Exploration and Validation of a Novel Inflammatory Response-Associated Gene Signature to Predict Osteosarcoma Prognosis and Immune Infiltration

Figures & data

Figure 1 The flow chart of the present study. (A) The osteosarcoma inflammatory response-associated DEmRNAs were identified in GSE21257 and MSigDB databases. (B) The inflammatory response-associated gene signature was established through univariate and multivariate cox regression analyses. (C) The application of inflammatory response-associated gene signature in clinic and validation in Target database. (D) Identification and functional analysis of DEmRNAs related to inflammatory response-associated risk. (E) The association between immune infiltration and inflammatory response.

Abbreviations: DEmRNA, differentially expressed messenger RNA; MSigDB, Molecular Signature Database.

Table 1 The Inflammatory Response-Associated DEmRNAs in GSE21257

Gene	Log2FC	P value
PDPN	−0.72	0.041
AXL	−0.75	0.0079
BST2	−0.69	0.022
C5AR1	−0.62	0.00014
STAB1	−0.78	0.0021
CXCL9	−0.94	0.015
TNFRSF1B	−0.79	0.018
CYBB	−0.88	0.031
CD48	−0.82	0.00028
CXCL10	−1.26	0.0034
LCP2	−0.77	0.00060
IL10RA	−0.71	0.000084
SERPINE1	−0.77	0.023
CLEC5A	−0.90	0.00000013
CCL5	−0.79	0.0046
OLR1	−0.91	0.0012
GNA15	−0.77	0.0020
IL1B	−0.85	0.000077
MYC	0.63	0.012
PLAUR	−0.80	0.00032
CCL2	−0.71	0.016
RGS1	−0.81	0.0090
FPR1	−0.82	0.00025
CD14	−1.29	0.000057

Abbreviation: DEmRNA, differentially expressed messenger RNA.

Figure 2 Identification of inflammatory response-associated DEmRNAs related to osteosarcoma metastasis. (A) DEmRNAs related to osteosarcoma metastasis in GSE21257 database. Red dots indicated up-regulated DEmRNAs (P < 0.05, FC > 1.5) and green dots indicated down-regulated DEmRNAs (P > 0.05, FC < −1.5). (B) Twenty-four inflammatory response-associated DEmRNAs were screened out in GSE21257 and MSigDB databases.

Abbreviations: DEmRNA, differentially expressed messenger RNA; FC, fold change; MSigDB, Molecular Signature Database.

Table 2 The Multivariate Cox Regression Analysis of GSE21257

Gene	Coefficient	HR	HR 95 Low	HR 95 High	P value
CLEC5A	−0.96	0.38	0.18	0.83	0.015
MYC	0.41	1.51	0.92	2.49	0.11

Abbreviation: HR, hazard ratio.

Figure 3 Construction and validation of the inflammatory response-associated prognostic signature in GSE21257 and Target datasets. (A) Three inflammatory response-associated prognostic genes were identified in univariate cox regression analysis. (B and E) Upper: The distribution and median value of risk scores in GSE21257 (B) and Target (E) datasets. Bottom: The scatter plot of patients’ risk scores and survival status in GSE21257 (B) and Target (E) datasets. (C and F) Kaplan-Meier survival plot of patients in high- and low-risk groups in GSE21257 (C) and Target (F) datasets. (D and G) Time-dependent ROC curve at 1, 3, and 5 years of inflammatory-associated prognostic signature in GSE21257 (D) and Target (G) datasets.

Abbreviations: Target, Therapeutically Applicable Research to Generate Effective Treatments; ROC, receiver operating characteristic; AUC, area under curve.

Figure 4 Relationships between inflammatory response-associated gene signature and clinical parameters in GSE21257. (A and B). Univariate and multivariate cox regression analyses revealed the risk score was the independent prognostic factor in GSE21257. (C–F). Boxplots of the relationship between CLEC5A expression and clinical characteristics. (G–J). The correlation between MYC expression and clinical characteristics.

Abbreviation: CLEC5A, C-type lectin domain family 5 member A.

Figure 5 Establishment of the nomogram in GSE21257. (A) The nomogram predicted the 3- and 5-year survival risk in osteosarcoma patients. (B) The calibration curve of the 3-year survival. (C) The calibration curve of the 5-year survival.

Figure 6 Identification and functional analyses of DEmRNAs related to inflammatory response-associated risks. (A) The heatmap of DEmRNAs associated with risk score in GSE21257. (B) The heatmap of risk score-associated DEmRNAs in Target dataset. (C) The overlapped DEmRNAs in GSE21257 and Target datasets. (D and E) The top 10 GO functional and top 30 KEGG pathway analyses of DEmRNAs.

Abbreviations: Target, Therapeutically Applicable Research to Generate Effective Treatments; DEmRNA, differentially expressed messenger RNA; GO, gene ontology; KEGG, Kyoto encyclopedia of genes and genomes.

Figure 7 The relationships between inflammatory response and immune infiltration in GSE21257. (A) The correlation between risk score and immune score. (B). The relationship between risk score and stromal score. (C) The infiltrative proportion of 22 immune cells in osteosarcoma with statistically significant. (D) The correlations between risk scores and different immune cells. (E) The association between risk scores and different immune functions. (F) The overlapped immune cells and functions in GSE21257 and Target datasets. *P < 0.05; **P < 0.01; ***P < 0.001.

Abbreviations: Target, Therapeutically Applicable Research to Generate Effective Treatments; NS, not significant; aDCs, activated dendritic cells; DCs, dendritic cells; iDCs, immature dendritic cells; NK, natural killer; aDCs, activated dendritic cells; pDCs, plasmacytoid dendritic cells; Tfh, T-follicular helper cells; Th, T helper; TIL, tumor infiltrating lymphocytes; Treg, T regulatory cells; APC, antigen-presenting cells; CCR, cytokine-cytokine receptor interaction; HLA, human leukocyte antigen; MHC, major histocompatibility complex; IFN, interferon.

Figure 8 The expression of MYC and CLEC5A in cell lines and osteosarcoma patients’ samples. (A and B) The expression of MYC (A) and CLEC5A (B) in osteosarcoma and osteoblast cell lines. (C and D) The expression of MYC (C) and CLEC5A (D) in high- and low-metastatic cell lines. (E and F) The expression of MYC (E) and CLEC5A (F) in metastasis and non-metastasis patients, scale bar: 20μm. CLEC5A: C-type lectin domain family 5 member A; *P < 0.05; **P < 0.01; ****P < 0.0001.