Article Figures & Data
Figures
- Figure 1.
The characterization of SEs. The pictures of transmission electron microscopy (A) and nanosight analysis with Flow NanoAnalyzer (A1) of SEs preprocessed from the HC group. The pictures of transmission electron microscopy (B) and nanosight analysis with Flow NanoAnalyzer (B1) of SEs preprocessed from the OSCC with LNM group. The pictures of transmission electron microscopy (C) and nanosight analysis with Flow NanoAnalyzer (C1) of SEs preprocessed from the OSCC with NLNM group. D, SE markers identified by Western blotting.
- Figure 2.
Bioinformatics analysis of DEPs. GO classification analysis (A1) and KEGG analysis (A2) of DEPs in OSCC-LNM relative to HC. GO classification analysis (B1) and KEGG analysis (B2) of DEPs in OSCC-LNM relative to OSCC-NLNM. GO classification analysis (C1) and KEGG analysis (C2) of all identified proteins. D, The hierarchical clustering representation of DEPs identified (D1, OSCC with LNM vs. HC; D2, OSCC with LNM vs. OSCC with NLNM). E, Common DEPs identified between OSCC with LNM vs. HC group and OSCC with LNM vs. OSCC with NLNM group. BP, biological processes; CC, cellular component; CP, cellular processes; EIP, environmental information processing; GIP, genetic information processing; M, metabolism; MF, molecular functions; OS, organismal systems.
- Figure 3.
Western blot validation of MS/MS data. Quantification of the expression levels of PF4V1, CXCL7, and F13A1 from Western blotting. “HC,” “LNM,” and “NLNM” represent the SEs of individual HCs, OSCC with LNM patients, and OSCC with NLNM patients, respectively.
- Figure 4.
ROC analysis of the newly developed biomarker panels. ROC curves of CXCL7 and ApoA1 expression levels in SE, serum, whole blood, and the combinations of the three specimens are shown for differentiating OSCC with LNM vs. OSCC with NLNM and HC group (A for CXCL7, C for ApoA1), and OSCC vs. HC groups (B for CXCL7, D for ApoA1). ROC curves of PF4V1, CXCL7, ApoA1, and the combinations of the three novel biomarkers are shown for differentiating OSCC with LNM vs. OSCC with NLNM and HC group (E), and OSCC vs. HC groups (F). ROC curves of F13A1 in whole blood, ApoA1 in serum, and the combinations are shown for differentiating OSCC with LNM vs. OSCC with NLNM and HC group (G), and OSCC vs. HC groups (H).
Tables
- Table 1.
Comparison of DEPs identified in SEs of the OSCC-LNM group versus the HC group
Number Accession number Abbreviation Full name Molecular weight (kDa) Sequence length Q valuea Scoreb OSCC-LNM/HC P value Expression 1 sp|P04275| VWF von Willebrand factor 309.26 2813 0 157.03 3.4326479 0.005 ↑ 2 tr|D3DP13| FGB Fibrinogen beta chain, isoform CRA_e 39.736 344 0 100.2 3.1698038 0.008 ↑ 3 tr|B4E1I8| LRG1 cDNA FLJ54228, highly similar to leucine-rich alpha-2-glycoprotein 36.494 330 0 56.239 2.8420843 0.029 ↑ 4 tr|Q6LAM1| CFI Heavy chain of factor I (fragment) 35.883 321 0 31.891 2.4030773 0.002 ↑ 5 tr|A2J1M2| ENSG00000223931 Rheumatoid factor RF-IP9 (fragment) 10.549 98 0 30.441 2.2035086 0.028 ↑ 6 sp|P02671| FGA Fibrinogen alpha chain 94.972 866 0 88.917 2.0509267 0.006 ↑ 7 tr|A0A125QYY9| ENSG00000223931 IBM-B2 heavy-chain variable region (fragment) 13.569 123 0 98.895 1.8935852 0.003 ↑ 8 tr|A0A0F7SYK7| ENSG00000223931 IGHV1-46 protein (fragment) 10.173 91 0 15.422 1.8274823 0.027 ↑ 9 tr|A0A024RAA7| C1QC Complement component 1, q subcomponent, C chain, isoform CRA_a 25.773 245 0 94.934 1.8169817 0.003 ↑ 10 tr|V9GYE7| CFHR2 Complement factor H-related protein 2 28.912 254 0 24.488 1.6460515 0.002 ↑ 11 tr|A8K2T4| C7 cDNA FLJ78207, highly similar to human complement protein component C7 mRNA 93.406 843 0 323.31 1.6075309 0.040 ↑ 12 sp|Q06033| ITIH3 Inter-alpha-trypsin inhibitor heavy-chain H3 99.848 890 0 82.334 1.5961385 0.019 ↑ 13 tr|Q6N091| ENSG00000223931 Uncharacterized protein DKFZp686C02220 (fragment) 54.159 500 0 63.267 1.5460653 0.030 ↑ 14 tr|A0A0 × 9UWK7| ENSG00000223931 MS-D4 heavy-chain variable region (fragment) 13.525 122 0 76.321 1.5418579 0.035 ↑ 15 tr|A8K008| ENSG00000223931 Uncharacterized protein 51.596 472 0 62.743 1.5019136 0.016 ↑ 16 sp|P10909| CLU Clusterin 52.494 449 0 286.73 0.6652614 0.008 ↓ 17 tr|H0YAC1| KLKB1 Plasma kallikrein (fragment) 76.773 686 0 148.39 0.6526857 0.005 ↓ 18 tr|Q5VY30| RBP4 Retinol-binding protein 4, plasma, isoform CRA_b 22.974 199 0 94.794 0.630816 0.029 ↓ 19 tr|B4E1B2| TF cDNA FLJ53691, highly similar to serotransferrin 74.831 678 0 323.31 0.6304394 0.039 ↓ 20 sp|P05546| SERPIND1 Heparin cofactor 2 57.07 499 0 130.1 0.6268114 0.004 ↓ 21 tr|Q53FV4| LUM Lumican variant (fragment) 38.413 338 0 21.207 0.6033161 0.001 ↓ 22 sp|A2NJV5| IGKV3D-7 Immunoglobulin kappa variable 2-29 13.085 120 0 23.778 0.5528563 0.006 ↓ 23 sp|P06727| APOA4 Apolipoprotein A-IV 45.398 396 0 261.29 0.5293484 0.030 ↓ 24 tr|A0A024R3E3| APOA1 Apolipoprotein A-I, isoform CRA_a 30.777 267 0 323.31 0.4976017 0.015 ↓ 25 tr|X6RLJ0| C1QA Complement C1q subcomponent subunit A (fragment) 23.351 220 0 23.923 0.4945711 0.005 ↑ 26 tr|E9KL36| TTR Transthyretin 15.887 147 0 239.06 0.4477275 0.009 ↑ 27 tr|V9GYM3| APOA2 Apolipoprotein A-II 14.914 133 0 54.723 0.4400852 0.008 ↑ 28 tr|D9YZU5| HBB Beta-globin 15.998 147 0 126.29 0.3773419 0.010 ↑ 29 tr|Q53H26| TF Transferrin variant (fragment) 77.079 698 0 81.553 0.3412472 0.021 ↓ 30 tr|A0A024R0T9| APOC2 Apolipoprotein C-II isoform 1 11.284 101 0 30.162 0.2207795 0.008 ↓ 31 tr|A0A0G2JRQ6| IGKV3D-7 Uncharacterized protein (fragment) 12.748 117 0.00303 11.523 Only detected in OSCC-LNM ↑ 32 tr|A0A193CHR0| ENSG00000223931 10E8 heavy-chain variable region (fragment) 14.429 131 0 18.246 Only detected in OSCC-LNM ↑ 33 tr|B2R6V9| F13A1 cDNA, FLJ93141, highly similar to Homo sapiens coagulation factor XIII, A1 polypeptide (F13A1), mRNA 83.24 732 0 17.861 Only detected in OSCC-LNM ↑ 34 sp|P02741| CRP C-reactive protein 25.038 224 0 29.616 Only detected in OSCC-LNM ↑ 35 sp|P0DOX4| ENSG00000223931 Immunoglobulin epsilon heavy chain 60.322 547 0 35.141 Only detected in OSCC-LNM ↑ 36 sp|P10720| PF4V1 Platelet factor 4 variant 11.553 104 0 20.13 Only detected in HC ↓ 37 tr|Q96SB0| IGKV3D-7 Anti-streptococcal/anti-myosin immunoglobulin lambda light-chain variable region (fragment) 11.594 108 0 35.763 Only detected in HC ↓ aQ value, the probability that the protein is a false hit.
bScore, the sum of the scores of the individual peptides.
