Patent Application
20240200141
Aspects of the invention relate to compositions, systems and methods for capturing extracellular vesicles, and more particularly to bioorthogonal ligation mediated extracellular vesicle capture.
Extracellular vesicles (EVs),[1,2] as a heterogeneous group of phospholipid bilayer-enclosed particles, can be released by all types of cells, especially tumor cells. Recently, the scientific community has begun to understand the importance of EVs as a mechanism and vehicle[3] of cellular interchange of bioactive molecules,[4] including proteins, DNA, and RNA.[5,6] Such interchanges can result in exchanges of genetic information and functional molecules,[7] leading to the subsequent reprogramming of the recipient cells.[8-10] Tumor-derived EVs are regarded as “biological shuttles”[11] capable of transporting biomolecules to mediate intracellular communication, microenvironment modulation, and cancer metastasis. Therefore, in addition to exploring the diagnostic values of tumor-derived EVs,[12-14] there is growing interest in performing functional studies of tumor-derived EVs in cellular communication,[15,16] (e.g., EV uptake and cargo transfer). Since tumor-derived EVs exist in a background of non-tumor-derived EVs, selectively purification of tumor-derived EVs—while retaining the integrity of their enclosed biomolecular cargos—has been identified as a major technical barrier to conducting the functional studies of tumor-derived EVs.
Conventionally, EVs can be isolated from blood plasma or serum based on their physical properties by using enrichment methods, e.g., ultracentrifugation,[17] precipitation,[18] filtrations,[19] size-based microfluidics,[20-22] and lipid-based nanoprobes.[23] However, these approaches are not suitable for specifically enriching tumor-derived EVs from EVs in the background. Significant research endeavors[24,25] have been devoted to exploring antibody or aptamer[26,27]-based techniques[28-30] to enrich and analyze tumor-derived EVs. For instance, GPC1 antibody-coated beads have been used to isolate pancreatic cancer-derived exosomes;[31] a herringbone microfluidic device (a.k.a. EVHB-Chip) functionalized with EGFRvIII antibody has been demonstrated to enrich glioblastoma-derived exosomes.[32] Previously, a “Nano Villi Chip”.[33] was developed in which densely packed, anti-epithelial cell adhesion molecule (anti-EpCAM)-grafted silicon (Si) nanowire arrays were engineered to achieve efficient and reproducible immune-affinity capture of tumor-derived EVs. However, due to the limited number of antigens present on the surface of individual EVs, immune-affinity EV capture approaches, which are driven by the dynamic binding between a pair of antigens (on EVs) and antibodies (on the substrates), often suffer from poor EV capture performance and high background. Moreover, conducting functional studies of tumor-derived EVs requires the purification of tumor-derived EVs with biological intactness. Therefore, it is necessary to develop novel purification systems with the capacity for both sensitive and specific capture of tumor-derived EVs and their subsequent release.
All publications and patent applications identified herein are incorporated by reference in their entirety and to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.
An embodiment of the invention relates to a method of selectively capturing an extracellular vesicle (EV) from a sample, including: functionalizing a capture agent for the EV with a first molecule from a first bioorthogonal functional group such that the capture agent remains attachable to the EV and the first molecule is also able to bond to a second molecule from a second bioorthogonal functional group, the second molecule being complementary to the first molecule; mixing the functionalized capture agent with the sample such that the functionalized capture agent binds to the EV and such that an activated sample is formed; functionalizing a capture surface with the second molecule; and depositing at least a portion of the activated sample on at least a portion of the functionalized capture surface to thereby selectively capture the EV by binding of the second molecule with the first molecule. In such an embodiment, the first molecule from the first bioorthogonal functional group and the capture agent are present in a molar ratio of between 2:1 to 10:1.
An embodiment of the invention relates to a method of assaying for a cancer in a subject including: selectively capturing an extracellular vesicle (EV) from a sample from the subject, the selectively capturing an EV including: functionalizing a capture agent for the EV with a first molecule from a first bioorthogonal functional group such that the capture agent remains attachable to the EV and the first molecule is also able to bond to a second molecule from a second bioorthogonal functional group, the second molecule being complementary to the first molecule; mixing the functionalized capture agent with the sample such that the functionalized capture agent binds to the EV and such an activated sample is formed; functionalizing a capture surface with the second molecule; and depositing at least a portion of the activated sample on at least a portion of the functionalized capture surface to thereby selectively capture the EV by binding of the molecule with the first molecule; releasing the EV from the capture surface; assaying a nucleic acid sequence from the EV; and determining from the assaying of the nucleic acid sequence from the EV whether the cancer is present in the subject. In such an embodiment, the first molecule from the first bioorthogonal functional group and the capture agent are present in a molar ratio of between 2:1 to 10:1.
An embodiment of the invention relates to a method of assaying for a cancer in a subject including: selectively capturing a plurality of extracellular vesicles (EVs) from a sample from the subject, where each extracellular vesicle (EV) of the plurality of EVs is selectively captured including: functionalizing a capture agent for an EV of the plurality of EVs with a first molecule from a first bioorthogonal functional group such that the capture agent remains attachable to the EV and the first molecule is also able to bond to a second molecule from a second bioorthogonal functional group, the second molecule being complementary to the first molecule; mixing the functionalized capture agent with the sample such that the functionalized capture agent binds to the EV and such an activated sample is formed; functionalizing a capture surface with the second molecule; and depositing at least a portion of the activated sample on at least a portion of the functionalized capture surface to thereby selectively capture the EV by binding of the molecule with the first molecule; releasing the plurality of EVs from the capture surface; assaying a plurality of nucleic acid sequences from the plurality of EVs; creating an expression profile of the plurality of nucleic acid sequences, the expression profile including a quantification of each of the plurality of nucleic acid sequences; comparing the expression profile with a control; and determining from the comparing of the expression profile with the control whether the cancer is present in the subject. In such an embodiment, the first molecule from the first bioorthogonal functional group and the capture agent are present in a molar ratio of between 2:1 to 10:1.
An embodiment of the invention relates to a kit for selectively capturing an extracellular vesicle (EV) from a sample, the kit including: a capture agent having a first molecule from a first bioorthogonal functional group; a substrate having a functionalized capture surface having a second molecule from a second bioorthogonal functional group, the second molecule being complementary to the first molecule; a cleaving agent; instructions for mixing the capture agent with the sample such that the capture agent binds to the EV and such that an activated sample is formed; instructions for depositing at least a portion of the activated sample on at least a portion of the functionalized capture surface to thereby selectively capture the EV by binding of the second molecule with the first molecule; and instructions for using the cleaving agent to release the EV from the capture surface. In such an embodiment, the first molecule from the first bioorthogonal functional group and the capture agent are present in a molar ratio of between 2:1 to 10:1.
Some embodiments of the current invention are discussed in detail below. In describing embodiments, specific terminology is employed for the sake of clarity. However, the invention is not intended to be limited to the specific terminology so selected. A person skilled in the relevant art will recognize that other equivalent components can be employed and other methods developed without departing from the broad concepts of the current invention. All references cited anywhere in this specification, including the Background and Detailed Description sections, are incorporated by reference as if each had been individually incorporated.
The term “bioorthogonal chemistry” refers to any chemical reaction that can occur inside of living systems without interfering with native biochemical processes.
The term capture agent can include any molecules, particles, etc. that selectively bind to particular rare cells such as, but not limited to antibodies.
A non-limiting example of a chemical reaction for use according to embodiments of the invention is the 1,3-dipolar cycloaddition between azides and cyclooctynes as described in the scheme below:
A non-limiting example of a chemical reaction for use according to embodiments of the invention is Nitrone Dipole Cycloaddition as described in the scheme below:
This cycloaddition between a nitrone and a cyclooctyne forms N-alkylated isoxazolines.
A non-limiting example of a chemical reaction for use according to embodiments of the invention is Norbornene Cycloaddition as described in the scheme below:
Nitrile oxide as a 1,3-dipole and a norbornene as a dipolarophile
A non-limiting example of a chemical reaction for use according to embodiments of the invention is Oxanorbornadiene Cycloaddition as described in the scheme below:
The oxanorbornadiene cycloaddition is a 1,3-dipolar cycloaddition followed by a retro-Diels Alder reaction to generate a triazole-linked conjugate with the elimination of a furan molecule.
A non-limiting example of a chemical reaction for use according to embodiments of the invention is Tetrazine Ligation as described in the scheme below:
The reaction of a trans-cyclooctene and an s-tetrazine in an inverse-demand Diels Alder reaction followed by a retro-Diels Alder reaction to eliminate nitrogen gas.
A non-limiting example of a chemical reaction for use according to embodiments of the invention is [4+1] Cycloaddition as described in the scheme below:
This isocyanide click reaction is a [4+1] cycloaddition followed by a retro-Diels Alder elimination of N2.
A non-limiting example of a chemical reaction for use according to embodiments of the invention is Quadricyclane Ligation as described in the scheme below:
Photoclick chemistry utilizes a photoinduced cycloreversion to release N2.
A non-limiting example of a chemical reaction for use according to embodiments of the invention is Quadricyclane Ligation as described below:
The quadricyclane ligation utilizes a highly strained quadricyclane to undergo [2+2+2] cycloaddition with π systems.
Some embodiments of the invention include a device for capturing an extracellular vesicle. Examples of such devices are described in U.S. Pat. No. 9,140,697 which is hereby incorporated by references in its entirety. A further non-limiting example of such a device is a Silicon Nanowire Substrates (SiNWS). In embodiments of the invention, the device includes a substrate; and a plurality of nanowires at least one of attached to or integral with a surface of the substrate such that each nanowire of the plurality of nanowires has an unattached end.
