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Journal of Experimental & Clinical Cancer Research
Published in: Journal of Experimental & Clinical Cancer Research 1/2020

01-12-2020 | Breast Cancer | Research

A novel culture method that sustains ERα signaling in human breast cancer tissue microstructures

Authors: Ana Luísa Cartaxo, Marta F. Estrada, Giacomo Domenici, Ruben Roque, Fernanda Silva, Emilio J. Gualda, Pablo Loza-Alvarez, George Sflomos, Cathrin Brisken, Paula M. Alves, Saudade André, Catarina Brito

Published in: Journal of Experimental & Clinical Cancer Research | Issue 1/2020

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Abstract

Background

Estrogen receptor α (ERα) signaling is a defining and driving event in most breast cancers; ERα is detected in malignant epithelial cells of 75% of all breast cancers (classified as ER-positive breast cancer) and, in these cases, ERα targeting is the main therapeutic strategy. However, the biological determinants of ERα heterogeneity and the mechanisms underlying therapeutic resistance are still elusive, hampered by the challenges in developing experimental models recapitulative of intra-tumoral heterogeneity and in which ERα signaling is sustained. Ex vivo cultures of human breast cancer tissue have been proposed to retain the original tissue architecture, epithelial and stromal cell components and ERα. However, loss of cellularity, viability and ERα expression are well-known culture-related phenomena.

Methods

BC samples were collected and brought to the laboratory. Then they were minced, enzymatically digested, entrapped in alginate and cultured for 1 month. The histological architecture, cellular composition and cell proliferation of tissue microstructures were assessed by immunohistochemistry. Cell viability was assessed by measurement of cell metabolic activity and histological evaluation. The presence of ERα was accessed by immunohistochemistry and RT-qPCR and its functionality evaluated by challenge with 17-β-estradiol and fulvestrant.

Results

We describe a strategy based on entrapment of breast cancer tissue microstructures in alginate capsules and their long-term culture under agitation, successfully applied to tissue obtained from 63 breast cancer patients. After 1 month in culture, the architectural features of the encapsulated tissue microstructures were similar to the original patient tumors: epithelial, stromal and endothelial compartments were maintained, with an average of 97% of cell viability compared to day 0. In ERα-positive cases, fibers of collagen, the main extracellular matrix component in vivo, were preserved. ERα expression was at least partially retained at gene and protein levels and response to ERα stimulation and inhibition was observed at the level of downstream targets, demonstrating active ER signaling.

