Patent Application
20250123297
There is an increasing appreciation for the heterogeneous nature of secreted extracellular vesicles (EVs) and non-vesicular nanoparticles (Jeppesen, D. K., et al. Cell 2019 177:428-445 e418; Zhang, H., et al. Nat Cell Biol 2018 20:332-343; Zhang, Q., et al. Cell Rep 2019 27:940-954 e946). Exosomes are 40-150 nm endosome-derived, lipid-bilayer enclosed, small extracellular vesicless (sEVs) (Jeppesen, D. K., et al. Cell 2019 177:428-445 e418; Mathieu, M., et al. Nat Cell Biol 2019 21:9-17; van Niel, G., et al. Nat Rev Mol Cell Biol 2018 19:213-228). Recently, a new type of small (<50 nm), non-membranous, extracellular nanoparticle, termed the exomere, was identified (Zhang, H., et al. Nat Cell Biol 2018 20:332-343). Both exosomes and exomeres are released by most cells and tissues under both physiological and pathological conditions. Their production and content appear to be altered in a number of disease states, including neoplastic, cardiovascular, immunological and neurological disorders. However, intrinsic heterogeneity and variable methods of isolation pose major challenges to realizing their clinical potential.
Disclosed herein is a newly identified secreted nanoparticle that is morphologically and molecularly distinct from the recently described nanoparticle termed an exomere. The disclosed nanoparticle is referred to herein as a supermere. Both exomeres and supermeres are amembranous in contrast to membrane-enclosed extracellular vesicles (EVs). Supermeres are smaller and morphologically distinct from exomeres. These supermeres contain cargo that contribute to the pathogenesis of colorectal cancer (CRC) and may be potential biomarkers for CRC as some of these cargo have been detected in the circulation of cancer patients. In addition, supermeres from cetuximab-resistant CRC cells are able to confer resistance when added to cetuximab-sensitive CRC cells. Moreover, the receptor for SARS-CoV-2, angiotensin-converting enzyme 2 (ACE2), is present in large amounts in supermeres isolated from conditioned medium of DiFi cells, a human CRC cell line. The isoforms detected in these supermeres are smaller than the full-length band detected in DiFi whole cell lysates and in small EVs; this smaller form corresponds to the ectodomain of ACE2. Moreover, ACE2 is α2,6-sialylated in DiFi cells, small EVs, exomeres and supermeres. A processed form of amyloid precursor protein (APP), a protein that is associated with the pathogenesis of Alzheimer's disease, was also detected in supermeres, as well as the receptor tyrosine kinase MET and glypican-1 (GPC1).
Supermeres can be obtained relatively quickly and in large amounts from conditioned medium and plasma. They contain a number of cargo that may serve as biomarkers for a number of disease states, including cancer, heart disease, and neurodegenerative disorders, amongst others. Because these are new secreted components of biofluids, they have not thus far been associated with disease states.
Supermeres contain a large fragment of the ectodomain of ACE2. As disclosed herein, this form of ACE2 may bind SARS-CoV-2 and reduce the severity of infection, as has been shown for soluble ACE2. ACE2 is downregulated during viral infection, most notably with coronavirus infection (SARS and COVID-19), and this process can drive proteins towards secretion. In addition, ACE2 present in the circulation may be relevant to disorders of Renin-Angiotensin Aldosterone System (RAS/RAAS) that is associated with cardiovascular disease and cardiovascular problems associated with viral infection. Therefore, ACE2 in supermeres may provide diagnostic information. By delivering ACE2 in different forms in supermeres to patients systemically and/or in an aerosolized form might provide a specific treatment for patients. In addition, disclosed herein is a strategy of delivering this form of ACE2 in nanoparticles systemically and in an aerosolized form into the lungs.
Also disclosed herein is a method for isolating secreted, non-membranous supermere nanoparticles from a biological sample or conditioned medium, the method involving: centrifuging the biological sample or conditioned medium at 250 to 350×g (e.g. 300×g) to produce a first supernatant free of cell debris; filtering the first supernatant with a 0.22 μm filter to produce a first filtrate with reduced microparticle contamination; ultracentrifuging the first filtrate with a 100,000 molecular weight cutoff centrifugal concentrator to produce a first concentrate; ultracentrifuging the first concentrate at 100,000 to 167,000×g for 1 to 4 hours to produce a first pellet that is enriched for extracellular vesicles and exosomes; removing this first supernatant above the pellet; ultracentrifuging the supernatant at 100,000 to 167,000×g for 16 to 18 hours to produce a second pellet that comprises exomeres and a second supernatant from above the exomere pellet that contains supermeres; ultracentrifuging the second supernatant at 300,000 to 400,000 (e.g. 368,000×g) for 16 to 18 hours to produce a third pellet that comprises supermeres; resuspending the third pellet in a physiological solution (e.g. PBS-HEPES (PBS-H) (25 mM vol/vol).
Also disclosed is a method for diagnosis colorectal cancer in a subject that involves isolating a biological sample from the subject; isolating supermeres from the sample according to the disclosed method; and assaying the supermeres for colorectal cancer biomarker.
The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.
Before the present disclosure is described in greater detail, it is to be understood that this disclosure is not limited to particular embodiments described, and as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present disclosure will be limited only by the appended claims.
Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the disclosure. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure, the preferred methods and materials are now described.
All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present disclosure is not entitled to antedate such publication by virtue of prior disclosure. Further, the dates of publication provided could be different from the actual publication dates that may need to be independently confirmed.
As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present disclosure. Any recited method can be carried out in the order of events recited or in any other order that is logically possible.
Embodiments of the present disclosure will employ, unless otherwise indicated, techniques of chemistry, biology, and the like, which are within the skill of the art.
The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to perform the methods and use the probes disclosed and claimed herein. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.), but some errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, temperature is in ° C., and pressure is at or near atmospheric. Standard temperature and pressure are defined as 20° C. and 1 atmosphere.
Before the embodiments of the present disclosure are described in detail, it is to be understood that, unless otherwise indicated, the present disclosure is not limited to particular materials, reagents, reaction materials, manufacturing processes, or the like, as such can vary. It is also to be understood that the terminology used herein is for purposes of describing particular embodiments only, and is not intended to be limiting. It is also possible in the present disclosure that steps can be executed in different sequence where this is logically possible.
It must be noted that, as used in the specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise.
The small (<50 nm), non-membranous nanoparticle, termed exomere, was recently identified by asymmetric flow field-flow fractionation (AF4) (Zhang et al., Nat. Cell Biol. 2018 20:332-343), which is incorporated by reference for the description of these isolation methods. An alternative method for isolating exomeres was described (Zhang et al. 2019. Cell Reports 27:940-954), which is incorporated by reference for the description of these isolation methods. Exomeres are highly enriched in metabolic enzymes and signature proteins involved in glycolysis and mTORC1 signaling (Zhang et al., 2018a). In addition to proteins, nucleic acids and lipids are also selectively secreted in exomeres.
Disclosed herein are a newly identified secreted, non-membranous nanoparticle, referred to herein as a “supermere,” which can be isolated from the supernatant used to produce exomeres.
Serum-free conditioned medium is removed and centrifuged for 15 minutes at 300×g to remove cellular debris. The resulting supernatant is then filtered through a 0.22-mm polyethersulfone filter (Nalgene, Rochester, NY) to reduce microparticle contamination. The filtrate is concentrated with a 100,000 molecular-weight cutoff centrifugal concentrator (Millipore). The concentrate is subjected to high-speed centrifugation at 167,000×g for 4 hours, and the resulting small EV (sEV)-enriched pellet is resuspended in PBS (e.g. containing 25 mM HEPES (pH 7.2)-PBSH) and washed. The remaining supernatant is subjected to centrifugation again at 167,000×g for at least 16 hours and not more than 18 hours; the resulting pellet is washed with fresh PBSH. This washed pellet contains exomeres. To isolate supermeres, the supernatant collected from the previous 16 to 18 hour ultracentrifugation step is ultracentrifuged at 368,000×g for at least 16 hours but not more than 18 hours. The resulting pellet is resuspended in PBS (e.g. containing 25 mM HEPES [pH 7.2]) and is designated supermeres.
Supermeres can therefore be isolated by first sequentially isolating EV-enriched material, next exomeres from cell-conditioned medium as previously described, or alternatively from plasma or other human or animal body fluids. The supernatant left over from the pelleting of exomeres can then be collected and subjected to ultracentrifugation at 368,000×g (rmax, 55000 RPM), e.g. using a Beckman Coulter SW55 Ti rotor for 16-18 hours. The resulting pellet represents supermeres. The supermeres can then be resuspended in a suitable liquid, such as PBS, and stored at 4° C.
A number of embodiments of the invention have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims.