In some embodiment. the device for capturing a cell includes a substrate having a nanostructured surface region. Also, in some embodiments, a plurality of binding agents are attached to the nanostructured surface region of the substrate. However, binding agents are not required for the device to bind to target extracellular vesicles. The nanostructured surface region includes a plurality of nanostructures, each having a longitudinal dimension and a lateral dimension. As a sample is placed on the device, extracellular vesicles are selectively captured by the binding agents and the plurality of nanostructures acting in cooperation (in embodiments having binding agents). When present, the binding agent or agents employed will depend on the type of extracellular vesicles being targeted. Conventional binding agents are suitable for use in some of the embodiments of the present invention. Non-limiting examples of binding agents include antibodies, nucleic acids, oligo- or polypeptides, cellular receptors, ligands, aptamers, biotin, avidin. Coordination complexes, synthetic polymers, and carbohydrates. In some embodiments of the present invention, binding agents are attached to the nanostructured surface region using conventional methods. The method employed will depend on the binding agents and the material used to construct the device. Non-limiting examples of attachment methods include non-specific adsorption to the surface, either of the binding agents or a compound to which the agent is attached or chemical binding, e.g., through self-assembled monolayers or silane chemistry. In some embodiments, the nanostructured surface region is coated with streptavidin and the binding agents are biotinylated, which facilitates attachment to the nanostructured surface region via interactions with the streptavidin molecules.
In some embodiments of the present invention, the nanostructures increase the surface area of the substrate and increase the probability that a given extracellular vesicle will come into contact. In these embodiments, the nanostructures can enhance binding of the target extracellular vesicles by interacting with surface components. In some embodiments, the nanostructures have a longitudinal dimension that is equal to its lateral dimension, where both the lateral dimension and the longitudinal dimension is less than 1 mm, i.e., nanoscale in size. In other embodiments, the nanostructures have a longitudinal dimension that is at least ten times greater than its lateral dimension. In further embodiments, the nanostructures have a longitudinal dimension that is at least twenty times greater, fifty times greater, or 100 times greater than its lateral dimension. In some embodiments, the lateral dimension is less than 1 mm. In other embodiments, the lateral dimension is between 1-500 nm. In further embodiments, the lateral dimension is between 30-400 nm. In still further embodiments, the lateral dimension is between 50-250 nm. In some embodiments, the longitudinal dimension is at least 1 mm long. In other embodiments, the longitudinal dimension is between 1-50 mm long. In other embodiments, the longitudinal dimension is 1-25 mm long. In further embodiments, the longitudinal dimension is 5-10 mm long. In still further embodiments, the longitudinal dimension is at least 6 mm long. The shape of the nanostructure is not critical. In some embodiments of the present invention, the nanostructure is a sphere or a bead. In other embodiments, the nanostructure is a strand, a wire, or a tube. In further embodiments, a plurality of nanostructure contains one or more of nanowires, nanofibers, nanotubes, nano-pillars, nanospheres, or nanoparticles.
An embodiment of the invention relates to a method of selectively capturing an extracellular vesicle (EV) from a sample, including: functionalizing a capture agent for the EV with a first molecule from a first bioorthogonal functional group such that the capture agent remains attachable to the EV and the first molecule is also able to bond to a second molecule from a second bioorthogonal functional group, the second molecule being complementary to the first molecule; mixing the functionalized capture agent with the sample such that the functionalized capture agent binds to the EV and such that an activated sample is formed; functionalizing a capture surface with the second molecule; and depositing at least a portion of the activated sample on at least a portion of the functionalized capture surface to thereby selectively capture the EV by binding of the second molecule with the first molecule. In such an embodiment, the first molecule from the first bioorthogonal functional group and the capture agent are present in a molar ratio of between 2:1 to 10:1.
An embodiment of the invention relates to the method above, where the first molecule from the biorthogonal functional group is selected from the list consisting of trans-cyclooctene (TCO), Alkyne, and a cyclooctyne derivative.
An embodiment of the invention relates to the method above, where the cyclooctyne derivative includes dibenzylcyclooctyne (DBCO) or biarylazacyclooctynone (BARAC).
An embodiment of the invention relates to the method above, where the second molecule from the second biorthogonal functional group is selected from the list consisting of tetrazine (TZ) and azide.
An embodiment of the invention relates to the method above, where the capture surface includes a nanostructured surface.
An embodiment of the invention relates to the method above, further including functionalizing a second capture agent for the EV with the first molecule such that the second capture agent remains attachable to the EV and the first molecule is also able to bond to the molecule including the second molecule from the second bioorthogonal functional group, and where the second capture agent is distinct from the capture agent.
An embodiment of the invention relates to a method of assaying for a cancer in a subject including: selectively capturing an extracellular vesicle (EV) from a sample from the subject, the selectively capturing an EV including: functionalizing a capture agent for the EV with a first molecule from a first bioorthogonal functional group such that the capture agent remains attachable to the EV and the first molecule is also able to bond to a second molecule from a second bioorthogonal functional group, the second molecule being complementary to the first molecule; mixing the functionalized capture agent with the sample such that the functionalized capture agent binds to the EV and such an activated sample is formed; functionalizing a capture surface with the second molecule; and depositing at least a portion of the activated sample on at least a portion of the functionalized capture surface to thereby selectively capture the EV by binding of the molecule with the first molecule; releasing the EV from the capture surface; assaying a nucleic acid sequence from the EV; and determining from the assaying of the nucleic acid sequence from the EV whether the cancer is present in the subject. In such an embodiment, the first molecule from the first bioorthogonal functional group and the capture agent are present in a molar ratio of between 2:1 to 10:1.
An embodiment of the invention relates to the method above, where the first molecule from the biorthogonal functional group is selected from the list consisting of trans-cyclooctene (TCO), Alkyne, and a cyclooctyne derivative.
An embodiment of the invention relates to the method above, where the cyclooctyne derivative includes dibenzylcyclooctyne (DBCO) or biarylazacyclooctynone (BARAC).
An embodiment of the invention relates to the method above, where the second molecule from the second biorthogonal functional group is selected from the list consisting of tetrazine (TZ) and azide.
An embodiment of the invention relates to the method above, where the capture surface includes a nanostructured surface.
An embodiment of the invention relates to the method above, further including functionalizing a second capture agent for the EV with the first molecule such that the second capture agent remains attachable to the EV and the first molecule is also able to bond to the molecule including the second molecule from the second bioorthogonal functional group, and where the second capture agent is distinct from the capture agent.
An embodiment of the invention relates to the method above, where the releasing the EV from the capture surface includes use of a cleaving agent.
An embodiment of the invention relates to a method of assaying for a cancer in a subject including: selectively capturing a plurality of extracellular vesicles (EVs) from a sample from the subject, where each extracellular vesicle (EV) of the plurality of EVs is selectively captured including: functionalizing a capture agent for an EV of the plurality of EVs with a first molecule from a first bioorthogonal functional group such that the capture agent remains attachable to the EV and the first molecule is also able to bond to a second molecule from a second bioorthogonal functional group, the second molecule being complementary to the first molecule; mixing the functionalized capture agent with the sample such that the functionalized capture agent binds to the EV and such an activated sample is formed; functionalizing a capture surface with the second molecule; and depositing at least a portion of the activated sample on at least a portion of the functionalized capture surface to thereby selectively capture the EV by binding of the molecule with the first molecule; releasing the plurality of EVs from the capture surface; assaying a plurality of nucleic acid sequences from the plurality of EVs; creating an expression profile of the plurality of nucleic acid sequences, the expression profile including a quantification of each of the plurality of nucleic acid sequences; comparing the expression profile with a control; and determining from the comparing of the expression profile with the control whether the cancer is present in the subject. In such an embodiment, the first molecule from the first bioorthogonal functional group and the capture agent are present in a molar ratio of between 2:1 to 10:1.
An embodiment of the invention relates to a kit for selectively capturing an extracellular vesicle (EV) from a sample, the kit including: a capture agent having a first molecule from a first bioorthogonal functional group; a substrate having a functionalized capture surface having a second molecule from a second bioorthogonal functional group, the second molecule being complementary to the first molecule; a cleaving agent; instructions for mixing the capture agent with the sample such that the capture agent binds to the EV and such that an activated sample is formed; instructions for depositing at least a portion of the activated sample on at least a portion of the functionalized capture surface to thereby selectively capture the EV by binding of the second molecule with the first molecule; and instructions for using the cleaving agent to release the EV from the capture surface. In such an embodiment, the first molecule from the first bioorthogonal functional group and the capture agent are present in a molar ratio of between 2:1 to 10:1.
An embodiment of the invention relates to the kit above, where the first molecule from the biorthogonal functional group is selected from the list consisting of trans-cyclooctene (TCO), Alkyne, and a cyclooctyne derivative.
An embodiment of the invention relates to the kit above, where the cyclooctyne derivative includes dibenzylcyclooctyne (DBCO) or biarylazacyclooctynone (BARAC).
An embodiment of the invention relates to the kit above, where the second molecule from the second biorthogonal functional group is selected from the list consisting of tetrazine (TZ) and azide.
An embodiment of the invention relates to the kit above, where the capture surface includes a nanostructured surface.
An embodiment of the invention relates to the kit above, further including a plurality of reagents for a nucleic acid test.
The embodiments illustrated and discussed in this specification are intended only to teach those skilled in the art how to make and use the invention. In describing embodiments of the invention, specific terminology is employed for the sake of clarity. However, the invention is not intended to be limited to the specific terminology so selected. The above-described embodiments of the invention may be modified or varied, without departing from the invention, as appreciated by those skilled in the art in light of the above teachings. It is therefore to be understood that, within the scope of the claims and their equivalents, the invention may be practiced otherwise than as specifically described.