Conclusions

The proposed model system is a new methodology to study ex vivo breast cancer biology, in particular ERα signaling. It is suitable for interrogating the long-term effects of anti-endocrine drugs in a set-up that closely resembles the original tumor microenvironment, with potential application in pre- and co-clinical assays of ERα-positive breast cancer.
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Literature
1.
Noone A, Cronin KA, Altekruse SF, Howlader N, Lewis R, Petkov VI, et al. Cancer incidence and survival trends by subtype using data from the surveillance epidemiology and end results program, 1992–2013. Cancer Epidemiol Biomark Prev. 2017;26(4):1–19.
2.
Diaz Bessone MI, Gattas MJ, Laporte T, Tanaka M, Simian M. The tumor microenvironment as a regulator of endocrine resistance in breast Cancer. Front Endocrinol (Lausanne). 2019;10(August):1–10.
3.
Rothenberger NJ, Somasundaram A, Stabile LP. The role of the estrogen pathway in the tumor microenvironment. Int J Mol Sci. 2018;19:2.
4.
Martelotto LG, Ng CKY, Piscuoglio S, Weigelt B, Reis-filho JS. Breast cancer intra-tumor heterogeneity. Breast Cancer Res. 2014;16(R48):1–11.
5.
Antoni D, Burckel H, Josset E, Noel G. Three-dimensional cell culture: a breakthrough in vivo. Int J Mol Sci. 2015;16:5517–27.PubMedPubMedCentral
6.
Sflomos G, Dormoy V, Metsalu T, Jeitziner R, Battista L, Scabia V, et al. A preclinical model for ERα-positive breast Cancer points to the epithelial microenvironment as determinant of luminal phenotype and hormone response. Cancer Cell. 2016;29(3):407–22.PubMed
7.
Tanos T, Sflomos G, Echeverria PC, Ayyanan A, Gutierrez M, Delaloye J, et al. Progesterone / RANKL is a major regulatory Axis in the human breast. Sci Transl Med. 2013;5(182):1–10.
8.
Muraro MG, Muenst S, Mele V, Quagliata L, Iezzi G, Tzankov A, et al. Ex-vivo assessment of drug response on breast cancer primary tissue with preserved microenvironments. Oncoimmunology. 2017;6(7):1–12.
9.
van der Kuip H, Mürdter TE, Sonnenberg M, McCellan M, Gutzeit S, Gerteis A, et al. Short term culture of breast cancer tissues to study the activity of the anticancer drug taxol in an intact tumor environment. BMC Cancer. 2006;6:1–11.
10.
Naipal KAT, Verkaik NS, Sánchez H, van Deurzen CHM, den Bakker MA, Hoeijmakers JHJ, et al. Tumor slice culture system to assess drug response of primary breast cancer. BMC Cancer. 2016;16(1):1–13.
11.
Campuzano S, Pelling AE. Scaffolds for 3D cell culture and cellular agriculture applications derived from non-animal sources. Front Sustain Food Syst. 2019;3(May):1–9.
12.
Rebelo SP, Pinto C, Martins TR, Harrer N, Estrada MF, Loza-Alvarez P, et al. 3D-3-culture: a tool to unveil macrophage plasticity in the tumour microenvironment. Biomaterials. 2018;163:185–97.PubMed
13.
Estrada MF, Rebelo SP, Davies EJ, Pinto MT, Pereira H, Santo VE, et al. Modelling the tumour microenvironment in long-term microencapsulated 3D co-cultures recapitulates phenotypic features of disease progression. Biomaterials. 2016;78:50–61.PubMed
14.
Mathew M, Santos SICO, Zalvidea D, Loza-Alvarez P. Multimodal optical workstation for simultaneous linear, nonlinear microscopy and nanomanipulation: upgrading a commercial confocal inverted microscope. Rev Sci Instrum. 2009;80(7):1–11.
15.
Kato S, Endoh H, Masuhiro Y, Kitamoto T, Uchiyama S, Sasaki H, et al. Activation of the Estrogen Receptor Through Phosphorylation by Mitogen-Activated Protein Kinase. Science (80- ). 1999;270:102–6.
16.