Isolation of Supermeres: Exomeres were isolated from DiFi conditioned medium as previously described (Zhang et al. 2019. Cell Reports). The supernatant left over from the pelleting of exomeres was collected and subjected to ultracentrifugation at 368,000×g (rmax, 55000 RPM) using a Beckman Coulter SW55 Ti rotor for 18-24 hours. The resulting pellet contained the isolated supermeres. The supermeres were resuspended in PBS and stored 4° C. for downstream analysis and functional assays, or for some analyses, immediately resuspended in lysis buffers.
AFM imaging and analysis: Five micro-liters of freshly isolated EV samples were incubated on freshly cleaved mica substrates (TedPella Inc, CA) for 5 min, washed with molecular grade de-ionized water to remove any unbound EV and air-dried overnight. Samples were imaged by Dimension Icon (Bruker Instruments, CA, USA) using TESP probes (Bruker Instruments, CA, USA) and images were recorded in amplitude mode-AFM at 1024 samples per line at 1 Hz as described previously (ACS Nano. 2010 Apr. 27; 4(4): 1921-1926; ACS Nano. 2019, 13, 9, 10499-10511). Over six different scan regions were randomly selected for imaging of particles. Images were processed for zero order plane flattening using SPIP™ software (Denmark). The surface height was determined using histogram function and was subtracted from the measured height of particles. The particles were then individually detected and sizes were quantified using grain analysis function. All samples were stored at 4° C. and imaged within one week of isolation.
Statistical methods All statistical analyses were performed using commercially available OriginLab 9.1 or R software. All values are expressed as the mean±SEM. Differences in means between two groups were analyzed using unpaired two-sided heteroscedastic t-tests with Welch's correction. The level of significance was taken as p <0.05.
The present studies were initially designed to provide a comprehensive proteomic and RNA analysis of clinically relevant cargo unique to exosomes and exomeres in a human colorectal cancer (CRC) cell line, DiFi, using an optimized strategy to purify exosomes (Jeppesen, D. K., et al. Cell 2019 177:428-445 e418) and a simplified method to isolate exomeres (Zhang, Q., et al. Cell Rep 2019 27:940-954 e946). High-speed ultracentrifugation of the sEV supernatant resulted in isolation of amembranous nanoparticles identical in morphology and content to that reported in the original characterization of exomeres using asymmetric flow field-flow fractionation (AF4) (Zhang, H., et al. Nat Cell Biol 2018 20:332-343). Early on in the study, it was speculated that an additional population of nanoparticles might be identified in the supernatant after exomere purification by high-speed ultracentrifugation. In fact, a morphologically distinct new nanoparticle was discovered that is termed the supermere (supernatant of exomeres).
Supermeres are morphologically and structurally distinct from exomeres and display different cellular uptake kinetics than sEVs and exomeres in vitro. There is a greater uptake of supermeres in most tissues in vivo compared to sEVs and exomeres, especially in the brain compared to the other fractions. Supermeres contain the majority of extracellular small RNA. Many of the clinically relevant proteins and extracellular RNA (exRNA), previously reported to be in exosomes (amyloid precursor protein [APP], MET, glypican-1 [GPC1], argonaute-2 [AGO2)], miR-1246, TGFβ-induced [TGFBI] and numerous glycolytic enzymes) are highly enriched in supermeres. Three functional properties of supermeres were identified: increased lactate secretion in recipient cells (a hallmark of the Warburg effect), transfer of cetuximab resistance, and altered liver metabolism following systemic injection. Supermeres are detectable by optimized flow cytometry, opening up another potential avenue of investigation in liquid biopsies alongside circulating tumor cells, DNA and EVs.
The most abundant protein in highly purified DiFi exosomes was the glycophosphatidylinositol (GPI)-linked dipeptidase DPEP1 that has been linked to CRC metastasis (Park, S. Y., et al. Oncotarget 2016 7:9501-9512). Diffuse overexpression of DPEP1 in a clinically well-annotated CRC tissue microarray portends a worse outcome, and DPEP1 is increased in sEVs isolated from the plasma of CRC patients compared to normal individuals. Moreover, a subpopulation of exosomes released from DiFi cells is highly enriched in DPEP1, two circulating biomarkers for CRC (CEA and EPCAM), and the ectonucleotidase CD73 that is involved in immunosuppression and is overexpressed in CRC (Goswami, S., et al. Nat Med 2020 26:39-46; Hammami, A., et al. Semin Immunol 2019 42:101304).
Taken together, this work identifies a new functional extracellular nanoparticle that is morphologically and molecularly distinct from exosomes and is replete with potential biomarkers and targets for drug discovery. Moreover, the ability to isolate and inventory the contents of distinct populations of sEVs and nanoparticles is demonstrated so as to assign cargo to their correct carriers. These findings have potential important implications for cancer, Alzheimer's disease, heart disease, and COVID-19 infection.
Supermeres are Novel Extracellular Nanoparticles with Distinct Uptake In Vitro and In Vivo
To determine if additional type(s) of nanoparticles could remain after exomere depletion, a sequential high-speed ultracentrifugation protocol (
To investigate uptake dynamics in vitro, sEVs, exomeres and supermeres derived from DiFi cells were fluorescently labeled and then used to treat MDA-MB-231 cells for 24 h. Supermeres and exomeres displayed significantly slower cellular uptake compared to sEVs (
ACE2, the critical entry receptor for the SARS-CoV-2 virus that causes COVID-19, was recently identified in sEVs and exomeres (Zhang, Q., et al. Gastroenterology 2021 160:958-961 e953). Here, it was found that supermeres derived from lung cancer (Calu-3) and CRC (LIM1215 and DiFi) cell lines contain similar levels of ACE2 as exomeres (
LC/MS-MS proteomics was next performed on gradient-purified sEVs, NVs, exomeres and supermeres. The proteomic profile of supermeres is clearly distinct from that of sEVs, NV and exomeres, while NV and exomeres are more similar (
Mutant KRAS exosomes derived from CRC cells can alter the metabolic state of the tumor microenvironment in a non-cell-autonomous fashion that is dependent, at least in part, on the transmembrane glucose transporter GLUT-1 (Zhang, Q., et al. Cell Mol Gastroenterol Hepatol 2018 5:627-629 e626). Since glycolytic enzymes were markedly enriched in supermeres (
Increased lactate secretion has been linked to EGFR and MET drug resistance (Apicella, M., et al. Cell Metab 2018 28:848-865 e846). Initially, the ability of supermeres from cetuximab-resistant cells (SC and CC-CR) to transfer resistance to cetuximab-sensitive cells (CC) cultured in a 3D environment of type-1 collagen was tested (Lu, Y., et al. Nat Med 2017 23:1331-1341; Li, C., et al. Proc Natl Acad Sci USA 2017 114:E2852-E2861). After exposure to CC cell-derived supermeres, CC cells in 3D culture remained sensitive to the growth inhibitory effects of cetuximab (
In summary, supermeres are functional extracellular nanoparticles. Supermeres are enriched in glycolytic enzymes and can increase lactate release in recipient cells, and supermeres derived from cetuximab-resistant cells are able to transfer resistance to cetuximab-sensitive recipient cells in a 3D cell culture system.
As there was a greater brain uptake of supermeres compared to both sEVs and exomeres (
MET, a receptor tyrosine kinase, is deregulated in many types of human cancers and mediates diverse biological responses, including metastasis (Comoglio, P. M., et al. Nat Rev Cancer 2018 18:341-358); MET transfer by exosomes has been proposed to increase the metastatic behavior of primary tumors (Peinado, H., et al. Nat Med 2012 18:883-891). Proteomics data indicated that full-length MET was present in sEVs, while only peptides covering the ectodomain were present for supermeres and exomeres (
GPC1 is a GPI-anchored cell surface heparan sulfate proteoglycan that is overexpressed in several cancers, including pancreatic cancer and CRC (Xu, R., et al. Nat Rev Clin Oncol 2018 15:617-638). GPC1 in plasma exosomes is reportedly a useful biomarker for the early detection of pancreatic cancer (Melo, S. A., et al. Nature 2015 523:177-182). However, different forms of GPC1 were far more associated with the non-vesicular exomere and supermere nanoparticles released from pancreatic cancer PANC1 cells and normal human renal epithelial cells (HREC) (
In summary, exomere and supermere nanoparticles are enriched in many shed, clinically relevant, membrane proteins, including APP, MET, GPC1, CEA, EGFR, AREG, ACE and ACE2, and can be detected by optimized flow cytometry.
