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Rapid intraoperative visualization of breast lesions with γ-glutamyl hydroxymethyl rhodamine green.

Ueo H, Shinden Y, Tobo T, Gamachi A, Udo M, Komatsu H, Nambara S, Saito T, Ueda M, Hirata H, Sakimura S, Takano Y, Uchi R, Kurashige J, Akiyoshi S, Iguchi T, Eguchi H, Sugimachi K, Kubota Y, Kai Y, Shibuta K, Kijima Y, Yoshinaka H, Natsugoe S, Mori M, Maehara Y, Sakabe M, Kamiya M, Kakareka JW, Pohida TJ, Choyke PL, Kobayashi H, Ueo H, Urano Y, Mimori K - Sci Rep (2015)

Bottom Line: Cleavage of the probe by GGT generates green fluorescence.We found that fluorescence derived from cleavage of gGlu-HMRG allowed easy discrimination of breast tumors, even those smaller than 1 mm in size, from normal mammary gland tissues, with 92% sensitivity and 94% specificity, within only 5 min after application.We believe this rapid, low-cost method represents a breakthrough in intraoperative margin assessment during breast-conserving surgery.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Surgery, Kyushu University Beppu Hospital, 4546 Tsurumihara, Beppu 874-0838 [2] Department of Surgery and Sciences, Graduate School of Medical Sciences, Kyushu University, Fukuoka 812-8582.

ABSTRACT
We previously developed γ-glutamyl hydroxymethyl rhodamine green (gGlu-HMRG) as a tool to detect viable cancer cells, based on the fact that the enzyme γ-glutamyltranspeptidase (GGT) is overexpressed on membranes of various cancer cells, but is not expressed in normal tissue. Cleavage of the probe by GGT generates green fluorescence. Here, we examined the feasibility of clinical application of gGlu-HMRG during breast-conserving surgery. We found that fluorescence derived from cleavage of gGlu-HMRG allowed easy discrimination of breast tumors, even those smaller than 1 mm in size, from normal mammary gland tissues, with 92% sensitivity and 94% specificity, within only 5 min after application. We believe this rapid, low-cost method represents a breakthrough in intraoperative margin assessment during breast-conserving surgery.

No MeSH data available.


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(a) Immunohistochemical staining of MCF-7 cell line for GGT. (b) Fluorescence image of MCF-7 cells obtained after administration of gGlu-HMRG. A differential interference contrast (DIC) image (left), a gGlu-HMRG fluorescence image (middle), and an image of cells pretreated with siRNA targeting GGT before application of gGlu-HMRG (right). (c) Time-dependent change of fluorescence intensity in MCF-7 cell line and MCF-7 cell line pretreated with siRNA targeting GGT. (d~f) Time-dependent fluorescence images of various breast tumor specimens [invasive ductal carcinoma (d), DCIS (e), intraductal papilloma (f)] after administration of gGlu-HMRG probe. In each of the specimens shown in (d–f), the time-dependent fluorescence intensities were measured at tumor lesions, normal mammary gland regions and fat (right column). (g, h) Comparisons of fluorescence localization (d and e) with pathological HE staining of the same specimen. (g) The cancer region is enclosed by a dotted line in the HE-staining image. Areas of increased fluorescence coincided well with pathologically cancerous region. Red arrows in (h) show fluorescence-positive area and malignant lesions. (i) Comparison of fluorescence increases (FI) after administration of gGlu-HMRG in breast lesions and normal tissues.
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f1: (a) Immunohistochemical staining of MCF-7 cell line for GGT. (b) Fluorescence image of MCF-7 cells obtained after administration of gGlu-HMRG. A differential interference contrast (DIC) image (left), a gGlu-HMRG fluorescence image (middle), and an image of cells pretreated with siRNA targeting GGT before application of gGlu-HMRG (right). (c) Time-dependent change of fluorescence intensity in MCF-7 cell line and MCF-7 cell line pretreated with siRNA targeting GGT. (d~f) Time-dependent fluorescence images of various breast tumor specimens [invasive ductal carcinoma (d), DCIS (e), intraductal papilloma (f)] after administration of gGlu-HMRG probe. In each of the specimens shown in (d–f), the time-dependent fluorescence intensities were measured at tumor lesions, normal mammary gland regions and fat (right column). (g, h) Comparisons of fluorescence localization (d and e) with pathological HE staining of the same specimen. (g) The cancer region is enclosed by a dotted line in the HE-staining image. Areas of increased fluorescence coincided well with pathologically cancerous region. Red arrows in (h) show fluorescence-positive area and malignant lesions. (i) Comparison of fluorescence increases (FI) after administration of gGlu-HMRG in breast lesions and normal tissues.

Mentions: First, we confirmed that activation of gGlu-HMRG fluorescence occurred in four types of BC cell lines (MCF7, MDA-MB-231, SK-BR-3 and CRL-1500) and one normal breast epithelium cell line (HMEC). Although HMEC is normal mammary gland cell line, it was immortalized and expressed GGT protein that was not expressed in normal mammary gland in vivo. Indeed, all five cell lines expressed GGT protein and large fluorescence increase was detected by means of fluorescence microscopy in all five-cell lines after application of gGlu-HMRG in the culture medium (Fig. 1a,b for MCF7; Supplementary Fig. 1 for the others). In accordance with our previous findings10, fluorescence was detected within 20 minutes after gGlu-HMRG administration and the signal continued to increase time-dependently (Fig. 1c). Further, the use of siRNA to knock down GGT blocked the fluorescence increase (Fig. 1b,c).


