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. 2014 Jul 14;26(1):77-91.
doi: 10.1016/j.ccr.2014.05.002.

Hematogenous metastasis of ovarian cancer: rethinking mode of spread

Sunila Pradeep  1 , Seung W Kim  2 , Sherry Y Wu  1 , Masato Nishimura  1 , Pradeep Chaluvally-Raghavan  3 , Takahito Miyake  1 , Chad V Pecot  4 , Sun-Jin Kim  2 , Hyun Jin Choi  1 , Farideh Z Bischoff  5 , Julie Ann Mayer  5 , Li Huang  2 , Alpa M Nick  1 , Carolyn S Hall  6 , Cristian Rodriguez-Aguayo  7 , Behrouz Zand  1 , Heather J Dalton  1 , Thiruvengadam Arumugam  2 , Ho Jeong Lee  2 , Hee Dong Han  8 , Min Soon Cho  9 , Rajesha Rupaimoole  1 , Lingegowda S Mangala  10 , Vasudha Sehgal  3 , Sang Cheul Oh  11 , Jinsong Liu  12 , Ju-Seog Lee  3 , Robert L Coleman  1 , Prahlad Ram  3 , Gabriel Lopez-Berestein  7 , Isaiah J Fidler  2 , Anil K Sood  13
Affiliations

Hematogenous metastasis of ovarian cancer: rethinking mode of spread

Sunila Pradeep et al. Cancer Cell. .

Abstract

Ovarian cancer has a clear predilection for metastasis to the omentum, but the underlying mechanisms involved in ovarian cancer spread are not well understood. Here, we used a parabiosis model that demonstrates preferential hematogenous metastasis of ovarian cancer to the omentum. Our studies revealed that the ErbB3-neuregulin 1 (NRG1) axis is a dominant pathway responsible for hematogenous omental metastasis. Elevated levels of ErbB3 in ovarian cancer cells and NRG1 in the omentum allowed for tumor cell localization and growth in the omentum. Depletion of ErbB3 in ovarian cancer impaired omental metastasis. Our results highlight hematogenous metastasis as an important mode of ovarian cancer metastasis. These findings have implications for designing alternative strategies aimed at preventing and treating ovarian cancer metastasis.

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Conflict of interest statement

The authors declare no competing financial interests.

