Cesar M. Castro

Label-free detection and molecular profiling of exosomes with a nano-plasmonic sensor

Exosomes show potential for cancer diagnostics because they transport molecular contents of the cells from which they originate. Detection and molecular profiling of exosomes is technically challenging and often requires extensive sample purification and labeling. Here we describe a label-free, high-throughput approach for quantitative analysis of exosomes. Our nano-plasmonic exosome (nPLEX) assay is based on transmission surface plasmon resonance through periodic nanohole arrays. Each array is functionalized with antibodies to enable profiling of exosome surface proteins and proteins present in exosome lysates. We show that this approach offers improved sensitivity over previous methods, enables portable operation when integrated with miniaturized optics and allows retrieval of exosomes for further study. Using nPLEX to analyze ascites samples from ovarian cancer patients, we find that exosomes derived from ovarian cancer cells can be identified by their expression of CD24 and EpCAM, suggesting the potential of exosomes for diagnostics.

A magneto-DNA nanoparticle system for rapid detection and phenotyping of bacteria

So far, although various diagnostic approaches for pathogen detection have been proposed, most are too expensive, lengthy or limited in specificity for clinical use. Nanoparticle systems with unique material properties, however, circumvent these problems and offer improved accuracy over current methods. Here, we present novel magneto-DNA probes capable of rapid and specific profiling of pathogens directly in clinical samples. A nanoparticle hybridization assay, involving ubiquitous and specific probes that target bacterial 16S rRNAs, was designed to detect amplified target DNAs using a miniaturized NMR device. Ultimately, the magneto-DNA platform will allow both universal and specific detection of various clinically relevant bacterial species, with sensitivity down to single bacteria. Furthermore, the assay is robust and rapid, simultaneously diagnosing a panel of 13 bacterial species in clinical specimens within 2 h. The generic platform described could be used to rapidly identify and phenotype pathogens for a variety of applications.

Micro-NMR for Rapid Molecular Analysis of Human Tumor Samples

A portable micro-NMR device enables rapid molecular diagnosis from scarce cancer cells in fine-needle aspirates from human tumors. A Micro-NMR Smart Phone for Detecting Cancer Smart phones may have provided millions of people with hours of mindless entertainment, but their real value may lie in the hands of clinicians at the patient’s bedside. Accurate diagnosis and staging of tumors through analysis of core biopsies is essential for ensuring that patients receive rapid treatment with the most appropriate cancer drugs. However, current techniques for analyzing tumor biopsies such as immunohistochemistry and flow cytometry are costly and time-consuming and require more tissue than biopsies often contain, necessitating that patients be subjected to repeat biopsies. Enter Weissleder and his team with their portable micro-NMR (nuclear magnetic resonance) device operated by smart phone technology that enables accurate diagnosis of malignant tumors from the scarce cancer cells present in fine-needle aspirate biopsies. Weissleder’s micro-NMR system uses nanoparticle-based magnetic affinity ligands to detect expression of protein markers that are associated with cancer. First, the investigators looked at the expression of nine protein markers in fine-needle aspirates taken from suspected malignant abdominal lesions in a cohort of 50 patients. Using detection of their nine-marker signature by micro-NMR, they correctly identified 44 patients as having malignant tumors, with verification of the diagnoses by standard techniques such as core biopsy, imaging, and clinical assessment. Using a four-protein marker signature (MUC-1, HER2, epidermal growth factor receptor, and EpCAM), Weissleder and his team were able to increase the diagnostic accuracy of their micro-NMR system still further to 96%. This exceeded the 84% accuracy of immunohistochemistry, a gold standard of cancer diagnosis, and was much faster, providing a diagnosis within 1 hour instead of 3 days. To validate their findings, the investigators tested their four-protein marker signature in another 20 patients with suspected abdominal malignancies and this time obtained a 100% accurate diagnosis. The micro-NMR cancer diagnostic system has the advantages of speed and accuracy, but the authors do point out several caveats. They discovered that protein marker expression is heterogeneous within and between tumors and varies depending on the location of the final needle aspirate site. They also point out that because of rapid protein degradation even at 4°C, the biopsies either should be analyzed immediately or should be carefully preserved. Despite these caveats, this portable micro-NMR device, which is easy to operate with smart phone technology, could improve the speed and accuracy of cancer diagnosis, leading to earlier and more effective treatments for cancer patients. Although tumor cells obtained from human patients by image-guided intervention are a valuable source for diagnosing cancer, conventional means of analysis are limited. Here, we report the development of a quantitative micro-NMR (nuclear magnetic resonance) system for rapid, multiplexed analysis of human tumors. We implemented the technology in a clinical setting to analyze cells obtained by fine-needle aspirates from suspected lesions in 50 patients and validated the results in an independent cohort of another 20 patients. Single fine-needle aspirates yielded sufficient numbers of cells to enable quantification of multiple protein markers in all patients within 60 min. Moreover, using a four-protein signature, we report a 96% accuracy for establishing a cancer diagnosis, surpassing conventional clinical analyses by immunohistochemistry. Our results also show that protein expression patterns decay with time, underscoring the need for rapid sampling and diagnosis close to the patient bedside. We also observed a surprising degree of heterogeneity in protein expression both across the different patient samples and even within the same tumor, which has important implications for molecular diagnostics and therapeutic drug targeting. Our quantitative point-of-care micro-NMR technique shows potential for cancer diagnosis in the clinic.

