ALOX5AP is highly expressed in glioma and correlates with clinical features
ALOX5AP expression in glioma was investigated using the TIMER2.0 database, comprising 153 GBM and five normal samples. The GEPIA database, including 163 GBM, 518 LGG, and 207 normal samples, was used to evaluate ALOX5AP differential expression between glioma and normal tissue. These results indicated that ALOX5AP was highly expressed in gliomas (Fig. 1A, B).
Expression of ALOX5AP. A, B Expression of ALOX5AP in the normal and tumor samples in TIMER2.0 and GEPIA online databases. Expression of ALOX5AP in gliomas of different C grade, D age, E sex, F IDH mutation status, G 1p19q codeletion status, H MGMTp methylation status, I radio status, J TMZ status based on CGGA database. *P < 0.05, **P < 0.01, ***P < 0.001
To examine the correlation with glioma clinical characteristics, we analyzed ALOX5AP expression in the CGGA cohort. ALOX5AP expression was higher in patients with WHO grade 4 GBM than in those with grade 2–3 glioma (P < 2.2e − 16) (Fig. 1C), in patients aged ≥ 40 years (P = 5.3e − 04) (Fig. 1D), in male (P = 9.8e − 06) (Fig. 1E), and in the IDH wild-type (P < 2.2e − 16) (Fig. 1F), 1p19q non-codeletion (P = 2.2e − 16) (Fig. 1G), and MGMTp unmethylated groups (P < 1.2e − 03) (Fig. 1H). However, ALOX5AP expression was not correlated with TMZ chemotherapy (P = 7.1e − 01) or radiotherapy (P = 3.2e − 01) (Fig. 1I, J).
ALOX5AP predicts worse survival in glioma
In the CGGA database, high ALOX5AP expression correlated with shorter OS in different subgroups, based on age, sex, 1p19q codeletion status, IDH mutation status, MGMTp methylation status, and grade (all P < 0.05) (Fig. 2A–F). For TCGA, higher ALOX5AP expression correlated with decreased OS in both TCGA-LGG (P < 0.001) (Fig. S1A) and TCGA-GBM samples (P = 0.018) (Fig. S1B). Multivariate COX proportional hazards regression analysis based on the TCGA cohort showed that ALOX5AP was an independent prognostic factor for glioma (Table. S2).
Prognostic analysis of ALOX5AP in glioma patients based on CGGA samples. Kaplan-Meier survival curve of CGGA samples in different subgroups, including A age, B sex, C 1p19q codeletion status, D IDH mutation status, E MGMTp methylation status and F WHO grades
Based on the area under the curve (AUC) of time-ROC, ALOX5AP expression accurately predicted glioma OS at 1 (CGGA: 0.612; TCGA: 0.796), 3 (CGGA: 0.663; TCGA: 0.772), 5 (CGGA:0.687; TCGA: 0.744), and 10 years (CGGA: 0.678; TCGA: 0.804) (Fig. 3A, B). A nomogram was created by combining ALOX5AP expression data with clinical prognostic factors, including WHO grade, age, sex, and IDH mutation, 1p19q codeletion, and MGMTp methylation status (Fig. 3C). Nomogram performance was evaluated by calibration, resulting in a concordance index of 0.739 for OS (Fig. 3D). Both ALOX5AP and nomogram findings demonstrated reliable predictive efficacy for OS, as evidenced by the elevated AUC and concordance index.
Prognostic prediction of ALOX5AP in glioma patients. A–B time-ROC curve predictive accuracy of ALOX5AP in CGGA cohort and TCGA cohort for OS at 1-, 3-, 5-, and 10-years. C The nomogram from CGGA cohort. D Prediction of the calibration curve from CGGA cohort
ALOX5AP is mainly expressed in TAMs but not in tumor cells
To assess ALOX5AP expression at the single-cell level, we conducted single-cell RNA-Seq analysis on two newly diagnosed GBM and two LGG samples based on GSE182109. Cells were divided into 25 clusters with 11 identified cell types: astrocytes, endothelial cells, macrophages, microglia, monocytes, neural stem cells, neurons, oligodendrocytes, pericytes, Schwann precursors, and T cells (Fig. 4A and Table S3). ALOX5AP was significantly expressed in the macrophages, microglia, monocytes, and T cells of both LGG and GBM (Fig. 4B). According to the GSE70630, GSE84465, GSE131928_10X, and GSE89567 datasets, the TISCH2 database also revealed significant ALOX5AP expression in GBM macrophages, monocytes, and T cells (Fig. 4C).
