Assessment of G Protein-Coupled Oestrogen Receptor Expression in Normal and Neoplastic Human Tissues Using a Novel Rabbit Monoclonal Antibody

In addition to the classical oestrogen receptors, ERα and ERβ, a G protein-coupled oestrogen receptor (GPER) has been identified that primarily mediates the rapid, non-genomic signalling of oestrogens. Data on GPER expression at the protein level are contradictory; therefore, the present study was conducted to re-evaluate GPER expression by immunohistochemistry to obtain broad GPER expression profiles in human non-neoplastic and neoplastic tissues, especially those not investigated in this respect so far. We developed and thoroughly characterised a novel rabbit monoclonal anti-human GPER antibody, 20H15L21, using Western blot analyses and immunocytochemistry. The antibody was then applied to a large series of formalin-fixed, paraffin-embedded human tissue samples. In normal tissue, GPER was identified in distinct cell populations of the cortex and the anterior pituitary; islets and pancreatic ducts; fundic glands of the stomach; the epithelium of the duodenum and gallbladder; hepatocytes; proximal tubules of the kidney; the adrenal medulla; and syncytiotrophoblasts and decidua cells of the placenta. GPER was also expressed in hepatocellular, pancreatic, renal, and endometrial cancers, pancreatic neuroendocrine tumours, and pheochromocytomas. The novel antibody 20H15L21 will serve as a valuable tool for basic research and the identification of GPER-expressing tumours during histopathological examinations.     

A Reliable Biomarker Derived from Plasmalogens to Evaluate Malignancy and Metastatic Capacity of Human Cancers

Antigen tumor markers employed in monitoring therapeutical approaches are limited by their specificity (Sp) and sensitivity (Se). The aim of this study was to investigate the suitability of a lipid tumor marker derived from ether-linked phospholipids and to compare it with others usually assayed in clinical practice. Complex lipids from normal and pathological breast, lung, and prostate tissue were isolated and analyzed by TLC and c-GLC methods. Results were compared as pooled samples, or by means of the averaged percent changes with respect to the composition observed in the normal tissue of the same patient. Sp, Se, negative-predictive (NPV) and positive- predictive values (PPV) were established for conventional markers and for the proposed lipid-derived marker. Results demonstrated that the content of monoenoic fatty acyl chains was significantly increased in total lipids, phosphatidylethanolamine, and especially in ethanolamine-containing ether lipids of neoplastic tissues with respect to their corresponding normal ones. Major changes were observed in the plasmalogen sub-fraction where the ratio monoenoic/saturated fatty acids can distinguish with high Se normal tissues from either benign or neoplastic tissues from breast, lung, or prostate lesions. Analyses of fatty acyl chains from ethanolamine-containing plasmalogens provided a reliable tumor marker that correlated with high Se and linearity with metastases spreading. This fact may be useful in prognosis of the most frequently observed human cancers.     

Reassessment of SST4 Somatostatin Receptor Expression Using SST4-eGFP Knockin Mice and the Novel Rabbit Monoclonal Anti-Human SST4 Antibody 7H49L61

Among the five somatostatin receptors (SST1–SST5), SST4 is the least characterized, which is in part due to the lack of specific monoclonal antibodies. We generated a knockin mouse model that expresses a carboxyl-terminal SST4-eGFP fusion protein. In addition, we extensively characterized the novel rabbit monoclonal anti-human SST4 antibody 7H49L61 using transfected cells and receptor-expressing tissues. 7H49L61 was then subjected to immunohistochemical staining of a series of formalin-fixed, paraffin-embedded normal and neoplastic human tissues. Characterization of SST4-eGFP mice revealed prominent SST4 expression in cortical pyramidal cells and trigeminal ganglion cells. In the human cortex, 7H49L61 disclosed a virtually identical staining pattern. Specificity of 7H49L61 was demonstrated by detection of a broad band migrating at 50–60 kDa in immunoblots. Tissue immunostaining was abolished by preadsorption of 7H49L61 with its immunizing peptide. In the subsequent immunohistochemical study, 7H49L61 yielded a predominant plasma membrane staining in adrenal cortex, exocrine pancreas, and placenta. SST4 was also found in glioblastomas, parathyroid adenomas, gastric and pancreatic adenocarcinomas, pheochromocytomas, and lymphomas. Altogether, we provide the first unequivocal localization of SST4 in normal and neoplastic human tissues. The monoclonal antibody 7H49L61 may also prove of great value for identifying SST4-expressing tumors during routine histopathological examinations.     

