Evolution of Experimental Models in the Study of Glioblastoma: Toward Finding Efficient Treatments

Evolution of Experimental Models in the Study of Glioblastoma: Toward Finding Efficient Treatments Ricardo Go ´mez-Oliva 1,2 , Samuel Domı ´nguez-Garcı ´a 1,2 , Livia Carrascal 2,3 , Jessica Abalos-Martı ´nez 1 , Ricardo Pardillo-Dı ´az 1,2 , Cristina Vera ´stegui 2,4 , Carmen Castro 1,2 , Pedro Nunez-Abades 2,3 *and Noelia Geribaldi-Dolda ´n 4,2 * 1 A ´ rea de Fisiologı ´a, Facultad de Medicina, Universidad de Ca ´diz, Ca ´diz, Spain, 2 Instituto de Investigacio ´n e Innovacio ´n Biome ´dica de Ca ´diz (INIBICA), Ca ´diz, Spain, 3 Departamento de Fisiologı ´a, Facultad de Farmacia, Universidad de Sevilla, Sevilla, Spain, 4 Departamento de Anatomı ´a y Embriologı ´a Humanas, Facultad de Medicina, Universidad de Ca ´diz, Ca ´diz, Spain Glioblastoma (GBM) is the most common form of brain tumor characterized by its resistance to conventional therapies, including temozolomide, the most widely used chemotherapeutic agent in the treatment of GBM. Within the tumor,thepresenceofgliomastemcells(GSC) seems to be the reason for drug resistance. The discovery of GSC has boosted the search for new experimental models to study GBM, which allow the development of new GBM treatments targeting these cells. In here, we describe different strategies currently in use to study GBM. Initial GBM investigations were focused in the development of xenograft assays. Thereafter, techniques advanced to dissociate tumor cells into single-cell suspensions, which generate aggregates referred to as neurospheres, thus facilitating their selective expansion. Concomitantly, the finding of genes involved in the initiation and progression of GBM tumors, led to the generation of mice models for the GBM. The latest advances have been the use of GBM organoids or 3D-bioprinted mini-brains. 3D bio-printing mimics tissue cytoarchitecture by combining different types of cells interacting with each other and with extracellular matrix components. These in vivo models faithfully replicate human diseases in which the effect of new drugs can easily be tested. Based on recent data from human glioblastoma, this review critically evaluates the different experimental models used in the study of GB, including cell cultures, mouse models, brain organoids, and 3D bioprinting focusing in the advantages and disadvantages of each approach to understand the mechanisms involved in the progression and treatment response of this devastating disease. Keywords: glioma stem cells, cell cultures of glioma cells, mouse models of glioblastoma, brain organoids, 3D bioprinting INTRODUCTION Glioblastoma (GBM) is the most common and aggressive tumor of the central nervous system (CNS) (1). It represents more than 60% of all brain tumors in adults and it is associated with a bad prognosis. Also, the Central Brain Tumor Registry of the United State (CBTRUS) registered in 2019 (including dates ranged between 2012 and 2016) that GBM represents the 14.6% of all malignant Frontiers in Oncology | www.frontiersin.org January 2021 | Volume 10 | Article 6142951 Edited by: Jose Ramon Pineda, University of the Basque Country, Spain Reviewed by: Holly Lindsay, Baylor College of Medicine, United States Rui Manuel Reis, Barretos Cancer Hospital, Brazil *Correspondence: Noelia Geribaldi-Dolda ´n [email protected] Pedro Nunez-Abades [email protected] Specialty section: This article was submitted to Neuro-Oncology and Neurosurgical Oncology, a section of the journal Frontiers in Oncology Received: 05 October 2020 Accepted: 14 December 2020 Published: 29 January 2021 Citation: Go ´mez-Oliva R, Domı ´nguez-Garcı ´aS, Carrascal L, Abalos-Martı ´nez J, Pardillo-Dı ´az R, Vera ´stegui C, Castro C, Nunez-Abades P and Geribaldi-Dolda ´n N (2021) Evolution of Experimental Models in the Study of Glioblastoma: Toward Finding Efficient Treatments. Front. Oncol. 10:614295. doi: 10.3389/fonc.2020.614295 MINI REVIEW published: 29 January 2021 doi: 10.3389/fonc.2020.614295 brain tumors being male population the most affected (1.5 times more than female) by this disease (2,3). Despite the efforts of scientists and clinicians to increase the life expectancy of GBM patients, survivors do not easily exceed the 15 th month (3–5) and the 5-year survival rate is as low as 5.8% (2,3,6). Furthermore, the median age is 65, and the most affected age ranges from 75 to 84 (3,6). Globally, the incidence of GBM is higher in some specific areas over others, such as North America, the west and north of Europe, and Australia (7). Several risk factors have been studied as critical for GBM development such as constitutive genetic factors, ionizing radiation, or reduced susceptibility to suffer allergies and asthma. However, some inconsistencies among the different studies reveal the need of further investigations (8–11). These inconsistencies are likely to be caused by the existence of different types of GBM, which behave differently. The World Health Organization (WHO) classified GBM in 2016 combining histopathological and molecular features (12). GBM are now classified into three subtypes based on the presence and absence of IDH mutations: GBM IDH-wild type (90% of tumors), IDH-mutant (10%), and GBM IDH-NOS in which a full IDH evaluation cannot be performed. GBM-IDH-wt include three variants: giant cell glioblastoma, gliosarcoma, and a novel and provisional variant, the epithelioid GBM characterized by large epithelioid cells and the presence of the BRAF V600E mutation (12,13). Based on the expressions of genes, GBM have been classified in Classical, Mesenchymal, Proneural, and Neural (14). Classical GBM is characterized by an amplification of the chromosome 7, the loss of chromosome 10, and an increase in EGFR expression. In Mesenchymal subtype a focal deletion of FN1 gene is observed affecting the AKT pathway, whereas the NF-kB pathway is highly expressed. The Proneural subtype is characterized by alterations of PDGFRA and point mutations in IDH. In this subtype some genes such as SOX, DCX, ASCL1 are affected. The Neural type is characterized by the presence of neural markers such as NEFL or GABRA1 (14). Several genetic alterations in GBM have been linked with recurrence and relapse. Thus, recurrent