Expression of ALOX5AP in single cell level. A Clustering and annotation of glioma in GSE180192 yielded 25 clusters and 11 cell types. B Expression of ALOX5AP in 25 clusters and 11 cell types. C Expression of ALOX5AP in various cell types in GSE70630, GSE84465, GSE131928_10X, and GSE89567 based on TISCH2 online database
ALOX5AP association with immune infiltration in glioma
In view of the fact that ALOX5AP is mainly expressed in immune cells, especially macrophages, rather than tumor cells in gliomas. We calculated the correlation between ALOX5AP and the immune, estimate, tumor purity, and stroma scores in the CCGA cohort using the ESTIMATE algorithm. High ALOX5AP expression was associated with higher immune, estimate, and stromal scores, albeit lower tumor purity (all P < 0.001) (Fig. 5A–D), suggestive of high levels of immune infiltration.
Relationship between ALOX5AP and immune infiltration in glioma. A–D Relationship between ALOX5AP and estimate score, immune score, tumor purity, and stroma score analyzed by ESTIMATE algorithm based on CGGA cohort. E, F Relationship between ALOX5AP and 22 types of immune cells analyzed by CIBERSORT algorithm based on CGGA cohort. G–I Relationship between ALOX5AP and immunosuppressive factors, chemokines, and chemokine receptors in the TISIDB database and scatterplots of the three highest correlation coefficients are presented. *P < 0.05, **P < 0.01, ***P < 0.001, ns = no significance
The correlation between ALOX5AP and 22 diverse types of immune cells in glioma was analyzed using the CIBERSORT algorithm based on the CGGA cohort. High ALOX5AP expression was associated with greater M2 macrophage (P < 0.001) and neutrophil (P < 0.01) proportions, whereas low ALOX5AP expression related to larger naive CD4 + T cell (P < 0.01), helper follicular cell (P < 0.05), activated mast cell (P < 0.01), and resting dendritic cell fractions (P < 0.05) (Fig. 5E).
The relationship between ALOX5AP and immunosuppressive factors, chemokines, and chemokine receptors in GBM was analyzed using the TISIDB database. The top three immunosuppressive factors (CSF1R, HAVCR2, and IL10), chemokine (CCL20, CCL16, and CXCL2), and chemokine receptor genes (CCR1, CCR2, and CCR5) correlating with ALOX5AP and the correlation coefficients between ALOX5AP and the immune component genes in the TISIDB database are shown in Fig. 5G–I and Table S4.
Correlation of ALOX5AP expression with immunotherapies and chemotherapeutics drugs sensitivity in glioma
It is believed that TMB is associated with response to immunotherapy in a wide range of tumors [35]. Mutations are processed into neoantigens and are presented to T cells by HLA molecules [36]. TMB, HLA molecules, and immune checkpoints can serve as a part of predicting the outcome of immunotherapy [36]. We calculated the correlation between ALOX5AP and several biomarkers, including HLA (HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, HLA-DMA, HLA-DMB, HLA-DOA, HLA-DOB, HLA-DPA1, HLA-DPB2, HLA-DQA1, HLA-DQB1, HLA-DRA, HLA-DRB1, and HLADRB5), and immune checkpoints (IDO1, CTLA4, LAG3, CD47, CD160, CD244, BTLA, ICOS, PDCD1, HAVCR2, CD276, TNFSF4, CD80, ARHGEF5, and VTCN1). ALOX5AP was significantly positively associated with most of the biomarkers which also showed upregulated in high ALOX5AP group in both CGGA (Fig. 6A–B) and TCGA samples (Fig. S2A–B).