Differential Expression of Small exRNAs Among sEVs, Exomeres and Supermeres
The RNA content in cells and extracellular carriers was next examined. EV-associated exRNAs, especially miRNAs, have attracted much attention due to their diverse biological functions and potential as cancer biomarkers (Jeppesen, D. K., et al. Cell 2019 177:428-445 e418; Srinivasan, S., et al. Cell 2019 177:446-462 e416; Murillo, O. D., et al. Cell 2019 177:463-477 e415; Slack, F. J. & Chinnaiyan, A. M. Cell 2019 179:1033-1055). The relative abundance of small exRNAs in supermeres was significantly higher than in exomeres and the sEV-P (
By far, the most abundant and most differentially expressed miRNA in supermeres was miR-1246 with a 210-fold change in expression levels compared to cells (
Several mechanisms have been proposed for sorting miRNAs into exosomes. The RNA-binding proteins, Y-box protein 1 (YBX1), sumoylated hnRNPA2B1 and Argonaute proteins (AGO1-4) have all been reported to mediate exosomal miRNA secretion (Shurtleff, M. J., et al. Elife 2016 5; Melo, S. A., et al. Cancer Cell 2014 26:707-721; Wu, B., et al. Nat Commun 2018 9:420). However, AGO1-4 are enriched in gradient-purified NV fractions and exomeres (Jeppesen, D. K., et al. Cell 2019 177:428-445 e418; Zhang, Q., et al. Cell Rep 2019 27:940-954 e946; Murillo, O. D., et al. Cell 2019 177:463-477 e415; Temoche-Diaz, M. M., et al. Elife 2019 8). The observed abundance of miRNAs in supermeres correlated with the proteomic data showing supermeres are highly enriched in ribonucleoproteins, including Argonaute proteins. AGO1 and AGO2 are highly enriched in DiFi cell-derived exomeres and supermeres but were not detected in high-resolution density gradient-purified sEVs (
In summary, supermeres display a distinct signature of small exRNAs with very high expression of specific miRNAs, including miR-1246, and supermeres are enriched for the miRNA-binding proteins AGO1, AGO2, hnRNPA2B1 and XPO5. High levels of AGO2 secretion in exomeres and supermeres may be a common feature of cancer cells.
Since supermeres are enriched for proteins involved in metabolism (
As the original intent of this project was to parse out the heterogeneity between sEVs and nanoparticles, the most abundant proteins in sEVs and exomeres of DiFi cells was next determined. DPEP1, a GPI-anchored zinc-dependent dipeptidase involved in glutathione metabolism, regulation of leukotriene activity (Nakagawa, H., et al. Cytogenet Cell Genet 1992 59:258-260) and neutrophil recruitment (Choudhury, S. R., et al. Cell 178:1205-1221 2019 e1217), as well as EGFR were the two proteins with highest spectral counts in gradient-purified DiFi sEVs (
Next, the clinical relevance of DPEP1 as a potential CRC biomarker was examined. Bioinformatic analysis of the U133Plus2 and TCGA Databases showed that DPEP1 is highly upregulated in CRC compared to normal colonic tissue (
DPEP1 has also been shown to be a receptor for neutrophil recruitment in various tissues. (Wang M. Nat Rev Nephrol. 2022 18(4): 199; Lau A, et al. Sci Adv. 2022 8 (5): eabm0142; Choudhury S R, et al. Cell. 2019 178(5): 1205-21 e17). DPEP1 on EVs could act as a decoy or a neutrophil recruitment factor in the tumor environment. Therefore, targeting DPEP1's ability to bind neutrophils as well as its enzymatic function could alter immune surveillance of tumors
Furthermore, sEVs derived from human cancer cell lines, DKO-1 and LS-174T (colon), MDA-MB-231 and its derivative LM2-4175 (breast), PANC-1 (pancreas), Gli36vIII (glioblastoma), Calu-3 (lung), as well as human normal renal epithelial cells (HREC), all had high levels of CD73 (
The next goal was to examine proteins that are highly enriched in exomeres and the NV fraction. The most abundant proteins detected in DiFi-derived exomeres and the NV fraction were β-actin and fatty acid synthase (FASN) (
In summary, DPEP1 and CD73 were identified in classical exosomes, as well as FASN in exomeres, to be potential CRC biomarkers and druggable targets. These results highlight the benefits of parsing distinct extracellular compartments to identify new biomolecules of clinical interest and to assign cargo to their correct carrier.
Heterogeneity of extracellular vesicles and nanoparticle populations is a major challenge in the EV field (Jeppesen, D. K., et al. Cell 2019 177:428-445 e418; Zhang, H., et al. Nat Cell Biol 2018 20:332-343; Zhang, Q., et al. Cell Rep 2019 27:940-954 e946; Mathieu, M., et al. Nat Cell Biol 2019 21:9-17; van Niel, G., et al. Nat Rev Mol Cell Biol 2018 19:213-228). This Example reports the isolation and characterization of a new extracellular nanoparticle that is termed the supermere. Supermeres are distinct from exomeres in terms of size, morphology, composition, cellular uptake dynamics, and tissue distribution. They contain many proteins previously reported to be associated with exosomes (van Niel, G., et al. Nat Rev Mol Cell Biol 2018 19:213-228). For example, TGFBI, the most abundant protein in supermeres, is purportedly a component of EVs from mesenchymal stromal cells (Ruiz, M., et al. Biomaterials 2020 226:119544). Based on the inverse correlation between high immunoreactivity for TGFBI in CRC and both overall and progression-free survival, as well as increased levels in supermeres isolated from the plasma of CRC patients compared to normal individuals, it is proposed that TGFBI levels may be a useful marker in liquid biopsies for CRC patients. Argonaute proteins, including AGO1 and AGO2, were presumed exosomal proteins, but refinements in purification demonstrate that these miRNA-binding proteins are predominantly non-vesicular (Jeppesen, D. K., et al. Cell 2019 177:428-445 e418; Temoche-Diaz, M. M., et al. Elife 2019 8), and it was shown that AGO1 and AGO2 are highly associated with supermeres. Other known RNA-binding proteins were also highly enriched in supermeres, highlighting the emerging appreciation that a significant proportion of extracellular RNAs (exRNAs) and RNA-binding proteins are not associated with EVs (Jeppesen, D. K., et al. Cell 2019 177:428-445 e418; Tosar, J. P., et al. Trends Biochem Sci 2021). Many miRNAs barely detectable or undetectable at the cellular level are highly and selectively enriched in supermeres. For example, miR-1246, which has been linked to serum exosomes in CRC patients (Cooks, T., et al. Nat Commun 2018 9:771), is the most highly expressed and highly enriched miRNA in supermeres. The strong staining of miR-1246 in CRC tissue compared to normal colonic mucosa supports a potential role for miR-1246 in the pathogenesis of CRC. Supermeres and exomeres are not the only non-vesicular extracellular nanoparticles capable of transporting miRNA. In particular, plasma and serum contain large amounts of 7-14 nm HDL particles, known to contain miRNA (Li, K., et al. Methods Mol Biol 2018 1740:139-153; Michell, D. L., et al. J Vis Exp 2016). All the cell line-derived supermere samples generated for this work were from serum-free conditions, and ApoA1 or ApoA2 (the most abundant proteins of HDL complexes) could not be detected in any of the samples by proteomic analysis. However, efficient purification from HDL-rich blood may benefit from additional approaches, perhaps utilizing a combination of high-resolution density gradient fractionation (Jeppesen, D. K., et al. Cell 2019 177:428-445 e418) and FPLC or size-exclusion chromatography (Li, K., et al. Methods Mol Biol 2018 1740:139-153; Michell, D. L., et al. J Vis Exp 2016), for improved separation of sEVs, exomeres, supermeres and HDL particles.
As disclosed herein, supermeres and exomeres isolated from cetuximab-resistant SC and CC-CR cells can transfer cetuximab resistance to cetuximab-sensitive cells. Activation of the receptor tyrosine kinases MET and RON induce de novo cetuximab resistance in SC cells (Li, C., et al. Proc Natl Acad Sci USA 2017 114:E2852-E2861). In CC-CR cells, upregulation of a long non-coding RNA (lncRNA), MIR100HG, and two embedded miRNAs, miR-100 and miR-125b, is responsible for this acquired mode of cetuximab resistance (Lu, Y., et al. Nat Med 2017 23:1331-1341). Thus, multiple cargos, including proteins and RNA (mRNA, miRNA, and lncRNA) carried by nanoparticles may contribute to these modes of drug resistance. The identity of these cargos, and whether they act independently or cooperatively in cetuximab resistance, await further investigation.
