Extracellular Vesicles from 3D Engineered Microtissues Harbor Disease‐Related Cargo Absent in EVs from 2D Cultures

Abstract

Engineered microtissues that recapitulate key properties of the tumor microenvironment can induce clinically relevant cancer phenotypes in vitro. However, their effect on molecular cargo of secreted extracellular vesicles (EVs) has not yet been investigated. Here, the impact of hydrogel-based 3D engineered microtissues on EVs secreted by benign and malignant prostate cells is assessed. Compared to 2D cultures, yield of EVs per cell is significantly increased for cancer cells cultured in 3D. Whole transcriptome sequencing and proteomics of 2D-EV and 3D-EV samples reveal stark contrasts in molecular cargo. For one cell type in particular, LNCaP, enrichment is observed exclusively in 3D-EVs of GDF15, FASN, and TOP1, known drivers of prostate cancer progression. Using imaging flow cytometry in a novel approach to validate a putative EV biomarker, colocalization in single EVs of GDF15 with CD9, a universal EV marker, is demonstrated. Finally, in functional assays it is observed that only 3D-EVs, unlike 2D-EVs, confer increased invasiveness and chemoresistance to cells in 2D. Collectively, this study highlights the value of engineered 3D microtissue cultures for the study of bona fide EV cargoes and their potential to identify biomarkers that are not detectable in EVs secreted by cells cultured in standard 2D conditions.

Sustained released of bioactive mesenchymal stromal cell‐derived extracellular vesicles from 3D‐printed gelatin methacrylate hydrogels

“…27,28 Hydrogel formulations using a variety of materials have been proposed for EV-controlled release in various applications. 19,24,29,30 We chose to investigate GelMA in these studies due to its widespread use as a 3D printable bioink based on its biocompatibility, biodegradability, and amenability to a wide array of chemical modifications. The GelMA synthesized for this work had comparable viscoelasticity to prior reports, 31 and the decreased pore size associated with increasing LAP content (Figure 2) is also consistent with a previous report showing an inverse relationship between photoinitiator (Irgacure 2959) concentration and GelMA hydrogel porosity.…”

A quick pipeline for the isolation of 3D cell culture‐derived extracellular vesicles

“…Culture conditions in 2D vs 3D (or bioreactor) impact EV production, and both the yield of EVs and their cargo have been shown to be altered (Millan et al., 2021 ; Palviainen et al., 2019 ; Rocha et al., 2018 ; Thippabhotla et al., 2019 ; Villasante et al., 2021 , Xie et al., 2017 ; Yang et al., 2020 ; Zhang et al., 2017 ). Critically in comparison to the EVs from monolayer cultures, 3D culture ‐derived EVs have been shown to be more akin to the EVs obtained from patients (Thippabhotla et al., 2019 ; Villasante et al., 2016 ).…”

Investigating the Consistency of Extracellular Vesicle Production from Breast Cancer Subtypes Using CELLine Adherent Bioreactors

“…To further assess the concept of 3D growth in the bioreactor altering EV protein cargo [70], we performed a comparison to a published proteomics dataset of EVs, isolated using simple differential centrifugation, from MDA-MB-231 and MCF7 grown as monolayers [71]. Quantitative comparison was not feasible due to differences in proteomics methodology so a simple comparison of their 100 most abundant EV proteins in each cell line/condition with those identified herein was performed.…”