人吲哚胺2,3-双加氧酶多克隆抗体的制备

背景与目的:吲哚胺2,3-双加氧酶(indoleamine 2,3-dioxygenase,IDO)是一种哺乳动物细胞质中的血红素蛋白,是催化色氨酸沿犬尿氨酸途径代谢的限速酶.它的表达不仅能抑制病原微生物的生长,还能抑制T细胞的应答,从而介导肿瘤免疫耐受.本研究的目的是表达和纯化His-hIDO融合蛋白,制备兔抗人IDO多克隆抗体,并用此抗体检测IDO在肿瘤细胞中的表达.方法:将编码人IDO全长cDNA克隆入pET30a( ),测序鉴定表达载体pET30a( )-hIDO,转化大肠杆菌BL21,IPTG诱导表达目的蛋白His-hIDO;用纯化的目的蛋白免疫新西兰大白兔,获得兔抗人IDO多克隆抗体;用Western blot检测该多克隆抗体与重组蛋白His-hIDO的反应性,并用该抗体分析表皮癌细胞A431和肝癌细胞HepG2经IFN-γ诱导前后的IDO表达情况.结果:His-hIDO融合蛋白可与抗His多克隆抗体特异性结合;兔抗人IDO多克隆抗体的效价高,特异性好,能检测肿瘤细胞经IFN-γ诱导后的IDO的表达.结论:兔抗人IDO多克隆抗体能够有效地识别体外表达的IDO蛋白,为研究IDO在肿瘤免疫耐受中的作用提供有力工具.     

间质纤维母细胞活化相关蛋白在大鼠胃移植瘤中的表达

目的 观察间质纤维母细胞CD34、成纤维细胞激活蛋白α（FAPα）、α-平滑肌肌动蛋白（α-SMA）在大鼠胃移植瘤与正常胃组织中的表达差异，探讨间质纤维母细胞活化状态在肿瘤发生、发展中的作用。方法 采用walker256细胞株建立大鼠胃移植瘤模型（n=12），以正常大鼠为对照（n=4），均在同等SPF级条件下饲养。16天后处死大鼠，取肿瘤组织、瘤周组织及正常饲养大鼠胃组织，免疫组化染色、Western blotting、RT-PCR法检测各组组织中CD34、FAPα、α-SMA的表达。结果 与正常对照组相比，肿瘤组织及瘤周组织中CD34蛋白及基因水平的表达均低于正常对照组，差异具有统计学意义（P＜0.05），而FAPα和α-SMA表达则明显高于正常对照组（P＜0.05）；肿瘤组织与瘤周组织之间FAPα和α-SMA表达差异亦具有统计学意义（P＜0.05）。结论 CD34、FAPα与α-SMA在肿瘤组织、瘤周组织及正常组织中的表达存在差异，提示间质纤维母细胞活化状态与肿瘤发生发展存在相关性。     

纤维母细胞活化蛋白（FAP—alpha）研究的临床意义

肿瘤间质纤维母细胞，不同于静息状态成人正常组织的纤维母细胞，特征性地表达一种糖蛋白-纤维母细胞活化蛋白（FAPα），FAPα具有二肽酰肽酶及胶原酶蛋白水解活性，因而可以降解二肽，也可以降解凝胶及Ⅰ型胶原，间质降解是肿瘤细胞侵袭的基本条件之一。以FAPα作为肿瘤生物标记以诊断及治疗肿瘤正在成为一个新兴领域。     

肿瘤来源外泌体在诱导肿瘤相关成纤维细胞生成中作用的研究进展

在肿瘤微环境(TME)中,肿瘤来源外泌体(TDE)可被成纤维细胞摄取,并诱导后者分化为肿瘤相关成纤维细胞(CAF),而CAF又可通过多种机制促进肿瘤的发展。了解TDE诱导CAF形成的作用机制,对基于TME理念的靶向治疗具有重要意义。目前,绝大多数研究均是采用超速离心或试剂盒提取等方法从人源性肿瘤细胞中提取TDE,将其与成纤维细胞进行共培养,再通过检测α-平滑肌肌动蛋白和成纤维细胞活化蛋白等特征蛋白的表达以鉴定CAF。进一步机制研究发现,TDE所携带的某些蛋白或miRNA可通过转化生长因子-β/SMAD、肿瘤坏死因子-α/核因子-κB、细胞外调节蛋白激酶1/2/血管细胞黏附蛋白-1和Ras超家族三磷酸鸟苷激酶-35/MMP1/MMP3等多条通路促进CAF的生成,其结果是促进肿瘤细胞的增殖和迁移、破坏细胞外基质成分、促进微血管生成,以及提高小鼠体内的成瘤能力。     

