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. 2016 Mar;238(4):519-530.
doi: 10.1002/path.4649. Epub 2016 Jan 9.

In vitro and in vivo correlates of physiological and neoplastic human Fallopian tube stem cells

Yusuke Yamamoto #  1 , Gang Ning #  1 , Brooke E Howitt  2 , Karishma Mehra  2 , Lingyan Wu  3 , Xia Wang  1 , Yue Hong  1 , Florian Kern  3 , Tay Seok Wei  3 , Ting Zhang  3 , Niranjan Nagarajan  3 , Debargha Basuli  4 , Suzy Torti  4 , Molly Brewer  5 , Mahesh Choolani  6 , Frank McKeon  1   3   7   8   9 , Christopher P Crum  2 , Wa Xian  1   2   7   10   11
Affiliations

In vitro and in vivo correlates of physiological and neoplastic human Fallopian tube stem cells

Yusuke Yamamoto et al. J Pathol. 2016 Mar.

Abstract

High-grade serous cancer (HGSC) progresses to advanced stages without symptoms and the 5-year survival rate is a dismal 30%. Recent studies of ovaries and Fallopian tubes in patients with BRCA1 or BRCA2 mutations have documented a pre-metastatic intramucosal neoplasm that is found almost exclusively in the Fallopian tube, termed 'serous tubal intraepithelial carcinoma' or STIC. Moreover, other proliferations, termed p53 signatures, secretory cell outgrowths (SCOUTs), and lower-grade serous tubal intraepithelial neoplasms (STINs) fall short of STIC but share similar alterations in expression, in keeping with an underpinning of genomic disturbances involved in, or occurring in parallel with, serous carcinogenesis. To gain insight into the cellular origins of this unique tubal pathway to high-grade serous cancer, we cloned and both immortalized and transformed Fallopian tube stem cells (FTSCs). We demonstrated that pedigrees of FTSCs were capable of multipotent differentiation and that the tumours derived from transformed FTSCs shared the histological and molecular features of HGSC. We also demonstrated that altered expression of some biomarkers seen in transformed FTSCs and HGSCs (stathmin, EZH2, CXCR4, CXCL12, and FOXM1) could be seen as well in immortalized cells and their in vivo counterparts SCOUTs and STINs. Thus, a whole-genome transcriptome analysis comparing FTSCs, immortalized FTSCs, and transformed FTSCs showed a clear molecular progression sequence that is recapitulated by the spectrum of accumulated perturbations characterizing the range of proliferations seen in vivo. Biomarkers unique to STIC relative to normal tubal epithelium provide a basis for novel detection approaches to early HGSC, but must be viewed critically given their potential expression in lesser proliferations. Perturbations shared by both immortalized and transformed FTSCs may provide unique early targets for prevention strategies. Central to these efforts has been the ability to clone and perpetuate multipotent FTSCs.

Keywords: Fallopian tubes; cell culture; neoplasia; ovary.

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Figures

Figure 1
Figure 1
Cloning, immortalization, and transformation of the Fallopian tube stem cells. (a) Cloned FTSCs with proliferation marker Ki67 (green) and ciliated marker FOXJ1 (red). Scale bar = 50 μm. (b) ALI differentiation culture of FTSCs stained with FOXJ1 (red) and acetylated tubulin (green). Scale bar = 25 μm. (c) RT-PCR of selected markers (n = 2; error bars, SD) (d) Heatmap of selected genes from whole-genome transcriptome analysis. (e) Schematic of FTSC immortalization and transformation in vitro. (f) Morphology of immortalized (FTSCi) and transformed FT stem cells (FTSCt) on plastic culture dishes and in 3D Matrigel assay. Scale bar = 25 μm. (g) Progressive change of gene expression among FTSCs, FTSCi, and FTSCt (n = 2 each). Genes with increased expression (> 1.5-fold and p < 0.05, 654 genes) following transformation were selected for heatmap production.
Figure 2
Figure 2
FTSCt xenograft tumour resembles human high-grade serous cancer. (a) Upper panel: 2000 FTSCt cells (PAX8, red) were injected into NSG mice and palpable tumour was observed at 2 weeks. Lower panel: xenograft tumour expressed HGSC hallmarks MUC4, p53, and PAX8. Scale bar = 50 μm. (b) Heatmap showing that FTSCt xenograft tumours and invasive SC share similar gene expression profiles (FTSCt tumour: n = 3; invasive SC: n = 10; and paired normal oviduct: n = 10; 2395 genes selected, > 2-fold and p < 0.05). (c) EZH2 protein in multiple stages of HGSC development. Scale bar = 1 mm. (d) EZH2 target genes in FTSCt xenograft tumours and invasive SC (n > 3; error bars, SD).
Figure 3
Figure 3
Molecular correlates of progression from STIC to invasive cancer. (a) Left: histology of the sections used for laser captured microdissection (LCM) of normal Fallopian tube epithelium, STIC, and invasive cancer. Right: p53 antibody staining showing high levels in STIC and invasive cancer. Scale bar = 1 mm. (b) Heatmaps showing progressive gene expression from STIC to invasive cancer in six individual patients (genes differentially expressed in invasive cancer compared with normal FT epithelium were selected, > 2-fold and p < 0.05). (c) Gene set enrichment analysis (GSEA) of invasive cancer versus STIC highlighting angiogenesis and regulation of cell adhesion in invasive cancer. (d) Plots of selected genes highly expressed in STIC and invasive cancer (normal Fallopian tube: n = 6; STIC: n = 6; invasive SC: n = 6; error bars: SD).
Figure 4
Figure 4
Early molecular changes associated with FTSC immortalization and STIC. (a) Heatmap showing 62 genes (> 2-fold, p < 0.05) commonly overexpressed between STIC and matched invasive serous cancer. (b) Representative images of CCNE1 and PTTG1 immunostaining on normal FT epithelium, STIC, and invasive serous cancer. Scale bar = 1 mm. (c) Venn diagram of genes overexpressed in STIC (> 1.5-fold, p < 0.05) and immortalized FTSCs (> 2-fold, p < 0.05). (d) Selected overlapping genes and fold change. n = 2; error bars: SD.
Figure 5
Figure 5
In vitro and in vivo correlations proposing a model of multi-step development of HGSC originating from Fallopian tube stem cells.
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