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Two-photon polymerization of miniaturised 3D scaffolds optimised for studies on glioblastoma multiforme in spaceflight-like microgravity conditions

ABSTRACT: The obtainment of innovative models recapitulating the complex tumor architecture and activity in vitro is a challenging drive in the understanding of pathology molecular bases, yet a crucial passage to the identification of targets for advanced oncotherapy. Topography tuning and gravitational unloading of cell culture can represent useful means in tumor biomimicry process: in this work, both were chosen for modelling of glioblastoma multiforme, a highly aggressive neoplasm that affects the central nervous system and that has poorly effective pharmacological and radiological countermeasures. Tumor modeling was pursued by the application of two-photon polymerization to fast prototyping of scaffolds suitable for U87-MG glioma cell culture, and by the exposure of cell-scaffold hybrids to simulated microgravity (s-µg) by rotation on random positioning machine. Glioma cell response was investigated by microscopy, quantitative reverse transcription-polymerase chain reaction and proteomic analyses, denoting synergic regulatory effects of cell scaffolding and s-µg on markers of tumor cell growth, metabolism and invasiveness.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Brain, Cell Culture

SUBMITTER: Martina Bartolucci

LAB HEAD: Andrea Petreto

PROVIDER: PXD051522 | Pride | 2025-02-27

REPOSITORIES: Pride

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			Action	DRS
	20221124_114607_Ciofani.sne	Other
	Ciofani_Genchi_1122_Flat_0g_060922_62.raw	Raw
	Ciofani_Genchi_1122_Flat_0g_130922_63.raw	Raw
	Ciofani_Genchi_1122_Flat_0g_220922_64.raw	Raw
	Ciofani_Genchi_1122_Flat_1g_060922_58.raw	Raw

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Project description:INTRODUCTION: Neuronal activity regulated by synaptic communication exerts an important role in tumorigenesis and progression in brain tumors. Genes for soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) annotated with the function ‘vesicle’ about synaptic connectivity were identified and one of these proteins, synaptosomal-associated protein 25 (SNAP25), was found to have discrepant expression levels in neuropathies. However, the specific mechanism and prognostic value of SNAP25 during glioma progression remain unclear. METHODS: Using RNA sequencing data from The Cancer Genome Atlas (TCGA) database, the differential synaptosis-related genes between LGG and GBM were identified as highly correlated. Cox proportional hazards regression analysis and survival analysis indicated that the candidate gene SNAP25 could differentiate the outcome of low- and high-risk patients, and the Chinese Glioma Genome Atlas (CGGA) cohort was used for validation of the data set. RT-qPCR, western blot, and immunohistochemistry assays were performed to examine the expression level of SNAP25 in glioma cells and samples. Functional assays were performed to identify the effects of SNAP25 knockdown and overexpression on cell viability, migration, and invasion. Then, an immunofluorescence assay of the xenograft tissue was applied to evaluate the expression of the neuronal dendron formation marker-MAP2. Liquid chromatography-high re solution mass spectrometry (LC-MS)-based metabolomics approach was presented for identifying crucial metabolic disturbances in glioma cells. In situ mouse xenograft model was used to investigate the role of SNAP25 in vivo.RESULTS: SNAP25 was down expressed in glioma tissues and cell lines and low-level SNAP25 indicated an unfavorable prognosis of glioma patients. SNAP25 inhibited cell proliferation, migration, invasion and fostered glutamate metabolism of glioma cells, exerting a tumor suppressor role. SNAP25 overexpression expressed lower expression of MAP2, indicating poor neuronal plasticity and connectivity. SNAP25 could interact with glutaminase（GLS）and GLS knockdown could rescue the anti-tumor effect of SNAP25 in glioma cells. Moreover, upregulation of SNAP25 also decreased tumor volume and prolonged the overall survival (OS) of the xenograft mouse.CONCLUSION: SNAP25 inhibited carcinogenesis of glioma via sponging glutamate metabolism by regulating GLS expression, as well as inhibiting dendritic formation, which could be considered as a molecular target for glioma diagnosis and therapy.

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Two-photon polymerization of miniaturised 3D scaffolds optimised for studies on glioblastoma multiforme in spaceflight-like microgravity conditions

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