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Insights into immune responses in oral cancer through proteomic analysis of saliva and salivary extracellular vesicles.

Winck FV, Prado Ribeiro AC, Ramos Domingues R, Ling LY, Riaño-Pachón DM, Rivera C, Brandão TB, Gouvea AF, Santos-Silva AR, Coletta RD, Paes Leme AF - Sci Rep (2015)

Bottom Line: Biological processes related to immune responses, peptidase inhibitor activity, iron coordination and protease binding were overrepresented in the group of differentially expressed proteins.The proteomics data were robust and could classify OSCC with 90% accuracy.The saliva proteome analysis revealed that immune processes are related to the presence of OSCC and indicate that proteomics data can contribute to determining OSCC prognosis.

View Article: PubMed Central - PubMed

Affiliation: Laboratório de Espectrometria de Massas, Laboratório Nacional de Biociências, LNBio, CNPEM, Campinas, SP, Brazil.

ABSTRACT
The development and progression of oral cavity squamous cell carcinoma (OSCC) involves complex cellular mechanisms that contribute to the low five-year survival rate of approximately 20% among diagnosed patients. However, the biological processes essential to tumor progression are not completely understood. Therefore, detecting alterations in the salivary proteome may assist in elucidating the cellular mechanisms modulated in OSCC and improve the clinical prognosis of the disease. The proteome of whole saliva and salivary extracellular vesicles (EVs) from patients with OSCC and healthy individuals were analyzed by LC-MS/MS and label-free protein quantification. Proteome data analysis was performed using statistical, machine learning and feature selection methods with additional functional annotation. Biological processes related to immune responses, peptidase inhibitor activity, iron coordination and protease binding were overrepresented in the group of differentially expressed proteins. Proteins related to the inflammatory system, transport of metals and cellular growth and proliferation were identified in the proteome of salivary EVs. The proteomics data were robust and could classify OSCC with 90% accuracy. The saliva proteome analysis revealed that immune processes are related to the presence of OSCC and indicate that proteomics data can contribute to determining OSCC prognosis.

No MeSH data available.


Related in: MedlinePlus

Extracellular vesicles isolated from saliva.Extracellular vesicles were isolated from the saliva of healthy and oral cancer patients using ultracentrifugation. (A) Nanoparticle tracking analysis of the extracellular vesicles isolated from healthy individuals (C4, C6, C10) and oral cancer patients (T2, T5, T16); (B) Immunochemical detection of the protein flotilin-1, a protein marker of extracellular vesicles, in the whole saliva proteome (S) and in the proteome of extracellular vesicles (EV); (C) Transmission electron microscopy of isolated extracellular vesicles using a negative staining method.
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f4: Extracellular vesicles isolated from saliva.Extracellular vesicles were isolated from the saliva of healthy and oral cancer patients using ultracentrifugation. (A) Nanoparticle tracking analysis of the extracellular vesicles isolated from healthy individuals (C4, C6, C10) and oral cancer patients (T2, T5, T16); (B) Immunochemical detection of the protein flotilin-1, a protein marker of extracellular vesicles, in the whole saliva proteome (S) and in the proteome of extracellular vesicles (EV); (C) Transmission electron microscopy of isolated extracellular vesicles using a negative staining method.

Mentions: The size distribution of salivary EV samples was measured using the NTA, revealing the presence of EVs with an average size of 177.19 nm (SD = 17.22 nm) (Fig. 4A). Western blotting analysis for flotilin-1 indicated a greater abundance of this marker protein in the isolates of EVs (Fig. 4B). Furthermore, the structure of isolated EVs was observed by transmission electron microscopy (TEM-HR), which revealed that the EVs were intact after isolation (Fig. 4C). A total of 381 proteins were identified by mass spectrometry in the EV proteome from the two groups of individuals (healthy (n = 10) and oral cancer (n = 7)) (Supplemental Table 6). Considering proteins that showed a minimum of three valid expression values in at least one group, 139 proteins were subjected to statistical analysis. Eight proteins from the EVs were differentially expressed (ANOVA, p < 0.05) between the two groups (Fig. 5). Among the total 381 proteins identified, 18 proteins were exclusively detected in the healthy group and 4 proteins were exclusively identified in the oral cancer group, considering proteins identified in at least two individuals. GO enrichment analysis revealed that the biological processes “antigen binding,” “enzyme inhibitor” and “endopeptidase regulatory functions” were overrepresented in the EVs proteome (Supplemental Figure 4, Supplemental Table 7).