成纤维细胞活化蛋白在乳腺癌组织的表达特征及临床意义

目的：观察基质成纤维细胞活化蛋白（fibroblast activation protein,FAP）在乳腺癌组织的分布特点、表达特征及其变化规律,探讨FAP在乳腺癌发生发展过程中的作用。方法：对术后病理证实为乳腺癌组织的82例病例进行回顾性分析,应用免疫组织化学法检测FAP的表达,探讨FAP在各临床病理参数组的表达分布特征。结果：免疫组化分析显示乳腺癌肿瘤细胞无FAP表达,FAP主要表达于肿瘤细胞周围基质成纤维细胞内。FAP表达与乳腺癌临床分期呈显著正相关（r=0．922,P=0．000）。非浸润性乳腺癌与浸润性乳腺癌FAP表达具有显著统计学差异（t=17．085P=0．000）。有淋巴结转移组较无淋巴结转移组FAP表达量高（t=11．003,P〈0．01）。结论：FAP特异性表达于乳腺癌瘤周基质成纤维细胞,FAP在女性乳腺癌发生、发展、侵袭转移方面起到了重要的作用。     

成纤维细胞激活蛋白促进肿瘤免疫逃逸和转化治疗研究进展

成纤维细胞激活蛋白（FAP）为Ⅱ型丝氨酸蛋白酶,在多种肿瘤间质中高表达,是肿瘤相关成纤维细胞（CAF）表面标志物之一,与肿瘤进展和总生存期缩短显著相关。近年研究发现,FAP具有介导肿瘤免疫逃逸的重要作用,通过影响天然免疫和适应性免疫反应中细胞种类、数量及功能的变化,调控多种趋化因子和细胞因子的分泌,重塑肿瘤免疫微环境并诱导肿瘤免疫治疗抵抗。通过综述FAP与肿瘤免疫逃逸的关系、作用机制以及在免疫治疗、特别是转化研究进展,可为优化肿瘤免疫治疗提供新思路。     

成纤维细胞活化蛋白（FAP）的研究进展

成纤维细胞活化蛋白（Fibroblast Activation Protein,FAP）存在于肿瘤基质成纤维细胞中,在细胞表面发挥作用,是一种膜丝氨酸肽酶,是II型丝氨酸蛋白酶家族成员之一,具有二肽肽酶及胶原酶活性,在肿瘤的生长中具有双重功能。FAP的蛋白水解酶活性可以增强肿瘤细胞对细胞外基质的侵袭力,同样也能促进肿瘤的生长和增殖。故以FAP作为靶点的分子成像以及作为肿瘤基质标志物的病理诊断、肿瘤基因治疗或免疫治疗将成为可能。近年来对FAP的研究进展进行如下综述。     

肝细胞癌组织中肿瘤相关成纤维细胞与血管生成拟态的相关性及临床意义

目的:探讨肝细胞癌(hepatocellular carcinoma,HCC)中α-SMA+肿瘤相关成纤维细胞（carcinoma-associated fibroblasts,CAFs）与血管生成拟态(vasculogenic mimicry,VM)的相关性及其临床意义。方法:收集60例临床资料完整的手术切除HCC标本,应用免疫组化法检测HCC间质中Vimentin、α-SMA表达情况,病理学方法计数Vimentin+的间质细胞及α-SMA+的CAFs。CD31联合PAS双重染色检测VM水平。统计学分析间质细胞中α-SMA+ CAFs表达与VM形成的相关性及其临床病理意义。结果:HCC间质细胞中α-SMA+ CAFs高表达率为61.7%（37/60）,VM阳性表达率为35.0%（21/60）;间质细胞α-SMA+ CAFs高表达及VM形成与肿瘤的大小、Edmondson分级、临床分期以及是否伴有肝硬化有关（P<0.05）;HCC中α-SMA+ CAFs表达与VM表达呈正相关(r=0.291,P=0.024)。结论:α-SMA+ CAFs和VM形成在肝细胞癌发展进程中发挥重要促进作用,α-SMA+ CAFs可能参与并促进VM形成。     

成纤维细胞活化蛋白与肿瘤

成纤维细胞活化蛋白(FAP)是基质中活化成纤维细胞表达的一种表面抗原,为Ⅱ型膜结合蛋白,属于丝氨酸蛋白水解酶家族,具有二肽肽酶(DPP)活性和胶原酶活性,可水解二肽肽酶、明胶和Ⅰ型胶原,在肿瘤的生长、侵袭和转移中发挥着重要作用.成纤维细胞活化蛋白还可以辅助临床诊断,而针对成纤维细胞活化蛋白的特异性抗体则为肿瘤免疫治疗开辟了新的途径.     

