The interest in the role of cancer stem cells in GBM persists due to their involvement in tumor drug resistance and relapse. GSCs are capable of both continuous self-renewal and enhanced capacity to initiate glioblastoma in vivo ( (Sharifzad et al. 2019). It is known that GSCs are a heterogeneous population present in both intratumoral perivascular and necrotic/ hypoxic niches (Ishii et al. 2016). These niches help to maintain the stemness property, regulate proliferation, self-renewal and fate and to protect the GSCs from environmental insults (Seidel et al. 2010; Fidoamore et al. 2016). An improved understanding of the molecular regulators and microenvironmental roles in tumor stem cells is crucial for designing treatment strategies aimed at the efficient eradication of this cell subpopulation. In like manner, in an effort to identify new targets for GBM diagnostics and therapeutics, studies have resulted in mounting interest in miRNAs. From our study, we were able to observe changes in the expression of miRNAs in the stem cells isolated from our parental GBM cell lines. We found miRs 34-5p, 128-3p, 181a-3p to be downregulated while miRNAs 221- 3p and 17-5p to be upregulated while a comparison was made between the stem-like and their parental cells. This expression pattern was similar to the observation made when the comparison was made between the parental GBMs and a non-GBM cell line from our previous study. To support this observation, it has been reported elsewhere that miRNA profiles in stem cells resemble those seen in cancer cells (Papagiannakopoulos and Kosik 2008).
While miRNAs have been reported to contribute to CSC properties such as tumorigenicity, asymmetric cell division and chemoresistance (Osaki et al. 2015), there are limited studies reporting expression levels of miRNAs using actual glioblastoma stem cells to make an accurate comparison. Instead, most studies report the miRNA altering a gene or a pathway that plays a role in the stemness property. For example, miR-128 has been reported to be downregulated in GBM and to target the oncogene BMI1 known to regulate proliferation, self-renewal and migration of GSCs associated with enhanced self-renewal of GSCs (Godlewski et al. 2008). The miR-34 has been reported downregulated in glioblastoma and it targets key stem pathways (NOTCH1, and NOTCH2). Its forced expression induces apoptosis and inhibits invasion (Li et al. 2009a; Guessous et al. 2010) and has been shown to reduce the CSC by inducing cell differentiation of glioma cells (Guessous et al. 2010). Ectopic expression of miR-17 has been reported to facilitate the enrichment of stem-like tumor cells in GBM cells stably transfected with miR-17 and cultured in serum-free medium for two weeks. The cells showed increased capacity to form colonies and neurospheres, and expressed higher levels of CD133 (Li and Yang 2012). Among the few studies reported using actual stems, a study using global miRNA expression analysis, revealed 51 most differentially expressed miRNAs between paired GSC and non-stem cell cultures. Nine miRNAs (miR-9-3p, miR-93-3p, miR-93-5p, miR-106b-5p, miR-124-3p, miR-153-3p, miR-301a-3p, miR-345-5p, and miR-652-3p) were strongly upregulated in GSCs when a comparison was made between the GSC cluster with more pronounced GSCs features and the non-stem cell cluster (Sana et al. 2018). Similarly, a study that evaluated the expression levels of miR-29a in GBM cells, stem cells (GSCs) and human tumors as well as normal astrocytes and normal brain by quantitative PCR found miR-29a to be downregulated in human GBM specimens, GSCs and GBM cell lines. Exogenous expression of miR-29a inhibited GSC and GBM cell growth and induced apoptosis (Yang et al. 2019). Together, these few evidences thus suggest that a stem-like state in glioblastoma can influence the expression pattern of a miRNA in vitro.
