Mónica Fernandes

Portugal

Functional Dissection of Stemness and EMT-Driven Invasion in Glioblastoma

Alexandra Matos1,2, Cátia Dias Correia1,2, Sofia Melo Calado1,3, Filipa Esteves1,4, José Bragança1,2, Marco António Campinho1,2, Mónica Teotónio Fernandes1,5

1. Algarve Biomedical Center Research Institute (ABC-RI), University of Algarve (UAlg), Faro, Portugal
2. Faculty of Medicine and Biomedical Sciences (FMCB), University of Algarve (UAlg), Faro, Portugal
3. Faculty of Sciences and Technology (FCT), University of Azores (UAc), Ponta Delgada, Portugal
4. ABC Collaborative Laboratory: Integrated Ageing and Rejuvenation Solutions (ABC CoLAB – Ageing Better), Loulé, Portugal
5. School of Health, University of Algarve (ESSUAlg), Faro, Portugal

Abstract

Background

Glioblastoma (GBM), the most aggressive primary brain tumor, offers limited therapeutic options and carries a dismal prognosis. GBM comprises a heterogeneous cellular landscape enriched in Glioblastoma Stem Cells (GSCs), which display self-renewal capacity, therapy resistance, and drive tumor recurrence. Targeting GSCs is therefore essential to improving patient outcomes, yet the regulatory mechanisms sustaining their maintenance remain incompletely understood.
CITED2 is a transcriptional co-regulator involved in self-renewal, quiescence, proliferation, and fate specification in embryonic and adult stem cells. Although CITED2 has been linked to tumor initiation, progression, and therapeutic resistance in several cancers, its contribution to GBM biology has not been previously characterized.

Methods

To elucidate the role of CITED2 in GBM, we used complementary 2D and 3D in vitro models. CITED2 expression was assessed across GBM cell lines and normal astrocytes. A GBM cell line with low endogenous CITED2 was engineered to overexpress CITED2 via lentiviral transduction. Functional assays evaluated the impact of CITED2 on self-renewal, proliferation, migration, and viability. GSC-enriched spheroids were generated under serum-free, anchorage-independent conditions, and bulk RNA sequencing was conducted to identify CITED2-driven transcriptomic alterations. CITED2-dependent effects on invasion were further examined using a human cerebral organoid-based in vitro model.

Results

CITED2 expression was consistently higher in GBM cell lines than in normal astrocytes. CITED2 overexpression enhanced stem-like properties, including clonogenicity and spheroid compactness. Transcriptomic profiling identified CITED2 as a driver of epithelial-to-mesenchymal transition (EMT) programs and therapy-resistance pathways. Consistent with the EMT signature, CITED2-overexpressing cells displayed markedly increased invasive behavior in a 3D in vitro model consisting of fused GSC spheroids and cerebral organoids.

Conclusions

In conclusion, CITED2 promotes GSC self-renewal and drives EMT-associated invasive behavior in GBM. These findings identify CITED2 as a previously unrecognized regulator of GSC biology and highlight its potential as a therapeutic target to impede GSC-mediated tumor progression and resistance to therapies.