Cell-based sensor for analysis of EGFR biomarker expression in oral cancer

Oral cancer is the sixth most common cancer worldwide and has been marked by high morbidity and poor survival rates that have changed little over the past few decades. Beyond prevention, early detection is the most crucial determinant for successful treatment and survival of cancer. Yet current methodologies for cancer diagnosis based upon pathological examination alone are insufficient for detecting early tumor progression and molecular transformation. To address this clinical need, we have developed a cell-based sensor to detect oral cancer biomarkers, such as the epidermal growth factor receptor (EGFR) whose over-expression is associated with early oral tumorigenesis and aggressive cancer phenotypes. The lab-on-a-chip (LOC) sensor utilizes an embedded track-etched membrane, which functions as a micro-sieve, to capture and enrich cells from complex biological fluids or biopsy suspensions. Once captured, "on-membrane" immunofluorescent assays reveal the presence and isotype of interrogated cells via automated microscopy and fluorescent image analysis. Using the LOC sensor system, with integrated capture and staining technique, EGFR assays were completed in less than 10 minutes with staining intensity, homogeneity, and cellular localization patterns comparable to conventional labeling methods. Further examination of EGFR expression in three oral cancer cell lines revealed a significant increase (p < 0.05) above control cells with EGFR expression similar to normal squamous epithelium. Results obtained in the microfluidic sensor system correlated well with flow cytometry (r(2) = 0.98), the "gold standard" in quantitative protein expression analysis. In addition, the LOC sensor detected significant differences between two of the oral cancer cell lines (p < 0.01), accounting for disparity of approximately 34 000 EGFR per cell according to quantitative flow cytometry. Taken together, these results support the LOC sensor system as a suitable platform for rapid detection of oral cancer biomarkers and characterization of EGFR over-expression in oral malignancies. Application of this technique may be clinically useful in cancer diagnostics for early detection, prognostic evaluation, and therapeutic selection. Having demonstrated the functionality of this integrated microfluidic sensor system, further studies using clinical samples from oral cancer patients are now warranted.     

Designing nanomaterial-enhanced electrochemical immunosensors for cancer biomarker proteins

Detection of multiple cancer biomarker proteins in human serum and tissue at point-of-care is a viable approach for early cancer detection, but presents a major challenge to bioanalytical device development. This article reviews recent approaches developed in our laboratories combining nanoparticle decorated electrodes and multilabeled secondary antibody labeled particles to achieve high sensitivity for the detection of cancer biomarker proteins. Two nanomaterial-based sensor platforms were used: (a) upright single wall carbon nanotube forests and (b) layers of densely packed 5 nm gold nanoparticles. Both platforms feature pendant carboxylate groups for easy attachment of enzymes or antibodies by amidization. In quality performance tests, the biocatalytic responses for determination of hydrogen peroxide of AuNP layers with attached horseradish peroxidase (HRP) on electrodes gave somewhat better detection limit and sensitivity than single wall carbon nanotube (SWNT) forest platforms with HRP attached. Evaluation of these sensors as platforms for sandwich immunoassays for cancer biomarker prostate specific antigen (PSA) in serum showed that both approaches gave accurate results for human serum samples from cancer patients. The best detection limit (0.5 pg mL^− 1) and sensitivity were obtained by combining the AuNP immunosensors with binding of 1 μm diameter magnetic particles decorated with secondary antibodies and 7500 HRP labels.     

In situ detection of live cancer cells by using bioprobes based on Au nanoparticles

We fabricate the high-performance probes based on Au nanoparticles (AuNP) for detection of live cancer cell. AuNP were synthesized with narrow sized distribution (ca. 10 nm) by Au salt reduction method and deposited onto the aminated substrate as a cross-linker and hot spot. Herein, AuNP has enabled the easy and efficient immobilization of the antibody (Cetuximab), which can selectively interact with epidermal growth factor receptor (EGFR) on the surface of epidermal cancer, as detecting moiety onto the AuNP-deposited substrate without nanolithography process. After conjugation of Cetuximab with AuNP-deposited substrate, Cetuximab-conjugated probe as a live cancer cell detector (LCCD) could detect EGFR-highexpressed A431 cells related to epithelial cancer with 54-times larger specificity and sensitivity in comparison with EGFR-deficient MCF7 cells. This implies that AuNP-based probes demonstrate abundant potentials for detection and separation of small biomolecules, cells and other chemicals.     