Hepatocellular carcinoma (HCC) is the fourth most common cause of cancer-related deaths worldwide1. The poor prognosis of HCC can be attributed to the fact that diagnosis is often made at a late stage in disease development2,3. Earlier detection of HCC is critical to reducing the high HCC mortality rates, as numerous potentially curative therapeutic interventions are available to treat early-stage HCC. Current American Association for the Study of Liver Disease (AASLD) guidelines3 recommend biannual liver ultrasonography with or without serum alpha-fetoprotein (AFP) for at-risk patients with cirrhosis and chronic liver disease; however, ultrasound is not sensitive enough to detect early lesions, and the reported performance of AFP varies widely4. Thus, the development of novel non-invasive diagnostics for early-stage HCC may significantly benefit at-risk patients at risk.
Among the three conventional liquid biopsy5,6 approaches in the context of oncology, i.e., circulating tumor cells (CTCs)7-9, circulating tumor DNA (ctDNA)10,11, and extracellular vesicles (EVs)12, EVs are present in circulation at relatively early stages of disease13 and persist across all disease stages. Furthermore, EVs' inherent stability guarantees the integrity of encapsulated biomolecular cargos, especially the extremely fragile mRNA. Therefore, tumor-derived EVs are regarded as “biomarker reservoirs”14, promising the implementation of downstream molecular analysis for non-invasive cancer diagnosis15. However, the conventional EV isolation methods, e.g., ultracentrifugation16 and precipitation processing, are based on EVs' physical properties (most notably density and solubility), which are incapable of separating tumor-derived EVs from total EVs. Since the majority of EVs in circulation are not of tumor origin, analyzing total EVs is of limited diagnostic power as a result of high background noise17. To overcome this issue, groups17,18,19,20 have developed various immunoaffinity-based approaches to enrich tumor-derived EVs. Evidence is emerging that EVs and their biomolecular cargos such as RNA have the potential to detect HCC21. Nonetheless, exploring the use of HCC EV-derived mRNA signatures as biomarkers for detecting HCC, especially early-stage HCC from at-risk chronic liver diseases (e.g., hepatitis and liver cirrhosis) is still limited by a number of challenges, including i) developing an EV purification system that can accommodate a multimarker cocktail to recognize, enrich, and recover HCC EVs secreted from the highly heterogeneous HCC22-24, ii) avoiding mRNA degradation by streamlining the EV purification process, and iii) seamlessly coupling a simple and quantitative downstream molecular assay with HCC EV purification systems.
The poor prognosis of hepatocellular carcinoma (HCC) is due to the fact that the majority of patients present with advanced stage disease. Among different tumor liquid biopsy approaches, extracellular vesicles (EVs) are present in circulation at relatively early stage of disease, thus opening up noninvasive diagnostic opportunity for HCC early detection. Here, a new HCC EV purification system (i.e., EV Click Chips) is described (
The covalent chemistry-mediated EV capture/release was built upon the combined use of click chemistry28-mediated EV capture and disulfide cleavage29-driven EV release in conjunction with an optimized multi-marker cocktail targeting three HCC-associated surface markers25, including EpCAM, ASGPR, and CD147. Further, the incorporation of densely packed silicon nanowires substrates (SiNWS) dramatically increases the device surface area26 contacting/interacting with EVs. Moreover, the microfluidic chaotic mixer facilitates repeated physical contact30 between SiNWS and the flow-through HCC EVs, further enhancing the performance of EV capture. In contrast to previous antibody-mediated EV capture18, a pair of highly reactive click chemistry motifs31 i.e., tetrazine (Tz) and trans-cyclooctene (TCO), were grafted onto EV capture substrates (i.e., SiNWS, via surface modification) and HCC EVs (via TCO-capture agent conjugation), respectively. The click chemistry reaction between Tz-grafted SiNWS and TCO-grafted HCC EVs is rapid specific, irreversible, and bioorthogonal31, resulting in immobilization of the HCC EVs with improved capture efficiency and reduced background. After click chemistry-mediated HCC EV capture, exposure to a disulfide cleavage agent, 1,4-dithiothreitol (DTT)32 leads to the prompt release of the HCC EVs from the SiNWS by cleaving the disulfide bond linking the Tz to the SiNWS. Recognizing that the field has been in dire need of practical methods capable of quantitatively assessing the performance (EV recovery yield and purity) of any given EV purification system, a quantitative evaluation method for assessing the performance of EV Click Chip is also described. By adopting this quantitative method throughout the optimization process, one is able to accurately determine the performance of EV Click Chips, achieving an optimal HCC EV purification condition that was used in pre-clinical studies.
The potential of a streamlined HCC EV-based mRNA assay was exploited by coupling i) EV Click Chips for purification of HCC EVs and ii) reverse-transcription droplet digital PCR (RT-ddPCR) for quantification of 10 well-validated HCC-specific mRNA transcripts33 using plasma samples of HCC patients and control cohorts. After conducting biostatistical analysis, HCC EV-derived 10-gene digital readouts exhibited a great potential for non-invasive early detection of HCC from at-risk cirrhotic patients.
The design and preparation of an EV Click Chip. An EV Click Chip (as seen in
Preparation of artificial plasma samples. To allow accurate evaluation of the performance of EV Click Chip throughout the optimization process, artificial plasma samples were prepared by spiking 10-μL aliquoted HepG2 cell-derived EVs (harvested by ultracentrifugation37,38) into 90-μL plasma from a female healthy donor. As shown in
In order to obtain the purity of the EVs recovered by EV Click Chips, the intrinsic ratios between C1orf101 and SRY transcripts in aliquoted HepG2 EVs were measured across a wide range of concentrations. The ratios between C1orf101 and SRY transcripts exhibited a consistent linear correlation (y=1.95 x, R2=0.999). With the C1orf101-to-SRY ratio determined as 1.95, the purity of the EVs harvested from EV Click Chips is then calculated as the ratio of the recovered SRY transcripts (contributed by recovered HepG2 EVs only) to the C1orf101 transcripts (contributed by both recovered HepG2 EVs and the non-specifically captured background plasma-derived EVs, denoted as C1orf101 gene rec-EV) using the following equation:
For HCC cell lines without SRY transcripts, cancer cell-derived EVs were spiked into the plasma from male donors, and the EV recovery yield and purity can be calculated using equations below:
Artificial plasma samples were prepared by spiking a) 10-μL aliquoted HepG2 cell-derived EVs into 90-μL plasma from a female healthy donor or female cirrhotic patient, b) 10-μL aliquoted SNU387 cell-derived EVs into 90-μL plasma from a male healthy donor or male cirrhotic patient and c) 10-μL aliquoted Hep 3B cell-derived EVs into 90-μL plasma from a male healthy donor or male cirrhotic patient. The EV recovery yield of the male HCC cell line (HepG2) observed for EV Click Chip can be obtained from the following equation (the copy numbers of SRY transcripts in the original 10-μL aliquoted HepG2 EVs and the EV Click Chip-recovered HepG2 EVs were denoted as SRY transcriptsori-EV and SRY transcriptsrec-EV, respectively):
The purities of the male HCC cell line (HepG2) EVs harvested from EV Click Chips were calculated as the ratio of recovered SRY transcripts (contributed by recovered HepG2 EVs only) to C1orf101 transcripts (contributed by both recovered HepG2 EVs and the non-specifically captured background female plasma-derived EVs, denoted as C1orf101transcriptsrec-EV) using the following equation:
For HCC cell lines without SRY transcripts (SNU 387, Hep3B), cancer cell-derived EVs were spiked into the plasma from a male donor or male cirrhotic patient, and the EV recovery yields and purities can be calculated using the following equations:
HCC EV purification with EV Click Chips. Prior to conducting HCC EV purification (capture/release) studies, TCO motif was covalently conjugated onto each antibody agent (
Surface marker selection and a multi-marker cocktail optimization for HCC EV capturing. Using data in the published literature40,41,25 that identified surface markers highly expressed in HCC EVs, HCC-CTCs, HCC cell lines, and primary tumor tissues of HCC patients, but virtually absent in white blood cells, 4 candidate antibodies, i.e. anti-EpCAM, anti-ASGPR, anti-CD147, and anti-GPC-3, directed against the corresponding surface markers were selected in order to achieve desired sensitivity and specificity of recognizing and capturing HCC EVs. The aforementioned RT-ddPCR assay was employed to assess the EV recover yield of EV Click Chips using artificial plasma samples in the presence of the individual antibodies and their cocktail mixtures. All experiments were performed at the optimum flow rate of 1 mL h−1 according to earlier experiences on developing NanoVilli EV Chip18. The number of TCO motif grafted on an antibody capture agent could affect the purification performance of EV Click Chips, thus first examined was how the TCO-to-anti-EpCAM mole ratios correlate with the EV recovery yields at 50-ng anti-EpCAM.
