Tim4‐Functionalized ^HBEV‐Chip by Isolating Plasma‐Derived Phosphatidylserine‐Positive Small Extracellular Vesicles for Pan‐Cancer Screening

Abstract

immune cells, histiocytes, and tumor cells) through the endolysosomal pathway. First, endocytosis of the cell membrane leads to formation of early endosomes, which fuse to form multivesicular bodies; then, the endocytosis of multivesicular bodies form exosomes secreted into extracellular space. [2] It has been reported that exosomes level in plasma of tumor patients is generally higher than that of healthy individuals and is closely associated with the type and progression of tumor. [3][4][5][6] Further studies have shown that exosomes carry multiple biological molecules, such as lipids, proteins and RNAs, which mirror the characteristics of source cells. [7] Therefore, tumor-derived exosomes are being recognized as potential cancer monitors in circulating liquids. As consensus on specific extracellular vesicle markers have not been reached yet, we took sEVs as a replacement term of exosomes in the follow-up paragraphs, in accordance with the MISEV2018 recommendations. [8] Pan-cancer screening is an approach to achieving universe cancer detection simultaneously from a single test, driven by biology and epidemiology. [9] As it would early detect the multiple cancer with pre-symptomy, pan-cancer screening contributes to decreasing the mortality rate of cancers, especially for the cancers without standard of care (SOC) screening method. And compared with a particular cancer type detection, it could increase the diagnostic efficiency for tumors derived from diverse organs. [10] But in liquid biopsy, sEVs are generally emerging as a diagnostics test for a particular cancer type and few applied in multiple cancer screening, which mainly focus on proteins and RNAs carried by sEVs. [11][12][13][14][15][16] Hence, there remains to be researched on lipids carried by sEVs in pan-cancer screening.Phosphatidylserine (PS) is an anionic lipid that is present in intracellular membranes in normal cells. However, in the tumor microenvironment, PS is affected by the influx of calcium ions, [17] flippase inactivation, [18] oxidative stress, [19] and radiochemotherapy, [20] resulting in abnormal flipping of PS to the outside of tumor cell membranes, such as in the non-small cell lung cancer cell lines A549 and H1975, pancreatic cancer cell line MIA PaCa-2, colorectal cancer cell line HT-29, and breast Thanks to containing tumor-related molecules, small extracellular vesicles (sEVs) are emerging as biomarkers in tumor liquid biopsy and commonly employed to diagnose a specific cancer. However, a pan-cancer screening method for pre-symptomatic patients is crucial to achieving the early cancer detection. Herein, a lipid-protein capture system on herringbone (HB) microfluidic chip ( HB EV-Chips) to isolate multiple tumor-derived sEVs is constructed. Phosphatidylserine (PS) is abnormally surface-supposed on tumor-derived sEVs and binds T-cell immunoglobulin domain and mucin domain-containing protein 4 (Tim4) in calcium-dependent manner. PS + sEVs isolated by the HB EV-Chips is perform on liquid chromatography electrospray ionization tandem...

A Versatile Design‐Enabled Analysis of Circulating Extracellular Vesicles in Disease Diagnosis

“…And for A549-derived EVs, the peak current obtained by using anti-CD63 was significantly lower than that using our design. These results not only revealed the lower CD63 expression at A549-derived EVs, which was verified by western blotting (Figure S17, Supporting Information) and was in line with the previous work [34] but also proved that our design was able to eliminate the negative effect of varied protein expressions among different cell lines by using the EV-specific lipid biomarkers.…”

Recent advances of liquid biopsy: Interdisciplinary strategies toward clinical decision‐making

“… 155 Deng et al. modified a herringbone microfluidic chip with TIM‐4 to isolate tumor‐derived PS‐positive EVs, 163 demonstrating that cancer patients have a higher number of PS‐positive EVs circulating in the bloodstream compared to healthy donors. Ye and colleagues presented a combination of TIM‐4 based specific isolation and a signal transduction strategy for the high‐sensitive detection of CD63 positive EVs, achieving an extremely low limit of detection around 4.39 × 10 3 particles/mL, which underscore the potential of PS‐specific approaches in enhancing the efficiency of EV isolation and detection 164 …”

Microfluidic Strategies for Extracellular Vesicle Isolation: Towards Clinical Applications

“…To increase the surface-to-volume ratio of channel walls, the inner surfaces of microfluidic chips with micro-and nano-structures are also chemically functionalized with antibodies to ensure the EV chemical affinity [110,111]. The most common microstructures are ordered rows of pillars [112][113][114], herringbone patterns [108,111,[115][116][117][118], and properly shaped microposts [119,120]. Meanwhile, in the case of nanopatterning, nanorods, nanowires, and more complex 3D structures [121] are typically used.…”

“…Electrokinetic force [98][99][100][101][102][103][104][105][106][107][152][153][154] Charge separation by electric fields Chemical: Fixed support Functionalized fixed support [108][109][110][111][112][113][114][115][116][117][118][119][120][121][122]130, EV capture by specific antibodies on fixed substrate Chemical: Floating Beads Magnetic beads [125][126][127][128][129][130][131][132][133][134][135][181][182][183][184][185][186] EV capture by specific antibodies on beads for magnetic manipulation…”