A crucial role of germ cell-like cell formation in tumour initiation
PGC-like cells were frequently observed in U251 and A172 mutant (A172 mut) glioma cell cultures but were barely observed in L229 and A172 wild-type (A172 wt) glioma cell cultures (Fig. 1B-D, Fig. S2A, B). We found that germ cell-like cell formation was positively correlated with tumour initiation in glioma cells, including U251, LN229, A172 wt and A172 mut cells (Fig. 1D and E). Notably, A172 wt cells without germ cell-like cell properties failed to form tumour within 120 days after grafting in nude mice. However, A172 mut cells that reobtained germ cell-like properties after treatment with a chemical carcinogen (3-methylcholanthrene, 3-MCA) gave rise to tumours within 2 weeks of grafting in nude mice (Fig. 1E). Interestingly, tumour tissues derived from A172 mut cells were teratocarcinomas (Fig. 1F), which provided strong support for the presence of germ cell-like cells since teratocarcinomas are thought to originate from germ cells [2, 5]. Germ cell-like tumour cells often grow above somatic tumour cells and can be easily separated from somatic tumour cells in vitro. We separated the upper cultures, which were enriched with PGC-like cells, from the bottom cultures, which contained a small proportion of PGC-like cells in A172mut cells, by shaking the flask rigorously. RT-PCR data showed that the expression of genes related to germ cells was much higher in the upper cells than in the bottom cells in A172 mut cultures (Fig. 1G). Compared to the bottom cells, the upper cells in A172 mut cultures showed increased tumour initiation abilities after injection into nude mice (Fig. 1G). Notably, SOX17 which links to the PGC state of human were reactivated in the upper cells of A172 mut cultures and expressed highly versus SOX2 which links to ES state of human (Fig. S2C). After culture, most single U251 and A172 mut cells generated clones containing PGC-like cells, indicating that PGC-like cells could be derived from somatic glioma cells (Fig. 1H).
We then investigated the relationship between germ cell formation and tumour initiation by deleting DAZL, which plays a crucial role in germ cell development from PGC specification, PGC fate determination to PGC further mature [34-36], with CRISPR-Cas9 technology. Since DAZL-/- glioma cells were not viable in culture possibly attributed to a crucial role of DAZL or germ cell fate in immortality of tumour cells, U251- DAZL+/-, LN229-DAZL+/- and A172mut- DAZL +/- cells were studied (Table S2). Our earlier study revealed that knockdown of DAZL inhibited tumour formation and increased the therapeutic sensitivity of cultured human glioma cells [24]. In this study, we further revealed that knockdown of DAZL greatly impaired germ cell formation and tumour initiation in U251, LN229 and A172 mut cells (Fig. 1I and J). These findings showed that activation of PGC-like cell formation from somatic tumour cells was essential in the initiation of glioma cell tumours, leading to tumourigenicity in new sites. Thus, activation of the PGC-like state might be necessary for a more aggressive stage of gliomas.
Genetic and epigenetic changes in genes related to embryonic/germ cell development in gliomas
PGC specification arises from ES cells [36-38], and findings in iPS cells highlight the possibility of somatic cell reprogramming [11]. This means that if a PGC-like fate truly occurs in gliomas, at least two key events are possibly involved: reprogramming and PGC specification (Fig. 1A). Therefore, we investigated whether the genes related to reprogramming, PGC specification and PGC development (including conversion of PGCs into EGCs and oogenesis from PGCs) were activated in gliomas and linked to the malignant prognosis of gliomas (Fig. 1A). The defined gene groups included those involved in reprogramming (inhibition: TP53; promotion: POU5F1, SOX2, MYC, KLF4), pluripotency (POU5F1, SOX2, MYC, KLF4, NOTCH1), induction of ES-PGC conversion by microenvironment (BMP2, BMP4, BMP8B, LIF), PGC specification (POUF51, PRDM1, SOX17, ACVR1, IFITM3), PGC fate maintenance (SOX17, PRDM1, KIT, Nanos3, DND1), PGC survival (DAZL, DDX4), proliferation/migration/survival (ITGB1, CXCR4, WNT5A, ROR2), meiosis (SYCP3, DMC1) , PGC-EGC conversion (inhibition: TP53, PTEN, BMP2, BMP4, SOX17; promotion: LIF, STAT3), oocytes (ZP3, GDF15) as well as early embryos (GDF15) [11, 33, 36, 37, 39-44].