Ultrasensitive Clinical Enumeration of Rare Cells ex Vivo Using a Micro-Hall Detector

A hybrid microfluidic/semiconductor chip analyzes single, immunomagnetically tagged ovarian cancer cells in unprocessed biological samples. Magnetic Microchip Counts Tumor Cells The idiom “looking for a needle in a haystack” could not be applied more appropriately in medicine than to describe the detection of circulating tumor cells (CTCs). Often, only 10 of these rare cells are present in 10 ml of blood; that’s about 1 CTC for every 1 billion blood cells. Despite their scarcity, these cells may hold a wealth of information to help guide treatment decisions for cancer patients. Undaunted, Issadore and colleagues developed a miniature device that combines microfluidics and magnets to measure CTCs in patient blood at single-cell resolution. The authors designed a micro-Hall detector (μHD) that senses the magnetic moment of particles. In this system, cells were first labeled with magnetic beads conjugated to antibodies directed at a target cell surface molecule. The magnetically labeled cells could then be flowed through the microfluidic channel, where tiny Hall detectors would sense their presence. Issadore et al. first tested the μHD with cells derived from human epithelial cancers (such as breast and brain), looking for three different cancer-related markers: human epidermal growth factor receptor 2 (HER2)/neu, epidermal growth factor receptor (EGFR), and EpCAM. Out of a mixture of blood cells—some labeled, some not—the authors only missed a cancer cell 10% of the time, compared with flow cytometry (the gold standard in the clinic), which had a false-negative rate of 81%. The μHD was also able to detect multiple biomarkers on individual cells simultaneously, which could work toward further refining subpopulations of rare cells according to surface expression. To show use in the clinic, Issadore and coauthors noted elevated numbers of CTCs in the blood of 20 ovarian cancer patients, but not in any of the 15 healthy volunteers. By comparison, CellSearch (the gold standard technology for CTC enumeration) only detected CTCs in 25% of the same patient samples. The μHD appears to be a more sensitive cell counter than existing devices, with the potential to change patient management and disease monitoring in the clinic. The needles are still there, but we now have a rapid way of sorting through the haystack. The ability to detect rare cells (<100 cells/ml whole blood) and obtain quantitative measurements of specific biomarkers on single cells is increasingly important in basic biomedical research. Implementing such methodology for widespread use in the clinic, however, has been hampered by low cell density, small sample sizes, and requisite sample purification. To overcome these challenges, we have developed a microfluidic chip–based micro-Hall detector (μHD), which can directly measure single, immunomagnetically tagged cells in whole blood. The μHD can detect single cells even in the presence of vast numbers of blood cells and unbound reactants, and does not require any washing or purification steps. In addition, the high bandwidth and sensitivity of the semiconductor technology used in the μHD enables high-throughput screening (currently ~107 cells/min). The clinical use of the μHD chip was demonstrated by detecting circulating tumor cells in whole blood of 20 ovarian cancer patients at higher sensitivity than currently possible with clinical standards. Furthermore, the use of a panel of magnetic nanoparticles, distinguished with unique magnetization properties and bio-orthogonal chemistry, allowed simultaneous detection of the biomarkers epithelial cell adhesion molecule (EpCAM), human epidermal growth factor receptor 2 (HER2)/neu, and epidermal growth factor receptor (EGFR) on individual cells. This cost-effective, single-cell analytical technique is well suited to perform molecular and cellular diagnosis of rare cells in the clinic.

Cancer cell profiling by barcoding allows multiplexed protein analysis in fine-needle aspirates.

