C1RL expression was upregulated in GBM, especially mesenchymal GBM, primary GBM and IDH1-wt GBM.
In this study, we employed 2120 glioma specimens and 23 non-tumour brain tissues from 5 datasets. The characteristics and clinical information of the 5 datasets were summarized in S Table 1.
C1RL expression was analysed according to the WHO classification, GBM subtype, GBM status and IDH1 mutation status. First, the expression of C1RL was always highest in GBM in the 5 datasets according to both the grading system and the histology system (Fig 1.A-H). However, the expression levels of C1RL in GBM samples varied greatly. Furthermore, C1RL expression among different subgroups of GBM was analysed. Among the four transcriptomic subgroups of GBM, C1RL expression was always highest in mesenchymal GBM (Fig 1.I-L). Secondary GBM was developed from lower grade glioma and exhibited lower C1RL expression than primary GBM (Fig 1.M-N). The IDH mutation status is a well-accepted marker for glioma classification. C1RL expression was higher in IDH1-wt GBM than in IDH1-mt GBM (Fig 1.O-Q). In addition, C1RL expression was higher in IDH1-wt LGG than in IDH1-mt LGG (Fig 1.P and Q). These results suggested that higher C1RL expression accompanies more advaced malignancy in glioma, especially in GBM.
C1RL-associated genes were enriched in the biological processes of the immune response.
The biological function of C1RL, especially in tumours, has not been clarified thoroughly. Therefore, we aimed to identify the possible biological function of C1RL through analysis of the biological functions of C1RL-associated genes. C1RL-associated genes were defined as genes with expression trends similar to those of C1RL in glioma samples. All the C1RL genes from each dataset were listed in S Table 2 and were evaluated by GO analysis. The biological processes are listed in reverse order of their p values. The GO analyses showed that C1RL-associated genes were mainly enriched in the biological processes of the immune response, inflammatory response, IFN-γ mediated signalling pathway, and innate immune response (Fig 2.A-D).
To determine whether C1RL plays a positive role in the anti-glioma immune response, the expression relationships between C1RL and existing biomarkers were analysed. The CD86 protein is the receptor of CTLA4 and is mainly expressed on dendritic cells and monocytes. Galectin-9, which is encoded by LGALS9, was identified as the ligand of Tim-3 and plays a key role in T cell apoptosis. TGFB1 encodes a secreted ligand in the transforming growth factor-beta (TGF-beta) superfamily of proteins. CD86[20], LGALS9[21], and TGFB1[22] play immunosuppressive roles in glioma. Our results showed that C1RL expression exhibited positive relations with CD86, LGALS9, and TGFB1 (Fig 3.A-L).
C1RL expression was correlated with reduced tumour purity and increased M2 macrophage infiltration
The immune response is based on the migration of immune cells. Both tumour purity and the infiltration of 22 types of leukocytes were assessed for each sample in the TCGA datasets. The samples are displayed in order of their C1RL expression level. Both the immune score and the stromal score exhibited a positive correlation with C1RL expression trends (Fig 4.A and B, top panels). In addition, tumour purity showed an inverse correlation with C1RL expression trends (Fig 4.A and B, middle panels). Moreover, C1RL expression was mostly related to the infiltration of M2 macrophages among the 22 types of leukocytes (Fig 4.A and B, bottom panels).
High expression of C1RL predicted unfavourable survival and therapeutic resistance in glioma
The median expression value of C1RL was used to separate samples into two subgroups. We evaluated the prognostic value of C1RL in the four glioma datasets. The patients with glioma exhibiting higher C1RL expression had significantly shorter survival times than their counterparts in the GSE16011mic, TCGAseq, CGGAmic, and CGGAseq datasets (Fig 5.A-D). However, the histopathology characteristics in the two subgroups are significantly different (S Table 3). The histopathology characteristics may contribute to the survival differences. Next, we compared survival times among GBM patients with different C1RL expression levels in the five datasets. All the survival curves exhibited significant differences (Fig 5.E-I). Moreover, the effectiveness of well-accepted treatment was evaluated in different C1RL expression groups. The primary GBM patients with lower C1RL expression showed better responses to resection, radiochemotherapy (temozolomide), and standard therapy (Fig 5.J-L). These results indicate that C1RL may contribute to therapeutic resistance.