Effect of dietary omega-3 fatty acids on castrate-resistant prostate cancer and tumor-associated macrophages

Abstract

Background: M2-like macrophages are associated with the pathogenesis of castrate-resistant prostate cancer (CRPC). We sought to determine if dietary omega-3 fatty acids (ω-3 FAs) delay the development and progression of CRPC and inhibit tumor-associated M2-like macrophages.

Methods: MycCap cells were grown subcutaneously in immunocompetent FVB mice. Mice were castrated when tumors reached 300 mm². To study effects of dietary ω-3 FAs on development of CRPC, ω-3 or ω-6 diets were started 2 days after castration and mice sacrificed after early regrowth of tumors. To study ω-3 FA effects on progression of CRPC, tumors were allowed to regrow after castration before starting the diets. M2 (CD206+) macrophages were isolated from allografts to examine ω-3 FA effects on macrophage function. Omega-3 fatty acid effects on androgen-deprived RAW264.7 M2 macrophages were studied by RT-qPCR and a migration/ invasion assay.

Results: The ω-3 diet combined with castration lead to greater MycCap tumor regression (tumor volume reduction: 182.2 ± 33.6 mm³) than the ω-6 diet (tumor volume reduction: 148.3 ± 35.2; p = 0.003) and significantly delayed the time to CRPC (p = 0.006). Likewise, the ω-3 diet significantly delayed progression of established castrate-resistant MycCaP tumors (p = 0.003). The ω-3 diet (as compared to the ω-6 diet) significantly reduced tumor-associated M2-like macrophage expression of CSF-1R in the CRPC development model, and matrix metallopeptidase-9 (MMP-9) and vascular endothelial growth factor (VEGF) in the CRPC progression model. Migration of androgen-depleted RAW264.7 M2 macrophages towards MycCaP cells was reversed by addition of docosahexaenoic acid (ω-3).

Conclusions: Dietary omega-3 FAs (as compared to omega-6 FAs) decreased the development and progression of CRPC in an immunocompetent mouse model, and had inhibitory effects on M2-like macrophage function. Clinical trials are warranted evaluating if a fish oil-based diet can delay the time to castration resistance in men on androgen deprivation therapy, whereas further preclinical studies are warranted evaluating fish oil for more advanced CRPC.

Figure 1:

Dietary ω−3 fatty acids (as compared to ω−6) delayed development of CRPC. (A) Tumor growth. Pre-castration tumor volume not shown. The numbers at each time point indicate the number of surviving mice in the two groups combined. The numbers at time points decreased as mice were sacrificed during the experiment as ω−6 group tumors reached 300 mm³. Tumor volume over time was compared between groups (Fig 1A and Fig 3A) using Generalized Estimating Equations (GEE) models. (B) Tumor weight. (C) Kaplan Meier curve showing time to development of CRPC. Data are means ±SEM (n=20 ω−3 diet, n=20 ω−6 diet); *p<0.05, **p<0.001.

Figure 2:

Dietary ω−3 fatty acids (as compared to ω−6) decreased tumor infiltration of M2-like macrophages, increased infiltration of M1-like and CD4⁺ T cells, and decreased gene expression of CSF-1R in M2-like macrophages in the development of CRPC mouse model. (A) Macrophage quantity and type by flow cytometry of tumor tissue (n=17 ω−3 diet, n=18 ω−6 diet). (B) Quantity of other immune cells by flow cytometry of tumor tissue (n=17 ω−3 diet, n=18 ω−6 diet). (C) M2-like macrophage gene expression in sorted M2 macrophages (n=3 ω−3 diet, n=2 ω−6 diet). Data are means ±SEM; *p<0.05, **p<0.01.

Figure 3:

Dietary ω−3 fatty acids (as compared to ω−6) decreased tumor growth in established CRPC and decreased expression of VEGF and MMP-9 in tumor infiltrating M2-like macrophages. (A) Tumor growth. Pre-castration tumor volume not shown (n=14 ω−3 diet, n=14 ω−6 diet). Tumor volume over time was compared between groups (Fig 1A and Fig 3A) using Generalized Estimating Equations (GEE) models. (B) Tumor weight (n=14 ω−3 diet, n=14 ω−6 diet). (C) Gene expression in M2-like macrophages isolated from tumor tissue (n=11 ω−3 diet, n=10 ω−6 diet). Data are means±SEM; *p<0.05, **p<0.01, ***p<0.003.

Figure 4:

DHA inhibited gene expression in M2 macrophages in FBS medium and charcoal stripped serum (CSS) medium in vitro. Murine macrophages Raw264.7 were polarized to M2 type macrophages and cultured in androgen-deprived conditions in CSS medium or non-androgen deprived conditions in FBS medium and treated with 50 μmol/L of DHA. Mean±SD, *p<0.05, **p<0.01.

Figure 5:

DHA inhibited RAW264.7-derived M2 macrophage migration towards MycCap prostate cancer cells in a co-culture system. Raw264.7 were polarized to M2-type macrophages and cultured in androgen-deprived conditions with CSS medium, plated in inserts and treated with 50 μmol/L of DHA. MycCap cells were cultured in androgen-deprived conditions with CSS medium, and plated in the lower chamber.

Figure 6:

Conceptual diagram of potential effects of ω−3 fatty acids on M2-like macrophages affecting CRPC development and progression. We propose that after castration, ω−3 FAs reduce CSF-1R gene expression in M2-like macrophages resulting in the inhibition of recruitment and polarization of M2 like macrophages towards tumor tissue, thus, delaying the development of CRPC. In the late stage after castration, ω−3 FAs decrease MMP9 and VEGF expression in M2-like macrophages resulting in the inhibition of migration and angiogenesis, thus, delaying the progression of CRPC.