To validate our hypothesis, Chinese Glioblastoma Genome Atlas (CGGA) data were analysed. Since tumourigenicity is thought to be the outcome of a series of genetic and epigenetic changes, we first investigated whether genetic and epigenetic changes in the gene groups were present in human gliomas. The whole-exome sequencing results of gliomas from the CGGA database showed that there were few or no genetic changes among a series of core genes and signalling pathways related to the embryonic/germ cell developmental axis in human gliomas (Fig. 2A). Additionally, the methylation analysis results revealed that most of the genes showed reduced levels of methylation (Fig. 2C). Of note, TP53 (46%), which inhibits reprogramming, PGC-EGC conversion and oocyte development [20, 36, 45, 46]; PTEN (7%), which strongly inhibits PGC-EGC conversion [9, 36]; and NOTCH1 (8%), which promotes pluripotency [36], were frequently detected among the genetic changes (Fig. 2A). TP53 and PTEN mutations were correlated with poor outcomes, while NOTCH1 mutations were associated with improved outcomes (Fig. 2B). The data indicated that epigenetic changes but not genetic changes, occurred in most of the core genes related to the embryonic/germ cell developmental axis in human gliomas, while the suppressor genes associated with reprogramming or PGC-EGC conversion frequently lost in gliomas with poor outcomes. Moreover, the reduced levels of methylation in the gene groups supported the activation of genes in gliomas.
Links of the embryonic/germ cell development-like axis to a malignant prognosis
Gliomas are pathologically classified as glioblastomas (GBM, grade WHO IV) and lower-grade gliomas (LGG, grades WHO II and WHO III) and have distinct malignant stages and outcomes [47]. We analysed the TCGA and GTEx dataset to compare the expression of several genes related with embryonic/germ cell development between gliomas and normal brain tissues. Compared to normal brain tissues, the mRNA level of SOX2, MYC, NOTCH1, STAT3, BMP2, ACVR1, ITGB1, WNT5A, CXCR4 and ZP3 increased significantly in both GBM and LGG as well as the mRNA levels of LIF, PRDM1, IFITM3 and GDF15 increased significantly in GBM but not in LGG (Fig. S3). However, there was no significant differentiation in the mRNA levels of BMP4, BMP8B, SOX17, KIT, NANOS3, DND1, DAZL, DDX4, ROR2, SYCP3 and DMC1 between gliomas and normal brain tissues (not shown). To further determine whether the gene groups were activated in gliomas and linked to pathologic classification, malignant prognosis and clinical outcomes, we analysed the mRNA sequencing results of gliomas from the CGGA database. The data showed that IPS reprogramming factors and pluripotency-related genes (POU5F1, SOX2, KLF4, MYC and NOTCH1) were detected in almost all histologic types and grades of gliomas (Fig. 3A, B, and Fig. S4A, S5A, Table S3-S5). While the mRNA levels of genes related to pluripotency (SOX2, KLF4, MYC and NOTCH1) were not obviously correlated with the clinical glioma grade or outcome, the mRNA level of POU5F1, which also plays a crucial role in PGC specification, was often linked to a higher glioma grade and poor outcome (Fig. 3A, 3B, 4A, 4C, 4D and Fig. S6A, Table S3-S5). These findings revealed that the activation of reprogramming-related genes was commonly seen even in low-grade gliomas; however, it might be insufficient to lead to a malignant prognosis.
As expected, the mRNA sequencing results of gliomas from the CGGA database showed that the mRNA levels of various genes related to germ cell development were also enriched in some gliomas, including LIF, PRDM1, BMP2, BMP4, BMP8, ACVR1, IFITM3, ITGB1, CXCR4, WNT5A, ROR2, ZP3, GDF15, SOX17, DAZL, DDX4, SYCP3 and DMC1 (Fig. 3A, and Fig. S4, S5B, S5C, Table S3, S4). The mRNA levels of PRDM1, a core germ cell specification gene, as well as its core upstream gene ACVR1 and downstream gene IFITM3 were totally associated with advanced pathologic grades and poor outcomes (Fig. 3B, 4B-D, and Fig. S5B, S6A, Table S3-S5). High mRNA levels of genes related to PGC proliferation/survival/migration (ITGB1, CXCR4, WNT5A and ROR2), ES-PGC conversion (BMP8B and LIF), PGC-EGC conversion (LIF and STAT3), PGC survival (DAZL) and oocytes/early embryos (GDF15) also showed totally correlations with an advanced pathologic grade and poor overall survival of patients (Fig. 3B, 4B-D, and Fig. S5B, S6A, Table S3-S5). Notably, POU5F1 partners with SOX2 in human ES cells and partners