Extent of Resection Seems to Show no Difference in Post-Operation Complications and Prognosis, While Inflammatory Response in ACP is Relevant to Poor Clinical Outcome
In 61 cases, total resection was performed in 36 cases and subtotal resection in 25 cases (residual part adhered to important structures, tumor or cystic part that is hard to separate) (Talbe.1). By comparing the differences of different resection methods and different surgical approaches, it was observed that there was no significant difference between postoperative complications and long-term no progress of children (Table. S1 and S2), in spite of relapse tissue shows more papillary humps embedded in tissue (Figure S1).
At the end of the follow-up, there were tumor recurrences in 18 patients among all the 59 survived, and the rest two died of electrocytes imbalance and hypothalamus reactions during admission. The relapse time was 6-70 (median 24) months, and the follow-up time was 1-120 (median 21) months. There was no significant difference in recurrence rate in the gender (P = 0.515), and in surgical approaches (P = 0.381). PFS calculated by Kaplan Meier analysis and log rank test revealed that there was no significant difference between the degree of tumor resection and PFS of patients with progression free survival (P = 0.693), that is, compared with the total resection group, the recurrence rate of patients with partial tumor resection was not increased (Figure. 1A).
Among the 25 patients who were diagnosed as ACP by postoperative pathology, there were 3 cases of low-grade inflammatory response, 12 cases of medium-grade and 10 cases of high-grade, χ 2 test showed that there was no significant difference in the degree of surgical resection between different inflammatory grades (P = 0.276), but there was a high correlation between the degree of surgical resection and tumor progression (P < 0.001). Meanwhile, the survival analysis showed that the prognosis of high-grade group was significantly worse than that of low-grade group (P = 0.022) (Figure. 1B). At the same time, in 6 cases of recurrent tumor tissue, 5 cases were high inflammatory infiltration state. Comparison before and after recurrence of the same case, showed more M2 activated phenotype of macrophage and microglia, while it’s interesting to find that Iba-1+ cells seem to decrease in relapse tumor (Figure. 1C and 1D). All these results suggested inflammatory response might influence the progress of ACP.
Macrophage and Serpins Family are Potential Players in TME of ACP
The results above hinted that inflammation might influence the progress of ACP and patients’ outcome. On the evidence that macrophage and microglia in brain are critical in TME re-building. In GSE94349, we used NetworkAnalyst and GSEA to screen potential functional molecules associated with TAMs and inflammation (Figure 2A; Figure S1B), and it indicated that SERPINs family (SERPINB2, SERPINB5, SERPINE1, SERPING1) were respectively involved in various functional gene sets like macrophage, microglia, inflammatory response and classic inflammation pathway NF-κB (Figure 2B). Apart from the four genes, we chose top 5 genes in each gene set to demonstrate their relationship. Correlation hotmap showed the potential relationship among those hub genes in different gene sets, and SERPINE1 had significant correlation with SERPINB2, SERPINB5. What’s more, SERPINE1 was also associated with either PRRG4, TNFSF10, DSC2 or SDC1, suggesting a close relationship of these five genes with tumor proliferation, invasion and metastasis (Figure 2D).
And it was worth noting that expression level of SERPING1 seemed to show negative correlation with other genes, suggesting it might participate in different pathways from SERPINE1. IL15, known as functional immune inducing and activation molecules, also showed pertinent connection with SERPINE1, SERPINB2 and SERPING1. We next used CIBERSORTx to verify the immune environment of ACP, PA and normal brain tissues, and the results showed high correlation and percentage of M0 macrophage, M2 macrophage, CD4+ T memory cells, which accounted for more than 60%, indicating involvement of macrophage in tumor environment (Figure 2C). Multiple comparisons between immune cells infraction of ACP (n=24), PA (n=10) and normal brain (n=27) tissues displayed macrophages M0, T cells CD4+ memory resting and plasma cells in ACP were significantly enriched. Macrophages M2 in tumor were all up-regulated, while macrophages M1 and T cells CD4+ memory activated were both less activated in ACP and PA (Figure 2E).
Comprehensive Analysis of SERPINs Family in Glioma and HNSC Shows SERPINs Prognostic Values
Based on the evidence that TME similarity with brain invasive tumor, and ACP are tendentious to malignantly transform into squamous cancer, we compared the transcriptional levels of SERPINs family in tumors with normal tissue in Oncomine database (P Value<0.01, Fold Change >1.5, Top Gene 10%, Data Type mRNA) (Figure 3B). It showed SERPINE1 was significantly upregulated in Brain, HNSC and Esophageal Cancer. SERPINB2 and SERPINB5 were more tendinous increasing in Lung Cancer and Gastrointestinal Cancer. The expression level of SERPING1 was higher in Brain, Esophageal Cancer and Lymphoma. The mRNA expression levels of SERPINE1 were significantly in patients of glioma and squamous cancer (Table S3).
In addition, Pan-Cancer analysis in TIMER also showed that the family increase in various cancer like COAD, HNSC, LUSC (P<0.001) (Figure 3A). We also tried to find if there exist similar expression patterns of SERPINs in other relevant cancer type. In GEPIA we chose GBM, LGG and HNSC to further explore transcriptional level of SERPINs between tumor and normal tissue (|Log2FC| Cutoff 1.5, q-value Cutoff 0.01). it was found that SERPINE1 and SERPING1 were significantly upregulated in all three cancers. In HNSC SERPINB5 was higher in tumor tissues while SERPINB2 was lower than normal tissue (Figure 3C).
Correlation between SERPINs expression level and tumor grades (stage) revealed that SERPINs had significant relevance with grades of GBM, while it seemed to have no influence in HNSC (Figure 4A). Kaplan–Meier curves demonstrated SERPINE1, SERPINB2 and SERPING1 were associated with RFS but not OS in GBM patients (P<0.05) (Figure 4B). And all SERPINs had no statistical difference both in OS and RFS in HNSC patients (Figure S3).
Predicted Functions and Pathways of the Changes among SERPINs and SERPINs in Macrophages Might Shares Similar Expression Pattern between ACP and GBM
Using TISCH, we sourced single-cell datasets GSE141982 and GSE131928 to reach a comprehensive exploration of SERPINs and TME. Cell cluster profiles showed that SERPINE1 evidently collocate in macrophage and mononuclear clusters, with slight enrichment in malignant cells and less in other cell types (Figure 5A). At the same time, SERPING1 had a minor expression level. SERPINB2 and SERPINB5 were both expressed with basic levels in all kinds of cells (Figure 5B). Gene-gene correlation showed SERPINE1 had a certain relation with SERPINB2 and SERPING1. Besides other SERPINs, SERPINE1 also showed significant correlation with CD68, CD86, CD163 and CD206 (Figure 5C). PPI network among SERPINs had been established to explore the relationship between upstream and downstream connection and probable pathway involved with tumor metastasis, invasion and activation of macrophage (Figure S2C). Enrichment pathway consist of various tumor inflammation pathway, such as p53, angiogenesis and so on. At last, we tried to conduct FISH to validate whether there exists colocation between SERPINEs and macrophage in ACP, and the result showed there existed evident accumulation of SERPINE1 in macrophage. It was notable that SERPINE1+ macrophages were more likely located surrounding solid tumor while SERPING1+ in cystic part. (Figure 5D).