Histology and immunohistochemistry of angiogenic factors in ANT and OSCC tissues
H&E stained ANT histological tissue section showed normal oral tissue histology. In contrast, H&E stained OSCC tissue section showed a distinct pattern of well-differentiated OSCC (Fig. 1A). CD31 immunohistochemistry tissue section showed few CD31-stained spots in ANT sections, and distinct CD31-stained microvessels in OSCC tissue sections (Fig. 1A). CK18 is a soluble cytokeratin present in proliferating cells and is highly concentrated in the G2-M phase of the cell cycle [27]. Since CK18 is highly expressed in OSCC tissues, we further performed CK18 immunohistochemistry. Brown granular staining in the nucleus of tumor cells was considered positive for CK18. ANT sections were hardly stained with CK18, but OSCC tissue sections showed intense CK18 staining (Fig. 1A). Quantitative analysis of CD31 stained microvessel structure revealed 2.5-fold higher MVD in OSCC tissue compared to ANT (Fig. 1B). Similarly, OSCC tissue showed a 5.06-fold higher expression of CK18 compared to ANT (Fig. 1C). mRNA expression analysis by RT-qPCR also showed upregulation of CD31 and CK18 in OSCC tissue compared to ANT (Fig. 1D and E).
mRNA expression of angiogenic factors in OSCC tissues and ANT
We further analyzed the mRNA level expression of angiogenic factors in OSCC and ANT tissue by RT-qPCR. The expression of ANG, ANG-2, HGF, PIGF, VEGF, PDGF-BB, and HB-EGF in the OSCC-group were 1.58-, 13.56-, 3.00-, 8.86-, 3.34-, 2.08-, and 4.63-fold higher, respectively compared to ANT-group (Fig. 1F-1N). LEP expression in OSCC-group was not significantly affected compared to ANT-group (Fig. 1O). The expression of bFGF was reduced in OSCC-group compared to ANT-group (Fig. 1H, 1M).
Protein level expression of angiogenic factors in OSCC tissues and ANT
We analyzed the protein level expression of angiogenic factors in OSCC and ANT tissue lysates by protein chip array. Among the angiogenic factors tested, the fluorescence intensities of angiogenin, ANG, ANG-2, HGF, PIGF, and VEGF were prominent in OSCC-group compared to ANT-group (Fig. 2A). Quantitative analysis revealed 1.80-, 6.37-, 2.21-, 3.47-, and 3.30-fold higher expression of ANG, ANG-2, HGF, PIGF, and VEGF in the OSCC-group, respectively compared to ANT-group (Fig. 2B-2G). Expressions of PDGF-BB, bFGF, HB-EGF, and leptin were not significantly changed in OSCC tissue compared to ANT (Fig. 2H-2K).
Protein level expression of angiogenic factors in saliva of OSCC patients and healthy controls
We further analyzed the protein level expression of angiogenic factors in the saliva of OSCC patients and healthy controls. The main aim of this part experiment was to further determine the potential application of angiogenic factors as non-invasive diagnostic and prognostic markers in OSCC. Among the angiogenic markers tested, 2.00-, 8.10-, and 1.38-, higher expression of HGF, PIGF, and VEGF, were upregulated in the saliva of OSCC patients compared to healthy controls (Fig. 3D-3H). Interestingly, PDGF-BB was not detected in healthy controls, while 62.5% of OSCC patients showed higher expression of PDGF-BB in saliva (Fig. 3H). Expressions of ANG, ANG-2, bFGF, HB-EGF, and leptin in saliva were not significantly different in OSCC compared to control (Fig. 3A, 3B, 3I-K). Expressions of PIGF, PDGF-BB, bFGF, HB-EGF, and leptin in saliva relatively low to visualize the fluorescence intensity by the naked eye. However, fluorescence signals of these proteins were obtained by an InnoScan 300 Microarray Scanner and quantified.
