Caspase-3 (R&D Systems AF835) and Ki67 (Abcam ab16667) antibody staining was performed on a Bond Max automated staining system (Leica Microsystems) using the Bond Intense R staining kit (Leica Microsystems DS9263). MDA-MB-231 tumors, significantly reducing tumor choline below baseline levels without observable systemic toxicity. These data introduce a new method to monitor therapeutically effective inhibitors of choline metabolism in breast cancer using a small molecule companion diagnostic. phospholipid biosynthesis leads to lower levels of pro-mitotic second messenger Kennedy pathway intermediates, increased ceramide levels, and de-stabilized endoplasmic reticulum [2, 17C19]. The most potent of these brokers, TCD-717, is usually a promising anti-cancer drug  that is being evaluated in clinical trials . MN58b, a [22, 23]. A limitation of this approach is usually that metabolite levels are also affected by the contributions of phospholipases, organic cation transporters, and sphingomyelinases [3, 24, 25]. Moreover, cell death can lead to deceptive decreases in tCho in MR spectra, requiring the measurement of secondary biomarkers [26, 27]. 18F and 11C choline PET tracers are useful for identifying ChoK inhibition , but choline tracer accumulation can be affected by choline transport inhibitors [29, 30] which have known toxicities . In addition, recent reports have shown that ChoK protein scaffolding, rather than the enzymatic function, may be critical for supporting cell viability [21, 32, 33]. Miyake and Parsons reported a c-Src-dependent link between ChoK and EGFR . More recent studies showed that small molecule non- symmetric ChoK inhibitors with low nM IC50s could substantially reduce the metabolic product TAK-285 PC but only cause reversible growth arrest with no effects on cell viability [21, 33]. Thus further development of fluorescence-based imaging strategies that report on enzyme expression rather than enzyme activity is needed. We have recently reported fluorescent small molecule choline mimetics (JAS239) that effectively attenuate choline phosphorylation. The structural similarity between symmetric, bis-heterocyclic ChoK inhibitors and a class of carbocyanine dyes used for optical imaging led to the development of these enzyme inhibitors with near infrared fluorescence (NIRF) . Within this wavelength range human tissue is relatively transparent [34C36] and NIRF optical imaging probes can be detected through several millimeters, and up to centimeters of tissue [37, 38]. Moreover, these probes exhibit a concentration dependent cellular uptake that cannot be TAK-285 attenuated in the presence of excess free choline indicating that they enter the cell independently of the choline transporters . There is a particular need for more specific agents to assist surgeons in distinguishing tumor from normal tissue [36, 39], and intraoperative imaging is an expanding field for which NIRF offers an inexpensive and effective method of delineating tumor margin and assessing lymph node involvement [40C42]. In this work we investigate JAS239 as a NIRF ChoK-targeted optical imaging probe in murine orthotopic breast tumors and compare this diagnostic method to MRS. growth (see Supplementary Figure 4AC4B) that could additionally be monitored TAK-285 with bioluminescence imaging. Bioluminescence in TAK-285 mice bearing orthotopic 4175-Luc+ tumors was measured 15 min following luciferin injection (Supplementary Figure 5A). This signal was used to delineate the tumor margins (in blue) and ACAD9 was used to confirm that the bioluminescence did not overlap with the NIR range (Supplementary Figure 5B). The next day no residual luminescence was detected, and mice were treated with control vehicle (Figure ?(Figure2A,2A, left mouse) or 20 nmol JAS239 in Tween-80/Tris buffer (Figure ?(Figure2A,2A, right mouse). After initial hepatic clearance (approximately 75 min), mice were injected i.p. with luciferin and imaged for bioluminescence and NIRF 15 min later. Bioluminescence was again used to delineate the tumor margin (Supplementary Figure 5C), and no NIRF signal was detected in the control animals (Figure ?(Figure2A,2A, left mouse; = 5). In the JAS239-injected animals, NIRF was emitted both from the tumor and from the kidneys (Figure ?(Figure2A,2A, right mouse; = 4). NIRF emission at.