We present a resource‐efficient approach to fabricate and operate a micro‐nanofluidic device that uses cross‐flow filtration to isolate and capture liposarcoma derived extracellular vesicles (EVs). The isolated extracellular vesicles were captured using EV‐specific protein markers to obtain vesicle enriched media, which was then eluted for further analysis. Therefore, the micro‐nanofluidic device integrates the unit operations of size‐based separation with CD63 antibody immunoaffinity‐based capture of extracellular vesicles in the same device to evaluate EV‐cargo content for liposarcoma. The eluted media collected showed ∼76% extracellular vesicle recovery from the liposarcoma cell conditioned media and ∼32% extracellular vesicle recovery from dedifferentiated liposarcoma patient serum when compared against state‐of‐art extracellular vesicle isolation and subsequent quantification by ultracentrifugation. The results reported here also show a five‐fold increase in amount of critical liposarcoma‐relevant extracellular vesicle cargo obtained in 30 min presenting a significant advance over existing state‐of‐art.

Introduction: Dedifferentiated liposarcoma (DDLPS) is frequently diagnosed late and patients typically respondpoorly to treatments bringing to an overall survival rate of only 10% at 10 years. Prognosis isenhanced by early detection of recurrent lesions, but current diagnostic methods rely on invasivetissue biopsies combined with whole or part-body imaging. These diagnostic and prognosticchallenges prompted us to explore the use of circulating biomolecules as potential biomarkersaccessible through bodily fluids. Extracellular Vesicles (EVs) are membrane vesicles containingproteins and nucleic acids that circulate in the blood, so they are an emerging class of easilyaccessible blood biomarkers. We recently identified extracellular vesicle EV-MDM2 as liquidbiopsy biomarker for disease identification in DDLPS. Unfortunately, due to these challenges forisolation and capture of EVs, despite the potential diagnostic and prognostic utility, the practicalrelevance of using EVs for routine analysis remains limited. Methods: A microfluidic device was fabricated via standard soft lithography in PDMS(polydimethylsiloxane). A 5 x 5 mm2 nanocapillary array membrane (nominal capillary diameterof 0.2 µm) was sandwiched between two microchannels for a vertically aligned configuration withan injection and separation channel (each 500 µm wide and 150 µm high) perpendicular to eachother. The separation channel surface was functionalized with the anti-CD63 antibody forcapturing EVs. Results: We present here a facile approach to fabricate and operate a micro-nanofluidic device that usescross-flow filtration to isolate and capture liposarcoma derived EVs. Our methodology integratesthe unit operations of size based separation with immunoaffinity-based capture of extracellularvesicles in the same device. The eluted media collected showed ~84% extracellular vesiclerecovery from the liposarcoma cell conditioned media and ~38% extracellular vesicle recoveryfrom dedifferentiated liposarcoma patient serum when compared to extracellular vesicle isolationand subsequent quantification by ultracentrifugation. The results reported here also show a five-fold increase in amount of critical liposarcoma relevant extracellular vesicle cargo obtained in30 minutes presenting a significant advance over existing state-of-art. Conclusion: In conclusion we developed a cross-flow filtration micro-nanofluidic device for isolation, selectivecapture and release of liposarcoma EVs. Citation Format: Lucia Casadei, Patricia Sarchet, Adarsh Choudhury, Shaurya Prakash, Raphael Pollock. Improving liposarcoma diagnosis through cross-flow microfiltration [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 475.