in our authorship and readership.In the past year, we had authors from across the globe (Figure 3), with China and the United States leading with the highest share of all published articles.We are also thrilled to see that Advanced NanoBiomed Research has readers all over the world, as shown in the country distribution of article downloads (Figure 4).One of the most exciting developments in Advanced NanoBiomed Research in the past year was the acceptance in the Emerging Sources Citation Index (ESCI) database of Web of Science, which is a testament to its quality and impact.

Melanoma is one of the most aggressive skin cancers due to its potential to metastasize widely in the body. The risk of metastasis is increased with later detection and increased thickness of the primary lesion, thus early identification and surgical removal is critical for higher survival rates for patients. However, even with appropriate treatment, some patients will develop recurrence which may be difficult to identify until advanced or causing symptoms. Recent advances in liquid biopsy have proposed less-invasive alternatives for cancer diagnosis and monitoring using minimal/zero invasion at sample collection, and circulating tumor cells(CTCs) have been considered a promising blood-based surrogate marker of primary tumors. However, previous CTC technologies relying on epithelial-cell adhesion molecules have limited to epithelial cells, thus hampering use of CTCs for non-epithelial cancers such as melanoma. Here, we used the Melanoma-specific OncoBean platform(MelanoBean) conjugated with melanoma specific antibodies(MCAM and MCSP). The device was used in comprehensive studies for diagnosing melanoma and evaluating surgery efficacy based on change in the number and characteristics of CTCs and CTC-clusters pre- and post-surgical treatment. Our study demonstrated that melanoma patients(n=45) at all stages(I-IV) have a noticeable number of MCTCs as well as MCTC-clusters compared to healthy donors(n=9)(P=0.0011), and surgical treatment leads to a significant decrease in the number of CTCs(P<0.0001). The CTCs recovered from the device underwent molecular profiling for melanoma-associated genes expression using multiplexed qRT-PCR, demonstrating the ability to monitor molecular signature through treatment. The presented MelanoBean and the comprehensive approach will empower prognostic value of CTCs in melanoma in much larger cohort studies.

This work presents chemically stable and biodegradable hydrogel beads for the isolation of circulating tumor cells (CTCs) and circulating exosomes in liquid biopsy. The liquid biopsy hydrogel beads ( LB beads) consisting of alginate and poly(vinyl alcohol) hydrogels show both chemical stability and stimuli-degradable characteristics. Unlike single-component hydrogels, this hybrid form is not easily degraded by buffers or cell culture media while its degradable characteristic remains; thus, it is useful in bio-applications requiring multi-step processes with various reagents and lengthy incubation periods. We applied our platform to clinical samples for isolating two promising circulating biomarkers for a liquid biopsy, CTCs and exosomes, by conjugating the hydrogel surface with anti-EpCAM and anti-CD63 antibodies, respectively, thus achieving 37.4 CTCs and comparable amount of exosome recovery per 1 milliliter of blood. The results show easy device-free isolation and retrieval of CTCs and exosomes, with recovered circulating biomarkers successfully analyzed by western blot analysis and fluorescence microscopy. We believe that this simple and versatile platform enables us to isolate prominent circulating biomarkers for clinical use in cancer diagnosis.

As the recognition between natural killer (NK) cells and cancer cells does not require antigen presentation, NK cells are being actively studied for use in adoptive cell therapies in the rapidly evolving armamentarium of cancer immunotherapy. In addition to utilizing NK cells, recent studies have shown that exosomes derived from NK cells also exhibit antitumor properties. Furthermore, these NK cell-derived exosomes exhibit higher stability, greater modification potentials and less immunogenicity compared to NK cells. Therefore, technologies that allow highly sensitive and specific isolation of NK cells and NK cell-derived exosomes can enable personalized NK-mediated cancer therapeutics in the future. Here, a novel microfluidic system to collect patient-specific NK cells and on-chip biogenesis of NK-exosomes is proposed. In a small cohort of non-small cell lung cancer (NSCLC) patients, both NK cells and circulating tumor cells (CTCs) were isolated, and it is found NSCLC patients have high numbers of NK and NK-exosomes compared with healthy donors, and these concentrations show a trend of positive and negative correlations with bloodborne CTC numbers, respectively. It is further demonstrated that the NK-exosomes harvested from NK-graphene oxide chip exhibit cytotoxic effect on CTCs. This versatile system is expected to be used for patient-specific NK-based immunotherapies along with CTCs for potential prognostic/diagnostic applications.

While significant advancements have been made in cancer therapeutics and treatments, early disease detection and diagnosis remains critical to ensuring favorable outcomes for patients. To that end, we propose a microfluidic based approach to the sensitive detection of an intriguing cancer biomarker, extracellular vesicles (EVs). Our extracellular vesicles on demand (EVOD) chip utilizes a catalyst-free click chemistry to rapidly and specifically isolate EVs of interest. This specific isolation is followed by subsequent dithiothreitol release of the isolated EVs for downstream functional analysis. This joint isolation and release provide a powerful tool for the screening and quantification of EVs of interest. By incorporating antibodies against cancer associated surface proteins into the click-chemistry, we were able to selectively recover cancer-associated exosomes, allowing for important insights into patient disease. This platform was also tested using non-small cell lung cancer (NSCLC) patient samples, where anti-epidermal growth factor receptor (EGFR) assisted platform were able to selectively isolate and release 76% more exosomes from NSCLC patients than from healthy donors. This matches the previously reported higher EGFR expression commonly found in NSCLC EVs. Through its rapid isolation kinetics and adaptability in marker targeting, the EVOD device provides a highly versatile liquid biopsy platform for clinicians to use in the fight against cancer.

