CALD1 Modulates Gliomas Progression via Facilitating Tumor Angiogenesis

Abstract

Angiogenesis is more prominent in anaplastic gliomas and glioblastoma (GBM) than that in pilocytic and diffuse gliomas. Caldesmon (CALD1) plays roles in cell adhesion, cytoskeletal organization, and vascularization. However, limited information is available on mechanisms underlying the effect of CALD1 on the microvascular facilitation and architecture in glioma. In this study, we explored the role of CALD1 in gliomas by integrating bulk RNA-seq analysis and single cell RNA-seq analysis. A positive correlation between CALD1 expression and the gliomas' pathological grade was noticed, according to the samples from the TCGA and CGGA database. Moreover, higher CALD1 expression samples showed worse clinical outcomes than lower CALD1 expression samples. Biofunction prediction suggested that CALD1 may affect glioma progression through modulating tumor angiogenesis. The map of the tumor microenvironment also depicted that more stromal cells, such as endothelial cells and pericytes, infiltrated in high CALD1 expression samples. CALD1 was found to be remarkably upregulated in neoplastic cells and was involved in tumorigenic processes of gliomas in single cell sequencing analysis. Histology and immunofluorescence analysis also indicated that CALD1 associates with vessel architecture, resulting in glioma grade progression. In conclusion, the present study implies that CALD1 may serve as putative marker monitoring the progress of glioma.

Keywords: CALD1; angiogenesis; brain tumor; glioma.

Figure 1

The expression profile, the overall survival analysis and pathways prediction based on CALD1 expression in the TCGA dataset. The distribution of CALD1 in cancer (A), WHO grade (B), IDH status (C), 1p19q status (D), MGMT status (E) and glioma subtype (F). The overall survival analysis based on the LGGGBM cohort (G), the LGG cohort (H), the GBM cohort (I), MGMT status (J) and radiotherapy (K). (L,M) The correlation between CALD1 expression and pathways from the GO/KEGG enrichment analysis. NS: no statistical difference; ***: p value < 0.001.

Figure 2

Stromal cells proportion in tumor microenvironment. (A) The correlation between CALD1 expression and the enrichment score of stromal cells. (B) The distribution of stromal cells in high and low CALD1 expression samples. (C) The correlation between the expression of CALD1 and genes from GO pathways. NS: no statistical difference; ***: p value < 0.001.

Figure 3

Single cell sequencing analysis in GBM microenvironment. (A) UMAP plot depicting eight cell clusters and the distribution of CALD1 expression levels within the eight cell clusters. (B) Violin plot exhibiting the relative expression level of CALD1 in eight cell clusters. (C) The single-cell trajectory of neoplastic cells contains four main branches. Cells are colored based on state (left), pseudotime (middle), and CALD1 (right). (D) GO enrichment analysis of CALD1 in BP in neoplastic cells. (E). GO enrichment analysis of CALD1 in MF in neoplastic cells. (F). KEGG enrichment analysis of CALD1 in neoplastic cells.

Figure 4

CALD1 facilitates GBM cells moveability in vitro and associates with tumor progression in pathological angiogenesis. (A) Immunofluorescent staining of l-CALD1 in cell line LN229. (B) Immunofluorescent staining of l-CALD1 in cell line U251. (C) Knock-out l-CALD1 affected mobility of GBM cells. (D) IHC staining of l-CALD1 in glioma cases according to WHO grades progression. (E) Comparing l-CALD1 in normal blood vessels and tumor angiogenesis. (F) Measurement of CRISPR/Cas9 knock-out l-CALD1 efficiency in U251 and LN229 by protein level. (G) Statistics of GBM cells mobility after koCALD1. (H) Statistics of comparing l-CALD1 expression level according to WHO grade progression. (I) Statistics of comparing l-CALD1 expression level in normal blood vessels and tumor angiogenesis. *: p value < 0.05; **: p value < 0.01.

Figure 5

The CI model based on genes encoding calmodulin-dependent proteins in the TCGA dataset. (A) The distribution of CI was illustrated by a heatmap, along with clinical features and the expression of genes encoding calmodulin-dependent proteins. The association with cancer (B), WHO grade (C), IDH status (D), 1p19q status (E), MGMT status (F) and glioma subtype (G). The overall survival analysis based on the LGGGBM cohort (H), the LGG cohort (I) and the GBM cohort (J). The overall survival analysis based on the subgroup of MGMT status (K) and radiotherapy (L). NS: no statistical difference; ***: p value < 0.001.

Figure 6

The GO/KEGG enrichment analysis and the GSEA analysis based on the CI model. (A) The relationship between CI and enrichment score of pathways from the GO/KEGG enrichment analysis. (B) The correlation between CI and pathways from the GO/KEGG enrichment analysis. (C) The GSEA analysis based on high and low CI samples.