Secreted clusterin inhibits tumorigenesis by modulating tumor cells and macrophages in human meningioma

Abstract

Background: Meningioma is the most common primary intracranial tumor with a high frequency of postoperative recurrence, yet the biology of the meningioma malignancy process is still obscure.

Methods: To identify potential therapeutic targets and tumor suppressors, we performed single-cell transcriptome analysis through meningioma malignancy, which included 18 samples spanning normal meninges, benign and high-grade in situ tumors, and lung metastases, for extensive transcriptome characterization. Tumor suppressor candidate gene and molecular mechanism were functionally validated at the animal model and cellular levels.

Results: Comprehensive analysis and validation in mice and clinical cohorts indicated clusterin (CLU) had suppressive function for meningioma tumorigenesis and malignancy by inducing mitochondria damage and triggering type 1 interferon pathway dependent on its secreted isoform, and the inhibition effect was enhanced by TNFα as TNFα also induced type 1 interferon pathway. Meanwhile, both intra- and extracellular CLU overexpression enhanced macrophage polarization towards M1 phenotype and TNFα production, thus promoting tumor killing and phagocytosis.

Conclusions: CLU might be a key brake of meningioma malignance by synchronously modulating tumor cells and their microenvironment. Our work provides comprehensive insights into meningioma malignancy and a potential therapeutic strategy.

Keywords: Clusterin; macrophage; meningioma; single-cell RNA-Seq; type 1 interferon.

Figure 1.

Delineation of major cell types within meningioma tissue samples. A. Schematic of meningioma sample collection for scRNA-Seq. Roman numerals indicate WHO tumor grade classification. B. UMAP (uniform manifold approximation and projection) visualization of single-cell clusters from all samples. Each cell cluster is color-coded and named based on cluster-specific marker genes in C. C. Dot plot of marker genes for each cell type (cluster). Color-scale indicates the mean of normalized expression of marker genes in each cell type, and the dot size is proportional to the percentage of cells within each cell cluster expressing the marker genes. The same applies to all dot plots in the rest of this article. D. Fraction of meninge-derived cell, myeloid, leukocytic cell, stromal cell and Macro-Tumor-Mixed cells in each assayed sample. Sample name suffixes (1, 2, and 3) correspond to the histological grading of the tumor sample. * marks samples from different sites of patient P2; ^ marks samples from different sites of patient P11; # marks samples from different sites of patient P13. E. Projection of expression levels of meninges and cell cycle phase marker genes on UMAP visualization. See also Supplementary Figure 1; Supplementary Table 1–4.

Figure 2.

Characterization of tumor cells in meningioma tissue samples. A. UMAP visualization of assayed single cells, split by normal and tumorous meninge samples. B. DDR (discriminative dimensionality reduction) tree visualization of tumor cell fate trajectory with pseudotime (left) and meningioma progression (right) information mapping. The analyzed cells include all the meninge-derived cells. Normal includes S1 and S2, low-grade includes S3–S10, and high-grade includes S11–S18. The same definition applies to the rest of this article. C. Heatmap visualization of expression levels of genes in meninge-derived cells (grouped by normal, low-grade and high-grade) with correlated and anti-correlated expression profile to trajectory pseudotime. From left to right, the cells are ordered according to increasing pseudotime. Gene expression levels across the pseudotime are z-score transformed and smoothed. D. DDR tree visualization of tumor cell fate trajectory with pseudotime (upper) and meningioma progression (lower) information mapping. The analyzed cells include all the meninge derived cells. Normal includes S1, S2; primary includes S1–S10; recurrent includes S11, S12, S15, and S16; metastasized includes S13, S14, S17, and S18. The same definition applies to the rest of this article. E. Heatmap visualization of expression levels of genes in meninge-derived cells (grouped by normal, primary, recurrent, and metastasized) with correlated and anti-correlated expression profile to trajectory pseudotime. From left to right, the cells are ordered according to increasing pseudotime. Gene expression levels across the pseudotime are z-score transformed and smoothed. F. Expression levels (y axis) of CLU in meninge-derived cells (left: grouped by normal, low-grade and high-grade; right: grouped by normal, primary, recurrent, and metastasized) aligned according to cell trajectory pseudotime (x axis). Each cell (dot) is color-coded according to the meningioma progression of the sample. The black line indicates the fitted expression trend over pseudotime. G. Proliferation of IOMM-LEE cells with different CLU expression conditions. IOMM-LEE: wildtype IOMM-LEE with empty lentivirus vector as control; CLU-oe: CLU overexpression; CLU-sg1, CLU-sg2 and CLU-sg3: CLU knock-out by CRISPR-cas9 with sg1, sg2 and sg3 guide RNA, respectively. On the fifth and sixth day, P-values for IOMM-LEE versus CLU-sg1, CLU-sg2, CLU-sg3 and CLU-oe all are < 0.0001. H. UMAP visualization of tumor cells with distinct gene expression signatures. I. Heatmap visualization of marker gene expression in distinct tumor cell types. J. Variation of the proportion of each defined tumor cell type through meningioma progression. K. Evaluation of the proliferation score of distinct tumor cell types (upper). Evaluation of potential differentiation level of tumor cells by cytoTRACE (lower). L. Representative immunohistochemistry staining of CLU and KI67 in grades 1–3 meningioma clinical paraffin samples (left). Photographs were amplified at ×40 under microscope. Expression comparison between CLU and KI67 in meningioma samples of grade 1 (n = 59), 2 (n = 21), and III (n = 22) by immunohistochemistry staining (right). The black line shows the fitted trend between CLU and KI67 expression. Spearman Rank Correlation Analysis was used for correlation analysis (r: Spearman’s correlation). See also Supplementary Figures 2–5; Supplementary Table 6.

