Sensitive β-galactosidase-targeting fluorescence probe for visualizing small peritoneal metastatic tumours in vivo

Fluorescence-guided diagnostics is one of the most promising approaches for facile detection of cancer in situ. Here we focus on β-galactosidase, which is overexpressed in primary ovarian cancers, as a molecular target for visualizing peritoneal metastases from ovarian cancers. As existing fluorescence probes are unsuitable, we have designed membrane-permeable HMRef-βGal, in which the optimized intramolecular spirocyclic function affords >1,400-fold fluorescence enhancement on activation. We confirm that HMRef-βGal sensitively detects intracellular β-galactosidase activity in several ovarian cancer lines. In vivo, this probe visualizes metastases as small as <1 mm in diameter in seven mouse models of disseminated human peritoneal ovarian cancer (SHIN3, SKOV3, OVK18, OVCAR3, OVCAR4, OVCAR5 and OVCAR8). Because of its high brightness, real-time detection of metastases with the naked eye is possible. Endoscopic fluorescence detection of metastases is also demonstrated. The results clearly indicate preclinical potential value of the probe for fluorescence-guided diagnosis of peritoneal metastases from ovarian cancers.


Supplementary Methods
Determination of pK cycl or pK a values of compounds. Absorption and fluorescence emission spectra of compounds were measured in 200 mM sodium phosphate buffer at different pH values.
For compounds with n acid-base equilibria (n = 1 or 2), pH profiles of absorbance (Abs) or fluorescence intensity (FI) were fitted to the following formula to determine pK a values.
MTT assay. SHIN3 cells were plated in flat-bottomed 96-well plates at the density of 5000 cells/well with 200 L/well RPMI1640 containing 10% FBS (culture medium). Following incubation (37 o C, 5% CO 2 in air) for 1 day, the medium was replaced with 200 L/well culture medium containing the indicated concentrations of HMR derivatives (1% DMSO as a cosolvent).
The cells were then incubated for 1 h. The medium was replaced with 200 L/well culture medium containing 50 g/mL MTT, and incubation was continued for 4 h. The medium was replaced with DMSO (100 L/well), and the absorbance at 570 nm was measured with a microplate reader (SH-9000; Corona, Electric Co., Ltd.).
In vivo toxicity test. Ten female BALB/c mice (8 weeks old) were assigned to two groups of five mice. One group received intraperitoneal injection of 7 mg/kg HMRef-Gal in PBS (pH 7.4), which is a ten times higher dose than that used for imaging applications, while the other group received vehicle alone. Body weight of each mouse was monitored for two weeks.