LncRNA RP3-525N10.2-NFKB1-PROS1 triplet-mediated low PROS1 expression is an onco-immunological biomarker in low-grade gliomas: a pan-cancer analysis with experimental verification

Glioma is the most common cancer in the central nervous system, and low grade gliomas are notorious for many types of tumors and heterogeneity. PROS1 not only plays an important role in the blood coagulation system, and recent studies have found that it was correlated with the development of tumors, especially related to tumor immune infiltration. However, the study of underlying role and mechanism of PROS1 in gliomas, especially in low-grade gliomas, is almost absent. We integrated the information of patients with LGG in The Cancer Genome Atlas (TCGA) cohort and Chinese Glioma Genome Atlas (CGGA) cohort. Then, we systematically demonstrated the differences and prognostic prognosis value of PROS1 based on multi-omics analyses. In addition, Cell counting kit-8 (CCK-8) assay, colony formation assay, 5-Ethynyl-2’-deoxyuridine (EdU) incorporation assay, and Transwell assays were performed to evaluate cell proliferation and invasion. qRT-PCR and immunohistochemistry were used to evaluate the expression of PROS1 in LGG. Various bioinformatics approaches revealed that PROS1 was a valuable prognostic marker and may influence tumour development via distinct mechanisms, including expression of DNA methyltransferase, RNA modification, and DNA mismatch repair system genes, copy number variation, single nucleotide variation frequency, genomic heterogeneity, cancer stemness, DNA methylation, and alternative PROS1 splicing. Our analyses indicated that the long non-coding RNA RP3-525N10.2 may “decoy” or “guide” the transcription factor NFKB1 and prevent its association with PROS1, thereby reducing PROS1 expression and improving poor LGG prognosis. PROS1 expression was also closely associated with tumour infiltration by immune cells, especially tumour-associated macrophages, as well as the expression of various immune checkpoint inhibitors, immunomodulators, and immune cell markers. long non-coding RNA RP3-525N10.2-NFKB1-PROS1 triplet-mediated PROS1 expression could serve as a biomarker for cancer diagnosis, prognosis, therapy selection, and follow-up in LGG patients.


Introduction
Glioma is the most common malignancy in the brain. Low-grade gliomas (LGGs, grade II and III), originating from neuroepithelial tissue [1], have high mortality in patients and are difficult to diagnose due to high intratumoural heterogeneity leading to distinct biological behaviour. To date, the standard treatment for LGG is maximal safe resection with adjuvant radiochemotherapy. Despite improvements in LGG treatment, more than half of the LGGs progress to therapy-resistant highgrade aggressive gliomas [2]. Several studies examined O-6-methylguanine-DNA methyltransferase (MGMT) methylation, codeletion of chromosome arms 1p and 19q, and isocitrate dehydrogenase (IDH) mutations to provide insight into LGG pathogenesis and advance cancer therapies for patients with LGGs [3,4]. However, these most widely utilised molecular biomarkers cannot adequately reflect individual heterogeneity and provide clinical stratification of LGG risk. Therefore, there is an urgent need to elucidate potential mechanisms of LGG progression, establish new drug targets, and identify effective biomarkers for patients at high risk of developing LGGs. Protein S1 (PROS1) is a well-known ligand of the TYRO3, AXL, and MERTK family of receptor tyrosine kinases. The duplication events that gave rise to these kinases may have occurred in the early metazoan evolution, about 6 million years ago [5]. PROS1 is a secreted water-soluble vitamin K-dependent protein that is γ-carboxylated within the N-terminal of the Gla domain. The Gla domain confers the ability of PROS1 to bind phosphatidylserine on the surface of apoptotic cells, and the C-terminal sex hormone-binding globulin-like module can bind and activate TYRO3 and MERTK [6]. PROS1 is a key plasma protein and plays critical roles in anticoagulation and the phagocytosis of apoptotic cells [7]. Tumour-secreted PROS1 can decrease the expression of macrophage M1 cytokines in vitro and in vivo [8]. PROS1 plays an important role in inflammatory diseases, including periodontitis and glomerular injury [9,10]. Moreover, PROS1 has been identified as a potential target gene in several types of human cancers, including papillary thyroid carcinoma, oral squamous cell carcinoma, malignant thyroid cancer, intrahepatic cholangiocellular carcinoma, and glioblastoma [11][12][13][14][15][16]. However, a comprehensive study on PROS1 expression, its prognostic value, and the underlying mechanisms in gliomas, especially in LGGs, is still missing. Additionally, the correlations of PROS1 expression with multi-omic data and tumour infiltration by immune cells in LGG remain undetermined.

