Generation and Characterization of a Tumor Stromal Microenvironment and Analysis of Its Interplay with Breast Cancer Cells: An In Vitro Model to Study Breast Cancer-Associated Fibroblast Inactivation
Abstract
:1. Introduction
2. Results
2.1. Characterization of Fibroblasts from Normal and Cancer Breast Tissues
2.2. Analysis of Human Primary BCAFs Grown as Monolayers or Spheroids
2.3. Reversion of BCAF Spheroids to Adhesion and Monolayer Growth
2.4. Evaluation of BC Cell Viability after Paracrine Interaction with BCAFs Grown as Monolayers, Spheroids or Reverted
2.5. Evaluation of BC Cell Migration after Paracrine Interaction with BCAF Monolayers and Reverted BCAFs
3. Discussion
4. Materials and Methods
4.1. Isolation and Culture of Primary Breast Fibroblasts
4.2. Immunofluorescence and Confocal Microscopy for Human Primary Breast Fibroblast Characterization
4.3. Cell Viability and Wound-Healing Assays for Human Primary Breast Fibroblast Characterization
4.4. Real Time PCR
4.5. Immunohistochemistry
4.6. Generation and Growth of BCAF Spheroids and Cell Culture
4.7. Total Cell Lysates and Western Blotting Analysis
4.8. MTT Assay
4.9. Analysis of BC Cell Migration
4.10. Statistical Analysis
5. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
- Ruocco, M.R.; Avagliano, A.; Granato, G.; Imparato, V.; Masone, S.; Masullo, M.; Nasso, R.; Montagnani, S.; Arcucci, A. Involvement of Breast Cancer-Associated Fibroblasts in Tumor Development, Therapy Resistance and Evaluation of Potential Therapeutic Strategies. Curr. Med. Chem. 2018, 25, 3414–3434. [Google Scholar] [CrossRef] [PubMed]
- Avagliano, A.; Fiume, G.; Ruocco, M.R.; Martucci, N.; Vecchio, E.; Insabato, L.; Russo, D.; Accurso, A.; Masone, S.; Montagnani, S.; et al. Influence of fibroblasts on mammary gland development, breast cancer microenvironment remodeling, and cancer cell dissemination. Cancers 2020, 12, 1697. [Google Scholar] [CrossRef] [PubMed]
- Avagliano, A.; Ruocco, M.R.; Aliotta, F.; Belviso, I.; Accurso, A.; Masone, S.; Montagnani, S.; Arcucci, A. Mitochondrial Flexibility of Breast Cancers: A Growth Advantage and a Therapeutic Opportunity. Cells 2019, 8, 401. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Li, C.I.; Anderson, B.O.; Daling, J.R.; Moe, R.E. Trends in Incidence Rates of Invasive Lobular and Ductal Breast Carcinoma. J. Am. Med. Assoc. 2003, 289, 1421–1424. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Makki, J. Diversity of breast carcinoma: Histological subtypes and clinical relevance. Clin. Med. Insights Pathol. 2015, 8, 23–31. [Google Scholar] [CrossRef] [Green Version]
- Salvatore, B.; Caprio, M.G.; Hill, B.S.; Sarnella, A.; Roviello, G.N.; Zannetti, A. Recent advances in nuclear imaging of receptor expression to guide targeted therapies in breast cancer. Cancers 2019, 11, 1614. [Google Scholar] [CrossRef] [Green Version]
- Arcucci, A.; Ruocco, M.R.; Granato, G.; Sacco, A.M.; Montagnani, S. Cancer: An Oxidative Crosstalk between Solid Tumor Cells and Cancer Associated Fibroblasts. Biomed Res. Int. 2016, 2016, 4502846. [Google Scholar] [CrossRef] [Green Version]