Moerkens M, Zhang Y, Wester L, van de Water B, Meerman JHN. Epidermal growth factor receptor signalling in human breast cancer cells operates parallel to estrogen receptor α signalling and results in tamoxifen insensitive proliferation. BMC Cancer. 2014;14(1):1–16.
17.
Karmakar S, Jin Y, Nagaich AK. Interaction of glucocorticoid receptor (GR) with estrogen receptor (ER) α and activator protein 1 (AP1) in dexamethasone-mediated interference of ERα activity. J Biol Chem. 2013;288(33):24020–34.PubMedPubMedCentral
18.
Lanzino M, Morelli C, Garofalo C, Panno ML, Mauro L, Ando S, et al. Interaction between estrogen receptor alpha and insulin/IGF signaling in breast cancer. Curr Cancer Drug Targets. 2008;8(7):597–610.PubMed
19.
Universi- JH, Chemistry HB. Ligand-independent activation of steroid hormone receptors. J Mol Med. 1998:469–79.
20.
Chen D, Washbrook E, Sarwar N, Bates GJ, Pace PE, Thirunuvakkarasu V, et al. Phosphorylation of human estrogen receptor α at serine 118 by two distinct signal transduction pathways revealed by phosphorylation-specific antisera. Oncogene. 2002;21(32):4921–31.PubMed
21.
Dolfi SC, Jäger AV, Medina DJ, Haffty BG, Yang J, Hirshfield KM. Fulvestrant treatment alters MDM2 protein turnover and sensitivity of human breast carcinoma cells to chemotherapeutic drugs. Cancer Lett. 2014;350(1–2):52–60.PubMedPubMedCentral
22.
Regan RMO, Osipo C, Ariazi E, Lee ES, Meeke K, Morris C, et al. Development and therapeutic options for the T reatment of Raloxifene-stimulated breast Cancer in Athymic mice. Cancer Ther Preclin. 2006;12(7):2255–64.
23.
Domenici G, Aurrekoetxea-Rodríguez I, Simões BM, Rábano M, Lee SY, Millán JS, et al. A Sox2–Sox9 signalling axis maintains human breast luminal progenitor and breast cancer stem cells. Oncogene. 2019;38(17):3151–69.PubMedPubMedCentral
24.
Dai X, Cheng H, Bai Z, Li J. Breast Cancer cell line classification and its relevance with breast tumor subtyping. J Cancer. 2017;8(16):3131–41.PubMedPubMedCentral
25.
Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real- time quantitative PCR and the 2/deltadeltaCt method. Methods. 2001;25(4):402–8.PubMed
26.
Matthew G, Heiden V, Cantley LC, Thompson CB. Understanding the Warburg Effect: The Metabolic Requirements of Cell Proliferation. Science (80- ). 2010;324(5930):1029–33.
27.
Gloushankova NA, Rubtsova SN, Zhitnyak IY. Cadherin-mediated cell-cell interactions in normal and cancer cells. Tissue Barriers. 2017;5(3):1–15.
28.
Altin JG, Sloan EK. The role of CD45 and CD45-associated molecules in T cell activation. Immunol Cell Biol. 1997;75(5):430–45.PubMed
29.
Buisseret L, Garaud S, De WA, Van Den EG, Boisson A, Solinas C. Tumor-infiltrating lymphocyte composition , organization and PD-1 / PD-L1 expression are linked in breast cancer, vol. 6; 2017. p. 1.
30.
Holl EK, Frazier VN, Landa K, Beasley GM, Hwang ES, Nair SK. Examining peripheral and tumor cellular Immunome in patients with Cancer. Front Immunol. 2019;10(July):1767.PubMedPubMedCentral
31.
Garaud S, Buisseret L, Solinas C, Gu-Trantien C, De Wind A, Van Den Eynden G, et al. Tumor-infiltrating B cells signal functional humoral immune responses in breast cancer. JCI Insight. 2019;4:18.
32.
Osborne CK, Wakeling A, Nicholson RI. Fulvestrant: an oestrogen receptor antagonist with a novel mechanism of action. Br J Cancer. 2004;90:2–6.
33.
Vergote I, Robertson JFR. Fulvestrant is an effective and well-tolerated endocrine therapy for postmenopausal women with advanced breast cancer: results from clinical trials. Br J Cancer. 2004;90:S11–4.PubMedPubMedCentral
34.