Sangerbox tools
The free data analysis platform Sangerbox (http:// www. sange rbox. com/ tool) was used to validate the pancancer expression of PROS1 and explore the correlation of PROS1 expression with the expression of DNA methyltransferases, RNA modification genes, and DNA mismatch repair system genes, as well as copy number variation, single nucleotide variation frequency, genomic heterogeneity, and cancer stemness using Spearman's or Pearson's method.

Gene expression profiling interactive analysis (GEPIA)
GEPIA (http:// gepia. cancer-pku. cn/ index. html) is a free web tool based on TCGA and GTEx data. In the current study, PROS1 expression, survival analysis, and possible involvements of lncRNAs and TFs were evaluated using the GEPIA modules "Expression DIY" and "Survival". In addition, the relationships between PROS1 and Gene markers were determined using Spearman's correlation coefficient in the module "Correlation analysis".

Tumor immune estimation resource (TIMER)
TIMER (https:// cistr ome. shiny apps. io/ timer/) is a web server for the comprehensive analysis of tumour-infiltrating immune cells. In our study, PROS1 expression and survival were evaluated using the "Gene" and "Survival" modules. TIMER was also applied to investigate the relationships between PROS1 expression and different gene marker sets of immune cells using the "Correlation" module [29].

Tissue sampling from glioma patients
Fresh glioma tissues from histologically confirmed cases were obtained from the Union Hospital, Tongji Medical Keywords: Carcinogenesis, Immunohistochemistry, Long noncoding rna, Transwell assays, Macrophages, Transcription factors, Tumor microenvironment Since there are no specific LGG cell lines, common glioma cell lines (U87, U251, and T98G) and the human astrocyte cell line NHA were used. Cells were cultured in Dulbecco's modified Eagle's medium (Gibco) containing 10% heat-inactivated foetal bovine serum and 1% penicillin/streptomycin. Real-time PCR was conducted to compare gene expression in 30 tumour samples with that in adjacent normal tissue. Real-time PCR was performed in triplicate using samples derived from three independent experiments. Primers for PROS1 (forward, 5'-GTG CCT TCC CTT GAA CCR RG-3' , reverse, 5'-CCA CGC TGA GTG ATC GAT AGA-3') and GAPDH (forward, 5'-AAA AGC ATC ACC CGG AGG AGAA-3' , reverse, 5'-AAG GAA ATG AAT GGG CAG CCG-3') were used for qPCRs. Ten formalin-fixed, paraffin-embedded LGG tissues and normal brain tissues were used for immunohistochemistry stainings.

lentivirus infection assay
Short hairpin RNA (shRNA) against PROS1 (shPROS1) and a negative control shRNA (sh NC) were designed and synthesised by genomeditech (Shanghai, China). In addition, the pcDNA3.1 vector (Vigene Biology) containing the full-length cDNA sequence of PROS1 was used to overexpress PROS1. The empty pcDNA3.1 vector was used as a negative control. The lentivirus pLent-shPROS1-GFP-Puro or its negative control (NC) pLent-GFP-Puro (genomeditech) was used to infect GBM cells with enhanced infection solution (genomeditech) according to the manufacturer's protocol. Similarly, pLent-PROS1-GFP-Puro lentivirus or empty vector (vector) pLent-GFP-Puro lentivirus (genomeditech) was used to overexpress genes. Seventy two hours after the cells were infected with lentivirus, 2 μg/ml puromycin was added to kill the cells that had not been transfected.

Cell counting Kit-8 assay
U87 and U251 cells was assessed with the Cell Counting Kit-8 (Dojindo Molecular Technologies, Kyushu, Japan) reagent according to the manufacturer's instructions. Cells were inoculated on 96-well plates at a density of 1000 cells per well with 100 μl of medium. Every 24 h for a total of 96 h, CCK8 solution (10 μl) was added to each well, and the cells were further incubated at 37 °C for 3 h. The absorbance of each well was measured at 450 nm with a spectrophotometer.

Colony formation assay
U87 and U251 Cells were prepared into a single-cell suspension, respectively, and seeded into a six-well plate (200 cells/well) for two-week incubation to form colonies. After staining with 0.01% crystal violet (Sigma), the colonies were subjected to microscopic examination. The rate of colony formation and survival fraction were calculated.

