The need for complex 3D culture models to unravel novel pathways and identify accurate biomarkers in breast cancer☆
Graphical abstract
Introduction
Breast cancer is a heterogeneous disease, encompassing multiple entities with distinct biological and clinical features [1]. The massively parallel sequencing endeavors performed by The Cancer Genome Atlas (TCGA; www.cancergenome.nih.gov), the International Cancer Genome Consortium (ICGC [2]) and individual investigators have provided a comprehensive characterization of breast cancer mainly at the genomic, but also the transcriptomic and epigenomic level. The use of this technology has demonstrated that breast cancers harbor heterogeneous constellations of somatic mutations and only few highly recurrently mutated driver genes [3], [4], [5], [6]. In fact, at base-pair resolution each breast cancer appears to be unique in its repertoire of genetic aberrations [6]. Despite this genetic heterogeneity seen between breast cancers, it is important to note that the number of specific signaling pathways activated in each molecular subtype of the disease seems to be limited [5]. In addition, massively parallel sequencing analyses of breast cancers have revealed intra-tumor genetic heterogeneity in a substantial proportion of cases [4], [7], [8]. In fact, it is currently accepted that at least a subset of breast cancers are composed of mosaics of tumor cell clones, which in addition to the founder genetic events present in all cells, also display additional genomic alterations.
It has been posited that the microenvironment exerts selective pressures on cancer cells, such as during the metastatic process or when the environment is changed due to an external selective pressure, such as drug treatment. Indeed, there is burgeoning evidence stemming from sequencing endeavors to demonstrate that primary breast cancers and their matched distant metastases are distinct in their mutational landscapes, and enrichment of populations of cancer cells harboring specific genetic alterations in the primary versus the metastatic site and vice versa has been observed [9], [10]. Also drug treatment has been reported to result in the selection of subclones, present in varying frequencies in the primary tumors, harboring mutations conferring resistance to the therapeutic agent [11], [12], [13], [14].
The impact of environmental cues on cancer is not restricted to biological or exogenous bottlenecks as exemplified above. In fact, it is plausible that throughout tumorigenesis and tumor progression, the microenvironment plays a pivotal role, as cancer cells are exposed to local selective pressures stemming from the structural and cellular microenvironment. In fact, a tumor cell is not an island [15]; instead, breast cancer cells interact with each other and with their surrounding non-malignant cells, hormones, secreted factors and the extracellular matrix (ECM). These complex microenvironmental interactions and forces contribute profoundly to the behavior, phenotype and evolution of cancer cells. For example, in estrogen receptor-negative breast cancer, increased expression levels of immune response pathway genes or increased presence of lymphocytic infiltration have been shown by independent investigators and studies to be the strongest predictor of outcome and, potentially, of chemotherapy benefit [16], [17], [18], [19].
Given the genomic complexity of breast cancer, understanding the epistatic interactions between mutations, as well as their effects on tissue function and endocrine, paracrine and autocrine signaling is germane for the development and validation of prognostic and predictive strategies. Most studies investigating the effect of genetic/epigenetic aberrations in vitro on specific aspects of cellular processes such as transformation, proliferation or signaling have been performed in oversimplified model systems, not taking alterations in tissue architecture, cell–cell interactions, or cell–microenvironment interactions into account. The understanding of the functional consequences of specific repertoires of genomic aberrations on signaling and pathway dependencies within and between the cancers cells but also with their surrounding microenvironment require model systems that truly recapitulate the disease. To date, the vast majority of functional studies using cancer cell lines are performed in traditional monolayer cultures, however, and culture systems that fully mirror human breast cancer, primary and metastatic, and its diverse cellular microenvironment have yet to be developed further.
Here, we provide an overview of the three-dimensional (3D) cell culture models currently being employed for the study of breast cancer, including co-culture systems. In addition, we discuss how these models can be used for the dissection of cell–cell and cell–stroma interactions and of the role of specific genetic aberrations or signaling pathways in normal and malignant mammary epithelial cells.
Section snippets
Three-dimensional cell culture models
The acini (also called alveoli in breast) and ducts of the normal mammary gland are highly organized structures, with a central lumen lined by polarized luminal epithelial cells and surrounded by an outer layer of myoepithelial cells. The epithelium is separated from the surrounding stromal ECM and stromal cells by a basement membrane (BM) (reviewed in [20]). In contrast, in invasive breast cancer, the neoplastic epithelial cells are in direct contact with the stroma [20] comprised of stromal
Heterotypic three-dimensional cell culture models
The most common 3D cell culture systems discussed above use monocultures, i.e. only one cell type. To fully recapitulate the histological complexity of the normal breast and invasive breast cancers, not only extracellular matrices and scaffolds are required but also stromal cells [71], which interact physically or via paracrine signaling with the epithelial cells [72].
