Screening of glycosidase-reactive
fluorescent probes for the detection
of breast tumors. (a) Flowchart of screening using human surgical
normal tissue and tumor specimens. (b) Example of screening using
surgically resected frozen human breast IDC and normal tissues. The
compound number of the applied probe is shown on each well. Probe
solution was prepared with PBS (−) containing 0.5% v/v DMSO
as a cosolvent. [fluorescent probe] = 50 μM. Scale bar, 2 cm.
(c) Fluorescence increase at 30 min in breast IDC tissues in the presence
and absence of each inhibitor. Black bars: fluorescence increase in
the absence of inhibitor. Gray bars: fluorescence increase in the
presence of inhibitor. [fluorescent probe] = 50 μM, [swainsonine]
= 500 μM for probe 5 (α-d-Man),
[PUGNAc] = 500 μM for probe 11 (β-d-GlcNAc). (d) Comprehensive analysis of intact glycosidase activities
in normal breast, IDC and DCIS tissues using 12 fluorescent probes
or gGlu-HMRG (N = 14–20 for normal, N = 5–7 for IDC, N = 3 for DCIS).
Fluorescence increase represent increase at 30 min from 1 min after
addition of fluorescent probes. Black, pink, and purple dots represent
fluorescence increases in normal breast, IDC and DCIS tissues, respectively.
(e) Time-dependent fluorescence increase of probe 5 (α-d-Man) and 11 (β-d-GlcNAc) in breast
normal and cancer tissues. Black lines represent fluorescence increases
in normal breast tissues. Pink lines represent fluorescence increases
in breast cancer (IDC + DCIS) tissues. Error bars represent s.d. *P < 0.05 by Welch’s t-test. (f)
DEG assay for IDC tissue using probe 5 (α-d-Man). MAN2C1 (116 kDa) was identified by peptide mass fingerprinting
analysis from the fluorescent spot on 2D gel. (g) IHC staining for
MAN2C1 in normal mammary gland, fat, IDC and DCIS tissues. Scale bars,
200 μm.