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. 2013 Sep 27:14:43.
doi: 10.1186/1471-2121-14-43.

In vitro three-dimensional modeling of fallopian tube secretory epithelial cells

Kate Lawrenson  1 , Maria Notaridou, Nathan Lee, Elizabeth Benjamin, Ian J Jacobs, Christopher Jones, Simon A Gayther
Affiliations

In vitro three-dimensional modeling of fallopian tube secretory epithelial cells

Kate Lawrenson et al. BMC Cell Biol. .

Abstract

Background: Fallopian tube secretory epithelial cells (FTSECs) have been implicated as a cell-of-origin for high-grade serous epithelial ovarian cancer. However, there are relatively few in vitro models of this tissue type available for use in studies of FTSEC biology and malignant transformation. In vitro three-dimensional (3D) cell culture models aim to recreate the architecture and geometry of tissues in vivo and restore the complex network of cell-cell/cell-matrix interactions that occur throughout the surface of the cell membrane.

Results: We have established and characterized 3D spheroid culture models of primary FTSECs. FTSEC spheroids contain central cores of hyaline matrix surrounded by mono- or multi-layer epithelial sheets. We found that 3D culturing alters the molecular characteristics of FTSECs compared to 2D cultures of the same cells. Gene expression profiling identified more than a thousand differentially expressed genes between 3D and 2D cultures of the same FTSEC lines. Pathways significantly under-represented in 3D FTSEC cultures were associated with cell cycle progression and DNA replication. This was also reflected in the reduced proliferative indices observed in 3D spheroids stained for the proliferation marker MIB1. Comparisons with gene expression profiles of fresh fallopian tube tissues revealed that 2D FTSEC cultures clustered with follicular phase tubal epithelium, whereas 3D FTSEC cultures clustered with luteal phase samples.

Conclusions: This 3D model of fallopian tube secretory epithelial cells will advance our ability to study the underlying biology and etiology of fallopian tube tissues and the pathogenesis of high-grade serous epithelial ovarian cancer.

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Figures

Figure 1
Figure 1
Characterization of primary FTSEC in vitro cultures. (a) Primary FTSEC cultures maintain expression of markers expressed by fallopian tube secretory epithelia in vivo (arrowheads indicate secretory, non-ciliated cells), the FTSEC cultures express cytokeratins, CA125, basal laminin, vimentin and nuclear PAX8. 90-100% of cells stain positive for PAX8 suggesting fallopian tube ciliated epithelial cells do not proliferate in vitro. Green stain shows positive staining, nuclei are counterstained with DAPI (blue), except in the case of PAX8. (b) Growth curves. FTSECs have a limited in vitro lifespan, as is typical for primary normal cells. (c) Modal karyotypes for 5/6 primary FTSEC cultures were normal and female (46,XX). # Two cells or fewer showed unbalanced structural chromosomal rearrangements or numerical abnormalities.
Figure 2
Figure 2
Histological analysis of 3D FTSEC spheroids. (a) Three dimensional spheroid cultures stained by hematoxylin and eosin. At (i) 15 days, the core of the spheroids contains abundant matrix material (*), covered by an epithelial cell monolayer (arrow) or cellular cap structure (arrowhead). (ii) After 40 days no viable cells remain within the core of the spheroids. A viable monolayer covers the surface of the spheroid (arrow), degenerate nuclear debris can be seen within the matrix core (arrowhead). (b) Electron microscopy of FTSEC spheroids. Features of epithelial cells in vivo are detected in 3D cultured FTSECs including (i) microvilli and cell-cell junction complexes including (ii,iii) tight junctions (white arrows and inset) and (iv) adherens junctions (white arrows and inset). Electron microscopy.
Figure 3
Figure 3
Immunohistochemical staining of 2D and 3D cultured FTSECs and primary fallopian tube tissue. Immunohistochemical staining of p53 (clone Do7) and a proliferation marker, MIB1, shows high expression of both markers in 2D cultured cells but low expression in 3D FTSEC cultures (at day 15), and fallopian tube epithelia in vivo. Laminin and vimentin are expressed in 2D cultured cells, 3D cultured cells and in vivo. Expression of fibronectin and collagen I was lower in 2D cultured cells than 3D cultures and fallopian tube epithelia in fresh tissue specimens. Collagen IV is predominantly expressed in the basal lamina (arrow) and stroma of fallopian tubes but was expressed at high levels by 2D cultured cells. Expression of this marker was low/absent in 3D cultured cells. * Fibronectin, collagen I and collagen IV were also examined in FTSEC01, expression patterns were highly similar to data for FTSEC283 and FTSEC03. Expression of collagen IV was restricted to the basal lamina and stroma. None of the markers examined were differentially expressed by ciliated and secretory fallopian tube epithelial cells in vivo. As our cell isolates were predominantly PAX8 positive (Figure 1), we do not expect ciliated cells to be present in the FTSEC spheroid cultures. Brown stain denotes positive antigen detection, cells are counterstained with eosin (blue). Light microscopy. Scale bars = 100 μm.
Figure 4
Figure 4
Gene expression microarray analysis of 2D and 3D cultured FTSECs. Hierarchical clustering of transcriptomic profiles of the 100 genes showing the most significant changes in expression between three 2D and 3D cultured FTSEC primary lines. Three independent samples were prepared for each cell line and each culture condition, each sample represents an individual microarray, technical replicates are shown. Cell lines cluster according to culture conditions, not patient.
Figure 5
Figure 5
Validation of genes identified through expression microarray profiling of 2D and 3D cultured FTSECs. The most significantly changing genes were validated by TaqMan qPCR in independent 2D and 3D cultures of FTSEC03, as well as in two additional cell lines from different patients (FTSEC01 and FTSEC284). Genes downregulated in 3D cultured cells (a)MARCH4 and (b)DIAPH3 were significantly downregulated in all 3 cell lines tested with the exception. (c) A similar trend was observed for GINS4 although this did not reach statistical significance. Genes upregulated in 3D cultured cells (d)C11orf96, (e)OLFM2A and (f)LRRK2 were all upregulated in all 3 samples following a transition to a 3D microenvironment. * P > 0.05, ** P > 0.01, *** P > 0.001, two-tailed paired Student’s T-tests were used to compare 2D and 3D cultured cells for each cell line individually.
Figure 6
Figure 6
FTSEC biomarker expression in 2D and 3D and cluster analysis comparing genome-wide transcriptomic profiles of 2D and 3D cultured FTSECs to follicular and luteal phase fallopian tube epithelium. (a) Expression of FTSEC biomarkers is typically higher in 3D than in 2D (i) oviductal glycoprotein 1, (ii) pregnanacy-associated plasma protein A and (iii) tissue factor pathway inhibitor 2. (b) Euclidean Pearson clustering analyses reveal that 2D cultured FTSECs cluster with follicular phase fallopian tube epithelium, whereas 3D cultured fallopian tube epithelial cells cluster with luteal phase tubal epithelium. Each point on the graph indicates an individual microarray profile, technical replicates of cultured cells are shown by colored circles, open circles denoting 2D cultured cells and closed circles denoting 3D cultured FTSECs. Black open circles indicate follicular phase fallopian tube epithelial samples, closed circles indicate luteal phase fallopian tube epithelium. Each point represents an individual patient. All patients from Georges et al. and Tone et al. [17,18] datasets are shown.
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