Amir E, Miller N, Geddie W, Freedman O, Kassam F, Simmons C, et al. Prospective study evaluating the impact of tissue confirmation of metastatic disease in patients with breast Cancer. J Clin Oncol. 2020;30(6):587–92.
35.
Burns KA, Korach KS. Estrogen receptors and human disease: an update. Arch Toxicol. 2016;86(10):1491–504.
36.
Lindstro LS, Karlsson E, Wilking UM, Johansson U, Hartman J, Lidbrink EK, et al. Clinically Used Breast Cancer Markers Such As Estrogen Receptor , Progesterone Receptor , and Human Epidermal Growth Factor Receptor 2 Are Unstable Throughout Tumor Progression. J Clin Oncol. 2020;30:21.
37.
Pontiggia O, Sampayo R, Raffo D, Motter A, Xu R, Bissell MJ, et al. The tumor microenvironment modulates tamoxifen resistance in breast cancer: a role for soluble stromal factors and fibronectin through β1 integrin. Breast Cancer Res Treat. 2012;133(2):459–71.PubMed
38.
Brechbuhl HM, Finlay-schultz J, Yamamoto TM, Gillen AE, Cittelly DM, Tan AC, et al. Fibroblast subtypes regulate responsiveness of luminal breast cancer to estrogen. Clin Cancer Res. 2018;23(7):1710–21.
39.
Morgan MM, Livingston MK, Warrick JW, Stanek EM, Alarid ET, Beebe DJ, et al. Mammary fibroblasts reduce apoptosis and speed estrogen-induced hyperplasia in an organotypic MCF7-derived duct model. Sci Rep. 2018;8(1):1–13.
40.
Jansen MPHM, Foekens JA, Van Staveren IL, Dirkzwager-Kiel MM, Ritstier K, Look MP, et al. Molecular classification of tamoxifen-resistant breast carcinomas by gene expression profiling. J Clin Oncol. 2005;23(4):732–40.PubMed
41.
Sampayo RG, Toscani AM, Rubashkin MG, Thi K, Masullo LA, Violi IL, et al. Fibronectin rescues estrogen receptor α from lysosomal degradation in breast cancer cells. J Cell Biol. 2018;217(8):2777–98.PubMedPubMedCentral
42.
Alco GUL, An ABG, Lu DSG, Pilanci KNUR, Tuzlali S, Ordu C, et al. Clinical and histopathological factors associated with Ki-67 expression in breast cancer patients. Oncol Lett. 2015;9:1046–54.PubMedPubMedCentral
43.
Davies EJ, Dong M, Gutekunst M, Närhi K, Van Zoggel HJAA, Blom S, et al. Capturing complex tumour biology in vitro: histological and molecular characterisation of precision cut slices. Sci Rep [Internet]. 2015;5(July):1–17 Available from: https://​doi.​org/​10.​1038/​srep17187.
44.
Mekala NK, Baadhe RR, Potumarthi R. Mass transfer aspects of 3D cell cultures in tissue engineering. Asia-Pacific J Chem Eng. 2014;9(3):318–29.
45.
Rouwkema J, Koopman BFJM, Blitterswijk CAV, Dhert WJA, Malda J. Supply of nutrients to cells in engineered tissues. Biotechnol Genet Eng Rev. 2009;26(1):163–78.
46.
Sun J, Tan H. Alginate-Based Biomaterials for Regenerative Medicine Applications. Materials (Basel). 2013;6:1285–309.
47.
Wilson JL, Najia MA, Saeed R, McDevitt TC. Alginate encapsulation parameters influence the differentiation of microencapsulated embryonic stem cell aggregates. Biotechnol Bioeng. 2014;111(3):618–31.PubMed
48.
Miranda JP, Rodrigues A, Tostões RM, Sofia Leite HZ, Carrondo MJT, Alves PM. Extending Hepatocyte Functionality for Drug-Testing Applications Using High-Viscosity Alginate–Encapsulated Three-Dimensional Cultures in Bioreactors. Tissue Eng Part C Methods. 2010;16(6).
49.
Kanta J. Collagen matrix as a tool in studying fibroblastic cell behavior. Cell Adhes Migr. 2015;9(4):308–16.
50.
Tilbury K, Campagnola PJ. Applications of second-harmonic generation imaging microscopy in ovarian and breast cancer. Perspect Medicin Chem. 2015;7:21–32.PubMedPubMedCentral
51.