人体组织库的建立及其在抗体组织特异性检测中的应用

共收集54种正常人体组织及人体肿瘤组织，建立人体组织库及适合本实验室的LSAB免疫组化方法，并用于6种抗体的组织特异性研究。实验表明，此组织库组织类型多，容量大，各组织的质量符合实验要求，能够用于各种抗体的组织特异性检测，同时证明所检6种抗体均有较强的特异性。     

Aberrant Expression of Posterior HOX Genes in Well Differentiated Histotypes of Thyroid Cancers

Molecular etiology of thyroid cancers has been widely studied, and several molecular alterations have been identified mainly associated with follicular and papillary histotypes. However, the molecular bases of the complex pathogenesis of thyroid carcinomas remain poorly understood. HOX genes regulate normal embryonic development, cell differentiation and other critical processes in eukaryotic cell life. Several studies have shown that HOX genes play a role in neoplastic transformation of several human tissues. In particular, the genes belonging to HOX paralogous group 13 seem to hold a relevant role in both tumor development and progression. We have identified a significant prognostic role of HOX D13 in pancreatic cancer and we have recently showed the strong and progressive over-expression of HOX C13 in melanoma metastases and deregulation of HOX B13 expression in bladder cancers. In this study we have investigated, by immunohistochemisty and quantitative Real Time PCR, the HOX paralogous group 13 genes/proteins expression in thyroid cancer evolution and progression, also evaluating its ability to discriminate between main histotypes. Our results showed an aberrant expression, both at gene and protein level, of all members belonging to paralogous group 13 (HOX A13, HOX B13, HOX C13 and HOX D13) in adenoma, papillary and follicular thyroid cancers samples. The data suggest a potential role of HOX paralogous group 13 genes in pathogenesis and differential diagnosis of thyroid cancers.     

Expression of Aldo-Keto Reductase Family 1 Member B10 in the Early Stages of Human Hepatocarcinogenesis

Aldo-keto reductase family 1, member B10 (AKR1B10), a cancer-related oxidoreductase, is expressed in well-differentiated hepatocellular carcinomas (HCCs). However, AKR1B10 levels are minimal in normal liver tissues (NLs), similar to the 70-kilodalton heat shock protein (HSP70) and glypican-3. Moreover, the role of AKR1B10 in chronic hepatitis or cirrhosis, which are considered preneoplastic conditions for HCC, has not been fully elucidated. The aim of this study was to evaluate the expression of AKR1B10, HSP70, and glypican-3 in 61 HCC tissue samples compared to corresponding non-tumorous liver tissues (NTs), comprising 42 chronic hepatitis and 19 cirrhosis cases to clarify the significance of molecular changes at the preneoplastic stages of HCC. Immunohistochemical analysis demonstrated that the median expression levels of AKR1B10 were higher in HCCs than in NTs (p < 0.001) and higher in NTs than NLs (p < 0.001) with 54.8%, 2.1%, and 0.3% expression in HCCs, NTs, and NLs, respectively. HSP70 and glypican-3 were expressed in HCCs, but minimally in NTs and NLs with no significant difference between expression in NTs and NLs. Furthermore, a multivariate analysis identified an association between hepatic steatosis and AKR1B10 expression in NTs (p = 0.020). Of the three protein expressed in well-differentiated HCCs, only AKR1B10 was upregulated in preneoplastic conditions, and a steatosis-related factor might influence its expression.     

