Behavior of glioblastoma brain tumor stem cells following a suborbital rocket flight: reaching the "edge" of outer space

The study investigates how brief exposure to suborbital rocket flight—encompassing hypergravity, microgravity, and increased ionizing radiation—affects the behavior of glioblastoma (GBM) brain tumor-initiating cells (BTICs). Space travel exposes biology to unique stressors, and prior work on the ISS and in simulated microgravity has shown gravity can alter proliferation, migration, and apoptosis of cancer cells, sometimes pushing cells toward pro- or anti-tumorigenic states. GBM is the most common malignant primary brain tumor with poor outcomes; BTICs are a stem-like, invasive, treatment-resistant subpopulation that drives recurrence. While simulated microgravity has been reported to reduce malignant GBM behaviors, the effects of actual rocket flight on patient-derived GBM cells are largely unknown. This study tests the hypothesis that suborbital rocket-flight exposure can induce lasting phenotypic changes in BTICs that influence aggressiveness in vitro and in vivo.

Prior research has demonstrated that altered gravity can modulate cancer cell biology. Studies aboard the ISS and parabolic rocket missions (e.g., TEXUS) showed changes in cytoskeletal dynamics, focal adhesions, proliferation, migration, and apoptosis in thyroid, breast, and ovarian cancer cells under microgravity and hypergravity. Simulated microgravity (e.g., rotating wall vessels, clinostats) has been reported to reduce malignant behavior in GBM models by increasing apoptosis and sensitizing cells to therapy. However, replicating the full environment of spaceflight on Earth is difficult, and there is limited knowledge about how actual suborbital flight exposure impacts patient-derived GBM BTICs. This study addresses that gap by evaluating phenotypic and tumorigenic outcomes after rocket-flight exposure.

Study material: Patient-derived GBM brain tumor–initiating cells (BTICs), line QNS108 (male patient). Cell culture: Cells grown on laminin-coated adherent flasks under normoxic conditions (37 °C, 5% CO2, 20% O2) in DMEM/F12 supplemented with 1% Anti-anti, 2% Gem21 NeuroPlex, FGF, and EGF; media changes every 2–3 days. Preparation and transport: BTICs passaged into two 7 mL Origen cell culture bags to grow as neurospheres. One bag designated ground control (GC), the other rocket flight (RF). Both kept at 37 °C in a portable incubator and transported by road over 48 hours from Jacksonville, FL, to Spaceport America (Las Cruces, NM). Payload integration: RF bag placed into a NASA BRIC (Biological Research In Canister) aluminum cylinder serving as payload; this BRIC had no gas control or temperature regulation. The BRIC was integrated into the SARGE II suborbital reusable launch vehicle (EXOS Aerospace). Flight profile and telemetry: Parabolic suborbital flight with total mission duration ~16 minutes from launch to landing; max altitude 25.74 km (84,448 ft), max velocity 598.60 m/s, max acceleration 13.32 m/s². Approximate hypergravity exposure of ~39 seconds at ~1.5 g during ascent; microgravity/minimum g recorded with acceleration −11.06 m/s² at ~176.38 s. Descent via parachute with guided landing and rapid recovery. Ionizing radiation exposure was not measured. Post-flight handling: Biologicals recovered at landing and returned to the portable incubator; RF and GC samples returned to the laboratory within ~24 hours post-flight. Both groups were expanded on adherent flasks for two weeks prior to assays and in vivo studies. In vitro assays: (1) Transwell migration assay using 6.5 mm inserts with 8.0 µm pores; 4×10^4 cells seeded in the top chamber with a 2% FBS gradient; 12-hour incubation at 37 °C/5% CO2; cells fixed with 4% PFA, non-migrated cells removed, migrated cells permeabilized (0.1% Triton/PBS), DAPI-stained; 9 random fields per membrane imaged on Zeiss LSM800; quantification with ImageJ; four technical replicates per run; experiments performed in triplicate. (2) Limiting dilution assay for stem cell frequency: cells plated at 900, 450, 225, 112, 56, 28, 14, 7 cells/well (12 wells per condition) in ultra-low attachment 96-well plates with 200 µL media; centrifuge 300×g 5 min; incubate 14 days with 25 µL media added every 5 days; spheroids counted and analyzed with ELDA webtool; experiments in triplicate. (3) Proliferation assays: Alamar Blue—3×10^3 cells/well (10 wells/condition) in 96-well plates; 10% Alamar Blue in media; absorbance at 570/600 nm measured at 4, 12, 24, 48, 72 h; blanks included. CellCyte X confluency—8×10^3 cells/well (12 wells/condition), two fields/well imaged every 6 h for 5 days at 37 °C/5% CO2/95% humidity; confluency quantified at endpoint. In vivo studies: RF and GC BTICs reseeded and grown two weeks post-flight, then orthotopically implanted into 6-week-old male nude mice (n=5 per group). Dose: 0.3×10^6 cells in right striatum (from bregma: lateral 1.34 mm, anterior 1.5 mm, depth 3.5 mm) via Hamilton syringe. Animals monitored and sacrificed at endpoint criteria. Brains fixed in 4% PFA, paraffin-embedded, stained with H&E and anti-human nuclei (HuNu) for human cell detection. Tumor-associated cyst measurement: All observable cystic lesions on intact brain sections were measured for total cyst area with ImageJ; torn sections excluded. Statistics: Cyst areas compared with Wilcoxon rank-sum test; survival analyzed by Kaplan–Meier and log-rank test; p<0.05 considered significant. Software: GraphPad Prism 8.0.0 and R Studio v4.2.2. Data/code availability: Source data in supplement; analysis code at https://github.com/cesarga0011/npj_Brain-Tumor-Stem-Cells-Following-a-Suborbital-Rocket-Flight.