glioblastoma shows a higher frequency of copy number variations in several genes, particularly cell cycle genes, an enrichment in the cyclin-dependent kinase inhibitor 2A/B (CDKN2A/B) loss, and an excessive activation of cell cycle pathway genes. Also, gen sets such as TERT promoter and IDH1 mutation or tumor protein 53 (TP53) and IDH1 mutation (15). Given the bad prognosis associated to this type of tumors, the search for therapeutic tools that represent a real increase in the survival rate has become the main goal in GBM research. Current GBM treatment includes the complete surgical resection of the tumor mass, followed by a combination of radiotherapy and chemotherapy (16). In this context, it is reasonable to say that the most significant development in clinical management of glioblastoma over the past two decades has been the groundbreaking trial of combining radiotherapy plus temozolomide (TMZ) (17), which resulted in an increase in the 2-year survival from 8% in patients with radiotherapy alone to 20% in patients with the combined therapy. Despite this improvement, effectiveness of treatment is variable from patient to patient. Apparently, effectiveness of treatment depends on several factors such as the tumor localization and size, or the brain anatomical structures affected (18). Essentially, one of the most relevant problems surrounding GBM is its infiltration into the healthy brain tissue, which makes practically impossible to perform a complete resection using surgical tools. In addition, the posterior radiation and chemotherapy do not completely eliminate all GBM cells (19). Thus, new insights in surgical tools are being used to allow visualization of cells within the tumor and improve the tumor mass resection. These are fluorescenceguided microsurgery (20) or intraoperative MRI, and ultrasound, which have been used in the surgical resection of CNS gliomas with the goal of maximizing extent of resection to improve patient outcomes (21). Regarding chemotherapy, TMZ is still the most effective so far, however, several other chemotherapeutic agents are being used, some of them directed to modulate the activation and suppression of signaling pathways altered in GBM. Examples of these new treatments are nelfinavir, tipifarnib, tamoxifen, or enzastaurin (22). These agents have proven not to be the most effective in individualized treatments, nonetheless, considering the molecular, cellular, histological, and genetic variances found in GBM, a deep molecular characterization of the different tumors could potentially allow the design of individualized therapies using these agents. A problem linked to the inefficacy of TMZ treatment in the long term is that some cells within the tumor have the ability to escape its action (23) as well as the complementary radiation (24). These are the glioma stem cells (GSC). These cells share many similarities with Neural Stem Cell (NSCs) present within the physiological neurogenic niche of the subventricular zone (SVZ). These similarities are principally self-renewal and differentiation capacity (25,26) in addition to several neurogenic markers, such as CD133 (27), nestin (28,29), CD15 (30,31), or some transcriptional factors including Sox2, Olig2, Nanog, and c-Myc (32). The first evidences on the role of the SVZ harboring malignant GBM cells were obtained using fluorescence guided resection of GBM using the commonly used fluorescent marker 5-aminolevulinic acid (5-ALA). These resections reveal the presence of fluorescent cells not only within the tumor mass but also in the adjacent SVZ, thus suggesting the presence of malignant cells in the SVZ of GBM patients (33). Clonal analysis of the stem cell populations suggested a GBM evolution as a result of multiple, genetically diverse clonal and sub-clonal populations involving both the SVZ and the tumor mass (33,34). The role of the SVZ as a place of origin of GBM has gained strength because of the similarities between GSC and NSC of the SVZ (35). Vasculature, hypoxia, and several growth factors that promote GSC proliferation have been deeply studied in order to clarify the role of the SVZ in the origin of GBM (36–38). The human SVZ is characterized by presenting a complex cytoarchitecture composed of layers that provide a good environment to proliferation and differentiation of NSC (39,40). In 2018, Lee et al. showed, using single-cell sequencing, that astrocyte-like NSC in the healthy SVZ tissue of Go ´mez-Oliva et al. Evolution of Experimental Models in the Study of Glioblastoma Frontiers in Oncology | www.frontiersin.org January 2021 | Volume 10 | Article 6142952 GBM patients hide GBM driver mutations, cells that are capable of migrating from the SVZ to lead the development of high-grade malignant gliomas in distant brain regions (35). Over the past years a vast set of methodologies have been used in the study of GBM, leading to most of our current knowledge on tumor development and prognosis (41)(Figure 1 and Tables 1,2). However, the past 10 years have been crucial in the development on new and innovative techniques, such as the growth of GBM organoids, which are leading to novel and individualized therapies (Figure 2 and Table 3)forthe treatment of this disorder (99,127). In here, we discuss some classical methodologies together with the description of the most recently developed techniques to study GBM. CLASSICAL APPROACHES IN THE STUDY OF GLIOBLASTOMA Cell Cultures of Glioblastoma Cells One of the most important tools in the study of GBM has been the use of cell cultures. Cell cultures provide the maintenance of cells in vitro for research and clinical studies, however it is important to choose appropriately the cell line and culture because none of the currently available cell-based glioma model systems is able to reproduce the complex microenvironment of glioma cells within the brain. Thus, each in vitro model has its advantages and disadvantages and it is necessary to select the cell system appropriate for each experimental question (128). Some of these systems have been widely used not only in the study of GSC but also in the study of NSC giving a great deal of information about the physiology of these cells (129–132)oraboutthefinding of small pharmacological