Cell invasion assays
2 × 10 4 cells were added into Matrigel-coated upper Transwell chambers for the invasion assay. The lower chambers were filled with DMEM containing 10% FBS. After incubation at 37 °C for 24 h, cells on the lower surface of the membrane were fixed in 100% methanol and stained with 0.1% crystal violet dye for 20 min at room temperature. Finally, after washing with PBS, cells were imaged in five randomly selected fields under a light microscope (Olympus Corporation) at × 100 magnification.

Survival analysis
The PrognoScan database (http:// www. progn oscan. org/) is a large collection of publicly available cancer microarray datasets [34]. The OncoLnc database (www. oncol nc. org), a tool for interactive explorations of survival correlations, contains survival data of 8,647 patients from 21 cancer studies supported by the TCGA. The relationships between PROS1 expression and patient prognosis (overall survival, disease-specific survival, disease-free interval, and progression-free interval) were visualised with forest plots and Kaplan-Meier curves.

Analysis of PROS1-interacting genes and proteins
The GeneMANIA database (http:// www. genem ania. org) was used to construct the PROS1 interaction network [35]. The STRING database (https:// string-db. org/) was used to construct the protein-protein interaction network of PROS1 [36].

GO, KEGG pathways, and GSEA of PROS1
GO and KEGG analyses were applied to explore the biological functions of PROS1 in LGG. GSEA was used to investigate potential PROS1 mechanisms. GO, KEGG, and GSEA analyses were analysed using the R package "ClusterProfiler".

Tumor immune dysfunction and exclusion (TIDE)
TIDE (http:// tide. dfci. harva rd. edu/), based on tumour pretreatment expression profiles, can estimate multiple published transcriptomic biomarkers to predict patient responses. It was used to investigate the association between PROS1 expression and therapy outcomes in clinical studies of immune checkpoint blockade [38].

Statistical analysis
Most analyses were conducted using R software, and the rest were GraphPad Prism 8.0. Logistic regression, univariate, and multivariate analyses were used to assess the influence of clinical variables on patient survival. Twotailed P-values less than 0.05 were considered statistically significant.

Analysis of PROS1 expression in patients with LGG
To explore possible anti-and pro-carcinogenic roles of PROS1, its mRNA expression was first analysed in human cancer and validated using the Sangerbox tools. . A separate analysis of PROS1 mRNA levels showed a significant difference between LGG and normal GTEx samples (Fig. 1C). Likewise, in the Gene Expression Profiling Interactive Analysis (GEPIA) database, higher PROS1 mRNA expression was found in LGG tissues than in normal brain tissues (Fig. 1D). Receiver operating characteristic curves were used to analyse the efficacy of PROS1 levels to distinguish between LGG samples and normal brain samples. The area under the curve (AUC) of 0.934 (0.922-0.946) suggests that the PROS1 gene may have the potential to identify LGG tissues. At the protein level, immunohistochemistry stainings were used to investigate the PROS1 expression in 15 paired tumour samples compared with adjacent normal tissues. The analysis demonstrated that PROS1 levels were substantially increased in LGG tissues ( Fig. 2A). Moreover, 30 paired samples and 4 cell lines were investigated by qRT-PCR. PROS1 mRNA expression was significantly upregulated in all 30 tumour samples compared to that in adjacent normal samples (Fig. 2B), as well as in 3 glioma cell lines (T98G, U87, and U251) compared to that in the non-malignant human astrocyte cell line NHA (Fig. 2C).

Identification of DEGs in LGG
PROS1-high samples and PROS1-low samples from HTSeq-Counts of TCGA-LGG were explored using the R package "DESeq2". In total, 2,050 DEGs showed statistically significant group differences, including 1,023 upregulated genes and 1,027 downregulated genes (Fig. 1F). LGG, which indicates that PROS1 may mediate tumorigenesis by epigenetic modification. DNA mismatches are potentially mutagenic and, thus, must be corrected by the mismatch repair system to maintain the integrity of the genetic information. As shown in Fig. 3C, we evaluated the correlation between the mutation levels of five mismatch repair system genes (MLH1, MSH2, MSH6, PMS2, and EPCAM) and PROS1, which suggested that the mismatch repair system may play a critical role in regulating tumorigenesis of PROS1. We analysed SNP data to detect variants and their frequencies in LGG. As shown in Fig. 3D, F missense mutations (0.2%) were the main type of SNPs. A frequency analysis of single nucleotide variations comparing groups with high and low PROS1 expression levels revealed IDH1, TP53, ATRX, CIC, TTN, FUBP1, MUC16, NOTCH1, PIK3CA, and EGFR as the top 10 mutated genes, with mutation percentages of 76%, 45%, 33%, 20%, 12%, 9%, 7%, 7%, 6%, and 6%, respectively ( Fig. 3F).Moreover, our analysis results showed that IDH1 and TP53 were the top2 mutation gene, and mutation frequency of IDH1 and TP53 in low PROS1 expression group was more than high PROS1 expression group, which also identified PROS1 may promote tumour progression and lead to poor prognosis in LGG patients. To identify alterations in copy number variation, data of PROS1 copy number variation extracted from the TCGA were explored using the R package "copynumber", and significant sample differences among the three types (491 neutral, 14 loss, and 3 gain) were detected by the Kruskal-Wallis test (Fig. 3E).