Barcoding technology enabled measurement of hundreds of cellular proteins from cancer patients with single-cell resolution. Fine-Tuning Single Cancer Cell Protein Analysis Fine-needle aspirates (FNAs) use thin needles to obtain cells from tumor masses. FNAs can give tremendous insight into malignancy, but the number of cells is so small that current technologies for protein analysis, such as immunohistochemistry, are insufficient. To address this technological gap, Ullal and colleagues developed the antibody barcoding with photocleavable DNA (ABCD) platform that allows for simultaneous analysis of many surface proteins on cells from cancer patients. The authors first isolated cancer cells within the FNAs of patients. These cells were then exposed to a cocktail of 90 antibodies, covering the hallmark processes in cancer (for example, apoptosis and DNA damage), and each containing a unique “barcode”—a single strand of DNA that could be released by light (photocleaved) and quantified using fluorescent complementary probes. After validating protein expression on human cancer cell lines with known protein composition, Ullal et al. moved to FNA samples from patients with lung adenocarcinoma. The protein profiles of 11 single cells taken from one patient showed low correlation with the bulk tumor sample, indicating high intratumor heterogeneity. The authors also noted high intertumor heterogeneity, because six patients with tumors that looked identical under a microscope had different proteomic profiles. By clustering the protein expression results and comparing to the patients’ genetic makeup, the authors suggest that therapies could be better personalized. The ABCD platform could help researchers to better understand tumor heterogeneity, as well as allow clinicians to personalize therapies and perhaps even track therapeutic response less invasively, as the authors demonstrated in vitro and in four patients taking kinase inhibitors. Technological challenges remain in making this platform validated and ready for the bedside, but successful early demonstrations in patients with lung cancer suggest that larger-scale clinical trials are in the near future. Immunohistochemistry-based clinical diagnoses require invasive core biopsies and use a limited number of protein stains to identify and classify cancers. We introduce a technology that allows analysis of hundreds of proteins from minimally invasive fine-needle aspirates (FNAs), which contain much smaller numbers of cells than core biopsies. The method capitalizes on DNA-barcoded antibody sensing, where barcodes can be photocleaved and digitally detected without any amplification steps. After extensive benchmarking in cell lines, this method showed high reproducibility and achieved single-cell sensitivity. We used this approach to profile ~90 proteins in cells from FNAs and subsequently map patient heterogeneity at the protein level. Additionally, we demonstrate how the method could be used as a clinical tool to identify pathway responses to molecularly targeted drugs and to predict drug response in patient samples. This technique combines specificity with ease of use to offer a new tool for understanding human cancers and designing future clinical trials.

Integrated Magneto–Electrochemical Sensor for Exosome Analysis

Extracellular vesicles, including exosomes, are nanoscale membrane particles that carry molecular information on parental cells. They are being pursued as biomarkers of cancers that are difficult to detect or serially follow. Here we present a compact sensor technology for rapid, on-site exosome screening. The sensor is based on an integrated magneto-electrochemical assay: exosomes are immunomagnetically captured from patient samples and profiled through electrochemical reaction. By combining magnetic enrichment and enzymatic amplification, the approach enables (i) highly sensitive, cell-specific exosome detection and (ii) sensor miniaturization and scale-up for high-throughput measurements. As a proof-of-concept, we implemented a portable, eight-channel device and applied it to screen extracellular vesicles in plasma samples from ovarian cancer patients. The sensor allowed for the simultaneous profiling of multiple protein markers within an hour, outperforming conventional methods in assay sensitivity and speed.

Magnetic Nanosensor for Detection and Profiling of Erythrocyte-Derived Microvesicles

During the course of their lifespan, erythrocytes actively shed phospholipid-bound microvesicles (MVs). In stored blood, the number of these erythrocyte-derived MVs has been observed to increase over time, suggesting their potential value as a quality metric for blood products. The lack of sensitive, standardized MV assays, however, poses a significant barrier to implementing MV analyses into clinical settings. Here, we report on a new nanotechnology platform capable of rapid and sensitive MV detection in packed red blood cell (pRBC) units. A filter-assisted microfluidic device was designed to enrich MVs directly from pRBC units, and label them with target-specific magnetic nanoparticles. Subsequent detection using a miniaturized nuclear magnetic resonance system enabled accurate MV quantification as well as the detection of key molecular markers (CD44, CD47, CD55). When the developed platform was applied, MVs in stored blood units could also be monitored longitudinally. Our results showed that MV counts increase over time and, thus, could serve as an effective metric of blood aging. Furthermore, our studies found that MVs have the capacity to generate oxidative stress and consume nitric oxide. By advancing our understanding of MV biology, we expect that the developed platform will lead to improved blood product quality and transfusion safety.

