To investigate the compartment-specific role of ketogenesis in breast cancer, we first overexpressed two key enzymes for ketone generation, HMGCS2 and BDH1, in hTERT-immortalized human fibroblasts. Figure 1 shows that HMGCS2 and BDH1 were highly overexpressed relative to empty vector control cells. Next, we evaluated if HMGCS2 and BDH1 overexpression is sufficient to mimic a starvation-state, inducing autophagy. To this end, HMGCS2, BDH1 and empty-vector control fibroblasts were analyzed by immunoblot with antibodies directed against autophagy markers.

Figure 1 shows that HMGCS2 and BDH1 overexpression both drive an autophagic program in fibroblasts, leading to the increased expression and activation of Cathepsin B (active form), Beclin-1 and LC3B (cleaved and active form).

Consistent with the idea that HMGCS2 and BDH1 overexpression in fibroblasts sensitizes them to the induction of autophagy, these fibroblasts were also more sensitive to the loss of Cav-1 upon co-culture with human breast cancer cells (MCF7) (Fig. 2). Previous studies have clearly documented that loss of Cav-1 expression under these co-culture conditions is due to its autophagic digestion, which is lysosome-dependent.^6,7

To examine the paracrine effects of HMGCS2- and BDH1-overexpressing fibroblasts on adjacent cancer cells, they were co-culultured with MCF7 cells under low mitogen conditions.

Figure 3 directly shows that, under co-culture conditions, HMGCS2- and BDH1-overexpressing fibroblasts stimulate an increase in mitochondrial mass and/or biogenesis in MCF7 cells. Note the dramatic increase in the expression of two well-established markers of mitochondrial mass, including a mitochondrial membrane protein and the mitochondrial chaperone, HSP60.

To examine the paracrine effects of ketogenic fibroblasts on MCF7 cell growth, we used quantitative FACS analysis. For this purpose, we assessed the cellular ratio between GFP(-) fibroblasts and GFP(+) MCF7 cells. Figure 4 shows that co-culture with HMGCS2- and BDH1-overexpressing fibroblasts, increases the proportion of MCF7 cells by > 2-fold, consistent with the idea that increased mitochondrial biogenesis in cancer cells “fuels” their proliferative capacity. FACS tracings are presented in Figure 4A, and bar graphs are shown in Figure 4B.

Since ketogenic fibroblasts appeared to increase the proliferation of human breast cancer cells, we next tested their in vivo activity on tumor growth. Briefly, to evaluate the tumor-promoting properties of HMGCS2 fibroblasts, we used a xenograft model. HMGCS2 fibroblasts were co-injected with MDA-MB-231 human breast cancer cells into the flanks of athymic nude mice and allowed to grow for 4.5 weeks. Figure 5A shows that tumor weight and volume are increased by 2.8-fold in tumors derived from HMGCS2-overexpressing fibroblasts, as compared with empty vector control fibroblasts. However, the tumor-promoting effects of HMGCS2 fibroblasts were not due to increased tumor angiogenesis, as no significant differences in CD31(+) vascular density were noted (Fig. 5B).

MDA-MB-231 breast cancer cells overexpressing ACAT1, ACAT2, OXCT1, OXCT2 or the empty vector were generated using a lentiviral approach. Figure 6 directly shows that we successfully overexpressed ACAT1/2 and OXCT1/2 in MDA-MB-231 cells.

Next, we assessed the tumorigenic effects of the overexpression expression ACAT isoforms or OXCT isoforms, using a xenograft model. As quantitatively similar results were obtained with both ACAT and OXCT isoforms, results are presented as ACAT1/2 and OXCT1/2, for simplicity.

Figure 7A shows that overexpression of ACAT1/2 and OXCT1/2 in human breast cancer cells directly promotes tumor growth. Note MDA-MB-231 cells overexpressing ACAT1/2 and OXCT1/2 show large increases in tumor growth, with up to a > 3-fold increase in tumor volume. However, little or no increases were observed in tumor angiogenesis (Fig. 7B). Thus, increased tumor vascularity could not account for the observed increases in tumor growth.

Finally, we used a lung colonization assay to assess possible effects on metastatic capacity. MDA-MB-231 cells overexpressing ACAT1/2 or OXCT1/2 were injected into the tail veins of athymic nude mice. After 8 weeks, the lungs were insuflated, and lung metastasis were scored. Figure 8 shows that MDA-MB-231 cells overexpressing ACAT1/2 greatly promote lung metastasis, as compared with control cells and OXCT1/2-overexpressing cells.