molecules that regulate these activities (133). Glioblastoma Cell Lines The first studies using cell-based glioma model systems used cell lines derived from induced Wistar/Furth rats and C57BL/6 mouse tumors of the central and peripheral nervous system (42,43). Later on, human GBM cells were also immortalized for its use in culture (44,45). This allowed the better understanding of glioma cell biology by simplifying the studies, since glioma cell lines provided an unlimited supply of cells available without ethical concerns and the possibility of obtaining reproducible results. Most commonly used human GBM immortalized cell lines are U87MG, U252, T98G, and LN-229. These cell lines show enrichment of cancer stem cells when grown as spheres in serum containing medium (134). To date, it is the fastest way to obtain preliminary results regarding the test of new anti-tumor drugs in vitro. GBM cell lines are easy to manipulate and to AB CDE FIGURE 1 | Drawing summary of classical models currently in use to study GBM. Tumor samples resected from patients upon surgery are used to (A) generate immortalized cell lines that can be maintained in time; (B) select GSC by culturing these cells as neurospheres under floating conditions; (C) produce xenograft models by transplanting either cultured isolated cells or tumor tissue; (D) perform genomic analysis to select candidate genes to produce genetically modified mice models of GBM; (E) canine models are good model for the study of GBM based on their analogies with human GBM. Go ´mez-Oliva et al. Evolution of Experimental Models in the Study of Glioblastoma Frontiers in Oncology | www.frontiersin.org January 2021 | Volume 10 | Article 6142953 TABLE 1 | Advantages and disadvantages of GBM experimental models. Method of study Advantage Disadvantage References GBM cell lines •Good method to understand GBM biology •Simple system provided with a single type of cells •Less ethical concerns •Reproducibility •Fast method to obtain preliminary results •Easy to manipulate •Does not allow studies about cell interactions or molecular mechanism in GBM development •Does not represent GBM microenvironment •Reduced genetic heterogeneity of cells compared with native tumor Fields et al. (42), Sundarraj et al. (43), de Ridder et al. (44), Notarangelo et al. (45), Weeber et al. (46), Torsvik et al. (47), Allen et al. (48). Patient-derived cells •Respect intratumor heterogenicity (GSC), tumor initiation, invasiveness process •Genotype and phenotype characteristics resemble those of primary tumors •Give more comparable results •Can be maintained in vitro •Cell cultures are limited to 20–30 passages before cells start exhibiting genomic and transcriptional changes •Quiescent GSC are deselected within the neurospheres upon the different passages •Introduce errors in terms of clonality, size, and number of neurospheres Sottoriva et al. (49), Jiang et al. (50), Linkous et al. (51), Lee et al. (52), Brewer et al. (53), Baskaran et al. (54), Jayakrishnan et al. (55), Wang et al. (56), Chen et al. (57), Mori et al. (58), Ladiwala et al. (59), Pollard et al. (60), Fael Al-Mayhani et al. (61), Rahman et al. (62). Xenograft model of GBM •Allow personalized drug efficiency tests in single patients •Maintain the original tumor architecture and histological characteristic •Genetically stable •A nude mouse is necessary to develop this model •Cells need to be cultured in spheroids forms before implantation •Does not reproduce the original niche. •It is not allow to test immunomodulatory therapies Shu et al. (63), Lee et al. (52), Tentler et al. (64), Joo et al. (65), Patrizii et al. (66), Ashizawa et al. (67), Hutchinson et al. (68), Son et al. (69), Khaddoui et al. (70), Lynes et al. (71). Genetically engineered and viral vectormediated transduction models •Modified models offer the ability to directly alter the genome of somatic cells in mouse tissues introducing or removing specific genes •Reproduce pre-clinical features •Easy approach to rapidly analyze therapeutic responses to drugs •Genetics and histology of the modified tumor models are often not representative of the original human tumor Furnari et al. (72), Holland et al. (73), Uhrbom et al. (74), Wei et al. (75), Baker et al. (76), Miyai et al. (77). Canine model of GBM •Similarities with human glioma •Good tool to perform preclinical studies •Ethical issues •Difficult to detect GBM in canine models Herranz et al. (78), Stoica et al. (79), Candolfiet al. (80), Chen et al. (81), Fernandez et al. (82). Organotypic cultures •Good model to study invasiveness •*Niche similarities •*Not reproduce interactions with blood flow factors or typical hypoxic conditions Eisemann et al. (83), Sliwa et al. (84); Marques-Torrejon et al. (85), Ravi et al. (86) Brain organoids •Provide a powerful tool for the ex vivo study of the molecular and cellular mechanisms •Maintain the architecture and organization of tissues •Difficult approach in terms of technology •Need biopsies of patients •Complex structures to maintain Lancaster and Knoblich (87), Clevers (88), Chen et al. (89), Sasai (90), Huch et al. (91); Hubert et al. (92), da Silva et al. (93), Ogawa et al. (94), Bian et al. (95), Krieger et al. (96), Linkous et al. (51), Hwang et al. (97), Jacob et al. (98), Zhang et al. (99), Perrin et al. (100), Vlachogiannis et al. (101), Ooft et al. (102), Ganesh et al. (103), da Hora et al. (104). (Continued) Go ´mez-Oliva et al. Evolution of Experimental Models in the Study of Glioblastoma Frontiers in Oncology | www.frontiersin.org January 2021 | Volume 10 | Article 6142954 maintain in cultures using serum-containing media. However, cell lines do not provide a reliable model to understand the cellular and molecular mechanism underlying the development of GBM or to evaluate therapeutic interactions because it fails to represent the native tumor microenvironment. In addition, the use of glioma cell cultures holds other issues intrinsic to immortalized cell lines. Successive cell passages select cells which have the highest proliferative potential, decreasing the genetic heterogeneity found in the parental tumor (46). It is likely that the selection imposed by the passages in in vitro cultures result in genetic drift, accumulation