Extracellular vesicles (EVs) are emerging as a potential diagnostic test for cancer. Owing to the recent advances in microfluidics, on‐chip EV isolation is showing promise with respect to improved recovery rates, smaller necessary sample volumes, and shorter processing times than ultracentrifugation. Immunoaffinity‐based microfluidic EV isolation using anti‐CD63 is widely used; however, anti‐CD63 is not specific to cancer‐EVs, and some cancers secrete EVs with low expression of CD63. Alternatively, phosphatidylserine (PS), usually expressed in the inner leaflet of the lipid bilayer of the cells, is shown to be expressed on the outer surface of cancer‐associated EVs. A new exosome isolation microfluidic device (newExoChip), conjugated with a PS‐specific protein, to isolate cancer‐associated exosomes from plasma, is presented. The device achieves 90% capture efficiency for cancer cell exosomes compared to 38% for healthy exosomes and isolates 35% more A549‐derived exosomes than an anti‐CD63‐conjugated device. Immobilized exosomes are then easily released using Ca2+ chelation. The recovered exosomes from clinical samples are characterized by electron microscopy and western‐blot analysis, revealing exosomal shapes and exosomal protein expressions. The newExoChip facilitates the isolation of a specific subset of exosomes, allowing the exploration of the undiscovered roles of exosomes in cancer progression and metastasis.

Small extracellular vesicles, often termed as “exosomes” carry informative cargo containing proteins and lipids, reflective of their cellular origin. Thus, they are promising biomarkers for early diagnosis of cancer. However, conventional profiling methods like quantitative polymerase chain reaction (qPCR) require complex procedures, thereby limiting the analytical sensitivity of exosomes for liquid biopsies. Here, we demonstrate a sensitive microfluidic device (CDEXO) that isolates and profiles cancer-associated exosomes directly from blood plasma using assembled chiral gold nanoparticles (AuNPs). The unique changes in chiral signals are associated with specific biomolecules on the exosomes’ membranes. Thus, we can distinguish exosomes of lung cancer patients from those of healthy individuals and detect mutated EGFR proteins on the membrane. Hence, this low-cost microfluidic device is an attractive technique for rapid, sensitive, and versatile profiling of various extracellular vesicles. Methods: The top layer of CDEXO devices were fabricated by soft lithography using polydimethylsiloxane (PDMS). The bottom glass slide was functionalized with a layer-by-layer assembly of cationic poly(dimethyl diallyl ammonium chloride) and anionic polystyrene sulfonate. AuNPs were prepared by adding gold nanoplates to a growth solution. Next, AuNPs were conjugated with biotinylated Annexin-V, using Neutravidin-biotin chemistry. Exosomes were harvested from lung cancer cell lines (A549, H1650, H3255) and lung fibroblasts (MRC5) and spiked into the CDEXO chip. EDTA was used to release the captured exosomes and quantified using nanoparticle tracking analysis (NTA). CD spectra were measured by spectrometry. Imaging of AuNPs and exosomes were done using scanning electron microscope (SEM). Results: CDEXO captures cancer-associated exosomes with an efficiency of 81.1±1.5%. H3255 derived exosomes that exhibited EGFR point mutation showed the greatest change in spectral signals from the baseline, followed by A549 (wild type), H1650, (EGFR exon19 deletion) and MRC5 (healthy). The CD responses were measurable at exosome numbers as low as 100. Further validation with 19 lung cancer patients showed an average 40% change in chiral signals from isolated exosomes that were 5.6 times higher in patients than healthy donors. Conclusions: A microfluidic device with chiral AuNPs allows sensitive and accurate detection of lung cancer-associated exosomes by conjugation with Annexin V. The resulting strong CD peaks that arise from specific interactions between exosomal surface proteins and chiral AuNPs facilitate in-depth profiling of target exosomes, including EGFR mutation expression. Citation Format: Yoon-Tae Kang, Ji-Young Kim, Emine Sumeyra Turali-Emre, Hee-Jeong Jang, Minjeong Cha, Abha Kumari, Colin Palacios-Rolston, Chitra Subramanian, Emma Purcell, Sarah Owen, Chung-Man Lim, Rishindra Reddy, Shruthi Jolly, Nithya Ramnath, Nicholas A. Kotov, Sunitha Nagrath. Chiroptical detection and mutation analysis of cancer-associated extracellular vesicles in microfluidic devices with oriented chiral nanoparticles [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 994.

Introduction: Extracellular vesicles (EV), recovered from a liquid biopsy, are emerging as a putative diagnostic test for cancer. To date, ultracentrifugation has been accepted as the gold standard for EV isolation despite its lengthy processing time and low recovery rate. Thanks to the recent technological advances in microfluidics, several microfluidic devices for EV isolation have been developed and are showing promise with respect to improved recovery rates, the ability to extract EVs from small sample volumes, and shorter processing time compared to ultracentrifugation. Among the techniques, immunoaffinity based microfluidic isolation using antibodies against tetraspanins, such as anti-CD63, has been widely applied for EV isolation in general, however, anti-CD63 is not specific to cancer-associated EVs and some cancer cells secrete exosomes with low expression of CD63, thus limiting exosome isolation for cancer study. Alternatively, one of the phospholipids, phosphatidylserine (PS), usually expressed in the inner leaflet of the lipid bilayer of the cells, has also been shown to be expressed on the outer surface of cancer-associated EVs during its vesiculation procedure. Here, we present a new exosome isolation device (newExoChip), conjugated with PS-specific protein, to isolate cancer-associated exosomes from plasma Methods: The newExoChip, having 30x60 circular channels conjugated with PS-specific protein, isolates cancer-associated exosomes in a Ca2+ dependent manner. The isolated exosomes using the device were easily released using Ca2+ chelation and the released exosomes has been analyzed quantitatively and qualitatively using nanoparticle tracking analysis (NTA) and western blot analysis, respectively. For clinical studies, we obtained non-small cell lung cancer (NSCLC) blood samples and each blood sample was centrifuged and the 30-100µl of plasma supernatant was flowed through the device using a syringe pump. Results: The newExoChip achieves 84.58% capture efficiency for NSCLC-derived exosomes compared to 38.43% for healthy exosomes and isolates averagely 47.4% more A549-derived exosomes than anti-tetraspanin devices (anti-CD63, 9, and 81). From a clinical study using 4 NSCLC blood samples, we recovered 1.47x109 EVs per 1 milliliter and 75.03% of vesicles was within the exosomal size range (30-150nm). The isolated exosomes from clinical samples were characterized by western blot using CD9 and flotillin-1, revealing exosomal protein expressions. Discussion and conclusion: Our results demonstrated that the isolated and released vesicles using the newExoChip show conventional features of exosomes, such as exosomal marker expressions and the exosomal size. We believe that the newExoChip facilitates the isolation of a specific subset of exosomes, allowing us to explore the undiscovered roles of exosomes in cancer. Citation Format: Yoon-Tae Kang, Emma Purcell, Colin Palacios-Rolston, Ting-Wen Lo, Nithya Ramnath, Shruti Jolly, Sunitha Nagrath. Microfluidic isolation (newExoChip) and profiling of cancer-associated exosomes using extracellular vesicular lipid-protein binding affinity [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 751.