Simultaneous Visualization of Multiple mRNAs and Matrix Metalloproteinases in Living Cells Using a Fluorescence Nanoprobe

Simultaneous monitoring of multiple tumour markers is of great significance for improving the accuracy of early cancer detection. In this study, a fluorescence nanoprobe has been prepared that can simultaneously monitor and visualize multiple mRNAs and matrix metalloproteinases (MMPs) in living cells. Confocal fluorescence imaging results indicate that the nanoprobe could effectively distinguish between cancer cells and normal cells even if one tumour maker of normal cells was overexpressed. Furthermore, it can detect changes in the expression levels of mRNAs and MMPs in living cells. The current approach could provide new tools for early cancer detection and monitoring the changes in expression levels of biomarkers during tumour progression.     

Aptamer/AuNP Biosensor for Colorimetric Profiling of Exosomal Proteins

Exosomes constitute an emerging biomarker for cancer diagnosis because they carry multiple proteins that reflect the origins of parent cells. Assessing exosome surface proteins provides a powerful means of identifying a combination of biomarkers for cancer diagnosis. We report a sensor platform that profiles exosome surface proteins in minutes by the naked eye. The sensor consists of a gold nanoparticle (AuNP) complexed with a panel of aptamers. The complexation of aptamers with AuNPs protects the nanoparticles from aggregating in a high‐salt solution. In the presence of exosomes, the non‐specific and weaker binding between aptamers and the AuNP is broken, and the specific and stronger binding between exosome surface protein and the aptamer displaces aptamers from the AuNP surface and results in AuNP aggregation. This aggregation results in a color change and generates patterns for the identification of multiple proteins on the exosome surface.     

pH响应型近红外菁类荧光探针的合成及细胞成像

癌症的早期诊治是提高癌症患者治愈率的关键。但传统的"always on"型显影剂存在自身背景干扰且易造成"假阳性"等问题。本文利用肿瘤细胞具有弱酸性这一特性,设计合成了p H激活型不对称菁类荧光探针,并选用氨基葡萄糖作为修饰基团以增强探针母体的水溶性并赋予其肿瘤靶向性。该探针具有p H调控的"off-on"可逆的近红外荧光特性,以及与肿瘤弱酸性微环境相吻合的p H响应范围。此外,探针的浓度高达1.0×10~(-5)mol/L时仍未表现出明显的细胞毒性。该探针在细胞水平实现了肿瘤细胞弱酸性微环境的特异性成像。     

Identification of Peptide tumor markers in a tumor graft model in immunodeficient mice

The medical demand for useful biomarkers is large and still increasing. This is especially true for cancer, because for this disease adequate diagnostic markers with high specificity and sensitivity are still lacking. Despite advances in imaging technologies for early detection of cancer, peptidomic multiplex techniques evolved in recent years will provide new opportunities for detection of low molecular weight (LMW) proteome biomarker (peptides) by mass spectrometry. Improvements in peptidomics research were made based on separation of peptides and/or proteins by their physico-chemical properties in combination with mass spectrometric detection, respectively identification, and sophisticated bioinformatic tools for data analysis. To evaluate the potential of serological tumor marker detection by differential peptide display (DPD) we analyzed plasma samples from a tumor graft model. After subcutaneous injection of HCT-116 cells in immunodeficient mice and their growth to a palpable tumor, plasma samples were analyzed by DPD. The comparison of obtained mass spectrometric data allows discovery of tumor specific peptides which fit well into the biological context of cancer pathogenesis and show a strong correlation to tumor growth. The identified peptides indicate events associated with hyper-proliferation and dedifferentiation of cells from an epithelial origin, which are typical characteristics of human carcinomas. We conclude that these findings are a "proof of principle" to detect differentially expressed, tumor-related peptides in plasma of tumor-bearing mice.     

Current status and need for standards in ion mobility spectrometry

Ion mobility spectrometry (IMS) is a well established technique for the detection of many compounds of interest based on the reduced mobility (K₀) values of their ions. While having the advantage of small size, weight, and power, IMS has been subject to low specificity and is subject to interferences that can cause false alarms in detectors used for security applications. The rate of false positive alarms is directly related to the detection window width required to maintain a high rate of true positive detections. These window widths are in turn a result of the historically available accuracy of reference measurements and the range of responses by multiple detectors. The windows cannot be arbitrarily reduced without risking an increase in the rate of false negative responses. Ongoing work has focused on high accuracy calibration as a means of decreasing the false alarm rates by reducing the variability between detectors which would allow for narrower detection windows. Central to the calibration procedure is the selection of an appropriate calibrant (or reference standard) that can be easily characterized and known with a high degree of certainty across a range of instrumental conditions. This review evaluates a number of previously proposed and potential calibrants against seven recommended criteria of suitability. We examine the sources of false positive alarms in IMS-based detectors and propose a calibration procedure based on high accuracy reference measurements. Initial results of applying this procedure in a post-processing manner are promising towards reducing detector variability and detection window width.     

