Abstract 1334: Dual-isolation of CTCs and cancer exosomes from blood samples with melanoma using immunoaffinity based OncoBean microfluidic devices
Authors: Kang Yoon-Tae , Lo Ting-Wen , Hadlock Thomas , Purcell Emma , Kramer Aaron , Reguera Monica De , McLean Scott Alan , Nagrath Sunitha 
Published: August 2019

Abstract
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.