Introduction of myoepithelial cells to the 3D collagen cultures of luminal epithelial cells leads to bilayered acinar structures
Signaling pathways and complex biological models
Signaling pathways that function in parallel in cells growing on cell culture plastic become reciprocally integrated or reprogrammed when cultured in lrECM or with stromal cells. In our laboratory, in addition to the usual breast cancer cell lines and, we have utilized the HMT3522 breast tumor progression series with the nonmalignant (S1), pre-malignant (S2, S3) and malignant (T4-2) human breast epithelial cell lines derived from the reduction mammoplasty of a woman [99], [100]. When cultured
Biomarkers and complex biological models
Cancer cell lines grown in conventional monolayer cultures have been used to link pharmacological data with genomic information and helped identify rare genotypes associated with targeted therapy response [120], [121], [122]. Some of these genomic response predictors identified in vitro have been or are in the process of being translated into the clinic (e.g. [123], [124], [125]). These data suggest that monolayer cultures are powerful tools identifying those subsets of cancers whose
Conclusion
The use of 3D lrECM culture systems to model normal mammary epithelial morphogenesis, dissect pathways involved in breast cancer progression and assess the effects of potential oncogenes or tumor suppressor genes on the polarized acinar-like structures has proven extremely valuable. In fact, the matrix stiffness of commercially available lrECM seems to closely mirror that of normal breast tissue [55], and mouse mammary epithelial cells cultured in 3D lrECM gels were shown to respond to
Conflict of interest
The authors have no conflicting financial interests.
Acknowledgments
We thank JS Reis-Filho for critical reading of the manuscript. The work from MJB's laboratory is supported by the National Cancer Institute (awards R37CA064786, U01CA143233, U54CA112970 and U01CA169538); by the U.S. Department of Defense (W81XWH0810736); by grants from the U.S. Department of Energy, Office of Biological and Environmental Research (contract no. DE-AC0205CH1123), and by a grant from the Breast Cancer Research Foundation.
References (147)
- et al.
Gene expression profiling in breast cancer: classification, prognostication, and prediction
Lancet
(2011) - et al.
The life history of 21 breast cancers
Cell
(2012) - et al.
Preexistence and clonal selection of MET amplification in EGFR mutant NSCLC
Cancer Cell
(2010) The differentiated state of normal and malignant cells or how to define a “normal” cell in culture
Int. Rev. Cytol.
(1981)- et al.
Glucose metabolite patterns as markers of functional differentiation in freshly isolated and cultured mouse mammary epithelial cells
Exp. Cell Res.
(1981) - et al.
Primary culture of parenchymal liver cells on collagen membranes. Morphological and biochemical observations
Exp. Cell Res.
(1975) - et al.
Collagenous substrata regulate the nature and distribution of glycosaminoglycans produced by differentiated cultures of mouse mammary epithelial cells
Exp. Cell Res.
(1985) - et al.
From laminin to lamin: regulation of tissue-specific gene expression by the ECM
Trends Cell Biol.
(1995) - et al.
How does the extracellular matrix direct gene expression?
J. Theor. Biol.
(1982) - et al.
Casein gene expression in mouse mammary epithelial cell lines: dependence upon extracellular matrix and cell type
Exp. Cell Res.
(1987)
Identification of multiple active growth factors in basement membrane Matrigel suggests caution in interpretation of cellular activity related to extracellular matrix components
Exp. Cell Res.
Morphogenesis and oncogenesis of MCF-10A mammary epithelial acini grown in three-dimensional basement membrane cultures
Methods
The morphologies of breast cancer cell lines in three-dimensional assays correlate with their profiles of gene expression
Mol. Oncol.
Unraveling the microenvironmental influences on the normal mammary gland and breast cancer
Semin. Cancer Biol.
Cancer cell migration within 3D layer-by-layer microfabricated photocrosslinked PEG scaffolds with tunable stiffness
Biomaterials
Tensional homeostasis and the malignant phenotype
Cancer Cell
Modeling dynamic reciprocity: engineering three-dimensional culture models of breast architecture, function, and neoplastic transformation
Semin. Cancer Biol.
