Effect of omega-3 fatty acid diet on prostate cancer progression and cholesterol efflux in tumor-associated macrophages-dependence on GPR120

Abstract

Background: Preclinical and clinical translational research supports the role of an ω-3 fatty acid diet for prostate cancer prevention and treatment. The anti-prostate cancer effects of an ω-3 diet require a functional host g-protein coupled receptor 120 (GPR120) but the underlying effects on the tumor microenvironment and host immune system are yet to be elucidated.

Methods: Friend leukemia virus B (FVB) mice received bone marrow from green fluorescent protein (GFP) labeled GPR120 wild-type (WT) or knockout (KO) mice followed by implanting Myc-driven mouse prostate cancer (MycCap) allografts and feeding an ω-3 or ω-6 diet. Tumor associated immune cells were characterized by flow cytometry, and CD206+ tumor infiltrating M2-like macrophages were isolated for gene expression studies. MycCap prostate cancer cell conditioned medium (CM) was used to stimulate murine macrophage cells (RAW264.7) and bone marrow-derived (BMD) macrophages to study the effects of docosahexanoic acid (DHA, fish-derived ω-3 fatty acid) on M2 macrophage function and cholesterol metabolism.

Results: The bone marrow transplantation study showed that an ω-3 as compared to an ω-6 diet inhibited MycCaP allograft tumor growth only in mice receiving GPR120 WT but not GPR120 KO bone marrow. In the ω-3 group, GPR120 WT BMD M2-like macrophages infiltrating the tumor were significantly reduced in number and gene expression of cholesterol transporters Abca1, Abca6, and Abcg1. RAW264.7 murine macrophages and BMDMs exposed to MycCaP cell CM had increased gene expression of cholesterol transporters, depleted cholesterol levels, and were converted to the M2 phenotype. These effects were inhibited by DHA through the GPR120 receptor.

Conclusion: Host bone marrow cells with functional GPR120 are essential for the anticancer effects of dietary ω-3 fatty acids, and a key target of the ω-3 diet are the M2-like CD206+ macrophages. Our preclinical findings provide rationale for clinical trials evaluating ω-3 fatty acids as a potential therapy for prostate cancer through inhibition of GPR120 functional M2-like macrophages.

Fig. 1. An ω-3 diet as compared to an ω-6 diet inhibited tumor growth in mice that received GPR120 WT bone marrow.

A Mouse model scheme. B–E Tumor volume by time growth curves of MycCaP allografts grown subcutaneously in FVB wild-type (WT-R = wild-type recipient) (n = 12 or 15 for ω-6 diet; n = 13 or 16 for ω-3 diet) or GPR120 knockout (KO-R = GPR120 knockout recipient) mice (n = 5 or 7 for ω-6 diet; n = 5 or 7 for ω-3 diet). The donor bone marrow is either from wild-type (WT-D = wild-type donor) or GPR120 knockout (KO-D = GPR120 knockout donor) FVB mice. F Tumor weights at time of sacrifice. Significance was determined by Student t test. (* <0.05 and ** <0.01). Tumor growth curves were steeper in the ω-6 group compared to ω-3 group (p < 0.001) in (B) and (D) but not significantly different in (C) (p = 0.570) or (E) (p = 0.916).

Fig. 2. The ω-3 as compared to the ω-6 diet decreased M2 macrophages in tumor tissue in mice that received GPR120 WT bone marrow.

A–D Percentage of macrophage subtypes from CD45+ cells in tumor tissue as determined by flow cytometry. Cells displayed in Fig. 2 are GFP+. Total macrophages = F4/80 + CD11b+, M1 macrophages = F4/80 + CD68+, and M2 macrophages = F4/80 + CD206 + . WT-R = wild-type recipient, KO-R = GPR120 knockout recipient, WT-D = wild-type donor, KO-D = GPR120 knockout donor. WT-R: n = 12 or 15 for ω-6 diet; n = 13 or 16 for ω-3 diet; KO-R: n = 5 or 7 for ω-6 diet; n = 5 or 7 for ω-3 diet. Significance determined by Student’s t test (* <0.05 and ** <0.01).

Fig. 3. The ω-3 diet regulated cholesterol efflux and polarization and function of macrophages in tumor tissue in mice that received GPR120 WT bone marrow.

For Figures (A–H), macrophages were isolated from the MycCaP tumors as described in the methods section. A–D Gene expression of M2 markers in macrophages isolated from MycCaP allografts. E–H Gene expression of cholesterol metabolism enzymes in macrophages isolated from MycCaP allografts. WT-R = wild-type recipient, KO-R = GPR120 knockout recipient, WT-D = wild-type donor, KO-D = GPR120 knockout donor. WT-R: n = 12 or 15 for ω-6 diet; n = 13 or 16 for ω-3 diet; KO-R: n = 5 or 7 for ω-6 diet; n = 5 or 7 for ω-3 diet. Significance determined by Student’s t test (* <0.05 and ** <0.01).

Fig. 4. MycCaP conditioned medium induced cholesterol efflux and conditioned medium induced gene expression of M2 markers was inhibited by DHA (ω-3 fatty acid).

A, C MycCaP conditioned medium effects on cholesterol transport and cholesterol levels in RAW 264.7 macrophages. B, D DHA inhibitory effects on cholesterol metabolism enzymes and cholesterol levels in MycCaP conditioned medium treated RAW 264.7 macrophages. E MycCaP conditioned medium induced expression of M2 markers in RAW 264.7 cells in response to IL-4 treatment. F DHA effects on MycCaP conditioned medium induced expression of M2 markers in response to IL-4 treatment (n = 3). Significance determined by Significance determine by Student’s t test (* <0.05 and ** <0.01).

Fig. 5. MycCaP conditioned medium induced cholesterol efflux and conditioned medium induced gene expression of M2 markers was inhibited by DHA in bone marrow derived macrophages (BMDMs) with a functional GPR120 receptor.

Bone marrow derived macrophages were generated as described in the methods section. A, B MycCaP conditioned media induced expression of cholesterol transporters in GPR120 WT and KO BMDMs. C, D DHA inhibition of cholesterol efflux enzymes induced by MycCaP conditioned media in GPR120 WT and KO BMDMs. E Effect of MycCaP conditioned media on cholesterol levels in GPR120 WT and KO BMDMs. F Effect of DHA on cholesterol levels in MycCaP conditioned media treated GRP120 WT and KO BMDMs. G–J Effect of MycCaP conditioned media on M2 markers in IL-4 treated GPR120 WT and KO BMDMs. K, L Effect of DHA on expression of M2 markers in MycCaP conditioned media and IL-4 treated GPR120 WT and KO BMDM’s. Significance determined by Student’s t test (* <0.05 and ** <0.01).