Picturing Breast Cancer Brain Metastasis Development to Unravel Molecular Players and Cellular Crosstalk
Abstract
:Simple Summary
Abstract
1. Introduction
2. Results
2.1. 4T1 Cell Injection Leads to the Establishment of Hippocampal BCBM with PDGF-B and Ki-67-Positive Cells
2.2. Malignant Cells Undergo a Mesenchymal–Epithelial Transition (MET)
2.3. Adhesion and Migration-Associated Signaling Molecules Are Involved in BCBM Formation
2.4. Cellular Crosstalk in BCBM Occurs via Gap Junctions
2.5. Establishment of BCBM Leads to Increased Vascularization and BBB Disruption
2.6. Pericytes Are Involved in the Formation of BCBMs
2.7. PLVAP Expression Occurs in BCBM
3. Discussion
4. Materials and Methods
4.1. Cell Culture and Mouse Model of BCBM
4.2. Collection of Brains
4.3. Hematoxylin-Eosin Staining, Immunohistochemistry and Immunofluorescence
4.4. Image Acquisition
4.5. Data Analysis
4.6. Statistical Analysis
5. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Marker | Antigen Retrieval | Permeabilization | Blocking | Primary Antibody | Secondary Antibody |
---|---|---|---|---|---|
α-SMA | 10 mM citrate buffer pH 6.0 | 0.5% Triton X-100 | 3% BSA + 0.5% Triton-X 100 | α-SMA (1:100) Abcam, #ab5694, Rabbit Pc | Alexa Fluor® 488 (1:500) Thermo Fisher Scientific, #A-11034, Goat anti-Rabbit |
β-catenin | 10 mM citrate buffer pH 6.0 | 0.5% Triton X-100 | 10% GS + 0.5% Triton-X 100 | β-catenin (1:20) Thermo Fisher Scientific #71-2700, Rabbit Pc | Alexa Fluor® 555 (1:100) Thermo Fisher Scientific, #A-21428, Goat anti-Rabbit |
β4 integrin | 10 mM citrate buffer pH 6.0 | 0.5% Triton X-100 | 3% BSA + 0.5% Triton-X 100 | β4 integrin (1:250) Santa Cruz Biotechnology #SC-514426, Mouse Mc | Alexa Fluor® 488 (1:500) Thermo Fisher Scientific, #A-11001, Goat anti-Mouse |
Caveolin-1 | 10 mM citrate buffer pH 6.0 | 0.01% saponin | 3% BSA + 0.01% saponin | Caveolin-1 (1:250) Cell Signalling #3238, Rabbit Pc | Alexa Fluor® 555 (1:500) Thermo Fisher Scientific, #A-21428, Goat anti-Rabbit |
Claudin-5 | 10 mM citrate buffer pH 6.0 | 0.5% Triton X-100 | 3% BSA + 0.5% Triton-X 100 | Claudin-5 (1:500) Thermo Fisher Scientific #35-2500, Mouse Mc | Alexa Fluor® 647 (1:500) Thermo Fisher Scientific, #A-21235, Goat anti-Mouse |
Cx43 | 10 mM citrate buffer pH 6.0 | 0.5% Triton X-100 | 10% BSA + 0.5% Triton-X 100 | Cx43 (1:50) Thermo Fisher Scientific #35-5000, Mouse Mc Cx43 (1:50) Abcam #ab11370, Rabbit Pc | Alexa Fluor® 647 (1:500) Thermo Fisher Scientific, #A-21235, Goat anti-Mouse Alexa Fluor® 555 (1:500) Thermo Fisher Scientific, #A-21428, Goat anti-Rabbit |
FAK | 10 mM citrate buffer pH 6.0 | 0.5% Triton X-100 | 3% BSA + 0.5% Triton-X 100 | FAK (1:250) Abcam #ab131435, Rabbit Pc | Alexa Fluor® 555 (1:500) Thermo Fisher Scientific, #A-21428 Goat anti-Rabbit |
GFAP | 10 mM citrate buffer pH 6.0 | 0.5% Triton X-100 | 3% BSA + 0.5% Triton-X 100 | GFAP (1:100) Sigma-Aldrich #G3893, Mouse Mc | Alexa Fluor® 488 (1:500) Thermo Fisher Scientific, #A-11001, Goat anti-Mouse |
Ki-67 | 10 mM citrate buffer pH 6.0 | 0.5% Triton X-100 | 3% BSA + 0.5% Triton-X 100 | Ki-67 (1:100) Thermo Fisher Scientific #PA5-19462, Rabbit | Alexa Fluor® 555 (1:500) Thermo Fisher Scientific, #A-21428 Goat anti-Rabbit |
