I14 - Studying the effect of long-term microgravity on glioblastoma-immune organoids on the International Space Station

Introduction: : Glioblastoma (GBM) is the most aggressive primary brain cancer with current treatments achieving an average overall survival of less than 2 years. This is due in part to the highly immunosuppressive tumor microenvironment (TME), a characteristic of many solid tumors that thwarts immunotherapy. However, the complex interactions between cancer cells and immune cells are difficult to recapitulate in culture models. Microgravity offers key advantages for cancer organoid development, including the ability to generate more robust, larger, complex, uniform, and reproducible spheroids that recapitulate cell-cell interactions more accurately than ground-based 3-D models, which are subject to gravitational forces leading to spheroid settling, disassociation, and heterogeneity. Microgravity may also serve as a method of creating accelerated disease models, with neural organoids displaying increased markers of maturation and aging. Additionally, microgravity has similar effects on the immune system as GBM. Astronauts experience elevated innate immune activity and impaired adaptive immunity. This mirrors what is observed in GBM tumors which are heavily infiltrated with innate immune cells (mostly macrophages) with reduced and impaired adaptive immune cells (T cells). The GBM TME also features abnormal compressive solid stress resulting from the growing tumor mass, which can be incorporated into our organoid model by embedding it in a confining agarose gel. To determine whether microgravity could allow us to create better GBM-immune organoid models, we sent individual small-scale, static experiments contained within Space Tango's High Sample Capacity CubeLab to the International Space Station (ISS) in March 2024 aboard the SpX-30 commercial resupply mission.

Materials and

Methods: : We first developed tumor-myeloid organoids to recreate the 3D spatial interactions in the TME. Multicellular spheroids with controllable size and cellular composition were created using the hanging drop method (Fig. 1A). Human U87 GBM cells were combined with THP-1 cells, either as monocytes (undifferentiated) or as macrophages (differentiated). After 48 hours, spheroids were transferred either directly into media and grown in suspension, or else embedded in a confining 1% agarose gel. These were grown in either regular culture medium, or medium with cytokines added to polarize the macrophages into pro-inflammatory (M1-like) or anti-inflammatory (M2-like) phenotypes.

The Space Tango CubeLab system supports temperature maintenance for individual experiments contained in sealed 1-mL cryovials with no gas or media exchange for a 30–45-day mission. We therefore optimized spheroid size and media conditions to result in viable spheroids after more than 45 days in sealed vials. We observed intact spheroids with viable cells beyond two months in sealed vials. The launched experiment contained spheroids with GBM cells alone, GBM cells + macrophages, GBM cells + monocytes, and GBM + macrophages with M1-like or M2-like polarizing agents, either embedded in 1% agarose or in free suspension. These were maintained in microgravity aboard the ISS for 40 days and were processed within 24 hours of their return to Earth. We collected RNA for bulk sequencing, media for secreted proteomics, and fixed intact spheroids for Xenium spatial transcriptomics, spatial proteomics, and histology.

Results, Conclusions, and Discussions:: Over time, the organoids reside in an acidic and hypoxic environment, with live cells at the organoid edges and a necrotic central core. This recapitulates the microenvironment of GBM and other tumors, which can be acidic, hypoxic, and necrotic. Agarose-embedded spheroids grew from their original size of ~150µm in diameter to a final size of ~600µm, displacing the surrounding gel and therefore generating solid stress. Spheroids grown in microgravity appeared to be more regular in shape (Fig. 1B). Conditioned media from GBM+monocyte organoids in microgravity had increased concentrations of CXCL12, OLR1, IL13, IL17A, and IL27, indicating alterations in the GBM-immune interaction, and mirroring cytokine expression in more advanced GBM tumors (Fig. 1C). These organoids had a strong transcriptional response to microgravity, upregulating TNFα signaling and inflammatory response pathways (Fig. 1D, E). They also upregulated LIF and PTGS2, genes associated with GBM progression and immunosuppression (Fig. 1F).

We are currently optimizing a protocol to perform Xenium spatial transcriptomics analysis on a panel of 5,000 genes. We will also analyze conditioned media using mass spectrometry and are exploring spatial mass spectrometry approaches for peptides and metabolites of interest. These methods to collect large amounts of high-quality data from organoid models contribute to efforts in the field of disease modeling to make organoid models as rigorous as animal models. This is the first study to our knowledge that characterizes the effects of microgravity on GBM cells, especially in combination with macrophages. We anticipate that these microgravity immune-tumoroids will provide valuable insight into the role of macrophages in GBM biology that would not be possible to obtain in Earth’s gravity. It may also serve as a valuable drug screening platform for patient-specific organoids or macrophage-targeting therapies. Upcoming planned flights (Including DCC-1, 2025) will include more complex organoids from patient-derived glioma cells and additional immune cell types.

Acknowledgements and/or References (Optional):: This work was funded by NSF/CASIS and AFOSR and made possible by SpaceTango, SpaceX, and the ISS National Laboratory. We also thank the Notre Dame Genomics and Bioinformatics Core Facility and the Notre Dame Mass Spectrometry and Proteomics facility.