Correlation of PROS1 expression with DNA methylation and alternative splicing
As shown above, PROS1 expression was significantly correlated with the expression of four DNA methyltransferases. To explore the potential mechanism between PROS1 expression and DNA methylation, MethSurv was utilised to investigate the impact of different methylation levels and PROS1 expression on the prognosis in LGG. As shown in a heatmap (Fig. 4A), we found high levels of methylation in some of the probe regions (cg03680898, cg10993409, cg10959048, cg03451959, and cg14753809) and low methylation levels in other probe regions (cg05897638, cg24305970, cg01408194, cg03168026, and cg09400966). The survival analysis indicated that cg14753809, cg05897638, and cg24305970 were associated with poor prognosis (Fig. 4B-D). Alternative splicing has been identified as a vital mechanism regulating phenotypic diversity, gene expression, and proteomics; it can be used in biomarker and drug resistance research, as well as a therapeutic target. We used the OncoSplicing website to explore differential alternative splicing events of the PROS1 gene in LGG. Two alternative promoters (PROS1_AP_65674 and PROS1_AP_65675; Fig. 4E, F) found in the TCGA SpliceSeq database were associated with poor prognosis (Fig. 4G-J).
These data indicate that PROS1 expression may influence cancer treatment and prognosis by affecting genomic heterogeneity and cancer stemness.

Correlation between PROS1 expression and clinical parameters of patients with LGG
To evaluate PROS1 expression among groups of patients with different clinicopathological characteristics, we analysed 510 LGG samples from the TCGA database with their clinical data. As shown in Additional file 1: Fig. S6 and Table S1, PROS1 expression was significantly correlated with the WHO grade, IDH status, primary therapy outcome, histological type, and outcome measures (overall survival, disease-specific survival, and progressionfree interval). All other examined clinical parameters were not significantly correlated with PROS1 expression (Additional file 1: Fig. S6). The results of the logistic regression analysis indicated that the categorical dependent variable PROS1 expression was correlated with poor prognostic clinical parameters, including WHO grade, IDH status, age, and histological type (Additional file 1: Table S2). Taken together, LGG with high PROS1 expression may be more likely to progress to a more advanced grade and less susceptible to IDH1 mutations than LGG with low PROS1 levels.

Correlation between PROS1 expression and the prognosis of patients with LGG
Since PROS1 expression levels were closely correlated with LGG tumour progression, we subsequently explored their prognostic significance. As shown in the forest plot (Fig. 5A), we first analysed the correlation between PROS1 expression and overall survival in 33 tumours from the TCGA database. The results indicated that high PROS1 expression was related to poor survival in several cancers (LGG, BCLA, STES, STAD, and UVM), and the pan-cancer analysis demonstrated similar results for disease-specific survival, disease-free interval, and progression-free interval (Additional file 1: Figs. S7-S9). Next, we analysed the correlations of PROS1 expression with overall survival and disease-specific survival (Fig. 5B, C respectively). These results were validated by data from various databases and websites, including the Chinese Glioma Genome Atlas (CGGA; Fig. 5D), GEPIA database (Fig. 5E), OncoLnc database (Fig. 5F), and Pro-gnoScan database (Fig. 5G, H). Based on receiver operating characteristic curves of TCGA (Fig. 5I) and CGGA (Fig. 5G) data, the associations of PROS1 expression with 1-, 2-, and 3 year were explored. The results suggested that PROS1 expression had a moderate prognostic value. Next, we conducted subgroup analyses of overall survival based on clinicopathological characteristics. The prognosis of LGG patients with high PROS1 levels was poor in women, age > 40 years, 1p/19q non-codeletion, SD&PD, and astrocytoma subgroups (Fig. 6A-E). Univariate ( Fig. 6F and Additional file 1: Table S3) and multivariate ( Fig. 6G and Additional file 1: Table S3) Cox regression analyses showed that PROS1 expression, WHO grade, and age were independent prognostic factors for overall survival of patients with LGG. Risk factor association (Fig. 6H) and Sankey (Fig. 6I) diagrams were used to visualise the overall prognostic trends of the groups with high and low PROS1 expression, as well as the relationships among the three independent factors in living status. Finally, a nomogram was constructed based on PROS1 expression, age, and WHO grade as a quantitative tool for predicting the prognosis of patients with LGG (Fig. 6J). The prediction efficiency of the nomogram was evaluated using the C-index (0.754, confidence interval: 0.727-0.782), which implied that this model had moderate prediction accuracy. Consistent with the former results, the agreement between predictions and observations was good (Fig. 6K).

