Immunoseroproteomic profiling in autoantibody to ENO1 as potential biomarker in immunodiagnosis of osteosarcoma by serological proteome analysis (SERPA) approach

Abstract

Osteosarcoma (OS) is the most common highly malignant primary solid bone tumor. Despite its relatively low incidence among cancers, it remains one of the most harmful primary malignant tumors in childhood and adolescence. It is now evident that serum autoantibodies against tumor-associated antigens (TAAs) could be used as serological cancer biomarkers in types of cancers. Serological proteome analysis (SERPA) approach was applied to profile anti-TAA autoantibody response in sera from patients with OS and normal human, as well as explore difference between this response. This approach can detect autoantibodies that could serve as clinical biomarkers and immunotherapeutic agents. Enzyme-linked immunosorbent assay (ELISA) and Western blotting were further used to validate the level of identified TAAs. ENO1 as a 47kD TAA in OS was identified and characterized by SERPA. Analysis of 172 serum samples with OS, osteochondroma (OC), and normal human sera (NHS) by ELISA showed higher frequency of anti-ENO1 autoantibodies in OS sera compared to others. Interestingly, decrease of ENO1 immunoreactivity was observed in most patients after treatments, which may imply a potential association between anti-ENO1 autoantibody titers and disease progression. Nine of twelve sera reacted strongly against purified ENO1, but three reacted weakly against purified ENO1, which indicated 75.0% sera with positive optimal density values from ELISA were consistently positive in Western blotting. The expression of ENO1 in OS tissues was evaluated by immunohistochemistry in tumor microarray. ENO1 was one of the autoantibodies that elicit autoimmune responses in OS and can be used as biomarkers in immunodiagnosis and progression of OS.

Keywords: ENO1; Osteosarcoma; immunodiagnosis; serological proteome analysis; tumor-associated antigen.

Figure 1.

Detection of autoantibodies against cellular protein antigens extracted from U2-OS and Saos-2 cell lines in sera from patients with OS, OC, and NHS. (A) 1-D Western blotting shows that autoantibodies against the protein antigens extracted from U2-OS cell line detected after probing with sera OS# 16, 19, 21, 25, 26 (Lanes 1-5) were significantly higher than that in sera OC# 24, 25, 26, 27, 28 (Lanes 6-10) and normal human sera N# 28, 29, 30, 31, 32 (Lances 11-15). (B) 1-D Western blotting shows that autoantibodies against the protein antigens extracted from Saos-2 cell line detected in sera from patients with OS# 16, 19, 21, 25, 26 (Lanes 1-5) were significantly higher than that in sera OC# 24, 25, 26, 27, 28 (Lanes 6-10) and normal human sera N# 1, 2, 3, 4, 5 (Lances 11-15). OS: Osteosarcoma; OC: Osteochondroma; NHS: Normal Human Sera

Figure 2.

The pattern of 47-kD proteins revealed by immunofluorescence analysis of Saos-2 cell substrate. Immunofluorescence was done using whole sera or sera purified with 47-kD antigen. Three representative OS sera (C, E,G) were used, and one normal human serum (A) was used as control. Left panel (A, C, E, G): whole sera; right panel (B, D, F, H): sera purified with 47-kD antigen

Figure 3.

2-DE Western blotting analysis of U2-OS and Saos-2 cells was probed with one representative OS serum sample and a pool of five 5 NHS used as control. (A) The 2-DE protein profile of U2-OS cells (left panel). Western blotting analysis of 2-DE gel was probed with one representative osteosarcoma serum sample OS (#26) which contains antibodies to the 47kD protein (middle panel). The nitrocellulose membrane transferred from 2-DE gel was stained with Ponceasu S (right panel). (B) The 2-DE protein profile of U2-OS cells (left panel). Western blotting analysis of 2-DE gel was probed with a pool of five 5 NHS (#3, 8, 7, 1, 36) (middle panel). The nitrocellulose membrane transferred from 2D gel was stained with Ponceasu S (right panel). (C) The 2-DE protein profile of Saos-2 cells (left panel). Western blotting analysis of 2-DE gel was probed with one representative osteosarcoma serum sample OS (#11) which contains antibodies to the 47kD protein (middle panel). The nitrocellulose membrane transferred from 2-DE gel was stained with Ponceasu S (right panel). (D) The 2-DE protein profile of Saos-2 cells (left panel). Western blot analysis of 2-DE gel was probed with a pool of five 5 NHS (#3, 8, 7, 1, 36) (middle panel). The nitrocellulose membrane transferred from 2D gel was stained with Ponceasu S (right panel)

Figure 4.

Prevalence of autoantibody to ENO1 in OS, OC and normal individuals. (A, B and C) Receiver operating characteristic (ROC) curve analysis of ENO1 expression to discriminate the OS group from the OC and NHS groups. (D) ELISA scatter dot plot shows OD values with solid lines representing the mean of each cohort, OS (n=52), OC (n=28) and NHS (n=49). The dotted line represents the mean value of the normal individuals’ cohort plus three standard deviations to determine levels and frequency of autoantibody reactivity against ENO1. (E) The graph shows higher frequency of anti-ENO1 antibodies in OS patient sera compared to OC and NHS sera. (F) The OD values of anti-ENO1 autoantibody over time in the serially collected at three time points: diagnosis, before surgery, and after surgery. (G, H and I) Serial study of anti-ENO1 antibody levels (OD value) during a 1-year period in three representative OC patients

Figure 5.

Evaluation of ENO1 protein expression in normal, osteosarcoma, and chondrosarcoma tissues by immunohistochemistry. (A, B) Negative staining of ENO1 expression in representative normal bone tissue at 100X and 400X magnification, respectively; (C, D) Positive staining of ENO1 expression in osteosarcoma tissue at 100X and 400X magnification, respectively; (E, F) Positive staining of ENO1 expression in chondrosarcoma tissue at 100X and 400X magnification, respectively

Figure 6.

Western blotting analysis with representative positive sera in ELISA. Representative sera from patients with OS (lanes 1-3), OC patients (lanes 4-6) and normal human sera (lanes 7-9) probed against purified ENO1 showed differences in immunoreactivity by Western blotting. 9 of 12 (75.0%) sera with positive OD values were consistently positive in Western blotting