of chromosomal aberrations, and phenotypic alterations in cell lines (47,48). Because of these drawbacks, biobanks containing annotated and validated cell lines derived from surgical samples of GBM patients that preserve GSC features are being developed. This strategy provides an open resource for the study of a large part of GBM diversity (135). Usually, the use of cell lines is well received as an approximation or as preliminary data but it is necessary the use of other models closer to reality to obtain more relevant and representative data. However, these cultures have been fundamental to solve and understand GBM biology (Figure 1A and Table 1). Patient-Derived Cells Most cancers, including GBM, display intratumor heterogeneity and this could be one of the reasons why some tumors lack of satisfactory treatment (49,50). Thus, the use of primary cell cultures derived from patients may facilitate the individualized study of GBM. Evidences show that the GSC subpopulation of GBM are very important to maintain tumor heterogeneity, as well as, the tumor initiation, maintenance, and invasion in vivo because of their capacity of self-renewal and differentiation (51). So far, GSC cultures have become the most accepted standard for studying GBM biology in vitro (52). One of the problems found when using patient derived cultures is the use of serum since the subpopulation of GSC within the GBM is not present in cell cultures after prolonged serum exposure. GSC differentiate under these conditions, losing many of primary tumor characteristics (52). To avoid this issue, patient-derived GSC can be maintained in vitro under floating conditions in a serum free medium in which they form characteristic aggregates referred to as neurospheres. The starting population of cells is usually plated as a single-cell suspension in a non-adhesive substrate containing defined media supplemented with fibroblast growth factor (bFGF) and/ or epidermal growth factor (EGF) (52,53). However, it is important to consider that useful life of patient-derived glioma cell cultures is limited. After 20–30 passages cells start exhibiting genomic and transcriptional changes in metabolic and signaling pathways such as ribosomal synthesis, telomere packaging, or Wnt signaling pathways among others (54). Interestingly, the success rates of neurosphere cultures from gliomas is dependent on tumor grade and genetics, being the low-grade glioma cell cultures the most difficult to culture as neurospheres (55). Despite its advantages, the neurosphere system presents a few drawbacks inherent to this cell culture method. Quiescent GSC may be lost with time since along the successive cell passages cells with the highest proliferative potential are selected and the subpopulation of quiescent GSC is deselected. This subpopulation is particularly important because it is believed to be responsible for chemotherapy and radiotherapy resistance (56,57). Moreover, to ensure clonality and multipotentiality of neurospheres, cells should be seeded as single cells per well (58). TABLE 1 | Continued Method of study Advantage Disadvantage References •Preserve important features of the original tumor heterogeneity •Useful to study GBM pathology and drug response •Good method to design personalized therapies and treatments and, also, in drug screening •Elevated cost 3D bioprinting •Provide a cell network that resembles reality in a very faithful way •Replicate the architecture of tissues •Good method to test drugs effectively •Good to analyze cell signaling •Good method to test drug sensibility scanning •Good method to study invasiveness process, immunologic interactions and cellular crosstalk •Need photocrosslinking •Need effective biomaterials, which do not affect normal tissue development •Printing resolution still needs to be improved •Need of a bioprinter •High cost Roseti et al. (105), Heinrich et al. (106), Ananthanarayanan et al. (107), Xiao et al. (108), Hermida et al. (109), Tang et al. (110) Go ´mez-Oliva et al. Evolution of Experimental Models in the Study of Glioblastoma Frontiers in Oncology | www.frontiersin.org January 2021 | Volume 10 | Article 6142955 However, this leads to a low number of cells because the concentration of paracrine and cell-to-cell signals required for cell growth and division are minimal under these conditions. On the contrary, neurospheres obtained after plating multiple cells per well show spontaneous locomotion resulting in cell aggregation and producing errors in terms of clonality, size, and number of neurospheres, thus leading to discrepancies in results and conclusions (59). Looking for an effective manner to cultivate GSC, some reports describe a methodology to attach these cells onto a surface to expand them in serum free medium as adherent 2D cultures in the presence of growth factors EGF and bFGF (60, 61). The use of adherent 2D-cultures increases the efficiency of culture expansion avoiding the differentiation and apoptosis associated to the sphere cultures. Attached cells are more exposed to growth factors used in cultures that maintain the proliferative capacity (60). Other authors maintain that both methods, sphere and adherent cultures, are equal and useful to study GBM (62) and it is likely that a combination of both models is the best strategy for the in vitro test of GBM drugs, or for the study of new specific markers of GBM malignancy and progression. Taking together all these facts prove the benefits of using this neurosphere cultures, starting with the capacity to preserve heterogeneity and also, to study the migration process and initiation of a new tumor. All these considerations are not present in cell lines models. However, the problem associated with the loss of quiescent GSC could be unfavorable in some studies. Nonetheless, this model allows the culture of cells from single individual patients, facilitating the study of interindividual differences (Figure 1B and Table 1). Xenograft Models of Glioblastoma In vivo transplants of GBM in animal models have long been used to study tumor development upon the engraftment of human cells into immunodeficient mice. Transplants of patient derived xenografts, in which dissociated tumor cells or tumor tissue fragments are implanted