- Table 2.
Comparison of the DEPs identified in SEs of the OSCC-LNM group versus the OSCC-NLNM group
Number Accession number Abbreviation Full name Molecular weight (kDa) Sequence length Q valuea Scoreb OSCC-LNM/OSCC-NLNM P value Expression 1 tr|A0A109PSY4| IGKV3D-7 MS-A1 light-chain variable region (fragment) 11.376 106 0.000 49.696 3.874 0.0003564 ↑ 2 sp|P04040| CAT Catalase 59.755 527 0.000 20.916 2.503 0.0193373 ↑ 3 sp|P04275| VWF von Willebrand factor 309.26 2813 0.000 157.030 1.807 0.0184345 ↑ 4 tr|V9GYE7| CFHR2 Complement factor H-related protein 2 28.912 254 0.000 24.488 1.762 0.0150898 ↑ 5 tr|B4DEF7| HSPA5 cDNA FLJ60062, highly similar to 78 kDa glucose-regulated protein 30.419 278 0.000 29.640 1.702 0.0380467 ↑ 6 tr|A0A0 × 9V9C4| ENSG00000223931 GCT-A8 heavy-chain variable region (fragment) 13.228 120 0.000 37.772 1.662 0.034641 ↑ 7 tr|B4DUV1| FBLN1 Fibulin-1 70.154 641 0.000 153.190 1.648 0.0107605 ↑ 8 sp|P80108| GPLD1 Phosphatidylinositol-glycan–specific phospholipase D 92.335 840 0.000 17.994 1.576 0.0105536 ↑ 9 tr|F2RM37| F9 Coagulation factor IX 51.744 461 0.000 25.634 1.556 0.0244641 ↑ 10 sp|Q03591| CFHR1 Complement factor H-related protein 1 37.65 330 0.000 117.800 1.552 0.0057128 ↑ 11 tr|Q68DS3| CLEC3B Uncharacterized protein DKFZp686H17246 (fragment) 14.708 133 0.000 71.101 1.517 0.0094758 ↑ 12 tr|Q6N030| ENSG00000223931 Uncharacterized protein 57.019 518 0.000 323.310 0.595 0.0352034 ↓ 13 sp|P07360| C8G Complement component C8 gamma chain 22.277 202 0.000 121.680 0.587 0.0305837 ↓ 14 tr|A0A024R3E3| APOA1 Apolipoprotein A-I, isoform CRA_a 30.777 267 0.000 323.310 0.539 0.0326735 ↓ 15 tr|A0A096LPE2| SAA4 SAA2-SAA4 readthrough 23.353 208 0.000 33.364 0.519 0.0309882 ↓ 16 tr|D3JV41| CXCL7 C-X-C motif chemokine (fragment) 13.708 126 0.000 156.490 0.340 0.0427637 ↓ 17 sp|P0DOX3| ENSG00000223931 Immunoglobulin delta heavy chain 56.224 512 0.000 164.380 0.243 0.0377757 ↓ 18 tr|A0A140TA29| C4B Complement C4-B 187.66 1698 0.000 63.495 0.166 0.0162487 ↓ 19 sp|A0A0C4DH39| ENSG00000223931 Immunoglobulin heavy variable 1–58 13.005 117 0.003 10.683 Only detected in OSCC-NLNM ↓ 20 tr|A0A0F7T6Q1| ENSG00000223931 IGHV1-2 protein (fragment) 11.911 105 0.000 78.857 Only detected in OSCC-NLNM ↓ 21 sp|P10720| PF4V1 Platelet factor 4 variant 11.553 104 0.000 20.130 Only detected in OSCC-NLNM ↓ 22 tr|Q5NV70| IGKV3D-7 V1-11 protein (fragment) 10.333 97 0.000 18.509 Only detected in OSCC-NLNM ↓ 23 tr|Q8TCF0| LBP LBP protein 52.933 477 0.000 19.414 Only detected in OSCC-NLNM ↓ 24 tr|Q96SA9| IGKV3D-7 Human anti-streptococcal/anti-myosin immunoglobulin kappa light-chain variable region (fragment) 11.52 107 0.000 12.329 Only detected in OSCC-NLNM ↓ 25 tr|A0A0G2JRQ6| IGKV3D-7 Uncharacterized protein (fragment) 12.748 117 0.003 11.523 Only detected in OSCC-LNM ↑ 26 tr|B2R6V9| F13A1 cDNA, FLJ93141, highly similar to Homo sapiens coagulation factor XIII, A1 polypeptide (F13A1), mRNA 83.24 732 0.000 17.861 Only detected in OSCC-LNM ↑ 27 tr|B2RA39| CFHR5 cDNA, FLJ94686, highly similar to Homo sapiens complement factor H-related 5 (CFHL5), mRNA 64.391 569 0.000 13.285 Only detected in OSCC-LNM ↑ 28 tr|Q0ZCH1| ENSG00000223931 Immunoglobulin heavy-chain variable region (fragment) 12.863 118 0.000 19.922 Only detected in OSCC-LNM ↑ aQ value, the probability that the protein is a false hit.