with SOX17 in human PGCs [33, 38]. Although the mRNA levels of SOX17, a core germ cell specification gene [38], were enriched in some gliomas, they did not show obvious correlations with advanced pathologic grades and poor outcomes (Fig. 3B, 3D, 4C, 4D and Fig. S5B, S6A, Table S3-S5), likely because SOX17 also inhibits PGC-EGC reprogramming [33]. The mRNA level of gene related with later meiosis (DMC1) was slightly associated with poor overall survival (Fig. 4C, 4D and Fig. S5B, S6A, Table S5). The mRNA levels of genes related to the activation of ES-PGC conversion but inhibition of PGC-EGC conversion (BMP2 and BMP4), late PGCs (DDX4), meiosis entry (SYCP3) as well as genes related to PGC fate determination (KIT, NANOS3 and DND1) showed no or negative correlations with advanced pathologic grades and poor outcomes (Fig. 3B, 4C and Fig. S5B, S6A, Table S3-S5). These data suggest that tumour cells arrested in the germ cell-like developmental stage (not early PGC-like stage) and that lost the ability to return to the embryonic cell-like state might not lead to a malignant prognosis of gliomas. Compared with LGG, GBM showed higher expression of POU5F1, PRDM1, BMP8B, LIF, STAT3, ACVR1, IFITM3, ITGB1, CXCR4, WNT5A, ROR2 and GDF15 (Fig. 3A, 3B, and Fig S2A, Table S4), indicating that both the activation of the PGC-like state and the return of germ cells to the embryo-like state were essential in the malignant behaviours of some gliomas via somatic parthenogenetic embryo-like cycle and/or somatic PGC-EGC/ES-like cycle. Compared with LGG, secondary GBM arising from LGG [48] showed higher expression of PRDM1, BMP8B, ACVR1, LIF, STAT3, ITGB1, WNT5A, CXCR4, MYC but decreased expression of genes related to germ cell fate, including Nanos3, DND1, BMP2, BMP4, SOX17, DDX4, SYCP3, DMC1 and ZP3 (Fig. 3A, 3C, and Fig. S4A, Table S4), indicating that the PGC-EGC/ES-like conversion pathway rather than the mature development pathway might be activated in secondary GBM and that the activation of PGC-EGC/ES-like cycle was linked to the malignant prognosis of LGG. Compared with primary GBM, secondary GBM showed higher expression of ITGB1, ACVR1, MYC, STAT3 and BMP2 but significantly decreased expression of genes related to germ cell mature (Nanos3, DND1, DDX4, SOX17, SYCP3, DMC1, ZP3 and GDF15) and genes related to inhibition of PGC-EGC conversion (BMP4 and SOX17) (Fig. 3A, 3D, and Fig. S4A, Table S3), further indicating that the activation of PGC-EGC/ES-like conversion was one of driving events to malignant prognosis of gliomas. In addition, the decreased expression of genes related to germ cell development was accompanied by the decreased expression of GDF15, consistent with the fact that activation of gene GDF15 links to the oocyte/early embryo-like state (Fig. 3A, 3D, and Table S3). Collectively, these findings indicated that the genes related to the activation of the parthenogenetic embryo-like cycle and activation of the PGC-EGC-like cycle were both linked to poor patient outcomes, which might represent two pathways that drive the malignant prognosis of gliomas.
Inhibition of embryonic/germ cell cycle-related gene expression among gliomas with 1p19q codeletion
LGG have wide survival ranges, from 1 to 15 years, and distinct therapeutic sensitivities [47]. LGG with deletion of chromosome arms 1p and 19q (1p19q codeletion) are often associated with impressive therapeutic sensitivities and favourable clinical outcomes [47]. Consequently, we determined whether genes related to the embryonic/germ cell developmental axis were inhibited in glioma samples with 1p19q codeletion. The combined mRNA sequencing data from the 325 and 694 datasets of CGGA showed that the mRNA levels of POU5F1, LIF, BMP8B, ROR2, CXCR4, IFITM3, ITGB1 and GDF15 were extremely low in almost all glioma samples with 1p19q codeletion (1p19q-codel) compared to glioma samples without 1p19q codeletion (1p19q-noncodel) (Fig. 5A-C, and Table S6). Overall, the mRNA levels of ACVR1, STAT3, WNT5A, KLF4 and DMC1 were obviously decreased in glioma samples with 1p/19q codeletion compared to those in glioma samples without 1p19q codeletion (Fig. 5A-C, and Fig. S7, Table S6). However, 1p19q codeletion did not inhibit the mRNA levels of SOX2, SOX17, SYCP3, MYC, ZP3, NOTCH1, DDX4, KIT, NANOS3, DND1, BMP2 and BMP4 (Fig. 5A-C, and Fig. S7, Table S6). These findings indicated that 1p19q codeletion robustly inhibited genes involved in promoting the embryonic/germ cell cycle.
