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. 2018 Nov 27;6(2):1801479.
doi: 10.1002/advs.201801479. eCollection 2019 Jan 23.

Cascade Reaction in Human Live Tissue Allows Clinically Applicable Diagnosis of Breast Cancer Morphology

Free PMC article

Cascade Reaction in Human Live Tissue Allows Clinically Applicable Diagnosis of Breast Cancer Morphology

Tomonori Tanei et al. Adv Sci (Weinh). .
Free PMC article


Clean operating margins in breast cancer surgery are important for preventing recurrence. However, the current methods for determining margins such as intraoperative frozen section analysis or imprint cytology are not satisfactory since they are time-consuming and cause a burden on the patient and on hospitals with a limited accuracy. A "click-to-sense" probe is developed based on the detection of acrolein, which is a substance released by oxidatively stressed cancer cells and can be visualized under fluorescence microscopy. Using live breast tissues resected from breast cancer patients, it is demonstrated that this method can quickly, selectively, and sensitively differentiate cancer lesion from normal breast gland or benign proliferative lesions. Since acrolein is accumulated in all types of cancers, this method could be used to quickly assess the surgical margins in other types of cancer.

Keywords: TAMRA phenyl azide; acrolein; breast cancer; breast‐conserving surgery; imaging.


Figure 1
Figure 1
Schematic presentation of intraoperative diagnosis. A) The pathology‐based method currently used in BCS. Frozen samples of breast cancer stumps are analyzed using microscopy with H&E staining. Further surgery is guided based on morphological analysis, i.e., depending on the IDC, DCIS, DH, and NBG regions. Several pathological procedures require about 1 h. B) Recently emerging techniques use CT, MRI, and chemical probes. The methods can image live cancer tissues, but still have problems associated with sensitivity and selectivity. Importantly, these methods cannot image the morphology of various cancer stages, and still require pathology to confirm their diagnosis (hence they are unlikely to replace the conventional H&E method as described in (A)). C) The “click‐to‐sense” method described in this paper allows the diagnosis of cancer morphology with high sensitivity and selectivity by simply treating the live tissues with chemical probe 1 for 5 min and then by fluorescent microscopy. Our new method could substitute for conventional pathological diagnosis.
Figure 2
Figure 2
A) “Click‐to‐sense” the cancer: method and mechanism. Fluorescently labeled azide 1 smoothly reacts with acrolein generated by cancer cells through a 1,3‐dipolar cycloaddition reaction (acrolein/azide “click” reaction). 1,2,3‐triazole as the “clicked” product decomposes into diazo compounds, which react with cell constituents to anchor the fluorescence label within the cells. The acrolein concentration is analyzed in a simple way by fluorescence readout at the whole‐cell level. B) Discrimination of cancer cells from normal cells by “click‐to‐sense” probe 1. Eleven cell lines were treated with our probe at 37 °C for 30 min. The cells were fixed and fluorescence was recorded by SpectraMax M2e, Molecular Devices. Fluorescence images of cells after incubation with 22.5 × 10−6 m of probe 1 (see Figure S1, Supporting Information, for images after treatment with other concentrations of 1). (a) TIG3; (b) HUVEC; (c) MCF10A; (d) SKBR3; (e) MDA‐MB‐231; (f) BxPC3; (g) HT29; (h) MCF7; (i) A549; (j) HeLa S3; and (k) PC3. The scale bar indicates 10 µm. C) Fluorescence intensity observed for each cell lines at the concentration range of 0–22.5 × 10−6 m of probe 1. Left to right: TIG3, HUVEC, MCF10A, SKBR3, MDA‐MB‐231, BxPC3, HT29, MCF7, A549, HeLa S3, and PC3. Fluorescence intensity was normalized as that emitted by 10 000 cells. D‐i) Total fluorescence intensity of HeLa S3 cell lysates (see method in Supporting Information); (ii) the SDS–PAGE images by fluorescence detection of HeLa S3 cell lysates treated with 10 × 10−6 m of probe 1 and control probe without azide group (see also Figure S2, Supporting Information). Cell constituents are labeled by the probe 1 based on the mechanism described in (A); (iii) the SDS–PAGE by coomassie staining (see method in Supporting Information). E) (i) ROS and (ii) FDP‐lysine analysis of the lysates of the selected cell lines (left to right: TIG3, BxPC3, A549, HeLa S3, and PC3). These methods (see Supporting Information) cannot selectively detect cancer.
Figure 3
Figure 3
A) Schematic procedure for labeling live tissues (breast cancer stumps) with probe 1. B) (i) A Tissue Matrix Chamber was used to slice fresh tissue; (ii) Keyence fluorescence microscope BZ‐X710; (iii) Optical sectioning algorithm system. C) The BZ‐X710 uses structured excitation light to scan the specimen, making it possible to capture clear images with no fluorescence blurring. (i) Excitation light from a metal halide lamp is passed through an electronic projection element, which projects the light onto the specimen in a grid pattern. The grid is only projected onto the focused areas of the specimen. (ii) Moving the grid causes the specimen to be scanned, allowing the capture of multiple images of the specimen. (iii) Extracting only the areas that the grid is projected onto from the multiple captured images prevents the effect of fluorescence blurring in the vertical direction. This produces clear images in which only the signal from the focused surface is extracted. Reproduced with permission. Copyright 2018, KEYENCE CORPORATION.
Figure 4
Figure 4
Microscopic images of live breast cancer (IDC, DCIS, DH) and normal breast gland (NBG) tissues labeled by “click‐to‐sense” probe 1 [for 5 min at 5 × 10−6, 10 × 10−6, and 20 × 10−6 m (red)] and Hoechst (blue). A) Pictures and double‐staining microscopic images of (i) IDC and NBG, (ii) DCIS and NBG. We defined ROI as shown in Figure S3 (Supporting Information). B) Statistical analysis of cancer sensitivity and selectivity among IDC, DCIS, DH, and NBG. Statistical test: P‐values, Mann‐Whitney U‐test; Bars, median. C) Expansion of images; Morphology of IDC, DCIS, DH, and NBG can clearly be detected by 20× and 200× (highly magnified fluorescent images) being labeled by probe 1 at 20 × 10−6 m. The detection of cancer morphology was consistent with H&E staining images of the frozen sections prepared from the same anonymized tissue samples. D) Analyzing 30 live cancer tissues (20 IDC and 10 DCIS), 30 adjacent live NBG tissues, and 5 adjacent live DH tissues from 30 breast cancer patients. Comparison of morphological analysis between “click‐to‐sense” method (by directly imaging the 1‐labeled live tissues by microscopy (20×) and (200×)) with conventional H&E pathological method, performed by pathologist in an anonymized form. H&E analysis was performed by preparing the frozen sections from the same tissue samples. E) Confocal microscopic images (400×) of the frozen sections from the same samples prepared in (C). For these experiments, we made a few slices from the same tumor block, and imaged the tumor cells using one slice for our “click‐to‐sense” and another for H&E methods. They look slightly different by the different sections, that is the difference caused by the “thickness” of the sections (each consecutively taken from the tumor block by 6 µm), but they are clinically regarded as the same section staining. We defined ROI for the analysis. F) Statistical analysis of cancer cell sensitivity and selectivity among IDC, DCIS, DH, and NBG. Statistical test: P‐values, Mann‐Whitney U‐test; Bars, median.

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