From 3D cell culture to organs-on-chips
Trends Cell Biol.
Patient-derived xenografts, the cancer stem cell paradigm, and cancer pathobiology in the 21st century
Lab. Invest.
Long-term expansion of epithelial organoids from human colon, adenoma, adenocarcinoma, and Barrett's epithelium
Gastroenterology
Tumors are unique organs defined by abnormal signaling and context
Semin. Cancer Biol.
Heterotypic signaling between epithelial tumor cells and fibroblasts in carcinoma formation
Exp. Cell Res.
Effect of androgens on different breast cancer cells co-cultured with or without breast adipose fibroblasts
J. Steroid Biochem. Mol. Biol.
A complex 3D human tissue culture system based on mammary stromal cells and silk scaffolds for modeling breast morphogenesis and function
Biomaterials
Engineering systems for the generation of patterned co-cultures for controlling cell–cell interactions
Biochim. Biophys. Acta
International network of cancer genome projects
Nature
Diverse somatic mutation patterns and pathway alterations in human cancers
Nature
The clonal and mutational evolution spectrum of primary triple-negative breast cancers
Nature
The Cancer Genome Atlas. Comprehensive molecular portraits of human breast tumours
Nature
The landscape of cancer genes and mutational processes in breast cancer
Nature
Tumour evolution inferred by single-cell sequencing
Nature
Genome remodelling in a basal-like breast cancer metastasis and xenograft
Nature
Mutational evolution in a lobular breast tumour profiled at single nucleotide resolution
Nature
Emergence of KRAS mutations and acquired resistance to anti-EGFR therapy in colorectal cancer
Nature
Whole-genome analysis informs breast cancer response to aromatase inhibition
Nature
The molecular evolution of acquired resistance to targeted EGFR blockade in colorectal cancers
Nature
An immune response gene expression module identifies a good prognosis subtype in estrogen receptor negative breast cancer
Genome Biol.
Tumor-associated lymphocytes as an independent predictor of response to neoadjuvant chemotherapy in breast cancer
J. Clin. Oncol.
Tumor-infiltrating CD8 + lymphocytes predict clinical outcome in breast cancer
J. Clin. Oncol.
Prognostic and predictive value of tumor-infiltrating lymphocytes in a phase III randomized adjuvant breast cancer trial in node-positive breast cancer comparing the addition of docetaxel to doxorubicin with doxorubicin-based chemotherapy: BIG 02-98
J. Clin. Oncol.
Cellular changes involved in conversion of normal to malignant breast: importance of the stromal reaction
Physiol. Rev.
Distribution of myoepithelial cells and basement membrane proteins in the normal breast and in benign and malignant breast diseases
Cancer Res.
Host microenvironment in breast cancer development: extracellular matrix-stromal cell contribution to neoplastic phenotype of epithelial cells in the breast
Breast Cancer Res.
Distinct role of macrophages in different tumor microenvironments
Cancer Res.
Paradoxical roles of the immune system during cancer development
Nat. Rev. Cancer
Collagen substrata for studies on cell behavior
J. Cell Biol.
Maintenance and induction of morphological differentiation in dissociated mammary epithelium on floating collagen membranes
In Vitro
Interaction of mouse mammary epithelial cells with collagen substrata: regulation of casein gene expression and secretion
Proc. Natl. Acad. Sci. U.S.A.
Expression of extracellular matrix components is regulated by substratum
J. Cell Biol.
Functional structure and composition of the extracellular matrix
J. Pathol.
Cited by (272)
Beyond the surface: Investigation of tumorsphere morphology using volume electron microscopy
2023, Journal of Structural BiologyPolyethyleneimine-decorated graphene oxide quantum dot as a carrier for suicide gene delivery to the breast cancer three-dimensional model
2023, Journal of Drug Delivery Science and TechnologyThe role of extracellular vesicles in non-small-cell lung cancer, the unknowns, and how new approach methodologies can support new knowledge generation in the field
2023, European Journal of Pharmaceutical SciencesComparative evaluation of the therapeutic strategies using a minimal model of luminal-A breast cancer
2023, Biochemical and Biophysical Research CommunicationsSynthesis, biological evaluation of novel iridium(III) complexes targeting mitochondria toward melanoma B16 cells
2023, European Journal of Medicinal Chemistry
- ☆
This review is part of the Advanced Drug Delivery Reviews theme issue on “Innovative tissue models for drug discovery and development”.