Central nervous system tumors and three-dimensional cell biology: Current and future perspectives in modeling

Table 1 Comparison between two-dimensional and three-dimensional tumor cell culture systems[14-18]

	2D tumor cell cultures	3D tumor cell cultures
Time required	Few days	Few weeks
Physiological relevance	Does not simulate in vivo tumor	Simulates in vivo tumor
Cell-cell and cell-ECM interactions	Low to no interactions	High level of interactions
Cell morphology	Flat cells expanding on 2D surface	Preserved in vivo cell shapes and growth patterns; multilayer growth
Oxygen and nutrients perfusion	Homogeneous	Heterogeneous due to the three-dimensional geometry
Response to drugs	More susceptible to drug actions	More resistant to drugs with a similar drug penetration profile to in vivo tumor counterparts
Gene expression	Many differences compared to in vivo tumor counterparts	Similar to in vivo tumor counterparts
Differentiation	Poor	Well differentiated cells
Cost	Cheap	Expensive
Technique difficulty	Low	High

2D: Two-dimensional; 3D: Three-dimensional; ECM: Extracellular matrix.

Table 2 Cancer stem cells biomarkers in brain tumors

Markers	Ref.
CD133	Gonçalves et al[12], Singh et al[13], Ogden et al[27], and Li et al[28]
A2B5	Ogden et al[27],
CD24	Gonçalves et al[12]
Aldehyde dehydrogenase (ALDH)	Gonçalves et al[12]
CD15	Li et al[28], and Son et al[29]
ABCG2	Li et al[28], Bleau et al[29], and Kondo et al[30]
Nestin	Rahman et al[32], and Pollard et al[33]
SOX2	Rahman et al[32], and Pollard et al[33]
CD44	Rahman et al[32], and Pollard et al[33]
OLIG2	Rahman et al[32], and Pollard et al[33]

Table 3 Characteristics of three-dimensional modeling technologies

Modeling technology	Methods of generation	Applications	Limitations
Spheroids[37-39]	Static suspension; Hanging drops; Spinner and rotational bioreactor; Magnetic levitation; Microfluidic system; Gel embedding (Matrigel, etc.)	Radioresistance through hypoxia and cell-cell contacts; Chemosensitivity and drug screening; Migration and invasion; Propagation and analysis of CSCs	Tumor heterogeneity; Immune system response; Interaction with normal non-tumor cells; Lack organ-like histology
Organoids[14,17,37,40,41]	Culturing done on matrices (Matrigel, collagen I, hyaluronic acid, etc.); Addition of culture supplements including FGF, EGF, Noggin, N2, B27, etc.	Disease mechanism; Drug discovery and toxicology; Developmental, stem cell biology and regenerative medicine; Infectious disease	Oxygen and nutrient distribution underdeveloped; Cellular microenvironments are challenging to replicate; Imaging difficulties; Expensive and time consuming assay

CSC: Cancer stem cell; EGF: Endothelial growth factor; FGF: Fibroblast growth factor.

