Characterization of NSG-70, a new glioblastoma stem cell line which harbours a marker isochromosome 9q (i9q)
NSG-70, a long-term cell line was derived from tumor tissue of a patient with recurrent glioma and has been cultured through more than 25 passages, whereas concurrently established normal brain cell cultures (NB) derived from epilepsy patient-derived biopsies could not be maintained beyond 5–6 passages. The adherent NSG-70 culture presented as small, translucent, spindle-shaped cells characteristic of Grade IV gliosarcoma tumors, that could also generate non-adherent organised neurospheres within 8–10 days in culture at a high frequency (55–60%), indicating a glioma stem cell (GSC)-like nature (Fig. 1a).
NSG-70 GSCs displayed a low proliferation potential with ~ 150h doubling time (determined through MTT assay and low Ki-67 expression, Figs.S1a;S1b). This slow-cycling (almost quiescent) nature was validated by PKH dye quenching assay that retained the dye in around 98.5% population even after six days in culture (Fig.S1c), and further reflected in high latency during xenograft formation, with all mice developing small tumors after four months of injection (106 cells; Fig.S1d). Immunohistochemistry of the NSG-70 xenografts displayed typical spindle shaped morphology of the tumor cells that ascertained the gliosarcoma phenotype, along with wild type ATRX and IDH1, and mutated p53 expression (Fig. 1b; Fig.S1e). Ki-67 and CD34 expression was restricted to < 40% cells, affirming its low proliferation index and indicating scanty tumor vessel density respectively.
To ascertain their stem-like nature, we profiled NSG-70 cells for expression of neural progenitor (SOX2, NESTIN and MSI1) as well as differentiation markers (glial fibrillary acidic protein – GFAP and S100 calcium-binding protein - S100β for astrocytic; TUJ1 and MAP2 for neuronal and OLIG2 for oligodendroglial lineages; Fig. 1c-i). Immunostaining of NSG-70 neurospheres also identified enhanced expression of stem cell markers CD133, SOX2, NESTIN and MSI1, while flow cytometry-based profiling and quantification indicated the presence of ~ 13% CD133 and 69.6% CD44-positive cells in adherent cultures (Figs. 1c-ii,d-I and d-ii). Presence of serum in the culture medium reduced the levels of stem cell marker expression (Fig. 1e). Xenograft tumors also expressed high levels of SOX2, CD44 and GFAP (Fig. 1f), confirming the presence of GSC-like cells along with differentiated cells. Further study of gross chromosomal rearrangements in NSG-70 cells in culture (serum as well as serum-free medium) indicated the cell line to be aneuploid with random loss and gain of various chromosomes in different metaphases, which lead to a near tetraploid complement (Fig. 1g-i). The most striking feature in a large majority of cells in the population was the presence of an isochromosome of the long arm of chromosome 9 (Isochromosome 9q or i9q). Chromosomal or cytogenomic microarray analysis that involves differential binding of specific probes to the site of genomic imbalances to enable identification of deletions/loss, duplications (gain/amplification), unbalanced translocations validated the presence of isochromosome 9q, with specific gain of cytoband q21.11-q34.3 (Fig. 1g-ii). Strikingly, this location harbors the NOTCH1 gene, which is of special interest in the context of GSC regulation and functions.
NSG-70 GSC culture is likely to represent an in vitro model of mixed lineage
In order to understand the molecular networks regulating NSG-70 cells, we profiled their gene expression along with two other GSC cell lines earlier established in the lab (KW and MUR) and further compared them with the expression of NB primary cultures. This profiling indicated significant alteration of specific genes and pathways in GSCs over normal, suggesting their possible contribution to gliomagenesis (Fig.S2a).
We hence oriented our study to compare the levels of stem cell and neural lineage markers in the GSC lines relative to hNSC and NB cells. All cells expressed considerable heterogeneity of these markers although high levels of CD44 expression in the 3 GSCs and lower levels of stem cell markers in NB cells were observed (NESTIN, OCT4 and NANOG; Fig. 2a; Fig.S2b) possibly indicating their roles in maintaining progenitor pool and multipotency. Significantly higher expression of oligodendrocyte lineage transcription factor 2 (OLIG2), neuronal precursor marker doublecortin (DCX), astrocytic marker (GFAP) was identified in GSCs compared to NB, the latter suggests astrocytes to be the predominant population (Fig. 2b). OLIG2 expression was highest in NSG-70 cells, suggesting that a subset of cells is committed to an oligodendroglial fate towards a proneural phenotype. The heterogeneity in the expression of stem cell and lineage markers was also reflected at the protein level (Figs. 2c; Fig.S2c).
Further, to delineate the subtype of GSC cultures, we profiled the expression levels of proneural and mesenchymal subtype-specific genes in the 3 GSC cell lines, which revealed KW to be predominantly proneural and MUR as mesenchymal, while NSG-70 presents with a mixed lineage (proneural and mesenchymal subtypes (Fig. 2d). In conclusion, NSG-70 is a new primary glioblastoma cell line representing a mixed (proneural and mesenchymal) subtype with high expression level of self-renewal molecules indicating its inherent stem-like nature with potential to differentiate into different lineages, along with a striking feature of a novel marker i9p marker.
