The most common primary malignant tumor in orthopedics is OS. It is characterized by a strong inclination toward local aggressiveness and early metastasis, which leads to a poor prognosis for patients with OS (Luetke, Meyers, Lewis, & Juergens, 2014). In recent years, predictive models based on multiple functional genomic methods for OS have become increasingly common in forecasting the prognosis of OS patients. Wang et al. analyzed the expression levels of cupping-associated long noncoding RNAs (lncRNAs) in OS and constructed a prognostic model for cupping-associated lncRNAs (X. Wang, Xie, & Lin, 2023). Yu et al. analyzed the gene expression levels of CD8 + lymphocytes in OS, identified six genes related to OS prognosis, and further constructed a prognostic model (Yu Chen et al., 2023). However, few studies have directly targeted OS tumor cells, and the cell type composition, dynamics, and characteristics of OS tumor foci are largely unknown. Consequently, we speculated that the detection of OS tumor cell-related genes is important for the prognosis of OS patients. Furthermore, these discoveries might help to identify prognostic biomarkers for OS tumors and develop more accurate therapeutic regimens and potential targeted drugs. Therefore, we constructed a risk model using HIS-OS-related genes in OS cell subsets to predict the prognosis of OS patients.
Within this research investigation, we established a bioinformatics prognostic model and validated its accuracy using HIS-OS genes. Seven HIS-OS-related genes were incorporated into a risk map to determine whether they could accurately predict OS prognosis. First, an alternative prognostic model comprising seven HIS-OS-associated genes was constructed by analyzing single-cell sequencing data of OS as well as differential gene expression analysis. These results proved that the risk profile is capable of accurately predicting an OS patient's prognosis. As a result, in the future clinical treatment of OS patients, the risk score can be calculated from our developed risk model, and the prognosis can be inferred from the calculated risk score. Furthermore, we examined the connection between the risk profile and the TME. Moreover, we investigated possible therapeutic targets and corresponding treatment drugs for this target. In conclusion, our HIS-OS-related gene-based prognostic model provides a valuable reference for evaluating the prognosis and treatment of OS.
Tumor purity is the proportion of tumor cells in a mixture and is closely related to the prognosis of tumor patients (Y. Mao et al., 2018). OS patients with increased tumor purity as well as decreased stromal scores, ESTIMATE scores, and immune scores tend to present a higher degree of malignancy, which often results in a negative prognosis (Yoshihara et al., 2013). Consistent with our study, the high-risk group had high tumor purity and lower ESTIMATE, immune, and stromal scores. Tumor-infiltrating immune cells (TIICs) in OS are considered to have a substantial influence on tumor advancement and prognosis, among other factors (Ying Chen, Zhao, & Wang, 2020; C. Zhang et al., 2020; Z. Zhang et al., 2022). To further understand the impact of TIIC on the prognosis of OS, we compared the abundance of 24 TIICs in the two risk groups. The immature dendritic cells (iDCs) and activated dendritic cells (aDCs) are the immature and activated dendritic cell (DC) subsets, respectively. Moreover, iDCs strongly phagocytose tumor antigens and differentiate into aDCs when they ingest antigens or are stimulated by certain factors (Gardner & Ruffell, 2016). aDCs recognize and process immune signals, transport tumor antigens, and present them to T cells, which activate the antitumor function of T cells (Wculek et al., 2019). CD8 + T cells and cytotoxic cells are considered to have important antitumor effects. They are considered to be the main T cells that exert antitumor effects; these cells can be activated by tumor antigens presented by aDCs and macrophages and thus recognize and kill tumor cells, exerting antitumor effects and thus significantly prolonging patient survival (Y. Sun et al., 2021; Thommen & Schumacher, 2018). Our findings were consistent with those of previous studies showing that aDCs, iDCs, CD8 + T cells, cytotoxic cells, T cells, and macrophages were significantly downregulated in the high-risk group compared with the low-risk group. However, macrophages are M2-type macrophages in most malignant tumors and promote tumor metastasis due to their proangiogenic and other effects (Huang et al., 2021). However, macrophages in highly differentiated OSs are composed of a mixture of both M1-type and M2-type cells. The greater the number of M1-type patients was compared to that of M2-type patients, the lower the risk of metastasis and the longer the lifespan expectancy of OS patients (Zhao, Zhang, Zhang, Ma, & Feng, 2021). Therefore, we suggest that infiltrating macrophages in the low-risk group were likely dominated by M1-type macrophages.
