Figure 1.
Gross morphology and histology of normal and cancerous chicken ovaries. A.
Normal ovary containing a hierarchy of pre-ovulatory follicles (F1–5), pre-hierarchical follicles (SYF), and post-ovulatory follicle (POF). B. Tumor mass (arrow) and a few atretic pre-ovulatory follicles (At). C. Abdominal viscera showing tumors nodules on intestinal wall and peritoneum (arrow heads) and a few ovarian follicles (OF). D-I. Photomicrographs of hematoxylin and eosin stained ovarian tissue sections. D. Normal ovarian stroma containing several undifferentiated follicles. E–I. Cancerous ovarian tissue section showing coarse fibrous stroma containing several acini and ducts (arrows heads; E), multiple cysts comprised of anaplastic cells (F), cystic spaces containing papillary projections of neoplastic cells (G), dyscohesive cells (H), and tumor-cell emboli in arterial lumen (I).
Table 1.
Characteristics of chickens with Stage III or IV ovarian tumor and associated chicken ovarian cancer (COVCAR) cell lines.
Figure 2.
Ascites-derived chicken ovarian cancer (COVCAR) cells in culture.
COVCAR cells were harvested from ascites and cultured as described in Materials and Methods section. Notice several translucent vesicles in the cytoplasm and spherical shape of the cells (A, B, C, and D). A few large spheroid mass of COVCAR cells were noticed as shown in A (arrow). Many cells had papilla-like or microvilli-like projections on the cell surface (C). Notice fibroelastic transformation of spherical COVCAR cells (D). E-F. Multiple layers of fully confluent COVCAR cells appearing as an interwoven mat. A few floating cells as shown in F (arrows) displayed translucent vesicles in the cytoplasm. G. COVCAR cells appearing as a network of tube-like structure. H. A few senescent cells showing cellular hypertrophy and stellate appearance.
Figure 3.
Anchorage-independent growth of chicken ovarian cancer (COVCAR) and normal ovarian surface epithelial (NOSE) cells.
COVCAR and NOSE cells were cultured in semi-solid media for 4 weeks as described in Materials and Methods section. A. NOSE cells remaining as single cells showing no detectable changes in morphology. B. COVCAR cells on day 6 of culture showing numerous projections on the surface and forming solid ball-like structure. C-D. Multiple colonies and a large sphere-like structure of COVCAR cells on day 11 of culture. E. Two acini composed of COVCAR cells on day 13 of culture. F. Proliferating COVCAR cells invading through agarose. G. A large sphere of COVCAR cells and several tube-like structures. Scale bars = 100 µm.
Figure 4.
Invasion of chicken ovarian cancer (COVCAR) cells in Matrigel extracellular matrix.
COVCAR cell lines (C5, C6, C11, and C19) and normal ovarian surface epithelial cells (NOSE) were layered on top of solidified Matrigel in transmembrane insert having 8 µm pores and cultured for 24 h as described in Materials and Methods section. The number of cells invaded through the Matrigel and pores (arrow heads in A and B) were counted in six fields after staining the bottom surface of the membrane. The histogram (top left) showing the number of COVCAR and NOSE (N) cells found on the bottom surface of the membrane. A–B. Representative photomicrographs of the membrane bottom surface showing many COVCAR cells (B) while NOSE cells were absent (A). C–F. Photomicrographs of COVCAR cells in Matrigel extracellular matrix. COVCAR cells formed sphere-like structures in Matrigel (C, D) that also led to outgrowth of cells (F) invading through the matrix. Stacks of COVCAR cells invading through the Matrigel matrix appeared as a rope-like structure (E). Numerous spherical COVCAR cells containing translucent vesicles (G and H) were found in the cell culture well beneath the Matrigel transmembrane insert. Many of these spherical COVCAR cells that migrated through the pores formed a layer of fibroelastic cells at the bottom of the well. Data in the histogram are represented as mean ± standard error of the mean from 3 replicates. +P<0.01, P≤0.0001 COVCAR vs NOSE.
Figure 5.
Migration and wound healing properties of chicken ovarian cancer (COVCAR) cells.
A vertical wound (700–900 µm width) was created in fully-confluent COVCAR cells followed by washing in culture medium and incubation at 37°C and 5% CO2. Photomicrographs were taken at 0, 8, 16 and 24 h post-wound creation to determine the wound area using image analysis software. A–D. Representative photomicrographs of the wound at 0, 8, 16, and 24 h after creating wound in C11 cell line in 4th passage. E–H. Wound area following 0, 8, 16, and 24 h in COVCAR cell lines C5 (E), C6 (F), C11 (G), and C19 (H). Data are represented as mean ± standard error of the mean from 3 replicates., P≤0.0001 COVCAR vs NOSE.
Figure 6.
Expression of cytoskeletal proteins in chicken ovarian cancer (COVCAR) cells.