NOTCH1 knockdown leads to reduction in stemness and mesenchymal markers, along with decreasing WNT5A expression
To elucidate the role of Notch pathway in self-renewal and differentiation of stem cells, we first evaluated the effects of NOTCH1 knockdown on stemness in the three GSC lines (NSG-70, KW and MUR). Our results demonstrated diminished expression of stemness as well as mesenchymal markers such as CD44, TWIST1 and FN1 in the 3 GSC lines (Figs. 2h-i; 2h-ii; 2i; Figs. S2g-i; 2g-ii; 2h). The cross-talk of Notch1 and Wnt signaling pathway has been shown as crucial in stem cell maintenance (Collu et al. 2014). However, recent reports suggested the role of Wnt5a in inducing mesenchymal-like characteristic in GSCs, there is a dearth of knowledge on the cross-talk of NOTCH1 and WNT5A. Hence, we sought to understand the significance of these signaling pathways in glioma. We identified significantly higher expression of NOTCH1 and WNT5A mRNA in primary GBM tumor tissues compared to normal tissues in the UALCAN database (Figs. 2e-i; 2e-ii). Further examining the expression levels of these two molecules in the three GSC lines, we observed that MUR cells expressed highest levels of both NOTCH1 and WNT5A, whereas KW and NSG-70 expressed moderate levels of NOTCH1 and very low levels of WNT5A (Figs. 2f; 2g). Interestingly, knockdown of WNT5A alone in WNT5A-high cells i.e., MUR also showed significantly decreased levels of stem cell and mesenchymal markers (Figs. 3a; 3b), suggesting coordinated action of NOTCH1 and WNT5A in maintaining stemness and promoting mesenchymal characteristics in GSCs.
WNT5A overexpression or induction in GSCs enhances NOTCH1 levels, stem cell and mesenchymal marker expression
In either case of NOTCH1 and WNT5A knockdown, reduction in the levels of stemness and mesenchymal markers at RNA and protein levels were concurrently detected in GSC lines; this suggests a coregulation of NOTCH1 and WNT5A in GSCs. Prompted by this observation, we assessed NOTCH1 expression upon overexpression of WNT5A in NSG-70 and KW (WNT5A-low GSCs) at RNA and protein levels, to explore if WNT5A wcould influence NOTCH1 levels. The data indicated elevated expression of NOTCH1 and its target gene HES1, stem markers (NESTIN, SOX2), and mesenchymal markers (FN1, TWIST1, VIMENTIN) in WNT5A overexpressed GSCs compared to their control counterparts (Figs. 3c; 3d). This suggests that components of WNT5A pathway synergize with NOTCH1, promoting GSC characteristics and thereby contributing to tumor progression.
WNT5A knockdown drives GSCs into proliferative phase
We also detected lower levels of p21 upon NOTCH1 knockdown in NSG-70 and MUR (Figs. 2h-ii ; 2i) whereas higher p21 levels after WNT5A overexpression (Fig. 3c), which indicated the involvement of WNT5A in cellular proliferation.
We further assessed the cell cycle progression of MUR (WNT5A silenced) as well as NSG-70 and KW (WNT5A overexpressed or rhWNT5A induced). Our data demonstrated that WNT5A knockdown in MUR facilitates cell proliferation marked by an increase in S phase cells (Fig. 3Sf). Flow cytometry and immunofluorescence to evaluate Ki67 positive population, along with MTT assay in MUR, also displayed similar results (Figs. 3e, f and g). All these data suggested that WNT5A knockdown drives GSCs into proliferative phase. Contrarily, the reduction in cellular proliferation upon WNT5A overexpression in WNT5A-low GSCs (NSG-70 and KW) as indicated by flow cytometry to measure the Ki67 positive population as well as MTT assay (Figs. 3h, i-i, and i-ii), supports the role of WNT5A in maintaining GSC quiescence.
NOTCH1 in association with WNT5A, promotes vasculogenic mimicry in GSCs besides maintaining stem cell vigor
The decreased levels of VEGFA and tumorigenicity observed in KW cells following NOTCH1 knockdown (Figs. 4a;4b; Fig. S3a), along with NOTCH1 being an important regulator of vasculogenic mimicry (Fig. 4d), we were curious to evaluate whether rhWNT5A too would have promoting vasculogenic mimicry in the WNT5A-low GSC (NSG-70) and on comparison with WNT5A-high GSCs (MUR). Our data showed almost 50% increase in the total number of junctions as well as the tube length in rhWNT5A treated cells compared to control. NOTCH1 knockdown significantly decreased both these parameters by approximately 75% whereas rhWNT5A treatment of NOTCH1 silenced cells effectively rescued the ability to form vessel like structures (Fig. 4d). Additionally, WNT5A induction of GSCs also resulted in higher expression of not only NOTCH1 but also other angiogenesis related genes including CD31, VEGFA and PDGFRα (Fig. 4c). This underlines the involvement of WNT5A in promoting vasculogenic mimicry in GSCs and might represent one of the crucial alternative ways by which GSCs promote tumor neovascularization.
Further to investigate whether silencing NOTCH1 and WNT5A expression was associated with reduced stem cell vigor of GSCs, we analysed the surface expression of the GSC marker CD44, by flow cytometry. NOTCH1 knockdown showed significant reduction in the CD44 expression in KW and MUR, while non-significant reduction in NSG-70 (Figs. 4e-i; 4e-ii, 4e-iii). WNT5A knockdown in MUR also showed decrease in CD44 levels (Fig. 4e-iv). Further to compare the frequencies of stem cells in control and NOTCH1 knockdown MUR cells, we compared their neurosphere formation ability and observed a significant reduction in the number as well as size of neurospheres generated from single cells (Fig. 4f). Similarly, we also observed a remarkable decrease in the adherent independent tumor cell growth and invasive ability of MUR upon NOTCH1 as well as WNT5A knockdown, as represented by the soft agar assay (Fig. 4g). All this together implies the collective role of NOTCH1 and WNT5A in maintaining stem cell characteristics in GSCs.