Digital diffraction analysis enables low-cost molecular diagnostics on a smartphone

Significance Smartphones and wearable electronics have advanced tremendously over the last several years but fall short of allowing their use for molecular diagnostics. We herein report a generic approach to enable molecular diagnostics on smartphones. The method utilizes molecular-specific microbeads to generate unique diffraction patterns of “blurry beads” which can be recorded and deconvoluted by digital processing. We applied the system to resolve individual precancerous and cancerous cells as well as to detect cancer-associated DNA targets. Because the system is compact, easy to operate, and readily integrated with the standard, portable smartphone, this approach could enable medical diagnostics in geographically and/or socioeconomically limited settings with pathology bottlenecks. The widespread distribution of smartphones, with their integrated sensors and communication capabilities, makes them an ideal platform for point-of-care (POC) diagnosis, especially in resource-limited settings. Molecular diagnostics, however, have been difficult to implement in smartphones. We herein report a diffraction-based approach that enables molecular and cellular diagnostics. The D3 (digital diffraction diagnosis) system uses microbeads to generate unique diffraction patterns which can be acquired by smartphones and processed by a remote server. We applied the D3 platform to screen for precancerous or cancerous cells in cervical specimens and to detect human papillomavirus (HPV) DNA. The D3 assay generated readouts within 45 min and showed excellent agreement with gold-standard pathology or HPV testing, respectively. This approach could have favorable global health applications where medical access is limited or when pathology bottlenecks challenge prompt diagnostic readouts.

Orthogonal Amplification of Nanoparticles for Improved Diagnostic Sensing

There remains an ongoing need for fast, highly sensitive, and quantitative technologies that can detect and profile rare cells in freshly harvested samples. Recent developments in nanomaterial-based detection platforms provide advantages over traditional approaches in terms of signal sensitivity, stability, and the possibility for performing multiplexed measurements. Here, we describe a bioorthogonal, nanoparticle amplification technique capable of rapid augmentation of detection sensitivities by up to 1-2 orders of magnitude over current methods. This improvement in sensitivity was achieved by (i) significantly reducing background noise arising from nonspecific nanoparticle binding, (ii) increasing nanomaterial binding through orthogonal rounds of amplification, and (iii) implementing a cleavage step to improve assay robustness. The developed method allowed sensitive detection and molecular profiling of scant tumor cells directly in unpurified human clinical samples such as ascites. With its high sensitivity and simplified assay steps, this technique will likely have broad utility in nanomaterial-based diagnostics.

Ascites analysis by a microfluidic chip allows tumor-cell profiling

Significance Serial molecular analyses of tumor cells during treatment- and biopsy-driven clinical trials are emerging norms for many cancers. Yet surgical and image-guided biopsies are expensive and invasive, explaining why alternative sources for tumor cells are being sought. In ovarian cancer (and other abdominopelvic cancers), abdominal fluid buildup (ascites) occurs frequently. We demonstrate that ascites tumor cells (ATCs) present a valuable source of tumor cells, rendering ascites another form of “liquid biopsy.” We evaluated 85 ovarian cancer-related markers and developed a unique, low cost miniaturized microfluidic ATC chip for on-chip enrichment and molecular profiling using small amounts of ascites. This approach could expand the utility of ATCs within cytotoxic and/or molecularly targeted ovarian cancer therapeutic trials. Ascites tumor cells (ATCs) represent a potentially valuable source of cells for monitoring treatment of ovarian cancer as it would obviate the need for more invasive surgical biopsies. The ability to perform longitudinal testing of ascites in a point-of-care setting could significantly impact clinical trials, drug development, and clinical care. Here, we developed a microfluidic chip platform to enrich ATCs from highly heterogeneous peritoneal fluid and then perform molecular analyses on these cells. We evaluated 85 putative ovarian cancer protein markers and found that nearly two-thirds were either nonspecific for malignant disease or had low abundance. Using four of the most promising markers, we prospectively studied 47 patients (33 ovarian cancer and 14 control). We show that a marker set (ATCdx) can sensitively and specifically map ATC numbers and, through its reliable enrichment, facilitate additional treatment-response measurements related to proliferation, protein translation, or pathway inhibition.