bScore, the sum of the scores of the individual peptides.
Supplementary Data
- Figure S1-S8 - Supplementary Fig. S1. The whole experimental workflow in this study. OSCC: oral squamous cell carcinomas; LNM: lymph node metastasis. Supplementary Fig. S2. The identified peptide fragments of four biomarkers. A: PF4V1; B: CXCL7; C: F13A1; D: ApoA1. Supplementary Fig. S3. The protein concentrations and mRNA expression levels assayed by ELISA and RT-PCR. A1-A2/B1-B2/C1-C2, CXCL4/CXCL7/ApoA1 concentrations in serum and SEs assayed by ELISA. A3-A4/B3-B4/C3-C4/D1-D2/D3-D4, CXCL4/CXCL7/ApoA1/PF4V1/F13A1 mRNA expression levels in tissues assayed by RT-PCR. SEs: serum exosomes; OSCC: oral squamous cell carcinomas; LNM: lymph node metastasis; NLNM: no lymph node metastasis; CT: cancerous tissues; CAT: corresponding adjacent tissues. Supplementary Fig. S4. The expression of PF4V1, CXCL7, F13A1 assayed by IHC. A, B and C represented the expression of PF4V1, CXCL7, F13A1 in cancer tissues and the corresponding paracancerous tissues at least five fields per tissue (amplification: 400Ã-), respectively. A1, B1, C1: cancer tissues; A2, A3, B2, C2: corresponding paracancerous tissues. Supplementary Fig. S5. Individual validation by RT-PCR. A1/A2, B1/B2, C1/C2, D1/D2 and E1/E2 represented the expressions of CXCL4, PF4V1, CXCL7, F13A1, ApoA1 in serum and whole blood. Supplementary Fig. S6. Clinicopathologic findings based on RT-PCR results of tissue samples. A1/A2/A3/A4/A5/A6, B1/B2/B3/B4/B5/B6, C1/C2/C3/C4/C5/C6, D1/D2/D3/D4/D5/D6 and E1/E2/E3/E4/E5/E6 represented the expressions of CXCL4, PF4V1, CXCL7, F13A1, ApoA1 in gender, nationality, tumor stage, tumor differentiation degree, smoking and drinking. WDSCC: well differentiated squamous-cell carcinoma; LDSCC: low differentiated squamous-cell carcinoma. Supplementary Fig. S7. Comparison of expression levels of differentially expressed proteins at different positive nodes of OSCC-LNM based on RT-PCR results. A1/A2, B1/B2/C1/C2, and D1/D2/E1/E2 represented the expression of PF4V1, CXCL7, F13A1, ApoA1 at different positive nodes of OSCC-LNM in serum exosomes, serum and tissue. Supplementary Fig. S8. Survival analysis provided by GEPIA. Survival analysis showed that PF4V1, CXCL7, F13A1 and ApoA1 had no connection with the overall survival of head and neck squamous cell carcinoma. A: PF4V1; B: CXCL7; C: F13A1; D: ApoA1.
- Supporting Information Figure Legends - Supporting Information Figure Legends
- Table S1 - The primer information in RT-PCR verification
- Table S2 - Compared to the HC group, DEPs identified in serum exosomes of the OSCC-NLNM group
- Table S3 - Clinicopathologic findings of four biomarkers in patients with OSCC
- Table S4 - Comparison of expression levels of DEPs at different phases of age
Volume 28, Issue 10