Figures

Figure 1
Figure 1. Parabiosis model shows that omental tumor cells can metastasize hematogenously
(A) Surgically attached parabionts 1 (#1), 15 (#2), and 60 (#3) minutes after the host mice were injected with 1% Evans blue dye. (B) Diagram of the parabiosis model where two mice were surgically joined. After 2 weeks, parental ovarian cancer cells were injected into the host mice. Once they became moribund, the parabionts were separated, and the host mice were euthanized. The guest mice were further observed. (C) Pictured is a moribund parabiont where SKOV3-OM1 cells were recycled (SKOV3-OM3) in the immunodeficient parabiosis model. (D) The dissected abdominal cavity of a representative parabiont two weeks after anastomosis. (E) CD31 and LYVE1 antibody staining of sections of the skin from between the anastamosed mice. Scale bars represent 100 μm. (F) Bioluminescence imaging of tumor cells injected into the ovary of the host mice on day 14 and of the guest mice of separated parabionts on days 25 and 45. (G) Localization of metastatic nodules was quantified and plotted. (H) Bioluminescence imaging of recycled SKOV3-OM3 cells introduced into the circulation via intra-cardiac injection. The color scale depicts the photon fluxes emitted from the tumor cells. (I) Hematoxylin and eosin (H&E) staining of normal omentum and tumor omentum. Scale bars represent 50 μm. (J) Omental metastasis in a patient with an isolated ovarian mass with an intact capsule. (K) H&E staining of regions of the omentum without gross disease from a patient. Scale bar represent 100 μm. See also Figure S1 and Table S1.
Figure 2
Figure 2. Activated ErbB3/NRG1 axis promotes hematogenous ovarian cancer metastasis
(A) Heat map represents normalized gene expression levels obtained from the microarray data of ovarian cancer cell lines SKOV3ip1 and SKOV3-OM3. Module of EGFR family genes differentially expressed in the SKOV3 and SKOV3-OM3 cells. (B) Phospho-RTK arrays probed with SKOV3ip1 and SKOV3-OM3 cell lysates. The red rectangles indicate the control and blue rectangles show EGF family receptors. (C) Immunoblot of total and phosphorylated (p) EGF receptors in SKOV3ip1 and SKOV3-OM3 cell lysates. Serum-starved SKOV3ip1 (left) and SKOV3-OM3 cells (right) were either treated with NRG1 (50 ng/mL) for different times or left untreated. Phosphorylation of EGF family receptors was determined using Western blotting with the indicated antibodies. (D) Phosphorylation of Src and PI3k/AKT was determined using Western blotting with the indicated antibodies in SKOV3ip1 and SKOV3-OM3 cell lysates. (E) Rac1 activation by NRG1 was measured by using a G-LISA kit. Data are represented in relative luminescence units (RLU). SKOV3ip1 and SKOV3-OM3 cells were serum starved for 24 hr and treated with antiErbB3 antibody (10 μg/mL), PI3K inhibitor (LY29004 μg/mL), or Src inhibitor (dasatinib (Dasa) 100 nM) and then stimulated with NRG1 (50 ng/mL) for 30 min. Mean ± SEM values are shown; *p < 0.05. (F) Phase contrast micrographs of 4-day 3DlrBM cultures of SKOV3ip1 and highly malignant SKOV3-OM3 cells. Inhibition of ErbB3 and PI3K restored the polarity of SKOV3-OM3 cells compared to untreated control. Scale bars represent 50 μm. See also Figure S2 and Table S2.
Figure 3
Figure 3. ErbB3/NRG causes mesenchymal transition and promotes CTCs
(A) Heat map from the microarray data shows that a group of genes involved in EMT were altered in SKOV3-OM3 cells compared with parental SKOV3ip1 cells. Heat map was generated to compare fold changes in the expression of genes between SKOV3ip1 and SKOV3-OM3 cells. (B) qRT-PCR for markers of EMT in SKOV3ip1 and SKOV3-OM3 cells. (C) Four-day 3DlrBM cultures of SKOV3ip1 and SKOV3-OM3 cells were stained with vimentin and SYTOX green. Scale bars represent 100 μm. (D) Cell lysates of SKOV3ip1 and SKOV3-OM3 cells were immunoblotted against vimentin. (E) Representative images show ErbB3+, CK+, and CK− CTCs within the microchannel in both SKOV3ip1 and SKOV3-OM3 in vivo models. Scale bars represent 10 μm. (F) Quantitative representations of total CTCs in the SKOV3ip1 and SKOV3-OM3 models. (G) ErbB3+ CTCs in the SKOV3-OM3 in vivo model. (H) Correlation of total tumor burden with enumeration of ErbB3+ CTCs in mice. Aggregate metastatic burden was determined by Pearson’s correlation using the aggregate tumor mass, and the number of CTCs was plotted for those specific samples. (I) Human omental sections stained for ErbB3 and CD31 show tumor cells within blood vessels. The adjacent slide was also stained with H&E. Scale bars represent 50 μm. (J) Representative immunohistochemical images of human ovarian tumors with low or high expression of ErbB3. Scale bars represent 50 μm. (K) Kaplan-Meier curves of disease-specific mortality for patients with epithelial ovarian carcinoma (n=217) based on ErbB3 expression. The log-rank test (two-sided) was used to compare differences between groups. Mean ± SEM values are shown. *p < 0.05; **p < 0.01; ****p <0.0001. See also Figure S3, Table S3, and Table S4.
