Maternal obesity increases offspring's mammary cancer recurrence and impairs tumor immune response

Abstract

Over 50% of women at a childbearing age in the United States are overweight or obese, and this can adversely affect their offspring. We studied if maternal obesity-inducing high fat diet (HFD) not only increases offspring's mammary cancer risk but also impairs response to antiestrogen tamoxifen. Female rat offspring of HFD and control diet-fed dams, in which estrogen receptor-positive (ER+) mammary tumors were induced with the carcinogen 7,12-dimethylbenz[a]anthracene (DMBA), exhibited similar initial responses to antiestrogen tamoxifen. However, after tamoxifen therapy was completed, almost all (91%) tumors recurred in HFD offspring, compared with only 29% in control offspring. The increase in local mammary tumor recurrence in HFD offspring was linked to an increase in the markers of immunosuppression (Il17f, Tgfβ1, VEGFR2) in the tumor microenvironment (TME). Protein and mRNA levels of the major histocompatibility complex II (MHC-II), but not MHC-I, were reduced in the recurring DMBA tumors of HFD offspring. Further, infiltration of CD8+ effector T cells and granzyme B+ (GZMB+) cells were lower in their recurring tumors. To determine if maternal HFD can pre-program similar changes in the TME of allografted E0771 mammary tumors in offspring of syngeneic mice, flow cytometry analysis was performed. E0771 mammary tumor growth was significantly accelerated in the HFD offspring, and a reduction in the numbers of GZMB and non-significant reduction of interferon γ (IFNγ) secreting CD8+ T cells in the TME was seen. Thus, consumption of a HFD during pregnancy increases susceptibility of the female rat and mouse offspring to tumor immune suppression and mammary tumor growth and recurrence.

Keywords: breast cancer; inflammation; local recurrence; tamoxifen therapy; tumor immune microenvironment.

Figure 1

Mammary tumorigenesis in the offspring of obesity-inducing high fat diet (HFD) fed dams. (A) Percentage of complete responses (green), partial responses (black), de novo resistant mammary tumors (red) to tamoxifen (TAM) therapy in the offspring of HFD (n = 50 tumors) or control (n = 37 tumors) diet fed dams. (B) TAM therapy ended after a complete response was maintained for 7 weeks, and then local mammary tumor recurrences (red) were monitored. Of the 14 CR tumors in the control offspring, 4 recurred (28.6%), whilst of the 22 CR tumors in the HFD offspring, 20 recurred (90.9%) (χ²; P < 0.001). (C) Growth of E0771 mammary tumors allografted to HFD (n = 33 tumors) and control offspring (n = 52 tumors). Means ± s.e.m. of tumor volumes are shown; *P < 0.05 at specific tumor measurement time-points.

Figure 2

Effect of maternal obesity-inducing high-fat diet (HFD) on cytokine mRNA levels in mammary tumors in the offspring. In DMBA tumors in rats, maternal HFD marginally increased (A) IL-6 and (B) IL17c, and (C) significantly IL17f (P = 0.04) levels in the TAM-treated tumors of offspring (red squares), compared with tumors in control offspring [C] (black circles). Means ± s.e.m., n = 4–10 are shown. In E0771 tumors in mice, (D) IL-6 levels were significantly down-regulated (P = 0.009) in the HFD offspring and there was no change in the expression of (E) IL-17c or (F) IL17f. Control [C]: black circle, HFD: red square. Means ± s.e.m., n = 7 are shown.

Figure 3

Effect of maternal obesity-inducing high-fat diet (HFD) on CD8A+ and granzyme B (GZMB) protein levels and markers of MHCII in mammary tumors in rat offspring. (A) Representative pictures of immunohistochemically stained CD8A+ and GZMB+ tumor-infiltrating lymphocytes (TILs) in TAM-treated and post-TAM recurring mammary tumors. 20×. Quantitative analysis of 10–30 areas of each slides (n = 4–6 for the two offspring groups) showed that maternal HFD diet significantly reduced the number of (B) CD8A+ TILs and (C) GZMB+ TILs in the recurring tumors. Means ± s.e.m., n = 4–10 offspring of both control and HFD groups are shown. *P < 0.05, **P < 0.01, ***P < 0.001. (D) Gene expression of RT1.Bb and (E) RT1.Da in rat mammary tumors from control (black circles) and HFD (red squares) offspring before TAM treatment, and in TAM-treated or in post-TAM recurring tumors. Means ± SEM, n = 3-8 offspring of both control and HFD groups are shown. (F) Representative pictures of immunohistochemically stained MHCII+ cells in rat mammary tumors before and during treatment and in recurring tumors from control and HFD offspring. 20×. (G) Quantitative analysis of 16–256 pictures captured from each slide depending on the tumor size (n = 5-9 for the two offspring groups) showed that maternal HFD significantly reduced the MCHII protein levels in recurring tumors compared to control offspring.

Figure 4

Effect of maternal obesity-inducing high fat diet (HFD) on tumor immunosuppressive markers in rat offspring. (A) Representative Western blots and quantitated protein levels of (B) FOXP3 and (D) VEGFR-2, in the mammary tumors of control [C] (black circles) and HFD (red squares) offspring before TAM treatment, and in TAM-treated or post-TAM recurring tumors. (C) Tgfβ1 mRNA levels in TAM-treated tumors of control and HFD offspring. Mean ± s.e.m., n = 4–10 offspring of both control and HFD groups are shown. *P < 0.05, **P < 0.01.

Figure 5

Effect of maternal obesity-inducing high fat diet (HFD) on markers of tumor immune cell infiltration of E0771 mammary tumors in mouse offspring. Frequency of (A) CD4+ T lymphocytes (CD4+CD3+), (B) T reg cells (FOXP3+CD4+CD3+), and (C) CD8+ T cells (CD8+CD3+). (D) CD8 T cells exhaustion (PD1+CD8+CD3+), and CD8 T cells activation (E) measured by GZMB (GZMB+CD8+CD3+) and (F) IFN-γ (IFN-γ+CD8+CD3+) in the mammary tumors from control [C] (black circles) and HFD (red squares) offspring. Mean ± s.e.m., n = 6–7 for both control and HFD groups are shown.

Figure 6

Effect of maternal obesity-inducing high fat diet (HFD) on markers of epithelial-to-mesenchymal transition (EMT) in offspring’s mammary tumors. (A) Protein levels of p38 and (B) Cdh1 gene expression in the rat mammary tumors of control [C] (black circles) and HFD (red squares) offspring before TAM treatment, and in TAM-treated or recurring tumors. Mean ± s.e.m., n = 4–10 offspring of both control and HFD groups are shown. (C) Cdh1 gene expression in allografts of E0771 tumors of control [C] (black circles) and HFD (red squares) mouse offspring. Mean ± s.e.m., n = 6–7 tumors of both control and HFD groups are shown.