Multiparity activates interferon pathways in peritoneal adipose tissue and decreases susceptibility to ovarian cancer metastasis in a murine allograft model

Abstract

Ovarian cancer is the fifth leading cause of cancer deaths in U.S. women and the deadliest gynecologic malignancy. This lethality is largely due to the fact that most cases are diagnosed at metastatic stages of the disease when the prognosis is poor. Epidemiologic studies consistently demonstrate that parous women have a reduced risk of developing ovarian cancer, with a greater number of births affording greater protection; however little is known about the impact of parity on ovarian cancer metastasis. Here we report that multiparous mice are less susceptible to ovarian cancer metastasis in an age-matched syngeneic murine allograft model. Interferon pathways were found to be upregulated in healthy adipose tissue of multiparous mice, suggesting a possible mechanism for the multiparous-related protective effect against metastasis. This protective effect was found to be lost with age. Based on this work, future studies exploring therapeutic strategies which harness the multiparity-associated protective effect demonstrated here are warranted.

Keywords: Adipose tissue; Interferons (IFNs); Murine age-matched model of parity; Ovarian cancer metastasis; Parity.

Fig. 1.. Multiparity decreases metastatic burden.

A. The C57Bl/6 parity mouse model. To investigate the role of parity in ovarian cancer metastasis, three parity statuses of C57Bl/6 mice were selected for this study. Nulliparous animals (P0) were not bred. Para 1 (P1) animals gave birth to 1 litter and Para 3 (P3) animals gave birth to 3 litters. The parous mice were bred beginning at 8 weeks of age. B. Abdominal tumor burden in situ. Mice were injected IP with 10⁷ ID8 mouse ovarian cancer cells tagged with RFP. After sacrifice at 8 weeks post IP injection, the abdominal cavity was exposed and imaged. C,D. Tumor area and intensity, respectively, were quantified as described in Materials and Methods. Abdominal tumor area and abdominal tumor intensity were calculated by dividing either the tumor area or the tumor intensity by the scale-adjusted body weight of each mouse. N = 9, 10, 10 for P0, P1,P3, respectively. For Tumor Area, p = 0.03 for P0–P3 and p = 0.1 for P1–P3. For Tumor Intensity, p = 0.05 for P0–P3 and p = 0.05 for P1–P3.

Fig. 2.. Evaluation of organ-specific tumor burden in P0, P1, and P3 mice.

A. Representative tumor burden images. Individual organs were dissected from the mouse peritoneal cavity and imaged. N = 9, 10, 10 for P0, P1, P3, respectively. Liv = liver, Stom = stomach, Omen = omentum/pancreas, Dia = diaphragm, PerL = parietal peritoneum left, SmI = small intestine, Spl = spleen, PerInj = parietal peritoneum injection side, Ovary = ovaries/uterus and periovarian adipose, Col = colon, Mes = mesentary, FatL = gonadal adipose tissue from the left of the uterus, FatR = gonadal adipose tissue from the right of the uterus. B. Quantification of organ-specific tumor burden. The Organ Area Fraction was calculated by dividing the tumor area by the organ area. Organs with significant differences between parity groups are shown, with the omentum as reference. For ovary, p = 0.01 P0–P3, p = 0.07 P1–P3; FatL, p = 0.03 P0–P3; PerL, p = 0.04 P0–P1, p = 0.001 P0–P3. C. Representative H&E staining of the periovarian adipose tissue. After imaging, the organs were fixed in 10% formalin and processed for histology. Scale bar equals 200 μm. Arrows denote fibrosis layer.

Fig. 3.. RNASeq analysis of gonadal adipose tissue.

A. Venn diagram representation of the upregulated and downregulated genes from RNAsequencing data for each pairwise comparison of parity groups. Gonadal adipose RNA of healthy non-tumor bearing mice (n = 4) from each parity group was isolated (Supplemental Fig. 2) and was subjected to RNA sequencing. This Venn diagram represents all genes with p-value less than 0.05. Each parity group comparison displayed a unique expression profile. B. Schematic of differentially expressed genes in interferon pathways. Metacore pathway analysis revealed enrichment of genes in IFN pathways. In this schematic, genes validated by qRT-PCR, which are IFN transcriptional activators or ISGs, are represented.

Fig. 4.. Impact of parity on abdominal tumor burden in situ in aged mice.

A. Aged mice (20.5-month old) of three parity statuses were injected with 9.6 × 10⁶ ID8-RFP cells. After sacrifice at 8 weeks post IP injection, the abdominal cavity was exposed and imaged. B. Tumor area was quantified as described in Materials and Methods. Abdominal tumor area was calculated by dividing the tumor area by the scale-adjusted body weight of each mouse. N = 5.

Fig. 5.. Evaluation of organ-specific tumor burden in aged P0, P1 and P3 mice.

A. Individual organs from aged mice were dissected from the peritoneal cavity and imaged. N = 5. B. Tumor area was quantified to determine the Organ Area Fraction by dividing the tumor area by the adjusted organ weight.