The Warburg effect, a cancer cell preference for glycolysis in the presence of oxygen, features enhanced lactate secretion that contributes to acidification of the tumor microenvironment and extracellular matrix degradation (Brooks, G. A. Cell Metab 2018 27:757-785). Increased lactate secretion also has been linked to resistance to drugs targeting EGFR and MET (Apicella, M., et al. Cell Metab 2018 28:848-865 e846). Cancer cell-derived supermeres contain large amounts of glycolytic enzymes and their addition to recipient cells results in increased lactate secretion. Furthermore, mice treated with supermeres resulted in reduced levels of lipids and glycogen in the liver. There was a supermere-selective striking decrease in liver to body weight ratio, a remarkable finding as the liver to body weight ratio is usually highly conserved. Nevertheless, there were common pathways downregulated and upregulated in the liver by both supermeres and exomeres. Notably, there was common downregulation of the mechanistic target of rapamycin complex 1 (mTORC1) pathway, a major nutrient-sensitive regulator of growth (Umemura, A., et al. Cell Metab 2014 20:133-144). The observe liver phenotype is similar to that reported with hepatic mTORC1 inhibition in which there was decreased hepatic steatosis and an increased inflammatory response (Umemura, A., et al. Cell Metab 2014 20:133-144).
Shedding or release of membrane receptors to the extracellular environment is associated with a number of disease states (Lichtenthaler, S. F., et al. EMBO J 2018 37) and drug resistance (Miller, M. A., et al. Clin Cancer Res 2017 23:623-629). Secretion of full-length transmembrane receptors is a distinctive feature of sEVs/exosomes but the ectodomain of many clinically relevant transmembrane receptors, including MET, GPC1, CEA, ACE, ACE2 and APP, are highly abundant in supermeres. As an important example, the secreted receptor ACE2 in sEVs and/or extracellular nanoparticles may act as a decoy for SARS-CoV-2 to attenuate infection, as has been demonstrated for human soluble recombinant ACE2 (Zhang, Q., et al. Gastroenterology 2021 160:958-961 e953; Monteil, V., et al. Cell 2020 181:905-913 e907). GPI attached to the C-terminus of a protein enables it to be anchored to the membrane of cells or EVs, and many GPI-anchored proteins of clinical importance, including GPC1, CEA, DPEP1 and CD73, have been detected in the extracellular space and ascribed to exosomes. However, as one example, upon closer inspection, GPC1 is less associated with exosomes, or other sEVs, but rather is enriched in exomeres and supermeres. The apparent molecular weight of a detached GPI-anchored protein will not differ noticeably from a membrane-embedded GPI-anchored protein, hence some of these proteins have been presumed to be associated with exosomes or other EVs. Yet other GPI-anchored proteins, e.g., DPEP1 and CD73, are strongly associated with EGFR/CD81-positive exosomes and do not appear to be liberated from their GPI anchor to any significant degree. DPEP1 was recently identified as an adhesion receptor on liver and lung endothelial cells for neutrophil recruitment, and targeting DPEP1 reduced mortality in murine models of sepsis (Choudhury, S. R., et al. Cell 178:1205-1221 2019 e1217). As disclosed herein, increased diffuse DPEP1 staining is associated with overall and progression-free survival in CRC and that there are significantly increased levels of DPEP1/CEA-positive exosomes in the plasma of CRC patients. High levels of CD73 have been linked to immune suppression and tumor progression due to the generation of extracellular adenosine (Antonioli, L., et al. Trends Cancer 2016 2:95-109). CD73 is upregulated in many cancers, including CRC (Hammami, A., et al. Semin Immunol 2019 42:101304; Yu, M., et al. Nat Commun 2020 11:515); there was increased CD73 in CRC tumor tissue as well as demonstrate that CD73-positive exosomes can be detected in CRC plasma.
Based on these findings, it is proposed that TGFBI, ENO1 and GPC1 may be useful markers for extracellular nanoparticles (exomeres and supermeres), while HSPA13 and ENO2 are more specifically associated with supermeres. Full-length CD73 may be a useful general marker for sEVs. Supermeres contain a number of clinically relevant biomolecules, such as PCSK9, ACE, ACE2, MET, GPC1 and APP, and supermeres are efficiently taken up in many organs compared to sEVs and exomeres, including the heart and brain. Some fruitful areas of future investigation will be determining the biogeneis of supermeres and exomeres and exploring the physiological and pathophysiological consequences of these nanoparticles and their cargo.
In summary, supermeres are a new type of circulating extracellular nanoparticle. Supermeres are enriched in proteins central to a number of disease states, including cancer, COVID-19, cardiovascular disease and Alzheimer's disease. Many of these proteins have previously ascribed to exosomes or other EVs. These findings serve to highlight the importance of parsing the exact extracellular compartment that contains a biomolecule of interest. Supermeres are also functional agents of intercellular communication that are efficiently taken up by multiple organs, including liver, lung, colon, heart and brain. Supermeres thus takes their place alongside exosomes, EVs and exomeres as attractive targets for liquid biopsies and potential therapeutic drug targets.
Cell lines. LS174T, PANC-1, Calu-3, and Hela cell lines were from the American Type Culture Collection (ATCC), Human primary renal proximal tubule epithelial cells (HREC) are from Innovative BioTherapies. HCA-7, its derivatives SC, CC and CC-CR, DiFi, and LIM1215 were maintained in the Coffey lab. DKO-1 cells were obtained from Dr. T. Sasazuki at Kyushu University, Gli36 cells were obtained from Dr. X. Breakefield at Harvard Medical School, and MDA-MB-231 and LM2-4175 cells were obtained from Dr. J. Massagué at Memorial Sloan-Kettering Cancer Center.
Cell culture. Human CRC cell lines, DiFi, DKO-1, HCA-7-derived SC (Li, C., et al. Proc Natl Acad Sci USA 2017 114:E2852-E2861), CC, CC-CR, LS174T, LIM1215, human breast cell lines MDA-MB-231 and LM2-4175, pancreatic cancer cell line PANC1, and lung cancer cell line Calu-3, human glioblastoma cell line Gli36vIII, and Hela cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% bovine growth serum, 1% glutamine, 1% non-essential amino acids, and 1% penicillin/streptomycin at 37° C. in a 5% CO2 humidified incubator. Cells were maintained by passage every 3-4 days at 70%-80% confluence and were routinely tested for mycoplasma contamination (Universal Mycoplasma Detection Kit, ATCC, Manassas, VA, USA). All cell culture media was purchased from Corning Cellgro (Manassas, VA), and all cell culture supplements were from Hyclone (Logan, UT) unless stated otherwise. Primary cultures of human renal proximal tubule epithelial cells (HREC) were generated from transplant discards purchased from Innovative BioTherapies (Ann Arbor, MI, USA). Primary cultures for production of extracellular vesicles were initiated at passage 2 as previously described (Jeppesen, D. K., et al. Cell 2019 177:428-445 e418), and cells were maintained in DMEM supplemented with 2 μg/ml Normocin, Insulin-Transferrin-Selenium (ITS), epidermal growth factor (EGF), hydrocortisone and T3 thyroid hormone. For 3D cultures, cells were cultured in type-1 collagen as previously described (Li, C., et al. Proc Natl Acad Sci USA 2017 114:E2852-E2861). Briefly, type-1 collagen was diluted at 2 mg/ml in DMEM containing 10% (vol/vol) FBS. Assays were set up using three collagen layers, 400 μl each, in 12-well culture dishes, with the middle layer containing the single-cell suspension at 5,000 cells/mL. Medium (400 μl) with or without reagents was added on top and changed every 2-3 days. Colonies were observed and counted after 14-17 days.
Extracellular vesicle and nanoparticle isolation from cultured cells grown in dishes. Extracellular nanoparticles were isolated from cell-conditioned medium as previously described (Zhang, Q., et al. Cell Rep 2019 27:940-954 e946), with minor modifications. Colon, breast, lung and pancreatic cells mentioned above were cultured in specific medium as described above until 80% confluent. The cells were then washed three times with PBS and cultured for 48 h in serum-free medium. For primary human kidney epithelial cells, cell-conditioned medium was collected from approximately 95% confluent cells grown for 96 h in cell culture flasks with DMEM without FBS. The serum-free conditioned medium was removed and centrifuged for 15 min at 1000×g to remove cellular debris, and the resulting supernatant was then filtered through a 0.22-μm polyethersulfone filter (Nalgene, Rochester, NY) to reduce microparticle contamination. The filtrate was concentrated with a 100,000 molecular-weight cutoff centrifugal concentrator (Millipore). The concentrate then was subjected to high-speed centrifugation at 167,000×g for 4 h in a SW 32 Ti Rotor Swinging Bucket rotor (k factor of 204, Beckman Coulter, Fullerton, CA), and the resulting sEV-enriched pellet was resuspended in PBS containing 25 mM HEPES (pH 7.2) and washed by centrifuging again at 167,000×g for 4 h. The washed pellet was designated as the sEV-P. To isolate exomeres, the supernatant collected from the 4 h ultracentrifugation was ultracentrifuged at 167,000×g for 16 h. The resulting pellet was resuspended in PBS containing 25 mM HEPES (pH 7.2) and washed by centrifuging again at 167,000×g for 16 h. The washed pellet was designated as exomeres. To isolate supermeres, the supernatant from the pelleting of exomeres was subjected to ultracentrifugation at 367,000×g (rmax, 55,000 RPM) using a Beckman Coulter SW55 Ti rotor (k factor of 48, Beckman Coulter, Fullerton, CA) for 16 h. The resulting pellet was resuspended in PBS containing 25 mM HEPES (pH 7.2) and was designated supermeres. The protein concentrations of the nanoparticles were determined with Direct Detect™ (Millipore, Burlington, MA). At no time during the process were samples subjected to temperatures below 4° C.
