In-depth proteomics analysis of sentinel lymph nodes from individuals with endometrial cancer

Abstract

Endometrial cancer (EC) is one of the most common gynecological cancers worldwide. Sentinel lymph node (SLN) status could be a major prognostic factor in evaluation of EC, but several prospective studies need to be performed. Here we report an in-depth proteomics analysis showing significant variations in the SLN protein landscape in EC. We show that SLNs are correlated to each tumor grade, which strengthens evidence of SLN involvement in EC. A few proteins are overexpressed specifically at each EC tumor grade and in the corresponding SLN. These proteins, which are significantly variable in both locations, should be considered potential markers of overall survival. Five major proteins for EC and SLN (PRSS3, PTX3, ASS1, ALDH2, and ANXA1) were identified in large-scale proteomics and validated by immunohistochemistry. This study improves stratification and diagnosis of individuals with EC as a result of proteomics profiling of SLNs.

Trial registration: ClinicalTrials.gov NCT02598219.

Keywords: alternative proteins; diagnosis; endometrial cancer; ghost proteome; protein mutations; proteomics; sentinel lymph nodes.

Conflict of interest statement

The authors declare no competing interests.

© 2021 The Authors.

Figures

Graphical abstract

Figure 1

Global workflow of spatially resolved analysis (A) Histopathological data obtained from immunocytochemistry performed with anti-P53 on tissue sections of sentinel nodes (SNs), either healthy (Aa) or cancerous (Ab, Ac, and Ad), from grade I (Ab) with magnification (Ab’ and Ab’’), grade II (Ac) with magnification (Ac’), and grade III (Ad) with magnification (Ad’). Similarly, results were obtained with their corresponding endometrial tissue, either healthy (Ae) or cancerous (Af, Ag, and Ah), at grade I (Af) with magnification (Af’ and Af’’), grade II (Ag) with magnification (Ag’), and grade III (Ah) with magnification (Ah’). See also IHC images in the Supplemental information). (B) Workflow for spatially resolved proteomics using IHC tissue sections. ROIs were subjected to enzymatic microdigestion using trypsin followed by liquid junction microextraction, and then subjected to shotgun proteomics analyses.

Figure 2

Proteomics analysis of SLNs (A) Hierarchical clustering of the most variable proteins between normal tissue and grades I–III (n = 3 for each category, ANOVA with permutation-based FDR 

Figure 3

Establishment of a molecular transition between SLN and EC grades (A–C) Volcano plots of proteins under- or overexpressed in (A) grade I SNs, (B) grade II SNs, and (C) grade III SNs. (D) Venn diagram representing specific proteins per grade versus normal and ones in common from SNs. (E) PCA PC1 versus PC2 of EC versus normal tissue. (F) Volcano plot and Hierarchical clustering of the most variable proteins between normal tissue and grade I–III EC (n = 3 for each category, ANOVA with permutation-based FDR 

Figure 4

Proteomics analysis of grade I–III EC (A–C) Volcano plots of proteins under- and overexpressed in (A) grade I EC, (B) grade II EC, and (C) grade III EC. (D) Hierarchical clustering of the most variable proteins between grade I–III EC (n = 3 for each category, ANOVA with permutation-based FDR 

Figure 5

Similar marker proteins highlighted between SLN and EC according to their grades (A) Hierarchical clustering of the most variable proteins between heathy tissue and grade I–III SNs and grade I–III EC (n = 3 for each category, ANOVA with permutation-based FDR 

Figure 6

IHC validation of the panel of survival markers identified Representative fluorescence images of the 5 proteins of EC grades. PRSS3, PTX3, and ASS1 are associated with poor outcome, and ALDH2 and ANX1 are considered positive outcome markers in (A) EC and (B) SLNs. Images were acquired with a confocal microscope at 40× magnification. Scale bars, 20 μm.

Figure 7

Mutation and alternative proteins analyses (A) MS/MS spectrum of mutated peptides. (B) Hierarchical clustering of the most variable alternative proteins between healthy tissue and grade I–III (a; n = 3 for each category, ANOVA with permutation-based FDR