Ovulation Enhances Intraperitoneal and Ovarian Seedings of High-Grade Serous Carcinoma Cells Originating from the Fallopian Tube: Confirmation in a Bursa-Free Mouse Xenograft Model

Background: Recently, new paradigms for the etiology and origin of ovarian high-grade serous carcinoma (HGSC) have emerged. The carcinogens released during ovulation transform fallopian tube epithelial cells, exfoliating and metastasizing to the peritoneal organs, including the ovaries. Solid in vivo evidence of the paradigms in a mouse model is urgently needed but is hampered by the differing tubo-ovarian structures. In mice, there is a bursa structure surrounding the distal oviduct and ovary. This, on one hand, prevents the direct influence of ovulatory follicular fluid (FF) on the exfoliated tumor cells. On the other hand, it hinders the seeding of exfoliated tumor cells into the ovary. Methods: In this study, we created a bursa-free mouse xenograft model to examine the effect of superovulation on peritoneal and ovarian metastases of transformed human tubal epithelial cells after intraperitoneal injection in NSG mice. Results: The bursa-free mouse model showed a better effect of ovulation on peritoneal metastasis. In this model, superovulation increased the number of transformed human tubal epithelial cell seedlings after intraperitoneal injection. Compared to the bursa-intact state, bursa-free ovaries were more vulnerable to external tumor seeding in either normal ovulation or superovulation state. Conclusions: This study provides the first in vivo evidence that intraperitoneal spreading of tubal HGSC cells is enhanced by ovulation. This study also demonstrated a mouse model for studying ovary-peritoneum interaction in cancer development.


Introduction
High-grade serous carcinoma (HGSC) is the most common type of epithelial ovarian cancer (EOC) and is a major killer. Nearly 80% of cases are diagnosed after intraperitoneal spread, leading to a poor prognosis. The problem has not improved over the past several decades, mainly because of the illusive etiological cause, mechanisms of malignant transformation, and even the tissue of origin. The puzzle of ovarian HGSC origin has been largely resolved. Multiple levels of evidence have indicated that secretory cells in the fallopian tube fimbrial epithelium (FTE) are the main origin of HGSC in the ovary, fallopian tube, and peritoneum [1][2][3].
Results from multiple epidemiological studies are consistent with the theory of incessant ovulation as the cause of EOC [4,5]. Factors that reduce the number of ovulation cycles, such as oral contraceptive use, full-term pregnancy, and breastfeeding, all showed a protective effect on HGSC [6]. Thus, a woman's number of ovulation cycles is associated with an increased EOC risk [7].
The mechanisms of the ovulation-induced FTE transformation are becoming increasingly clear. Ovulatory follicular fluid (FF) contains high levels of various carcinogens, such as reactive oxygen species (ROS), insulin-like growth factor (IGF) axis proteins, and coagulation factor-hepatocyte growth factor (HGF) activation cascade proteinases, which confer a range of cell transformation phenotypes, including DNA double-strand breaks [8,9], stemness activation, clonal expansion [10], anchorage-independent growth, and xenograft tumorigenesis [10,11]. The transformation effect of ovulatory FF is not limited to the FTE transformation. It also confers metastatic seeding of transformed FTE cells by promoting anoikis resistance, peritoneal attachment, proliferation, migration, and invasion [12]. These results suggest that ovulation not only promotes the malignant transformation of FTE but also the peritoneal spreading of tubal intraepithelial carcinoma (STIC), the immediate precursor of HGSC [13,14].
In all the previous studies, the pro-metastasis activities of ovulation were demonstrated by human FF exposure in vitro or ex vivo [8,10,11] or by co-injection of FF, transforming human FTE cells into the peritoneal cavity of immune-compromised mice [9,12]. An in vivo demonstration of the effect of ovulation on HGSC development is lacking. Before experimental proof is feasible, an anatomic barrier in the murine reproductive system must be overcome. In rodents, a bursa structure envelops the ovaries and distal oviduct [15] and interferes with the direct communication of FF with the peritoneal cavity after ovulation [16]. The presence of ovarian bursa in mouse not only prevents the direct release of ovulatory FF into the peritoneum but may also hinder the seeding of exfoliated STIC cells into the ovary.
In the present study, we established a corresponding model to overcome the bursa barrier and demonstrated the metastasis-enhancing effect of ovulation in vivo for the first time. Figure 1A shows the timeline of the surgical removal of the ovarian bursa and superovulation in the studied mice. To avoid surgical wound disturbance [17], an i.p. xenograft was performed ten days after the operation. In the superovulation group, FEXT2-LUC cells were injected 6 h after hCG administration or 6 h before the expected ovulation time. Intraperitoneal tumor growth was monitored weekly using an IVIS for 6 weeks. Mice were sacrificed in the seventh week. Representative images of bursa-intact and bursa-free ovaries are shown in Figure 1B.  As shown in Figure 2A and Table 1, bursa-free and bursa-intact mice did not show differences in intraperitoneal tumor luminescence signal throughout the follow-up period. There was no difference in the number or total weight of intraperitoneal tumors examined at the seventh week (data not shown). As expected, the removal of the ovarian bursa led to an increase in periovarian seeding of PAX8(+) tumors in both normal-ovulation and superovulation mice (p = 0.009 and p = 0.02, respectively, and p= 0.0006 as a pool) ( Figure 2B,C, Table 1).

