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Taking Biomarker Discovery From 2D to 3D: Increased Biological Activity of EVs Isolated From 3D Prostate Cancer Cultures

Fri, 09/24/2021 - 10:03

3D rendering of extracellular vesicles released from cells as a banner image.

Jamshed Arslan, Pharm D, PhD

Tissues within the human body are made of a three-dimensional (3D) arrangement of cells working together to perform vital functions. The commonly used 2D monolayer cultures have limited expandability and cell-cell communication, making them less relevant to human physiology. By contrast, in 3D cultures, such as spheroids or organoids, cells can easily permeate through the scaffold allowing the cells to stretch out and have better organization and cellular communication. Likewise, 3D engineered microtissues can truly mimic the tumor microenvironment (TME). Recently, a team in Switzerland reported that 3D microtissues offer clinically relevant phenotypes and allows the identification of biomarkers that the 2D cultures could not detect. They discovered that the quality and quantity of extracellular vesicles (EVs) from 3D microtissues are superior to the traditional 2D cultures. EVs are lipid bound vesicles (exosomes, microvesicles, or apoptotic bodies) that are actively secreted from cells into the extracellular space and are categorized based on their size, biogenesis, and function. The researchers found that the protein and nucleic acid cargo of EVs from 3D cultures yields more information and is of greater utility than their 2D counterparts.

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3D Culture Conditions Enrich Molecular Cargo of Prostate Cells EVs

To identify cargo of EVs associated with metastasis, the team produced 3D microtissues of benign and malignant prostate cells using an inert hydrogel, a 3D network of hydrophilic polymer. Simple Western analysis showed that 3D cultures of malignant cells enriched prostate-specific membrane antigen (PSMA), CD44, and vimentin, indicating an increase in relapse risk, proliferation, and aggressiveness, respectively. Transmission electron microscopy, nanoparticle tracking analysis, Simple Western, and size exclusion chromatography together indicated that 3D cultures enhanced EV production without significantly affecting or altering EV markers or morphology. Analyzing RNAseq data revealed that these EVs had higher expression of cancer-specific genes such as matrix metalloproteinases (MMPs) and the tumorigenesis markers mucin 1 (MUC1) and early growth response protein-1 (EGR1). Quantitative proteomic evaluation by LC-MS/MS delineated that 3D culture conditions also enrich the proteins related to vesicle transport.

After highlighting the differential effect of 3D culture on molecular cargo, the researchers further explored the biological significance of such changes.

Simple Western showing exosomes isolated from A549 cells and HT-29 cells, and whole cell lysate of COLO 205 cells, probed with Rabbit Anti-Flotillin-1 Polyclonal Antibody highlighting Flotillin-1 as an exosome marker.


Flotillin-1 is a canonical marker of the exosome subset of EVs. Simple Western lane view of Flotillin-1 expression in exosomes isolated from A549 cells (NBP3-11645), HT-29 cells (NBP3-11685), and whole cell lysate of COLO 205 cells. Samples were probed with Rabbit Anti-Flotilin-1 Polyclonal Antibody (NBP1-79022) and a single band was detected ~50 kDa. Lysates were run under reducing conditions using the 12-230 kDa system.




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Functional Relevance of Extracellular Vesicles From 3D Microtissues

The proteomic data showed enhanced levels of GDF15 in the 3D-EVs from prostate cancer cells. Imaging flow cytometry and western blotting verified GDF15, a protein linked to prostate cancer progression, to be a bona fide EV protein in clinically relevant 3D microtissues. To observe what effects 3D-EVs impart on 2D cultured cells in terms of cancerous properties, the team exposed naïve prostate cancer PC3 cells to various conditions. Experiments using matrigel-coated membrane indicated that only the 3D-EVs from cancer cells significantly promoted invasiveness. Similarly, naïve prostate cancer 2D cells exhibited chemoresistance to the chemotherapy drug doxorubicin only when incubated with prostate cancer 3D-EVs, but not the 2D-EVs.

In other words, 3D-EVs have physiologically relevant RNA and protein cargo that the 2D-EVs do not possess. Furthermore, the malignant 3D microtissues can impart their cancerous features and functionality to naïve, 2D cultured cells through isolated EVs.

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The Tissue Clearing Pro-Organoid Kit used on liver spheroids to stain for the visualization of multiple structures including DNA, MRPII, and MDRI (upper left); DNA, CD68, Albumin, and Vimentin (upper right); DNA, panCK, and CD31 (lower left); and DNA, panCK. and Cytochrome P450 3A4 (lower right).


The Tissue Clearing Pro-Organoid Kit (7390-NOV) permits for tissue clearing and staining of 3D cultures, organoids, and microtissues. This kit was applied to open liver spheroids for visualization of stained structures: Upper left- DNA, MRPII, and MDRI; Upper right- DNA, CD68, Albumin, and Vimentin; Lower Left- DNA, panCK, and CD31; Lower Right- DNA, panCK. and Cytochrome P450 3A4.




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This article presents 3D microtissues as a biologically relevant culture system to enhance our understanding of cancer-related changes in EV cargo. Using Simple Western analysis, the team identified three biomarkers present only in the EVs from 3D prostate cancer cells. The research team’s use of imaging flow cytometry to verify GDF15 as an EV biomarker is an unprecedented approach. Future studies on EVs isolated from plasma of prostate cancer patients can further validate the clinical importance of these findings.

Millan, C. et al. (2021) used Mouse Anti-GAPDH Monoclonal Antibody (NB300-221), Mouse Anti-PMSA Monoclonal Antibody (NBP2-02045), Sheep Anti-CD44 Polyclonal Antibody (AF3660), Goat Anti-Vimentin Polyclonal Antibody (AF2105), Rabbit Anti-Alix Polyclonal Antibody (NBP1-90201), Rabbit Anti-TSG101 Polyclonal Antibody (NBP1-80659), Rabbit Anti-Calnexin Polyclonal Antibody (NB100-1965), Rabbit Anti-Ago2 Monoclonal Antibody (NBP2-67121), Goat Anti-GDF-15 Polyclonal Antibody (AF957), Rabbit Anti-TOP1 Monoclonal Antibody (NBP2-67606), and Mouse Anti-FASN Monoclonal Antibody (MAB5927).

Jamshed ArslanJamshed Arslan, Pharm D, PhD   
Dr Arslan is an Assistant Professor at Salim Habib University (formerly, Barrett Hodgson University), Pakistan. His interest lies in neuropharmacology and preparing future pharmacists.


Research in Focus

Millan, C., Prause, L., Vallmajo-Martin, Q., Hensky, N., & Eberli, D. (2021). Extracellular Vesicles from 3D Engineered Microtissues Harbor Disease-Related Cargo Absent in EVs from 2D CulturesAdvanced healthcare materials, e2002067. Advance online publication.

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