Identification of PROS1-interacting genes and proteins
The GeneMANIA database was used to analyse the gene-gene interaction network of PROS1 and identify altered neighbouring genes. The results showed that 20 genes were most closely related to PROS1, including MERTK, C4BPB, F5, and F8 (Fig. 7A). Similarly, the STRING database was used to assess the PROS1 protein-protein interaction network which contained 29 edges and 11 nodes, including TYRO3, PROC, and GGCX (Fig. 7B). Taking the above results into account, the relationships between coagulation-related genes and PROS1 was explored in LGG data from the TCGA. The significant results of this analysis demonstrated that in LGG, PROS1 was positively correlated with F2R, FGG, and PROC but negatively correlated with F5 (Fig. 7C).

Analysis of long noncoding RNA (lncRNA)-transcription factor (TF)-PROS1 triplets
To better understand the potential regulatory mechanisms of PROS1 in LGG, we examined the regulation of PROS1 expression by lncRNA-TF-gene triplets. We used the LncMAP database to explore possible PROS1containing triplet formations in LGG. The results showed that seven TFs may modulate PROS1 expression in LGG (Fig. 8A). To identify the TFs most likely regulating the PROS1 gene, we conducted an expression, prognosis, and correlation analysis of these seven TFs (Fig. 8B).
Among the four TFs (STAT1, SPI1, NFKB1, and CEBPA) with high expression and prognostic significance in LGG (Fig. 8C-I), the correlation coefficient was the highest (R = 0.514) between NFKB1 and PROS1. Subsequently, we explored lncRNAs possibly acting on NFKB1 and identified 31 lncRNAs that can interact with NFKB1 (Fig. 8J). Differential expression and prognostic analyses were used to determine the target lncRNAs. Ultimately, the two lncRNAs RP3-525N10.2 and MIR497HG were significantly differentially expressed in LGG (Fig. 8K-L), but only RP3-525N10.2 was significantly downregulated, suggesting a good prognosis. In summary, we identified the triplet RP3-525N10.2-NFKB1-PROS1 and, considering the downregulated expression of RP3-525N10.2 in LGG, suggest its possible role as a tumour suppressor gene affecting the prognosis in LGG. We propose that the lncRNA RP3-525N10.2 may act as a "decoy" that binds to the TF NFKB1 or guide the TF, and prevents its association with the target PROS1, which can reduce PROS1 expression, thereby improving the prognosis in patients with LGG.

Gene ontology (GO), kyoto encyclopedia of genes and genomes (KEGG) pathways, and gene set enrichment analysis (GSEA) of PROS1 in patients with LGG
GO and KEGG enrichment analyses were used to investigate PROS1-related biological pathways and functions. Circular plots depict the top 10 significant terms of BP, MF, CC, and KEGG enrichment analyses (Fig. 9A-D). Notably, in all analysis results, PROS1 was enriched in immune response-related processes or pathways, such as complement activation, B cell-mediated immunity, immunoglobulin-mediated immune response, and lymphocyte-mediated immunity in BP term, immunoglobulin complex, T-cell receptor complex, and MHC class II protein complex in CC term, and immunoglobulin receptor binding in MF term. In the KEGG pathway analysis, several immune-related pathways were highly correlated with PROS1 expression, including Th17 cell differentiation, Th1 and Th2 cell differentiation, primary immunodeficiency, T-cell receptor signalling pathway, and natural killer (NK) cell-mediated cytotoxicity. Consistent with the results of the GO and KEGG analyses, GSEA, including KEGG (Fig. 9E) and Reactome (Fig. 9F) pathways, showed that the top eight signalling pathways affected by PROS1 were enriched mainly in immune-related activities, including NK cell-mediated cytotoxicity, T-cell receptor signalling pathways, immunoregulatory interactions between lymphoid and non-lymphoid cells, signalling by the B-cell receptor BCR, and programmed death 1 (PD1) signalling. These results strongly imply that PROS1 participates in the regulation of immune responses in LGG.