Fibroblast activation protein regulates tumor-associated fibroblasts and epithelial ovarian cancer cells

The fibroblast activation protein (FAP) is a cell surface serine protease which has emerged as a specific marker of tumor-associated fibroblasts (TAFs). FAP has been shown to have both in?vitro dipeptidyl peptidase and collagenase activity. However, the biological function of FAP in the tumor microenvironment is largely unknown. In this study, we first show that TAFs isolated from ovarian cancer samples have the characteristics of stem cells. To explore the functional role of FAP, the protein was silenced by siRNA lentiviral vector transfection. FAP silencing inhibited the growth of TAFs in?vitro, accompanied with cell cycle arrest at the G2 and S?phase in TAFs. FAP silencing also reduced the stem cell marker gene expression in TAFs. SKOV3 cells do not express FAP. Although FAP-silenced SKOV3 cells induced ovarian tumors, the rate of tumor growth was significantly decreased, as shown in the xenograft mouse model. TAF phenotypes in the xenograft tumor tissues were further assayed by immunohistochemistry. The expression of TAF markers, including fibroblast-specific protein, FAP, smooth muscle actin, desmin, vascular endothelial growth factor and fibroblast growth factor was decreased in the tumor stroma induced by FAP-silenced SKOV3 cells. In conclusion, FAP is an important regulator of the microenvironment in tumor formation and targeting FAP is a potential therapeutic strategy to combat ovarian cancer.     

FAP和TGFβ1在宫颈癌中的表达及其意义

[目的]检测FAP和TGβ1在宫颈癌中的表达及其意义。[方法]应用免疫组织化学方法对54例宫颈癌进行FAP和TGFβ1的检测。[结果]在54例宫颈癌间质中FAP表达阳性率为88．89％,正常宫颈组织中无表达。FAP表达与宫颈癌的临床分期有关,与淋巴结转移和病理分级无关。在54例宫颈癌组织中,TGFβ1表达阳性率为87．04％。TGFβ1表达与宫颈癌的临床分期和病理分级有关,与淋巴结转移无关。FAP表达与TGFβ1阳性表达呈正相关（r=0．546,P=0．000）。[结论]FAP的表达与宫颈癌演进有关。TGFβ1可诱导癌组织中反应性间质产生．从而促进FAP表达和肿瘤演进。     

Effective immunoconjugate therapy in cancer models targeting a serine protease of tumor fibroblasts

PURPOSE: Invasion and metastasis of malignant epithelial cells into normal tissues is accompanied by adaptive changes in the mesenchyme-derived supporting stroma of the target organs. Altered gene expression in these nontransformed stromal cells provides potential targets for therapy. The present study was undertaken to determine the antitumor effects of an antibody-conjugate against fibroblast activation protein-alpha, a cell surface protease of activated tumor fibroblasts. EXPERIMENTAL DESIGN: A novel antibody-maytansinoid conjugate, monoclonal antibody (mAb) FAP5-DM1, was developed to target a shared epitope of human, mouse, and cynomolgus monkey fibroblast activation protein-alpha, enabling preclinical efficacy and tolerability assessments. We have used stroma-rich models in immunodeficient mice, which recapitulate the histotypic arrangement found in human epithelial cancers. RESULTS: Treatment with mAb FAP5-DM1 induced long-lasting inhibition of tumor growth and complete regressions in xenograft models of lung, pancreas, and head and neck cancers with no signs of intolerability. Analysis of chemically distinct conjugates, resistance models, and biomarkers implicates a unique mode of action, with mitotic arrest and apoptosis of malignant epithelial cells coupled to disruption of fibroblastic and vascular structures. CONCLUSIONS: We show that mAb FAP5-DM1 combines excellent efficacy and tolerability and provides a first assessment of the mode of action of a novel drug candidate for tumor stroma targeting, thus encouraging further development toward clinical testing of this treatment paradigm.     

