Supporting Breast Cancer Research with Microgravity

Introduction

According to the Global Cancer Observatory, there were 2.3 million new cases of breast cancer and over half a million deaths worldwide in 2020 (Sung, 2021). 

Litegrav is proud to have collaborated with the University of Pavia to further research in breast cancer with its simulated microgravity platform.   

Drug resistance and unwanted complications make it imperative to explore new therapeutic avenues. Unusual and extreme conditions like microgravity can affect a variety of cancer cells by curbing their growth, survival, and diffusion (Nassef, 2020).

Microgravity is complementary to existing interventions. Because cells respond to mechanical forces, it influences stress-related changes. As ways to better understand cancer, extreme biology is opening inroads for further inquiries. 

Human breast cancer cells (HBCCs) behave differently in real and simulated microgravity, where they readily form 3D structures, called multicellular spheroids. These structures resemble tumors found in the body, giving researchers more realistic models to study. These can point researchers to promising new drug targets (Grimm, 2022).

Breast Cancer Cells in Space

A study by Nassef et al. examined how the weightlessness in space affects MCF-7 breast cancer cells. They focused on alterations in the structure and expression of genes related to the cytoskeleton, cell attachment, signaling molecules, and the extracellular matrix. The group used special marker proteins to emphasize parts of the cellular skeleton and captured changes with a fluorescence microscope designed for microgravity (Nassef, 2019).

They later measured changes in RNA activity and cell shape in a second experiment. Breast cancer cells displayed significant structural reconfigurations, including gaps in the tubulin network, clusters of tubulin, and cytoskeleton changes shortly after microgravity exposure. Changes in gene activity were also noted, with some displaying more activity and others less. Additionally, proteins that prevent metastasis were reduced (Nassef, 2019).

In a separate study HBCCs were sent into space in a photon capsule, fixed at different time points. The cells were then compared with controls remaining in normal gravity. After the mission, scientists implemented fluorescent markers for studying cell proliferation, cytoskeletal structure, and chromatin. 

The primary observations in real microgravity included the cytokeratin network surrounding the nucleus and chromatin structure were looser, cell division was delayed, cellular growth slowed due to interference in the cell cycle, and microtubules were altered in many cells (Vassy, 2003).

Their results align with predictions from the cellular tensegrity model, suggesting cells rely on internal tension and balance to maintain their structure. Prolonged mitosis is probably connected with changes in the microtubules. 

Two possible reasons for the change were offered: the disruptions in the cells’ organic self-organization, modeled using reaction-diffusion processes or the activation or deactivation of proteins stabilizing microtubules, also affecting other parts of the cellular structure. 

Altogether, the findings were foundational in showing how microgravity impacts cellular processes and structure (Vassy, 2003).

Enter Simulated Microgravity

When HBCCs, particularly MCF-7 and MDA-MB-231 lines, are exposed to simulated microgravity using a Random Positioning Machine (RPM), they transform from 2D flat layers into 3D spheroid clusters. This shift is accompanied by changes in proteins forming the cellular skeleton, along with components of the extracellular matrix, and focal adhesion molecules, which assists the cells in adhering to their surroundings (Sahana, 2021).

Among the major discoveries are those related to gene expression. After 24 hours on the RPM, the MDA-MB-231 cells exhibited  two genes (ITGB and LAMA3) that experienced significant reduction in both flat cells and 3D spheroids. Another gene, VCL, was reduced in 3D spheroids (Sahana, 2021).

In MCF-7 cells, genes linked to adhesion and cell structure were drastically reduced in flat cells. In 3D spheroids, other genes experience a reduction, although VCL and LAMA3 were unchanged. These analyses highlighted the centrality of fibronectin, vinculin, and E-cadherin in the formation of 3D spheroids (Sahana, 2021).

The research also involved imaging MCF-7 cells engineered to track β-catenin, a signaling protein. It was present in the nucleus in normal gravity and RPM-exposed flat cells. RPM exposure changed the activity of certain target genes in signaling pathways (Sahana, 2021).

Vinculin and β-catenin are crucial for the formation of 3D spheroids during 24 hours of simulated microgravity; these findings provide researchers with novel insights into how breast cancer cells adapt in microgravity.

A study using Gene Array Technology discovered significant increases in the activity of several 3D spheroid genes in comparison to controls in normal gravity and flat cells. Many were connected with cellular survival, stress response, and cellular death processes (Kopp, 2018).

When breast cancer cells were exposed to the RPM for 24 hours and treated with the drug dexamethasone (a NFκB activity inhibitor) there was a dose-dependent significant reduction in 3D spheroid formation. This suggests the NFκB has a substantial role in tumor spheroid formation (Kopp, 2018).