Single-cell analysis of PROS1 functions in patients and experimental verification in GBM cell lines
To explore the functions of PROS1 in LGG tissues at the single-cell level, CancerSEA was used to explore phenotypes possibly regulated by PROS1. As shown in t-SNE and box plots (Additional file 1: Fig. S10), PROS1 was upregulated at the single-cell level in EXP0059 glioma cell groups.
The results of the functional analyses (Additional file 1: Fig. S10) demonstrated that PROS1 positively influenced cell invasion in two glioma single-cell sequencing datasets (BCH1126 and BCH836). As shown in Fig. 10, CCK8 assays depicted that knocking down PROS1 expression significantly reduced the proliferation ability of U87 cells and U251 cells; conversely, overexpressing PROS1 increased cell proliferation (Fig. 10B). Transwell assays were used to assess the invasion ability of cells. PROS1 knockdown resulted in a lower U87 cell and U251 cell invasion rate, while PROS1 over-expression showed the opposite results (Fig. 2D). In addition, colony formation assay (Fig. 10C) and 5-Ethynyl-2'-deoxyuridine (EdU) incorporation assay (Fig. 11) also suggested that knockdown of PROS1 inhibited cell proliferation. In contrast, over-expression of PROS1 increased cell proliferation rate. In summary, PROS1 increased the proliferation and invasion of GBM cells.

PROS1 is correlated with immune cell infiltration and tumour microenvironment in LGG
The functional enrichment results indicated that PROS1 may play a critical role in the immune system. Therefore, we investigated the relationships between PROS1 expression and immune cell infiltration. According to analyses of the Tumor Immune Estimation Resource (TIMER) database, six types of immune cells (B cells, CD8 + T cells, CD4 + T cells, macrophages, neutrophils, and dendritic cells) were associated with PROS1 expression and   prognosis in patients with LGG (Fig. 12A, B). Further analyses using the ssGSEA (Fig. 12C, D) showed that PROS1 expression levels were significantly positively associated with the infiltration of B cells, Tem, DC, Tgd, CD8 + T cells, NK cells, Th2 cells, NK CD56dim cells, eosinophils, T cells, Th17 cells, neutrophils, cytotoxic cells, iDC, aDC, T helper cells, and macrophages, but significantly negatively correlated with infiltration of CD56bright cells, pDC, and TReg. Moreover, various copy numbers of PROS1 were not significantly correlated with immune cell infiltration levels in LGG (Fig. 12E). To analyse the possible influence of PROS1 expression on the tumour microenvironment during tumour development, we analysed immune and stromal scores of LGG samples using the R package "Estimate". As shown in Fig. 10F, H PROS1 was positively correlated with immune score (R = 0.440, P < 0.001), stromal score (R = 0.430, P < 0.001), and estimated immune score (R = 0.440, P < 0.001).

Correlation of PROS1 expression with the expression of immune cell markers, immune checkpoint inhibitors, and immunomodulators
To better understand the interaction between PROS1 expression and immune responses, the TIMER and GEPIA databases were utilised to validate the association between PROS1 expression and immune cell markers in LGG. The genes listed in Table 1 represent various immune cells, including B cells, T cells, CD8 + T cells, monocytes, tumour-associated macrophages (TAMs), M1 macrophages, M2 macrophages, neutrophils, NK cells, and dendritic cells. In LGG, expression of PROS1 was significantly correlated with that of most immune cell markers (Table 1). Some studies have shown that PROS1 can influence the activation of TAMs [8,[17][18][19][20]; therefore, we explored the relationships between PROS1 expression and TAMs, including microglia and monocyte-derived macrophages (Table 1). According to the analyses of the TIMER and GEPIA databases, PROS1 expression levels had a significant positive correlation with 10 of the 13 immune cell markers in LGG (Additional file 1: Fig. S11). The correlation analysis between immunomodulators and PROS1 expression may reveal the types of cancers that benefit from immunotherapy targeting PROS1. In LGG, PROS1 was positively correlated with most of the 150 analysed immunomodulators (Fig. 13A). Based on the improved understanding of the functions of novel human immune checkpoint inhibitors [21], 60 immune checkpoint genes (24 inhibitory, 36 stimulatory) were explored regarding their correlations with PROS1 in different cancer types (Fig. 13B). Notably, more than 50 immune checkpoint markers were significantly associated with PROS1 expression in LGG. The scatter plots of five selected common immune checkpoint genes (CD274, CTLA4, HAVCR2, IDO1, and PDCD1) in the TIMER and GEPIA databases are shown in Fig. 11C-G. Collectively, these results strongly imply that PROS1 may play a critical role in LGG immunity and may be a potential immunotherapy target.

