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Research article
Geovanni Dantas Cassali
Universidade Federal de Minas Gerais
Corresponding Author
ORCiD: https://orcid.org/0000-0002-5650-6743
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This work is licensed under a CC BY 4.0 License. Read Full License
Background: Invasive micropapillary carcinoma (IMPC) is a rare malignant breast tumor and a variant form of invasive ductal carcinoma that is an aggressive neoplasm of the human breast and canine mammary gland. The importance of the tumor microenvironment in cancer development has gradually been recognized, but little is known about the cell types outlining the cystic space of canine IMPC. This study aimed to characterize the neoplastic cells outlining the cystic space of IMPC.
Methods: Immunohistochemistry (IHC), immunofluorescence (IF), superresolution and transmission electron microscopy (TEM) were used to assess the cell types in the cystic areas of IMPCs.
Results: Cells expressing the mesenchymal markers alpha-smooth muscle actin (aSMA), Vimentin, and S100A4 outlined the cystic space of IMPC. Furthermore, loss of epithelial cell polarity in IMPC was shown by the localization of MUC1 at the stroma-facing surface. This protein modulates lumen formation and inhibits the cell-stroma interaction. Immunohistochemical and IF staining for the myoepithelial cell marker p63 were negative in IMPC samples, and TEM revealed morphological aspects of myoepithelial-like cells with thin cytoplasmic extensions outlining the cystic space.
Conclusions: The cells outlining the cystic space of IMPC in the canine mammary gland were characterized using IHC, IF and TEM. The presence of cells expressing aSMA, Vimentin, and S100A4 in the IMPC stroma suggested a role for tumor-associated fibroblasts in the IMPC microenvironment. The reversal of cell polarity revealed by the limited basal localization of MUC1 may be an important factor contributing to the invasiveness of IMPC. For the first time, the cystic space of canine mammary gland IMPC was shown to be delimited by myoepithelial-like cells that had lost p63 expression. These findings may enhance our understanding of the cellular microenvironment of invasive tumors to improve cancer diagnosis and treatment.
Keywords
Invasive micropapillary carcinoma, super-resolution microscopy, transmission electron microscopy, canine mammary gland, cancer, tumor microenvironment
Figure 1
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Figure 6
This is a list of supplementary files associated with this preprint. Click to download.
- Supplementaryfigure1.tif
Supplementary Figure 1. Photomicrographs illustrating p63, CD31, Vimentin and Cytokeratin (AE1/AE3) immunostaining in normal canine mammary glands. (a) Positive staining for normal myoepithelial cell markers is observed in cells lining the mammary gland ducts. (b) Positive staining for CD31 on the plasma membrane of endothelial cells. (c) Positive staining for Vimentin in stromal cells. (d) Representative image of IHC staining in the negative control (NC) sample. (e) Positive staining for CK (AE1/AE3) in ductal epithelial cells. Scale bars = 20 µm and 50 µm. (Novolink™ Polymer Detection System, counterstained with Harris’s hematoxylin).
- Supplementaryfigure2.tif
Supplementary Figure 2. Representative images of S100A4, αSMA, Vimentin and MUC1 expression in invasive areas of canine IMPCs. (a-c) Superresolution images of the nuclear envelope markers Lamin B1/B2 (green), S100A4, αSMA, and Vimentin (red); nuclei were stained with Hoechst (blue) in invasive areas. (d) Stroma-facing positive staining for MUC1 (red) in canine IMPCs. The merged images show the overlapping signals. Representative images from n=4 discrete primary tumor cases are shown, and 5 images were acquired from invasive areas in each sample. Scale bars = 20 μm.
- Supplementaryfigure3.tif
Supplementary Figure 3. Negative staining for von Willebrand factor, CD31, and p63 in cells outlining the cystic spaces of invasive micropapillary areas in the canine mammary gland. (a) Superresolution images of the nuclear envelope markers Lamin B1/B2 (green), von Willebrand factor, CD31 (b), and p63 (c) (red); nuclei were stained with Hoechst (blue). Note that staining is negative for endothelial and lymphatic cell markers (CD31 and von Willebrand factor) in micropapillary areas. Staining for the myoepithelial cell marker p63 is positive in the normal mammary gland control sample (d). The merged images show the overlapping signals. € Representative image of IF staining in the negative control sample. Representative images from n=4 discrete primary tumor cases are shown, and 5 images were acquired from invasive areas in each sample. Scale bars = 20 μm.