Figure 3.

In vivo and in vitro validation of meningioma suppression by CLU. A. Western-blot image of CLU in culture supernatant of IOMM-LEE cells with control vectors, CLU overexpression (CLU-oe) or knockout (CLU-sg3) condition. B. Tumors grown from CLU-oe, IOMM-LEE and CLU-sg3 cells as shown by Spectrum in vivo Imaging System and photographs. The same luminescence color scale applies to G. C. Growth curve statistics of B (n = 10), 2-sided paired t-test. D. Survival curve of CLU-oe, IOMM-LEE and CLU-sg3 cells bearing mice (n = 20). E. Photographs of tumors grown from IOMM-LEE cells with control vector (TET-Vector) and Tet-CLU induced with doxycycline (left). Growth curve statistics of the left (n = 8) (right), 2-sided paired t-test. F. H&E staining of mouse brain tissues with tumor induced by IOMM-LEE with control vector (Vector), CLU-oe and CLU-sg3 cells. Photographs were amplified at ×40 under microscope. G. Lung metastasis of tumors induced by IOMM-LEE with control vector (Vector), CLU-oe and CLU-sg3 cells as detected by Spectrum in vivo Imaging System. H. H&E staining of mouse lung tissues with metastases induced by IOMM-LEE with control vector (Vector), CLU-oe and CLU-sg3 cells. Photographs were amplified at ×40 under microscope. I. Left: Tumor spheres of IOMM-LEE with control vector (Vector), CLU-oe and CLU-sg3 cultured in nonserum DMEM containing EGF/bFGF. Right: Cell colonies of IOMM-LEE with control vector (Vector), CLU-oe and CLU-sg3 grown on the dish (upper); Giemsa staining of IOMM-LEE with control vector (Vector), CLU-oe and CLU-sg3 cells transmigrated the Transwell (lower). J. From left to right, statistics of I. K. Proliferation of IOMM-LEE cells in vitro when cocultured with supernatant from IOMM-LEE cells with (CLU-sup) or without (IOMM-LEE-sup) CLU overexpression. L. Proliferation of IOMM-LEE cells with control vector (Vector) and IOMM-LEE cells expressing CLU with defective signaling peptide (nsCLU). See also Supplementary Figure 6.

Figure 4.

Meningioma tumor cell suppression by CLU through innate immunity signaling. A. Transmission electron microscope image (×2500, ×7000, and ×15 000) of mitochondria in tet-vector and tet-CLU tumor tissue. B. Relative abundance of MT-LOOP and MT-CO2 by qPCR in IOMM-LEE cells with control vector (Vector). C. Western-blot images of autophagy marker (P62, LC3B) expression in IOMM-LEE with control vector (Vector), CLU-oe, CLU-sg3 and nsCLU cells. D. Heatmap of differentially expressed genes between wildtype and IOMM-LEE cells with control vector (Vector), CLU overexpression (CLU-oe), CLU knock-out (CLU-sg3). E. Volcano plot for differentially expressed genes between wildtype and IOMM-LEE cells with control vector (Vector) and CLU overexpression (CLU-oe). F. KEGG pathway enrichment for genes significantly upregulated in CLU-oe over IOMM-LEE cells with control vector (Vector). G. Relative expression of DDX58, OASL, IFIT1, and IFNB1 by qPCR in IOMM-LEE cells with control vector (Vector) and CLU overexpression (CLU-oe). H. Western-blot images of DDX58, TRAF1, p-TBK1, p-STING, STING, p-P65, and P65 in IOMM-LEE with control vector (Vector) and CLU overexpression (CLU-oe). See also Supplementary Figure 6, Supplementary Tables 5 and 7.