• Flight environment: RF cells experienced a suborbital parabolic flight reaching max altitude 25.74 km, max velocity 598.60 m/s, and max acceleration 13.32 m/s² over ~16 minutes; ~39 s hypergravity (~1.5 g) during ascent; microgravity/minimum g recorded as −11.06 m/s² at ~176 s. - In vitro phenotypes: Compared with ground controls, RF-exposed QNS108 BTICs showed significantly higher migration (Transwell assay: p<0.0001) and higher stemness (limiting dilution assay: p<0.01; 1/(stem cell frequency) confidence intervals 27.2–60.8 [GC] vs 10.5–22.6 [RF]). No significant differences in proliferation were detected by Alamar Blue or CellCyte X confluency assays. - In vivo outcomes: Orthotopic implantation (n=5/group) demonstrated larger tumor-associated cystic areas in RF group (median 6.57 mm²; IQR 4.23–8.98) vs GC (median 2.04 mm²; IQR 1.53–3.20); p=0.00029. Survival was decreased in the RF group (median 97 days) compared to GC (median 130 days); log-rank p=0.0172.

The study shows that brief exposure to the stresses of suborbital rocket flight can induce lasting, more aggressive phenotypes in patient-derived GBM BTICs, manifested as increased migration and stemness in vitro and larger cystic lesions with reduced survival in vivo. Prior spaceflight studies indicate that cells sense altered gravity via cytoskeletal and focal adhesion dynamics, potentially transducing biomechanical cues into biochemical signals that reprogram behavior. In this work, RF cells likely underwent cytoskeletal and signaling changes during cycles of hypergravity and microgravity. Additional unregulated environmental factors—such as hypoxia and temperature fluctuations—likely occurred because the BRIC payload lacked gas and temperature control, and the launch area had no shelter. Hypoxia is known to enrich the glioma stem cell niche via HIF1/2 pathways, which could contribute to the sustained increase in stemness and invasion observed post-flight. The results suggest selection pressures and/or reprogramming during flight may expand more aggressive clonal populations that persist for weeks. Notably, these findings contrast with some simulated microgravity studies that reported reduced GBM aggressiveness, highlighting differences between real flight conditions (including hypergravity cycles and unmeasured radiation) and ground-based simulations. The persistence of phenotypic changes echoes observations from the NASA Twins Study, where some molecular alterations lasted months after spaceflight. The authors note potential sex-specific effects not addressed here (male-derived cells, male mice) and propose future work to track phenotypic and genomic evolution of male and female GBM lines in true microgravity (ISS) or repeat suborbital missions. Overall, expanding space research can inform both astronaut health and terrestrial oncology.

Brief suborbital rocket-flight exposure of patient-derived GBM BTICs led to sustained increases in migratory capacity and stemness in vitro, and to larger cystic tumor growth and reduced survival in vivo, indicating a shift toward a more aggressive phenotype. These results demonstrate that real spaceflight environments—even of short duration—can durably influence tumor cell behavior. Future research should identify the molecular and genetic drivers of these changes, quantify and control environmental variables (e.g., radiation, oxygen, temperature), assess reproducibility across multiple GBM lines and both sexes, and leverage controlled space platforms (e.g., ISS) or repeat flights to dissect the contributions of hypergravity, microgravity, and radiation. Such work will advance understanding of human biology in space and may yield insights for treating aggressive cancers on Earth.

• Environmental variables during flight were not fully controlled or measured: the BRIC lacked gas (oxygen/CO2) control and temperature regulation; ionizing radiation exposure was not recorded. - Cross-country transport and launch conditions may have introduced hypoxia and temperature fluctuations for both GC and RF groups. - Single cell line (QNS108) derived from a male patient; in vivo studies used male mice only, limiting generalizability and precluding assessment of sex differences. - Small animal cohort (n=5 per group) limits statistical power. - Mechanistic drivers (genetic, epigenetic, cytoskeletal, signaling) underlying observed phenotypic changes were not delineated. - Real flight conditions combine hypergravity, microgravity, and other stressors, making it difficult to attribute effects to a single factor or to directly compare with simulated microgravity studies.