PTX3 mediates the infiltration, migration, and inflammation-resolving-polarization of macrophages in glioblastoma

Abstract

Introduction: Pentraxin 3 (PTX3) is an essential regulator of the immune system. However, the immune-modulatory role of PTX3 in the tumor microenvironment of glioma has not been elucidated.

Methods: The RNA seq samples were obtained from The Cancer Genome Atlas (TCGA) and the China Glioma Genome Atlas (CGGA) datasets. The single-cell sequencing data of glioblastoma (GBM) samples were obtained from the Single Cell Portal platform (http://singlecell.broadinstitute.org). Immunohistochemistry was used to assess PTX3 expression, HAVCR2, PD-1, PD-L1, and CD276 in glioma sections from the Xiangya cohort (n = 60). Multiplex immunofluorescence staining of PTX3, CD68, and CD163 was performed in several solid cancer types, including GBM. HMC3 was cocultured with U251 and U87, and transwell assay and flow cytometry assay were performed to explore the migration and polarization activity of HMC3.

Results: PTX3 expression is significantly increased in GBM. PTX3 expression predicts worse survival in the Xiangya cohort. PTX3 is closely related to the expression of PD-1, PD-L1, CD276, and HAVCR2 in the tumor microenvironment. Additionally, PTX3 is involved in tumorigenic and immunogenic processes, especially the activity of macrophages based on various signaling pathways in cellular communications and critical transcription factors. Specifically, PTX3 actively mediates macrophages' infiltration, migration, and inflammation-resolving-polarization. PTX3 could also predict immunotherapy response.

Conclusion: PTX3 is critically involved in macrophage infiltration, migration, and inflammation-resolving-polarization and modulates an immunosuppressive microenvironment.

Keywords: PTX3; cellular communication; glioma microenvironment; macrophage; transcription factor.

FIGURE 1

Inter‐tumor and intra‐tumor heterogeneous expression characteristics of PTX3 in gliomas. (A) PTX3 expression in proneural, neural, classical, and mesenchymal subtypes in the TCGA dataset. (B) The ROC curve indicates the sensitivity and specificity of PTX3 expression in predicting the ME and CL subtypes. (C) PTX3 expression in primary, secondary, and recurrent gliomas in the TCGA dataset. (D) Intra‐tumor distribution of PTX3 in LE (Leading Edge), IT (Infiltrating Tumor), CT (Cellular Tumor), PAN (Pseudopalisading Cells Around Necrosis), PNZ (Perinecrotic Zone), MVP (Microvascular Proliferation), and HBV (Hyperplastic Blood Vessels) regions based on Ivy GBM RNA‐seq data. (E) PTX3 expression in different radiographical regions in the TCGA dataset. CE, contrast‐enhanced; NCE, non‐contrast‐enhanced; NT, normal tissue. (F) The ROC curves indicate the sensitivity and specificity of PTX3 in predicting 3‐year and 5‐year survival in the TCGA and CGGA datasets. (G) Representative images of IHC staining for PTX3 in different pathological grades of gliomas in Xiangya cohort. (H) Statistical analysis of H‐score regarding PTX3 expression in Xiangya cohort. (I) Kaplan–Meier analysis of OS of glioma patients based on high vs. low expression of PTX3 (H‐score) in Xiangya cohort.

FIGURE 2

IHC staining for classical immune checkpoints. (A) Representative images of IHC staining for HAVCR2 in different pathological grades of gliomas. (B) Scattering plot depicting the correlation between HAVCR2 and PTX3 based on the H‐score. (C) Representative images of IHC staining for PD‐1 in different pathological grades of gliomas. (D) Scattering plot depicting the correlation between PD‐1and PTX3 based on the H‐score. (E) Representative images of IHC staining for PD‐L1 in different pathological grades of gliomas. (F) Scattering plot depicting the correlation between PD‐L1and PTX3 based on the H‐score. (G) Representative images of IHC staining for CD276 in different pathological grades of gliomas. (H) Scattering plot depicting the correlation between CD276 and PTX3 based on the H‐score. (I) Statistical analysis of H‐score regarding HAVCR2, PD‐1, PD‐L1, and CD276 expression in Xiangya cohort.

FIGURE 3

Molecular features of PTX3 at the single‐cell level. (A) t‐SNE for the dimension reduction and visualization of aneuploid cells, diploid cells, and other 14 cell types within the tumor microenvironment. (B) UMAP for the dimension reduction and visualization of cells with high or low PTX3 expression. (C) Relative proportion of four cell types in cells with high or low PTX3 expression. (D) The differentially expressed genes among the identified 14 cell types. E. PTX3 expression in three cell states based on pseudotime analysis. (F) Pseudotime trajectory analysis based on PTX3 expression. (G) Top six differentially expressed genes between high and low PTX3 expression. (H) GO and KEGG enrichment analysis of differentially expressed genes between high and low PTX3 expression.

FIGURE 4

Cellular interaction within the two neoplastic cell clusters with different PTX3 expressions. The cellular interaction network identified cell clusters in various signaling pathways, including A. VEGF, B. VISFATIN, C. LT, D. FSH, E. IL17, and F. IL10 signaling pathways.

FIGURE 5

The relationship between PTX3 expression and transcription factors. (A) The heatmap for the distribution of eleven modules of transcription factors in identified malignant cells. B. Violin plot for the regulon levels in malignant cells with high or low PTX3 expression in each regulon module. C. Scattering plot for the distribution of transcription factors in malignant cells based on PTX3 expression. D. t‐SNE plot for the dimension reduction of regulon modules. E. The different regulon levels of malignant cells with high or low PTX3 expression in each module. F. GO enrichment analysis of top‐ranked regulons. G. KEGG enrichment analysis of top‐ranked regulons

FIGURE 6

Multiplex immunofluorescence staining of CD68, CD163, PTX3, and DAPI. Multiplex immunofluorescence staining of CD68 (pink), CD163 (red), PTX3 (yellow), and DAPI (blue) in GBM samples from Xiangya cohort (10X and 40X), scale bar 100 and 20 um, respectively.

FIGURE 7

U251‐derived PTX3 mediated the migration and polarization of HMC3. (A) Western blotting results of PTX3 expression in NC and siRNA groups. (B) qPCR results of PTX3 expression in NC and siRNA groups. (C) Study design of the coculture system between HMC3 and U251 cells for transwell assay. (D) Representative images of transwell assay for migration of HMC3 in NC and siRNA groups in different time points. (E) Statistical analysis of transwell assay. (F) Study design of the coculture system between HMC3 and U251 cells for flow cytometry assay. (G) Flow cytometry assay results of CD68 and CD163 expression in NC and siRNA groups. (H) Statistical analysis of flow cytometry assay.

FIGURE 8

U87‐derived PTX3 mediated the migration and polarization of HMC3. (A) Western blotting results of PTX3 expression in NC and siRNA groups. (B) qPCR results of PTX3 expression in NC and siRNA groups. (C) Representative images of transwell assay for migration of HMC3 in NC and siRNA groups in different time points. (D) Statistical analysis of transwell assay. (E) Flow cytometry assay results of CD68 and CD163 expression in NC and siRNA groups. (F) Statistical analysis of flow cytometry assay.