Regulatory role of exosomes in colorectal cancer progression and potential as biomarkers

Colorectal cancer(CRC) remains an enormous challenge to human health worldwide. Unfortunately, the mechanism underlying CRC progression is not well understood. Mounting evidence has confirmed that exosomes play a vital role in CRC progression, which has attracted extensive attention among researchers. In addition to acting as messengers between CRC cells, exosomes also participate in the CRC immunomodulatory process and reshape immune function. As stable message carriers and liquid biopsy option...     

外泌体作为结直肠癌诊断标志物的研究进展

外泌体是一种由细胞分泌至胞外的直径为30~150 nm的纳米囊泡,广泛分布于人体血液、尿液、唾液、汗液、乳汁和脑脊液等多种体液中。结直肠癌是常见的消化道恶性肿瘤,其发病率在全球范围内位居第三,严重威胁着人类健康。结直肠癌来源的外泌体中含有一些肿瘤特异性蛋白、微小RNAs（microRNAs,miRNAs）和长链非编码 RNAs（long non-coding RNAs,lncRNAs）等,与结直肠癌的发生和发展具有相关性,因此有望作为结直肠癌诊断的生物标志物。本文主要对外泌体在结直肠癌诊断中的应用前景作一综述。     

Molecular lipid species in urinary exosomes as potential prostate cancer biomarkers

BackgroundExosomes have recently appeared as a novel source of noninvasive cancer biomarkers, since these nanovesicles contain molecules from cancer cells and can be detected in biofluids. We have here investigated the potential use of lipids in urinary exosomes as prostate cancer biomarkers.MethodsA high-throughput mass spectrometry quantitative lipidomic analysis was performed to reveal the lipid composition of urinary exosomes in prostate cancer patients and healthy controls.ResultsControl samples were first analysed to characterise the lipidome of urinary exosomes and test the reproducibility of the method. In total, 107 lipid species were quantified in urinary exosomes. Several differences, for example, in cholesterol and phosphatidylcholine, were found between urinary exosomes and exosomes derived from cell lines, thus showing the importance of in vivo studies for biomarker analysis. The 36 most abundant lipid species in urinary exosomes were then quantified in 15 prostate cancer patients and 13 healthy controls. Interestingly, the levels of nine lipids species were found to be significantly different when the two groups were compared. The highest significance was shown for phosphatidylserine (PS) 18:1/18:1 and lactosylceramide (d18:1/16:0), the latter also showed the highest patient-to-control ratio. Furthermore, combinations of these lipid species and PS 18:0-18:2 distinguished the two groups with 93% sensitivity and 100% specificity. Finally, in agreement with the reported dysregulation of sphingolipid metabolism in cancer cells, alteration in specific sphingolipid lipid classes were observed.ConclusionThis study shows for the first time the potential use of exosomal lipid species in urine as prostate cancer biomarkers.     

外泌体在胰腺癌中的研究现状与进展

外泌体是一种双层脂质膜连接囊泡样小体，存在于各种体液中，参与细胞及肿瘤微环境之间的物质运输和信号传递。外泌体含有多种生物活性分子，包括脂质、蛋白质、DNA、mRNA以及非编码RNA，可以通过这些活性分子影响肿瘤的发生和发展，甚至可以影响肿瘤的治疗。胰腺癌是一种常见的恶性肿瘤，侵袭性强，预后较差，死亡率高。胰腺癌来源的外泌体是胰腺肿瘤微环境中的重要组成部分，促使胰腺癌细胞成功逃避细胞凋亡的重要因素，并且可以促进肝脏转移微环境的形成。近年来，与胰腺癌相关的外泌体逐渐成为新的研究热点，研究发现外泌体有望成为早期胰腺癌筛查的新型生物学标志，并将为胰腺癌靶向治疗提供可行的技术基础。     

Dysregulation of microRNAs in breast cancer and their potential role as prognostic and predictive biomarkers in patient management

MicroRNAs (miRNAs) are an emerging class of gene expression modulators with relevant roles in several biological processes, including cell differentiation, development, apoptosis, and regulation of the cell cycle. Deregulation of those tiny RNA molecules has been described frequently as a major determinant for the initiation and progression of diseases, including cancer. Not only miRNAs but also the enzymes responsible for miRNA processing could be deregulated in cancer. In this review, we address the role of miRNAs in the pathogenesis of breast cancer, since there are oncogenic, tumor-suppressive, and metastatic-influencing miRNAs. Additionally, the different detection platforms and normalization strategies for miRNAs will be discussed. The major part of this review, however, will focus on the capability of miRNAs to act as diagnostic, predictive, or prognostic biomarkers. We will give an overview of their potential to correlate with response to or benefit from a given treatment and we will consider their ability to give information on prognosis in breast cancer. We will focus on miRNAs validated by more than one study or verified in independent cohorts or where results rely on preclinical as well as clinical evidence. As such, we will discuss their potential use in the personalized management of breast cancer.     