Analysis of HLA and immune checkpoints based on CGGA cohort. A Differential expression of HLA in ALOX5AP high and low groups and the correlation between ALOX5AP and HLA. B Differential expression of immune checkpoints in ALOX5AP high and low groups and the correlation of ALOX5AP and immune checkpoints. *P < 0.05, **P < 0.01,***P < 0.001.
Afterward, we explored TMB in the ALOX5AP high and low expression groups. The result showed that the ALOX5AP high expression group had a higher TMB (P = 2.9e − 16) (Fig. 7A). We used the TCIA database to evaluate the immunotherapy response of TCGA-GBM samples through IPS, and a higher IPS signifies a better response to immunotherapy. The results revealed that the IPS of CTLA-4 negative PD-1 negative and CTLA-4 negative PD-1 positive groups in the low ALOX5AP expression group were significantly higher than that in the high ALOX5AP expression group (Fig. 7B, C), which strongly predicted that GBM patients with lower ALOX5AP expression would benefit immunotherapy. In comparison, the IPS of CTLA-4 positive PD-1 negative and CTLA-4 positive PD-1 positive groups did not differ significantly between ALOX5PA high and low groups (Fig. 7D, E). Based on TCGA and CGGA samples, the high ALOX5AP expression group had higher TIDE scores (TCGA: P = 3.8e − 07; CGGA: P = 1.5e − 09) (Fig. 7F, G), suggesting that ALOX5AP is associated with immune evasion. Moreover, the expression of ALOX5AP was positively correlated with TIDE score (TCGA: R = 0.23, P = 1.3e − 09; CGGA: R = 0.26, P = 2.2e − 16) (Fig. 7H, I).
The relationship of ALOX5AP to immunotherapy response and drug sensitivity. A The significantly different distributions of the TMB in ALOX5AP high and low groups based on TCGA samples. B–E Comparison of IPS of patients with different risk groups. F, G The significantly different distributions of the TIDE score in ALOX5AP high and low groups based on TCGA and CGGA samples. H, I Correlation between ALOX5AP expression and TIDE score based on TCGA and CGGA samples. J, K Comparison of the sensitivity of patients with different risk groups to common chemotherapeutics based on TCGA and CGGA samples. ( Half maximal inhibitory concentration: IC50) *P < 0.05, **P < 0.01, ***P < 0.001, ns = no significance
Given that chemotherapy is one of the critical means of glioma treatment in the clinic, we next calculated the sensitivity of glioma to 13 drugs, including camptothecin, vincristine, cisplatin, cytarabine, docetaxel, 5-fluorouracil, paclitaxel, irinotecan, oxaliplatin, gemcitabine, temozolomide, epirubicin, cyclophosphamide, and carmustine. The ALOX5AP high expression group was less sensitive to vincristine, cytarabine, temozolomide, and carmustine, and more sensitive to 5-fluorouracil and irinotecan. Glioma sensitivity to docetaxel and gemcitabine was not associated with ALOX5AP (Fig. 7J, K).
The signaling pathway associated with ALOX5AP
To investigate the biological function of ALOX5AP in glioma, GSEA was conducted based on the TCGA database. In GBM, the top five signaling pathways of GO terms are chemokine receptor binding, immunoglobulin complex circulating, immunoglobulin receptor binding, phagocytosis recognition, and response to chemokine (Fig. 8A); the top five signaling pathways of KEGG terms are graft versus host disease, hematopoietic cell lineage, intestinal immune network for IGA production, leishmania infection, and NOD-like receptor signaling pathway (Fig. 8B). In LGG, the top five signaling pathway of GO terms are B cell-mediated immunity, human immune response mediated circulating immunoglobulin, immunoglobulin complex circulating, and immunoglobulin receptor binding (Fig. 8C); the top five signaling pathway of KEGG terms are allograft rejection, cytokine-cytokine receptor, graft versus host disease, hematopoietic cell lineage, leishmania infection (Fig. 8D).