Superovulation Enhanced the Early Peritoneal Seeding and The Later Growth of Transformed FTE Cells, Which Was Revealed Only in the Bursa-Free Xenograft Model
On the third day after intraperitoneal injection, bursa-intact mice, with or without superovulation, did not show significant differences in the tumorigenesis signal. However, in the bursa-removed group, superovulation mice showed a significantly higher intraperitoneal tumor signal than normal ovulation mice ( Figure 3A, Table 1), and the differences increased over time ( Figure 3B). Seven weeks after i.p. injection, there was also a higher average tumor weight (8.9 folds, p = 0.049) and tumor number (2.0 folds, p = 0.053) in the superovulation group than in the normal ovulation group ( Figure 4A). This was again observed only in bursa-removed mice.

Superovulation in the Bursa-Free Status Did Not Further Increase the Periovarian Tumor Seeding
Unexpectedly, under the bursa-free setup, there was no statistical difference in the periovarian seeding of xenograft tumors between the normal ovulation and superovulation groups ( Figure 4B).

Discussion
There is a bursa structure in rodents enclosing the ovary and distal oviduct. It helps to confine the ovulated oocytes and facilitate the catch-up by the oviduct infundibulum. Although the small opening (of approximately 0.04 to 0.12 cm 2 [15]) of the foramen of the ovarian bursa located at the proximal mesosalpinx allows limited drainage, the majority of ovulatory FF and oviduct fluid flowing into the uterine cavity instead of the peritoneal cavity after ovulation [15,18]. It is thus assumed that the bursa structure, on one hand, interferes with the release of FF-carcinogens into the peritoneum and, on the other hand, with the seeding of exfoliated cancer cells into the ovary. Indeed, the present study showed an increase in local ovarian seeding after bursa removal.
The presence of the bursa structure retards the release of pro-metastatic FF factors into the peritoneum, thereby reducing intraperitoneal seeding of intraperitoneally injected tumor cells. In the STIC-to-HGSC stage of cancer development, STIC cells frequently detach from the epithelium into the lumen of the fallopian tube. Detached cancer cells usually float in the peritoneal fluid as multicellular spheroids [19]. Due to their miniature size, how these exfoliated STIC cells metastasize intraperitoneally is largely unknown. We previously discovered that i.p. co-injection of human FF with HGSC cells or transforming FTE cells promotes intraperitoneal metastasis [12]. FF harbors a pro-metastasis activity, which favors anoikis resistance, anchorage-independent growth, peritoneal attachment, proliferation, and migration of HGSC cells and STIC-mimicking partially transformed FTE cells [12].
The present study provides the first in vivo evidence that endogenous ovulation promotes intraperitoneal metastasis of transformed FTE cells.
In our bursa-free i.p. injection xenograft model, all seven tested mice grew tumors in the normal ovulation state. Superovulation, while also achieving a 100% tumorigenesis rate, resulted in a similar severity of local tumorigenesis as measured by the area of PAX8+ tumor lesions ( Figure 2C). We suppose that under the oncogenic influence of normal ovulation, orthotopic tumorigenesis from ovarian seeding is already saturated, and no further increase can be achieved by superovulation.