The IPS is an excellent biomarker for identifying responders to immunotherapy and consists of four main factors: immunosuppressive cells, effector cells, MHC molecules, and immune checkpoints (Charoentong et al., 2017). Increased IPS scores are associated with increased immunogenicity (Hajiran et al., 2021). In our study, IPS was found to be higher in both risk groups, without any notable difference between them, suggesting that OS is a highly immunogenic tumor that could benefit from immunotherapy.
Immune checkpoint molecules are often found at higher levels in the TME of a variety of malignant tumors (Arum et al., 2010; Toor, Sasidharan Nair, Decock, & Elkord, 2020), and immune checkpoint inhibitor treatment has demonstrated encouraging clinical outcomes (Meftahpour, Aghebati-Maleki, Fotouhi, Safarzadeh, & Aghebati-Maleki, 2022; Tang et al., 2022). However, little information about this therapeutic strategy has been published for OS. Therefore, we compared the expression levels of 38 immune checkpoint molecules in the two risk groups. CD80 and CD86 are mostly found on the plasma membrane of antigen-presenting cells, namely, DCs and monocytes/macrophages, and their important function is to bind to and costimulate signals with CD28 receptor proteins on the surface of T cells, which in turn activate the proliferation and differentiation of T cells and contribute to antitumor immunity (Bolandi et al., 2021; Esensten, Helou, Chopra, Weiss, & Bluestone, 2016; Fleischer et al., 1996). CTLA4 is expressed at higher levels on activated T cells and interacts with the ligands CD80 and CD86 to provide coinhibitory signals that suppress T-cell activation and multiplication (Engelhardt, Sullivan, & Allison, 2006; Krummel & Allison, 1995). Multiple studies in the field of cancer have demonstrated that elevated levels of CD80, CD86, and CD28 are associated with the suppression of OS cell proliferation and metastasis (Li et al., 2022). Consistent with these findings, the expression levels of CD80, CD86, and CD28 were notably elevated in our study compared with those in the low-risk group. However, the low-risk group exhibited significant overexpression of CTLA4, which we suggested was associated with secondary upregulation of CTLA4 on the T-cell surface due to T-cell activation.
TIDE is highly regarded for its usefulness in identifying the effectiveness of immune checkpoint inhibitors, and numerous reports have suggested that CTLA-4 and PD-1 inhibitors are essential for treating many intermediate malignancies. Additionally, combining medications that target CTLA-4 and PD-1 receptors may have additive advantages when used with anticancer immunotherapies (Rotte, 2019). New research has indicated that CTLA-4 and PD-1 could have functions in the treatment of OS, but it is still uncertain how individuals with varying prognostic risks respond to this kind of immunotherapy (S.-D. Wang et al., 2016). Therefore, the TIDE score was chosen for this study to evaluate the effectiveness of PD-1 and CTLA-4 immunotherapy in both the high-risk and low-risk patients. The findings of our study indicate that individuals with a low risk of death exhibit positive responses to immune checkpoint therapy and may benefit from immunotherapy. The high-risk group had modest levels of immunosuppressive checkpoint expression, which remained unresponsive to immune checkpoint therapy. This finding is consistent with our conclusions and speculations above.