Insights into immune responses in oral cancer through proteomic analysis of saliva and salivary extracellular vesicles.

Winck FV, Prado Ribeiro AC, Ramos Domingues R, Ling LY, Riaño-Pachón DM, Rivera C, Brandão TB, Gouvea AF, Santos-Silva AR, Coletta RD, Paes Leme AF - Sci Rep (2015)

Extracellular vesicles isolated from saliva.Extracellular vesicles were isolated from the saliva of healthy and oral cancer patients using ultracentrifugation. (A) Nanoparticle tracking analysis of the extracellular vesicles isolated from healthy individuals (C4, C6, C10) and oral cancer patients (T2, T5, T16); (B) Immunochemical detection of the protein flotilin-1, a protein marker of extracellular vesicles, in the whole saliva proteome (S) and in the proteome of extracellular vesicles (EV); (C) Transmission electron microscopy of isolated extracellular vesicles using a negative staining method.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4633731&req=5

f4: Extracellular vesicles isolated from saliva.Extracellular vesicles were isolated from the saliva of healthy and oral cancer patients using ultracentrifugation. (A) Nanoparticle tracking analysis of the extracellular vesicles isolated from healthy individuals (C4, C6, C10) and oral cancer patients (T2, T5, T16); (B) Immunochemical detection of the protein flotilin-1, a protein marker of extracellular vesicles, in the whole saliva proteome (S) and in the proteome of extracellular vesicles (EV); (C) Transmission electron microscopy of isolated extracellular vesicles using a negative staining method.
Mentions: The size distribution of salivary EV samples was measured using the NTA, revealing the presence of EVs with an average size of 177.19 nm (SD = 17.22 nm) (Fig. 4A). Western blotting analysis for flotilin-1 indicated a greater abundance of this marker protein in the isolates of EVs (Fig. 4B). Furthermore, the structure of isolated EVs was observed by transmission electron microscopy (TEM-HR), which revealed that the EVs were intact after isolation (Fig. 4C). A total of 381 proteins were identified by mass spectrometry in the EV proteome from the two groups of individuals (healthy (n = 10) and oral cancer (n = 7)) (Supplemental Table 6). Considering proteins that showed a minimum of three valid expression values in at least one group, 139 proteins were subjected to statistical analysis. Eight proteins from the EVs were differentially expressed (ANOVA, p < 0.05) between the two groups (Fig. 5). Among the total 381 proteins identified, 18 proteins were exclusively detected in the healthy group and 4 proteins were exclusively identified in the oral cancer group, considering proteins identified in at least two individuals. GO enrichment analysis revealed that the biological processes “antigen binding,” “enzyme inhibitor” and “endopeptidase regulatory functions” were overrepresented in the EVs proteome (Supplemental Figure 4, Supplemental Table 7).

Bottom Line: Biological processes related to immune responses, peptidase inhibitor activity, iron coordination and protease binding were overrepresented in the group of differentially expressed proteins.The proteomics data were robust and could classify OSCC with 90% accuracy.The saliva proteome analysis revealed that immune processes are related to the presence of OSCC and indicate that proteomics data can contribute to determining OSCC prognosis.

View Article: PubMed Central - PubMed

Affiliation: Laboratório de Espectrometria de Massas, Laboratório Nacional de Biociências, LNBio, CNPEM, Campinas, SP, Brazil.

ABSTRACT
The development and progression of oral cavity squamous cell carcinoma (OSCC) involves complex cellular mechanisms that contribute to the low five-year survival rate of approximately 20% among diagnosed patients. However, the biological processes essential to tumor progression are not completely understood. Therefore, detecting alterations in the salivary proteome may assist in elucidating the cellular mechanisms modulated in OSCC and improve the clinical prognosis of the disease. The proteome of whole saliva and salivary extracellular vesicles (EVs) from patients with OSCC and healthy individuals were analyzed by LC-MS/MS and label-free protein quantification. Proteome data analysis was performed using statistical, machine learning and feature selection methods with additional functional annotation. Biological processes related to immune responses, peptidase inhibitor activity, iron coordination and protease binding were overrepresented in the group of differentially expressed proteins. Proteins related to the inflammatory system, transport of metals and cellular growth and proliferation were identified in the proteome of salivary EVs. The proteomics data were robust and could classify OSCC with 90% accuracy. The saliva proteome analysis revealed that immune processes are related to the presence of OSCC and indicate that proteomics data can contribute to determining OSCC prognosis.

No MeSH data available.


Related in: MedlinePlus