Expression of fatty acid synthase (FASN), the key enzyme in de novo synthesis of long-chain fatty acids, is normally low but increases in cancer. Consequently, FASN is a novel target for cancer therapy. However, because FASN inhibitors can lead to tumor stasis rather than shrinkage, noninvasive methods for assessing FASN inhibition are needed. To this end, we combined 1 H, 31 P, and 13 C magnetic resonance spectroscopy (MRS) (a) to monitor the metabolic consequences of FASN inhibition and (b) to identify MRS-detectable metabolic biomarkers of response. Treatment of PC-3 cells with the FASN inhibitor Orlistat for up to 48 h resulted in inhibition of FASN activity by 70%, correlating with 74% inhibition of fatty acid synthesis. Furthermore, we have determined that FASN inhibition results not only in lower phosphatidylcholine levels but also in a 59% drop in the phospholipid precursor phosphocholine (PCho). This drop resulted from inhibition in PCho synthesis as a result of a reduction in the cellular activity of its synthetic enzyme choline kinase.

Histone deacetylase (HDAC) inhibitors are new and promising antineoplastic agents. Current methods for monitoring early response rely on invasive biopsies or indirect blood-derived markers. Our goal was to develop a magnetic resonance spectroscopy (MRS) -based method to detect HDAC inhibition. The fluorinated lysine derivative Boc-Lys-(Tfa)-OH (BLT) was investigated as a 19 F MRS molecular marker of HDAC activity together with 31 P MRS of endogenous metabolites. In silico modeling of the BLT-HDAC interaction and in vitro MRS studies of BLT cleavage by HDAC confirmed BLT as a HDAC substrate. BLT did not affect cell viability or HDAC activity in PC3 prostate cancer cells. PC3 cells were treated, in the presence of BLT, with the HDAC inhibitor p-fluorosuberoylanilide hydroxamic acid (FSAHA) over the range of 0 to 10 Mmol/L, and HDAC activity and MRS spectra were monitored. Following FSAHA treatment, HDAC activity dropped, reaching 53% of control at 10 Mmol/L FSAHA. In parallel, a steady increase in intracellular BLT from 14 to 32 fmol/cell was observed. BLT levels negatively correlated with HDAC activity consistent with higher levels of uncleaved BLT in cells with inhibited HDAC. Phosphocholine, detected by 31

The anti-vascular function of platelet-derived growth factor receptor (PDGFR) inhibitor imatinib combined with paclitaxel has been demonstrated by invasive immunohistochemistry. The purpose of this study was 1. to noninvasively monitor the response to anti-PDGFR treatment and 2. to understand the underlying mechanism of this response. Thus, response to treatment was studied in a prostate cancer bone metastasis model using macromolecular (biotin-BSA-GdDTPA) dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI). PC-3MM2 bone metastases that caused osteolysis and grew in neighboring muscle showed high blood volume fraction (fBV) and vascular permeability (PS) at the tumor periphery, compared to muscle tissue and intraosseous tumor. Imatinib alone or with paclitaxel significantly reduced PS by 35% (one-tailed paired t-test p=0.045) and 40% (p=0.0003) respectively, whereas paclitaxel alone or no treatment had no effect. Based on changes in PS we hypothesized that imatinib interferes with the signaling pathway of vascular endothelial growth factor (VEGF). This mechanism was verified by immunohistochemistry. It demonstrated reduced activation of both PDGFR-β and VEGFR2 in imatinib-treated mice. Our study therefore demonstrates that macromolecular DCE-MRI can be used to detect early vascular effects associated with response to therapy targeted to PDGFR and provides insight into the role played by VEGF in anti-PDGFR therapy.

Histone deacetylase inhibitors (HDACis) are emerging as promising and selective antitumor agents. However, HDACis can lead to tumor stasis rather than shrinkage, in which case, traditional imaging methods are not adequate to monitor response. Consequently, novel approaches are needed. We have shown in cells that (19)F magnetic resonance spectroscopy (MRS)-detectable levels of the HDAC substrate Boc-Lys-TFA-OH (BLT) are inversely correlated with HDAC activity. We extended our investigations to a tumor xenograft model. Following intraperitoneal injection of BLT, its accumulation within the tumor was monitored by in vivo (19)F MRS. In animals treated with the HDACi suberoylanilide hydroxamic acid (SAHA), tumoral BLT levels were higher by 77% and 132% on days 2 and 7 of treatment compared with pretreatment levels (n = 6; p < .05). In contrast, tumoral BLT levels remained unchanged in control animals and in normal tissue. Thus, (19)F MRS of BLT detected the effect of HDACi treatment as early as day 2 of treatment. Importantly, tumor size confirmed that SAHA treatment leads to inhibition of tumor growth. However, difference in tumor size reached significance only on day 6 of treatment. Thus, this work identifies BLT as a potential molecular imaging agent for the early noninvasive MRS detection of HDAC inhibition in vivo.