Differential Expression of Peroxisomal Proteins in Distinct Types of Parotid Gland Tumors

Salivary gland cancers are rare but aggressive tumors that have poor prognosis and lack effective cure. Of those, parotid tumors constitute the majority. Functioning as metabolic machinery contributing to cellular redox balance, peroxisomes have emerged as crucial players in tumorigenesis. Studies on murine and human cells have examined the role of peroxisomes in carcinogenesis with conflicting results. These studies either examined the consequences of altered peroxisomal proliferators or compared their expression in healthy and neoplastic tissues. None, however, examined such differences exclusively in human parotid tissue or extended comparison to peroxisomal proteins and their associated gene expressions. Therefore, we examined differences in peroxisomal dynamics in parotid tumors of different morphologies. Using immunofluorescence and quantitative PCR, we compared the expression levels of key peroxisomal enzymes and proliferators in healthy and neoplastic parotid tissue samples. Three parotid tumor subtypes were examined: pleomorphic adenoma, mucoepidermoid carcinoma and acinic cell carcinoma. We observed higher expression of peroxisomal matrix proteins in neoplastic samples with exceptional down regulation of certain enzymes; however, the degree of expression varied between tumor subtypes. Our findings confirm previous experimental results on other organ tissues and suggest peroxisomes as possible therapeutic targets or markers in all or certain subtypes of parotid neoplasms.     

Quantitative Real-Time PCR Analysis of YKL-40 and Its Comparison with Mammalian Chitinase mRNAs in Normal Human Tissues Using a Single Standard DNA

YKL-40 (YKL for the first three N-terminal residues of a 40 kDa protein) belongs to a group of human chitinase-like proteins (CLPs), which are similar to chitinases but lack chitinolytic activity. YKL-40 mRNA and its protein levels have been reported elevated in multiple disorders including asthma, cystic fibrosis, rheumatoid arthritis and malignant tumors. Here, we quantified the YKL-40 mRNA levels and compared them with chitinases and housekeeping genes in normal human tissues. To establish the quantitative real-time PCR (qPCR) system for evaluation of relative YKL-40 mRNA levels, we constructed a human standard DNA molecule by ligating cDNAs of YKL-40, two mammalian chitinases and two housekeeping genes in a one-to-one ratio. We generated cDNAs from various normal human tissues and analyzed the YKL-40 mRNA expression levels using a qPCR system with the standard DNA. We found that YKL-40 mRNA is present widely in human tissues while its expression patterns exhibit clear tissue specificity. Highest YKL-40 mRNA levels were detected in the liver, followed by kidney, trachea and lung. The levels of YKL-40 mRNA in the kidney and liver were more than 100-times higher than those of chitotriosidase mRNA. Our study provides for the first time a comprehensive analysis of the relative expression levels of YKL-40 mRNA versus mammalian chitinases in normal human tissues.     

Involvement of MicroRNA-1-FAM83A Axis Dysfunction in the Growth and Motility of Lung Cancer Cells

Lung cancer is the most prevalent types of cancer and the leading cause of cancer-related deaths worldwide. Among all cancers, lung cancer has the highest incidence, accompanied by a high mortality rate at the advanced stage. Favorable prognostic biomarkers can effectively increase the survival rate in lung cancer. Our results revealed FAM83A (Family with sequence similarity 83, member A) overexpression in lung cancer tissues compared with adjacent normal tissues. Furthermore, high FAM83A expression was closely associated with poor lung cancer survival. Here, through siRNA transfection, we effectively inhibited FAM83A expression in the lung cancer cell lines H1355 and A549. FAM83A knockdown significantly suppressed the proliferation, migration, and invasion ability of these cells. Furthermore, FAM83A knockdown could suppress Epidermal growth factor receptor (EGFR)/Mitogen-activated protein kinase (MAPK)/Choline kinase alpha (CHKA) signaling activation in A549 and H1355. By using a bioinformatics approach, we found that FAM83A overexpression in lung cancer may result from miR-1-3p downregulation. In summary, we identified a novel miR-1-FAM83A axis could partially modulate the EGFR/choline phospholipid metabolism signaling pathway, which suppressed lung cancer growth and motility. Our findings provide new insights for the development of lung cancer therapeutics.     