- Avagliano, A.; Fiume, G.; Pelagalli, A.; Sanità, G.; Ruocco, M.R.; Montagnani, S.; Arcucci, A. Metabolic Plasticity of Melanoma Cells and Their Crosstalk With Tumor Microenvironment. Front. Oncol. 2020, 10, 722. [Google Scholar] [CrossRef]
- Ruocco, M.R.; Avagliano, A.; Granato, G.; Vigliar, E.; Masone, S.; Montagnani, S.; Arcucci, A. Metabolic flexibility in melanoma: A potential therapeutic target. Semin. Cancer Biol. 2019, 59, 187–207. [Google Scholar] [CrossRef] [PubMed]
- Avagliano, A.; Granato, G.; Ruocco, M.R.; Romano, V.; Belviso, I.; Carfora, A.; Montagnani, S.; Arcucci, A. Metabolic Reprogramming of Cancer Associated Fibroblasts: The Slavery of Stromal Fibroblasts. Biomed Res. Int. 2018, 2018, 6075403. [Google Scholar] [CrossRef] [Green Version]
- Weigelt, B.; Peterse, J.L.; Van’t Veer, L.J. Breast cancer metastasis: Markers and models. Nat. Rev. Cancer 2005, 5, 591–602. [Google Scholar] [CrossRef]
- Romano, V.; Belviso, I.; Venuta, A.; Ruocco, M.R.; Masone, S.; Aliotta, F.; Fiume, G.; Montagnani, S.; Avagliano, A.; Arcucci, A. Influence of Tumor Microenvironment and Fibroblast Population Plasticity on Melanoma Growth, Therapy Resistance and Immunoescape. Int. J. Mol. Sci. 2021, 22, 5283. [Google Scholar] [CrossRef]
- Shiga, K.; Hara, M.; Nagasaki, T.; Sato, T.; Takahashi, H.; Takeyama, H. Cancer-associated fibroblasts: Their characteristics and their roles in tumor growth. Cancers 2015, 7, 2443–2458. [Google Scholar] [CrossRef]
- Avagliano, A.; Ruocco, M.R.; Nasso, R.; Aliotta, F.; Sanità, G.; Iaccarino, A.; Bellevicine, C.; Calì, G.; Fiume, G.; Masone, S.; et al. Development of a Stromal Microenvironment Experimental Model Containing Proto-Myofibroblast Like Cells and Analysis of Its Crosstalk with Melanoma Cells: A New Tool to Potentiate and Stabilize Tumor Suppressor Phenotype of Dermal Myofibroblasts. Cells 2019, 8, 1435. [Google Scholar] [CrossRef] [Green Version]
- Hinz, B.; Celetta, G.; Tomasek, J.J.; Gabbiani, G.; Chaponnier, C. Alpha-smooth muscle actin expression upregulates fibroblast contractile activity. Mol. Biol. Cell 2001, 12, 2730–2741. [Google Scholar] [CrossRef] [Green Version]
- Schreiber, H. Fibroblasts: Dangerous travel companions. J. Exp. Med. 2019, 216, 479–481. [Google Scholar] [CrossRef]
- Duda, D.G.; Duyverman, A.M.M.J.; Kohno, M.; Snuderl, M.; Steller, E.J.A.; Fukumura, D.; Jain, R.K. Malignant cells facilitate lung metastasis by bringing their own soil. Proc. Natl. Acad. Sci. USA 2010, 107, 21677–21682. [Google Scholar] [CrossRef] [Green Version]
- Gao, Q.; Yang, Z.; Xu, S.; Li, X.; Yang, X.; Jin, P.; Liu, Y.; Zhou, X.; Zhang, T.; Gong, C.; et al. Heterotypic CAF-tumor spheroids promote early peritoneal metastatis of ovarian cancer. J. Exp. Med. 2019, 216, 688–703. [Google Scholar] [CrossRef] [Green Version]
- Ortiz-Otero, N.; Clinch, A.B.; Hope, J.; Wang, W.; Reinhart-King, C.A.; King, M.R. Cancer associated fibroblasts confer shear resistance to circulating tumor cells during prostate cancer metastatic progression. Oncotarget 2020, 11, 1037–1050. [Google Scholar] [CrossRef] [Green Version]
