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. 2025 Oct 28;44(10):116287.
doi: 10.1016/j.celrep.2025.116287. Epub 2025 Sep 17.

A comprehensive analysis of supermere, exomere, and extracellular vesicle isolation and cargo in colorectal cancer

Oleg S Tutanov  1 , Clark Massick  1 , Marisol Ramirez  2 , James N Higginbotham  1 , Lizandra Jimenez  3 , Mark Castleberry  1 , Zheng Cao  1 , Eliana John  1 , Maxwell S Hamilton  4 , Qin Zhang  1 , Dennis K Jeppesen  1 , Danielle L Michell  1 , Sarah E Glass  3 , Purvi Patel  5 , Kristie L Rose  6 , Evan Krystofiak  3 , Hua-Chang Chen  2 , Quanhu Sheng  2 , Qi Liu  2 , James G Patton  7 , Alissa M Weaver  8 , Jeffrey L Franklin  9 , Kasey C Vickers  10 , Robert J Coffey  11
Affiliations

A comprehensive analysis of supermere, exomere, and extracellular vesicle isolation and cargo in colorectal cancer

Oleg S Tutanov et al. Cell Rep. .

Abstract

Biofluids contain a heterogeneous mixture of extracellular vesicles and non-vesicular nanoparticles (including exomeres and supermeres) that transport a diverse array of proteins, RNA, and lipids. Our previous efforts to characterize the contents of these carriers in colorectal cancer relied on 2D culture systems requiring large-scale setups and time-consuming ultracentrifugation-based isolation. To streamline this process, we have combined 3D hollow-fiber bioreactor production and fast-protein liquid chromatography-based size-exclusion chromatography. Here, we compare the impact of culture methods and purification strategies on small extracellular vesicle, exomere, and supermere cargo. Proteomic analyses show consistently distinct profiles for extracellular vesicles, exomeres, and supermeres regardless of culture conditions or isolation method. In contrast, these two variables influence small RNAs, their base modifications, and lipidomic profiles. We present an online tool to query these and future secretome datasets (https://superomics.shinyapps.io/browse).

Keywords: CP: Cancer; CP: Genomics; EV; FPLC; NVEP; RNA-seq; SEC; exomeres; exosomes; extracellular RNA; extracellular vesicles; hollow fiber bioreactor; lipidomics; non-vesicular extracellular nanoparticles; proteomics; sEV; secreted RNAs; supermeres.

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Conflict of interest statement

Declaration of interests The authors declare no competing interests.

Figures

Figure 1.
Figure 1.. Characterization of EV and NVEP samples
(A–F) Characterization of FPLC-SEC purification of exomeres and supermeres. (A) Protein profile of FPLC-SEC fractionated UC-purified exomeres (green) and supermeres (blue). (B–F) Protein (B), lipid (C), rhodamine-PE (D), RNA (E), and TGFBi (F) profiles of FPLC-SEC fractionated DiFi cell concentrated media. (G and I) EV marker validation by immunoblot of EV and NVEP samples from wild-type DiFi cells (G) and DiFi cells expressing TGFBi-NG-His (I). (H) Electron micrographs of EV and NVEP samples. Fields are at a direct magnification of 30,000×, dotted boxes depict additional 2× magnification. Scale bar, 100 nm. (J) Nanoparticle tracking analysis of C-DGUC-purified media from DiFi cells expressing TGFBi-NG-His. (E and J) Data are represented as mean ± SEM. See also Figures S1 and S10 and Table S1.
Figure 2.
Figure 2.. Proteomic analysis of NVEPs and EVs
(A) Heatmap of the top 20 most abundant proteins from 3D-derived EV and NVEP samples; log2-transformed median normalized spectral counts. (B) PCA of the proteins identified in EV and NVEPs samples. Samples EVp 2D/3D, exomere 2D/3D, and supermere 2D/3D were purified with UC from 2D and 3D, respectively; sEV, hEV, and NV were purified by C-DGUC; samples marked FPLC purified by FPLC-SEC; and samples marked FPLC/His purified with a nickel column after FPLC-SEC. (C) UpSet plot of supermere samples’ proteomics results. (D–F) Venn diagrams of the top 100 most abundant proteins from EV (D), exomere (E), and supermere (F) samples. Proteins comprising the overlap between all samples for a given carrier are listed. (G) FAVS analysis of 2D UC supermeres using TGFBi and ENO1. See also Figures S2–S4 and Tables S2–S4.
Figure 3.
Figure 3.. Analysis of small RNA content of NVEPs and EVs
(A) Proportion of mapped reads in different sequencing categories. (B) Proportion of different RNA species. Shown are exomeres (Exo), supermeres (Sup), EVp and C-DGUC-purified EVs, hEVs, and NVs from 3D material. (C) PCA of miRNA composition in different samples. EV, hEV, and NV purified by C-DGUC; samples marked FPLC purified by FPLC-SEC; and all others purified by ultracentrifugation. The line separates 2D and 3D-derived samples. (D and E) Volcano plots comparing miRNA (D) and yDR (E) in 3D- versus 2D-derived UC supermeres. (F–I) Volcano plots comparing miRNA (F), yDR (G), tDR (H), and rDR (I) in FPLC- versus UC-isolated supermeres. Gray dot, non-significant; green, log2FC significant; red, log10 adjusted p value (adjp) and log2FC significant; FC, fold change, p < 0.05. See also Figures S5 and S6 and Table S5.
Figure 4.
Figure 4.. Epitranscriptomics of NVEPs and EVs
(A–L) Ribonucleotide and modified base distribution in exomeres and supermeres for 2D and 3D fractions using UC and FPLC. Distribution of G, C, A, U, I (A–E) and modified (F–L) bases. Data are represented as violin plots with mean, *p < 0.05; **p < 0.01; and ***p < 0.001. See also Figures S7 and S8 and Table S7.
Figure 5.
Figure 5.. Lipidomics of NVEPs and EVs
(A–D) Lipid concentrations (top) and proportions (bottom) for top 10 categories (A), main classes (B), sub-classes (C), and species (D). (E) PCA of the lipid species identified in EV and NVEP samples. (F–I) Volcano plots comparing lipid species (F, H) and subclasses (G, I) in 3D- versus 2D-derived (F, G) or UC- versus FPLC-SEC-isolated (H, I) supermeres. Gray dot, non-significant; green, log2FC significant; red, log10 p value and log2FC significant; FC, fold change, p < 0.05. See also Figure S9 and Table S6.
Figure 6.
Figure 6.. Superomics web portal
(A) Web portal encompassing multiomics results generated in this study. (A–D) Examples of querying proteomics (A), sRNA-seq (B), and lipidomics (C, D) data for samples grouped by EV/NVEP type (A–C) or individual (D) samples, with faceting for isolation method (B), growth conditions (D), or both (A, C).
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