High intratumoral expression of fibroblast activation protein (FAP) in colon cancer is associated with poorer patient prognosis

-An active stroma is important for cancer cell invasion and metastasis. We investigated the expression of fibroblast activation protein (FAP) in relation to patient prognosis in colorectal cancer. Colorectal cancer specimens from 449 patients were immunohistochemically stained with a FAP antibody and evaluated in the tumor center and tumor front using a semiquantitative four-level scale. FAP was expressed by fibroblasts in 85–90 % of the tumors examined. High versus no/low expression in the tumor center was associated with poor prognosis (multivariate hazard ratio, HR?=?1.72; 95 % CI 1.07–2.77, p?=?0.025). FAP expression in the tumor front, though more frequent than in the tumor center, was not associated with prognosis. FAP expression in the tumor center was more common in specimens with positive microsatellite instability (MSI) screening status and in patients with high CpG island methylator phenotype (CIMP) status. However, inclusion of MSI screening status and CIMP status in the multivariate analysis strengthened the risk estimates for high FAP expression in the tumor center (HR?=?1.89; 95 % CI 1.13–3.14; p?=?0.014), emphasizing the role of FAP as an independent prognostic factor. Stromal FAP expression is common in colorectal cancer, and we conclude that high FAP expression in the tumor center, but not the tumor front, is an independent negative prognostic factor.     

Tumor immunotherapy targeting fibroblast activation protein, a product expressed in tumor-associated fibroblasts

Murine studies have shown that immunologic targeting of the tumor vasculature, a key element of the tumor stroma, can lead to protective immunity in the absence of significant pathology. In the current study, we expand the scope of stroma-targeted immunotherapy to antigens expressed in tumor-associated fibroblasts, the predominant component of the stroma in most types of cancer. Mice were immunized against fibroblast activation protein (FAP), a product up-regulated in tumor-associated fibroblasts, using dendritic cells transfected with FAP mRNA. Using melanoma, carcinoma, and lymphoma models, we show that tumor growth was inhibited in tumor-bearing mice vaccinated against FAP and that the magnitude of the antitumor response was comparable to that of vaccination against tumor cell-expressed antigens. Both s.c. implanted tumors and lung metastases were susceptible to anti-FAP immunotherapy. The antitumor response could be further enhanced by augmenting the CD4+ T-cell arm of the anti-FAP immune response, achieved by using a lysosomal targeting sequence to redirect the translated FAP product into the class II presentation pathway, or by covaccination against FAP and a tumor cell-expressed antigen, tyrosinase-related protein 2. No morbidity or mortality was associated with anti-FAP vaccination except for a small delay in wound healing. The study suggests that FAP, a product which is preferentially expressed in tumor-associated fibroblasts, could function as a tumor rejection antigen in a broad range of cancers.     

成纤维细胞活化蛋白在乳腺癌组织的表达特征及临床意义

目的:观察基质成纤维细胞活化蛋白(fibroblast activation protein,FAP)在乳腺癌组织的分布特点、表达特征及其变化规律,探讨FAP在乳腺癌发生发展过程中的作用。方法:对术后病理证实为乳腺癌组织的82例病例进行回顾性分析,应用免疫组织化学法检测FAP的表达,探讨FAP在各临床病理参数组的表达分布特征。结果:免疫组化分析显示乳腺癌肿瘤细胞无FAP表达,FAP主要表达于肿瘤细胞周围基质成纤维细胞内。FAP表达与乳腺癌临床分期呈显著正相关(r=0.922,P=0.000)。非浸润性乳腺癌与浸润性乳腺癌FAP表达具有显著统计学差异(t=17.085P=0.000)。有淋巴结转移组较无淋巴结转移组FAP表达量高(t=11.003,P<0.01)。结论:FAP特异性表达于乳腺癌瘤周基质成纤维细胞,FAP在女性乳腺癌发生、发展、侵袭转移方面起到了重要的作用。     

APC +/- alters colonic fibroblast proteome in FAP

Here we compared the proteomes of primary fibroblast cultures derived from morphologically normal colonic mucosa of familial adenomatous polyposis (FAP) patients with those obtained from unaffected controls. The expression signature of about 19% of total fibroblast proteins separates FAP mutation carriers from unaffected controls (P < 0.01). More than 4,000 protein spots were quantified by 2D PAGE analysis, identifying 368 non-redundant proteins and 400 of their isoforms. Specifically, all three classes of cytoskeletal filaments and their regulatory proteins were altered as were oxidative stress response proteins. Given that FAP fibroblasts showed heightened sensitivity to transformation by KiMSV and SV40 including elevated levels of the p53 protein, events controlled in large measure by the Ras suppressor protein-1 (RSU-1) and oncogenic DJ-1, here we show decreased RSU1 and augmented DJ-1 expression in both fibroblasts and crypt-derived epithelial cells from morphologically normal colonic mucosa of FAP gene-carriers. The results indicate that heterozygosity for a mutant APC tumor suppressor gene alters the proteomes of both colon-derived normal fibroblasts in a gene-specific manner, consistent with a "one-hit" effect.