See the published version of this preprint at Cell & Bioscience.
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Wei Shi
Shandong University of Traditional Chinese Medicine Affiliated Hospital
Corresponding Author
ORCiD: https://orcid.org/0000-0001-9485-0038
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Background: Adenomyosis (AM) is a common benign chronic gynaecological disorder; however, the precise pathogenesis of adenomyosis is still poorly understood. Single-cell RNA sequencing (scRNA-seq) can uncover rare subpopulations, explore genetic and functional heterogeneity, and reveal the uniqueness of each cell. It provides us a new approach to reveal biological issues from a more detailed and microscopic perspective. Here, we utilize this revolutionary technology to identify the changes of gene expression patterns between ectopic lesions and the eutopic endometrium at the single-cell level and explore a potential novel pathogenesis of AM.
Methods: A control endometrium (sample with leiomyoma excluding endometrial disorders, n=1), eutopic endometrium and ectopic lesion (from a patient with adenomyosis, n=1) samples were analysed by scRNA-seq, and additional leiomyoma (n=3) and adenomyosis (n=3) samples were used to confirm colocalization and vasculogenic mimicry (VM) formation. Protein colocalization was visualized by immunofluorescence, and CD34-periodic acid-Schiff (PAS) double staining was used to assess the formation of VM.
Results: The scRNA-seq results suggest that cancer-, cell motility- and inflammation- (CMI) associated terms, cell proliferation and angiogenesis play important roles in the progression of AM. Moreover, the colocalization of EPCAM and PECAM1 increased significantly in the ectopic endometrium group (P < 0.05), cell subpopulation with high copy number variation (CNV) levels possessing tumour-like features existed in the ectopic lesion sample, and VNN1- and EPCAM-positive cell subcluster displayed active cell motility in endometrial epithelial cells. Furthermore, the epithelial cells transformed to endothelial cells with the obvious accumulation of vasculogenic mimicry formations (positively stained with PAS but not CD34, P < 0.05) in ectopic lesions.
Conclusions: In the present study, our results support the theory of adenomyosis derived from the invasion and migration of the endometrium. Moreover, cell subcluster with high CNV level and tumour-associated characteristics is identified. Furthermore, epithelial-endothelial transition (EET) and the formation of VM in tumours, the latter of which facilitates the blood supply and plays an important role in maintaining cell growth, were also confirmed to occur in AM. These results indicated that the inhibition of EET and VM formation may be a potential strategy for AM management.
Keywords
Adenomyosis, Single-cell RNA sequencing, Malignant tumour characteristic, Epithelial-endothelial transition, Vasculogenic mimicry
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
This is a list of supplementary files associated with this preprint. Click to download.
- Additionalfile2TableS1.xlsx
- Additionalfile3TableS2.xlsx
- Additionalfile4TableS3.xlsx
- Additionalfile5TableS4.xlsx
- FigureS1.jpg
Figure S1 HE staining and QC. (A) Samples from the AM_CTRL, AM_EM and AM_EC groups were HE stained, and the black arrow shows the gland invading the muscular layer. After QC, the proportion of mitochondrial genes (B), the number of genes expressed (C), and the number of UMIs (D) in each cell are shown in the violin plots. (E) The mean proportion of mitochondrial genes, mean number of genes expressed, mean number of UMIs in each cell and cell number of the three sample groups before and after QC are shown. HE, Hematoxylin-Eosin; QC, Quality Control.
- FigureS2.jpg
Figure S2 Cell type identification and heatmap of gene expression in clusters. (A) Seventeen clusters were displayed in the AM_CTRL, EAM_EM and AM_EC groups. (B) Heatmap showing the expression levels of specific markers in each cluster. (C) The cell number and percentage corresponding to each cell type were counted. Complementary representative markers of different cell types (D) and corresponding violin plots (E) are shown.
- FigureS3.jpg
Figure S3 Colocalization of epithelial cell and endothelial cell markers in cluster 1. (A, B) Complementary epithelial cell markers (CDH1 and KRT7), endothelial cell markers (VWF and CDH5) and colocalization of the two cell type markers are displayed in the t-SNE map. (C) Confirmatory colocalization of EPCAM and PECAM1 was conducted in additional AM_CTRL (V) (n=3), AM_EM (V) (n=3), AM_EC (V) (n=3) samples, EPCAM (red), PECAM1 (green) and nuclei (blue) were stained. The white arrows show the colocalized cells containing EPCAM and PECAM1, scale bars = 20 μm, *P < 0.05. GO (D) and KEGG (E) analyses of upregulated genes in cluster 1 compared with all other clusters (cluster 2 to cluster 17). Representative GO (F) and KEGG (G) terms of upregulated genes in cluster 1 compared with the epithelial cell population (cluster 7 and cluster 17) are displayed. GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes.