into mouse brains have successfully been used in murine models (Figure 1C). Mainly, threetypesofmousehavebeenused,whichareclassified TABLE 2 | Current trends in mouse xenografts and allografts. Reference Implantation type Cell type Tumor type Animal model Strategy used Tateishi et al. (111) Orthotopic xenograft MGG152 Recurrent GBM SCID mice NAMPT inhibitor HT1080 Fibrosarcoma Ashizawa et al. (67) Heterotopic xenograft GB-SCC010 Primary GBM NOD-SCID mice and NOG mice STAT3 inhibitor GB-SCC026 Primary GBM Szabo et al. (112) Orthotopic xenograft LNT-229 GBM Nude mice Neutralization with VEGF or PlGF antibodyLN-308 GBM Sharpe et al. (113) Orthotopic xenograft BT111 Primary GBM Nude mice Monoamine oxidase B-activated pro-drugBT116 Primary GBM Zhang et al. (114) Orthotopic xenograft LN-319 GBM NSG mice ErbB2/HER2-Specific NK CellsOrthotopic allograft GL261 GBM Parrish et al. (115) Heterotopic and orthotopic xenograft GBM12 Primary GBM Mdr1a/b −/− Bcrp1 −/− knockout and wild type mice PARP inhibitors and temozolomide Garros-Regulez et al. (116) Heterotopic xenograft U251 GBM Nude mice mTOR inhibition with Rapamycin and Temozolomide Karpel-Massler et al. (117) Orthotopic xenograft GBM164 Primary GBM SCID mice Bcl-xL inhibition with ABT263 U87MG GBM Yuan et al. (118) Orthotopic xenograft Patient derived brain tumor initiating cells Primary GBM SCID mice ABT-888 and temozolomide treatment Chang et al. (119) Orthotopic xenograft LN229 GBM Nude mice Pyr3 treatment U87MG GBM Sun et al. (120) Orthotopic xenograft TT150630 Primary GBM B-NDG mice Palbociclib in treatment TT150728 Primary GBM Bejarano et al. (121) Heterotopic xenograft h676 Primary GBM Nude mice TRF1 Chemical Inhibitors h543 Primary GBM Guo et al. (122) Orthotopic allograft GL261, GL261 Red- FLuc and GL261-Luc2 GBM C57BL mice FTY720 treatment Gravina et al. (123) Heterotopic and orthotopic xenograft U87MG GBM Nude mice RES529, a TORC1/TORC2 dissociative inhibitor Zalles et al. (124) Orthotopic xenograft G55 GBM Nude mice Neutralization with ELTD1 antibody Jensen et al. (125) Orthotopic xenograft patient-derived GB brain tumor stem cells Primary GBM SCID mice Afatinib and pacritinib treatment Yang et al. (126) Heterotopic and orthotopic allograft GL261 GBM C57BL/6 mice Bip inhibition and ionizing radiation Go ´mez-Oliva et al. Evolution of Experimental Models in the Study of Glioblastoma Frontiers in Oncology | www.frontiersin.org January 2021 | Volume 10 | Article 6142956 according to their immune response: i) nude mice which are unable to produce T cells; ii) non-obese diabetic severe combined immunodeficiency (NOD-SCID) and SCID-beige mice, which lack T and B cells; and iii) NOD-SCID IL2R-gnull (NSG or NOG) mice, which lack T, B, or NK cell activity (136). GBM implantation is usually done in the subcutaneous flank location (heterotopic implantation) facilitating the visual observation of tumor development, which allows testing the efficacy of antitumor drugs by analyzing the tumor dimensions. However, an important limitation to heterotopic models is that the established microenvironment has an important role in GBM tumors. For this reason, xenografts based on the implantation of GBM in the brain (orthotopic implantation) are more extensively used nowadays because it provides a CNS microenvironment, and preserves the integrity of tumor-initiating cells (63). We have summarized in Table 2 series of studies involving PDX models, showing how orthotopic or heterotopic xenografts have been used in different immunodeficient mouse strains to perform preclinical studies on the efficacy of novel drug treatments. Invasive xenografts have been established from surgical specimens that were first maintained as tissue spheroids in short-term culture to obtain cell lines (137,138). Many human and mouse cell lines have been used in PDX models (111,112, 139,140). The patient-derived cells are engrafted into immunocompromised mice to maintain the histopathologic, genomic, and phenotypic characteristic of the primary tumor across early passages (64–66). Lee et al. used GSC lines to initiate tumors in mice and they found a high percentage of engraftments compared with patient derived tumor xenografts. This type of tumor induction was much more potent and maintained the heterogeneity typical of GBM (35,67). While NSC implantation into nude mice does not result in tumor formation, GSC implantation leads to the generation of tumors in a healthy brain, preserving tumor heterogeneity. This strategy opens the possibility to development of personalized drugs facilitating individual drug-screening and the search for resistance mechanisms. Notwithstanding, several drugs showing favorable results in pre-clinical studies using PDX have failed posteriorly in clinical trials (68). Indeed, this is not completely surprising since orthotropic implantation in PDX models does not reproduce the conditions of the niche of origin. The human stroma and microenvironment are not similar to those in the mouse, limiting the study to tumor biology and therapy resistance (69). Moreover, since these mouse models lack inflammatory responsive cells or an intact immune system it does not allow testing for immunomodulatory therapies. This limitation is currently critical since immune therapeutics have recently been very successful in treating a diverse group of A B E CD FIGURE 2 | Drawing summary of novel models currently in use to study GBM. (A) 3D Cultures of cells embedded in Matrigel have been key techniques in the development of brain organoids; (B) growth of 3D structures using microfluidic systems replicates the changing microenvironment surrounding GBM in the human brain; (C) GBM cells can be grafted in vitro in human brain slices grown as organotypic cultures replicating the natural environment of a GBM tumor; (D) culture GBM organoids by either using glioma cells or by inserting GSC into brain organoids; (E) bio-print GBM 3D-spheroids using extracellular matrix materials and other cell types. Go ´mez-Oliva et al. Evolution of Experimental Models in the Study of Glioblastoma Frontiers in Oncology | www.frontiersin.org January 2021 | Volume 10 | Article 6142957 cancerous lesions and there has been an explosion in the study of immune therapeutics for cancer