MicroRNAs in stool samples as potential screening biomarkers for pancreatic ductal adenocarcinoma cancer

Pancreatic ductal adenocarcinoma (PDAC) accounts for approximately 90-95% exocrine malignant tumors of the pancreas. The high prevalence of metastasis and the difficulty of early diagnosis lead to a dismal prognosis. MicroRNAs (miRNAs) play a critical role in extensive biological processes. The purpose of this study was to evaluate the feasibility of stool miRNAs as novel biomarker for PDAC screening. MiRNAs were extracted from clinical specimens which included cancer and matched adjacent benign pancreatic tissues of 30 PDAC patients, pancreatic juice of 20 from the 30 PDAC patients and 10 chronic pancreatitis (CP) patients, stool samples of the 30 PDAC patients, the 10 CP patients and 15 healthy volunteers. Relative expression of a panel of 5 dysregulated miRNAs (miR-21, miR-155, miR-196a, miR-216 and miR-217) was analyzed with qRT-PCR. Receiver operating characteristic curve (ROC) analysis was performed to assess the diagnosing value of stool miRNAs in PDAC patients. The study showed that our methods of extracting and detecting miRNAs from pancreatic juice and stool specimens had high reproducibility. Compared to matched adjacent benign pancreatic tissues and pancreatic juice of CP patients, the expression of miR-21 (P = 0.0021 and P = 0.0027) as well as miR-155 (P = 0.0087 and P = 0.0067) was significantly higher and the expression of miR-216 (P < 0.0001 and P = 0.0044) was significantly lower in primary tumor tissues and pancreatic juice of PDAC patients. PDAC patients had a significantly higher stool miR-21 and miR-155 (P = 0.0049 and P = 0.0112) and lower miR-216 level (P = 0.0002) compared to normal controls. The same results were obtained in the expression levels of stool miR-21, miR-155 and miR-216 between PDAC and CP patients (P = 0.0337, P = 0.0388 and P = 0.0117, respectively). Receiver operating characteristic (ROC) analysis by using stool miRNAs expression indicated that combination of miR-21 and miR-155 had best sensitivity of 93.33% while the combination of miR-21, miR-155 and miR-216 would be best for detecting and screening PDAC with area under the curve (AUC) of 0.8667 (95% CI: 0.7722-0.9612) and a better balance of sensitivity and specificity (83.33% vs. 83.33%). Our data indicate that miRNAs could be extracted and detected from pancreatic juice and stool efficiently and reproducibly. MiR-21, miR-155 and miR-216 in stool have the potential of becoming biomarkers for screening PDAC.     

Lipidomic profiling of exosomes from colorectal cancer cells and patients reveals potential biomarkers

Strong evidence suggests that differences in the molecular composition of lipids in exosomes depend on the cell type and has an influence on cancer initiation and progression. Here, we analyzed by liquid chromatography–mass spectrometry (LC‐MS) the lipidomic signature of exosomes derived from the human cell lines normal colon mucosa (NCM460D), and colorectal cancer (CRC) nonmetastatic (HCT116) and metastatic (SW620), and exosomes isolated from the plasma of nonmetastatic and metastatic CRC patients and healthy donors. Analysis of this exhaustive lipid study highlighted changes in some molecular species that were found in the cell lines and confirmed in the patients. For example, exosomes from primary cancer patients and nonmetastatic cells compared with healthy donors and control cells displayed a common marked increase in phosphatidylcholine (PC) 34 : 1, phosphatidylethanolamine (PE) 36 : 2, sphingomyelin (SM) d18 : 1/16 : 0, hexosylceramide (HexCer) d18 : 1/24 : 0 and HexCer d18 : 1/24 : 1. Interestingly, these same lipids species were decreased in the metastatic cell line and patients. Further, levels of PE 34 : 2, PE 36 : 2, and phosphorylated PE p16 : 0/20 : 4 were also significantly decreased in metastatic conditions when compared to the nonmetastatic counterparts. The only molecule species found markedly increased in metastatic conditions (in both patients and cells) when compared to controls was ceramide (Cer) d18 : 1/24 : 1. These decreases in lipid species in the extracellular vesicles might reflect function‐associated changes in the metastatic cell membrane. Although these potential biomarkers need to be validated in a larger cohort, they provide new insight toward the use of clusters of lipid biomarkers rather than a single molecule for the diagnosis of different stages of CRC.     