- FigureS4.jpg
Figure S4 Gene expression changes and functional analyses of the AM_CTRL, AM_EM and AM_EC groups. Representative GO (A) and KEGG (C) terms of upregulated DEGs in the AM_EC group compared with the AM_EM group are shown. Representative GO (B) and KEGG (D) terms of upregulated genes in the AM_EM group compared with those in the AM_CTRL group are shown. (E) Venn diagram showing the common change genes between AM_EM versus AM_CTRL and AM_EC versus AM_EM. (F) Enriched GO terms of common changed genes in (E) are shown. GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes.
- FigureS5.jpg
Figure S5 Identification of subclusters in epithelial cell populations. (A) The t-SNE map shows 7 subclusters in the epithelial cell group. Histograms show the proportion of subcluster 1 in AM_EM and AM_EC (B), and the proportion of subcluster 4 is shown in (C). (D) The heatmap displays the expression levels of specific markers in each subcluster. Representative GO (E) and KEGG (F) terms of the top 500 genes expressed in subcluster 1 are indicated. The corresponding GO (G) and KEGG (H) analyses of subcluster 4 are also presented. GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes.
- FigureS6.jpg
Figure S6 Change in marker distribution on the pseudotime trajectory and verification of VM formation. (A) Distribution of groups on the pseudotime trajectory. (B) Cell type, including epithelial cell, cluster 1 and endothelial cell, distributed on the pseudotime trajectory. (C, D) Epithelial cell markers (EPCAM, CDH1 and KRT7) and endothelial cell markers (PECAM1, VWF and CDH5) distributed on the pseudotime trajectory. (E) Confirmatory VM channel formations were conducted in additional AM_CTRL (V) (n=3), AM_EM (V) (n=3), AM_EC (V) (n=3) samples, VM channels were positive for PAS staining but negative for CD34 (black arrows); red arrows represent channels positive for CD34; scale bars = 50 μm. (F) Histogram indicates the number of VM channels in different groups; ns represents not significant, *P < 0.05. VM, Vasculogenic Mimicry. PAS, Periodic Acid-Schiff.
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See the published version of this preprint at Cell & Bioscience.
This is a preprint, a preliminary version of a manuscript that has not completed peer review at a journal. Research Square does not conduct peer review prior to posting preprints. The posting of a preprint on this server should not be interpreted as an endorsement of its validity or suitability for dissemination as established information or for guiding clinical practice.
Learn more about In Review
Research
Wei Shi
Shandong University of Traditional Chinese Medicine Affiliated Hospital
Corresponding Author
ORCiD: https://orcid.org/0000-0001-9485-0038
Badges
Prescreen
This manuscript passed our Prescreen assessment
Complete author information
Appropriate declaration statements
Potential risks to human health
Prescreen
Peer Review Timeline
CURRENT STATUS: Published
View the published article at Cell & Bioscience.
Version 1
Posted 16 Dec, 2020
No community comments so far
Editorial decision: Major revision
On 12 Jan, 2021
Review #2 received
Received 11 Jan, 2021
Reviewer #2 agreed
On 23 Dec, 2020
Review #1 received
Received 23 Dec, 2020
Reviewers invited
Invitations sent on 14 Dec, 2020
Reviewer #1 agreed
On 14 Dec, 2020
Editor assigned
On 08 Dec, 2020
Submission checks complete
On 08 Dec, 2020
Editor invited
On 08 Dec, 2020
First submitted
On 03 Dec, 2020
Metrics
Comments: 0
PDF Downloads: ...
HTML Views: ...
Subject Areas
General Cell Biology & Physiology
License:
This work is licensed under a CC BY 4.0 License. Read Full License
View the published article at Cell & Bioscience.
Background: Adenomyosis (AM) is a common benign chronic gynaecological disorder; however, the precise pathogenesis of adenomyosis is still poorly understood. Single-cell RNA sequencing (scRNA-seq) can uncover rare subpopulations, explore genetic and functional heterogeneity, and reveal the uniqueness of each cell. It provides us a new approach to reveal biological issues from a more detailed and microscopic perspective. Here, we utilize this revolutionary technology to identify the changes of gene expression patterns between ectopic lesions and the eutopic endometrium at the single-cell level and explore a potential novel pathogenesis of AM.
Methods: A control endometrium (sample with leiomyoma excluding endometrial disorders, n=1), eutopic endometrium and ectopic lesion (from a patient with adenomyosis, n=1) samples were analysed by scRNA-seq, and additional leiomyoma (n=3) and adenomyosis (n=3) samples were used to confirm colocalization and vasculogenic mimicry (VM) formation. Protein colocalization was visualized by immunofluorescence, and CD34-periodic acid-Schiff (PAS) double staining was used to assess the formation of VM.
Results: The scRNA-seq results suggest that cancer-, cell motility- and inflammation- (CMI) associated terms, cell proliferation and angiogenesis play important roles in the progression of AM. Moreover, the colocalization of EPCAM and PECAM1 increased significantly in the ectopic endometrium group (P < 0.05), cell subpopulation with high copy number variation (CNV) levels possessing tumour-like features existed in the ectopic lesion sample, and VNN1- and EPCAM-positive cell subcluster displayed active cell motility in endometrial epithelial cells. Furthermore, the epithelial cells transformed to endothelial cells with the obvious accumulation of vasculogenic mimicry formations (positively stained with PAS but not CD34, P < 0.05) in ectopic lesions.