Abstract Melanoma is among the most aggressive cancers, and its rate of incidence continues to grow. Early detection of melanoma has been hampered due to the lack of promising markers for testing. Recent advances in liquid biopsy have proposed noninvasive alternatives for cancer diagnosis and monitoring. Circulating tumor cells (CTCs) and cancer‐exosomes are gaining influence as promising biomarkers because of their cancer‐associated molecular markers and signatures. However, technologies that offer the dual‐isolation of CTCs and exosomes using a single sample have not been thoroughly developed. The dual‐utilization OncoBean (DUO) device is conjugated with melanoma specific antibodies, MCAM and MCSP, enabling simultaneous CTC and exosome isolations. Using blood samples from patients, CTCs and exosomes are specifically isolated from a single sample and then undergo molecular profiling for comprehensive study. Melanoma patients have 0–17CTCs mL−1 and 299 µg exosomal protein mL−1 while healthy donors display fewer than 2CTCs and 75.6 µg of exosomes mL−1, respectively. It is also demonstrated that both markers express melanoma‐associated genes using multiplex qRT‐PCR to test for expression pattern of a 96 gene panel. The dual isolation and molecular characterization will allow for further research into melanoma to identify viable markers for disease progression and treatment efficacy.

Introduction: Melanoma is among the most aggressive cancers, and its incidence continues to grow. Due to the lack of promising markers to predict the disease and onset of metastasis, early detection and evaluation of treatment efficacy have been hampered. Recent advances in liquid biopsy have proposed alternatives for diagnosing disease with the merits of enabling continuous monitoring and non-invasiveness. CTCs and cancer exosomes are evolving as promising biomarkers due to their innate capability of having cancer-associated molecules and signatures. However, simultaneous isolation of CTCs and exosomes using the same methods from the identical samples has not been studied yet. Here, we propose the use of the OncoBean microfluidic device conjugated with melanoma specific antibodies, MCAM and MCSP for the simultaneous isolation. Using whole blood samples from patients, CTCs and exosomes are specifically isolated from the same samples and then undergo molecular profiling for comprehensive studies. Methods: OncoBean devices were fabricated by soft lithography using polydimethylsiloxane (PDMS). The fabricated devices were conjugated with biotinylated MCSP and MCAM, using Neutralavidin-biotin chemistry. Using 6ml blood samples, 3ml of whole blood and 3ml of plasma were used for CTC isolation and melanoma-associated exosome isolation, respectively. In order to remove cellular debris, the plasma sample was filtered using a 200nm filter. After CTC isolation, captured cells were stained with fluorescent antibodies for melanoma specific (Melan-A and S100), leukocyte (CD45), and nucleus (DAPI) markers. The captured and stained CTCs were enumerated with consideration of size, nucleus to cytoplasm ratio and melanoma marker expression. The isolated exosomes were examined by scanning electron microscopy (SEM) to evaluate their abundance and size. The protein concentration and exosomal protein expression were evaluated by protein extraction using RIPA buffer. Results: All melanoma patient samples showed 1-35 CTCs per 3ml of blood. Healthy donors had no CTCs. The abundance of isolated exosomes was evaluated by SEM and showed sizes ranging from 117-143nm. The average exosomal protein from melanoma was 28.6±19.2μg/ml, considerably higher than that of healthy donor, 11.0±3.1μg/ml. The exosomes isolated by OncoBean tested positively for the common exosomal protein, CD9, implying that the present device isolated melanoma exosomes specifically. Discussion and conclusion: We showed that melanoma patients have both circulating tumor cells and cancerous exosomes in their blood samples. Even though their absolute CTC number and exosomal protein concentration showed no high correlation, simultaneous isolation using the identical microfluidic devices will be helpful for further verification of disease and cross validation according to their roles in disease. Citation Format: Yoon-Tae Kang, Ting-Wen Lo, Thomas Hadlock, Emma Purcell, Aaron Kramer, Monica De Reguera, Scott Alan McLean, Sunitha Nagrath. Dual-isolation of CTCs and cancer exosomes from blood samples with melanoma using immunoaffinity based OncoBean microfluidic devices [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1334.

Profiling of extracellular vesicles (EVs) is an emerging area in the field of liquid biopsies because of their innate significance in diseases and abundant information reflecting disease status. However, unbiased enrichment of EVs and thorough profiling of EVs is challenging. In this paper, we present a simple strategy to immobilize and analyze EVs for multiple markers on a single microfluidic device and perform differentiated immunostaining-based characterization of extracellular vesicles (DICE). This device, composed of four quadrants with a single inlet, captures biotinylated EVs efficiently and facilitates multiplexed immunostaining to profile their extracellular proteins, allowing for a multiplexed approach for non-invasive cancer diagnostics in the future. From controlled sample experiments using cancer cell line derived EVs and specific fluorescence staining with lipophilic dyes, we identified that the DICE device is capable of isolating biotinylated EVs with 84.4% immobilization efficiency. We extended our study to profile EVs of 9 clinical samples from non-small cell lung cancer (NSCLC) patients and healthy donors and found that the DICE device successfully facilitates immunofluorescent staining for both the NSCLC patients and the healthy control. This versatile and simple method to profile EVs could be extended to EVs of any biological origin, promoting discoveries of the role of EVs in disease diagnostics and monitoring.