- Ao, Z.; Shah, S.H.; Machlin, L.M.; Parajuli, R.; Miller, P.C.; Rawal, S.; Williams, A.J.; Cote, R.J.; Lippman, M.E.; Datar, R.H.; et al. Identification of cancer-associated fibroblasts in circulating blood from patients with metastatic breast cancer. Cancer Res. 2015, 75, 4681–4687. [Google Scholar] [CrossRef] [Green Version]
- Richardson, A.M.; Havel, L.S.; Koyen, A.E.; Konen, J.M.; Shupe, J.; Wiles, W.G.; David Martin, W.; Grossniklaus, H.E.; Sica, G.; Gilbert-Ross, M.; et al. Vimentin Is Required for Lung Adenocarcinoma Metastasis via Heterotypic Tumor Cell-Cancer-Associated Fibroblast Interactions during Collective Invasion. Clin. Cancer Res. 2018, 24, 420–432. [Google Scholar] [CrossRef] [Green Version]
- Jones, M.L.; Siddiqui, J.; Pienta, K.J.; Getzenberg, R.H. Circulating fibroblast-like cells in men with metastatic prostate cancer. Prostate 2013, 73, 176–181. [Google Scholar] [CrossRef] [Green Version]
- Langhans, S.A. Three-Dimensional in Vitro Cell Culture Models in Drug Discovery and Drug Repositioning. Front. Pharmacol. 2018, 9, 6. [Google Scholar] [CrossRef]
- da Rocha-Azevedo, B.; Grinnell, F. Fibroblast morphogenesis on 3D collagen matrices: The balance between cell clustering and cell migration. Exp. Cell Res. 2013, 319, 2440–2446. [Google Scholar] [CrossRef] [Green Version]
- Kunz-Schughart, L.A.; Knuechel, R. Tumor-associated fibroblasts (part I): Active stromal participants in tumor development and progression? Histol. Histopathol. 2002, 17, 599–621. [Google Scholar] [CrossRef]
- Ware, M.J.; Colbert, K.; Keshishian, V.; Ho, J.; Corr, S.J.; Curley, S.A.; Godin, B. Generation of Homogenous Three-Dimensional Pancreatic Cancer Cell Spheroids Using an Improved Hanging Drop Technique. Tissue Eng. Part C. Methods 2016, 22, 312–321. [Google Scholar] [CrossRef] [Green Version]
- Kunz-Schughart, L.A.; Kreutz, M.; Knuechel, R. Multicellular spheroids: A three-dimensional in vitro culture system to study tumour biology. Int. J. Exp. Pathol. 1998, 79, 1–23. [Google Scholar] [CrossRef]
- Nii, T.; Makino, K.; Tabata, Y. A Cancer Invasion Model Combined with Cancer-Associated Fibroblasts Aggregates Incorporating Gelatin Hydrogel Microspheres Containing a p53 Inhibitor. Tissue Eng. Part C Methods 2019, 25, 711–720. [Google Scholar] [CrossRef]
- Xia, L.; Sakban, R.B.; Qu, Y.; Hong, X.; Zhang, W.; Nugraha, B.; Tong, W.H.; Ananthanarayanan, A.; Zheng, B.; Chau, I.Y.Y.; et al. Tethered spheroids as an in vitro hepatocyte model for drug safety screening. Biomaterials 2012, 33, 2165–2176. [Google Scholar] [CrossRef]
- Friedrich, J.; Seidel, C.; Ebner, R.; Kunz-Schughart, L.A. Spheroid-based drug screen: Considerations and practical approach. Nat. Protoc. 2009, 4, 309–324. [Google Scholar] [CrossRef]
- Di Modugno, F.; Colosi, C.; Trono, P.; Antonacci, G.; Ruocco, G.; Nisticò, P. 3D models in the new era of immune oncology: Focus on T cells, CAF and ECM. J. Exp. Clin. Cancer Res. 2019, 38, 117. [Google Scholar] [CrossRef] [Green Version]