An electrochemical method to detect folate receptor positive tumor cells

This paper reports an electrochemical method to detect folate receptor positive tumor cells by making use of the interaction between folic acid immobilized on gold nanoparticles and its receptor over-expressed on tumor cell membrane. Experimental results have shown that a gold electrode modified with folic acid functionalized gold nanoparticles can clearly denote folate receptor positive tumor cells, such as ovarian tumor cells and human cervical cancer cells. So, electrochemical technique has been introduced for cancer cells detection and a simple method to detect folate receptor positive tumor cells has been developed.     

Using aptamers to visualize and capture cancer cells

Since diseased cells exist in exceedingly low concentration at the early stage of cancer, highly sensitive imaging and detection methods are required. By improving the methods for capturing and visualizing cancer cells, clinicians can diagnose metastatic relapse, stratify patients for therapeutic purposes, monitor response to drugs and therapies, and track tumor progression. Therefore, using advanced biotechnological and analytical methods combined with cell-SELEX (systematic evolution of ligands by exponential enrichment)-based aptamers, we improved the capture and visualization of diseased cells in a manner that is inexpensive, simple, sensitive, and fast. This multiplexed cancer detection platform therefore improves our control over a range of clinical exigencies, including cancer diagnosis, therapeutic modalities, and drug delivery systems.     

Detection of prolactin inducible protein mRNA, a biomarker for breast cancer metastasis, using a molecular beacon-based assay

Mortality due to breast cancer is increasingly linked to early, undetected metastasis, making methods for earlier detection acutely necessary. We describe the development of an assay based on molecular beacon (MB) chemistry with fluorescence detection to monitor a breast cancer biomarker for the analysis of breast cancer metastasis. The MB assay is based on the complementary base-pairing interactions of the MB nucleic acid with mRNA indicative of breast cancer metastasis. The presence of mRNA is characterized by an increase in the fluorescence intensity of the molecular beacon. The assay gives a linear, reproducible response to prolactin inducible protein mRNA, with a limit of detection in the high picomolar range. This method sensitively and specifically identifies a biomarker directly in serum samples in minimal time and with a straightforward procedure, dramatically reducing the total time for sample analysis over current methods from days to hours. The potential impact of this work in detection and understanding of breast cancer metastasis lies in improvements in simplicity, accuracy, and speed over current methods, which could allow for improved patient treatment and prognoses. Ultimately, additional sample throughput will result in better understanding of disease progression.     

PAUF promotes adhesiveness of pancreatic cancer cells by modulating focal adhesion kinase

Pancreatic cancer is a notorious disease with a poor prognosis and low survival rates, which is due to limited advances in understanding of the molecular mechanism and inadequate development of effective treatment options over the decades. In previous studies, we demonstrated that a novel soluble protein named pancreatic adenocarcinoma up-regulated factor (PAUF) acts on tumor and immune cells and plays an important role in metastasis and progression of pancreatic cancer. Here we show that PAUF promotes adhesiveness of pancreatic cancer cells to various extracellular matrix (ECM). Our results further support a positive correlation of activation and expression of focal adhesion kinase (FAK), a key player in tumor cell metastasis and survival, with PAUF expression. PAUF-mediated adhesiveness was significantly attenuated upon blockade of the FAK pathway. Moreover, PAUF appeared to enhance resistance of pancreatic cancer cells to anoikis via modulation of FAK. Our results suggest that PAUF-mediated FAK activation plays an important role in pancreatic cancer progression.     