The effect of hypoxia on glioblastoma cell lines has been expounded in detail while less attention has been paid to the influence of hypoxia on growth and differentiation using actual cancer stem cells (Keith and Simon 2007). In this regard, our study investigated the interaction between GSCs and the hypoxia microenvironment to understand the effect of hypoxia on the in vitro stemness in glioblastoma. We observed that when the stem-like cells were grown under hypoxic conditions, they were larger in diameter as compared to their normoxia counterparts. Hypoxia has already been reported to enhance neurosphere formation and cell proliferation. A study that treated T4121 glioma stem and non-stem cells in hypoxia for several days noted distinct growth advantages. Cells grown in hypoxia had increased growth than the cells grown in normoxia (Heddleston et al. 2009). We were able to observe that some neurospheres under the normoxia microenvironment had zones. This observation was made via some immunocytochemistry results where we observed that the inner zones of some spheres seemed to have higher expression of the markers under study suggesting a hypoxic situation in the middle of these spheres. It has been hypothesized that the presence of low oxygen tensions in stem cell niches offers a selective advantage that is well suited to their particular biological roles (Cipolleschi et al. 1993). Collectively, this highlights the importance of the hypoxia microenvironment in favoring the growth of the stem-like cells suggesting a cellular response sensitive to changes in the microenvironment. Most cellular adaptations under hypoxia are commonly regulated by HIFs. We observed an elevated expression of both HIF-1α and HIF-2α in our stem-like cells grown under normoxia and hypoxia while HIF-1α was significantly expressed under hypoxia. It has been reported that when GBM-derived neurosphere cultures were grown in 1% oxygen, HIF1α protein levels increased dramatically and the mRNA encoding HIF-2, was induced over 10-fold (Bar et al. 2010). It has been highlighted that HIF-2α is preferentially expressed by CD133 + putative GSCs under both hypoxic and normoxic conditions, whereas HIF-1α is induced under hypoxia not only in CD133 + but also in CD133- glioma cells (Li et al. 2009b). Similarly, it was observed that the knockdown of HIF-2α in a primary GBM010 line completely blocked the upregulation of the side population signature genes, greatly suppressed the increase of CD133 levels and also abrogated the hypoxia-mediated increase in the sphere-forming capacity following hypoxia. The silencing of HIF-1α, on the other hand, had no significant effect on CD133 levels (Seidel et al. 2010). These evidences support HIFs to play a role in cellular adaptations under hypoxia.
The miRNAs could help to modulate the survival mechanisms of the stem cells under the hypoxia microenvironment. When we compared the expression of miRNAs in the actual stem cell cultured under normoxia and hypoxia, we found miR-34-5p, 128a-3p and 181a-3p to be downregulated by a fold difference of 3.5 to 5, 2 to 5 and 2 to 4 for respectively while miR-221-3p and 17-5p to be upregulated by a fold difference of 3.5 to 4 and 2.5 to 4 respectively. There are few evidences elucidating the underlying the effect of hypoxia on miRNAs using actual GSCs. However, most evidence is based on glioblastoma cells. HIF1 has been associated with the down-regulation of miRNAs. The miR-17-92 cluster was reported to be reduced in hypoxia-treated cells. The down-regulation of miR-17-92 by hypoxia stabilized HIF1 because this HRM is able to repress the expression of HIF1 through direct targeting (Yan et al. 2009). The miR-128 can modulate angiogenesis, functioning through suppression of P70S6K1, a kinase upstream of HIF-1a and VEGF. miR-128 is decreased in gliomas which correlates with increased angiogenesis, cell proliferation, and tumor growth. Induced expression of miR-128 is able to attenuate these effects, while forced expression of P70S6K1 can partly rescue the inhibitory function of miR-128 on cancer growth (Shi et al. 2012). Elsewhere, a study found 73 miRNAs to be differentially expressed by microarray analysis between MCF-7 spheroids cells and MCF-7-cancer stem cells as compared with their MCF-7 parental cells among which miR-210 was the most expressed (Tang et al. 2018). Upregulation of miR-210 a known hypoxia-induced miRNA, (Huang et al. 2009) indicated the presence of a hypoxic microenvironment in the MCF-7 spheroids cells and MCF-7-cancer stem cells. Elsewhere, a study that did miRNA profiling in GBM spheroid cultures grown in either 2 or 21% oxygen found miR-210 to be significantly upregulated in hypoxia in patient-derived spheroids (Rosenberg et al. 2015). These evidences show that miRNAs may represent a therapeutic target although it is not clear from the results whether this miRNA maybe related to specific cancer stem cell functions.