- Shao, H.; Moller, M.; Wang, D.; Ting, A.; Boulina, M.; Liu, Z.J. A Novel Stromal Fibroblast-Modulated 3D Tumor Spheroid Model for Studying Tumor-Stroma Interaction and Drug Discovery. J. Vis. Exp. 2020, 2020, e60660. [Google Scholar] [CrossRef]
- Granato, G.; Ruocco, M.R.; Iaccarino, A.; Masone, S.; Calì, G.; Avagliano, A.; Russo, V.; Bellevicine, C.; Di Spigna, G.; Fiume, G.; et al. Generation and analysis of spheroids from human primary skin myofibroblasts: An experimental system to study myofibroblasts deactivation. Cell Death Discov. 2017, 3, 17038. [Google Scholar] [CrossRef] [Green Version]
- Bremnes, R.M.; Dønnem, T.; Al-Saad, S.; Al-Shibli, K.; Andersen, S.; Sirera, R.; Camps, C.; Marinez, I.; Busund, L.T. The Role of Tumor Stroma in Cancer Progression and Prognosis: Emphasis on Carcinoma-Associated Fibroblasts and Non-small Cell Lung Cancer. J. Thorac. Oncol. 2011, 6, 209–217. [Google Scholar] [CrossRef] [Green Version]
- Qiao, A.; Gu, F.; Guo, X.; Zhang, X.; Fu, L. Breast cancer-associated fibroblasts: Their roles in tumor initiation, progression and clinical applications. Front. Med. 2016, 10, 33–40. [Google Scholar] [CrossRef]
- Stylianou, A.; Gkretsi, V.; Stylianopoulos, T. Transforming growth factor-β modulates pancreatic cancer associated fibroblasts cell shape, stiffness and invasion. Biochim. Biophys. Acta-Gen. Subj. 2018, 1862, 1537–1546. [Google Scholar] [CrossRef]
- Santi, A.; Kugeratski, F.G.; Zanivan, S. Cancer Associated Fibroblasts: The Architects of Stroma Remodeling. Proteomics 2018, 18, 1700167. [Google Scholar] [CrossRef]
- Fu, Z.; Song, P.; Li, D.; Yi, C.; Chen, H.; Ruan, S.; Shi, Z.; Xu, W.; Fu, X.; Zheng, S. Cancer-associated fibroblasts from invasive breast cancer have an attenuated capacity to secrete collagens. Int. J. Oncol. 2014, 45, 1479–1488. [Google Scholar] [CrossRef] [Green Version]
- Amornsupak, K.; Jamjuntra, P.; Warnnissorn, M.; O-Charoenrat, P.; Sa-nguanraksa, D.; Thuwajit, P.; Eccles, S.A.; Thuwajit, C. High ASMA + Fibroblasts and Low Cytoplasmic HMGB1 + Breast Cancer Cells Predict Poor Prognosis. Clin. Breast Cancer 2017, 17, 441–452.e2. [Google Scholar] [CrossRef] [Green Version]
- Hinz, B.; Gabbiani, G. Mechanisms of force generation and transmission by myofibroblasts. Curr. Opin. Biotechnol. 2003, 14, 538–546. [Google Scholar] [CrossRef]
- Hecker, L.; Jagirdar, R.; Jin, T.; Thannickal, V.J. Reversible differentiation of myofibroblasts by MyoD. Exp. Cell Res. 2011, 317, 1914–1921. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Kisseleva, T.; Cong, M.; Paik, Y.H.; Scholten, D.; Jiang, C.; Benner, C.; Iwaisako, K.; Moore-Morris, T.; Scott, B.; Tsukamoto, H.; et al. Myofibroblasts revert to an inactive phenotype during regression of liver fibrosis. Proc. Natl. Acad. Sci. USA 2012, 109, 9448–9453. [Google Scholar] [CrossRef] [Green Version]
- Kalluri, R.; Zeisberg, M. Fibroblasts in cancer. Nat. Rev. Cancer 2006, 6, 392–401. [Google Scholar] [CrossRef]