Extracellular vesicle and nanoparticle isolation from cultured cells grown in bioreactors. DKO-1 cells were maintained in CELLine Adhere 1000 (CLAD1000) bioreactors (INTEGRA Biosciences AG, Zizers, Switzerland) at 37° C. in a 5% CO2 humidified incubator, as previously described (Jeppesen, D. K., et al. Cell 2019 177:428-445 e418). Cell-conditioned medium was harvested from bioreactors every 48 h, starting from one week after inoculation of the bioreactor and continuing for a period of 4 weeks. Pellets of sEVs were generated as previously described (Jeppesen, D. K., et al. Cell 2019 177:428-445 e418). Exomeres and supermeres were isolated as described above. At no time during the process were samples subjected to temperatures below 4° C.
Extracellular vesicle and nanoparticle isolation from human plasma samples. All procedures on human peripheral blood specimens were approved and performed in accordance with the Vanderbilt University Medical Center Institutional Review Board (IRB #161529 and #151721). All human participants provided informed consent (clinical trial registration number: NCT03263429). Blood was drawn into BD Vacutainer Blood Collection Tubes (BD Bioscience) containing buffered sodium citrate as anticoagulants. The first tube drawn was discarded. Further processing of samples was initiated within 2 h of blood draw. Plasma was generated by centrifugation of the blood at 1,500×g for 15 min and then a second round of centrifugation of the supernatant at 3,000×g for 15 min to ensure that no platelets remained. The resulting plasma samples were diluted immediately approximately 1:20 in ice cold PBS and spun at 20,000×g for 30 min to pellet and remove large EVs and microparticles. Clarified supernatants were subjected to ultracentrifugation at 167,000×g for 4 h in a SW 32 Ti Swinging Bucket rotor (k factor of 204, Beckman Coulter, Fullerton, CA) to sediment the sEV pellet (sEV-P). Pellets of crude sEVs were resuspended in ice-cold PBS, tubes were filled with PBS-H (25 mM HEPES), and then subjected to ultracentrifugation at 167,000×g for 4 h. The washed pellet was resuspended in ice-cold PBS-H. Pellets of exomeres and supermeres were generated as described above. The protein concentrations of the nanoparticles were determined with Direct Detect™ (Millipore, Burlington, MA). At no time during the process were plasma or plasma sEVs subjected to temperatures below 4° C.
High-resolution (12-36%) iodixanol density gradient fractionation. The gradient fractionation was performed as previously described (Jeppesen, D. K., et al. Cell 2019 177:428-445 e418). Twelve individual fractions of 1 ml were collected from the top of the gradient. Fractions 4 and 5, and fractions 8 and 9 were separately pooled. These two pools were then diluted 12-fold in PBS and subjected to ultracentrifugation at 120,000×g for 4 h at 4° C. using a SW41 TI Swinging Bucket rotor. The resulting pellets were lysed in cell lysis buffer for further proteomic and immunoblotting analysis.
Atomic Force Microscopy (AFM) imaging and analysis. Twenty microliters of isolated sEVs, the non-vesicular fraction (NV), exomeres and supermeres were diluted 1:1 with PBS and then incubated over (3-Aminopropyl) triethoxysilane (AP)-modified mica substrates (Ted Pella Inc, CA) for 3 min. To remove unbound particles, substrates were washed twice with 50 μL PBS and imaged in PBS at room temperature. Measurements were conducted in PBS using a Dimension FastScan Microscope (Bruker Instruments, Santa Barbara, CA) in off-resonance tapping mode, with ScanAsyst Fluid+tips (Bruker, CA) with nominal radius ˜2 nm and experimentally determined spring constants of 0.7 N/m. AFM images were taken at 256 samples per line, at 0.75 Hz. Images were exported offline and processed using Gwyddion or custom R software.
For statistical analysis, data were expressed as mean values±standard deviations. Statistical significance was identified by the Student's t-test for the differences among different samples. P values of less than 0.01 was considered to be statistically significant.
Negative stain transmission electron microscopy. Highly purified sEV fractions, NV fractions, exomeres and supermeres were prepared for transmission electron microscopy (TEM) as previously described (Jeppesen, D. K., et al. Cell 2019 177:428-445 e418).
Proteomics. Gradient fractionated sEVs, NV, exomeres and supermeres derived from DiFi cells or from PANC-1 and MDA-MB-231 cells were lysed in RIPA buffer, and equal amounts of protein were run on a NuPAGE Bis-Tris gel. LC/MS/MS was performed as previously described (Zhang, Q., et al. Cell Rep 2019 27:940-954 e946).
Proteomic analysis. Proteins with average count ≥1 in each fraction were considered detectable. Spectral counts of proteins were normalized by the total spectral counts and were log 2-transformed. Principal component analysis was performed to assess the similarity between samples. Differential expression between sEVs, NV, exomeres and supermeres was identified using Limma. Proteins with a fold change of >2 and a false discovery rate (FDR)<0.05 were considered to be significantly differentially expressed. Gene set enrichment analysis (GSEA) was implemented against three reference gene sets from the Molecular Signatures database (MSigDB v6.1): (H) hallmark gene sets (50 gene sets); (C2) KEGG gene sets (186 gene sets), and (C5) all gene ontology (GO) gene sets (5,917 gene sets). Default parameters were used to identify significantly enriched gene sets (min size 15, max size 500, FDR <0.25).
Structured illumination microscopy (SIM). 3D SIM imaging and processing was performed on a Nikon N-SIM structured illumination platform equipped with an Andor DU-897 EMCCD camera and a SR Apo TIRF 100× (1.49 NA, WD 0.12) oil immersion objective. Samples were imaged in PBS at room temperature. For calibration, 100 nm fluorescent (360/430 nm, 505/515 nm, 560/580 nm and 660/680 nm) beads (TetraSpeck™ Microspheres, Thermo Fisher Scientific, Waltham, MA, USA) were fixed and imaged. Images were analyzed using ImageJ software (National Institutes of Health, Bethesda, MD, USA).
Immunofluorescence staining for SIM. DiFi cells were cultured on 35-mm culture dishes with a 1.5 coverslip and 14-mm glass diameter (P35G-0.170-14-C, MatTek Corporation, Ashland, MA, USA) to approximately 50% confluence. Cells were fixed with 4% paraformaldehyde in PBS at room temperature for 20 min and then extracted for 5 min with 1% Triton X-100 in 4% paraformaldehyde in PBS as previously described (Jeppesen, D. K., et al. Cell 2019 177:428-445 e418). Cells were washed three times in PBS and blocked in 10% bovine serum albumin (BSA) in PBS. Cells were incubated with primary antibodies diluted in 10% BSA overnight at 4° C., washed with PBS for three times. The secondary Alexa Fluor antibodies (anti-rabbit conjugated to Alexa Fluor 488, and anti-mouse conjugated to Alexa Fluor 568) were prepared in blocking buffer and centrifuged at 13,000 rpm for 10 min before incubation on cells for 1 h at room temperature. Primary antibodies used were anti-DPEP1 (Sigma, HPA012783, 1:50), anti-CD63 (BD, 556019, 1:50).
Immunofluorescence staining for confocal microscopy. DiFi cells (2×105) were cultured on 6-well plates for 2 days. Cells were then washed with PBS three times and fixed with 4% paraformaldehyde (PFA) for 10 min at room temperature and permeabilized with 0.5% Triton X-100 for 5 min at room temperature. Fixed cells were blocked for 2 h in 5% BSA at 4° C. and subsequently incubated overnight at 4° C. with primary antibodies in 5% BSA in PBS (anti-DPEP1, Sigma, HPA012783, 1:100; anti-CD63, BD, 556019, 1:100; anti-Na/KATPase a, Cell Signaling Technology, 3010, 1:500) The cells were washed three times in PBS and then incubated overnight with secondary antibodies in 5% BSA in PBS (anti-rabbit conjugated to Alexa Fluor 488, Cy3, anti-rabbit conjugated to Alexa Fluor 647). Immunofluorescence was analysed using a Zeiss LSM 710 confocal microscope. Microscopy was performed within the Vanderbilt Cell Imaging Shared Resource (CISR). All micrographs were taken using a 63× oil immersion objective lens.
Protein isolation from cells and all isolated fractions. Proteins were isolated as previously described (Zhang, Q., et al. Cell Rep 2019 27:940-954 e946).