AGR2, a mucinous ovarian cancer marker, promotes cell proliferation and migration

Ovarian cancer is a leading cause of death in women. Early detection of ovarian cancer is essential to decrease mortality. However, the early diagnosis of ovarian cancer is difficult due to a lack of clinical symptoms and suitable molecular diagnostic markers. Thus, identification of meaningful tumor biomarkers with potential clinical application is clearly needed. To search for a biomarker for the early detection of ovarian cancer, we identified human anterior gradient 2 (AGR2) from our systematic analysis of paired normal and ovarian tumor tissue cDNA microarray. We noted a marked overexpression of AGR2 mRNA and protein in early stage mucinous ovarian tumors compared to normal ovarian tissues and serous type ovarian tumors by Western blot analysis and immunohistochemistry. To further elucidate the role of AGR2 in ovarian tumorigenesis, stable 2774 human ovarian cancer cell lines overexpressing AGR2 were established. Forced expression of AGR2 in 2774 cells enhanced the growth and migration of ovarian cancer cells. AGR2 protein was detected in the serum of mucinous ovarian cancer patients by Western blot and ELISA analysis. Thus, AGR2 is a potential biomarker for the diagnosis of mucinous ovarian cancer and an ELISA assay may facilitate the early detection of mucinous ovarian cancer using patient serum.     

Application of cycling gradient capillary electrophoresis to detection of APC, K-ras, and DCC point mutations in patients with sporadic colorectal tumors

A previously introduced technique of cycling gradient capillary electrophoresis (CGCE) was applied to monitoring of molecular changes during adenoma-carcinoma transition in progression of sporadic colorectal cancer. The purpose of this work was optimization of separation parameters for selected mutation regions in tumor suppressor genes involved in the early stages of colorectal carcinogenesis, followed by scanning for these mutations in clinical tissue samples from patients with adenomatous polyps and early carcinomas. A total of 47 colorectal tumors in various stages of progression were examined. Main emphasis was given to evaluation of mutation detection sensitivity and specificity required for effective early disease detection. A total of 7 different somatic mutations was identified among 32 K-ras mutant samples, 1 inherited mutation and 5 somatic mutations were identified among 15 adenomatous polyposis coli (APC) mutated samples. None of the two previously reported "deleted in colorectal carcinomas" (DCC) mutations was found in any of the clinical samples. In addition to simple optimization of running conditions, CGCE has demonstrated sensitivity and selectivity allowing detecting small mutant fractions as well as combination of multiple mutants within a single target sequence.     

Analysis of the NCI-60 dataset for cancer-related microRNA and mRNA using expression profiles

BackgroundRecent studies have indicated that microRNA (miRNA) may play an oncogenic or tumor suppressor role in human cancer. To study the regulatory role of miRNAs in tumorigenesis, an integrated platform has been set up to provide a user friendly interface for query. The main advantage of the present platform is that all the miRNA target genes’ information and disease records are drawn from experimentally verified or high confidence records.ResultsMiRNA target gene results are annotated with reference to the disease gene as well as the pathway database. The correlation strength between miRNA and target gene expression profile is quantified by computing the correlation coefficient using the NCI-60 expression profiling data. Comprehensive analysis of the NCI-60 data found that the cumulative percentage of negative correlation coefficients for cleavage regulation is slightly higher than its positive counterpart; which indicated that the mRNA degradation mechanism is slightly dominant. In addition, the RNAHybrid and TargetScans scores are computed which potentially served as quantitative estimators for miRNA–mRNA binding events.Three scores are defined for each miRNA–mRNA pair, which are based on the disease gene and pathway information. These three scores allow user to sort out high confidence cancer-related miRNA–mRNA pairs.Statistical tests were applied to investigate the relations of three chromosomal features, i.e., CpG island, fragile site, and miRNA cluster, with cancer-related miRNAs. A web-based interface has been set up for query, which can be accessed at: http://ppi.bioinfo.asia.edu.tw/mirna_target/ConclusionsThe main advantage of the present platform on miRNA–mRNA targeting information is that all the target genes’ information and disease records are experimentally verified. Although this may limit the number of miRNA–mRNA relationships, the results provided here are more solid and have fewer false positive events. Certain novel cancer-related miRNA–mRNA pairs are identified and confirmed in the literature. Fisher's exact test suggests that CpG island and fragile site associated miRNAs tend to associate with cancer formation. In summary, the present platform provides an easy means of investigating cancer-related miRNAs.     