Association between PROS1 expression and therapy outcomes in clinical studies of immune checkpoint blockade (ICB)
Based on the predictive power of response outcomes and overall survival among ICB subcohorts, we evaluated the relevance of PROS1 as a biomarker by comparing its performance with those of standardised biomarkers. We found that PROS1 alone had an AUC value > 0.5 in 8 of the 20 examined ICB subcohorts (Fig. 14A). PROS1 exhibited a higher predictive value than B.Clonality, which gave AUC values > 0.5 in 6 ICB subcohorts. However, as a predictive biomarker, PROS1 was comparable to the combination of TMB score and B.Clonality (AUC > 0.5 in 8 ICB subcohorts) but inferior to MSI, CD27A, TIDE, IFNG, Merck18, and CD8. Our results also indicated that high PROS1 expression was correlated with worse PD1 outcomes in kidney renal clear cell carcinoma (Braun2020_PD1 Clear), melanoma (Liu2019_PD1 Ipi_Naive, Gide2019_ PD1, and Riaz2017_PD1 Ipi_Naive), PD-ligand 1 (LI) (BladderMariathasan2018_PDL1 mUC), ACT melanoma (Lauss2017_ACT), and CTLA4 (Nathan-son2017_CTLA4 Post) but achieved good CTLA4 therapeutic outcomes in melanoma (Nathanson2017_ CTLA4 Pre), and kidney renal clear cell carcinoma (Miao2018_ICB Clear) cancer cohorts (Fig. 14B). Screening results of knockout phenotypes also implied that PROS1 knockout may positively impact lymphocyte-mediated tumour killing in MC38 colon cancer (Kearney2018_T_PD1) models (Fig. 14C). Moreover, the MXD3 expression levels were correlated with overall survival (Fig. 14D-H)  (Nutt_Glioma@PRECOG, TCGA, and GSE16011@ PRECOG) and likewise with CTL levels (Fig. 14D-H) in 3/4 of the glioma cohorts (ca00037@PRECOG, TCGA, and GSE16011@PRECOG). CTL expression levels in the GSE16011@PRECOG cohort were also correlated with overall survival in PROS1 mRNA expression and copy number alteration data (Fig. 14I, J). Finally, the Enrichr platform was used to identify drug molecules, collected from the DSigDB database, targeting PROS1. The results showed that spiperone PC3, thapsigargin PC3, quercetin CTD 00006679, and pentabromodiphenyl ether CTD 00003077 are the four drug molecules with which most DEGs interact (Fig. 14K).