treatment over the past few years (70,71). Among the advantages of these xenograft models three can be highlighted: i) they allow personalized drug efficiency tests in single patients, ii) the original tumor architecture and histological characteristic are preserved and iii) they are genetically stable (Tables 1 and 2). However, there are important caveats that need to be addressed in xenograft models: i) immunodeficient rodents may not respond to certain drugs, ii) the surrounding microenvironment of mouse origin may interfere with drug response, and iii) they do not allow the test of immune therapies, thus limiting the type of drugs to be tested. Genetically Engineered and Viral Vector- Mediated Transduction Mouse Models As mentioned before, several genetic alterations have been found after the analysis of large numbers of uncultured GBM tissue samples removed from patients, leading to the discovery of several genes commonly mutated in GBM. Some of these mutations are already present in common cancer genes, such as EGFR, BRAF, RAS, PIK3CA, PIK3R1, PTEN (141,142), particularly, EGFR variant III (EGFRvIII) is the most common active EGFR mutant in GBM. The presence of this mutant correlates with a poor patient prognosis due to its ability to extend downstream signaling (143). Accordingly, kinases like v- Src, which regulate the activity of these type of receptors have also been involved proliferation and migration of glioma cells (144). Additionally, a very clinically relevant discovery was the finding of the IDH1 mutation appears in a high percentage of secondary GBM and a small percentage of primary GBM (142). While PTEN loss, and EGFR amplification, are associated with primary glioblastoma, IDH1 mutation is common in secondary glioblastoma and show a higher survival rate. The study of genetic alterations has led to the development of genetically engineered mouse models of GBM (77). Transgenic mouse models and knockouts as well as vector-mediated genetic approaches accurately reproduce pre-clinical features of GBM including the accumulation of genetic and epigenetic alterations in tumor suppressor genes or to the activation of oncogenic pathways, which lead to the progression of tumors (72,142,145). There are several examples employed in the development of mouse glioma modelling of aberrant expression of relevant downstream signaling pathways, resulting from i) expressing vsrc kinase under the control of glial fibrillary acidic protein TABLE 3 | Current preclinical trials using organoids for the study of GBM. Reference GBM organoid from Contribution to GBM study Potential clinical use Advantage Disadvantage Hubert et al. (92) Patient-derived GBSC Microenvironmental impact and cancer stem cell biology Tumor sensitivity to radiochemotherapy Preserve phenotypic heterogenicity and tumorigenic capacity No vessels No immune cells Slow growth No interaction with no-tumor cell Ogawa et al. (94) hESC-derived cerebral organoid genetically modified by introducing HRas G12V through CRISPR/Cas9 Model for tumor formation and transplantation Platform to test human cancer phenotypes and personalized therapy Interactions between tumor and nontumor cell No vessels No immune cells Slow growth Bian et al. (95) hESC/iPSC-derived cerebral organoid genetically modified by introducing several mutation combinations through CRISPR/Cas9 GBM invasiveness and evaluation of drug response Personalized therapy Interactions between tumor and nontumor cell No vessels No immune cells Slow growth Da Silva et al. (93) Co-culture of human GBM spheroids with mouse ESC Identification of anti-GBM invasion strategies Drug screening. Tumor sensitivity to radiochemotherapy Interactions between tumor and nontumor cell No vessels No immune cells Linkous et al. (51) Co-culture of patient derived GBM stem cells with hESC-derived cerebral organoid GBM biology in the human brain environment. Drug screening Tumor sensitivity to radiochemotherapy Interactions between tumor and nontumor cell No vessels No immune cells Krieger et al. (96) GBM cells co-cultured with hESC-organoid cells Invasion and transcriptional heterogeneity Drug screening Tumor sensitivity to radiochemotherapy Interactions between tumor and nontumor cell No vessels No immune cells Jacob et al. (98) Patient-derived resected glioblastoma tumor tissue GBM cells heterogeneity, tumor microenvironment and GBM infiltration Useful to test therapy response including immunotherapy Construction of a living organism bank Biobank Fast growth Vessels and immune cells No interaction with nontumor cell Hwang et al. (97) Patient derived- IPSC with c-met mutation Reproduction a genomic network described in the most aggressive primary human GBM Therapy response for GBM with c-met mutation Good model to study progression and aggressiveness No vessels No immune cells Slow growth No interaction with nontumor cell Go ´mez-Oliva et al. Evolution of Experimental Models in the Study of Glioblastoma Frontiers in Oncology | www.frontiersin.org January 2021 | Volume 10 | Article 6142958 (GFAP) gene regulatory elements (77,146), ii) aberrantly activating the p21-RAS signaling pathway mimicking the effect of EFGRvIII mutation (147,148)toiii)overexpressionof IDH1 R132H in the SVZ of the adult mouse brain (149). In addition transduction of genes with viral vectors have been used to efficiently reproduced GBM development: i) retroviruses engineered to express relevant gain-of-function genes that result from overexpression of the viral oncogene v-sis, the cellular counterpart of which is c-sis or PDGF-B (73,74); ii) adenovirus containing the EGFRvIII mutant into mice harboring activated RAS, which led to the efficient formation of glioblastoma (75); or iii) lentivirus expressing oncogenes such as HRAS or AKT (150,151). Models used in the study of GBM are the knockout of p53 tumor suppressor gene harboring a conditional allele of the tumor suppressor Nf1 (152), a model that displays an upregulation of Ras signaling; the Cdkn2a knockout mice combined with Kras and Akt upregulation by viral induction (74); the combination of both p53 and Cdkn2a knockout; the introduction of EGFRvIII or Pten loss in the glioma-prone mouse strain RasB8 mice with activated HRAS; the combined conditional