- Supplementaryfigure4.tif
Supplementary Figure 4. Representative images of S100A4, αSMA, Vimentin and MUC1 expression in invasive areas of human IMPCs. (a-c) Superresolution images of the nuclear envelope markers Lamin B1/B2 (green), S100A4, αSMA, and Vimentin (red); nuclei were stained with Hoechst (blue) in invasive areas. (d) Stroma-facing positive staining for MUC1 (red) is observed in human IMPC. The merged images show the overlapping signals. Representative images from one primary tumor case are shown, and 5 images of staining with each antibody were acquired from invasive areas. Scale bars = 20 μm.
- Supplementaryfigure5.tif
Supplementary Figure 5. Negative staining for von Willebrand factor, CD31, and p63 in cells outlining the cystic spaces of invasive micropapillary areas in the human breast. (a) Superresolution images of the nuclear envelope markers Lamin B1/B2 (green), von Willebrand factor, CD31 (b), and p63 (c) (red channel); nuclei were stained with Hoechst (blue). Note that the staining is negative for endothelial and lymphatic cell markers (CD31 or von Willebrand factor) in micropapillary areas. (d) Representative images of IF staining in the negative control sample. The merged images show the overlapping signals. Representative images from one primary tumor case are shown, and 5 images of staining with each antibody were acquired from invasive areas. Scale bars = 20 μm.
- SupplementaryTable1.docx
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View the published article at BMC Veterinary Research.
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See the published version of this preprint at BMC Veterinary Research.
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 article
Geovanni Dantas Cassali
Universidade Federal de Minas Gerais
Corresponding Author
ORCiD: https://orcid.org/0000-0002-5650-6743
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 BMC Veterinary Research.
Version 2
Posted 07 Jan, 2021
No community comments so far
Editorial decision: Minor revision
On 29 Jan, 2021
Review #1 received
Received 15 Jan, 2021
Review #2 received
Received 15 Jan, 2021
Reviewer #2 agreed
On 27 Dec, 2020
Reviewers invited
Invitations sent on 27 Dec, 2020
Reviewer #1 agreed
On 27 Dec, 2020
Editor assigned
On 21 Dec, 2020
Submission checks complete
On 21 Dec, 2020
Editor invited
On 21 Dec, 2020
No comments provided
Editorial decision: Major revision
On 07 Oct, 2020
Review #2 received
Received 02 Oct, 2020
Review #1 received
Received 10 Sep, 2020
Reviewer #2 agreed
On 27 Aug, 2020
Reviewer #1 agreed
On 21 Aug, 2020
Reviewers invited
Invitations sent on 09 Aug, 2020
Editor assigned
On 21 Jul, 2020
Submission checks complete
On 20 Jul, 2020
Editor invited
On 20 Jul, 2020
First submitted
On 20 Jul, 2020
Metrics
Comments: 0
PDF Downloads: ...
HTML Views: ...
License:
This work is licensed under a CC BY 4.0 License. Read Full License
View the published article at BMC Veterinary Research.
Background: Invasive micropapillary carcinoma (IMPC) is a rare malignant breast tumor and a variant form of invasive ductal carcinoma that is an aggressive neoplasm of the human breast and canine mammary gland. The importance of the tumor microenvironment in cancer development has gradually been recognized, but little is known about the cell types outlining the cystic space of canine IMPC. This study aimed to characterize the neoplastic cells outlining the cystic space of IMPC.
Methods: Immunohistochemistry (IHC), immunofluorescence (IF), superresolution and transmission electron microscopy (TEM) were used to assess the cell types in the cystic areas of IMPCs.
Results: Cells expressing the mesenchymal markers alpha-smooth muscle actin (aSMA), Vimentin, and S100A4 outlined the cystic space of IMPC. Furthermore, loss of epithelial cell polarity in IMPC was shown by the localization of MUC1 at the stroma-facing surface. This protein modulates lumen formation and inhibits the cell-stroma interaction. Immunohistochemical and IF staining for the myoepithelial cell marker p63 were negative in IMPC samples, and TEM revealed morphological aspects of myoepithelial-like cells with thin cytoplasmic extensions outlining the cystic space.