Immunoblot analysis. Lysed samples were prepared in 5× sample buffer, heated to 70° C. for 10 min, or boiled for 5 min before being loaded on gels. The samples (30 μg) were separated on 4-12% SDS-PAGE Bis-Tris gels (Life Technologies) under either reducing or non-reducing conditions, depending on the subsequent use of primary antibody, before being transferred to nitrocellulose membranes (GE Healthcare, Pittsburgh, PA). Membranes were blocked for 1 h in 5% non-fat dry milk, or 5% bovine serum albumin, depending on the primary antibody subsequently used. Membranes was incubated with primary antibodies overnight at 4° C. After incubation with HRP-coupled secondary antibodies for 1 h, immunoblots were developed using chemiluminescence (Western Lightning Plus-ECL, PerkinElmer, Waltham, MA). The primary antibodies used were the following: anti-EEF1A1 (ab157455), anti-A33 (ab108938), anti-EPCAM (ab32392), anti-AGO2 (ab186733), anti-Syntenin-1 (ab133267), anti-ACE2 (ab108252), anti-APP (ab32136), anti-GPC1 (ab199343), anti-CEACAM5/CEA (ab133633), anti-TPI1 (ab96696), anti-LDHB (ab85319), anti-GPI (ab66340), anti-HSPA8 (ab51052), anti-PCSK9 (ab181142), anti-VPS35 (ab157220) and anti-MVP (ab175239) are from Abcam. Anti-MET (8198), anti-CEACAM5/CEA (2383), anti-CD73 (13160), anti-FASN (3180), anti-ACLY (4332), anti-AGO1 (5053), anti-XPO5 (12565), anti-HNRNPA2B1 (9304), anti-Alix (2171), anti-ALDOA (8060), anti-ENO1 (3810), anti-ENO2 (8171), anti-HK1 (2024), anti-PKM1/2 (3190), anti-LDHA (3582), anti-pAKT (9271), anti-AKT (9272), anti-pERK1/2 (9101), anti-ERK1/2 (9102) and anti-HSP90 (C45G5) are from Cell Signaling Technology. Anti-HSPA13 (sc-398297), anti-ACE (sc-271860), anti-FASN (sc-48357), and anti-CD9 (SC-13118) are from Santa Cruz. Anti-APP (Millipore, MAB348), anti-EGFR (Millipore, 06-847), anti-GPC-1 (Invitrogen, PA5-28055), anti-MET (R&D, AF276), anti-CD81 (R &D Systems, MAB4615), anti-AREG (6R1C2.4, Bristol-Myers Squibb Research Institute), anti-TGFBI (Proteintech, 10188-1-AP), anti-β-Actin (Sigma, A5316), anti-DPEP1 (sigma, HPA012783), anti-FLOT1 (BD, 610820), anti-β1-Integrin (BD, 610467) and anti-CD63 (BD, 556019).
ELISA for TGFBI. TGFBI concentrations in the sEV-P, exomeres, and supermeres derived from human cancer cell lines and human platelet-poor plasma were determined by ELISA kit (R&D Systems, DY29350) according to the manufacturer's instructions. Briefly, 96-well microplates were coated overnight in the dark at room temperature with 100 μl/well of capture antibody (Ab) at 4.0 g/ml in neat DPBS (Life Technologies, 14190-144). Coated plates were washed 3 times with PBS supplemented with 0.05% tween-20 and then blocked with 1% BSA in PBS (Research Products International, A30075) for 1 h at room temperature in the dark. Plates were then washed 3 times and samples diluted appropriately in blocking buffer were captured for 2 h at room temperature in the dark. Plates were washed 3 times and 100 μl/well of biotinylated detection Ab (200 ng/ml in blocking buffer) was added for 2 h at room temperature in the dark. Plates were washed 3 times and 100 μl/well of streptavidin-HRP diluted 1/200 in blocking buffer was added for 20 min at room temperature in the dark. Plates were washed 3 times and 100 μl/well of substrate solution (R&D Systems, DY999) was added and allowed to develop for 20 min at room temperature in the dark and 50 μl/well of stop solution (2 N H2SO4) was added and OD 450 nm was determined immediately.
Fluorescence-activated vesicle sorting (FAVS) staining, sorting and analysis. The small EV pellet (sEV-P) derived from DiFi cells was stained and sorted as previously described (Zhang, Q., et al. Cell Rep 2019 27:940-954 e946; Higginbotham, J. N., et al. J Extracell Vesicles 2016 5:29254). Equal number of sorted sEVs were lysed for immunoblotting as previously described.
For FAVS staining and analysis of the sEV-P, exomeres and supermeres derived from DiFi cells or human plasma, 100 μg of samples were blocked and processed as described above. For samples that were incubated with directly-conjugated primary antibodies, the samples were washed three times in PBS-H and centrifuged at 304,000×g with a S100-AT4 fixed angle rotor (75,000 rpm, effective k factor of 29) for 30 min unless stated otherwise. For samples that were stained with unconjugated primary antibodies, after incubation for overnight at 4° C., the samples were washed twice, then incubated with secondary antibody for 1 h at room temperature and then washed three times in PBS-H for single color analysis. For dual-color stained samples with one directly conjugated and one un-conjugated primary antibody, samples were stained with unconjugated primary antibody first, and then washed as described above except that after incubation with the secondary antibody, the samples were washed only twice and then the samples were stained with the directly conjugated primary antibody for the second color and washed three times in PBS-H as described above. The samples are then ready to be analyzed. Nanoparticles incubated with secondary antibody only were used as negative controls. Primary antibodies used as directly conjugated antibodies were: anti-DPEP1 (LSBio, LS-A109972), anti-FASN (Santa Cruz, SC-48357), anti-c-MET (R&D, FAB3582R), anti-CD81 (R&D, FAB4615P), and anti-EGFR (CTX) (chimeric mouse/human, Lilly, AF-647-conjugated). Un-conjugated primary antibodies used were: anti-TGFBI (Proteintech, 10188-1-AP), anti-GPC1 (Abcam, ab199343), anti-CEACAM5/CEA (Abcam, ab133633), anti-AGO2 (Abcam, ab186733), and anti-APP (Millipore, MAB348). Secondary antibodies used were: Goat anti-rabbit (H+L) (Invitrogen A32733), donkey anti-goat (H+L) (Invitrogen, A32814), and goat anti-mouse (H+L) (Invitrogen, A865).
RNA purification from cells, sEV-P, exomeres and supermeres. RNA was purified using the miRNeasy Mini Kit (QIAGEN, 217004) according to the manufacturer's protocol with elution in a volume of 30 μl. For extracellular nanoparticle RNA isolation, QIAzol Lysis Reagent was incubated with concentrated samples for an extended 15 min incubation prior to chloroform extraction. The concentration and integrity of the RNA were estimated using the Quant-It RiboGreen RNA Assay Kit (Thermo Fisher Scientific) and High Sensitivity RNA kit on the 5300 Fragment analyzer (Agilent Technologies), respectively.
Small RNA library preparation and sequencing. All RNA sequencing was performed at Hudson Alpha (Huntsville, Al, USA). The concentration and integrity of the RNA were estimated using the Quant-It RiboGreen RNA Assay Kit (Thermo Fisher Scientific) and High Sensitivity RNA kit on the 5300 Fragment analyzer (Agilent Technologies), respectively. Total RNA from each sample was taken into a small RNA library preparation protocol using the Automated NEXTflex® Small RNA-Seq Kit v3 (Bioo Scientific, PerkinElmer) for Illumina® Libraries on PerkinElmer® Scilone® G3 NGS workstation according to manufacturer's protocol. Briefly, a 3′ 4N adenylated adapters mix was ligated to total input RNA followed by removal of excess adapters using adapter inactivation buffers. Post-ligation purification was done two times using the NEXTflex Cleanup beads, and the purified material was eluted in 10 μl of Nuclease Free water. 5′ 4N adapters then were ligated to the RNA samples. Reverse transcription (RT) was done using M-MuLV reverse transcriptase for 30 min at 42° C. and then for 10 min at 90° C. After the RT step the samples were cooled at 4° C. in the thermal cycler. Post-RT samples were spun down at 2,000 rpm for 2 min and then stored in −20° C. freezer overnight. The following day, post-RT material was purified using NEXTflex Cleanup beads, with elution in 22.5 μl Nuclease Free water into NEXTflex barcoded PCR Primer Mix. PCR setup was done using NEXTflex Small RNA PCR Master Mix, and the amplification was performed at 95° C. for 2 min followed by 20 cycles of 95° C. for 20 s, 60° C. for 30 s and 72° C. for 15 s; the final elongation was done at 72° C. for 2 min. Post-PCR dual size gel free size selection was done on the Scilone G3 using NEXTflex Cleanup Beads with final elution made in 15 μl NEXtflex Resuspension buffer. From the post-PCR purified final libraries, 2 μl of each library was taken for quality analysis, a 2× dilution plate was made and the final library concentration and profile were assessed using Quant-iT Picogreen dsDNA Assay Kit (Thermo Fisher Scientific) and High Sensitivity (HS) DNA Assay on the Caliper LabChip Gx (PerkinElmer Inc.), respectively. qPCR was performed on final libraries using KAPA Biosystems Library Quantification kit (Kapa Biosystems, Inc.) to determine the exact nano molar concentration. Each library was diluted to a final concentration of 1.5 nM and pooled in equimolar ratios. Single End (SE) sequencing (50 bp) was performed on an Illumina NovaSeq 6000 sequencer (Illumina, Inc).