knockout of tumor suppressors p53, Nf1 and Pten genes (151). Genetically engineered or viral vector-mediated models offer the ability to directly modify the genome of somatic cells in mouse tissues (by targeting the genomic alterations driving tumor behavior) for a rapid generation of complex mouse tumor models, that harbor specific genetic alterations providing the chance to potentially study specificdrugs interfering with the function of such genes (76,77)(Figure 1D and Table 1). Canine Models The use of canine models to understand GBM biology represents a good option to simulate human conditions. The incidence of brain tumors in dogs is high and its characteristics are similar to those found in human (78,79). Also, the comparison of GBM histopathological features in dog versus those in murine models leads to the conclusion of dog models as a good tool to perform preclinical studies (80,81). The advantages of using the canine models of glioma are based on the similarities with human gliomas including the presence of several neural precursor markers such as nestin (82) and the capacity to form spheres (78). In contrast, an intrinsic problem to its use is the difficulty of detecting canine brain tumors and the ethical issues involved and the need to comply with the 3R (refinement, replacement, and reduction) for animal use in experimentation (Figure 1E and Table 1). NOVEL TECHNICAL APPROACHES IN THE STUDY OF GLIOBLASTOMA As it can be inferred from the studies discussed above, in vitro cell culture systems for the study of GBM do not exactly reproduce the real conditions surrounding brain tumors and the use of animal models may not be the best approach either to reproduce the particular niche in which GBM cells reside inside a human brain. Thus, a refinement of the in vitro techniques was required to produce humanized GBM models based on the threedimensional (3D) culture of GBM cells in a system that reproduced the microenvironment of human brain tumors (Figure 2). 2D vs 3D Co-Cultures Studies on GBM require the culture of patient derived GBM reproducing in vitro the conditions that establish interactions between different types of cells and between the cells with the extracellular matrix. GBM include a combination of fibroblasts, endothelial cells, and stem cells that release signaling ligands that determinate the characteristic. One of the challenges in cell culture for GBM has been the search for coating elements that reproduce these conditions as opposed to the effect of plastic. In this context a collection of hydrogels have been produced from ECM derived polymers that replicate the GBM microenvironment such hyaluronic acid, chitosan, chondroitin sulfate polysaccharides, alginates, and collagen/gelatin proteins (153). Matrigel (a mix of mouse collagen, laminin, and ECM-associated growth factors) is a hydrogel widely used in cultures of GBM, which allows cells to grow interacting on multiple sides (154). However, these conditions do not faithfully reflect the situation in vivo since proper tissue architecture and cell-cell contacts may be lost in such 2D systems as well as the contacts with the extracellular matrix. Thus, recent works aimed to produce 3D GBM structures by culturing GBM cells onto hydrogel coated 3D scaffolds in which GSC or pieces of patient derived GBM are cultured reproducing a more real tumor environment (Figure 2A). These systems reproduce cell growth environment of GBM cells in combination with more than one type of cell, soluble signaling factors as well as the extracellular matrix signaling. Matrigelcoated 3D polystyrene scaffolds have already been successfully used to test drug efficacy (155), morphological structures in human tumors (156) and invasion (reviewed in Caragher et al., 2019) (154). The limitations of these scaffolds are the substrate stiffness, the selection across passages of cells that attach more loosely to the scaffold, which in turn are more invasive and apparently show expression patterns more similar to GSC. Also, the mouse origin of Matrigel makes it different from human brain ECM and more humanized Matrigel is being developed (154) together with other new promising biomaterials (157). Microfluidic Technology in Glioblastoma GBM tumors are structures surrounded by a constantly changing microenvironment, which leads its development, however, the particular invariable conditions of the medium in 3D cultures do not reproduce this important attribute. Microfluidic technologies solve the problem of GBM cells growing in static medium (Figure 2B). These systems allow liquid media to be continually delivered to growing cells (158,159). GBM primary cells can be grown in a scaffold of hydrogel tubes with circulating medium. Cells are pumped into the tubes in a solution containing brain ECM elements (160). The latest advances in Go ´mez-Oliva et al. Evolution of Experimental Models in the Study of Glioblastoma Frontiers in Oncology | www.frontiersin.org January 2021 | Volume 10 | Article 6142959 Representing All Molecular Subtypes. EBioMedicine (2015) 2(10):1351–63. doi: 10.1016/j.ebiom.2015.08.026 136. Yoshida GJ. Applications of patient-derived tumor xenograft models and tumor organoids. JHematolOncol(2020) 13(1):4. doi: 10.1186/s13045-019-0829-z 137. Engebraaten O, Hjortland GO, Hirschberg H, Fodstad O. Growth of precultured human glioma specimens in nude rat brain. J Neurosurg (1999) 90(1):125–32. doi: 10.3171/jns.1999.90.1.0125 138. Mahesparan R, Read TA, Lund-Johansen M, Skaftnesmo KO, Bjerkvig R, Engebraaten O. Expression of extracellular matrix components in a highly infiltrative in vivo glioma model. Acta Neuropathol (2003) 105(1):49–57. doi: 10.1007/s00401-002-0610-0 139. Plowman J, Waud WR, Koutsoukos AD, Rubinstein LV, Moore TD, Grever MR. Preclinical antitumor activity of temozolomide in mice: efficacy against human brain tumor xenografts and synergism with 1,3-bis(2-chloroethyl)-1- nitrosourea. Cancer Res (1994) 54(14):3793–9. 