Prolonged Exposure to Simulated Microgravity Changes Release of Small Extracellular Vesicle in Breast Cancer Cells

Abstract

Breast cancer is the leading cause of cancer incidence worldwide and among the five leading causes of cancer mortality. Despite major improvements in early detection and new treatment approaches, the need for better outcomes and quality of life for patients is still high. Extracellular vesicles play an important role in tumor biology, as they are able to transfer information between cells of different origins and locations. Their potential value as biomarkers or for targeted tumor therapy is apparent. In this study, we analyzed the supernatants of MCF-7 breast cancer cells, which were harvested following 5 or 10 days of simulated microgravity on a Random Positioning Machine (RPM). The primary results showed a substantial increase in released vesicles following incubation under simulated microgravity at both time points. The distribution of subpopulations regarding their surface protein expression is also altered; the minimal changes between the time points hint at an early adaption. This is the first step in gaining further insight into the mechanisms of tumor progression, metastasis, the education of the tumor microenvironments, and preparation of the metastatic niche. Additionally, this may lighten up the processes of the rapid cellular adaptions in the organisms of space travelers during spaceflights.

Keywords: breast cancer; cell-cell communication; exosomes; extracellular vesicles; microgravity; tetraspanins.

Figure 1

The spectrum of RPMs in our laboratory ranges from a small desktop RPM that remains in an incubator during operation (right) to a large RPM with an integrated incubator (left and center). The iRPM was designed and constructed by Jörg Seckler and Simon L. Wuest, Institute for Automation Engineering, University of Applied Sciences and Arts Northwestern Switzerland (FHNW), Brugg-Windisch, Aargau, Switzerland. The desktop RPM was purchased from Airbus Defense and Space (ADS), Leiden, The Netherlands.

Figure 2

Particle count via Interferometry of particles >50 nm. Values were measured in triplicates of all three samples per experimental condition and timeline. Displayed are counts per capture spot.

Figure 3

Particle size distribution by the interferometric analysis of all sample sets. Measurements were taken in triplicates from three samples each; the results were normalized with the IgG control, and the size range spans from 50–200 nm.

Figure 4

Particle number by fluorescence analysis via counterstain of captured small EVs with the tetraspanins CD81, CD63, and CD9. Measurements were taken in triplicates from three samples each; the results were normalized with the isotype control; the size includes particles below 50 nm. (a) Total number of particles from all capture spots. (b) Number of particles captured on the CD81 spot. (c) Number of particles captured on the CD63 spot. (d) Number of particles captured on the CD9 spot. ** is defined as p ≤ 0.01, *** as p ≤ 0.001.

Figure 5

Visual representation of the colocalization analysis of the CD81 capture spot. All possible combinations are displayed: CD81, CD9/CD81, CD63/CD81 and CD9/CD63/CD81. None of the presented changes are significant.

Figure 6

Visual representation of the colocalization analysis of the CD63 capture spot. All possible combinations are displayed: CD63, CD9/CD63, CD63/CD81 and CD9/CD63/CD81. The increase of CD63-only vesicles is significantly increased after 5 d of s-μg vs. 1 g. * is defined as p ≤ 0.05.

Figure 7

Visual representation of the colocalization analysis of the CD9 capture spot. All possible combinations are displayed: CD9, CD9/CD63, CD9/CD81 and CD9/CD63/CD81. None of the presented changes are significant.

Figure 8

Fluorescence colocalization analysis via counterstain of captured small EVs with the tetraspanins CD81, CD63, and CD9. (a) Complete overview of exosome populations on the different tetraspanin spots at all experimental conditions. (b) Population detail of the three capture spots at 5 d 1 g. (c) Population detail of the three capture spots at 5 d RPM. (d) Population detail of the three capture spots at 10 d 1 g. (e) Population detail of the three capture spots at 10 d RPM.

Figure 9

Graphical Abstract displaying the increase of small EV release in MCF-7 breast cancer cells following exposure to s-μg on a Random Positioning Machine (RPM) (Created with BioRender.com, license number: JM24RW7M01).