Optimization of Click Chips for HCC EV purification. To further optimize the device performance, how the flow rates affect the recovery yield of HepG2 EVs was studied. 100-μL artificial plasma samples pre-incubated with the optimal antibody cocktail were introduced into EV Click Chips at flow rates ranging from 0.2 to 2.0 mL h−1, and >85% average recovery yields were observed at the flow rates of 0.2 to 1.0 mL h−1 (
Characterization of HCC EVs purified by EV Click Chips. To better understand the working mechanisms associated with the click chemistry-mediated EV capture and disulfide cleavage-driven EV release, fluorescence microscopy, transmission electron microscopy (TEM), dynamic light scattering (DLS), and/or scanning electron microscopy (SEM) were employed to characterize the EV sizes and EV/SiNWS interfaces during the EV purification process, in which freshly harvested HepG2 EVs in PBS were used as a model system. To allow direct tracking of the capture and release processes of HCC EVs in EV Click Chips, HepG2 EVs were first labeled (
Quantification of 10 HCC-specific genes using HCC EVs purified from clinical samples. Under the optimal HCC EV purification condition, a workflow was achieved (
HCC EV Z Scores for HCC detection. HCC EV Z Scores for each sample were computed based on the expression of 10 genes in purified HCC EVs using the weighted Z-score method45. The copy numbers of the 10 genes were combined into the single HCC EV Z Scores. As depicted in the box plot (
HCC largely occurs in pre-existing chronic liver diseases46, but can also develop without such pre-conditions. Thus, subgroup analysis was performed to test the feasibility of distinguishing HCC patients from the Non-cancer group (Liver Cirrhosis, Chronic Hepatitis and healthy donors) using ROC curve. The AUC for the HCC EV Z Score for distinguishing HCC from Non-cancer was 0.86 (95% CI, 0.79 to 0.93; sensitivity=85.2%, specificity=80.1%,
HCC EV Z Scores for early HCC detection. HCC occurs in the background of liver cirrhosis in over 80% of cases46, emphasizing the need to develop an early detection method to identify localized HCC from at-risk liver cirrhosis populations, in turn providing great hope for curative therapy. Among all patients with HCC, the HCC EV Z Scores were highly differentiated between early-stage HCC and advanced-stage HCC according to BCLC staging42 (P<0.01), Milan criteria43 (P<0.01), and UNOS DS Criteria44 (P<0.01). These results indicate promise that the HCC EV Z Score generated from HCC EVs purified by EV Click Chips may serve as a noninvasive predictor for early detection of HCC. To explore the potential for the EV Click Chip-based HCC-EV Assay to detect early-stage HCC, the feasibility of distinguishing early-stage HCC patients (defined according to BCLC staging, Milan criteria, or UNOS DS Criteria) from at-risk liver cirrhosis patients (where HCC prevalence is higher) using ROC analysis was tested. Since serum AFP levels were available for all HCC patients and at-risk chronic liver disease patients, the performance of the HCC EV Z Score with the clinical AFP test for differentiating early-stage HCC (BCLC Stage 0-A, within Milan Criteria, or within UNOS DS Criteria) vs. at-risk liver cirrhosis patients was compared. (
The results above demonstrate development of a new HCC EV purification system, i.e. EV Click Chips, by uniquely integrating several novel strategies including covalent chemistry-mediated EV capture/release, a multimarker antibody cocktail, nanostructured substrates, and a microfluidic chaotic mixer, promising rapid and effective purification of HCC EVs with intact mRNA cargo. By coupling EV Click Chips with a downstream RT-ddPCR assay designated to quantify 10 well-validated HCC-specific mRNA transcripts33, the resulting HCC EV-derived 10-gene digital readouts exhibited great potential for non-invasive early detection of HCC. A unique feature of EV Click Chips is the exploration of the covalent chemistry-mediated EV purification (capture/release) process through two consecutive steps: i) click chemistry-mediated EV capture and ii) disulfide cleavage-driven EV release. Click chemistry is a class of rapid bioorthogonal organic reactions frequently used for bioconjugation, e.g., coupling of biomolecules with substrates of interest (e.g., reporter molecules). Due to the very low number of antigens present on the surface of individual EVs, immunoaffinity-based EV capture approaches, which are driven by the dynamic binding between a pair of antigen (on EV) and antibody (on the substrate), often suffer from poor EV capture performance and high background issues. This problem can be resolved by replacing immunoaffinity-mediated capture to click-chemistry-mediated capture. Among different click chemistry reactions, the inverse-electron-demand Diels-Alder cycloaddition47 between Tz and TCO motifs (a rate constant39 of 104 M−1·s−1) was selected given their balanced chemical properties concerning both stability and reactivity, and the lack of a need for the presence of a catalyst. The ligation between Tz-grafted SiNWS and TCO-grafted EVs is rapid, specific, irreversible, and insensitive to biomolecules, water, and oxygen, leading to specific, rapid, and irreversible immobilization of the EVs with improved capture efficiency and reduced nonspecific trapping of particles in the background. Furthermore, considering the fact that HCC EVs are secreted by highly heterogeneous HCC22-24 cells, it is conceivable that no single capture agent can achieve sufficient performance to capture HCC EVs. Therefore, it is necessary to develop an antibody cocktail to recognize and capture HCC EVs from clinical samples, allowing for sensitive and specific detection of HCC-derived EVs across all disease stages. The experimental data using both artificial and clinical plasma samples showed that significantly greater EV capture yield and purity was achieved when utilizing a 3-antibody combination cocktail compared to each single antibody alone (i.e., anti-EpCAM, anti-ASGPR, and anti-CD147). Moreover, based on previous experience in exploring the combined use of nanostructured immunoaffinity substrates and PDMS microfluidic chaotic mixers to achieve highly efficient capture of targeted particles (i.e., CTCs and EVs) in peripheral blood, integrating this device configuration with click chemistry-mediated EV capture and a multimarker antibody cocktail offers the most sensitive and specific technology for capturing HCC EVs with a minimal level of background. This approach also allows for more effective conjugation of the TCO-grafted antibody cocktail onto the majority of HCC EVs in a small volume of solution, facilitating the click-chemistry-mediated HCC EV capture onto Click Chips. Following the click chemistry-mediated capture of EV Click Chips, the subsequent disulfide cleavage-driven HCC EV release confers the second layer of specificity to the HCC EVs purification process, further improving the purity of recovered HCC EVs.
The combined use of a multimarker antibody cocktail and EV Click Chips could possibly lead to recovering EVs which are not of HCC origin. For example, anti-EpCAM could capture EVs from other epithelial tissues. To address this concern, the RT-ddPCR assay capable of quantifying 10 HCC-specific genes as a downstream readout for the purified HCC EVs was adopted. These 10 HCC-specific genes were selected from tissue lineage-associated transcripts expressed in liver cells but absent in the EVs released from blood cells and other tissues. The resulting 10-gene digital readouts were predominantly contributed by HCC EVs, thus conferring the third layer of specificity for detecting HCC EVs.
In the process of optimizing the EV Click Chip, a novel quantitative evaluation method was developed that has addressed the dire need of assessing the purification performance (EV recovery yield and purity) of the EV Click Chip. Due to the lack of highly prevalent mutations in HCC, a novel system was devised where the SRY gene encoded on Chromosome Y from a male HCC cell line would be utilized as an artificial HCC biomarker. An artificial plasma sample was first prepared by spiking EVs from a male HCC cell line into plasma from a female healthy donor, and utilized quantification of the SRY transcript as a means for distinguishing and quantifying spiked HCC EVs. RT-ddPCR assay was then adopted for counting the copy numbers of the SRY and C1orf101 transcripts (in Chromosome Y and Chromosome 1, respectively) in the purified HCC EVs. This method is more convenient and quantitative than the existing methods17 that required pre-labeling or pre-transfection of EVs with specific transcripts messages. Moreover, this method is broadly applicable to the optimization of any other tumor-derived EV purification platforms before clinical study.
Current diagnostics for HCC fall into two main categories: radiologic imaging and blood-based biomarker tests48. However, the diagnostic performance of these modalities (i.e., ultrasonography and serum AFP) is inadequate, particularly for the diagnosis of early-stage HCC49. When liquid biopsies emerged, they were hailed as a possible screening tool for cancer, but proved to lack sufficient specificity and sensitivity for early detection of cancer50,51, The EV Click Chip-based HCC EV Assay for HCC diagnosis was applied, where the early detection strategies are currently unsatisfactory. There have been promises on the horizon for emerging liquid biopsy-based HCC diagnostics such as ctDNA-based methylation for HCC detection52 and CTC-based RNA signature for HCC detection33. Although ctDNA methylation profile using whole genome bisulfite sequencing can detect early-stage HCC52, its use in HCC screening may be challenging because of the relatively high cost and long turnaround time. Further, an inherent limitation of ctDNA-based methylation is its fragmentation, and it is released predominantly by cell death into the bloodstream, amid the background of DNA released from normal cells53. Moreover, although CTCs enable high specificity detection of HCC-specific mRNA signatures, the sensitivity of the CTC-based 10-gene assay for early detection of HCC is limited due to the fact that fewer CTCs are present in earlier stages of cancer54. The 10-gene panel originally developed by the MGH group33 for HCC CTC detection was adopted for the EV Click Chip, which takes advantage of HCC EVs. These small membrane-bound particles encapsulate HCC-specific mRNA which can be selectively isolated from total EVs even at an early stage in satisfactory quantities. The analysis of the isolated pooled HCC EVs has allowed mRNA-based detection of HCC-specific gene signatures, paving the way for early detection of HCC.
In this example, the potential clinical utilities of EV Click Chips in HCC for (i) differentiating HCC from Non-HCC, (ii) differentiating HCC from other cancers with or without metastasis to liver, and (iii) distinguishing early-stage HCC from at-risk liver cirrhosis is demonstrated. EV Click Chips exhibit dramatically improved recovery yield and purity of HCC EVs compared to commonly used EV isolation methods (i.e. ultracentrifugation). Beyond the early diagnosis of HCC from at-risk CLD patients, the resulting HCC EV digital score generated by the assay also showed the potential for HCC staging consistent with BCLC and Milan criteria and significantly augment the ability of current diagnosis and staging criteria to realize early detection of HCC and longitudinal monitoring of disease progression. The platform for HCC early diagnosis is broadly applicable to other cancer types. Since tumor-derived EVs can be efficiently isolated by targeting multiple surface markers and can carry tumor-specific genes that are absent in normal blood components, they hold considerable promise for the early detection of cancer.
