CD97 is associated with mitogenic pathway activation, metabolic reprogramming, and immune microenvironment changes in glioblastoma

Abstract

Glioblastoma (GBM) is the most common primary brain tumor with a median survival under two years. Using in silico and in vitro techniques, we demonstrate heterogeneous expression of CD97, a leukocyte adhesion marker, in human GBM. Beyond its previous demonstrated role in tumor invasion, we show that CD97 is also associated with upregulation of the mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/Erk) and phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) pathways in GBM. While CD97 knockout decreased Akt activation, CD97 targeting did not alter MAPK/Erk activation, did not slow GBM cell proliferation in culture, and increased levels of glycolytic and oxidative phosphorylation metabolites. Treatment with a soluble CD97 inhibitor did not alter activation of the MAPK/Erk and PI3K/Akt pathways. Tumors with high CD97 expression were associated with immune microenvironment changes including increased naïve macrophages, regulatory T cells, and resting natural killer (NK) cells. These data suggest that, while CD97 expression is associated with conflicting effects on tumor cell proliferative and metabolic pathways that overall do not affect tumor cell proliferation, CD97 exerts pro-tumoral effects on the tumor immune microenvironment, which along with the pro-invasive effects of CD97 we previously demonstrated, provides impetus to continue exploring CD97 as a therapeutic target in GBM.

Figure 1

Single-cell analysis of CD97-expressing glioblastoma cells. Glioblastoma (GBM) cells from 14 IDH-wild type cases were subjected to single-cell analysis. (A) Shown is a t-SNE plot classifying cells from these 14 cases into 19 clusters. (B) Neoplastic versus non-neoplastic cells were identified and highlighted in pink via copy number variation (CNV) analysis, with stromal cells shown in cyan. (C) Neoplastic cells (n = 2530) were clustered into 19 distinct cell populations based off differential gene expression. (D) CD97⁺ and CD97⁻ cells are highlighted among neoplastic cells from these 19 clusters from 14 GBM patient samples. (E) Percentage of CD97⁺ cells among the 19 clusters derived from 2,530 tumor cells.

Figure 2

Proliferative pathways are activated in CD97-expressing patient glioblastoma cells and are suppressed with CD97 knockdown in a human GBM cell line. Proliferative pathways are activated in CD97-expressing patient GBM cells (A–D). (A) Variable feature plots illustrating the top 10 differentially expressed genes in CD97⁺ cells versus CD97^- cells demonstrate a bias toward NFKB pathway in CD97⁺ cells and higher expression of immune genes in CD97^- cells. (B) Genes associated with NFKB signaling were upregulated in CD97⁺ cells compared to CD97^- cells. (C) CD97 + tumor cells had higher expression of several receptors that utilize PI3K/Akt signaling, cytoplasmic activators of Akt, and downstream targets of Akt. (D) CD97⁺ tumor cells had higher expression of genes associated with the MAPK pathway activity score (MPAS) than CD97^- tumor cells. (E) CD97⁺ tumor cells had higher expression of genes associated with GBM stem cells than CD97^- tumor cells. (F) WT U251 cells demonstrate higher protein expression of phosphorylated AKT (pAKT) compared to CD97KO U251 cells (P < 0.01), while AKT, ERK, and phosphorylated ERK (pERK) did not demonstrate difference in protein expression between the two groups. Full western blots are shown in Supplementary Figs. S12 and S13. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 3

Transcriptional programs activated in CD97-expressing GBM cells and in CD97^hi patient GBMs. (A) Ontologic analysis of the top 25 genes with higher expression in CD97⁺ versus CD97^- single GBM cells from 14 patient GBMs reveal associations with second-messenger signaling and extracellular matrix organization. (B) Ontologic analysis of the 133 genes with fold change > 2 and adjusted P < 0.05 in bulk RNA-seq of CD97^hi GBMs relative to CD97^lo GBMs revealed that upregulated genes were associated with pathways involved in extracellular matrix organization, extracellular structure organization, and glycosaminoglycan binding. (C,D) Heatmap (C) and bar plot (D) showing expression of the 10 genes that comprise the MPAS gene signature in bulk RNA-seq data across 151 patients reveals significantly higher expression of MPAS genes in CD97^hi versus CD97^lo patient GBMs. (E) Volcano plot showing differentially expressed genes in U251 cells with CD97 knockout (CD97 KO) versus wild-type U251 (CD97 WT) cells. Genes on the left are upregulated in CD97 WT/downregulated in CD97 KO, while genes on the right are upregulated in CD97 KO/downregulated in CD97 KO. (F) Compared to wild-type U251 GBM cells, those with CD97 knockout demonstrated significant reduction in gene expression associated with pathways related to cancer, focal adhesion, PI3K pathway, and ERK/MAPK pathways. *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 4

CD97 alters the GBM immune microenvironment. Compared to CD97^lo GBMs, CD97^hi GBM tumors exhibited significantly greater proportion of (A) nonpolarized M0 macrophages, (B) regulatory T cells, and (C) resting NK cells. In contrast, compared to CD97^hi GBM tumors, CD97^lo GBM tumors had higher proportions of (D) monocytes. (E) THP-1 monocytes co-cultures with wild type U251 GBM cells had significantly higher macrophage differentiation compared to those co-cultured with U251 CD97-knockdown cells (P < 0.001). *P < 0.05; **P < 0.01; ***P < 0.001.