Conclusions: The cells outlining the cystic space of IMPC in the canine mammary gland were characterized using IHC, IF and TEM. The presence of cells expressing aSMA, Vimentin, and S100A4 in the IMPC stroma suggested a role for tumor-associated fibroblasts in the IMPC microenvironment. The reversal of cell polarity revealed by the limited basal localization of MUC1 may be an important factor contributing to the invasiveness of IMPC. For the first time, the cystic space of canine mammary gland IMPC was shown to be delimited by myoepithelial-like cells that had lost p63 expression. These findings may enhance our understanding of the cellular microenvironment of invasive tumors to improve cancer diagnosis and treatment.
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
This is a list of supplementary files associated with this preprint. Click to download.
- Supplementaryfigure1.tif
Supplementary Figure 1. Photomicrographs illustrating p63, CD31, Vimentin and Cytokeratin (AE1/AE3) immunostaining in normal canine mammary glands. (a) Positive staining for normal myoepithelial cell markers is observed in cells lining the mammary gland ducts. (b) Positive staining for CD31 on the plasma membrane of endothelial cells. (c) Positive staining for Vimentin in stromal cells. (d) Representative image of IHC staining in the negative control (NC) sample. (e) Positive staining for CK (AE1/AE3) in ductal epithelial cells. Scale bars = 20 µm and 50 µm. (Novolink™ Polymer Detection System, counterstained with Harris’s hematoxylin).
- Supplementaryfigure2.tif
Supplementary Figure 2. Representative images of S100A4, αSMA, Vimentin and MUC1 expression in invasive areas of canine IMPCs. (a-c) Superresolution images of the nuclear envelope markers Lamin B1/B2 (green), S100A4, αSMA, and Vimentin (red); nuclei were stained with Hoechst (blue) in invasive areas. (d) Stroma-facing positive staining for MUC1 (red) in canine IMPCs. The merged images show the overlapping signals. Representative images from n=4 discrete primary tumor cases are shown, and 5 images were acquired from invasive areas in each sample. Scale bars = 20 μm.
- Supplementaryfigure3.tif
Supplementary Figure 3. Negative staining for von Willebrand factor, CD31, and p63 in cells outlining the cystic spaces of invasive micropapillary areas in the canine mammary gland. (a) Superresolution images of the nuclear envelope markers Lamin B1/B2 (green), von Willebrand factor, CD31 (b), and p63 (c) (red); nuclei were stained with Hoechst (blue). Note that staining is negative for endothelial and lymphatic cell markers (CD31 and von Willebrand factor) in micropapillary areas. Staining for the myoepithelial cell marker p63 is positive in the normal mammary gland control sample (d). The merged images show the overlapping signals. € Representative image of IF staining in the negative control sample. Representative images from n=4 discrete primary tumor cases are shown, and 5 images were acquired from invasive areas in each sample. Scale bars = 20 μm.
- Supplementaryfigure4.tif
Supplementary Figure 4. Representative images of S100A4, αSMA, Vimentin and MUC1 expression in invasive areas of human IMPCs. (a-c) Superresolution images of the nuclear envelope markers Lamin B1/B2 (green), S100A4, αSMA, and Vimentin (red); nuclei were stained with Hoechst (blue) in invasive areas. (d) Stroma-facing positive staining for MUC1 (red) is observed in human IMPC. The merged images show the overlapping signals. Representative images from one primary tumor case are shown, and 5 images of staining with each antibody were acquired from invasive areas. Scale bars = 20 μm.
- Supplementaryfigure5.tif
Supplementary Figure 5. Negative staining for von Willebrand factor, CD31, and p63 in cells outlining the cystic spaces of invasive micropapillary areas in the human breast. (a) Superresolution images of the nuclear envelope markers Lamin B1/B2 (green), von Willebrand factor, CD31 (b), and p63 (c) (red channel); nuclei were stained with Hoechst (blue). Note that the staining is negative for endothelial and lymphatic cell markers (CD31 or von Willebrand factor) in micropapillary areas. (d) Representative images of IF staining in the negative control sample. The merged images show the overlapping signals. Representative images from one primary tumor case are shown, and 5 images of staining with each antibody were acquired from invasive areas. Scale bars = 20 μm.
- SupplementaryTable1.docx
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