10-15 μm Si nanowires (diameter=100-200 nm) were introduced onto Tz-grafted SiNWS via a fabrication process combining photolithographic patterning and silver (Ag) nanoparticle-templated wet etching55, offering approximately 30 times more surface areas (in contrast to a flat substrate) for facilitating click chemistry-mediated EV capture. According to the protocols published in previous study34 SiNWS were fabricated by combining the photolithographic patterning and Ag nanoparticle-templated wet etching55. In short, a p-type Si (100) wafer (Silicon Quest Int'l) was spin-coated with a thin film photoresist (AZ 5214, AZ Electronic Materials USA Corp.) using a resistivity of 10-20 Ω·cm. The Si wafer was then immersed into the etching solution containing HF (4.6 M, Sigma-Aldrich), AgNO3 (0.2 M, Sigma-Aldrich) and deionized (DI) water after being exposed to ultraviolet light. Finally, the Ag nanoparticle-templates were removed by immersing these Si wafer into boiling aqua regia (HCl/HNO3, 3:1 (v/v), Sigma-Aldrich) for 15 min. The SiNWS were then treated with acetone (≥99.5%, Sigma-Aldrich), followed by ethanol anhydrous (Sigma-Aldrich) wash. A disulfide linker was used to couple the Tz motifs grafted on the chips by designing a three-step chemical modification procedure: (i) Silanization: The SiNWS were first immersed in a freshly prepared piranha solution (H2SO4/H2O2, 2:1 (v/v), Sigma-Aldrich) for 1 hour, followed by rinsing with DI water and ethanol successively for three times. After drying under nitrogen flow, the resultant SiNWS were sealed in a vacuum desiccator for treatment (3-mercaptopropyl) trimethoxysilane vapor (211.4 mg, 200 μL, Sigma-Aldrich) for 45 min to introduce thiol groups onto the SiNWS. (ii) Incorporation of disulfide bond: OPSS-PEG-NH2 (0.30 mg, 3.8 mM, Nanocs Inc.) was incubated with freshly prepared HS-SiNWS in dimethyl sulfoxide (DMSO, 200 μL) solution for 2 hours to introduce disulfide linkers with terminal amine groups. Then the amine-terminated SiNWS (H2N-SiNWS) were rinsed with ethanol three times. (iii) To graft Tz motifs, the H2N-SiNWS was incubated with Tz-sulfo-NHS ester (0.32 mg. 3.8 mM, Click Chemistry Tools Bioconjugate Technology Company) in PBS (200 μL. PH=8.5) for 1 h. The resulting Tz-grafted SiNWS were rinsed with DI water three times. After drying under nitrogen flow, the Tz-grafted SiNWS were stored at −20° C. To confirm successful preparation of Tz-grafted SiNWS, X-ray photoelectron spectroscopy (XPS) was employed to monitor functional group transformation step-by-step.
Goat anti human EpCAM (R&D Systems, Inc.), goat anti human CD147 (R&D Systems, Inc.), rabbit anti human ASGPR (LifeSpan BioSciences, Inc.), and sheep anti human GPC3 (R&D Systems, Inc.) were incubated with TCO-PEG+-NHS ester (0.5 mM, Click Chemistry Tools Bioconjugate Technology Company) in PBS according to different mole ratios at room temperature for 30 min respectively. The individual TCO-antibody conjugates were prepared freshly before their use.
HepG2, Hep 3B cell line were purchased from American Type Culture Collection and cultured in Eagle's Minimum Essential Medium with 10% fetal bovine serum (FBS), 1% GlutaMAX-I and 100 U mL−1 penicillin-streptomycin (Thermo Fisher Scientific) in a humidified incubator with 5% CO2. SNU 387 cell line was purchased from American Type Culture Collection and cultured in RPMI-1640 Medium with 10% FBS, 1% GlutaMAX-I and 100 U mL−1 penicillin-streptomycin in a humidified incubator with 5% CO2.
HepG2, Hep 3B, SNU 387 cells were cultured in 18 Nunc EasYDish dishes (145 cm2. Thermo Fisher Scientific) for 72 hours. Then the culture medium was switched to serum-free culture medium (Thermo Fisher Scientific) to starve the cells for 24-48 hours. The serum-free culture medium incubated with cells was finally collected for EV isolation. After first centrifugation at 300 g (4° C. for 10 min to remove cells and cell debris, the supernatant was collected and transferred to new tubes and centrifuged at 2800 g (4° C.) for 10 min to further eliminate the remaining cellular debris and large particles. The supernatant was carefully transferred to Ultra-Clear Tubes (38.5 mL, Beckman Coulter, Inc., USA), followed by ultracentrifugation using an Optima L-100 XP Ultracentrifuge (Beckman Coulter, Inc, USA) at 100 000 g (4° C.) for 70 min. The EV pellets at the bottom of the tubes were carefully collected and resuspended in 200 μL fresh PBS. For the artificial plasma samples, each 10 μL aliquot of EV pellets was spiked into 90 μL healthy donor's plasma or cirrhotic patients' plasma.
For SEM characterization of HepG2 EVs, 10 μL pure HepG2 EVs in 100 μL PBS were run through the chips. The SiNWS were then cut to expose the cross sections of the silicon nanowire arrays. The severed SiNWS with captured HepG2 EVs were fixed in 4% PFA for 1 hour, followed by sequential dehydration through 30, 50, 75, 85, 95, and 100% ethanol solutions for 10 min each. After overnight lyophilization, the samples were sputter-coated with gold at room temperature. The images were visualized and taken under a ZEISS Supra 40VP SEM at an accelerating voltage of 10 520 keV.
For TEM characterization of HepG2 EVs, 10 μL freshly harvested HepG2 EVs or purified HepG2 EVs were deposited on the 200-mesh formvar-carbon coated EM grids for 20 min, and then the grids were transferred (membrane side down) to a 100-μl drop of 4% PFA for 10 min. After 3 times water-drop washing, the grids were treated with 2% uranyl acetate for 5 min and excess fluid was blotted by filter paper. The grids air dried before TEM imaging by JEM1200-EX (JEOL USA Inc.) at 80 kV.
For the fluorescent labeling of captured EVs, 10 μL pure HepG2 EVs in 100 μL PBS were run through the chips. 1.2 μL PKH26 dye was added into 200 μL Diluent C and mixed continuously for 30 seconds by gentle pipetting. The severed SiNWS with captured HepG2 EVs were incubated with this PKH26 dye solution at room temperature for 10 minutes. The EV Click Chips after HCC EV capture and release were observed by a fluorescence microscopy.
After chip assembly and leak testing according to previously described protocols18, the artificial plasma samples (100 μL) or the clinical plasma samples (500 μL) incubated with TCO-antibodies were then injected into EV Click Chip microfluidic devices. For EV release, 100 μL DTT solution (50 mM) was injected into the EV Click Chips at 1.0 mL/h and the released EVs were collected in 1.5 mL RNase-free Eppendorf tubes for subsequent RNA extraction.
The HCC EVs recovered from EV Click Chips were lysed by 700 μL QIAzol Lysis Reagent. RNA was extracted using a miRNeasy Micro Kit (Qiagen, German) according to the manufacturer's instructions. Then the complementary DNA (cDNA) was synthesized using a Thermo Scientific Maxima H Minus Reverse Transcriptase Kit according to the manufacturer's instructions. For the optimization experiments, cDNA was subjected to detect SRY transcripts and C1orf101 transcripts using duplex ddPCR in one tube with two fluorescence filters (i.e. FAM and VIC). For the clinical samples, 10 μL of total cDNA (12 μL) was divided into 5 tubes to detect the 10 genes with two fluorescence filters in each tube. ddPCR experiments were performed on a QX 200 system (Bio-Rad Laboratories, Inc.) according to the manufacturer's instructions. Data were analyzed using the QuantaSoft™ software to quantify the corresponding copy numbers of gene transcripts detected in each assay.
All the participants in this study were enrolled between October 2016-October 2019. Treatment—naïve HCC patients across all stages were enrolled in this study. HCC patients who had other malignant tumors, or severe mental diseases were excluded. The control cohorts consisted of patients with chronic liver disease, other cancers with or without metastasis to liver, and healthy donors. A detailed description of each control cohort and clinical characteristics can be found in the supporting information. All patients and healthy donors provided written informed consent for this study according to the IRB protocol (#14-000197) at UCLA and (STUDY00000066) at Cedars-Sinai Medical Center.
Peripheral venous blood samples were collected from fasting patients or healthy donors with the written informed consent from each patient or healthy donor according to the institutional review board (IRB) protocols at UCLA and Cedars-Sinai Medical Center. Each 8.0 mL blood sample was collected in a BD Vacutainer glass tube (BD Medical, Fisher Cat. #02-684-26) with acid citrate dextrose. Samples were processed according to the manufacturer's protocol within 4 h of collection.
The final plasma samples were collected for the HCC EV study after centrifugation at 10,000 g for 10 min. 500 μL plasma samples were then incubated with TCO conjugated anti-EpCAM (250 ng), anti-ASGPR (125 ng) and anti-CD147 (125 ng) at room temperature for 30 min before being loaded into the EV Click Chips for the HCC EV purification.
The EV recovery yields and purities are expressed as Mean±S.E.M. Significant differences between different groups were evaluated using one-way ANOVA. The 10-gene HCC EV Z Score, which represents the likelihood estimate of 10-gene activation, was computed from the RNA expression of the 10 genes using a weighted Z score method45 in R studio. After mean centering of expression data across the samples, HCC EV Z Scores were computed by the error-weighted mean of the expression values of the 10 genes in a sample. ROC curve was applied to evaluate the diagnostic performance for each parameter using MedCalc software.