Clinical significance of embryonic/germ cell cycle-related molecular groups among gliomas
Histopathological classification is often performed in gliomas; however, this method is not sufficient to predict clinical outcomes [47]. Consequently, we performed an analysis of the molecular group related to the embryonic/germ cell cycle (including LIF, STAT3, PRDM1, IFITM3, ACVR1, CXCR4, WNT5A, ROR2, ITGB1, POU5F1, GDF15 and BMP8B) (Table S7) to determine whether we could identify glioma outcomes more accurately based on the germ cell-related molecular group than based on the histologic class. The combined mRNA sequencing data of 325 and 694 CGGA datasets showed that patients with higher expression of genes in the molecular group had much poorer overall survival than patients with lower expression of genes in the gene groups (Fig. 6A). The median survival of patients in the two groups was 443 days (high) and 3,411 days (low) respectively (Fig. 6A, and Table S8). Notably, increased expression of genes in the molecular group was frequently observed in the same gliomas (Fig. 6B). Approximately 71.92% of gliomas with increased expression of genes in the molecular group had at least two genes with increased expression (Fig. 6B). Among grade II-primary, II-recurrence, III-primary, II-recurrence, IV-primary, IV-recurrence gliomas and IV-secondary gliomas, the total ratio of gene groups with high expression of a single gene was ~24.71%, ~52.50%, ~40.88%, ~58.3%, ~83.98%, ~89.91% and ~100%, respectively (Fig. 6C, and Table S9), further indicating that activation of the embryonic/germ cell-like developmental axis may be associated with the pathological grade, recurrence and progression of gliomas. Interestingly, the mRNA profiles of the molecular group could be used to separate poor outcomes from improved outcomes among patients who harboured gliomas with the same WHO grade, especially among the patients with grade III gliomas (Fig. 6C, and Table S8, S10). Median survival was 344 days (higher) and 710 days (lower) among patients with grade IV gliomas, 544 days (higher) and 2,633 days (lower) among patients with grade III gliomas, and 2,219 days (higher) and more than 5,000 days (lower) among patients with grade II gliomas (Fig. 6D, and Table S8). Notably, glioma classification based on the WHO grade and molecular group could predict clinical behaviours more accurately (Fig. 6E, Fig. S8A and Table S8, S10). Subtype grade II gliomas with lower expression of genes in the molecular group had favourable clinical outcomes (Fig. 6E and Table S8). Grade III gliomas with higher expression of genes in the molecular group were similar to grade IV gliomas in terms of clinical outcomes (Fig. 6E, and Table S8).
Among patients who had gliomas with 1p19q codeletion in the combined dataset, patients with higher expression of genes in the gene group (median survival=1265 days) had much poorer overall survival than patients with lower expression of genes in the molecular group (median survival> 5000 days) (Fig. 6F and Table S8). Among the gliomas without 1p19q codeletion in the combined dataset, the median survival of the two groups of patients was 415 days (higher) and 2382 days (lower) (Fig. 6F and Table S8) respectively. Classification by combining the 1p19q status and the molecular group could subdivide gliomas into four subtypes with distinct clinical outcomes (Fig. 6F, G and Table S8), suggesting a possible molecular method to predict clinical behaviour. These findings indicate that the embryonic/germ cell cycle-related molecular group can be used as a good marker for predicting clinical outcomes and may be associated to the low-glioma progression to advanced gliomas.
Appearance of embryonic/germ cell-like cells in gliomas
We then investigated whether embryonic/germ cell-like cells were present in human gliomas and linked to malignant traits. HE and immune staining showed that germ cell-like cells could be observed in some human glioma tissues, and the appearance of germ cell-like cells was associated with the pathological grade of gliomas (Fig. 7A, and Fig. S8). Compared with LGG, germ cell-like cells were easily observed in GBM (Table S11). Notably, a series of embryonic/germ cell-like cells at different developmental stages could be observed in the same tumour tissues among some grade IV gliomas (approximately 40%), including PGC-like cells, oocyte-like cells and parthenogenetic preimplantation embryo-like cells, indicating that a somatic parthenogenetic embryo-like cycle might be present in some GBM (Fig. 7B, and Fig. S8). It was documented that polyploid giant cancer cells (PGCCs) were blastomere-like cells [1, 49]. Taken together, it is possible that activation of the embryonic/germ cell-like developmental axis occurs during tumour initiation and malignant prognosis.