Emerging role of BAD and DAD1 as potential targets and biomarkers in cancer

As key regulators of apoptosis, BAD and defender against apoptotic cell death 1 (DAD1) are associated with cancer initiation and progression. Multiple studies have demonstrated that BAD and DAD1 serve critical roles in several types of cancer and perform various functions, such as participating in cellular apoptosis, invasion and chemosensitivity, as well as their role in diagnostic/prognostic judgement, etc. Investigating the detailed mechanisms of the cancerous effects of the two proteins will contribute to enriching the options for targeted therapy, and may improve clinical treatment of cancer. The present review summarizes research advances regarding the associations of BAD and DAD1 with cancer, and a hypothesis on the feasible relationship and interaction mechanism between the two proteins is proposed. Furthermore, the present review highlights the potential of the two proteins as therapeutic targets and valuable diagnostic and prognostic biomarkers.     

Role of exosomes in pancreatic cancer

Pancreatic cancer (PC) is a malignant tumour of the human digestive system that has a poor prognosis. Exosomes contain proteins and nucleic acids, and constitute a class of extracellular vesicles defined as membrane-bound nanovesicles of endocytic origin, with a diameter of 40–150 nm. Exosomes are potential diagnostic markers of PC; however, their roles in cancer initiation and progression remain unclear. Previous studies have focused on the molecular mechanisms and functions of exosomes that allow them to accelerate PC cell proliferation, migration and invasion. The present review discusses the interactions between exosomes and the pathophysiology of PC. The potential clinical applications of exosomes are also discussed.     

Expression profile of long non-coding RNAs in pancreatic cancer and their clinical significance as biomarkers

Long non-coding RNAs (lncRNAs) have shown great potential as powerful and non-invasive tumor markers. However, little is known about their value as biomarkers in pancreatic cancer (PC). We applied an Arraystar Human LncRNA Microarray which targeting 7419 lncRNAs to determine the lncRNA expression profile in PC and to screen the potential biomarkers. The most increased lncRNAs in PC tissues were HOTTIP-005, XLOC_006390, and RP11-567G11.1. Increased HOTTIP-005 and RP11-567G11.1 expression were poor prognostic factors for patients with PC (n = 144, p < 0.0001). The expression patterns of HOTTIP splice variants in PC were also detected. HOTTIP-005 and HOTTIP-001 were the first and second most increased HOTTIP splice variants, respectively. Plasma HDRF and RDRF (HOTTIP-005 and RP11-567G11.1 derived RNA fragments in plasma/serum) were present in stable form. Their levels were significantly increased in the patients with PC as compared to the healthy controls (n = 127 and 122 respectively, p < 0.0001) and the high levels were derived from PC. HDRF and RDRF levels are promising indicators for distinguishing patients with PC from those without PC. This study identified HOTTIP-005 and RP11-567G11.1 and their plasma fragments with the potential to be used as prognostic and diagnostic biomarkers of PC. Further large-scale prospective studies are needed to confirm our findings.     

Identification of prostate cancer biomarkers in urinary exosomes

Exosomes have recently appeared as a novel source of non-invasive cancer biomarkers since tumour-specific molecules can be found in exosomes isolated from biological fluids. We have here investigated the proteome of urinary exosomes by using mass spectrometry to identify proteins differentially expressed in prostate cancer patients compared to healthy male controls. In total, 15 control and 16 prostate cancer samples of urinary exosomes were analyzed. Importantly, 246 proteins were differentially expressed in the two groups. The majority of these proteins (221) were up-regulated in exosomes from prostate cancer patients. These proteins were analyzed according to specific criteria to create a focus list that contained 37 proteins. At 100% specificity, 17 of these proteins displayed individual sensitivities above 60%. Even though several of these proteins showed high sensitivity and specificity for prostate cancer as individual biomarkers, combining them in a multi-panel test has the potential for full differentiation of prostate cancer from non-disease controls. The highest sensitivity, 94%, was observed for transmembrane protein 256 (TM256; chromosome 17 open reading frame 61). LAMTOR proteins were also distinctly enriched with very high specificity for patient samples. TM256 and LAMTOR1 could be used to augment the sensitivity to 100%. Other prominent proteins were V-type proton ATPase 16 kDa proteolipid subunit (VATL), adipogenesis regulatory factor (ADIRF), and several Rab-class members and proteasomal proteins. In conclusion, this study clearly shows the potential of using urinary exosomes in the diagnosis and clinical management of prostate cancer.     