Exploring different Click Chemistry motifs. The Click Chemistry motifs (i.e., Tz and TCO) used in the current version of EV Click Chips demonstrated good HCC EV capture performance. Additional Click Chemistry motifs investigated include methylated Tz/TCO motif, strain-promoted Azide-Alkyne reaction (SPAAC), and Cu(I)-catalyzed Azide-Alkyne reaction (CuAAC) (
Ewing sarcoma (ES) is a highly aggressive cancer that ranks as the second most frequent bone cancer during childhood and adolescence and is known for frequent metastases and poor prognoses.[34] Recently, ES EVs have been identified to be secreted by ES cells, actively participating in the tumorigenesis, progression, and metastasis of ES by not only reprogramming surrounding normal stromal cells but also promoting intercellular communication within the tumor cells themselves.[35,36] At present, few research efforts focus on isolating ES EVs, likely due to the lack of specific surface biomarkers to target. It is technically challenging to develop an efficient method for isolating ES EVs. As a result, only conventional methods—ultracentrifugation[37] and filtration[38]—have been adopted for their isolation. However, these are incapable of purifying ES EVs out of the non-ES EV background. Recently, an integrated microfluidic digital analysis chip with a dual-probe hybridization assay was developed for the detection of ES-EV mRNA,[39] demonstrating the presence of EWS-rearranged mRNA in ES EVs. However, this platform was not designed for the specific enrichment of ES EVs and is incapable of recovering intact ES EVs for downstream functional study.
To pave the way for conducting functional studies of ES EVs, a novel ES-EV purification system (i.e., “ES-EV Click Chip”) was introduced by coupling covalent chemistry-mediated EV capture/release within a nanostructure-embedded microchip (
To determine the specificity of LINGO-1 (a transmembrane signaling protein considered as a new marker and therapeutic target expressed on ES tumor surface[40]) as an ES cell surface marker, immunofluorescence staining was used to evaluate the expression of LINGO-1 on ES cell lines (e.g., A673, ES-5838, and SK-ES-1 cell lines), and white blood cells (WBCs) isolated from healthy donors' blood. For comparison, expression of CD99 (a transmembrane glycoprotein commonly used as an ES cell surface marker[52,53]) on ES cell lines and WBCs was also evaluated. The fluorescent images (
ES-EV Click Chip is composed of two components: (i) a Tz-grafted SiNWS and (ii) a PDMS-based chaotic mixer with a serpentine microchannel. Si nanowires with diameters of 100-200 nm and lengths of 3-5 or 7-10 μm were fabricated via a combination of photolithographic patterning and silver (Ag) nanoparticle-templated wet etching.[54] The densely packed Si nanowires (spacings=200-400 nm) provide large surface areas for immobilizing Tz moieties. Through a 3-step modification process[55]: (i) vapor deposition of (3-mercaptopropyl) trimethoxysilane (MPS), (ii) incorporation of a disulfide linker via ortho-pyridyl disulfide polyethylene glycol amine (OPSS-PEG-NH2), and (iii) NHS ester reaction between Tz-sulfo-NHS ester and the terminal primary amine group on SiNWS, abundant Tz moicties were tethered onto the Si nanowires to generate the Tz-grafted SiNWS. PDMS-based chaotic mixers were fabricated with herringbone patterns by inductively coupled plasma-reactive ion etching (ICP-RIE).[45,56] The herringbone pattern spacings and microchannel widths/lengths/heights (2 mm×60 mm×70 μm) were configured to facilitate direct physical contact[56] between the functional SiNWS and EVs. Finally, a microfluidic chip holder was used to combine the PDMS-based chaotic mixer with the Tz-grafted SiNWS to make a complete ES-EV Click Chip, and an automated fluidic handler was employed to handle EV samples.
Prior to EV capture studies, the complementary click chemistry moieties—TCO—were conjugated[57] onto goat anti-LINGO-1 via the NHS ester reaction between TCO-PEG4-NHS ester and the primary amine groups on anti-LINGO-1 to produce TCO-anti-LINGO-1 conjugate. As a model system for testing the EV-capture/release performance of ES-EV Click Chips, A673 EV samples were prepared by homogeneously re-suspending A673 EV pellets into serum-free medium and divided into several replicates (each 100 μL). The TCO-anti-LINGO-1 conjugate was pre-incubated with A673 EV samples to allow the specific antigen-antibody interaction. Then the obtained TCO-anti-LINGO-1-grafted A673 EV sample was run through ES-EV Click Chip, resulting in the efficient, chemoselective, and irreversible capture of A673 EVs on the Tz-grafted SiNWS via the IEDDA cycloaddition[46] between Tz and TCO moicties. Afterward, to release the captured EVs, DTT (50 mM, 50 μL) was injected into ES-EV Click Chip. DTT-mediated thiol-disulfide exchange reactions cause the reduction and cleavage of the disulfide bonds linking ES EVs or spare Tz moicties to SiNWS, resulting in the prompt release of captured ES EVs from the SiNWS.
To demonstrate the feasibility of click chemistry-mediated EV capture on SiNWS, PKH26 red-fluorescent dye was used to label A673 EVs (
To characterize the release process of ES EVs, DTT was injected into the ES-EV Click Chip, which had captured PKH26-labeled A673 EVs. As shown in
To optimize the ES-EV capture performance of ES-EV Click Chip (
Then the effects of different concentrations of anti-LINGO-1 conjugates on the capture efficiencies of ES-EV Click Chips and Nano Villi Chips was compared. The schematic diagram of
After EV capture, DTT solution was injected into ES-EV Click Chips to release ES EVs from the SiNWS (
2.5. Detection of EWS Rearrangements in ES EVs by Coupling ES-EV Click Chips with Reverse Transcription Droplet Digital PCR
To demonstrate the feasibility of detecting EWS rearrangements using reverse transcription droplet digital PCR (RT-ddPCR) in ES EVs purified by the ES-EV Click Chips, artificial ES-EV plasma samples were prepared by homogeneously re-suspending ES EV pellets into healthy donors' blood plasma (containing a significant quantity of normal cell-derived EVs) and divided into several replicates (each 100 μL). As illustrated in
Then, the isolation efficiency and specificity of ES-EV Click Chips was compared using 1 pmol of TCO-anti-LINGO-1, TCO-anti-CD99, and TCO-anti-CD63 conjugates, because CD99 had been used as an ES cell surface marker to isolate circulating tumor cells[52,53] and CD63 was used as a surface marker to isolate EVs (preferentially small EVs[58]). As shown in
The isolation performance of ES-EV Click Chips was compared to immunomagnetic beads[60] and ultracentrifugation[37] (two commonly used EV enrichment methods), as well as the ExoQuick ULTRA EV Isolation Kit for Serum and Plasma (non-specifically isolating total EVs using an EV precipitation mechanism). The results summarized in
The purified ES EVs can be co-cultured with recipient cells and studied for EV uptake and RNA cargo transfer (
To visualize the EV uptake process, PKH26-labeled ES-5838 EVs were purified by ES-EV Click Chips and co-cultured with A673 cells at 37° C. for 1, 2, and 4 h, respectively. A673 cells alone served as the negative controls (0 h). In parallel, the PKH26 negative control samples (without ES-5838 EVs) were also purified by ES-EV Click Chips and co-cultured with A673 cells. For static fluorescence imaging, A673 cells were washed with DPBS three times, fixed with 4% paraformaldehyde (PFA), stained with 4′,6-diamidino-2-phenylindole (DAPI), and imaged using a 40× objective lens on a Nikon Eclipse Ti fluorescence microscope under bright field, lasers 405 nm (DAPI) and 561 nm (PKH26). As shown in
Furthermore, it has been recognized that EVs are able to transfer their RNA cargoes to recipient cells both in vitro and in vivo.[8] Because the male ES-5838 cell-derived EVs harbor unique EWS-ERG rearrangement and sex-determining region of the Y-chromosome (SRY) transcripts, which are not present in female A673 cells, the EWS-ERG rearrangement and SRY expression could be used as specific molecular markers for quantification of ES-5838 EVs that were internalized by A673 cells. Therefore, after co-culturing with ES-5838 EVs for 1, 2, and 4 h, A673 cells in wells were washed with DPBS three times, treated with 0.25% trypsin-EDTA at 37° C. for 1 min and washed thoroughly with the citric acid buffer to remove the unbound EVs and cell surface-bound EVs. After centrifugation at 300 g for 10 min, A673 cell pellets were lysed by 700 μL of QIAzol lysis reagent and purified with miRNeasy Mini Kits (Qiagen). The purified RNA was subjected to RT-ddPCR quantification. Both EWS-ERG rearrangement and SRY transcript were detectable in A673 cells with ES-5838 EV uptake. As summarized in
A novel ES-EV purification system—ES-EV Click Chip—has been developed by coupling covalent chemistry-mediated EV capture/release within a nanostructure-embedded microchip. This device exploits anti-LINGO-1-specific recognition, sensitive click chemistry-mediated EV capture, and disulfide cleavage-driven EV release on a SiNWS-embedded microfluidic platform, realizing the highly efficient purification of ES EVs while maintaining their well-preserved integrity and biological activity. Fluorescence microscopy, TEM, SEM, and DLS characterization was adopted to demonstrate the EV capture and release features of ES-EV Click Chip. ES-EV Click Chip has several distinct advantages. (i) ES-EV Click Chips were optimized to have higher capture efficiency and lower antibody consumption compared with the previously reported Nano Villi Chips.[33] This improvement is attributed to the rapid, chemoselective, and irreversible click chemistry-mediated capture mechanism, as well as the significantly increased number of click reaction sites between TCO moicties grafted on EVs and Tz moicties functionalized on Si nanowire arrays. (ii) Compared to other potential capture agents, such as anti-CD99 and anti-CD63, the use of anti-LINGO-1 in ES-EV Click Chips significantly improves the efficiency and specificity of ES-EV enrichment. (iii) Furthermore, the mild reagent DTT-mediated disulfide bond cleavage enables the subsequent release of ES EVs with high efficiency. Compared with other EV-capture and release strategies on nanostructured substrates (e.g., the immune-affinity EV capture/proteinase K and temperature-responsive dual EV release strategy[32] and the non-specific exosome trapping/porous silicon nanowire dissolving strategy[63]), ES-EV Click Chips could purify ES EVs under milder conditions with high specificity and isolation efficiency, enhanced reproducibility, reduced cost and time consumptions, as well as recovering tumor-derived EVs with well-preserved integrity for downstream functional studies. It was demonstrated that ES-EV Click Chip could purify ES EVs without any size bias and recover them with well-preserved viability and RNA cargo contents. The recovered ES EVs can be rapidly internalized and shuttle their RNA cargoes to recipient cells, which can be leveraged to explore their physiologic and potential pathologic roles in intercellular communication.