Figure 5.

CLU increased interferon pathway through TLR2. A. Left: Western-blot images of P65, TLR2, P105, P50, and IκBα in IOMM-LEE with control vector (Vector), CLU-oe, CLU-sg3 and nsCLU cells; Right: Western-blot images of p-P65 (Thr254), p-P65 (Ser276), and p-P65 (Ser281) in IOMM-LEE with control vector (Vector) and CLU overexpression (CLU-oe). B. Western-blot images of P65 in IOMM-LEE with control vector (Vector), CLU-oe, CLU αchain overexpression (CLU-α), and CLU βchain overexpression (CLU-β) cells. C. Confocal microscopy imaging of P65 and mitochondria in IOMM-LEE with control vector (Vector), CLU-oe cells (upper). Western-blot images of P65 subcellular expression in IOMM-LEE with control vector (Vector), CLU-oe. D. CXCL10 expression by qPCR in IOMM-LEE with control vector (Vector), CLU-oe and CLU-sg3 cells. E. TNFα mRNA expression by qPCR in IOMM-LEE with control vector (Vector), CLU-oe cells. F. Statistics of apoptosis assay by FCM for IOMM-LEE, CLU-oe and CLU-sg3 cells treated with/without TNFα. G. Western-blot images of DDX58, TRAF1, p-TBK1, p-STING, STING, p-P65, and P65 in IOMM-LEE cells treated with/without TNFα. H. CXCL10 expression by qPCR in IOMM-LEE cells treated with/without TNFα. I. Molecular docking simulation of CLU-TLR2 binding. J. Confocal microscopy imaging of CLU (green) and TLR2 (red) in meningioma tissue. K. Western-blot images of DDX58, TRAF1, p-TBK1, P65, TLR2, and IκBα in IOMM-LEE with control vector (Vector), CLU-oe, CLU-oe with TLR2 knockout (CLU-oe-TLR2-sgRNA) and CLU-sg3 cells. L. CXCL10 expression by qPCR in CLU-oe, CLU-oe with TLR2 knockout (CLU-oe-TLR2-sgRNA) cells. See also Supplementary Figure 7 and Supplementary Table 8.

Figure 6.

Characterization of myeloid cells in meningioma tissue samples. A. UMAP visualization of myeloid lineage cells. The re-clustered cells here included Monocytes, Macrophages, DC and neutrophils in Figure 1B. Macro: macrophage; Mono: monocyte; DC: dendritic cell; Neu: neutrophil. B. Dot plot of marker genes for myeloid cell clusters. C. The bar chart shows the variation of the proportion of each defined myeloid cell subtype through meningioma progression. D. M1/M2 macrophage evaluation of each macrophage cluster according to gene expression signature. E. Boxplots for comparing CLU and TNF mRNA expression in Macro_IL1B, Macro_VSIG4 and Macro_CX3CR1. F. Relative expression of TNF, CD86, CD163 by qPCR from M0 macrophages with (CLU-oe) or without (vector) CLU overexpression. M0: unpolarized macrophages produced by treating THP1 monocytes with phorbol ester. G. Relative expression of TNF, CD86, CD163 by qPCR from LPS treated macrophages with (CLU-oe) or without (vector) CLU overexpression. M1: polarized macrophages with M1 phenotype. H. Relative expression of TNF mRNA by qPCR in M1 macrophages when cultured with recombinant CLU (re-CLU) or supernatant from IOMM-LEE or 293T cells with/without CLU overexpression, respectively. I. TNFα protein expression by ELISA in M1 macrophage when cultured with recombinant CLU (re-CLU) or supernatant from IOMM-LEE or 293T cells with/without CLU overexpression, respectively. J. Statistics of phagocytosis rate of M1 macrophages to IOMM-LEE, CLU-oe and CLU-sg3 cells. K. Proposed model of CLU suppressing meningioma. Histone deacetylase inhibition could promote CLU expression in meningioma tumor cells. Secreted CLU protein binds to TLR2 on the cell surface of tumor cells, causing mitochondria damage, and promotes type 1 interferon pathway and P65 expression. Secreted CLU protein could also stimulate TNFα production and phagocytosis function by M1 macrophages. TNFα also promotes type 1 interferon pathway in meningioma. Collectively, CLU prevents meningioma malignancy by suppressing tumor cells and polarizing M1 macrophage. See also Supplementary Figure 8; Supplementary Tables 3–5.