Development of a Multiplex Immunohistochemistry Workflow to Investigate the Immune Microenvironment in Mouse Models of Inflammatory Bowel Disease and Colon Cancer

Multiplex immunohistochemistry (mIHC) enables simultaneous staining of multiple immune markers on a single tissue section. Mounting studies have demonstrated the versatility of mIHC in evaluating immune infiltrates in different diseases and the tumour microenvironment (TME). However, the majority of published studies are limited to the analysis of human patient samples. Performing mIHC on formalin-fixed paraffin-embedded (FFPE) mouse tissues, particularly with sensitive antigens, remain challenging. The aim of our study was to develop a robust and reproducible protocol to uncover the immune landscape in mouse FFPE tissues. Effective antibody stripping while maintaining sensitivity to antigens and tissue adhesion to the glass slide is critical in developing an mIHC panel to allow successive rounds of staining. Thus, we identified a highly efficient stripping method that preserves signal intensity and antigenicity to allow multiple rounds of staining. We subsequently optimised an mIHC workflow with antibodies specific against CD4, CD8α, FOXP3 and B220 to identify distinct T and B cell populations on mouse FFPE tissues. Lastly, the application of this mIHC panel was validated in a mouse model of inflammatory bowel cancer, two allograft mouse models of spontaneous colon adenocarcinoma and a sporadic mouse model of colon cancer. Together, these demonstrate the utility of the aforementioned protocol in establishing the quantity and spatial localisation of immune cells in different pathological tissues.     

实时荧光定量PCR方法检测人癌组织中RFP蛋白的表达

目的检测RFP(Ret finger protein)蛋白在人正常组织及人癌组织中的分布。方法采用实时荧光定量PCR方法,以18 S rRNA为内对照,检测RFP在人正常组织及人癌组织中的表达水平。结果RFP表达水平子宫颈鳞状细胞癌组织高于正常子宫颈组织,子宫内膜腺癌组织高于正常子宫内膜组织,胃腺癌组织高于正常胃组织,食管鳞状细胞癌组织高于正常食管组织,而子宫内膜腺癌组织高于子宫颈鳞状细胞癌组织,胃腺癌组织高于食管鳞状细胞癌组织,脑癌组织高于正常脑组织。结论RFP可能成为治疗恶性肿瘤的一个潜在的分子靶点。     

Time to Classify Tumours of the Stomach and the Kidneys According to Cell of Origin

Malignant tumours are traditionally classified according to their organ of origin and whether they are of epithelial (carcinomas) or mesenchymal (sarcomas) origin. By histological appearance the site of origin may often be confirmed. Using same treatment for tumours from the same organ is rational only when there is no principal heterogeneity between the tumours of that organ. Organ tumour heterogeneity is typical for the lungs with small cell and non-small cell tumours, for the kidneys where clear cell renal carcinoma (CCRCC) is the dominating type among other subgroups, and in the stomach with adenocarcinomas of intestinal and diffuse types. In addition, a separate type of neuroendocrine tumours (NETs) is found in most organs. Every cell type able to divide may develop into a tumour, and the different subtypes most often reflect different cell origin. In this article the focus is on the cells of origin in tumours arising in the stomach and kidneys and the close relationship between normal neuroendocrine cells and NETs. Furthermore, that the erythropoietin producing cell may be the cell of origin of CCRCC (a cancer with many similarities to NETs), and that gastric carcinomas of diffuse type may originate from the ECL cell, whereas the endodermal stem cell most probably gives rise to cancers of intestinal type.     