Fabrication of ES-EV Click Chip Devices: ES-EV Click Chip device consists of (i) a Tz-grafted SiNWS and (ii) a PDMS-based chaotic mixer. Firstly, SiNWS with densely packed Si nanowires (diameters=100-200 nm, spacings=200-400 nm, lengths of 3-5 or 7-10 μm) were prepared by a combination of photolithographic patterning and AgNP-templated wet etching[54] according to the following procedures: (i) (100) p-type Si wafers (Silicon Quest International) were spin-coated with a thin-film photoresist (AZ 5214, AZ Electronic Materials USA Corp.) and exposed to ultraviolet light; (ii) the wafers were immersed into etching solution with hydrofluoric acid (4.6 M, Sigma-Aldrich) and silver nitrate (0.2 M, Sigma-Aldrich); and (iii) the wafers were treated with boiling aqua regia [i.e., hydrochloric acid/nitric acid, 3:1 (v/v), Sigma Aldrich] to remove the silver film. The resultant SiNWS were incubated with a piranha solution [sulfuric acid/hydrogen peroxide, 2:1 (v/v), Sigma-Aldrich]. Next, Tz moieties with disulfide linkers were functionalized onto the SiNWS via a 3-step chemical modification[55] process: (i) exposing the SiNWS to silane vapor of MPS (95%, 200 μL, Sigma-Aldrich) in a scaled vacuum desiccator for 45 min; (ii) incubating the SiNW with 200 μL of dimethyl sulfoxide (DMSO) solution containing OPSS-PEG-NH2 (3.8 mM, Nanocs Inc.) for 2 h at room temperature; and (iii) further incubating the SiNW with 200 μL of PBS solution containing Tz-sulfo-NHS ester (3.8 mM; Click Chemistry Tools) for 1 h at room temperature. Thus, Tz-grafted SiNWS were produced and ready to use.
Secondly, PDMS-based microfluidic chaotic mixers[45] were prepared by ICP-RIE.[56] Briefly, (i) a master wafer was photolithographically prepared by spin-coating a layer of negative photoresist (MicroChem Corp.) with a thickness of 75 μm onto a silicon wafer; (ii) after exposure to UV light with a photomask containing a 2.0-mm-width serpentine rectangular microfluidic channel, the second layer of negative photoresist was spin-coated with a thickness of 40 μm; (iii) using a Mask Aligner (Karl Suss America Inc.), the second photomask containing herringbone ridges was aligned between the former pattern and the one to be imprinted; (iv) the Si master was exposed to trimethylchlorosilane (99%, Sigma-Aldrich) vapor for 1 min and transferred to a petri dish; (v) for replica molding, well-mixed PDMS precursor (RTV 615 A and B in a 10:1 ratio; GE Silicones) was filled into the petri dish, degassed, and incubated in an oven at 80° C.to make a 5-mm-thick chip; and (vi) the produced PDMS-based chaotic mixer was peeled off and punched with two through-holes at the ends of the serpentine rectangular microfluidic channel for insertion of tubing. Finally, the above Tz-grafted SiNWS and PDMS-based chaotic mixer were combined in a custom-designed chip holder to give an ES-EV Click chip device. Then, ES-EV Click chip device was placed in an automated digital fluidic handler to control the loading and flow of reagents and EV samples.
Preparation of TCO-Antibody Conjugates: The TCO-anti-LINGO-1 conjugate was produced by incubating TCO-PEG+-NHS ester (4 μM, Click Chemistry Tools) with polyclonal goat IgG human LINGO-1 antibody (1 μM, R&D Systems Inc.) in PBS solution (pH 7.4) at room temperature for 30 min. TCO-anti-CD99 and TCO-anti-CD63 conjugates were prepared accordingly by incubating TCO-PEG4-NHS ester (4 μM, Click Chemistry Tools) with polyclonal goat IgG human CD99 antibody (1 μM, R&D Systems Inc.) and Monoclonal Mouse IgG1 human CD63 antibody (1 μM, R&D Systems Inc.), respectively. The resultant TCO-antibody conjugates (1 μM) in PBS solution were stored at −20° C. until use.
Culture of ES Cell Lines: ES cell lines, i.e., A673 cells (female origin, harboring EWS-FLI1 type 1 rearrangement) and SK-ES-1 cells (male origin, harboring EWS-FLI1 type 2 rearrangement) were obtained from the American Type Culture Collection (ATCC) and regularly tested negative for mycoplasma contamination. ES-5838 cells (male origin, harboring EWS-ERG rearrangement) were provided by Dr. James S. Tomlinson's Lab (UCLA). These cells were grown in 18 Nunc EasYDish dishes (150 mm, Thermo Fisher Scientific) with Dulbecco's Modified Eagle's Medium (DMEM, Thermo Fisher Scientific), fetal bovine serum (FBS, 10% (v/v), Thermo Fisher Scientific), GlutaMAX-I (1% (v/v), Thermo Fisher Scientific), and penicillin-streptomycin (100 U mL−1, Thermo Fisher Scientific) in a humidified incubator with 5% CO2 at 37° C. for three days.
Immunofluorescence Characterization of LINGO-1 and CD99 Expression on Cells: To demonstrate the specificity of LINGO-1 expression on ES cells, WBCs were isolated from the peripheral venous blood sample of a healthy donor with approval from UCLA Institutional Review Board (IRB, #00000173) and served as the control group of ES cells. A673 cells, ES-5838 cells, and WBCs on glass coverslips were detected with the following immunocytochemistry (ICC) protocol. First, cells were fixed with 4% PFA fixative solution (Electron Microscopy Sciences) for 20 min and subsequently incubated with 0.1% Triton X-100 for 10 min at room temperature. Next, these cells were incubated overnight at 4° C. with the primary antibody, i.e., polyclonal goat IgG human LINGO-1 antibody [1:100 (v/v)] or polyclonal goat IgG human CD99 antibody [1:40 (v/v)], in 200 μL of PBS containing 2% donkey serum (Jackson ImmunoResearch). After washing with PBS, these cells were incubated with the secondary antibody, i.e., donkey anti-goat IgG (H+L) [Alexa Fluor 647, 1:500 (v/v); Invitrogen] in 200 μL of PBS containing 2% donkey serum at room temperature for 1 h. After washing with PBS, these cells were treated with DAPI solution [1:1000 (v/v), Invitrogen]. Thereafter, these cells were imaged using a 40× objective lens on a Nikon Eclipse 90i fluorescence microscope.
Isolation and Preparation of ES-EV Samples: ES cells were cultured in serum-free medium for 24 h. A total of 234 mL of medium was collected in six Falcon 50 ml Conical Centrifuge Tubes (Thermo Fisher Scientific) and centrifuged at 4° C. and 300 g for 10 min to remove cells and cellular debris. The supernatant was centrifuged at 4° C. and 4,600 g for 30 min to eliminate large particles. Thereafter, the supernatant was transferred to six Ultra-Clear Tubes (38.5 mL, Beckman Coulter, Inc., USA) and centrifuged at 4° C. and 100,000 g for 2 h using Optima L-100 XP Ultracentrifuge (Beckman Coulter, Inc. USA). For making model EV samples, the resultant EV pellet was resuspended in 2 mL of serum-free medium and divided into 20 equal parts (each 100 μL). For making artificial EV plasma samples, the EV pellet was resuspended in 2 mL of blood plasma collected from a female healthy donor with approval from the UCLA Institutional Review Board (IRB, #00000173), and divided into 20 aliquots (each 100 μL). These ES-EV samples were stored at −80° C. for future use. EV Labeling with PKH26 Red-Fluorescent Dye: ES EVs were labeled with PKH26 red fluorescent cell linker kit (Sigma-Aldrich) according to the instructions with some modifications.[64] Briefly, EV pellets were resuspended in 500 μL of Diluent C. Separately, 500 μL Diluent C was mixed with 2 μL of PKH26 red-fluorescent dye (1 mM) to prepare a 2× dye (4 μM) solution. After mixing the EV and PKH26 solution for 5 min at 4° C., 1 mL of 1% bovine serum albumin (BSA, Sigma-Aldrich) was added to bind excess dye. Then, the PKH26-labeled EVs were washed with PBS through ultracentrifugation at 4° C. and 100,000 g for 2 h to remove the free PKH26 dye. The pellet was resuspended in PBS and divided into several replicates. Meanwhile, as a negative control, PKH26 dye alone (without ES EVs) was also washed with PBS by ultracentrifugation and diluted in PBS to make the PKH26 negative control sample.
ES-EV Capture and Release by ES-EV Click Chips: Prior to capture, the ES-EV sample (100 μL) was pre-incubated with the TCO-LINGO-1 conjugate for 20 min at room temperature. Meanwhile, 200 μL of PBS was injected into ES-EV Click Chip at a flow rate of 1 mL h−1 to test leaks. The resultant TCO-grafted EV sample was then introduced into ES-EV Click Chip at an optimal flow rate of 0.2 mL h−1 and captured on the Tz-grafted SiNWS via the click chemistry-mediated EV capture. Afterward, to release the EVs captured on chips, a DPBS solution (50-100 μL) containing DTT (50 mM) was injected into ES-EV Click Chip at an optimal flow rate of 0.2 mL h−1. The released EVs were collected into a 1.5-mL ribonuclease (RNase)-free Eppendorf tube.