Figure 4
Figure 4. Knock-down of ErbB3 reduces tumor growth and metastasis in vivo.
(A) Nude mice that received intra-cardiac injections of SKOV3-OM3 cells were randomly assigned to one of two groups (control siRNA-DOPC or ErbB3 siRNA-DOPC). Bioluminescence imaging was performed on day 28. The color scale bars depicting the photon fluxes emitted from the tumor cells are shown. (B) Quantitative representation of bioluminescence. (C) Representative images of the extent of metastatic spread in control vs. ErbB3 siRNA-DOPC treated mice. Metastatic areas are outlined with dotted white lines. (D) Bar graph shows the percentage of animals in each study arm with metastases to intraperitoneal and distant organ sites. The average tumor weight (E) and number of tumor nodules (F) are shown for SKOV3-OM3 and IG10-OM2 models 50 days after intra-cardiac injection. (G) SKOV3-OM3 cells were injected into the ovary of host parabiont mice; five weeks later the mice were imaged using bioluminescence imaging. (H) Tumor weight was plotted after ErbB3 siRNA-DOPC treatment. (I) The percentage of animals in each study arm with metastases to intraperitoneal and distant organ sites was plotted. Mean ± SEM values are shown. *p < 0.05; **p < 0.01; ***p <0.001. See also Figure S4.
Figure 5
Figure 5. Antibody mediated perturbation of ErbB3 inhibits ovarian cancer metastasis
(A) Nude mice that received intra-cardiac injection of SKOV3-OM3-Luc cells were randomly assigned to one of two groups (10 mice per group): group 1 was administered control (phosphate-buffered saline), and group 2 received the MM121 antibody. Bioluminescence imaging was performed on day 28. The color scales depicting the photon fluxes emitted from the tumor cells are shown. (B) Quantitative representation of bioluminescence. (C, D) At the end of the study, mice were euthanized, tumors were harvested and average tumor weight and number of nodules is shown. (E) Bar graph shows the percentage of animals in each study arm with metastases to intraperitoneal and distant organ sites. (F) Tumor weight in both host and guest mice were plotted after control or MM121 treatment. (G) Percentage of mice with metastases in distant organs in host and guest mice were plotted. (H) OVCA432 cells were injected into the ovary of host mice. Tumor weight in both host and guest mice were plotted after control siRNA-DOPC or ErbB3 siRNA-DOPC treatment (I) Percentage of mice with metastases in distant organs in host and guest mice. Mean ± SEM values are shown. *p < 0.05; **p < 0.01; ***p <0.001. See also Figure S5.
Figure 6
Figure 6. ErbB3 expression promotes hematogenous omental metastasis
(A) Nude mice that received intra-cardiac injections of either HeyA8-EV or HeyA8-ErbB3 cells were necropsied after three weeks. Tumor weights in both groups were plotted. (B) Percentage of mice with metastases in different organs. (C) Bioluminescence imaging after HeyA8-EV and HeyA8-ErbB3 cell injection into the host ovary. (D) Quantification of tumor weight at necropsy. (E) Pattern of metastases in host and guest mice. Mean ± SEM values are shown. **p < 0.01; ***p <0.001. See also Figure S6.
Figure 7
Figure 7. NRG1 activates ErbB3 signaling and enhances omental metastasis
Percentage of EDU-positive (A) SKOV3-OM3, (B) OVCA432 or (C) HeyA8-ErbB3 cells treated with either control siRNA (siCon) or ErbB3 siRNA (siErbB3). (D) SKOV3-OM3 cells were injected subcutaneously and treated with either control siRNA-DOPC or ErbB3 siRNA-DOPC for four weeks. (E) Ki67 staining on subcutaneous tumor sections. Scale bars represent 100 μm. (F) HeyA8-Ev and HeyA8-ErbB3 cells on soft agar in the presence or absence of NRG1. (G) Quantification of the average colony size from experiment shown in panel F. (H) Nude mice were treated with control siRNA-CH or NRG1 siRNA-CH nanoparticles and injected with SKOV3-OM3 cells. Aggregate tumor weight was assessed four weeks later. (I) The average number of tumor nodules in the omentum was plotted. (J) H&E staining on omental sections after siRNA treatments. Scale bars represent 100 μm. (K) Nude mice were treated with control siRNA-CH or NRG1 siRNA-CH nanoparticles and injected with HeyA8-ErbB3 cells into the heart. Three weeks later, the mice were necropsied. Aggregate tumor weight and (L) number of nodules in the omentum were plotted. Mean ± SEM values are shown. *p < 0.05; **p < 0.01; ***p <0.001; ****p <0.0001. See also Figure S7.
Figure 8
Figure 8. Model of ovarian cancer metastasis to omentum
Schematic representation of hematogenous metastasis to the omentum. Traditionally, epithelial ovarian cancer is thought to metastasize via direct surface spread (top). Our results point to an alternative pathway that also involves hematogenous metastasis with strong tropism toward the omentum (bottom).
See this image and copyright information in PMC

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