抑制乳腺癌耐药蛋白基因逆转肝癌多药耐药的体内实验

目的探讨小片段RNA干扰抑制乳腺癌耐药蛋白(BCRP)基因及其蛋白表达在裸鼠体内逆转人肝癌组织多药耐药(MDR)的可行性。方法建立裸鼠多药耐药肝细胞癌模型,随机分为A组和B组,A组(对照)注射生理盐水40μl+Lipo-fectamine200010μl,B组注射BCRP基因RNAi质粒pSUPER-BCRP40μl+Lipofectamine200010μl,两组均行瘤内注射1次。5d后,各组均经腹腔注射阿霉素5mg/kg化疗,每5d给药1次,共4次。彩色B超测量肿瘤体积;化疗结束后1周处死裸鼠,RT-PCR及Westernblot法检测各组裸鼠肿瘤组织中BCRP基因mRNA的转录水平及其蛋白的表达水平。结果B组每次化疗后肿瘤体积均明显缩小;除第1次化疗外,其余各次化疗后肿瘤体积均小于A组;化疗结束后,B组与A组相比,肿瘤组织中BCRP基因mRNA的转录水平和BCRP蛋白的表达水平均明显降低。结论BCRP基因RNAi质粒pSUPER-BCRP可有效降低裸鼠肝癌组织BCRP基因mRNA的转录水平及其蛋白的表达水平,在一定程度上逆转MDR,为从基因水平逆转MDR提供了初步的实验依据。     

Expression in COS cells of a mouse--human chimaeric B72.3 antibody

B72.3 is a mouse hybndoma cell-line secreting an IgG1 antibodywhich recognises an epitope on a tumour-associated antigen,TAG-72. This high molecular weight mucin-like molecule is foundon a variety of human neoplasms, including colon, breast andovarian carcinomas. Chimaeric Immunoglobulin genes with theB72.3 specificity have been constructed by joining the mousevariable regions from cDNA clones to human genomic constantregions using recombinant DNA techniques. The chimaeric heavyand light chain Immunoglobulin genes were placed under the controlof a strong viral promoter, and co-transfected into COS-1 cells.SDS–PAGE analysis of the ³⁵S-labelled products demonstratedthat the transiently expressed antibodies were correctly synthesisedand assembled. The specific binding characteristics of the parentB72.3 antibody were retained by the chimaeric antibody in anantigen-based ELISA. This system gave sufficiently high transientexpression of the chlmaerlc antibody molecules to allow rapidphysical and Immunological characterisation of the engineeredgene products.     

HE4 Transcription- and Splice Variants-Specific Expression in Endometrial Cancer and Correlation with Patient Survival

We investigated the HE4 variant-specific expression patterns in various normal tissues as well as in normal and malignant endometrial tissues. The relationships between mRNA variants and age, body weight, or survival are analyzed. ICAT-labeled normal and endometrial cancer (EC) tissues were analyzed with multidimensional liquid chromatography followed by tandem mass spectrometry. Levels of HE4 mRNA variants were measured by real-time PCR. Mean mRNA levels were compared among 16 normal endometrial samples, 14 grade 1 and 14 grade 3 endometrioid EC, 15 papillary serous EC, and 14 normal human tissue samples. The relationship between levels of HE4 variants and EC patient characteristics was analyzed with the use of Pearson correlation test. We found that, although all five HE4 mRNA variants are detectable in normal tissue samples, their expression is highly tissue-specific, with epididymis, trachea, breast and endometrium containing the highest levels. HE4-V0, -V1, and -V3 are the most abundant variants in both normal and malignant tissues. All variants are significantly increased in both endometrioid and papillary serous EC, with higher levels observed in grade 3 endometrioid EC. In the EC group, HE4-V1, -V3, and -V4 levels inversely correlate with EC patient survival, whereas HE4-V0 levels positively correlate with age. HE4 variants exhibit tissue-specific expression, suggesting that each variant may exert distinct functions in normal and malignant cells. HE4 levels appear to correlate with EC patient survival in a variant-specific manner. When using HE4 as a biomarker for EC management, the effects of age should be considered.     