To assess the possible phenotypic effects of ketone body utilization on parental MDA-MD-231, they were treated with 3-hydroxy-butyrate. The results of this analysis are summarized in Table S1.

Note that treatment with 3-hydroxy-butyrate quantitatively increases the expression of protein markers associated with (1) protein synthesis, (2) an epithelial-mesenchymal transition (EMT), (3) protein folding and calcium homeostasis, as well as certain (4) neuronal proteins. Interestingly, neurons are known to be one of the few cell types that express the rare enzymes associated with ketone utilization (ACAT1/2 and OXCT1/2).^1,3

Antibodies were as follows: HMGCS2 (AV41562, Sigma); ACAT1 (HPA007569, Sigma); ACAT2 (HPA025736, Sigma); BDH1 (ab6834, Abcam); HMGCL (WH03155, Sigma); cathepsin B (sc13985, Santa Cruz Biotech); Beclin-1 (NBPI-00085, Novus Biologicals); LC3B (ab48394, Abcam); Cav-1 (sc-894, Santa Cruz Biotech); HSP60 (ab46798, Abcam); cytokeratin 8/18 (20R-CP004, Fitzgerald Industries International); surface of intact mitochondria (MAB1273, Millipore); β-tubulin and β-actin (Sigma); secondary antibodies for immunofluorescence were Alexa-Green 488 nm and Alexa Orange-Red 546 nm (Invitrogen). Other reagents were as follows: 4,6-diamidino-2- phenylindole (DAPI) (D3571), Prolong Gold Antifade mounting reagent (P36930) (Invitrogen).

Human skin fibroblasts immortalized with telomerase reverse transcriptase protein (hTERT-BJ-1) were originally purchased from Clontech, Inc. The breast cancer cell lines MCF7 and MDA-MB-231 were purchased from ATCC. All cells were maintained in DMEM with 10% Fetal Bovine Serum (FBS) and Penicillin 100 units/mL-Streptomycin 100 µg/mL.

For lentivirus production, 293Ta-packaging cells (Genecopoeia) were transfected with different lentiviral plasmids (all from Genecopoeia). The following plasmids were used: EX-neg-Lv105 (the empty vector control), EX-V0811-Lv105 (encoding BDH1), EX-I0546-Lv105 (encoding HMGCS2), EX-C0185-Lv105 (encoding ACAT1), EX-C0676-Lv105 (encoding ACAT2), EX-M0222-Lv105 (encoding OXCT1) and EX-T1301-Lv105 (encoding OXCT2). All plasmids contained a puromycin resistance marker. The resulting lentiviral particles were then used to infect fibroblasts and MDA-MB-231 cells to obtain stable cell lines overexpressing the target gene of interest.

Cells were harvested in lysis buffer (10 mM TRIS-HCl pH 7.5, 150 mM NaCl, 1% Triton X-100, 60 mM octylglucoside) containing protease inhibitors (Roche Applied Science) and phosphatase inhibitors (Roche Applied Science) and centrifuged at 13,000 × g for 15 min at 4°C to remove insoluble debris. Protein concentrations were analyzed using the BCA reagent (Pierce). Thirty μg of proteins were loaded and separated by SDS-PAGE and transferred to a 0.2 μm nitrocellulose membrane (Fisher Scientific). After blocking for 30 min in TBST (10 mM TRIS-HCl pH 8.0, 150 mM NaCl, 0.05% Tween-20) with 5% nonfat dry milk, membranes were incubated with the primary antibody for 1 h, washed and incubated for 30 min with horseradish peroxidase-conjugated secondary antibodies. The membranes were washed and incubated with an enhanced chemiluminescence substrate (ECL; Thermo Scientific).

hTERTfibroblasts and MCF7 cells were plated on glass coverslips in 12-well plates in 1 ml of complete media. MCF7 cells were plated within 2 h of fibroblast plating. The total number of cells per well was 1 ×10⁵, at a 5:1 fibroblast-to-epithelial cell ratio. The day after plating, media was changed to DMEM with 10% NuSerum (a low-protein alternative to FBS; BD Biosciences) and Pen-Strep. Cells were maintained at 37°C in a humidified atmosphere containing 5% CO₂ for 5 d.