Periostin and CA242 as potential diagnostic serum biomarkers complementing CA19.9 in detecting pancreatic cancer

Pancreatic ductal adenocarcinoma (PDAC) is a highly malignant tumor with few biomarkers to guide treatment options. Carbohydrate antigen 19.9 (CA19.9), the most frequently used biomarker for PDAC, is not sensitive and specific enough for the detection of the disease. This study aimed to evaluate serum periostin (POSTN) and CA242 as potential diagnostic biomarkers complementing CA19.9 in detecting pancreatic cancer. Blood samples were from 362 participants, including 213 patients with different stages of PDAC, 75 patients with benign pancreatic disease, and 74 healthy individuals. All samples were randomly divided into a training set and a validation set. Carbohydrate antigen 19.9, CA242, POSTN, as well as carcinoembryonic antigen, were measured by ELISA or automated immunoassay. The receiver operating characteristic curve analysis revealed that the performance of CA19.9 in the validation group were improved by the marker panel composed of CA19.9, POSTN, and CA242, to discriminate early stage PDAC not only from healthy controls (area under the curve [AUC]_CA19.9 = 0.94 vs AUC_{CA19.9 + POSTN + CA242} = 0.98, P < .05) but also from benign conditions (AUC_CA19.9 = 0.87 vs AUC_{CA19.9 + POSTN + CA242} = 0.90, P < .05). In addition, POSTN retained significant diagnostic capabilities to distinguish PDAC CA19.9‐negative from healthy controls (AUC_POSTN = 0.87) as well as from benign conditions (AUC_POSTN = 0.84) in the whole set. This study suggested that POSTN and CA242 are potential diagnostic serum biomarkers complementing CA19.9 in detecting early pancreatic cancer.     

Autoantibodies as potential biomarkers for breast cancer

Introduction

Only a limited number of tumor markers for breast cancer are currently available. Antibodies to tumor-associated proteins may expand the number of available tumor markers for breast cancer and may be used together in a serum profile to enhance sensitivity and specificity.

Methods

In the present study, we interrogated a breast cancer cDNA T7 phage library for tumor-associated proteins using biopan enrichment techniques with sera from normal individuals and from breast cancer patients. The enrichment of tumor-associated proteins after biopanning was tested using a plaque-lift assay and immunochemical detection. The putative tumor-associated phage clones were collected for PCR and sequencing analysis. Unique and open reading frame phage-expressed proteins were then used to develop phage protein ELISAs to measure corresponding autoantibodies using 87 breast cancer patients and 87 normal serum samples. A logistic regression model and leave-one-out validation were used to evaluate predictive accuracies with a single marker as well as with combined markers. Identities of those selected proteins were revealed through the sequence BLAST program.

Results

We harvested 100 putative tumor-associated phage clones after biopan enrichment. Sequencing analysis revealed that six phage proteins were inframe and unique. Antibodies to these six phage-expressed proteins were measured by ELISAs, and the results showed that three of the phage clones had statistical significance in discriminating patients from normal individuals. BLAST results of the three proteins showed great matches to ASB-9, SERAC1, and RELT. Measurements of the three predictive phage proteins were combined in a logistic regression model that achieved 80% sensitivity and 100% specificity in prediction of sample status, whereas leave-one-out validation achieved 77.0% sensitivity and 82.8% specificity among 87 patient samples and 87 control samples. Receiver operating characteristic curve analysis and the leave-one-out method both showed that combined measurements of the three antibodies were more predictive of disease than any of the single antibodies studied, underscoring the importance of identifying multiple potential markers.

Conclusion

Serum autoantibody profiling is a promising approach for early detection and diagnosis of breast cancer. Rather than one autoantibody, a panel of autoantibodies appears preferable to achieve superior accuracy. Further refinements will need to be made to further improve the accuracy. Once refined, the assay must be applied to a prospective patient population to demonstrate applicability.     

