The still young and developing space age, characterized by lunar and Martian exploration and the vision of extraterrestrial settlements, presents a unique environment to study the impact of microgravity (µg) on human physiology and disease development. Cancer research is currently a key focus of international space science, as µg fundamentally impacts cellular processes like differentiation, adhesion, migration, proliferation, survival, cell death, or growth of cancer cells as well as the cytoskeleton and the extracellular matrix (ECM). By creating three-dimensional (3D) tumor models in a µg-environment, like multicellular spheroids (MCS), researchers can expedite drug discovery and development, reducing the need for animal testing. This concise review analyses the latest knowledge on the influence of µg on cancer cells and MCS formation. We will focus on cells from brain tumors, lung, breast, thyroid, prostate, gastrointestinal, and skin cancer exposed to real (r-) and simulated (s-) µg-conditions.

Cancer is defined as a group of diseases characterized by abnormal cell growth, expansion, and progression with metastasis. Various signaling pathways are involved in its development. Malignant tumors exhibit a high morbidity and mortality. Cancer research increased our knowledge about some of the underlying mechanisms, but to this day, our understanding of this disease is unclear. High throughput omics technology and bioinformatics were successful in detecting some of the unknown cancer mechanisms. However, novel groundbreaking research and ideas are necessary. A stay in orbit causes biochemical and molecular biological changes in human cancer cells which are first, and above all, due to microgravity (µg). The µg-environment provides conditions that are not reachable on Earth, which allow researchers to focus on signaling pathways controlling cell growth and metastasis. Cancer research in space already demonstrated how cancer cell-exposure to µg influenced several biological processes being involved in cancer. This novel approach has the potential to fight cancer and to develop future cancer strategies. Space research has been shown to impact biological processes in cancer cells like proliferation, apoptosis, cell survival, adhesion, migration, the cytoskeleton, the extracellular matrix, focal adhesion, and growth factors, among others. This concise review focuses on publications related to genetic, transcriptional, epigenetic, proteomic, and metabolomic studies on tumor cells exposed to real space conditions or to simulated µg using simulation devices. We discuss all omics studies investigating different tumor cell types from the brain and hematological system, sarcomas, as well as thyroid, prostate, breast, gynecologic, gastrointestinal, and lung cancers, in order to gain new and innovative ideas for understanding the basic biology of cancer.

Extracellular vesicle (EV) surface proteins, expressed by primary tumours, are important biomarkers for early cancer diagnosis. However, the detection of these EV proteins is complicated by their low abundance and interference from non-EV components in clinical samples. Herein, we present a MEmbrane-Specific Separation and two-step Cascade AmpLificatioN (MESS2CAN) strategy for direct detection of EV surface proteins within 4 h. MESS2CAN utilises novel lipid probes (long chains linked by PEG2K with biotin at one end, and DSPE at the other end) and streptavidin-coated magnetic beads, permitting a 49.6% EV recovery rate within 1 h. A dual amplification strategy with a primer exchange reaction (PER) cascaded by the Cas12a system then allows sensitive detection of the target protein at 10 EV particles per microliter. Using 4 cell lines and 90 clinical test samples, we demonstrate MESS2CAN for analysing HER2, EpCAM and EGFR expression on EVs derived from cells and patient plasma. MESS2CAN reports the desired specificity and sensitivity of EGFR (AUC = 0.98) and of HER2 (AUC = 1) for discriminating between HER2-positive breast cancer, triple-negative breast cancer and healthy donors. MESS2CAN is a pioneering method for highly sensitive in vitro EV diagnostics, applicable to clinical samples with trace amounts of EVs.

Microgravity changes the gene expression pattern in various cell types. This study focuses on the breast cancer cell lines MCF-7 (less invasive) and MDA-MB-231 (triple-negative, highly invasive). The cells were cultured for 14 days under simulated microgravity (s-µg) conditions using a random positioning machine (RPM). We investigated cytoskeletal and extracellular matrix (ECM) factors as well as focal adhesion (FA) and the transmembrane proteins involved in different cellular signaling pathways (MAPK, PAM and VEGF). The mRNA expressions of 24 genes of interest (TUBB, ACTB, COL1A1, COL4A5, LAMA3, ITGB1, CD44, VEGF, FLK1, EGFR, SRC, FAK1, RAF1, AKT1, ERK1, MAPK14, MAP2K1, MTOR, RICTOR, VCL, PXN, CDKN1, CTNNA1 and CTNNB1) were determined by quantitative real-time PCR (qPCR) and studied using STRING interaction analysis. Histochemical staining was carried out to investigate the morphology of the adherent cells (ADs) and the multicellular spheroids (MCSs) after RPM exposure. To better understand this experimental model in the context of breast cancer patients, a weighted gene co-expression network analysis (WGCNA) was conducted to obtain the expression profiles of 35 breast cell lines from the HMS LINCS Database. The qPCR-verified genes were searched in the mammalian phenotype database and the human genome-wide association studies (GWAS) Catalog. The results demonstrated the positive association between the real metastatic microtumor environment and MCSs with respect to the extracellular matrix, cytoskeleton, morphology, different cellular signaling pathway key proteins and several other components. In summary, the microgravity-engineered three-dimensional MCS model can be utilized to study breast cancer cell behavior and to assess the therapeutic efficacies of drugs against breast cancer in the future.

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.

: A long-term stay of humans in space causes a large number of well-known health problems and changes in protists and plants. Deep space exploration will increase the time humans or rodents will spend in microgravity (µg). Moreover, they are exposed to cosmic radiation, hypodynamia, and isolation. OMICS investigations will increase our knowledge of the underlying mechanisms of µg-induced alterations in vivo and in vitro.

: We summarize the findings over the recent 3 years on µg-induced changes in the proteome of protists, plants, rodent and human cells. Considering the thematic orientation of microgravity-related publications in that time frame, we focus on medicine-associated findings such as the µg-induced antibiotic resistance of bacteria, the myocardial consequences of µg-induced calpain activation and the role of MMP13 in osteoarthritis. All these point to the fact that µg is an extreme stressor that could not be evolutionarily addressed on Earth.

: In conclusion, when interpreting µg-experiments, the direct, mostly unspecific stress response, must be distinguished from specific µg-effects. For this reason, recent studies often do not consider single protein findings but place them in the context of protein-protein interactions. This enables an estimation of functional relationships, especially if these are supported by epigenetic and transcriptional data (multi-omics).

In this review article, we discuss the current state of knowledge in cancer research under real and simulated microgravity conditions and point out further research directions in this field. Outer space is an extremely hostile environment for human life, with radiation, microgravity, and vacuum posing significant hazards. Although the risk for cancer in astronauts is not clear, microgravity plays a thought-provoking role in the carcinogenesis of normal and cancer cells, causing such effects as multicellular spheroid formation, cytoskeleton rearrangement, alteration of gene expression and protein synthesis, and apoptosis. Furthermore, deleterious effects of radiation on cells seem to be accentuated under microgravity. Ground-based facilities have been used to study microgravity effects in addition to laborious experiments during parabolic flights or on space stations. Some potential 'gravisensors' have already been detected, and further identification of these mechanisms of mechanosensitivity could open up ways for therapeutic influence on cancer growth and apoptosis. These novel findings may help to find new effective cancer treatments and to provide health protection for humans on future long-term spaceflights and exploration of outer space.