Immunocytochemistry was performed as previously described.^14,15 All steps were performed at room temperature. Briefly, after 30 min fixation in 2% paraformaldehyde, cells were permeabilized for 10 min with immunofluorescence (IF) buffer (PBS, 0.2% BSA, 0.1% TritonX-100). Then, cells were incubated for 10 min with NH₄Cl in PBS to quench free aldehyde groups. Primary antibodies were incubated in IF buffer for 1 h. After washing with IF buffer (3×, 10 min each), cells were incubated for 30 min with fluorochrome-conjugated secondary antibodies diluted in IF buffer. Finally, slides were washed with IF buffer (3×, 10 min each), incubated with the DAPI nuclear stain and mounted.

Images were collected with a Zeiss LSM510 meta confocal system using a 405 nm Diode excitation laser with a band pass filter of 420–480 nm, a 488 nm Argon excitation laser with a band pass filter of 505–550 nm, and a 543 nm HeNe excitation laser with a 561–604 nm filter. Images were acquired with 40× or 63× objectives, as stated in the figure legends.

GFP (+) MCF7 cells were grown in co-culture with hTERTfibroblasts for 5 d. Then, cells were trypsinized and analyzed by flow cytometry. The GFP (+) MCF7 cell population was identified using the 488 nm laser. Fibroblasts were identified as the GFP (-) cell population. The percentage of GFP (+) cells over the total cell population was calculated using FlowJo 8.8 software.

Animals were housed and maintained in a pathogen-free environment/barrier facility at the Kimmel Cancer Center at Thomas Jefferson University under National Institutes of Health (NIH) guidelines. Mice were kept on a 12-h light/dark cycle with ad libitum access to chow and water. Approval for all animal protocols used for this study was reviewed and approved by the Institutional Animal Care and Use Committee (IACUC). Briefly, MDA-MB-231 tumor cells (1 × 10⁶ cells) alone or mixed with fibroblasts (3 × 10⁵ cells) were resuspended in 100 μl of sterile PBS and were co-injected into the flanks of athymic NCr nude mice (NCRNU; Taconic Farms; 6–8 weeks of age). After 4–5 weeks, mice were sacrificed; tumors were excised, measured and frozen in liquid nitrogen cooled isopentane.

Six-μm tumor frozen sections were fixed with 4% paraformaldehyde in PBS for 10 min at 4°C and washed 3× with PBS. A three-step biotin-streptavidin-horseradish peroxidase method was used for antibody detection. After fixation, the sections were blocked with 10% rabbit serum and incubated overnight at 4°C with rat monoclonal CD31 antibody (550274, BD Biosciences). The sections were then incubated with biotinylated rabbit anti-rat IgG (Vector Labs) and streptavidin-HRP (Dako). Immunoreactivity was revealed with 3, 3′ diaminobenzidine. For quantitation of vessels, CD31-positive vessels were counted in 4–6 fields of each tumor using a 20× objective lens and an ocular grid (0.25 mm² per field). The total numbers of vessel per unit area was calculated, and the data was represented graphically.

2D DIGE (two-dimensional difference gel electrophoresis) and mass spectrometry protein identification were run by Applied Biomics. Image scans were performed immediately following the SDS-PAGE using Typhoon TRIO (Amersham BioSciences) following the protocols provided. The scanned images were then analyzed by Image QuantTL software (GE-Healthcare) and then subjected to in-gel analysis and cross-gel analysis using DeCyder software version 6.5 (GE-Healthcare). The ratio of protein differential expression was obtained from in-gel DeCyder software analysis. The selected spots were picked by an Ettan Spot Picker (GE-Healthcare), following the DeCyder software analysis and spot picking design. The selected protein spots were subjected to in-gel trypsin digestion, peptides extraction, desalting and followed by MALDI-TOF/TOF (Applied Biosystems) analysis to determine the protein identity.

To analyze metastatic potentials, a lung colonization assay was performed. Briefly, MDA-MB-231 cancer cells (1 × 10⁶ cells) were re-suspended in 100 μl of sterile PBS and injected in the tail vein of athymic NCr nude mice (NCRNU; Taconic Farms; at 6–8 weeks of age). After 8 weeks, mice were sacrificed, and the lungs were insuflated with 15% India ink dye, washed in water and bleached in Fekete’s solution (70% ethanol, 3.7% paraformaldehyde, 0.75 M glacial acetic acid). A low-power stereo microscope was then used to determine the number of negatively stained metastatic colonies per lung lobe.