外泌体在肺癌中的研究进展

外泌体（exosomes）是由多种细胞分泌的具有脂质双分子层结构的纳米级膜囊小泡,其内含有蛋白质、脂质、核酸等大量生物活性物质。研究表明,外泌体在肺癌的发生发展过程中发挥着重要作用,尤其是在肺癌的早期诊断、侵袭和转移、预后评估以及治疗等方面的作用成为目前研究的热点。因此,关于外泌体的研究将在转化医学的研究模式下,为肺癌临床早期诊断、治疗及预后评估带来新的契机。本文对外泌体在肺癌中的研究进展作一综述。     

癌源性外泌体对乳腺癌MCF-7细胞生物学行为的影响

目的 探究乳腺癌细胞来源的外泌体对乳腺癌MCF-7细胞生物学行为的影响。方法 使用超速离心法对人乳腺癌MCF-7细胞培养液中的外泌体进行提取和纯化,通过透射电子显微镜观察外泌体的形态和大小,Western blot实验鉴定外泌体的标志蛋白。以外泌体与MCF-7细胞共培养48 h后的细胞为实验组,以未处理的MCF-7细胞为对照组。用MTT法、克隆形成实验、细胞划痕实验、细胞侵袭实验、流式细胞术实验和Western blot实验检测乳腺癌源性外泌体对MCF-7细胞增殖、克隆形成、迁移、侵袭、凋亡及上皮-间充质转化(Epithelial-mesenchymal transition,EMT)能力的影响。结果 透射电子显微镜下外泌体的形状为典型的杯托状或者凹的半球状结构,直径40~110 nm;Western blot实验结果显示外泌体标志性蛋白CD9及CD81均明显表达;MTT和克隆形成实验结果显示,实验组细胞增殖及克隆形成能力较对照组明显增强(P＜0.05);划痕实验和侵袭实验结果显示,与对照组相比,实验组细胞迁移及侵袭能力明显增强(P＜0.001);流式细胞术分析显示,在外泌体刺激的乳腺癌细胞中,凋亡细胞的百分比显著低于对照组(P＜0.05);Western blot结果显示,乳腺癌来源的外泌体增加了N-cadherin和Vimentin的表达,降低了E-cadherin表达,促进了乳腺癌EMT过程(P＜0.05)。结论 乳腺癌细胞来源的外泌体能增强肿瘤细胞的细胞增殖、克隆形成、迁移和侵袭能力,促进乳腺癌EMT过程,并抑制肿瘤细胞凋亡。表明肿瘤来源外泌体可能作为细胞间信号转导的介质,在促进肿瘤生长和转移的信息传递方面发挥重要作用。     

外泌体在多发性骨髓瘤中的作用及其临床应用

外泌体是由细胞分泌的内含蛋白或核酸等活性物质、直径为35~120 nm的脂质双分子层囊泡。外泌体通过调控细胞间通讯,不仅参与调控细胞的正常生理过程,同时参与包括肿瘤在内的多种疾病的病理过程。肿瘤来源的外泌体参与肿瘤细胞与微环境的相互作用,并通过与转移、免疫抑制等相关信号通路刺激肿瘤的发生与发展。多发性骨髓瘤（multiple myeloma,MM）是最为常见的血液系恶性肿瘤之一,其具体发病机制尚未完全清楚,且缺乏安全、高效的诊疗手段。而外泌体因携带丰富的生物学信息,为肿瘤的治疗提供了一个新的靶点。因此,本文就外泌体在MM发生和发展中的作用及其以外泌体为基础的诊疗新方向作一综述。     

The role of tumor-associated macrophages in gastric cancer development and their potential as a therapeutic target

Gastric cancer (GC) represents the fifth cause of cancer-related death worldwide. Molecular biology has become a central area of research in GC and there are currently at least three major classifications available to elucidate the mechanisms that drive GC oncogenesis. Further, tumor microenvironment seems to play a crucial role, and tumor-associated macrophages (TAMs) are emerging as key players in GC development. TAMs are cells derived from circulating chemokine- receptor-type 2 (CCR2) inflammatory monocytes in blood and can be divided into two main types, M1 and M2 TAMs. M2 TAMs play an important role in tumor progression, promoting a pro-angiogenic and immunosuppressive signal in the tumor. The diffuse GC subtype, in particular, seems to be strongly characterized by an immuno-suppressive and pro-angiogenic phenotype. No molecular targets in this subgroup have yet been identified. There is an urgent need to understand the molecular pathways and tumor microenvironment features in the GC molecular subtypes. The role of anti-angiogenics and checkpoint inhibitors has recently been clinically validated in GC. Both ramucirumab, a fully humanized IgG1 monoclonal anti-vascular endothelial growth factor receptor 2 (VEGFR2) antibody, and checkpoint inhibitors in Epstein Bar Virus (EBV) and Microsatellite Instable (MSI) subtypes, have proved beneficial in advanced GC. Nevertheless, there is a need to identify predictive markers of response to anti-angiogenics and immunotherapy in clinical practice for a personalized treatment approach. The importance of M2 TAMs in development of solid tumors is currently gaining increasing interest. In this literature review we analyze immune microenvironment composition and signaling related to M1 and M2 TAMs in GC as well as its potential role as a therapeutic target.