MLCK | 10 mM citrate buffer pH 6.0 | 0.5% Triton X-100 | 3% BSA + 0.5% Triton-X 100 | MLCK (1:50) Thermo Fisher Scientific #PA5-15177, Rabbit Pc | Alexa Fluor® 555 (1:500) Thermo Fisher Scientific, #A-21428, Goat anti-Rabbit |
N-cadherin | 10 mM citrate buffer pH 6.0 | 0.5% Triton X-100 | 3% BSA + 0.5% Triton-X 100 | N-cadherin (1:100) Thermo Fisher Scientific #PA5-19486, Rabbit Pc | Alexa Fluor® 555 (1:500) Thermo Fisher Scientific, #A-21428, Goat anti-Rabbit |
Pan cytokeratin | 10 mM citrate buffer pH 6.0 | 0.5% Triton X-100 | 3% BSA + 0.5% Triton-X 100 | Pan Cytokeratin (1:100) Thermo Fisher Scientific #MA5-12231, Mouse Mc | Alexa Fluor® 647 (1:500) Thermo Fisher Scientific, #A-21235, Goat anti-Mouse |
PLVAP | 10 mM citrate buffer pH 6.0 | 0.5% Triton X-100 | 3% BSA + 0.5% Triton-X 100 | PLVAP (1:20) Santa Cruz Biotechnology #sc-50168, Goat Pc | IgG-FITC (1:100) Santa Cruz Biotechnology, #sc-2024, Donkey anti-Goat |
Rac1 | 10 mM citrate buffer pH 6.0 | 0.5% Triton X-100 | 5% GS in 3% BSA + 0.5% Triton-X 100 | Rac1 (1:25) Thermo Fisher Scientific #PA1-091, Rabbit Pc | Alexa Fluor® 555 (1:100) Thermo Fisher Scientific, #A-21428, Goat anti-Rabbit |
RhoA | 10 mM Tris-borate EDTA buffer pH 8.0 | 0.5% Triton X-100 | 10% BSA + 0.5% Triton-X 100 | RhoA (1:10) Thermo Fisher Scientific #OSR00266W, Rabbit Pc | Alexa Fluor® 555 (1:100) Thermo Fisher Scientific, #A-21428, Goat anti-Rabbit |
Thrombin | 10 mM Tris-borate EDTA buffer pH 8.0 | - | 3% BSA | Thrombin (1:200) Santa Cruz Biotechnology #sc-271449, Mouse Mc | Alexa Fluor® 647 (1:500) Thermo Fisher Scientific, #A-21235, Goat anti-Mouse |
Tomato lectin | 10 mM citrate buffer pH 6.0 | 0.5% Triton X-100 | 3%BSA + 0.5% Triton-X 100 | - | Tomato lectin (1:500) Vector Laboratories #FL-1171 |
Vimentin | 10 mM citrate buffer pH 6.0 | 0.5% Triton X-100 | 3%BSA + 0.5% Triton-X 100 | Vimentin (1:100) Thermo Fisher Scientific #PA5-27231, Rabbit Pc | Alexa Fluor® 555 (1:500) Thermo Fisher Scientific, #A-21428, Goat anti-Rabbit |
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Figueira, I.; Galego, S.; Custódio-Santos, T.; Vicente, R.; Molnár, K.; Haskó, J.; Malhó, R.; Videira, M.; Wilhelm, I.; Krizbai, I.; et al. Picturing Breast Cancer Brain Metastasis Development to Unravel Molecular Players and Cellular Crosstalk. Cancers 2021, 13, 910. https://doi.org/10.3390/cancers13040910
Figueira I, Galego S, Custódio-Santos T, Vicente R, Molnár K, Haskó J, Malhó R, Videira M, Wilhelm I, Krizbai I, et al. Picturing Breast Cancer Brain Metastasis Development to Unravel Molecular Players and Cellular Crosstalk. Cancers. 2021; 13(4):910. https://doi.org/10.3390/cancers13040910
Chicago/Turabian StyleFigueira, Inês, Sofia Galego, Tânia Custódio-Santos, Raquel Vicente, Kinga Molnár, Janos Haskó, Rui Malhó, Mafalda Videira, Imola Wilhelm, István Krizbai, and et al. 2021. "Picturing Breast Cancer Brain Metastasis Development to Unravel Molecular Players and Cellular Crosstalk" Cancers 13, no. 4: 910. https://doi.org/10.3390/cancers13040910