Simulated Microgravity and Thyroid Cancer: Spheroid Formation, Drug Response, and Therapeutic Insights

Thyroid cancer is the most common malignancy of the endocrine system, comprising several subtypes—papillary, follicular, medullary, anaplastic, and Hürthle cell carcinomas—each with different biological behaviors and treatment responses. 

Many thyroid cancers can still absorb iodine, making radioactive iodine therapy an effective first line treatment. However, dedifferentiated tumors lose their iodine uptake capacity, limiting therapeutic options and necessitating alternative approaches.

Freed from the constraints of normal gravity, cancer cells spontaneously organize into three-dimensional multicellular spheroids that more faithfully recapitulate real tumors than conventional cultures. 

These experiments, initially conducted during spaceflight missions, revealed fundamental insights into how mechanical forces shape cancer cell behavior—insights now becoming accessible through ground-based simulation platforms like Litegrav’s PIROUETTE. 

Oncology in Three-Dimensions

Cancer research currently relies on two-dimensional monolayer cultures, where cells grow as flat sheets on plastic surfaces. While convenient and reproducible, this approach does not capture the three-dimensional architecture of real tumors. Cells in monolayers experience uniform nutrient and oxygen exposure, lack the cell-cell contacts found in solid tumors, and don't produce the extensive extracellular matrix of tumor microenvironments.

Scaffold-based approaches embed cells in materials like collagen or Matrigel, while liquid-overlay techniques and hanging drop methods encourage cells to aggregate through surface tension. Spinner flasks provide mechanical agitation to promote aggregation. Each method produces multicellular spheroids, spherical clusters that better approximate tumor structure, but with limitations.

Necrosis is the central challenge. As spheroids grow beyond a few hundred micrometers in diameter, their cores are starved of oxygen and nutrients, forming necrotic zones of dead cells. 

While this mimics aspects of poorly vascularized tumors, the rapid onset of necrosis limits spheroid size and complicates experimental interpretation. Distinguishing drug effects from simple nutrient deprivation becomes difficult when necrotic cores dominate the structure.

Spheroids also exhibit drug resistance patterns more similar to actual tumors than monolayer cultures do. The three-dimensional architecture creates diffusion barriers that limit drug penetration. Cells in spheroid interiors experience hypoxia, activating stress response pathways that confer resistance. 

Cell-cell contacts and extracellular matrix production trigger survival signaling absent in monolayers. These features make spheroids valuable for preclinical drug testing, but only if they can be grown large enough and maintained long enough to conduct meaningful experiments.

Early Spaceflight Experiments

The 2008 TEXUS-44 sounding rocket mission provided the first systematic look at thyroid cells in microgravity. Normal thyroid follicular cells were exposed to approximately six minutes of weightlessness during the rocket's parabolic trajectory. Even this brief exposure triggered striking cellular changes. The cells adopted unusual morphologies, their nuclear chromatin condensed into more compact configurations, and their plasma membranes underwent structural reorganization.

Most intriguingly, the cells released fragments of the thyrotropin receptor, the key protein through which thyroid-stimulating hormone regulates thyroid function, into their surrounding medium. Several proteins associated with cellular stress responses and metabolic adaptation showed heightened expression. These changes suggested that thyroid cells actively sense and respond to altered gravitational forces.

A subsequent experiment took a different approach, exposing follicular thyroid carcinoma cells to 31 repeated periods of microgravity during a parabolic flight campaign. Each parabola provided approximately 22 seconds of weightlessness, separated by periods of normal and hypergravity as the aircraft climbed and dove. This intermittent exposure pattern revealed changes in cytoskeletal organization, the internal protein scaffolding that gives cells their shape and mechanical properties.

Gene expression analysis showed that production of β-actin (ACTB) and keratin 80 (KRT80) increased significantly compared to ground controls. Both proteins play crucial roles in maintaining cell structure and regulating cell migration. β-actin forms the dynamic filaments that drive cell movement and shape changes, while KRT80 is an intermediate filament protein that provides mechanical stability. 

Their upregulation suggested that cancer cells were actively remodeling their cytoskeleton in response to altered mechanical forces—potentially relevant to metastatic behavior, where cancer cells must deform to squeeze through tissue barriers and blood vessel walls.

Sustained Microgravity Simulation on Earth

While spaceflight experiments provided proof of concept, their limited duration and accessibility constrained systematic investigation. Random Positioning Machines (RPMs)—ground-based devices that continuously reorient samples to average the gravitational vector over time—enabled longer-duration studies with better experimental control.