Sahana, et al. observed that in simulated microgravity, there is also a reduction in E-cadherin, and the presence of c-Src, a protein linked to cancer progression and cellular growth. The formation of 3D spheroids was halted when c-Src was inhibited but enhanced when E-cadherin activity was blocked with antibodies. 

These findings indicate that c-Src and E-cadherin are major components in the formation and growth of tumor spheroids under microgravity conditions (Sahana, 2018).

Vamvakidou et al. created a 3D tumor model using a combination of HBCCs cultivated together in a rotating wall vessel. The most significant finding was that the 3D spheroids developed a layered structure consisting of centralized dead cells with contrarily actively dividing cells on the periphery (Vamvakidou, 2007).

Models like this are invaluable for testing novel therapeutics in the lab. 

Conclusion

Breast cancer remains the leading cause of cancer-related deaths in women. 

More alarmingly, cases continue to climb. Microgravity and other extreme environments let us explore options that were once unavailable. Despite remarkable leaps forward in medicine and technology, no definite protocol is yet in sight.

Yet with ongoing effort and new tools, the emperor of all maladies may become more manageable. 

References and Works Cited:

Grimm D, Schulz H, Krüger M, Cortés-Sánchez JL, Egli M, Kraus A, Sahana J, Corydon TJ, Hemmersbach R, Wise PM, Infanger M, Wehland M. The Fight against Cancer by Microgravity: The Multicellular Spheroid as a Metastasis Model. Int J Mol Sci. 2022 Mar 12;23(6):3073. doi: 10.3390/ijms23063073. PMID: 35328492; PMCID: PMC8953941.

Kopp S, Sahana J, Islam T, Petersen AG, Bauer J, Corydon TJ, Schulz H, Saar K, Huebner N, Slumstrup L, Riwaldt S, Wehland M, Infanger M, Luetzenberg R, Grimm D. The role of NFκB in spheroid formation of human breast cancer cells cultured on the Random Positioning Machine. Sci Rep. 2018 Jan 17;8(1):921. doi: 10.1038/s41598-017-18556-8. PMID: 29343717; PMCID: PMC5772637.

Nassef MZ, Kopp S, Wehland M, Melnik D, Sahana J, Krüger M, Corydon TJ, Oltmann H, Schmitz B, Schütte A, Bauer TJ, Infanger M, Grimm D. Real Microgravity Influences the Cytoskeleton and Focal Adhesions in Human Breast Cancer Cells. Int J Mol Sci. 2019 Jun 28;20(13):3156. doi: 10.3390/ijms20133156. PMID: 31261642; PMCID: PMC6651518.

Nassef MZ, Melnik D, Kopp S, Sahana J, Infanger M, Lützenberg R, Relja B, Wehland M, Grimm D, Krüger M. Breast Cancer Cells in Microgravity: New Aspects for Cancer Research. Int J Mol Sci. 2020 Oct 5;21(19):7345. doi: 10.3390/ijms21197345. PMID: 33027908; PMCID: PMC7582256.

Sahana J, Nassef MZ, Wehland M, Kopp S, Krüger M, Corydon TJ, Infanger M, Bauer J, Grimm D. Decreased E-Cadherin in MCF7 Human Breast Cancer Cells Forming Multicellular Spheroids Exposed to Simulated Microgravity. Proteomics. 2018 Jul;18(13):e1800015. doi: 10.1002/pmic.201800015. Epub 2018 Jun 19. PMID: 29785723.

Sahana J, Corydon TJ, Wehland M, Krüger M, Kopp S, Melnik D, Kahlert S, Relja B, Infanger M, Grimm D. Alterations of Growth and Focal Adhesion Molecules in Human Breast Cancer Cells Exposed to the Random Positioning Machine. Front Cell Dev Biol. 2021 Jun 30;9:672098. doi: 10.3389/fcell.2021.672098. PMID: 34277614; PMCID: PMC8278480.

Sung H, Ferlay J, Siegel RL, Laversanne M, Soerjomataram I, Jemal A, Bray F. Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries. CA Cancer J Clin. 2021 May;71(3):209-249. doi: 10.3322/caac.21660. Epub 2021 Feb 4. PMID: 33538338.

Vamvakidou AP, Mondrinos MJ, Petushi SP, Garcia FU, Lelkes PI, Tozeren A. Heterogeneous breast tumoroids: An in vitro assay for investigating cellular heterogeneity and drug delivery. J Biomol Screen. 2007 Feb;12(1):13-20. doi: 10.1177/1087057106296482. Epub 2006 Dec 8. PMID: 17166827.

Vassy J, Portet S, Beil M, Millot G, Fauvel-Lafève F, Gasset G, Schoevaert D. Weightlessness acts on human breast cancer cell line MCF-7. Adv Space Res. 2003;32(8):1595-603. doi: 10.1016/S0273-1177(03)90400-5. PMID: 15002416.

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