Small RNA-seq analysis. Cutadapt was used to trim adapters. TIGER, was used to perform small RNA-seq analysis, including reads mapping, miRNA quantification and differential analysis. Specifically, Bowtie was used to map reads to the human miRNAs from miRBase v22 and the human reference genome hg19. Data were normalized by the total number of reads in each sample. Principal component analysis was performed to assess the similarity between samples. DESeq2 was used to detect differential expression between cells, the sEV-P, exomeres and supermeres. miRNAs with fold change >2 and a false discovery rate (FDR)<0.05 were considered to be significantly differentially expressed.
Quantitative RT-PCR. Analysis of miRNA levels was performed with the TaqMan small RNA assays (Cat #: 4366596, Applied Biosystems) and TaqMan Fast Advanced Master Mix (Cat #: 4444556, Applied Biosystems) according to the manufacturer's instructions, with U6 small nuclear RNA (U6 snRNA) as the internal control. Briefly, 10 ng of total RNA was used per individual RT reaction (total 15 μl per reaction); 0.5 μl of the resultant cDNA was used in 20 μL qPCR reactions. Quantitative real-time PCR was performed on the Bio-Rad CFX96 C1000 Touch Thermal cycler by using the iQ SYBR Green Supermix (Bio-Rad). Relative measurement of gene expression was calculated following manufacturer's instructions using the ΔΔCt method. U6 was used to calculate normalized fold-change. The following reagents were used: hsa-miR-1246 (catalog #4427975, assay ID: 462575_mat, thermoFisher Scientific), hsa-miR-675 (catalog #4427975, assay ID: 002005, thermoFisher Scientific), and U6 snRNA (catalog #4427975, assay ID: 001973, thermoFisher Scientific).
Fluorescence in situ hybridization (FISH) for hsa-miR-1246. Five-micrometer paraffin-embedded sections of colonic tissue and TMAs were deparaffinized and rehydrated. In situ hybridization process was performed and the TSA Plus fluorescence system was used as previously described (Shimizu, T., et al. Cell Mol Gastroenterol Hepatol 2020 9:61-78) as well as the manufacturer's protocol for the miRCURY LNA™ microRNA ISH Optimization Kit (QIAGEN). Briefly, the slides were incubated with proteinase-K (15 μg/ml) at 37° C. for 10 min and were washed three times with PBS. The slides were incubated with peroxidase block (Vector Laboratories, SP-6000) at room temperature for 10 min to block endogenous peroxidase activity. After in situ hybridization for 1 h at 55° C. with locked nucleic acid probes (0.4 nM for hsa-miR-1246, 1 nM of U6 snRNA and 40 nM of Scramble-miR probe), the slides were washed and blocked in blocking solution (2% sheep serum, 1% BSA, 0.1% Tween, PBS) at room temperture for 15 min and incubated with anti-digoxigenin-POD antibody (1:400, Roche, 11207733910) in antibody dilutant solution (1% sheep serum, 1% BSA, PBS, 0.05% Tween) at room temperature for 1 h. To detect digoxigenin, the TSA Plus Cy5 substrate (1:200, PerkinElmer, NEL745001KT) was applied to the slides and incubated at room temperature for 10 min. After washing three times in PBS, the slides were incubated with DAPI for 5 min, and slides were mounted with ProLong Gold Antifade Reagent (Invitrogen, P36934). Slides were scanned by Vanderbilt University Digital Histology Shared Resource Core. The Lan miRNA detection probes consist of hsa-miR-1246 (Qiagen, Cat #: 33911 YD00610948-BCG); U6 snRNA (Qiagen, Cat #: YD00699002) and scramble-miR probe (Qiagen, Cat #: YD00699004).
Treatment of recipient cells with sEV-P, exomeres and supermeres in 3D culture. Two thousand CC or DiFi cells were incubated with indicated concentrations of the sEV-P, exomeres or supermeres derived from CC, SC, CC-CR or DiFi cells at 37° C. for 30 min. Then the cells were grown in type-1 collagen as described above for 2 weeks. Fresh medium was added with or without cetuximab (CTX, 0.3 μg/ml) and/or indicated concentrations of extracellular nanoparticles every 3-4 days as indicated. Colonies were counted using the GelCount (Oxford Optronix) with identical acquisition and analysis settings and represented as mean from triplicates±s.e.m. The images of the colonies were taken using the EVOS Fluorescence Microscope (ThermoFisher).
Lactate release measurement. Lactate release into the medium was measured using the Glycolysis cell-based assay kit (Cayman chemical, catalog #: 600450) according to the instructions. Two thousand CC cells were grown in type-1 collagen in 12-well plate and treated with or without indicated amounts of extracellular nanoparticles as described above for 14 days. The medium was collected and used for the assay.
Immunohistochemistry (IHC). Tumor xenografts were fixed in neutralized formalin and embedded in paraffin. Slices were deparaffinized with serial histoclear and ethanol. Antigen retrieval was performed in citrate buffer (pH 6.0) with high pressure at 110° C. for 15 min, then quenched in 0.03% H2O2 with sodium azide for 5 min. The slides were incubated with primary antibodies at room temperature for 60 min and then incubated in Dako Envision+System-HRP labeled Polymer at room temperature for 30 min. Signal was detected by incubating in DAB+ Substrate Chromogen System at room temperature for 5 min. Primary antibodies used were the following: anti-DPEP1 (rabbit, 1:1,000, Sigma, HPA012783); anti-CD73 (rabbit, 1:300, Cell Signaling Technology, 13160), anti-TGFBI (rabbit, 1:300, Abcam, ab170874), anti-FASN (mouse, 1:500, Santa Cruz, sc48357), and anti-AGO2 (rabbit, 1:500, Abcam, ab57113).
Labeling and uptake of sEV-P, exomeres and supermeres in vitro. The sEV-P and extracellular nanoparticles derived from DiFi cells were labeled with Alexa Fluor 647 (A20173, Invitrogen) according to the manufacturer's instructions. For monitoring the uptake of the sEV-P, exomeres and supermeres over time, MDA-MB-231 cells were seeded at 20,000 cells per well on a 35-mm dish (P35G-0.170-14-C, MatTek Corporation) in DMEM culture media overnight. Then the cells were treated with either DMSO control or the Alexa Fluor 647-labeled sEV-P, exomeres and supermeres (40 μg/ml) in serum-free DMEM media. Images were acquired using a 60× objective NA on a VisiTech iSIM with a Nikon Ti base. Fluorescence (640 Far Red, 10% laser power, 100 ms exposure time) images were taken of 3 fields of view, each with several cells. Three z-slices 1 μm apart were taken of each fluorescent field and the maximum Z-projection was analyzed. Cells were imaged every 15 minutes for 24 h. For each field of view, the average intensity of the far-red channel was measured. Each field of view for each treatment (DMSO, sEV-P, exomere and supermere) was averaged and normalized to the starting value (n=1). Images shown are of one representative cell.
For imaging cells treated with LysoTracker, MDA-MB-231 cells were seeded and treated with labeled supermeres (40 μg/ml) as described above. Twenty-four hours later after supermere treatment, the cells were washed twice with PBS and lysotracker Red DND-99 (L7528, Molecular Probes, 100 nm) was applied to the cells for 1 h. Images were acquired with iSIM.
For inhibitor treatment before supermere uptake, MDA-MB-231 cells were seeded at 20,000 cells per well or Hela cells at 25,000 cells per well on a 35-mm dish (P35G-0.170-14-C, MatTek Corporation) in DMEM culture media. Twenty-four hours later, cells were pre-incubated with the inhibitors in serum free DMEM media for 30 min. The following inhibitors were used: 100 nM bafilomycin A (Sigma, SML1661-. 1ML), 20 μM dynasore (Sigma, D7693-5 MG), 25 μM CK666 (Sigma, SML0006), 5 μM cytochalasin D (Sigma, C2618-200 ul). The Alexa Fluor 647-labeled supermeres (40 μg/ml) were added to the cells for 24 h in the presence of indicated inhibitors. Images were acquired using a 60× objective NA on a VisiTech iSIM with a Nikon Ti base. Brightfield (30 ms exposure time) and fluorescence (640 Far Red, 10% laser power, 100 ms exposure time) images were taken of 10 or more fields of view, each with several cells per field. Three z-slices 1 μm apart were taken of each fluorescent field and the brightest slice was analyzed.