TEM Characterization: The ES EVs in solution or Si nanowires mechanically detached from the SiNWS after EV capture/release were fixed in 4% PFA for 30 min at room temperature. Next, 5 μL of samples were placed onto formvar and carbon-coated copper grids (200-mesh) and incubated for 5 min. After blotting the excess samples with filter paper, grids were negatively stained with 2% uranyl acetate for 10 min. After rinsing with deionized water three times, samples were dried and imaged by JEM1200-EX (JEOL USA Inc.) at 80 kV.
For immunogold-TEM, 5 μL of samples were placed onto formvar and carbon-coated nickel grids (200-mesh) and incubated for 5 min. After wiping off the excess samples, grids were blocked in a blocking solution containing 0.4% BSA for 30 min and rinsed with deionized water three times. Then samples were incubated with monoclonal mouse IgG1 human LINGO-1 antibody [clone #332237, 1:1000 (v/v), R&D Systems Inc.] or monoclonal mouse anti-CD63 (1:500 (v/v), Abcam] for 1 h. Meanwhile, samples were incubated with the blocking solution as negative controls. After rinsing with deionized water three times, the samples were incubated with goat anti-mouse IgG H&L 10-nm gold [1:40 (v/v), Abcam] for 1 h. Thereafter, grids were rinsed and negatively stained with 2% uranyl acetate, followed by drying and TEM imaging.
SEM Characterization: To characterize the distribution of EVs on Si nanowire arrays after capture/release, SiNWS were cut to expose the cross-sections of Si nanowire arrays and incubated with 4% PFA for 1 h at room temperature. Next, the substrates were dehydrated by sequentially immersing in 30%, 50%, 75%, 85%, 95%, and 100% ethanol solutions for 10 min per solution. After drying, the substrates were sputter-coated with gold and imaged under a ZEISS Supra 40VP SEM at an accelerating voltage of 10 keV.
DLS Characterization: The size distributions of EVs before capture and after release were measured using Malvern Zetasizer Nano ZS. EV samples were diluted 1:10 or 1:20 in the cuvette and analyzed by Malvern Zetasizer Nano ZS to give the size distribution.
Extraction and Quantification of RNA from ES EVs: For EVs captured on ES-EV Click Chip, RNA was extracted by introducing 700 μL of QIAzol lysis reagent at a flow rate of 0.5 mL h−1 for 200 μL and then 60 mL h−1 for the leftover 500 μL. The outflow was collected in a 1.5-mL ribonuclease (RNase)-free Eppendorf tube. For EVs before capture and after release in solution, 700 μL of QIAzol lysis reagent was added to lyse EVs in 1.5-mL ribonuclease (RNase)-free Eppendorf tubes. The extracted EV-derived RNA was purified with miRNeasy Micro Kits (Qiagen), according to the manufacturer's protocol. During the RNA purification process, DNase I (RNase-free, Thermo Fisher Scientific) was used to digest DNA for 15 min at room temperature. Finally, RNA was dissolved in DNase/RNase-free water and centrifuged off the RNeasy MinElute Spin Columns into 1.5-mL ribonuclease (RNase)-free collection tubes. The RNA was quantified with Qubit 3.0 Fluorometer (Thermo Fisher Scientific, USA) and Qubit RNA HS Assay according to the manufacturer's instructions.
RT-ddPCR Detection: RNA was reverse-transcribed to cDNA with a Maxima H Minus Reverse Transcriptase Kit (Thermo Fisher Scientific, USA) according to the manufacturer's protocol. The reverse transcription reaction was performed at 55° C. for 30 min and 85° C. for 5 min. Thereafter, cDNA was detected with ddPCR Supermix for Probes (No dUTP, Bio-Rad). EWS rearrangements were detected using self-designed primers and probes. SRY transcript was detected using a commercial primer/probe kit (Catalog #4331182; Assay ID: Hs00976796_s1, Thermo Fisher Scientific). Droplets containing ddPCR reaction were transferred into a 96-well plate and sealed. ddPCR reaction was performed at 96° C. for 10 min, followed by 40 cycles (94° C. for 30 s and 60° C. for 60 s) and 98° C. for 10 min. The DNA amplicons contained in droplets were detected by a QX200 Droplet Reader in combination with a QuantaSoft™ software package.
Comparison with Immunomagnetic Beads, Ultracentrifugation, and ExoQuick ULTRA EV Isolation Kit: For immunomagnetic bead separation, Tz-grafted magnetic beads were prepared by incubating 2.8 μm Dynabeads™ M-270 Amine (2×108 beads, 100 μL, Thermo Fisher Scientific) with Tz-sulfo-NHS ester (0.32 mg, Click Chemistry Tools, USA) in PBS buffer for 1 h at room temperature. Each artificial A673 EV plasma sample was pre-incubated with the TCO-LINGO-1 conjugate (1 pmol) for 20 min and incubated with Tz-grafted magnetic beads (2×107 beads) at room temperature for 30 min to isolate A673 EVs. For ultracentrifugation, each artificial A673 EV plasma sample was centrifuged at 100,000 g for 2 h using Optima L-100 XP Ultracentrifuge. For the commercially used EV isolation assay, each artificial A673 EV plasma sample was isolated and purified using the ExoQuick ULTRA EV Isolation Kit (System Biosciences) according to the manufacturer's protocol. For all the methods, RNA was extracted from the isolated EVs and quantified using Qubit 3.0 Fluorometer (Thermo Fisher Scientific), followed by quantification of EWS-FLI1 type 1 rearrangement using RT-ddPCR detection. Healthy-donor plasma samples without A673 EVs were processed in parallel to give the systems' RNA background.
CCK-8 Cell Viability Assay: A673 cells (5×103 cell/well) were evenly plated into a sterile 96-well cell culture plate with the cell culture medium (250 μL per well) and pre-incubated in a humidified incubator with 5% CO2 at 37° C. for 24 h. For the DTTox effluent-added group, 50 μL of DTTox effluent was added into each well and incubated with A673 cells for 24 h. For the negative control, 50 μL of DPBS solution was incubated with A673 cells for 24 h. Thereafter, CCK-8 (Sigma-Aldrich) assay was used to test the effect of DTTox effluent on cell viability. The cell culture medium of A673 cells in each well was replaced with 10 μL of CCK-8 solution and 100 μL of serum-free medium. Meanwhile, a blank well without A673 cells was also added with 10 μL of CCK-8 solution and 100 μL of serum-free medium to serve as the blank of CCK-8 assay. After incubating for 4 h, the solution of each well was transferred to a Costar 96 Flat Transparent plate and placed into the Tecan Infinite 200 PRO. The optical density (OD, absorbance) at 450 nm was measured with an i-control Microplate Reader. The cell viability (%) was calculated as the ratio of the OD450 value of the DTTox effluent-added group (deducting the blank OD450 value) to that of the negative control group (deducting the blank OD450 value).
Downstream Functional Studies: A673 cells (5×103 cell/well) were evenly plated into a sterile 96-well cell culture plate with the cell culture medium (250 μL per well) and pre-incubated in a humidified incubator with 5% CO2 at 37° C. for 24-48 h. Then the cell culture medium was replaced with serum-free medium (250 μL per well) for EV uptake study. Before the addition of ES-5838 EVs, the wells with A673 cells alone served as the negative controls (0 h). After being released from ES-EV Click Chip, PKH26-labeled ES-5838 EVs (50 μL) were added to the wells and co-cultured with A673 cells at 37° C. for 1, 2, and 4 h, respectively. In parallel, the PKH26 negative control sample was also purified by ES-EV Click Chips and co-cultured with A673 cells at 37° C. for 1, 2, and 4 h, respectively.
Fluorescence Imaging of Uptake Process of ES EVs into Recipient Cells: For static fluorescence imaging, A673 cells were washed with DPBS three times, fixed with 4% PFA for 10 min, stained with DAPI [1:1000 (v/v)] for 10 min, and imaged using a 40× objective lens on a Nikon Eclipse Ti fluorescence microscope under bright field, lasers 405 nm (DAPI) and 561 nm (PKH26). For dynamic monitoring of the ES-5838 EV uptake and internalization process by live A673 cells, the 96-well cell culture plate was placed on the 3D automatic objective table and photographed once every 15 min for 90 min using a 40× objective lens on the Nikon Eclipse Ti fluorescence microscope under bright field and laser 561 nm (PKH26).
Detection of Gene Transcripts in Recipient Cells After EV Uptake. For detecting EWS-ERG rearrangement and SRY transcript of ES-5838 EVs internalized by A673 cells after co-culturing, A673 cells were washed with DPBS three times and treated with 0.25% trypsin-EDTA (Thermo Fisher Scientific) at 37° C. for 1 min and washed thoroughly with the citric acid buffer to remove the unbound EVs and cell surface-bound EVs. A673 cells were centrifuged at 300 g for 10 min. The cell pellets were lysed by 700 μL of QIAzol lysis reagent and purified with miRNeasy Mini Kits (Qiagen) according to the manufacturer's protocol. The purified RNA was subjected to RT-ddPCR detection.
This application is a U.S. National Stage of PCT/US2021/024454, filed Mar. 26, 2021, which claims priority to U.S. Provisional Application No. 63/000,776 filed Mar. 27, 2020; the entire contents of which are hereby incorporated by reference.
This invention was made with government support under Grant Number CA198900 and CA235340, awarded by the National Institutes of Health. The government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2021/024454 | 3/6/2021 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63000776 | Mar 2020 | US |