Provenzano PP, Inman DR, Eliceiri KW, Knittel JG, Yan L, Rueden CT, et al. Collagen density promotes mammary tumor initiation and progression. BMC Med. 2008;6:1–15.
52.
Li T, Sun L, Miller N, Nicklee T, Woo J, Hulse-Smith L, et al. The association of measured breast tissue characteristics with mammographic density and other risk factors for breast cancer. Cancer Epidemiol Biomark Prev. 2005;14(2):343–9.
53.
Netti PA, Berk DA, Swartz MA, Grodzinsky AJ, Jain RK. Role of extracellular matrix assembly in interstitial transport in solid tumors. Cancer Res. 2000;60(9):2497–503.PubMed
54.
Rijal G. Native-mimicking in vitro microenvironment: an elusive and seductive future for tumor modeling and tissue engineering. J Biol Eng. 2018:1–22.
55.
56.
Wang X, Mori I, Tang W, Nakamura M, Nakamura Y, Sato M. p63 Expression in Normal , Hyperplastic and Malignant Breast Tissues. Breast cancer. 2002;9(3):0–3.
57.
Ribeiro-silva A, Ramalho LNZ. The relationship between p63 and p53 expression in Normal and neoplastic breast tissue. Arch Pathol Lab Med. 2003;127(3):336–40.PubMed
58.
Makki J. Diversity of breast carcinoma: histological subtypes and clinical relevance. Clin Med Insights Pathol. 2015;8(1):23–31.PubMedPubMedCentral
59.
Kang KS, Morita I, Cruz A, Jeon YJ, Trosko JE, Chang CC. Expression of estrogen receptors in a normal human breast epithelial cell type with luminal and stem cell characteristics and its neoplastically transformed cell lines. Carcinogenesis. 1997;18(2):251–7.PubMed
60.
Jia M, Dahlman-Wright K, Gustafsson JÅ. Estrogen receptor alpha and beta in health and disease. Best Pract Res Clin Endocrinol Metab. 2015;29(4):557–68.PubMed
61.
Lindstro LS, Yau C, Czene K, Thompson CK, Hoadley KA, Van Veer LJ, et al. Intratumor heterogeneity of the estrogen receptor and the long-term risk of fatal breast Cancer. JNCI J Natl Cancer Inst. 2018;110(7):726–33.
62.
Lin C-Y, Ström A, Vega VB, Kong SL, Yeo AL, Thomsen JS, et al. Discovery of estrogen receptor alpha target genes and response elements in breast tumor cells. Genome Biol. 2004;5(9):R66.PubMedPubMedCentral
63.
Kim J, Lee J, Jang SY, Kim C, Choi Y, Kim A. Anticancer effect of metformin on estrogen receptor-positive and tamoxifen-resistant breast cancer cell lines. Oncol Rep. 2016;35(5):2553–60.PubMedPubMedCentral
64.
Fleury L, Gerus M, Lavigne AC, Richard-Foy H, Bystricky K. Eliminating epigenetic barriers induces transient hormone-regulated gene expression in estrogen receptor negative breast cancer cells. Oncogene. 2008;27(29):4075–85.PubMed
65.
Buchsbaum RJ, Oh SY. Breast cancer-associated fibroblasts: where we are and where we need to go. Cancers (Basel). 2016;8(2):1–19.
66.
Kurobe M, Furukawa S, Hayashi K. Synthesis and secretion of an epidermal growth factor (EGF) by human fibroblast cells in culture. Biochem Biophys Res Commun. 1985;131(3):1080–5.PubMed
67.
Majety M, Pradel LP, Gies M, Ries CH. Fibroblasts influence survival and therapeutic response in a 3D co-culture model. PLoS One. 2015;10(6):1–18.
Metadata
Title
A novel culture method that sustains ERα signaling in human breast cancer tissue microstructures
Authors
Ana Luísa Cartaxo
Marta F. Estrada
Giacomo Domenici
Ruben Roque
Fernanda Silva
Emilio J. Gualda
Pablo Loza-Alvarez
George Sflomos
Cathrin Brisken
Paula M. Alves
Saudade André
Catarina Brito
Publication date
01-12-2020
Publisher
BioMed Central
Published in
Journal of Experimental & Clinical Cancer Research / Issue 1/2020
Electronic ISSN: 1756-9966
DOI
https://doi.org/10.1186/s13046-020-01653-4

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