background: Reliable prognostic indicators for accurately predicting postoperative outcomes in Hepatocellular carcinoma (HCC) patients are lacking. Although cancer stem-like cells (CSCs) and tumor-associated macrophages (TAMs) in tumor microenvironment are implicated in the occurrence and development of HCC, whether the combination of CSC biomarkers and TAM populations could achieve better performance in predicting the prognosis of patients with HCC has been rarely reported.Methods: A total of 306 HCC patients were randomly divided into the training and validation cohorts at a 1:1 ratio, and the expression of OV6 and CD68 was assessed using immunohistochemistry in HCC samples. The prognostic value of these biomarkers for post-surgical survival and recurrence were evaluated by the curve of receiver operating characteristic and multivariate Cox regression analyses.Results: The density of OV6⁺ CSCs was positively correlated with the infiltration of CD68⁺ TAMs in HCC. Both high OV6 expression and CD68⁺ TAM infiltration was closely associated with poor overall survival (OS) and progression-free survival (PFS) of HCC patients. Moreover, overexpression of OV6 and infiltration of CD68⁺ TAMs were identified as independent prognostic factors for OS and PFS after liver resection. The integration of OV6 and CD68 with tumor size and microvascular invasion exhibited highest C-index value for survival predictivity in HCC patients than any other biomarkers or clinical indicators alone.Conclusion: Incorporating intratumoral OV6 expression and CD68⁺ TAMs infiltration with established clinical indicators may serve as a promising prognostic signature for HCC, and could more accurately predict the clinical outcomes for HCC patients after liver resection.     

Adipose tissue inflammation and liver fat in patients with highly active antiretroviral therapy-associated lipodystrophy

In this cross-sectional study, we sought to determine whether gene expression of macrophage markers and inflammatory chemokines in lipoatrophic subcutaneous abdominal adipose tissue and liver fat content are increased and interrelated in human immunodeficiency virus (HIV)-1-positive, highly active antiretroviral therapy (HAART)-treated patients with lipodystrophy (HAART+LD+; n = 27) compared with those without (HAART+LD-; n = 13). The study groups were comparable with respect to age, gender, and body mass index. The HAART+LD+ group had twofold more intra-abdominal (P = 0.01) and 1.5-fold less subcutaneous (P = 0.091) fat than the HAART+LD- group. As we have reported previously, liver fat was 10-fold higher in the HAART+LD+ compared with the HAART+LD- group (P = 0.00003). Inflammatory gene expression was increased in HAART-lipodystrophy: CD68 4.5-fold (P = 0.000013), tumor necrosis factor (TNF)-alpha 2-fold (P = 0.0094), chemokine (C-C motif) ligand (CCL) 2 2.5-fold (P = 0.0024), CCL3 7-fold (P = 0.0000017), integrin alphaM (ITGAM) 3-fold (P = 0.00067), epidermal growth factor-like module containing, mucin-like, hormone receptor-like (EMR)1 2.5-fold (P = 0.0038), and a disintegrin and metalloproteinase domain (ADAM)8 3.5-fold (P = 0.00057) higher in the HAART+LD+ compared with the HAART+LD- group. mRNA concentration of CD68 (r = 0.37, P = 0.019), ITGAM (r = 0.35, P = 0.025), CCL2 (r = 0.39, P = 0.012), and CCL3 (r = 0.54, P = 0.0003) correlated with liver fat content. In conclusion, gene expression of markers of macrophage infiltration and adipose tissue inflammation is increased in lipoatrophic subcutaneous abdominal adipose tissue of patients with HAART-associated lipodystrophy compared with those without. CD68, ITGAM, CCL2, and CCL3 expression is significantly associated with accumulation of liver fat.     

BTN3A2 expression in epithelial ovarian cancer is associated with higher tumor infiltrating T cells and a better prognosis

BTN3A2/BT3.2 butyrophilin mRNA expression by tumoral cells was previously identified as a prognostic factor in a small cohort of high grade serous epithelial ovarian cancer (HG-EOC). Here, we evaluated the prognostic value of BT3.2 at the protein level in specimen from 199 HG-EOC patients. As the only known role of butyrophilin proteins is in immune regulation, we evaluated the association between BT3.2 expression and intratumoral infiltration of immune cells by immunohistochemistry with specific antibodies against BT3.2, CD3, CD4, CD8, CD20, CD68 and CD206. Epithelial BT3.2 expression was significantly associated with longer overall survival and lower risk of disease progression (HR?=?0.651, p?=?0.006 and HR?=?0.642, p?=?0.002, respectively) and significantly associated with a higher density of infiltrating T cells, particularly CD4+ cells (0.272, p<0.001). We also observed a strong association between the relative density of CD206+ cells, as evaluated by the ratio of intratumoral CD206+/CD68+ expression, and risk of disease progression (HR?=?1.355 p?=?0.044, respectively). In conclusion, BT3.2 protein is a potential prognostic biomarker for the identification of HG-EOC patients with better outcome. In contrast, high CD206+/CD68+ expression is associated with high risk of disease progression. While the role of BT3.2 is still unknown, our result suggest that BT3.2 expression by epithelial cells may modulates the intratumoral infiltration of immune cells.     

Prognostic value of polarized macrophages in patients with hepatocellular carcinoma after curative resection

As the most predominant tumour‐infiltrating immune cells, tumour‐associated macrophages (TAMs) are significant for fostering tumour growth, progression and metastasis. CD68‐positive TAMs display dissimilarly polarized programmes comprising CD11c‐positive pro‐inflammatory macrophages (M1) and CD206‐positive immunosuppressive macrophages (M2). The aim of this study is to determine the prognostic significance of diametrically polarized TAMs in hepatocellular carcinoma (HCC) and their application to risk stratification of patients according to their specific prognostic values. This study included 80 consecutive patients with HCC, and we evaluated diametrically polarized functional status of macrophages by immunohistochemical staining of CD68, CD11c and CD206. Prognostic values and clinicopathologic features were assessed in these patients. High versus low CD11c‐positive TAM density (P = 0.005) and low versus high CD206‐positive TAM density (P = 0.002) were associated with better overall survival, whereas CD68‐positive TAM density had no prognostic significance (low versus high, P = 0.065). Furthermore, the presence of these positive staining macrophages did not show any prognostic significance for recurrence‐free survival (all P > 0.05). Multivariate Cox regression analysis identified CD11c‐positive and CD206‐positive TAMs as an independent prognostic factor (P < 0.001, P = 0.031, respectively). Intratumoural infiltration of diametrically polarized TAMs, a novel identified independent prognostic factor for survival in patients with HCC, could be combined with the TNM stage and the Barcelona Clinic Liver Cancer stage to improve a risk stratification system.