- Toullec, A.; Gerald, D.; Despouy, G.; Bourachot, B.; Cardon, M.; Lefort, S.; Richardson, M.; Rigaill, G.; Parrini, M.C.; Lucchesi, C.; et al. Oxidative stress promotes myofibroblast differentiation and tumour spreading. EMBO Mol. Med. 2010, 2, 211–230. [Google Scholar] [CrossRef]
- Benyahia, Z.; Dussault, N.; Cayol, M.; Sigaud, R.; Berenguer-Daizé, C.; Delfino, C.; Tounsi, A.; Garcia, S.; Martin, P.M.; Mabrouk, K.; et al. Stromal fibroblasts present in breast carcinomas promote tumor growth and angiogenesis through adrenomedullin secretion. Oncotarget 2017, 8, 15744–15762. [Google Scholar] [CrossRef] [Green Version]
- Bu, L.; Baba, H.; Yasuda, T.; Uchihara, T.; Ishimoto, T. Functional diversity of cancer-associated fibroblasts in modulating drug resistance. Cancer Sci. 2020, 111, 3468–3477. [Google Scholar] [CrossRef]
- Fiori, M.E.; Di Franco, S.; Villanova, L.; Bianca, P.; Stassi, G.; De Maria, R. Cancer-associated fibroblasts as abettors of tumor progression at the crossroads of EMT and therapy resistance. Mol. Cancer 2019, 18, 70. [Google Scholar] [CrossRef] [Green Version]
- Liu, X.; Xu, J.; Brenner, D.A.; Kisseleva, T. Reversibility of Liver Fibrosis and Inactivation of Fibrogenic Myofibroblasts. Curr. Pathobiol. Rep. 2013, 1, 209–214. [Google Scholar] [CrossRef] [Green Version]
- Wang, Z.; Tang, Y.; Tan, Y.; Wei, Q.; Yu, W. Cancer-associated fibroblasts in radiotherapy: Challenges and new opportunities. Cell Commun. Signal. 2019, 17, 47. [Google Scholar] [CrossRef] [Green Version]
- Tyan, S.W.; Kuo, W.H.; Huang, C.K.; Pan, C.C.; Shew, J.Y.; Chang, K.J.; Lee, E.Y.H.P.; Lee, W.H. Breast cancer cells induce cancer-associated fibroblasts to secrete hepatocyte growth factor to enhance breast tumorigenesis. PLoS ONE 2011, 6, e15313. [Google Scholar] [CrossRef] [Green Version]
- Shekhar, M.; Werdell, J.; Santner, S.; Pauley, R.; Tait, L. Breast Stroma Plays a Dominant Regulatory Role in Breast Epithelial Growth and Differentiation: Implications for Tumor Development and Progression-PubMed. Available online: https://pubmed.ncbi.nlm.nih.gov/11245428/ (accessed on 26 July 2021).
- Arcucci, A.; Ruocco, M.R.; Albano, F.; Granato, G.; Romano, V.; Corso, G.; Bancone, C.; De Vendittis, E.; Della Corte, A.; Montagnani, S. Analysis of extracellular superoxide dismutase and Akt in ascending aortic aneurysm with tricuspid or bicuspid aortic valve. Eur. J. Histochem. 2014, 58, 200–206. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Arcucci, A.; Ruocco, M.R.; Amatruda, N.; Riccio, A.; Tarantino, G.; Albano, F.; Mele, V.; Montagnani, S. Analysis of extracellular superoxide dismutase in fibroblasts from patients with systemic sclerosis. J. Biol. Regul. Homeost. Agents 2011, 25, 647–654. [Google Scholar]
- Lebret, S.C.; Newgreen, D.F.; Thompson, E.W.; Ackland, M.L. Induction of epithelial to mesenchymal transition in PMC42-LA human breast carcinoma cells by carcinoma-associated fibroblast secreted factors. Breast Cancer Res. 2007, 9, R19. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Aboussekhra, A. Role of cancer-associated fibroblasts in breast cancer development and prognosis. Int. J. Dev. Biol. 2011, 55, 841–849. [Google Scholar] [CrossRef] [Green Version]