140. Kessler JD, Kahle KT, Sun T, Meerbrey KL, Schlabach MR, Schmitt EM, et al. A SUMOylation-dependent transcriptional subprogram is required for Mycdriven tumorigenesis. Science (2012) 335(6066):348–53. doi: 10.1126/ science.1212728 141. Network TC. Corrigendum: Comprehensive genomic characterization defines human glioblastoma genes and core pathways. Nature (2013) 494 (7438):506. doi: 10.1038/nature11903 142. Parsons DW, Jones S, Zhang X, Lin JC, Leary RJ, Angenendt P, et al. An integrated genomic analysis of human glioblastoma multiforme. Science (2008) 321(5897):1807–12. doi: 10.1126/science.1164382 143. An Z, Aksoy O, Zheng T, Fan QW, Weiss WA. Epidermal growth factor receptor and EGFRvIII in glioblastoma: signaling pathways and targeted therapies. Oncogene (2018) 37(12):1561–75. doi: 10.1038/s41388-017-0045-7 144. de Groot J, Milano V. Improving the prognosis for patients with glioblastoma: the rationale for targeting Src. JNeurooncol(2009) 95 (2):151–63. doi: 10.1007/s11060-009-9916-2 145. Cancer Genome Atlas Research N. Comprehensive genomic characterization defines human glioblastoma genes and core pathways. Nature (2008) 455 (7216):1061–8. doi: 10.1038/nature07385 146. Weissenberger J, Steinbach JP, Malin G, Spada S, Rulicke T, Aguzzi A. Development and malignant progression of astrocytomas in GFAP-v-src transgenic mice. Oncogene (1997) 14(17):2005–13. doi: 10.1038/sj.onc.1201168 147. Ding H, Roncari L, Shannon P, Wu X, Lau N, Karaskova J, et al. Astrocytespecific expression of activated p21-ras results in malignant astrocytoma formation in a transgenic mouse model of human gliomas. Cancer Res (2001) 61(9):3826–36. 148. Ding H, Shannon P, Lau N, Wu X, Roncari L, Baldwin RL, et al. Oligodendrogliomas result from the expression of an activated mutant epidermal growth factor receptor in a RAS transgenic mouse astrocytoma model. Cancer Res (2003) 63(5):1106–13. 149. Bardella C, Al-Dalahmah O, Krell D, Brazauskas P, Al-Qahtani K, Tomkova M, et al. Expression of Idh1(R132H) in the Murine Subventricular Zone Stem Cell Niche Recapitulates Features of Early Gliomagenesis. Cancer Cell (2016) 30 (4):578–94. doi: 10.1016/j.ccell.2016.08.017 150. Marumoto T, Tashiro A, Friedmann-Morvinski D, Scadeng M, Soda Y, Gage FH, et al. Development of a novel mouse glioma model using lentiviral vectors. Nat Med (2009) 15(1):110–6. doi: 10.1038/nm.1863 151. Alcantara Llaguno S, Chen J, Kwon CH, Jackson EL, Li Y, Burns DK, et al. Malignant astrocytomas originate from neural stem/progenitor cells in a somatic tumor suppressor mouse model. Cancer Cell (2009) 15(1):45–56. doi: 10.1016/j.ccr.2008.12.006 152. Zhu Y, Guignard F, Zhao D, Liu L, Burns DK, Mason RP, et al. Early inactivation of p53 tumor suppressor gene cooperating with NF1 loss induces malignant astrocytoma. Cancer Cell (2005) 8(2):119–30. doi: 10.1016/j.ccr.2005.07.004 153. Xiao W, Sohrabi A, Seidlits SK. Integrating the glioblastoma microenvironment into engineered experimental models. Future Sci OA (2017) 3(3):FSO189. doi: 10.4155/fsoa-2016-0094 154. Caragher SP, Shireman JM, Huang M, Miska J, Atashi F, Baisiwala S, et al. Activation of Dopamine Receptor 2 Prompts Transcriptomic and Metabolic Plasticity in Glioblastoma. J Neurosci (2019) 39(11):1982–93. doi: 10.1523/ JNEUROSCI.1589-18.2018 155. Gomez-Roman N, Stevenson K, Gilmour L, Hamilton G, Chalmers AJ. A novel 3D human glioblastoma cell culture system for modeling drug and radiation responses. Neuro Oncol (2017) 19(2):229–41. doi: 10.1093/neuonc/ now164 156. Weil S, Osswald M, Solecki G, Grosch J, Jung E, Lemke D, et al. Tumor microtubes convey resistance to surgical lesions and chemotherapy in gliomas. Neuro Oncol (2017) 19(10):1316–26. doi: 10.1093/neuonc/nox070 157. Rape A, Ananthanarayanan B, Kumar S. Engineering strategies to mimic the glioblastoma microenvironment. Adv Drug Deliv Rev (2014) 79-80:172–83. doi: 10.1016/j.addr.2014.08.012 158. Bolivar JM, Wiesbauer J, Nidetzky B. Biotransformations in microstructured reactors: more than flowing with the stream? Trends Biotechnol (2011) 29 (7):333–42. doi: 10.1016/j.tibtech.2011.03.005 159. Olubajo F, Achawal S, Greenman J. Development of a Microfluidic Culture Paradigm for Ex Vivo Maintenance of Human Glioblastoma Tissue: A New Glioblastoma Model? Transl Oncol (2020) 13(1):1–10. doi: 10.1016/ j.tranon.2019.09.002 160. Li Q, Lin H, Rauch J, Deleyrolle LP, Reynolds BA, Viljoen HJ, et al. Scalable Culturing of Primary Human Glioblastoma Tumor-Initiating Cells with a Cell-Friendly Culture System. Sci Rep (2018) 8(1):3531. doi: 10.1038/s41598- 018-21927-4 161. Ayuso JM, Monge R, Martinez-Gonzalez A, Virumbrales-Munoz M, Llamazares GA, Berganzo J, et al. Glioblastoma on a microfluidic chip: Generating pseudopalisades and enhancing aggressiveness through blood vessel obstruction events. Neuro Oncol (2017) 19(4):503–13. doi: 10.1093/ neuonc/now230 162. Lancaster MA, Knoblich JA. Generation of cerebral organoids from human pluripotent stem cells. Nat Protoc (2014) 9(10):2329–40. doi: 10.1038/ nprot.2014.158 163. Lancaster MA, Renner M, Martin CA, Wenzel D, Bicknell LS, Hurles ME, et al. Cerebral organoids model human brain development and microcephaly. Nature (2013) 501(7467):373–9. doi: 10.1038/nature 12517 164. Qian X, Nguyen HN, Song MM, Hadiono C, Ogden SC, Hammack C, et al. Brain-Region-Specific Organoids Using Mini-bioreactors for Modeling ZIKV Exposure. Cell (2016) 165(5):1238–54. doi: 10.1016/j.cell.2016.04.032 165. Roesch S, Rapp C, Dettling S, Herold-Mende C. When Immune Cells Turn Bad-Tumor-Associated Microglia/Macrophages in Glioma. Int J Mol Sci (2018) 19(2):436. doi: 10.3390/ijms19020436 166. Malesci A, Bianchi P, Celesti G, Basso G, Marchesi F, Grizzi F, et al. Tumorassociated macrophages and response to 5-fluorouracil adjuvant therapy in stage III colorectal cancer. Oncoimmunology (2017) 6(12):e1342918. doi: 10.1080/2162402X.2017.1342918 167. Allavena P, Sica A, Garlanda C, Mantovani A. The Yin-Yang of tumorassociated macrophages in neoplastic progression and immune surveillance. Immunol Rev (2008) 222:155–61. doi: 10.1111/j.1600-065X.2008.00607.x Conflict of Interest: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Copyright © 2021 Go mez-Oliva, Domı  nguez-Garcı  a, Carrascal, Abalos-Martı  nez, Pardillo-Dı  az, Vera stegui, Castro, Nunez-Abades and Geribaldi-Dolda n. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Go ´mez-Oliva et al. Evolution of Experimental Models in the Study of Glioblastoma Frontiers in Oncology | www.frontiersin.org January 2021 | Volume 10 | Article 61429516