Stem cell-derived neural organoids as platforms to investigate glioblastoma invasion and migration: A systematic review

Table 4 Analysis of the invasion of glioblastoma model into neural organoids

Ref.	Co-culture method	Co-culture time (days)	Groups evaluated	Invasion analysis method	Invasion analysis results	General purpose
Van De Looverbosch et al[33]	GSCs cells were plated onto the NOs	13	1000 GSCs	Fluorescence images and computing mapping (with clearing)	For both samples, most isolated GSCs were found dispersed between 25 and 200 μm from their surface	To demonstrate the value of computing mapping based on deep learning for counting cells in spheroids, identifying differentially labeled subpopulations in spheroids, and mapping the invasion of GBM cells into NOs
			2000 GSCs
Ferreira et al[34]	GBM cells were plated onto the NOs ULA 6-well plates with shaker (65 rpm)	14	Mock group (7 and 14 days)	Confocal fluorescence images and flow cytometry	Significant reduction in tumor cell proportion on ZIKV group and all CNS tumor cell lines reached the inner regions of the NOs after two weeks	To establish a co-culture model of human NOs with cancer cells from various CNS tumors and utilize these assemblies to investigate the oncolytic effects of ZIKV
			ZIKV infection group (20000 PFU) (7 and 14 days)
Pedrosa et al[25]	GBM cells or spheroids were plated onto the NOs	41	2000 GBM cells	Confocal fluorescence images and phase-contrast images	GFP-GICs started to integrate 24 hours after co-culture began. By 15 days, GFP-GIC7 and GFP-PG88 attached to and entered the Nos. By 41 days, both had successfully invaded and multiplied inside the NOs	To evaluate 5-ALA-mediated PDT for GBM treatment, aiming to eliminate infiltrating tumor cells while preserving normal tissue using GBM-initiating cells (GIC7 and PG88) in NOs
			2 tumor spheres
Fedorova et al[35]	GBM spheroids were placed on top of NOs (inclined at 45°)	30, 60, and 90	Inclined plane	Confocal fluorescence images (with clearing)	GBM migration into NOs increases over time. Migration distance and cell number are significantly higher at 60 and 90 days, with the highest number of migrating cells at 60 days. Matrigel and Geltrex increased GBM cell migration to NOs compared to the system without ECM. Matrigel showed a higher number of cells distant from the GBM/NOs border compared to Geltrex	To propose a GLICO model to study GBM growth and migration in NOs, highlighting the impact of ECM proteins
	GBM spheroids were placed of NOs embedded in a droplet of Matrigel or Geltrex in a ULA dishes with orbital shaker (0.035 g)		Matrigel
			Geltrex
Bassot et al[36]	GSCs cells were plated onto the NOs	5	Control (miR-ctrl)	Immunofluorescence and confocal imagens	Observed invasion of the GSCs into the NOs in the miR-Ctrl condition and a stronger signal for Ki-67 in the invasive single cells distant from their primary site, indicating that miRNAs can penetrate the NOs and affect GBM invasive capacity	To identify miR-17-3p, miR-222, and miR-340 as key regulators of GBM aggressiveness. Their combined modulation inhibits tumor growth, induces cell death, and shows therapeutic potential in GBM models
			Transfected (miR-17-3p, miR-222, miR-340)
Goranci-Buzhala et al[37]	GSCs cells were plated onto the NOs in a ULA-Lumox dish	20	Nek2-KD protein expressed with DOX	Immunofluorescence and confocal imagens (with clearing)	Nek2-KD-expressing GSCs failed to enter brain organoids. Naive cells diffused and recapitulated the known characteristics of invading GSCs, such as establishing protrusion-like processes in the form of microtubes. Nek2-KD-expressing U3047MG cells exhibited impaired invasion and failed to grow in NOs	To explore how GSCs suppress ciliogenesis to maintain self-renewal and tumor progression. Restoring cilia formation induces GSC differentiation, reducing tumor infiltration. The study suggests cilium induction as a potential therapy for GBM
			Naíve (control)
Azzarelli et al[14]	GSCs cells were plated in ULA 96-well plate onto NOs 6 cm dishes	7	10000 cells	Confocal fluorescence images	On low density, the GSCs group shows some individual cells dispersed sparsely across NOs, the invasion by interconnected streams was more prominent in high-density GSCs group. The behavior of the two cell lines was comparable (genetic profile, classified as RTKI and present PDGFRα amplification)	Followed the behavior of two different cell lines in the GOC system and found that GSCs with similar genetic alterations exhibit comparable behaviors upon organoid engraftment
			50000 cells
Krieger et al[38]	GBM cells were plated onto the NOs in a GravityTRAP-ULA + centrifuged (100 g per 3 minutes)	2	Patient F2	Immunohistochemistry and confocal images (with clearing)	To similar NOs size, the fraction of NOs volume taken up by tumor cells was similar across the 4 patient-derived cell lines, with similar sizes. Tumor cells spread widely in all cases. Invasion depths exceeded 100 μm in the majority, with some cells detected at 300 μm from the surface. Cells from patients F6 and F9 were less invasive than cells from F2 and F3	To investigate GBM invasion in a human-derived model using NOs as a scaffold. This model provides a clinically relevant platform for studying GBM
			Patient F3
			Patient F6
			Patient F9
Goranci-Buzhala et al[23]	GSCs spheres were plated onto the NOs	3-10	20 days-old organoids	Fluorescence (with clearing) and time-lapse images	GSCs integrated faster into older organoids, suggesting that mature NOs create a favorable environment for GSC growth, likely driven by neuronal activity and factors like NLGN3. Additionally, differences in invasive patterns were shown between primary and recurrent GSCs	To establish three different methods to assess GSC invasion in NOs and develop imaging techniques to validate organoids as reliable models. The assays reveal GSC affinities for organoids at different stages and capture key invasion aspects
			40 days-old organoids
			60 days-old organoids
Linkous et al[24]	GSCs cells were plated in 24-well plate onto NOs with shaker	14	Control group	Fluorescence (with clearing), luciferase activity and histopathological	GSC-827 exhibited infiltrative edges with tumor cells invading normal NOs, while GSC-923 showed a diffuse invasion pattern, resembling GBM patient samples. Both cell types (827 and 923) formed a network of tumor microtubes that facilitated infiltration into NOs	To train GLICO models for high throughput drug screening
			TMZ group
			BCNU group
			Ionizing radiation (0, 5 and 10 Gy)

GSCs: Glioblastoma stem cells; NOs: Neural organoids; GBM: Glioblastoma; ULA: Ultra-low attachment; ZIKV: Zika virus; PFU: Plaque-forming units; CNS: Central nervous system; GFP-GICs: Glioblastoma cell line with green fluorescent protein expression; 5-ALA: 5-aminolevulinic acid; PDT: Photodynamic therapy; GLICO: Glioma-cerebral organoid; ECM: Extracellular matrix; Ki67: Proliferation marker; Nek2-KD: Nek-knockdown; DOX: Doxycycline; RTKI: Receptor tyrosine kinase I; PDGFRα: Platelet-derived growth factor receptor alpha; GOC: Glioblastoma-organoid co-cultures; NLGN3: Neuroligin-3; TMZ: Temozolomide; BCNU: Carmustine.