肝癌组织中Wnt通路上下游因子的表达及其临床意义

目的探讨Wnt通路Wif-1、C-myc、Cycling-D1mRNA及蛋白在正常肝组织、肝硬化及肝癌组织中的表达及其与肝癌临床病理参数的相关性。方法运用免疫组织化学SP法与原位杂交技术检测100份肝癌组织、50份肝硬化组织、50例正常肝组织中Wif-1、C-myc、Cycling-D1蛋白及mRNA的表达,并测定肝癌组织中的Wif-1、C-myc和Cycling-D1的阳性率、敏感性及特异性。结果肝硬化与肝癌组织中Wif-1 mRNA及蛋白的阳性细胞表达率明显低于正常肝组织(P<0.05),C-myc和Cycling-D1mRNA及蛋白的阳性细胞表达率明显高于正常肝组织(P<0.05)。肝癌组织中Wif-1、C-myc和Cycling-D1mRNA及蛋白的阳性表达均与HBV分级、TNM分期及β-catenin异质表达显著相关(P<0.05)。Wif-1、C-myc和Cycling-D1mRNA及蛋白在肝癌组织中的表达呈正相关(rWif-1=0.892,rC-myc=0.354,rCycling-D1=0.378)。肝癌组织中mRNA及蛋白的敏感性:Wif-1:9.09%、11.11%,C-myc:91.38%、89.66%,Cycling-D1:91.67%、82.5%,特异性:Wif-1:97.75%、95.6%,C-myc:82.5%、85.71%,Cycling-D1:92.86%、81.82%。结论肝癌的侵润和β-catenin的异质表达均与Wif-1下降,C-myc及Cycling-D1上调相关。     

Expression of Irisin/FNDC5 in Breast Cancer

Irisin is a myokine formed from fibronectin type III domain-containing protein 5 (FNDC5), which can be found in various cancer tissues. FNDC5 and irisin levels have been poorly studied in the tumor tissues of breast cancer (BC). The aim of this study was to determine the levels of irisin expression in BC tissues and compare them to clinicopathological factors and Ki-67 and PGC-1α expression levels. Tissue microarrays (TMAs) with 541 BC tissues and 61 samples of non-malignant breast disease (NMBD; control) were used to perform immunohistochemical reactions. FNDC5 gene expression was measured in 40 BC tissue samples, 40 samples from the cancer margin, and 16 NMBD samples. RT-PCR was performed for the detection of FNDC5 gene expression. Higher irisin expression was found in BC patients compared to normal breast tissue. FNDC5/irisin expression was higher in patients without lymph node metastases. Longer overall survival was observed in patients with higher irisin expression levels. FNDC5/irisin expression was increased in BC tissues and its high level was a good prognostic factor for survival in BC patients.     

Rapid screening and identification of dominant B cell epitopes of HBV surface antigen by quantum dot-based fluorescence polarization assay

A method for quickly screening and identifying dominant B cell epitopes was developed using hepatitis B virus (HBV) surface antigen as a target. Eleven amino acid fragments from HBV surface antigen were synthesized by 9-fluorenylmethoxy carbonyl solid-phase peptide synthesis strategy, and then CdTe quantum dots were used to label the N-terminals of all peptides. After optimizing the factors for fluorescence polarization (FP) immunoassay, the antigenicities of synthetic peptides were determined by analyzing the recognition and combination of peptides and standard antibody samples. The results of FP assays confirmed that 10 of 11 synthetic peptides have distinct antigenicities. In order to screen dominant antigenic peptides, the FP assays were carried out to investigate the antibodies against the 10 synthetic peptides of HBV surface antigen respectively in 159 samples of anti-HBV surface antigen-positive antiserum. The results showed that 3 of the 10 antigenic peptides may be immunodominant because the antibodies against them existed more widely among the samples and their antibody titers were higher than those of other peptides. Using three dominant antigenic peptides, 293 serum samples were detected for HBV infection by FP assays; the results showed that the antibody-positive ratio was 51.9% and the sensitivity and specificity were 84.3% and 98.2%, respectively. In conclusion, a quantum dot-based FP assay is a very simple, rapid, and convenient method for determining immunodominant antigenic peptides and has great potential in applications such as epitope mapping, vaccine designing, or clinical disease diagnosis in the future.