The bursa-free mouse model, although essentially mimicking the ovulatory FF release in humans, is still limited by the fundamental anatomic and physiological differences between mice and humans. First, although we did not find a regeneration of the bursa up to seventh weeks after the removal, whether the bursa-free state will affect ovarian physiology, such as the efficiency of ovulation and the postovulatory wound healing, is not known. The second concern is whether the direct drainage of FF into the peritoneal cavity will change the constitution of peritoneal fluid or affect the peritoneal environment. Third, after mimicking bursa-free ovulation, a robust model simulating menstruation and its backflow is still lacking, although there is a way to induce decidualization and blood-shedding in rodent endometrium by hormone and sesame oil treatment [40]. Finally, the current in vivo model, although conveying the effect of ovulation on the peritoneal metastasis, is still not a spontaneous orthotopic tumorigenesis model of HGSC. A genetically engineered mouse model targeting the initiating driver mutations of TP53 and CCNE1 in the LGR-expressing cancer initiation cells in FTE will be an ideal model to test the effect of ovulation on the genesis and metastasis of HGSC.
In conclusion, the bursa-free mouse model better simulates human ovulation and facilitates the observation of the effect of ovulation on cancer metastasis. This study provides the first in vivo proof that superovulation enhances the peritoneal seeding of transformed FTE cells. A scenario of metastatic peritoneal seeding of STIC cells promoted by ovulation can thus be validated. The established bursa-free mouse model may provide an important tool for the study of various postovulatory impacts, such as ovulation-related wound healing and adhesions, as well as the development of endometriosis and cancer metastasis.

Bursa Removal Surgery and Xenograft Model
To analyze the abdominal dissemination of transformed human FTE cells, we performed bursectomy and i.p. xenografts of FEXT2-LUC cells in 8-to 12-week-old female NOD/Shi-scid/IL-2Rγ null (NSG) mice (Jackson Laboratory, Bar Harbor, ME, USA). Briefly, after hair removal and disinfection, the uterine horn was exposed by a 1-cm incision at the dorsal skin. Under a four-fold dissecting surgical microscope, the fat behind the ovary was held with forceps, and the bursa was peeled off with forceps ( Figure 1B). The surgical wound was closed with a skin nail, and the same procedures were performed on the other side. The entire procedure takes approximately 10-15 min. In the sham operation, the uterine horn was exposed using the same laparotomy procedure and was left in the air for the same duration.
Seven days after the operation, when the wounds were completely healed, mice were superovulated with 5 IU PMSG and 5 IU hCG (given 46 h apart) in a 4-day cycle for 6 weeks. Six hours after the first hCG injection, FEXT2 cells (1 × 10 4 ) were mixed with 200 µL PBS and injected into the peritoneal cavity (as shown in Figure 1A). Based on the status of bursa and ovulation, mice were grouped into bursa-intact normal ovulation (BINO), or superovulation (BISO), and bursa-removed normal ovulation (BRNO) or superovulation (BRSO) groups, and followed weekly by an in vivo imaging system (IVIS ® , PerkinElmer, Shelton, CT, USA). All mouse study procedures were performed according to protocols approved by the Institutional Animal Care and Use Committee of Tzu-Chi University (Approval ID: 108-25).

Statistics
All data are presented as the mean ± standard error. Statistical analyses were performed using GraphPad Prism version 8.0 (GraphPad Software, La Jolla, CA, USA) and Microsoft Excel 2019. Detailed information on the statistical analysis is provided in the figure legends.