Functional enrichment analysis of ALOX5AP. A The top 5 enriched pathways of GBM in GSEA based on GO terms. B The top 5 enriched pathways of GBM in GSEA based on KEGG terms. C The top 5 enriched pathways of LGG in GSEA based on GO terms. D The top 5 enriched pathways of LGG in GSEA based on KEGG terms. E Top 71 proteins with the highest correlation coefficient with ALOX5AP from STRING database; F GO enrichment analysis of ALOX5AP and ALOX5AP related genes; G KEGG enrichment analysis of ALOX5AP and ALOX5AP related genes
The interacting proteins of ALOX5AP were screened using the STRING database; the 71 strongest interacting proteins were visualized using Cytoscape (Fig. 8E). GO and KEGG enrichment analyses were performed for the 72 respective protein-encoding genes, including ALOX5AP. The top five BP, CC, and MF terms according to P values are shown in Fig. 8F. The top 20 KEGG-enriched pathways were eicosanoid metabolism, arachidonic acid metabolism, fatty acid biosynthesis, myeloid leukocyte activation, neuroinflammatory response, glial cell activation, microglia activation, and leukocyte activation involved in inflammatory response, etc. (Fig. 8G). These results suggest that ALOX5AP plays a fundamental role in TME.
Validation of ALOX5AP expression in glioma tissue
Our bioinformatics analyses demonstrated that ALOX5AP was expressed in macrophages and positively correlated with the M2 macrophage fraction. To confirm the bioinformatics findings, we employed multiplex immunofluorescence staining of ALOX5AP, CD163 (M2 macrophage marker), and CD68 (M0 macrophage marker) in a glioma tissue microarray (Fig. 9A). ALOX5AP + and CD163 + cell percentages were increased in WHO grade 4 compared to grade 2–3 gliomas (all P < 0.05) (Fig. 9B, C), whereas CD68 + cell percentage showed no grade-specific difference (Fig. 9D). Moreover, ALOX5AP co-localized with CD68 and CD163 in gliomas (Fig. 10A). The group with high ALOX5AP + cell percentage had higher CD68 + and CD163 + cell percentages than those in the low-percentage ALOX5AP + cells in both WHO grade 2–3 (all P < 0.05) and grade 4 gliomas (all P < 0.05) (Fig. 10B–E). Spearman’s correlation analysis showed that ALOX5AP + cell percentage positively correlated with CD163 + cell percentage (R = 0.4, P = 1.8e–05) and CD68 + cell percentage (R = 0.43, P = 3.6e–06) in the glioma tissue microarray (Fig. 10F, G).
Multiplex immunofluorescence staining of ALOX5AP (red), CD163 (green) and CD68 (yellow) protein in glioma tissue microarray with different grades. A Representative immunofluorescence image of ALOX5AP, CD163, and CD68 expression in WHO grade 2, grade 3, and grade 4 glioma. (The scale bar is 200 μm). B–D Quantitative analysis of ALOX5AP+, CD163+, and CD68 + cells percentage in WHO grade 2–3 and grade 4 glioma samples. *P < 0.05, ns = no significance
Validation of ALOX5AP in relationship to macrophage markers and prognosis in glioma tissue microarray. A The colocalization of ALOX5AP (red), CD163 (green), and CD68 (yellow) in glioma (The scale bar is 200 μm). B, C Difference analysis of CD163 + cell percentage in high and low ALOX5AP + cell percentage group. D, E Difference analysis of CD68 + cell percentage in high and low ALOX5AP + cell percentage group. F, G The correlation between the ALOX5AP + cells percentage and the CD163 + cells percentage and CD68 + cell percentage. H Kaplan-Meier curve; I time-ROC curve. *P < 0.05, **P < 0.01, ***P < 0.001
To verify the prognostic value of ALOX5AP in glioma, we performed Kaplan–Meier analysis and time-ROC curve based on the glioma tissue microarray. High ALOX5AP + cell percentage correlated with worse OS in 109 glioma samples (P = 0.023) (Fig. 10H). In the glioma tissue microarray, ALOX5AP + cell percentage similarly accurately predicted glioma OS at 1 (AUC = 0.665), 3 (AUC = 0.637), and 5years (AUC = 0.611) (Fig. 10I).
Add Comment