Rapid intraoperative visualization of breast lesions with γ-glutamyl hydroxymethyl rhodamine green.

Ueo H, Shinden Y, Tobo T, Gamachi A, Udo M, Komatsu H, Nambara S, Saito T, Ueda M, Hirata H, Sakimura S, Takano Y, Uchi R, Kurashige J, Akiyoshi S, Iguchi T, Eguchi H, Sugimachi K, Kubota Y, Kai Y, Shibuta K, Kijima Y, Yoshinaka H, Natsugoe S, Mori M, Maehara Y, Sakabe M, Kamiya M, Kakareka JW, Pohida TJ, Choyke PL, Kobayashi H, Ueo H, Urano Y, Mimori K - Sci Rep (2015)

(a) Immunohistochemical staining of MCF-7 cell line for GGT. (b) Fluorescence image of MCF-7 cells obtained after administration of gGlu-HMRG. A differential interference contrast (DIC) image (left), a gGlu-HMRG fluorescence image (middle), and an image of cells pretreated with siRNA targeting GGT before application of gGlu-HMRG (right). (c) Time-dependent change of fluorescence intensity in MCF-7 cell line and MCF-7 cell line pretreated with siRNA targeting GGT. (d~f) Time-dependent fluorescence images of various breast tumor specimens [invasive ductal carcinoma (d), DCIS (e), intraductal papilloma (f)] after administration of gGlu-HMRG probe. In each of the specimens shown in (d–f), the time-dependent fluorescence intensities were measured at tumor lesions, normal mammary gland regions and fat (right column). (g, h) Comparisons of fluorescence localization (d and e) with pathological HE staining of the same specimen. (g) The cancer region is enclosed by a dotted line in the HE-staining image. Areas of increased fluorescence coincided well with pathologically cancerous region. Red arrows in (h) show fluorescence-positive area and malignant lesions. (i) Comparison of fluorescence increases (FI) after administration of gGlu-HMRG in breast lesions and normal tissues.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4499838&req=5

f1: (a) Immunohistochemical staining of MCF-7 cell line for GGT. (b) Fluorescence image of MCF-7 cells obtained after administration of gGlu-HMRG. A differential interference contrast (DIC) image (left), a gGlu-HMRG fluorescence image (middle), and an image of cells pretreated with siRNA targeting GGT before application of gGlu-HMRG (right). (c) Time-dependent change of fluorescence intensity in MCF-7 cell line and MCF-7 cell line pretreated with siRNA targeting GGT. (d~f) Time-dependent fluorescence images of various breast tumor specimens [invasive ductal carcinoma (d), DCIS (e), intraductal papilloma (f)] after administration of gGlu-HMRG probe. In each of the specimens shown in (d–f), the time-dependent fluorescence intensities were measured at tumor lesions, normal mammary gland regions and fat (right column). (g, h) Comparisons of fluorescence localization (d and e) with pathological HE staining of the same specimen. (g) The cancer region is enclosed by a dotted line in the HE-staining image. Areas of increased fluorescence coincided well with pathologically cancerous region. Red arrows in (h) show fluorescence-positive area and malignant lesions. (i) Comparison of fluorescence increases (FI) after administration of gGlu-HMRG in breast lesions and normal tissues.
Mentions: First, we confirmed that activation of gGlu-HMRG fluorescence occurred in four types of BC cell lines (MCF7, MDA-MB-231, SK-BR-3 and CRL-1500) and one normal breast epithelium cell line (HMEC). Although HMEC is normal mammary gland cell line, it was immortalized and expressed GGT protein that was not expressed in normal mammary gland in vivo. Indeed, all five cell lines expressed GGT protein and large fluorescence increase was detected by means of fluorescence microscopy in all five-cell lines after application of gGlu-HMRG in the culture medium (Fig. 1a,b for MCF7; Supplementary Fig. 1 for the others). In accordance with our previous findings10, fluorescence was detected within 20 minutes after gGlu-HMRG administration and the signal continued to increase time-dependently (Fig. 1c). Further, the use of siRNA to knock down GGT blocked the fluorescence increase (Fig. 1b,c).

Bottom Line: Cleavage of the probe by GGT generates green fluorescence.We found that fluorescence derived from cleavage of gGlu-HMRG allowed easy discrimination of breast tumors, even those smaller than 1 mm in size, from normal mammary gland tissues, with 92% sensitivity and 94% specificity, within only 5 min after application.We believe this rapid, low-cost method represents a breakthrough in intraoperative margin assessment during breast-conserving surgery.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Surgery, Kyushu University Beppu Hospital, 4546 Tsurumihara, Beppu 874-0838 [2] Department of Surgery and Sciences, Graduate School of Medical Sciences, Kyushu University, Fukuoka 812-8582.

ABSTRACT
We previously developed γ-glutamyl hydroxymethyl rhodamine green (gGlu-HMRG) as a tool to detect viable cancer cells, based on the fact that the enzyme γ-glutamyltranspeptidase (GGT) is overexpressed on membranes of various cancer cells, but is not expressed in normal tissue. Cleavage of the probe by GGT generates green fluorescence. Here, we examined the feasibility of clinical application of gGlu-HMRG during breast-conserving surgery. We found that fluorescence derived from cleavage of gGlu-HMRG allowed easy discrimination of breast tumors, even those smaller than 1 mm in size, from normal mammary gland tissues, with 92% sensitivity and 94% specificity, within only 5 min after application. We believe this rapid, low-cost method represents a breakthrough in intraoperative margin assessment during breast-conserving surgery.

No MeSH data available.


Related in: MedlinePlus