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Review
. 2014 Jul 10;33(28):3619-33.
doi: 10.1038/onc.2013.321. Epub 2013 Aug 12.

Epithelial ovarian cancer experimental models

E Lengyel  1 , J E Burdette  2 , H A Kenny  1 , D Matei  3 , J Pilrose  4 , P Haluska  5 , K P Nephew  4 , D B Hales  6 , M S Stack  7
Affiliations
Review

Epithelial ovarian cancer experimental models

E Lengyel et al. Oncogene. .

Abstract

Epithelial ovarian cancer (OvCa) is associated with high mortality and, as the majority (>75%) of women with OvCa have metastatic disease at the time of diagnosis, rates of survival have not changed appreciably over 30 years. A mechanistic understanding of OvCa initiation and progression is hindered by the complexity of genetic and/or environmental initiating events and lack of clarity regarding the cell(s) or tissue(s) of origin. Metastasis of OvCa involves direct extension or exfoliation of cells and cellular aggregates into the peritoneal cavity, survival of matrix-detached cells in a complex ascites fluid phase and subsequent adhesion to the mesothelium lining covering abdominal organs to establish secondary lesions containing host stromal and inflammatory components. Development of experimental models to recapitulate this unique mechanism of metastasis presents a remarkable scientific challenge, and many approaches used to study other solid tumors (for example, lung, colon and breast) are not transferable to OvCa research given the distinct metastasis pattern and unique tumor microenvironment (TME). This review will discuss recent progress in the development and refinement of experimental models to study OvCa. Novel cellular, three-dimensional organotypic, and ex vivo models are considered and the current in vivo models summarized. The review critically evaluates currently available genetic mouse models of OvCa, the emergence of xenopatients and the utility of the hen model to study OvCa prevention, tumorigenesis, metastasis and chemoresistance. As these new approaches more accurately recapitulate the complex TME, it is predicted that new opportunities for enhanced understanding of disease progression, metastasis and therapeutic response will emerge VSports手机版. .

