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Introduction: Analytical methods of extracellular vesicles (EVs) is becoming an increasingly promising field of study due to them being an effective biomarker for cancers. EVs are present in various types of body fluids, which can be easily used for diagnostic purpose. Prior research has explored numerous techniques for isolating and analyzing these EVs based on their physical and biochemical properties, however, the complexity of biological samples makes conventional EV isolation difficult to exclusively extract EVs of a certain type of cell. Current downstream analysis methods lack the ability to differentiate exosomes of different origins in a sample. Recent studies suggested the presence of certain proteins in cancer exosomes that facilitates preferential uptake of the exosomes by organ-specific cancer cells, called organotropism. Using this unique property, we devised a microfluidic platform to examine the uptake of specific exosomes onto their respective progenitor cell lines, thus aiming to use the interaction for cancer diagnosis purposes. Methods: The CellExoChip was prepared following the standard PDMS-based soft lithography method. The device consists of an inlet and outlet, with dimensions of 22x22 mm with a cell capture area of about 50 mm2. The microfluidic device was functionalized by Streptavidin and the cancer cells were biotinylated with EZ-Link-NHS-Biotin to create an intense affinity between the cells and the device. We further injected dyed exosomes with different origins through the device and evaluated their specific uptake using fluorescence microscope. Results: The prepared CellExoChip successfully immobilized over 1,500 cells onto the surface and viability evaluation demonstrated that only 6.79% of the initial cells were sacrificed during the biotinylation and on-chip binding process. The average on-chip cell viability showed 75.47±7.68%. The uptake of lung cancer cell exosomes into three different cancer cell lines (lung, bladder and breast) was measured on chip. The relative uptake of lung cell exosomes by the respective lung cells was 100% compared to the bladder cells and breast cell which were 15.87% and 40.31%, respectively. We extended this specific uptake evaluation to other LungCell-LungExo combinations using H1650, A549, and in-house CTC cell line, CTCR5, and the results demonstrated the organotropism nature of the exosomes in lung cancer. Discussion and conclusion: We present a novel screening method to accurately characterize specific cancer-derived EVs using a microfluidic platform. This process bypasses the requirement of analyzing and profiling these embedded proteins prior to EV isolation. Our microfluidic device facilitates this interaction between cells and exosomes through the diagnosis process of liquid biopsy. Citation Format: Kruthi Srinivasa Raju, Zeqi Niu, Joseph Marvar, Shawn Fortna, Nna-Emeka Onukwugha, Yoon-Tae Kang, Sunitha Nagrath. On-chip evaluation of cancer cell-extracellular vesicle interactions using a novel microfluidic microsystem (CellExoChip) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2792.

communication. Their membrane contains and protects proteins, nucleic acids, and metabolites. Hence, they can serve as active cargo delivery vehicles and messengers of genetic information. [1] There has been an increased interest in the clinical use of EVs as biomarkers due to their significance in cellular signaling, disease progression, and therapeutics. The isolation and characterization of exosomes allow their use as reliable biomarkers for minimally invasive disease diagnosis, called liquid biopsy. One of the promising opportunities for disease diagnosis is the diagnosis of cancer since EV secretion is increased by many cancer types. Their presence in a variety of biosamples -that is, blood, urine, saliva -makes them an attractive avenue for exploration for liquid biopsy to provide a simple, in vitro analysis of a patient's tumor status. [2,3] Deregulated cellular metabolism has been established as a hallmark of cancer which supports the use of metabolite characterization as a sensitive, inexpensive, and origin-agnostic tool for minimally-invasive diagnosis of cancer. [4] However, the large amounts of biosample contaminants and the size of the desired endosome-derived small EVs (sEV), also called exosomes, at just 30-150 nm diameter makes isolation difficult and challenging [5] using conventional EV isolation methods.Polydimethylsiloxane (PDMS) is an inexpensive robust polymer that is commonly used as the fundamental fabrication material for soft-lithography-based microfluidic devices. Owing to its versatile material properties, there are some attempts to use PDMS as a porous 3D structure for sensing. However, reliable and easy fabrication has been challenging along with the inherent hydrophobic nature of PDMS hindering its use in biomedical sensing applications. Herein, a cleanroom-free inexpensive method to create 3D porous PDMS structures, "ExoSponge" and the effective surface modification to functionalize its 3D porous structure is reported. The ability of ExoSponge to recover cancer-associated extracellular vesicles (EVs) from complex biological samples of up to 10 mL in volume is demonstrated. When compared to ultracentrifugation, the ExoSponge shows a significant increase in cancer EV isolation of more than 210%. Targeted ultra-high pressure liquid chromatography-tandem mass spectrometry (LC-MS/MS) is further employed to profile 70 metabolites in the EVs recovered from the lung cancer patient's plasma. The resulting profiles reveal the potential intraexosomal metabolite biomarker, phenylacetylglutamine (PAG), in non-small cell lung cancer. The high sensitivity, simple usage, and cost-effectiveness of the ExoSponge platform creates huge potential for rapid, economical and yet specific isolation of exosomes enabling future diagnostic applications of EVs in cancers.