When thyroid cancer cells are cultured on RPMs for days to weeks, they undergo comprehensive phenotypic changes affecting cytoskeletal organization, extracellular matrix composition, cell adhesion molecule expression, proliferation rates, migration behavior, and apoptosis. Most dramatically, the cells spontaneously aggregate into large three-dimensional spheroids without requiring scaffolds, hanging drops, or special surface treatments.

These simulated microgravity spheroids grow larger before developing necrotic cores, suggesting improved nutrient distribution. Their cellular organization more closely resembles in vivo tumor architecture, with distinct outer proliferative zones and inner quiescent regions. The extracellular matrix composition and cell-cell junction proteins expressed in these spheroids more accurately reflect those found in thyroid tumors removed during surgery.

Similar spheroid formation has been observed across multiple cancer types exposed to altered gravity, suggesting a common mechanobiological response. When freed from gravity, cancer cells appear to default to a more "natural" three-dimensional organization. The altered mechanical environment may permit cell-cell interactions and signaling events that are disrupted in standard culture conditions.

Drug Responses and Therapeutic Insights

The improved physiological relevance of microgravity-grown spheroids makes them valuable for drug screening and mechanistic studies. One unexpected finding emerged from dexamethasone experiments. 

Dexamethasone, a synthetic corticosteroid commonly used to reduce inflammation and manage side effects during cancer treatment, prevented spheroid formation when thyroid cancer cells were exposed to simulated microgravity in its presence.

This observation hints at complex interactions between glucocorticoid signaling and the cellular processes driving three-dimensional organization under altered gravity. 

Dexamethasone affects numerous cellular pathways—modulating cytoskeletal dynamics, altering cell adhesion molecule expression, and influencing extracellular matrix production. Any or all of these effects might interfere with the mechanical and biochemical cues cells use to aggregate and organize in microgravity.

From a therapeutic perspective, this raises intriguing questions. If corticosteroids can disrupt the three-dimensional organization of cancer cells under specific mechanical conditions, might this reveal new intervention strategies? 

Could manipulating the mechanical environment enhance or diminish drug efficacy in ways not apparent in conventional culture? 

These questions exemplify how altered gravity experiments can uncover relationships between mechanical forces, cellular organization, and drug responses.

The spheroids also serve as models for studying metastasis. Metastatic cells must detach from primary tumors, survive in circulation, extravasate through blood vessel walls, and establish secondary tumors in distant organs. 

Each step involves dramatic changes in cell adhesion, cytoskeletal organization, and mechanical properties. Spheroids grown in altered gravity—with their more physiological cell-cell contacts and matrix organization—may better recapitulate the mechanical challenges metastatic cells face than monolayer cultures or conventionally-grown spheroids.

From Parabolic Flights to Programmable Platforms

While parabolic flights and orbital experiments validated the concept, their limited accessibility and high costs constrain research pace. Ground-based platforms are democratizing altered-gravity research by making simulated microgravity available to laboratories worldwide at a fraction of the cost and complexity of spaceflight.

Litegrav's PIROUETTE represents this new generation of research tools. As a programmable microgravity platform, researchers can explore specific gravitational regimes and their effects on tumor biology with unprecedented flexibility.  

The platform is already advancing cancer research through collaboration with the University of Pavia's Department of Molecular Medicine. 

In partnership with Litegrav, researchers have been investigating how simulated microgravity affects breast cancer cell lines, observing significant morphological changes and activation of genetic pathways linked to tumor growth and metastasis. 

Microgravity is now undoubtedly an indispensable new tool in the war on cancer. 

References

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Grimm, D. et al. Simulated microgravity alters differentiation and increases apoptosis in human follicular thyroid carcinoma cells. FASEB J. 16, 604–606 (2002).

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Groebe, K. & Mueller-Klieser, W. On the relation between size of necrosis and diameter of tumor spheroids. Int. J. Radiat. Oncol. Biol. Phys. 34, 395–401 (1996).

Kopp, S. et al. Mechanisms of three-dimensional growth of thyroid cells during long-term simulated microgravity. Sci. Rep. 5, 16691 (2015).

Melnik, D. et al. Dexamethasone inhibits spheroid formation of thyroid cancer cells exposed to simulated microgravity. Cells 9, 367 (2020).

Svejgaard, B. et al. Common effects on cancer cells exerted by a random positioning machine and a 2D clinostat. PLoS One 10, e0135157 (2015).

Ulbrich, C. et al. Differential gene regulation under altered gravity conditions in follicular thyroid cancer cells: relationship between the extracellular matrix and the cytoskeleton. Cell. Physiol. Biochem. 28, 185–198 (2011).

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