Besides HIF-1α and HIF-2α, the expression of SOX2, OCT4, GLUT-1, BCL-2 and survivin were also found to upregulated in the stem-like cells under normoxia and even further upon hypoxia induction. This observation could have been attributed to the transcription factor HIF that has been reported to regulate the expression of these genes (Covello 2006; Gordan et al. 2007; Seidel et al. 2010). Restricted oxygen conditions have been reported to expand the fraction of cells positive for a cancer stem cell marker or the side population in established cancer cell lines (Blazek et al. 2007; McCord et al. 2009). Notably, Oct4, and Sox2 are already confirmed factors that contribute to the survival and self-renewal of brain tumor stem cells (Gangemi et al. 2009; Du et al. 2009). Cancer stem cells have been shown to regulate Glut1, a transcriptional target of HIF, under hypoxia to a greater degree than non-stem cells (Li et al. 2009b). A study found Bcl-2 mRNA and its protein to be highly expressed in brain glioma stem cells when compared to their corresponding primary glioma cells. This observation was confirmed by a different study that also showed the brain cancer stem cells expressing higher levels of BCL2 protein when compared with CD133 negative cells. Therefore, as an anti-apoptotic gene, Bcl-2 assigns immortality characteristics to cells (Wang 2012; Qi et al. 2015). Upregulation of survivin has been observed in well-characterized patient-derived glioblastoma stem cell (GSC) lines on comparing the mRNA expression between GSC and non-GSC. An observation suggesting the possibility of the protein to contribute to therapy resistance in GSCs and its prognostic value in predicting postsurgical survival (Guvenc et al. 2013). Using the selected genes, we show the stem-like state and the hypoxia microenvironments to be playing a significant role in their expression that may be indirectly or directly through HIF-1α or HIF-2α or via the miRNAs.
The inconsistencies between the in vitro and in vivo culture models have forced the researchers to develop physiologically relevant in vitro assays that better reflect the tumor-microenvironment. The 3-D culture system can provide a simple realistic model to culture spheres that recapitulate the microenvironmental aspects creating the cell niches that can be used to study cellular interactions in depth. When we cultured our GBM03 and T98G cell lines under our modified 3D culture system, they were all able to form spheroids by the 12th day. The spheroid morphology was not so different between the cells cultured in NS34 or DMEM/FBS. The spheroids grown in both culture mediums expressed HIF-2α, Glut1, survivin and OCT4 by immunocytochemistry except for HIF-2 α and survivin that were suggestively upregulated in the spheroids cultured in NS34 media. Similarly, these spheroids had outstanding staining patterns highlighting an intricate cellular matrix connection. A study that compared the expression of OCT4 between 2D, neurosphere and 3D models found OCT4 to was upregulated in the 3D assays compared to the other two models (Musah-Eroje and Watson 2019). A study using MCF-7 found the cells exhibiting BCSC-like properties as shown by high expression of sox2 and oct4 when cultured as spheroid cells 3D-semisolid culture system as compared to monolayer culture (Tang et al. 2018). Both HIF-1α and HIF-2α have been shown to be crucially involved in the promotion of in vitro sphere formation, cell growth, and the survival of CD133 + glioma cells (Li et al., 2009). These evidences show that the tumour microenvironment could have been influenced reprograming of the cell towards a stem-like phenotype (Heddleston et al. 2009; Poli et al. 2018). Subject to further confirmation, these evidences suggest that maybe 3D culture model could be used as an alternative cheaper culture model. This is because the culture of neurospheres is time consuming as compared to the spheroid culture. Neurosperes take about four weeks to be fully formed while the spheroid culture takes less than twelve days related to the fact that true stem cells grow slower than their differentiated progeny(Kawai et al. 2016). Both culture models result in cell aggregation that can capture the hypoxia microenvironment possibly in the inner zones. Similarly, the components to making the NS34 media are expensive and to using the conventional culture media.