- Madsen, C.D.; Pedersen, J.T.; Venning, F.A.; Singh, L.B.; Moeendarbary, E.; Charras, G.; Cox, T.R.; Sahai, E.; Erler, J.T. Hypoxia and loss of PHD 2 inactivate stromal fibroblasts to decrease tumour stiffness and metastasis. EMBO Rep. 2015, 16, 1394–1408. [Google Scholar] [CrossRef]
- Ene-Obong, A.; Clear, A.J.; Watt, J.; Wang, J.; Fatah, R.; Riches, J.C.; Marshall, J.F.; Chin-Aleong, J.; Chelala, C.; Gribben, J.G.; et al. Activated pancreatic stellate cells sequester CD8+ T cells to reduce their infiltration of the juxtatumoral compartment of pancreatic ductal adenocarcinoma. Gastroenterology 2013, 145, 1121–1132. [Google Scholar] [CrossRef] [Green Version]
- Froeling, F.E.M.; Feig, C.; Chelala, C.; Dobson, R.; Mein, C.E.; Tuveson, D.A.; Clevers, H.; Hart, I.R.; Kocher, H.M. Retinoic acid-induced pancreatic stellate cell quiescence reduces paracrine Wnt-β-catenin signaling to slow tumor progression. Gastroenterology 2011, 141, 1486–1497.e14. [Google Scholar] [CrossRef]
- Sherman, M.H.; Yu, R.T.; Engle, D.D.; Ding, N.; Atkins, A.R.; Tiriac, H.; Collisson, E.A.; Connor, F.; Van Dyke, T.; Kozlov, S.; et al. Vitamin D receptor-mediated stromal reprogramming suppresses pancreatitis and enhances pancreatic cancer therapy. Cell 2014, 159, 80–93. [Google Scholar] [CrossRef] [Green Version]
- Sahai, E.; Astsaturov, I.; Cukierman, E.; DeNardo, D.G.; Egeblad, M.; Evans, R.M.; Fearon, D.; Greten, F.R.; Hingorani, S.R.; Hunter, T.; et al. A framework for advancing our understanding of cancer-associated fibroblasts. Nat. Rev. Cancer 2020, 20, 174–186. [Google Scholar] [CrossRef] [Green Version]
- Öhlund, D.; Handly-Santana, A.; Biffi, G.; Elyada, E.; Ponz-Sarvise, M.; Corbo, V.; Oni, T.; Hearn, S.; Lee, E.; Chio, I.; et al. Distinct populations of inflammatory fibroblasts and myofibroblasts in pancreatic cancer. J. Exp. Med. 2017, 214, 579–596. [Google Scholar] [CrossRef]
- Costa, A.; Kieffer, Y.; Scholer-Dahirel, A.; Pelon, F.; Bourachot, B.; Cardon, M.; Sirven, P.; Magagna, I.; Fuhrmann, L.; Bernard, C.; et al. Fibroblast Heterogeneity and Immunosuppressive Environment in Human Breast Cancer. Cancer Cell 2018, 33, 463–479.e10. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Hill, B.S.; Sarnella, A.; D’Avino, G.; Zannetti, A. Recruitment of stromal cells into tumour microenvironment promote the metastatic spread of breast cancer. Semin. Cancer Biol. 2020, 60, 202–213. [Google Scholar] [CrossRef] [PubMed]
- Busch, S.; Andersson, D.; Bom, E.; Walsch, C.; Stahlberg, A.; Landberg, G. Cellular organization and molecular differentiation model of breast cancer-associated fibroblasts. Mol. Cancer 2017, 16, 73. [Google Scholar] [CrossRef] [Green Version]
- Barbato, A.; Iuliano, A.; Volpe, M.; D’alterio, R.; Brillante, S.; Massa, F.; De Cegli, R.; Carrella, S.; Salati, M.; Russo, A.; et al. Integrated Genomics Identifies miR-181/TFAM Pathway as a Critical Driver of Drug Resistance in Melanoma. Int. J. Mol. Sci. 2021, 22, 1801. [Google Scholar] [CrossRef]