According to our functional analysis, GSEA revealed that genes associated with biological processes such as the inflammatory response and IFN-γ response were enriched predominantly in patients at low risk for OS. The inflammatory response has dual functions in tumor progression and can either promote or inhibit tumor progression (Greten & Grivennikov, 2019; Philip, Rowley, & Schreiber, 2004). Current research suggests that the induction of acute inflammation promotes immune cell maturation and antigen presentation, leading to an antitumor immune response (H. Zhao et al., 2021). IFN-γ has numerous antitumor effects, including promoting inflammatory responses, modulating antigen presentation, inhibiting angiogenesis, and promoting tumor dormancy and apoptosis to inhibit tumor growth (Burke & Young, 2019; Mauldin et al., 2016). In addition, it has been shown that enhanced IFN-γ secretion could reduce the development of tumorigenic M2 macrophages, thereby inhibiting the growth of OS (Kang et al., 2017). GO enrichment analysis revealed that these genes were involved mostly in biological processes such as ECM organization, external encapsulating structure organization, and ossification. Several investigations have indicated that the tumor-associated ECM is involved in promoting tumor cell growth, invasion, metastasis, and angiogenesis and that it resists cell death and drug diffusion (Najafi, Farhood, & Mortezaee, 2018; Theocharis, Skandalis, Gialeli, & Karamanos, 2016). The collective influence of these EMC-associated biological processes may serve as a significant catalyst for OS cell migration. In addition, KEGG analysis revealed that these genes were enriched in pathways including the PI3k-Akt signaling pathway, The ribosome pathway, and the focal adhesion pathway. The PI3K-Akt signaling pathway is an intracellular signaling pathway that responds to extracellular signals to promote metabolism, proliferation, cell survival, growth, and angiogenesis (Yang et al., 2019). Ribosome synthesis is increased in cancer cells as a reaction to increased protein synthesis and maintenance of unrestricted growth, and several articles have revealed that the ribosome pathway is related to the development of tumors and a negative prognosis (El Khoury & Nasr, 2021; Figueiredo & McCarthy, 2021). High expression of the focal adhesion pathway has been shown to be closely associated with tumor metastasis, and inhibition of related genes improves the survival of tumor patients (Legerstee & Houtsmuller, 2021; Lu, Linares, Xu, & Rui, 2021). The above evidence demonstrated that pathways enriched for HIS-OS-related genes included those that promote tumor proliferation, migration, and invasion, ultimately resulting in a worse prognosis among patients with OS.
We examined the gene expression levels in both the tumor and normal groups and the association between gene expression and survival prognosis to further determine the role of the above prognosis-related genes in OS. Analysis of the findings revealed a substantial increase in SERPINE2 expression in the tumor group, and these patients had a poorer prognosis. SERPINE2 encodes a serpin protein that belongs to a family of proteins that inhibit serine proteases and are effective against trypsin, thrombin, and plasma proteases (Buchholz et al., 2003). In other malignancies, the role of SERPINE2 in inhibiting serine proteases and promoting tumor progression has been widely reported, especially in cases of tumor metastasis, such as breast, gastric, and lung adenocarcinomas (Dokuni et al., 2020; Fayard et al., 2009; K. Wang et al., 2014). Experimental investigations have demonstrated that SERPINE2 is significantly upregulated in OS tissues, particularly in stage II-III patients with metastases and tumor nodal metastases (M. Mao & Wang, 2016). High expression of SERPINE2 in OS stimulates tumor cell proliferation, promotes drug resistance, and leads to poor survival through regulation of CDK4 and cell cycle protein D. The aforementioned information indicates that SERPINE2 might serve as a promising target for therapeutic intervention in patients with OS.
The PDB structure of SERPINE2 was shown to dock well with that of IITZ-01 in virtual screening and molecular docking simulations. We further conducted CCK-8 and migration invasion tests to confirm the inhibitory impact of IITZ-01 on OS cells. Specifically, we observed significant inhibition of OS cell proliferation, migration, and invasion. However, the exact underlying mechanism requires further investigation. Furthermore, future research will need animal trials to confirm the inhibitory impact of IITZ-01 on OS in living organisms. In conclusion, our findings suggest that SERPINE2 might serve as a promising therapeutic target for OS and that IITZ-01, an inhibitor of SERPINE2, shows promise as a therapeutic drug for OS.