Brightfield images were used to identify cell boundaries and an ROI was drawn manually around each cell in each field of view. These region of interests (ROIs) were then opened on the fluorescent image and the mean fluorescence intensity of each ROI (cell) was measured. For each field of view, a background ROI was drawn in a region with no cells, and this background value was subtracted from each cell fluorescence mean in the field of view. Images shown in the figure are representative of the average fluorescence intensity. Dark shadows in the lower right hand corner of brightfield images represent a bypass filter physically impeding the image and not any data or cell information.
Animal studies. Male C57Bl/6 mice (6-10 weeks old) were purchased from Jackson Laboratories. Mice were injected with exomeres (100 μg or 300 μg) or supermeres (100 μg or 300 μg) derived from DiFi cells diluted in 100 μl phosphate buffered saline pH 7.4 (PBS, Gibco, 70011-044) into the tail vein. The control group received vehicle (PBS) only. Mice received daily injections for 3 consecutive days and were sacrificed 24 h after the last injection. All animal studies and procedures were approved by the Animal Care and Use Committee of Vanderbilt University.
Biodistribution of extracellular samples in vivo. The sEV-P and extracellular nanoparticles derived from DiFi cells were labeled with IRDye 800 CW NHS Ester (P/N: 929-70020, LI-COR) according to the manufacturer's protocol. The labeled sEV-P was pelleted by centrifuging at 304,000×g with a S100-AT4 fixed angle rotor (75,000 rpm, effective k factor of 29) for 40 min. The labeled exomeres were pelleted by centrifugation at 167,000×g in a SW 32 Ti Rotor Swinging Bucket rotor (k factor of 204, Beckman Coulter, Fullerton, CA) for 16 h. The supermeres were pelleted by centrifugation at 367,000×g (rmax, 55,000 RPM) using a Beckman Coulter SW55 Ti rotor (k factor of 48, Beckman Coulter, Fullerton, CA) for 16 h. The samples were resuspended and washed in PBS (pH 7.4) and then pelleted again as described above. Two hundred micrograms of labeled sample in 500 μl of PBS were injected intraperitoneally into 10-week-old male C57Bl/6 mice. Twenty-two hours after injection, organs were harvested and imaged using the Odyssey imaging system (LI-COR Biosciences). All animal studies and procedures were approved by the Animal Care and Use Committee of Vanderbilt University.
Histochemistry. Liver samples were fixed in 10% formalin overnight and transferred into 70% ethanol prior to paraffin embedding. Formalin-fixed, paraffin-embedded (FFPE) sections (4 μm) were stained with Gill 2 Hematoxylin (Richard-Allan Scientific, 72504) and Eosin (Sigma-Aldrich, HT110316) (H&E). The percentage of surface area composed of large hepatocytes with increased cytoplasmic vacuolations was estimated for each slide by a liver pathologist (VQT). Fresh frozen, optimal cutting temperature (OCT) compound (Fisher Health Care, 4585)-embedded liver sections (8 μm) were stained with Oil red O (Sigma-Aldrich, 0625). Briefly, liver sections were fixed in 10% neutral buffered formalin for 10 min, washed with double-distilled water and equilibrated with 60% isopropanol. Oil red O was dissolved in isopropanol (0.5% w/v), filtered (0.22 μm) and diluted with distilled water (3:2) immediately prior to staining. Liver sections were stained for 15 min at room temperature, washed with 60% isopropanol, counterstained with Gill 2 Hematoxylin (Richard-Allan Scientific, 72504) and mounted with Vectamount (Vector Laboratories, H-5501). Stained sections were scanned using the Aperio Versa 200 (Leica Microsystems GmbH) in the Digital Histology Shared Resource at Vanderbilt University Medical Center. Positive surface area was automatically assessed with Tissue IA v2.0 integrated into the Leica Digital Image Hub slide manager platform (Leica Biosystems). ORO staining was scored independently by two liver pathologists (VQT, WJH) for lipid vesicles in a 4-tier scheme as follows: score 0 for no vesicles; score 1 for rare inconspicuous vesicles in the centrilobular vein (CV) area; score 2 for present conspicuous vesicles in the CV area; score 3 for confluent vesicles in the CV area; score 4 for confluent vesicles in the CV area, extending between separate CVs.
FFPE sections (4 μm) were stained with periodic acid Schiff (PAS) with and without diastase to highlight polysaccharides such as glycogen. Briefly, FFPE sections were dewaxed and dehydrated, oxidized for 10 min with periodic acid (Acros Organics, 19840-0050), washed in lukewarm distilled water for 5 min, stained with Schiff reagent (Acros Organics, 61117-5000) for 10 min, washed in lukewarm water for 5 min, counterstained in Gill 2 Hematoxylin (Richard-Allan Scientific, 72504) for 4 min, dehydrated, and mounted with Acrytol (Electron Microscopy Sciences, 13518). All PAS-only slides were scored double-blinded and independently by two liver pathologists (VQT, WJH) for the presence of dark magenta deposits suggestive of glycogen deposition in a 3-tier scheme as follows: score 1 for 0-33% of hepatocytes with dense deposits; score 2 for 34-66%; score 3 for 67-100%. Diastase-treated slides were treated for 20 min with α-Amylase from porcine pancreas Type VIB (0.5% in ddH2O, Sigma-Aldrich, A1376-5000KU) prior to the periodic acid staining step, to confirm that the dark magenta deposits were polymeric carbohydrates such as hepatic glycogen. Statistics were performed in R with the Wilcoxon rank sum test for two-group analyses and Kruskal Wallis one-way analysis of variance for more than two groups.
Liver triglyceride analysis. Snap frozen liver tissues (50 mg) were homogenized with ceramic beads using the PowerLyzer (Qiagen). Liver triglyceride content was quantified by the Triglyceride Assay kit (Abcam, ab65336) per the manufacturer's instructions. Samples were measured on a microplate reader at OD570 nm.
RNA isolation from liver tissue. Liver tissue samples were immediately stored in RNAlater (Ambion) until homogenization with ceramic beads using the PowerLyzer (Qiagen) and RNA was extracted using the RNeasy Kit (Qiagen) according to the manufacturer's instructions.
RNA-seq library preparation for liver-derived RNA. RNA-seq libraries were prepared using 300 ng of RNA and the NEBNext Ultra II Directional RNA Library Prep kit (NEB, Cat: E7760L). Fragmentation, cDNA synthesis, end repair/dA-tailing, adaptor ligation and PCR enrichment were performed per manufacturer's instructions. Individual libraries were assessed for quality using the Agilent 2100 Bioanalyzer and quantified with a Qubit Fluorometer. The adapter ligated material was evaluated using qPCR prior to normalization and pooling for sequencing. The libraries were sequenced using the NovaSeq 6000 with 150 bp paired-end reads. RTA (version 2.4.11; Illumina) was used for base calling and data QC was completed using MultiQC v1.7 by the Vanderbilt Technologies for Advanced Genomics (VANTAGE) core (Vanderbilt University, Nashville, TN).
RNA-seq analysis of liver-derived RNA. Adapters were trimmed by Cutadapt. After trimming, RNA-seq reads were mapped to the mouse genome mm10 using STAR, and quantified by featureCounts. DESeq2 was used to detect differential expression between supermere-treated/exomere-treated and PBS. Genes with a fold change of >1.5 and a false discovery rate (FDR)<0.1 were considered to be significantly differentially expressed. GSEA was used to perform functional enrichment analysis against Hallmark gene sets from MSigDB.
Statistical analyses were performed using the SPSS Statistical Analysis System (version 22.0; SPSS, Chicago, IL), R (The R foundation) and GraphPad Prism for Windows (version 9.0; GraphPad Software). Data are presented as mean±s.e.m. All statistical tests were two-sided, and a P value of less than 0.05 was considered statistically significant. Statistical tests are indicated in figure legends. Adjustment for multiple comparisons of significance between groups was performed by the Holm-Bonferroni procedure for ANOVA or Dunn's multiple comparison test for Kruskal-Wallis, as indicated in corresponding figure legends.
Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of skill in the art to which the disclosed invention belongs. Publications cited herein and the materials for which they are cited are specifically incorporated by reference.
Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.
This application claims benefit of U.S. Provisional Application No. 63/253,945, filed Oct. 8, 2021, which is hereby incorporated herein by reference in its entirety.
This invention was made with Government Support under Grant No. CA197570, CA179514, and CA241685 awarded by the National Institutes of Health. The Government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2022/077513 | 10/4/2022 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63253945 | Oct 2021 | US |