- Bradford, M. A Rapid and Sensitive Method for the Quantitation of Microgram Quantities of Protein Utilizing the Principle of Protein-Dye Binding. Anal. Biochem. 1976, 72, 248–254. [Google Scholar] [CrossRef]
- Ruocco, M.R.; Lamberti, A.; Serrano, M.J.; Fiume, G.; Arcucci, A. Editorial: Tumor Microenvironment and Cancer Cell Interactions in Solid Tumor Growth and Therapy Resistance. Front. cell Dev. Biol. 2022, 10, 896194. [Google Scholar] [CrossRef] [PubMed]
Mean Age ± SD (Years) | 60.25 ± 6.31 |
---|---|
Tumor Grade | |
G1 | 1 |
G2 | 2 |
G3 | 5 |
Tumor Stage | |
T0 | 0 |
T1 | 3 |
T2 | 5 |
T3 | 0 |
T4 | 0 |
Lymph Node status | |
N0 | 7 |
N1 | 1 |
ER status | |
positive | 8 |
negative | 0 |
PR status | |
positive | 8 |
negative | 0 |
HER2 status | |
positive | 0 |
negative | 8 |
Gene | Sequence |
---|---|
SPARC | Forward: 5′-TCTTCCCTGTACACTGGCAGTTC-3′ Reverse: 5′-AGCTCGGTGTGGGAGAGGTA-3′ |
FAP | Forward: 5′- GGAAGTGCCTGTTCCAGCAATG-3′ Reverse: 5′- TGTCTGCCAGTCTTCCCTGAAG-3′ |
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. |
© 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
Share and Cite
Romano, V.; Ruocco, M.R.; Carotenuto, P.; Barbato, A.; Venuta, A.; Acampora, V.; De Lella, S.; Vigliar, E.; Iaccarino, A.; Troncone, G.; et al. Generation and Characterization of a Tumor Stromal Microenvironment and Analysis of Its Interplay with Breast Cancer Cells: An In Vitro Model to Study Breast Cancer-Associated Fibroblast Inactivation. Int. J. Mol. Sci. 2022, 23, 6875. https://doi.org/10.3390/ijms23126875
Romano V, Ruocco MR, Carotenuto P, Barbato A, Venuta A, Acampora V, De Lella S, Vigliar E, Iaccarino A, Troncone G, et al. Generation and Characterization of a Tumor Stromal Microenvironment and Analysis of Its Interplay with Breast Cancer Cells: An In Vitro Model to Study Breast Cancer-Associated Fibroblast Inactivation. International Journal of Molecular Sciences. 2022; 23(12):6875. https://doi.org/10.3390/ijms23126875
Chicago/Turabian StyleRomano, Veronica, Maria Rosaria Ruocco, Pietro Carotenuto, Anna Barbato, Alessandro Venuta, Vittoria Acampora, Sabrina De Lella, Elena Vigliar, Antonino Iaccarino, Giancarlo Troncone, and et al. 2022. "Generation and Characterization of a Tumor Stromal Microenvironment and Analysis of Its Interplay with Breast Cancer Cells: An In Vitro Model to Study Breast Cancer-Associated Fibroblast Inactivation" International Journal of Molecular Sciences 23, no. 12: 6875. https://doi.org/10.3390/ijms23126875
APA StyleRomano, V., Ruocco, M. R., Carotenuto, P., Barbato, A., Venuta, A., Acampora, V., De Lella, S., Vigliar, E., Iaccarino, A., Troncone, G., Calì, G., Insabato, L., Russo, D., Franco, B., Masone, S., Velotti, N., Accurso, A., Pellegrino, T., Fiume, G., ... Arcucci, A. (2022). Generation and Characterization of a Tumor Stromal Microenvironment and Analysis of Its Interplay with Breast Cancer Cells: An In Vitro Model to Study Breast Cancer-Associated Fibroblast Inactivation. International Journal of Molecular Sciences, 23(12), 6875. https://doi.org/10.3390/ijms23126875