Immunohistochemistry of MMPs in ANT and OSCC tissues
In addition, the expression pattern of MMPs in ANT and OSCC tissues was evaluated. MMP-2, MMP-3, and MMP-13 antibody-stained immunohistochemistry tissue section showed few brown stained spots in ANT sections, while distinct brown stained cell cytoplasm in OSCC tissue sections (Fig. 4A). Quantitative analysis of the staining showed higher expression of MMP-2, MMP-3, and MMP-13 in OSCC tissue than ANT (Fig. 4B, 4C, 4D).
mRNA expression of MMPs in OSCC tissues and ANT
We further analyzed the mRNA level expression of MMPs in OSCC and ANT tissue by RT-qPCR. The expressions of MMP-1, MMP-2, MMP-3, MMP-8, MMP-9, MMP-10, MMP-13, TIMP-1, and TIMP-2 in OSCC-group were 58.01-, 7.92-, 87.54-, 7.69-, 37.51-, 146.29-, 115.20-, 7.60-, and 3.73-fold higher, respectively compared to ANT-group (Fig. 4E-4M).
Protein level expression of MMPs in OSCC tissues and ANT
To determine the protein levels of MMPs in tissues, we performed a semi-quantitative protein chip array. The results showed that the fluorescence intensities of MMP-1, MMP-2, MMP3, MMP-8, MMP-9, MMP-10, MMP-13, TIMP-1, and TIMP-2 in the OSCC group were significantly increased compared with the ANT-group (Fig. 5A). The mean fluorescence intensity of MMP-1, MMP-2, MMP3, MMP-8, MMP-9, MMP-10, MMP-13, TIMP-1 and TIMP-2 in OSCC tissue were 13.80-, 1.21-, 2.44-, 3.96-, 7.50-, 15.83-, 59.75-, 3.29-, and 1.89-fold higher than ANT-group, respectively (Fig. 5B-J). The result of protein level expression of MMPs was in accordance with the results of RT-qPCR (Fig. 4).
Protein level expression of MMPs in saliva of OSCC patients and healthy controls
The protein level expression of MMPs in the saliva of OSCC patients and healthy controls was further analyzed. It demonstrated that the expression levels of MMP-1, MMP-3, MMP-8, MMP-9, MMP-10 and MMP-13 in saliva were relatively high to visualize the green fluorescence (Fig. 6A). Quantification analysis revealed that the MMP-1, MMP-3, MMP-8, MMP-9, MMP-10, MMP-13, and TIMP-2 were 111.71-, 256.32-, 2.66-, 5.48-, 16.31-, 174.20-, 1.34-fold upregulated in the saliva of OSCC patients, respectively, compared to healthy controls (Fig. 6B, D-H and J). Intriguingly, MMP-1, MMP-3 and MMP-13 were highly expressed in the saliva of the OSCC group, but their expressions were too low to be detected in the healthy individual.
The correlation between angiogenic factors and MMPs expression with the functional states in HNSCC
The Figure 7 depicts the correlation between the expression patterns of ANGs and MMPs expression with the functional states in HNSCC. ANG2 and PIGF expression positively correlate with cancer cell stemness. HB-EGF expression positively correlates with inflammation, cancer cell quiescence and stemness. The expression of leptin positively correlates with cancer cell stemness. MMPs expression was positively correlated with angiogenesis, EMT and metastasis.
Survival analysis
We further analyzed the survival curve from 124~248 HNSCC patients (25~50%) with higher expression of ANGs and MMPs and 124~248 patients (25~50%) with lower expression of ANGs and MMPs. HNSCC patients with lower expression of ANG (p=0.0094), PIGF (p=0.0426), PDGF-BB (p=0.0285), HB-EGF (p=0.0091), MMP-8 (p=0.0214) and TIMP-1 (p=0.0023) showed significantly higher 5 years survival rate compared to those HNSCC patients with higher expression of these factors (Fig. 8A, D, F, H, M, Q). Similarly, HNSCC patients with lower expression of MMP-1 (p=0.1056), MMP-2 (p=0.3013), MMP-3 (p=0.2857), MMP-9 (p=0.1032), and TIMP-2 (p=0.1719) showed higher 5 years survival rate compared to those HNSCC patients with higher expression of these factors but there was no significant different between lower and higher groups (Fig. 8J, K, L, R).