Oral cancer diagnostics based on infrared spectral markers and wax physisorption kinetics

Infrared microspectroscopy is an emerging approach for disease analysis owing to its capability for in situ chemical characterization of pathological processes. Synchrotron-based infrared microspectroscopy (SR-IMS) provides ultra-high spatial resolution for profiling biochemical events associated with disease progression. Spectral alterations were observed in cultured oral cells derived from healthy, precancerous, primary, and metastatic cancers. An innovative wax-physisorption-based kinetic FTIR imaging method for the detection of oral precancer and cancer was demonstrated successfully. The approach is based on determining the residual amount of paraffin wax (C₂₅H₅₂) or beeswax (C₄₆H₉₂O₂) on a sample surface after xylene washing. This amount is used as a signpost of the degree of physisorption that altered during malignant transformation. The results of linear discriminant analysis (LDA) of oral cell lines indicated that the methylene (CH₂) and methyl group (CH₃) stretching vibrations in the range of 3,000–2,800 cm^?1 have the highest accuracy rate (89.6 %) to discriminate the healthy keratinocytes (NHOK) from cancer cells. The results of wax-physisorption-based FTIR imaging showed a stronger physisorption with beeswax in oral precancerous and cancer cells as compared with that of NHOK, which showed a strong capability with paraffin wax. The infrared kinetic study of oral cavity tissue showed a consistency in the wax physisorption of the cell lines. On the basis of our findings, these results show the potential use of wax-physisorption-based kinetic FTIR imaging for the early screening of oral cancer lesions and the chemical changes during oral carcinogenesis.

Aggregation-induced emission luminogen for specific identification of malignant tumour in vivo

Colorectal cancer(CRC) is still a major killer,mainly due to the lack of early detection biomarker with sensitivity/specificity.Conventional histopathology is the gold standard approach in practice,but there are still some issues(false diagnosis and long waiting period).Aggregation-induced emission(AIE) is a novel photophysical phenomenon which has been widely utilized for biological imaging in vitro,including malignant cells,showing remarkable imaging contrast and enhanced emission in high concentrated state.However,it is unclear if AIE dyes can identify malignant cells specifically in vivo.A great challenge for specific labeling of malignant cells is probably ascribed to the complex compositions in tumor tissues.Herein,an AIE dye-based mitochondria-targeted histological staining strategy was employed to localize cancer and the metastatic track in the fresh colorectal cancer.This novel approach holds great potential to revolutionize the clinical diagnosis and intervention for devastating malignancy.     

A NIR Programmable In Vivo miRNA Magnifier for NIR-II Imaging of Early Stage Cancer

Aberrant expressions of biomolecules occur much earlier than tumor visualized size and morphology change, but their common measurement strategies such as biopsy suffer from invasive sampling process. In vivo imaging of slight biomolecule expression difference is urgently needed for early cancer detection. Fluorescence of rare earth nanoparticles (RENPs) in second near-infrared (NIR-II) region makes them appropriate tool for in vivo imaging. However, the incapacity to couple with signal amplification strategies, especially programmable signal amplification strategies, limited their application in lowly expressed biomarkers imaging. Here we develop a 980/808 nm NIR programmed in vivo microRNAs (miRNAs) magnifier by conjugating activatable DNAzyme walker set to RENPs, which achieves more effective NIR-II imaging of early stage tumor than size monitoring imaging technique. Dye FD1080 (FD1080) modified substrate DNA quenches NIR-II downconversion emission of RENPs under 808 nm excitation. The miRNA recognition region in DNAzyme walker is sealed by a photo-cleavable strand to avoid “false positive” signal in systemic circulation. Upconversion emission of RENPs under 980 nm irradiation activates DNAzyme walker for miRNA recognition and amplifies NIR-II fluorescence recovery of RENPs via DNAzyme catalytic reaction to achieve in vivo miRNA imaging. This strategy demonstrates good application potential in the field of early cancer detection.     

Graphene-Based Biosensors with High Sensitivity for Detection of Ovarian Cancer Cells

Ovarian cancer has the highest mortality rate in the world. Therefore, it is urgent but still challenging to develop an efficient circulating tumor cell (CTC) detection method to sensitively detect ovarian cancer. To address such issues, herein, for the first time, we present a novel CTC detection method for ovarian cancer cells by designing sensitive and rapid graphene-based biosensors. This graphene-based sensor, consisting of a cell pool and two electrodes, can be prepared by a conventional chip fabrication process. It demonstrates high-sensitivity detection even for several ovarian cancer cells by comparing the electrical signal before and after adding cell solution. Moreover, the graphene-based biosensors can perform rapid detection with good repeatability. This suggests that this novel method is possible to use for the early detection of ovarian cancer with very low CTC cell concentration. This work provides a novel and quick strategy to detect ovarian cancer and further judge or predict the risk of the transfer of ovarian cancer.