Table 4 Organoid and organoid-on-a-chip models

Ref.	Model system	System cell origin	Tumor type	Relevant genes	Results summary
Bian et al[38], 2018	neoCOR	hESCs	CNS-PNET; GBM	Amplified expression of MYC; Differential expression of GBM associated gene aberrations (GBM-1, GBM-2, and GBM-3)	neoCOR used to test gain- and loss of-function mutations, singly or in combination; generation of CNS-PNET or GBM xeno-transplantable tumors
Ogawa et al[58], 2018	Human cerebral organoids	hESC	GBM	RAS activation and TP53 deletion	Generation of tumors in cerebral organoids using CRISPR/Cas9 technology; tumors exhibited invasive phenotype and replicated the hallmarks of tumorigenesis in vivo
Ballabio et al[59], 2020	Human cerebellar organoids	Human induced pluripotent stem cell (iPSC)	Medulloblastoma (MB) subgroup 3	Overexpression of GFI1/c-MYC (GM) and OTX2/c-MYC (OM) gene combinations	OM as a novel driver gene combination required for Group 3 MB tumorigenesis; GM and OM overexpression induces tumor formation in mouse cerebellum; SMARCA4 and Tazemetostat reduces OM tumorigenesis
Linkous et al[60], 2019	GLICO	hESCs; iPSCs	GBM	-	GLICO recapitulate primary human GBM with in a primitive brain microenvironment; GSCs exhibit high resistance to drug and radiation-inducedgenotoxic stress; GSCs form tumor by relocating to the human cerebral organoid, invasion and proliferation within themicroenvironment of the GLICO
Akay et al[61], 2018	Microfluidic chip	Patient primary human GBM multiforme specimens	GBM	-	Generation of brain cancer chip that exhibit diffusion prevention mechanism to culture GBM-patient derived 3D spheroids; treatment with TMZ and bevacizumab (Avastin, BEV) in combination enhanced GBM cell death compared to TMZ alone
Ayuso et al[62], 2017	Microfluidic chip	U-251 MG human GBM cell line	GBM	-	Generation of microfluidic device to behavior models that simulate blood flow through the tumor; deprivation of nutrients and oxygen induces pseudopalisade formation; pseudopalisading process renders GBM cells to become of more aggressive behavior
Cui et al[63], 2018	Microfluidic chip	GL261 and CT-2A mouse glioma cell lines	GBM	-	Generation of microfluidic angiogenesis model that simulate GBM tumor angiogenesis and macrophage-associated immunosuppression within GBM tumor microenvironment; GL261 and CT-2A GBM-like tumors promote angiogenesis through driving M2-like macrophage polarization; TGF-b1, and surface integrin (avb3) endothelial-macrophage interactions regulates inflammation-mediated angiogenesis through Src-PI3K-YAP signaling; inhibition of integrin (avb3) and cytokine receptor (TGFb-R1) repress GBM tumor neovascularization
Lin et al[64], 2018	Microfluidic chip	Patient derived GSCs	GBM	-	Generation of glioma perivascular niches on a chip; Perivascular niches maintain the pluripotent state of GSCs; Stronger chemoresistance of GSCs against TMZ associates with endothelial cell co-culturing, GSCs neurosphere formation and the expression of 6-O-methylguanine and Bmi-1 gene
Yi et al[65], 2019	Bio-printed chip	Patient primary human GBM specimens	GBM	-	Generation of complex cancerous-tissue constructs constituting brain ECM composition, oxygen gradient-generating system, cancer-stroma structure; exhibited patient-specific response upon the treatment with drug combinations, chemoradiation and TMZ

2-D: Two dimensional; 3-D: Three dimensional; CNS-PNET: Central nervous system primitive neuroectodermal; CSC: Cancer stem cell; GBM: Glioblastoma; GLICO: Cerebral organoid glioma; GSCs: Glioma stem cells; hESC: Human embryonic stem cells; NeoCOR: Neoplastic cerebral organoid; PI3K: Phosphatidylinositol 3 kinase; PNET: Primary neuroectodermal tumor; TGF: Transforming growth factor; TMZ: Temozolomide.

Table 5 Features and characteristics comparison between spheroids and organoids

	Spheroids	Organoids
Cells used	Cell lines or CSCs	Embryonic stem cells, induced pluripotent stem cells or CSCs
Physiologic relevance	Lower	Higher
Tumor heterogeneity	Lower	Higher
Technique difficulty	Lower	Higher
Cost	Lower	Higher
Time	Weeks	1-3 mo
Genetic manipulation	Moderately available	Moderately available
Biobanks	Not available (cells are difficult to maintain long-term)	Available
Advantages	Cost effective; Highly accessible; Good for high throughput drug testing	Retains tumor heterogeneity; Better simulation of the physiological environment
Disadvantages	Hard to maintain long-term; Not as representative of the physiologic environment	More complex; Higher failure rate; May give variable results

CSC: Cancer stem cell.