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Figures

FIGURE 1
FIGURE 1. Three-dimensional organotypic and ex vivo models of ovarian cancer metastasis
A) Three-dimensional (3D) culture models of cellular contact with sub-mesothelial collagen. (a) Following initial intra-peritoneal adhesion, mesothelial cell retraction exposes the sub-mesothelial interstitial collagen-rich matrix to which ovarian cancer (OvCa) cells avidly adhere. Scanning electron micrograph of murine submesothelial collagen matrix. (b) Initial multivalent cell-matrix contact of OvCa cell (left) with collagen (right) is visualized by SEM. (c) Culturing cells atop 3D collagen (as in b) followed by cDNA microarray analysis can reveal changes in gene expression that result from the initial adhesive contact. (d) Metastasizing cells migrate into the 3D collagen matrix to anchor metastatic lesions. Confocal reflectance microscopy overlaid with fluorescence microscopy visualizes cells within the 3D matrix. (e) Cells seeded into a 3D matrix proliferate in a matrix metalloproteinase-dependent manner to form expansive matrix-anchored multicellular aggregates. Micrographs shown in a, b are courtesy of Yueying Liu, University of Notre Dame and Dr. Katarina Wolf, Radboud University Nijmegen, The Netherlands. Photographs and micrographs shown in e are courtesy of Dr. Natalie Moss, Northwestern University. B) Multicellular aggregate (MCA) cultures mimic non-adherent OvCa cells in ascites. (a) To generate MCA cultures, cells are suspended at a concentration of 100,000 cells/ml and seeded as 20 μl droplets on the inner surface of a tissue culture dish lid. After addition of PBS to the culture dish to maintain humidity, the lid is gently inverted. Following aggregation, individual MCAs may be subcultured for use in additional assays. (b) Light micrograph of individual MCA generated using DOV13 cells. (c) Dispersal of MCA generated from DOV13 cells. An individual MCA was subcultured onto a coverslip coated with type I collagen and photographed after 12 hours. Dispersal can be quantified by DAPI staining and measuring inter-nuclear distance. (d) Fluorescence micrograph of individual MCA generated using CellTracker Red-labeled DOV13 cells. (Scale bar 30 μm) (e) Scanning electron micrograph of MCA generated using DOV13 cells. MCAs were placed in primary fixative (2% glutaraldehyde, 2% paraformaldehyde in 0.1 M Cacodylate buffer pH 7.35) followed by fixation with osmium tetroxide, dehydration in ethanol, and critical point drying. Platinum coated samples were examined using a Hitachi S-4700 Field Emission Scanning Electron Microscope (scale bar 50 μm). (f) Transmission electron micrograph of MCA generated using DOV13 cells. Following primary fixation as described in e, samples were encapsulated in HistoGel, fixed with osmium tetroxide, dehydrated in ethanol and acetone, infiltrated with Epon/Spurr’s resin and cut into ultrathin sections. Sections were mounted on nickel grids, stained with uranyl acetate and Sato’s Triple Lead stain and examined using a JEOL 1400 transmission electron microscope (scale bar 10 μm). Micrographs shown are courtesy of Yuliya Klymenko, University of Notre Dame. C) Ovarian cancer in vitro models recapitulate peritoneal metastasis. (a) Ovarian cancer growing as spheroids in a 3D matrix. (b) A 3D peritoneal culture using primary human peritoneal mesothelial cells and extracellular matrix to investigate OvCa cell adhesion. (c) A 3D organotypic “meso-mimetic” culture of the peritoneal cavity. Primary human omental fibroblasts are embedded in extracellular matrix and a layer of primary human omental mesothelial cells plated on top. Ovarian cancer cells are added to the culture and adhesion, invasion, and proliferation of the cancer cells is investigated. Using fluorescently-labeled OvCa cells, changes in gene and protein expression can be individually evaluated in the cancer and stromal cells. D) Ex vivo peritoneal explant model of early events in intra-peritoneal adhesion. (a) Optically clear silastic resin is generated using a Sylgard® 184 silicone elastomer kit (Fisher), using approximately 6 ml per well of a 6-well culture plate. Dissected murine peritoneal tissue obtained from a midline incision is pinned mesothelial-side up to the silastic resin and immersed in PBS. Tissue integrity is maintained for up to 48 h. Addition of fluorescently tagged tumor cells to the explant enables monitoring by (b) relative fluorescence of cell lysate, (c) fluorescence microscopy of frozen sections, (d) confocal microscopy, or (e) scanning electron microscopy. Beneath the tumor cells in (e, round), the cobblestone mesothelium is visible. Note that images in panels (b–d) are representative of a 2 h time point following addition of OvCa cells to the explant. Photographs and micrographs shown are courtesy of Yueying Liu, University of Notre Dame.
FIGURE 2
FIGURE 2. Cell line-based and patient-derived in vivo xenograft models
A) Optical imaging of disseminated i.p. metastasis. (a) SKOV3ip.1 cells were transduced using a lentiviral vector to express red fluorescent protein (RFP), followed by i.p. injection (5×106 cells) into nude mice. After 24 days, mice were sacrificed, the peritoneal cavity opened and metastatic nodules visually enumerated. (b) Fluorescent images of peritoneal organs in situ were taken using a Xenogen IVIS Lumina imaging system. (c) Dissected organs were imaged using a Xenogen IVIS Lumina system. Yellow arrow indicates dissected omentum. Photographs shown are courtesy of Yueying Liu, University of Notre Dame. B) Distinct in vivo growth patterns of ovarian cancer cells mimic human metastatic dissemination. (a) OVCAR-5 cells (1×106) were injected i.p. into nude mice. After 45 days, mice were sacrificed. Blue lines outline widely disseminated growth of the xenograft as small peritoneal nodules. (b) HeyA8 cells (1×106) were injected i.p. into nude mice. After 28 days, mice were sacrificed. Blue lines outline the growth of large tumors. (c) HeyA8 cells (0.5×105) expressing luciferase were injected s.c. into the mouse flank in the absence (blue circle) or presence (red circle) of carcinoma-associated fibroblasts (CAFs, 1×105). After 14 days, mice were subjected to longitudinal in vivo bioluminescence imaging using the Xenogen IVIS 200 Imaging System. Photographs shown are courtesy of Dr. Marion Zillhardt, University of Chicago. C) Histological comparison of patient tumor and “xenopatient” graft in the mouse. Following surgical removal of a serous ovarian tumor, a 1 cm fragment was finely minced and injected i.p. into SCID/Beige mice. At about 20 weeks, mice were sacrificed and tumors collected. Mouse tumor grafts (a,c,e) and patient tumors (b,d,f) were subjected to staining with hematoxylin and eosin (H&E, a,b), pan-cytokeratin (c,d), or Ki67 (e,f), as indicated. Photographs shown are courtesy of Saravut John Weroha, Mayo Clinic. D) Formation of ovarian tumors in LSL-KRasG12D; Ptenfl/fl mice. Tumors were initiated by injection of adenovirus expressing cre recombinase in the right ovarian bursa. The left ovary was not injected and served as an internal control. Mice were sacrificed after 8 weeks and the primary tumor was excised (a), embedded in paraffin, and subjected to staining with H&E (b). Photographs shown are courtesy of Dr. Iris Romero, University of Chicago.
FIGURE 3
FIGURE 3. The domestic egg laying hen model of ovarian cancer
A) Normal ovary of a domestic laying hen. The ovary contains a set of 4 large pre-ovulatory hierarchical follicles, small developing follicles, and a post-ovulatory follicle. B) Primary malignant ovarian tumor in laying hen with stage IV OvCa. The tumor was classified as a serous OvCa on an endometrioid background. The tumor has metastasized to distant organs with profuse ascites and had an endometrioid histotype. Multiple solid tumor masses are seen. C) Histological types of malignant ovarian tumors in hens. (a) Ovarian serous carcinoma showing sheets of lacelike papillary folding and cells with large pleomorphic nuclei with mitotic bodies. (b) Ovarian endometrioid carcinoma with confluent back-to-back glands. Glands contain a single layer of epithelial cells with sharp luminal margin. (c) Ovarian mucinous carcinoma with crowded glands in clusters without intervening stroma surrounded by a fibromuscular layer. The epithelium contains columnar and intercalated ciliated goblet cells. The nuclei are separated from the basement membrane and have moved toward the apical surface with occasional stratification and luminal secretion. (d) Poorly differentiated ovarian clear cell carcinoma showing vacuolated cells containing high-grade nuclear atypia that invade the stroma and theca layer of stromal follicles. Deposition of eosinophilic hyalinized matrix in the stroma and necrotic bodies are also seen. Hematoxylin and eosin staining (original magnification 40X). D) Immunohistochemical localization of E-cadherin expression in human (a,b) and laying hen (c,d) normal ovarian and cancerous tissue. The uninvolved epithelial cells in normal human ovarian tissue (a) and in normal laying hen ovarian tissue (c) lack E-cadherin, while early neoplastic transformation (black arrow) of adjacent ‘normal’ ovarian surface epithelial cells and mild to moderate dysplastic cells (white arrow) are marked by an increase in E-cadherin expression. Tumor cells of a human endometrioid type tumor (b) and a primary hen tumor (d) express high E-cadherin levels in distinct patterns throughout the tissue (bar= 50μm).
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