Discussion
In patients with LGG, the correlations of PROS1 expression with prognosis and multi-omics data have not been described yet. This is the first comprehensive evaluation of PROS1-related mechanisms possibly underlying carcinogenesis in patients with LGG. In this study, we first analysed PROS1 gene expression across various human cancers with a focus on LGG using in vitro techniques (qRT-PCRs with 30 pairs of tumour and adjacent tissues, as well as with conventional cell lines, and PROS1 immunohistochemistry with 15 paired tumour and adjacent tissues). Immune cell infiltration and tumour microenvironment, immune checkpoint inhibitors, immunomodulators, immune cell markers, and therapeutic outcomes in clinical studies of immune checkpoint blockade were subsequently explored, and we identified significant correlations with PROS1 expression. Notably, a connection between PROS1 expression and TAMs was revealed. Moreover, PROS1 functions at the single-cell level, as well as the involvement of lncRNA-TF-gene triplets, were explored in LGG. Finally, PROS1 expression was comprehensively analysed in combination with clinical diagnosis, treatment, and multi-omics data, including DNA methyltransferase expression and functional DNA methylation, expression of RNA modification and DNA mismatch repair system genes, copy number variation and single nucleotide variation frequency, PROS1 alternative splicing, tumour genomic heterogeneity, and cancer stemness. These results suggest the involvement of PROS1 in glioma carcinogenesis. This conclusion is supported by a study indicating that PROS1 may play a vital role in the development of GBM by influencing cellular proliferation, migration, invasion, and apoptosis [11]. Because lncRNAs and TFs have been widely found to play indispensable roles in the occurrence and progression of tumours, the LncMAP database was used to explore TFs and lncRNAs that may regulate PROS1 expression in LGG. Among seven TFs that may interact with PROS1, NFKB1 was identified based on expression, survival, and correlation analyses. A previous study has shown that the lncRNA SLC26A4-AS1 promotes NPTX1 transcriptional activity by recruiting NFKB1, thereby exerting antiangiogenic effects in glioma cells [22]. In the present study, we considered lncRNAs that were positively correlated with PROS1 and NFKB1, but only lncRNA RP3-525N10.2 met all screening conditions, was significantly upregulated in LGG, and had the potential to improve the prognosis of patients with LGG. Taken together, our study findings suggest that lncRNA RP3-525N10.2 may decoy or guide NFKB1, thereby reducing PROS1 expression and improving the poor prognosis in patients with LGG.
In view of the finite studies on PROS1 functions, we annotated its functions using GO, KEGG pathways, and GESA. The results demonstrated that PROS1-related genes were focused on well-known processes, including T-cell receptor signalling, KEGG cancer pathways, transforming growth factor-β signalling, JAK-STAT signalling, and various other immune-related pathways. Functional analyses at the single-cell level showed that PROS1 was upregulated in glioma cells, promoting cell invasion. Based on these results, we explored the relationships between PROS1related genes and the prognosis of patients with LGG.
Immune cell infiltration in the tumour microenvironment and sufficient expression of immune checkpoint genes can alter the efficacy of chemotherapy, radiotherapy, or immunotherapy, thereby influencing the prognosis of patients with cancer. Our results demonstrated that PROS1 expression was significantly positively correlated with the presence of most immune cells in LGG tissue, the expression of biomarkers by tumour-infiltrating immune cells, and the expression of immune checkpoint genes. Among these immune cells, TAMs are a rich cellular component of the brain TME that possess both tumour-promoting and immunosuppressive capacities [23]. Ubil et al. reported that PROS1 decreases M1 macrophage cytokine expression in vitro and in vivo [8]. Maimon et al. found that PROS1-deficient bone marrow-derived macrophages led to elevated TNF-α, IL-6, NOS2, and IL-10 levels via modulation of the SOCS3/NF-κB pathway [17]. Sadahiro et al. showed that PROS1 is secreted by TAMs/microglia and subsequently physically associates with and activates AXL in mesenchymal glioma sphere cultures [20]. The TAM population can genetically be divided into at least two main groups: tissue-resident microglia/macrophages of embryonic origin and tissue-invading monocyte-derived macrophages [24,25]. Their biomarkers extracted from CellMarker websites and the literature [26] are listed in Table 5, which shows that PROS1 expression is significantly correlated with TAMs and might partially account for PROS1-mediated oncogenic mechanisms in LGG. Taken together, TAMs play a unique role in tumours, and LGG is no exception. Additionally, the significant association between immune checkpoint genes and PROS1 indicated that targeting PROS1 might increase the efficacy of immunotherapy in LGG, which was consistent with the Tumor Immune Dysfunction and Exclusion (TIDE) results. According to the DSigDB database, spiperone PC3, thapsigargin PC3, quercetin CTD 00006679, and pentabromodiphenyl ether CTD 00003077 were the best drug candidates. This study has some limitations. First, the TCGA and CGGA databases inevitably neglect the inner tumour heterogeneity in different databases. Second, the possible mechanisms of DNMTs, MMRs, DNA methylation, alternative splicing, the identified lncRNA-TF-gene triplet and the roles of PROS1 in tumour migration, immune cell infiltration, and tumour escape should be explored in more detail in future studies. Third, although targeting PROS1 in patients with LGG is promising, a concern may be that PROS1 inhibits BBB breakdown, as it exerts a protective effect at the BBB [27].
In conclusion, this study elucidated that PROS1 is upregulated in LGG and various other types of human cancers and may serve as a novel prognostic biomarker in RP3-525N10.2

Tumorigenesis
LGG Brain

Decoy Guide
Tumor Immune Infiltration