Conclusions: In the present study, our results support the theory of adenomyosis derived from the invasion and migration of the endometrium. Moreover, cell subcluster with high CNV level and tumour-associated characteristics is identified. Furthermore, epithelial-endothelial transition (EET) and the formation of VM in tumours, the latter of which facilitates the blood supply and plays an important role in maintaining cell growth, were also confirmed to occur in AM. These results indicated that the inhibition of EET and VM formation may be a potential strategy for AM management.
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
This is a list of supplementary files associated with this preprint. Click to download.
- Additionalfile2TableS1.xlsx
- Additionalfile3TableS2.xlsx
- Additionalfile4TableS3.xlsx
- Additionalfile5TableS4.xlsx
- FigureS1.jpg
Figure S1 HE staining and QC. (A) Samples from the AM_CTRL, AM_EM and AM_EC groups were HE stained, and the black arrow shows the gland invading the muscular layer. After QC, the proportion of mitochondrial genes (B), the number of genes expressed (C), and the number of UMIs (D) in each cell are shown in the violin plots. (E) The mean proportion of mitochondrial genes, mean number of genes expressed, mean number of UMIs in each cell and cell number of the three sample groups before and after QC are shown. HE, Hematoxylin-Eosin; QC, Quality Control.
- FigureS2.jpg
Figure S2 Cell type identification and heatmap of gene expression in clusters. (A) Seventeen clusters were displayed in the AM_CTRL, EAM_EM and AM_EC groups. (B) Heatmap showing the expression levels of specific markers in each cluster. (C) The cell number and percentage corresponding to each cell type were counted. Complementary representative markers of different cell types (D) and corresponding violin plots (E) are shown.
- FigureS3.jpg
Figure S3 Colocalization of epithelial cell and endothelial cell markers in cluster 1. (A, B) Complementary epithelial cell markers (CDH1 and KRT7), endothelial cell markers (VWF and CDH5) and colocalization of the two cell type markers are displayed in the t-SNE map. (C) Confirmatory colocalization of EPCAM and PECAM1 was conducted in additional AM_CTRL (V) (n=3), AM_EM (V) (n=3), AM_EC (V) (n=3) samples, EPCAM (red), PECAM1 (green) and nuclei (blue) were stained. The white arrows show the colocalized cells containing EPCAM and PECAM1, scale bars = 20 μm, *P < 0.05. GO (D) and KEGG (E) analyses of upregulated genes in cluster 1 compared with all other clusters (cluster 2 to cluster 17). Representative GO (F) and KEGG (G) terms of upregulated genes in cluster 1 compared with the epithelial cell population (cluster 7 and cluster 17) are displayed. GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes.
- FigureS4.jpg
Figure S4 Gene expression changes and functional analyses of the AM_CTRL, AM_EM and AM_EC groups. Representative GO (A) and KEGG (C) terms of upregulated DEGs in the AM_EC group compared with the AM_EM group are shown. Representative GO (B) and KEGG (D) terms of upregulated genes in the AM_EM group compared with those in the AM_CTRL group are shown. (E) Venn diagram showing the common change genes between AM_EM versus AM_CTRL and AM_EC versus AM_EM. (F) Enriched GO terms of common changed genes in (E) are shown. GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes.
- FigureS5.jpg
Figure S5 Identification of subclusters in epithelial cell populations. (A) The t-SNE map shows 7 subclusters in the epithelial cell group. Histograms show the proportion of subcluster 1 in AM_EM and AM_EC (B), and the proportion of subcluster 4 is shown in (C). (D) The heatmap displays the expression levels of specific markers in each subcluster. Representative GO (E) and KEGG (F) terms of the top 500 genes expressed in subcluster 1 are indicated. The corresponding GO (G) and KEGG (H) analyses of subcluster 4 are also presented. GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes.
- FigureS6.jpg
Figure S6 Change in marker distribution on the pseudotime trajectory and verification of VM formation. (A) Distribution of groups on the pseudotime trajectory. (B) Cell type, including epithelial cell, cluster 1 and endothelial cell, distributed on the pseudotime trajectory. (C, D) Epithelial cell markers (EPCAM, CDH1 and KRT7) and endothelial cell markers (PECAM1, VWF and CDH5) distributed on the pseudotime trajectory. (E) Confirmatory VM channel formations were conducted in additional AM_CTRL (V) (n=3), AM_EM (V) (n=3), AM_EC (V) (n=3) samples, VM channels were positive for PAS staining but negative for CD34 (black arrows); red arrows represent channels positive for CD34; scale bars = 50 μm. (F) Histogram indicates the number of VM channels in different groups; ns represents not significant, *P < 0.05. VM, Vasculogenic Mimicry. PAS, Periodic Acid-Schiff.
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