Photomicrographs of chicken ovarian cancer (COVCAR) cells showing E-cadherin (A), α-smooth muscle actin (C; SMA), and cytokeratin (E) immunostaining. Paraformaldehyde-fixed COVCAR cells were immunostained as described in Materials and Methods section. Fixed COVCAR cells were incubated with anti-mouse IgG (G) in place of primary antibody as negative control. Nuclei were visualized with DAPI staining (B, D, F, and H) on cells immunostained with E-cadherin, SMA, or cytokeratin, respectively. Scale bars-10 µm.
Figure 7.
Expression of genes in chicken ovarian cancer cells (COVCAR) and normal ovarian surface epithelial cells (NOSE).
RT-PCR analyses for expression of various cytoskeletal proteins, growth factors and receptors, protein/enzymes related to steroid hormone synthesis, gonadal hormone and hormone receptors in chicken ovarian cancer cell lines (C5, C6, C7, C11, C19) and normal ovarian surface epithelial cells (NOSE; n = 5 animals). Total RNA was extracted from cultured cells in passages 3–4 and treated with deoxyribonuclease-I. Approximately 250 ng of cDNA (+RT) was used as template to amplify the gene products. Contamination controls consisted of reverse transcribed RNA without reverse transcriptase (-RT). M- DNA size marker.
Table 2.
Expression of selected gene transcripts in chicken ovarian cancer (COVCAR) cell lines and normal ovarian surface epithelial (NOSE) cells and sequences of the oligonucleotide primers used for amplification.
Figure 8.
Quantification of vimentin, N-cadherin, cytokeratin, ZEB1, and VEGF mRNA in chicken ovarian cancer (COVCAR) cells.
Vimentin mRNA (A), N-cadherin mRNA (B), cytokeratin mRNA (C), ZEB1 mRNA (D), and VEGF mRNA (E) abundance in normal ovarian surface epithelial cells (N; n = 5 animals) and COVCAR cell lines (C5, C6, C7, C11, C19). Total RNA was extracted from cultured cells in passages 3–4 and treated with deoxyribonuclease-I. Following reverse transcription, approximately 50 ng of cDNA was used in quantitative real-time PCR using SYBR® green as the dye to quantify vimentin mRNA, N-cadherin mRNA, cytokeratin mRNA, ZEB1 mRNA, VEGF mRNA, or β-actin mRNA in separate reactions. Each reaction was run in triplicate per cell line and the critical threshold (CT) values were subtracted from that of β-actin mRNA, averaged and converted from log-linear to linear term. *P<0.05, +P<0.01, P≤0.0001 COVCAR vs NOSE; n = 3/cell line.
Figure 9.
Quantification of E-cadherin in cancerous ovaries and chicken ovarian cancer (COVCAR) cells.
Western blot analysis to determine the levels of E-cadherin (A, C) and cleavage product of E-cadherin (cE-cadherin; B, D) in ovaries (A and B) or in ovarian cancer cell lines (C and D). Protein extracts from cancerous ovaries or chicken ovarian cancer cell lines (COVCAR; C5, C6, C7, C11, and C19) and normal ovaries (NO; n = 5 animals) or ovarian surface epithelial cells (N; n = 5 animals) were treated with reducing agent, heat denatured, separated by electrophoresis and blotted onto PVDF membrane. E-cadherin and cE-cadherin were detected by immunostaining using mouse anti-human E-cadherin antibody. E-cadherin or cE-cadherin levels were represented as a proportion of a-tubulin levels. Data are represented as mean ± standard error of the mean from at least 3 replicates. *P<0.05, +P<0.01 Cancerous vs Normal or COVCAR vs NOSE.
Figure 10.
Ovalbumin expression in cancerous ovaries and chicken ovarian cancer (COVCAR) cells. A.
Western blot analysis of ovalbumin levels in cancerous (C5, C6, C7, C11, C19) and normal ovaries (N; n = 5 animals) of the chicken. Protein extracts from normal and cancerous ovaries were treated with reducing agent, heat denatured, separated by electrophoresis and blotted onto PVDF membrane. Ovalbumin was detected by immunostaining using mouse anti-chicken ovalbumin antibody. Ovalbumin levels were represented as a proportion to α-tubulin levels. Data are represented as mean ± standard error of the mean from at least 3 replicates. +P<0.01, P≤0.0001 Cancerous vs Normal. B. RT-PCR analyses for ovalbumin mRNA expression in chicken ovarian cancer cell lines (COVCAR; C5, C6, C7, C11, and C19) and normal ovarian surface epithelial cells (NOSE). Approximately 250 ng of cDNA (+RT) prepared from COVCAR cell lines and NOSE cells (from 5 chickens) was used as template to amplify a partial ovalbumin cDNA. Contamination controls consisted of COVCAR or NOSE cell RNA untreated with reverse transcriptase (−RT). C. Western blot analysis to detect ovalbumin expression in COVCAR cell lines (C5, C6, C7, C11, and C19) and NOSE cells (N1, N2, N3, N4, N5). Cellular protein lysates were subjected to immunoblotting as described above to detect ovalbumin. α-tubulin immunostaining was used as positive control.
Table 3.
Sequences of the oligonucleotide primers used for amplification and quantification of selected gene transcripts in chicken ovarian cancer (COVCAR) cell lines and normal ovarian surface epithelial (NOSE) cells.