Influence of omega-6 PUFA arachidonic acid and bone marrow adipocytes on metastatic spread from prostate cancer

Abstract

Background: Prostate cancer (CaP) preferentially metastasises to the bone, and we have previously shown that the poly-unsaturated fatty acid (PUFA) arachidonic acid (AA) is a potent stimulator of CaP invasion. Here we present that AA promotes CaP invasion by inducing bone marrow adipocyte formation.

Methods: Boyden invasion-chamber assays assessed the ability of dietary oils, their PUFA components, and specific PUFA-loaded adipocytes to induce PC-3 invasion. Lipid transfer and metabolism was followed using deuterated AA and Fourier Transform Infrared spectroscopy (FTIR).

Results: Poly-unsaturated fatty acid constituents, but not their corresponding dietary oils, induced PC-3 invasion. PUFAs induce bone marrow adipocyte (BM-Ad) differentiation with AA inducing higher levels of BM-Ad differentiation, as compared with other PUFAs (3998+/-514.4 vs 932+/-265.8; P=0.00002), which stimulated greater PC-3 invasion than free AA (22 408.5+/-607.4 vs 16 236+/-313.9; P=0.01111) or adipocytes generated in the presence of other PUFAs. In bone marrow co-culture PC-3 and BM-Ad interactions result in direct uptake and metabolism of AA by PC-3 cells, destruction of the adipocyte and subsequent formation of a bone metastasis.

Conclusion: The data supports the hypothesis that AA not only promotes CaP invasion, it also prepares the 'soil', making it more supportive for implantation and propagation of the migrating metastatic cell.

Figure 1

Poly-unsaturated fatty acid (PUFA) components, but not heterogeneous dietary oils, are stimulators of prostate cancer (CaP) invasion. 2 × 10⁵ PC-3-GFP cells were seeded in modified Boyden chambers above either (A) 1 μg ml⁻¹ dietary lipids or increasing levels of (B) linoleic acid (LA) (C) α-linolenic acid (ALA) (D) oleic acid encaged in methyl-β-cyclodextrin. Levels of invasion are proportional to fluorescence detected by a bottom reading BMG FLUOstar OPTIMA plate reader at 488/520 nm (excitation/emission filter). In each assay (n=3) a serum free RPMI 1640 (TCP)^–ve control, human BMS^+ve control and a 3 μg ml⁻¹ arachidonic acid (AA) control were included.

Figure 2

Arachidonic acid (AA) primes the ‘soil’ for prostate cancer (CaP) invasion. Mesenchymal cells isolated from primary human bone marrow (BM) were grown in the presence of 50 μM linoleic acid (LA), arachidonic acid (AA), α-linolenic acid (ALA), eicosapentaenoic acid (EPA) or oleic acid in basal media supplemented with 5 × 10⁻⁷ M hydrocortisone to induce adipocyte differentiation. Control cultures include differentiation in proprietary adipogenic media and long-term bone marrow stroma (BMS) growth media. (A) Phase contrast photo-micrographic mosaics comprising 11 × 8 overlapping fields of view merged using the Mosaic J plug-in (ImageJ). Adipocytes are distinguished from dark stromal background as phase-bright cells. Scale bar=1 mm. (B) Histogram showing number of adipocytes formed in the presence of each lipid (n=3). (C) Histogram showing the invasive stimulus of specific lipid-laden adipocytes derived from human BM mesenchymal stem cells (MSC) with 5 × 10⁻⁷ M hydrocortisone. 2 × 10⁵ PC-3-GFP cells were seeded in modified Boyden chambers and invasion was measured after 18 h using a bottom reading BMG FLUOstar OPTIMA plate reader at 488/520 nm (excitation/emission filter).

Figure 3

Localisation of D₈-AA within bone marrow (BM) adipocytes. Deuterated signal overlaid onto phase contrast photomicrograph of a D₈-AA loaded BM adipocyte. BM mesenchymal stem cells (MSC) were differentiated into adipocytes with 50 μM D₈-AA. Phase contrast image of an adipocyte overlaid with (A) υ(C–D) Fourier Transform Infrared spectroscopy (FTIR) spectral image and (B) υ(=C–D) spectral image to show localisation of D₈-AA.

Figure 4

PC-3 cells target and take-up arachidonic acid (AA) from AA-loaded human bone marrow adipocyte (BM-Ad). (A) Interaction of PC-3 cells co-cultured with AA-pulsed adipocytes from human BM mesenchymal cells followed by time-lapse video-microscopy. Adipocyte=white arrow, PC-3 cells=green arrows. (B) Photomicrographs showing separate phase contrast, total lipid and D8-AA Fourier Transform Infrared spectroscopy (FTIR) spectral image and a phase contrast D₈-AA overlaid image of a PC-3 D₈-AA loaded BM adipocyte (Adp) co-culture. (C) Fourier Transform Infrared Spectroscopy spectra of four regions defined in the phase contrast and total lipid spectra photomicrograph shown in B, with inset high-resolution spectra of the 1789–2627 cm⁻¹ region corresponding to D₈-AA. Bkg=background.

Figure 5

Arachidonic acid (AA) localises around the nucleus following uptake by PC-3 cells. (Ai) Phase contrast image of a PC-3/bone marrow adipocyte (BM-Ad) co-culture showing a PC-3 cell flanked top and bottom by D₈-AA loaded adipocytes; (Aii) Fourier Transform Infrared spectroscopy (FTIR)-spectral micrograph of the υ(C–D) signal overlaid on to the optical photomicrograph, with increasing signal absorbance depicted by a shift towards red/white colours; (Aiii) FTIR-spectral micrograph of the total lipid hydrocarbon signal overlaid onto the optical photomicrograph with increasing signal absorbance depicted by a shift towards red/white colours. (B) Fourier Transform Infrared spectroscopy-spectral signatures from i) PC-3 cell, (ii+iii) background as defined in Figure 6Ai and Aii. Fourier Transform Infrared spectroscopy Spectral profiles (inset) concentrate on the 1793.4–2643.5 cm⁻¹ region encompassing the (C, D) spectral region (highlighted by the green vertical line).

Figure 6

Chemometric analysis of arachidonic acid (AA) metabolism in PC-3 cells. Endogenous (A) lipid and (B) phosphorylation signals in PC-3 cells following exposure to 25 μM and 100 μM D₈-AA or no D₈-AA (control). Fourier Transform Infrared spectroscopy (FTIR) measurements over 3 h.