Rapid visualization of mammary gland tumor lesions of dogs using the enzyme-activated fluorogenic probe; γ-glutamyl hydroxymethyl rhodamine green.

Since gamma-glutamyl transpeptidase (GGT) is highly and locally expressed in human breast cancer, a GGT-enzymatically activatable fluorescent probe, gamma-glutamyl hydroxymethyl rhodamine green (gGlu-HMRG), has been developed to detect the human breast cancer area with high performance. In this study, GGT expression and the efficacy of gGlu-HMRG on visualization were investigated in canine mammary gland tumors (MGT). Seventeen non-fixed fresh-frozen MGT specimens and each peritumoral control tissue were utilized. The GGT mRNA levels were highly observed in the tumor specimens compared with the control. GGT immunostaining was mostly observed on the cell membrane and cytosol of the alveolar and duct mammary epithelium of MGT tissues. These signals were strongly positive in several cases while they were mild to not observed in other cases. When gGlu-HMRG solution was dropped to the non-fixed tissue pieces of MGT or control tissues, the fluorescence intensities (FIs) were measured using Maestro in-vivo imaging device. FIs in MGT tissues were significantly higher than each control tissue 20 min after treatment. Based on Youden index method, the maximum sensitivity and specificity of FI was 82.4% and 82.4%. These findings suggest that GGT is highly expressed in several MGTs in dogs and gGlu-HMRG could visualize at least a part of MGT tissues in dogs. Nevertheless, it should be needed to assess the false-negative areas more carefully in canine than human cases.

Mammary gland tumor (MGT) is the most common tumor in female dogs. Approximately 50% of them are malignant [16] and 58% of those which had regional mastectomy have a recurrence in the ipsilateral mammary chain until one year after the first surgery [21]. Based on 2016 AAHA oncology guidelines, wide surgical excision with around 2 cm margins for single malignant MGT is recommended in dogs [2]. Therefore, it is important to determine the precise margins for appropriate tumor resection [2, 20]. Although intraoperative frozen section analysis is quite effective to make a rapid diagnosis and consider surgical margins [6], it is not feasible in terms of manpower, cost and time to conduct total-circumferential examination especially in the veterinary field. Then, other methods to visualize tumor lesions and determine the appropriate margins for surgery are eagerly being developed.

As a new option of intraoperative examination to guide the appropriate points of surgery, enzyme-activated fluorescent reagents are being developed [5]. These reagents are reported to detect specific cell types with high sensitivity, portability, real-time capabilities and absence of ionizing radiation [25]. In human, gamma-glutamyl transpeptidase (GGT), one of the hydrolytic enzymes, is mainly expressed on the membrane of the breast cancer cells [9]. Based on this characteristic, gamma-glutamyl hydroxymethyl rhodamine green (gGlu-HMRG), a GGT-enzymatically activatable fluorescent probe, was attempted to detect human breast cancer tissues [24]. This reagent is chemically produced based on fluorophore rhodamine green. It does not have fluorescence under the intact condition in most organs including human normal mammary gland and comes out the high green fluorescent color by cleavage of a GGT-specific action site. According to Urano et al., fluorescent activation of gGlu-HMRG by GGT occurred on the various human breast cancer types [24].

In this study, therefore, the efficacy of gGlu-HMRG on visualization of canine MGT was investigated. To examine the expression of GGT in canine MGT, the quantity of GGT mRNA was determined by qPCR and its distribution was observed using immunohistochemistry. The efficacy of gGlu-HMRG was investigated by fluorescence test using not-fixed frozen canine MGT tissues.

MATERIALS AND METHODS

Sample collection

All specimens in this study were obtained from the canine MGT patients at the University of Tokyo-Veterinary Medical Center (UT-VMC) from 2014 to 2017. The tumor and perilesional normal tissues were taken intraoperatively and separated in half respectively. A half of them were fixed in formalin and embedded paraffin for pathological assessment and immunohistochemical staining. The other pieces of them were used for fresh-frozen tissues for to evaluate GGT mRNA expression and the effect of gGlu-HMRG. They were carved out into several small pieces and immediately stored at −80°C. The perilesional normal tissues,mammary and epidermal tissues, were confirmed based on the pathological assessment and utilized as control. Some tumors, which could not be divided in half due to the small size, were excluded from this study.

The qPCR method

Unfixed fresh-frozen tissue pieces were used. The expression levels of GGT mRNA of the tumors and controls were quantified by qPCR method. Total RNA of a tissue specimen was extracted using RNeasy Mini Kit (QIAGEN, Hilden, Germany). The total RNA was reverse-transcripted using ReverTra Ace qPCR RT Master Mix (TOYOBO, Osaka, Japan). Canine GGT and Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA levels as an internal control were quantified with the use of qPCR method. The qPCR method was performed by StepOnePlus (Applied Biosystems, Forster City, CA, USA) using Thunderbird SYBR qPCR Mix (TOYOBO). The conditions for qPCR were as follows: 95°C for 10 min, 40 cycles of 95°C for 15 sec and 60°C for 1 min. Primer sequences used for this experiment are shown in Table 1. All reactions were replicated, and each primer set had the same single peak of melting curve in all the samples. The quantification was performed by StepOne software v2.3 based on the standard curve which was established by cDNA of kidney tissue from one healthy dog. The GGT mRNA expression levels of tissues were normalized using GAPDH as an internal control [3, 11, 12, 22].

Table 1.

Primer Sequences for qPCR

Primer set		Sequence (5′ to 3′)	Position	Annotation No.
GGT	Forward	GGC TTT TGT TGC CAT CCC TG	293–531	XM_005636554.3
	Reverse	ACA TCC CTC CCA ATC TCC GA
GAPDH	Forward	GCT GCC AAA TAT GAC GAC ATCA	748–822	NM_001003142
	Reverse	GTA GCC CAG GAT TTT GAG

Immunohistochemical staining

Expression of GGT protein was observed by immunohistochemical staining. Paraffin sections were deparaffinized and subsequently rehydrated. The sections were activated with Tris-EDTA (pH 9.0) at 98°C for 30 min and at room temperature for 30 min. Endogenous peroxidase activity was blocked with Dako Rael Peroxidase-Blocking (Agilent, Santa Clara, CA, USA) for 10 min at room temperature. Non-specific protein binding was blocked with 8% skim milk for 40 min at 37°C. Then, sections were incubated with primary antibodies at 4°C overnight. The primary antibody used was GGT1 antibody (3E6) sc-100746 (Santa Cruz Biotechnology, Santa Cruz, CA, USA, diluted at 1:500). The efficacy of the antibody for canine GGT was confirmed using canine kidney tissue lysate which has much of GGT (data not shown). The sections were visualized by using Dako EnVision + System (Agilent) and Dako Liquid DAB + Substrate Chromogen System (Agilent). The sections were also counterstained with hematoxylin.

GGT immunoresponse intensity was subjectively evaluated from (−) to (+++) based on the previous study [7]. The scoring system used was: -, no GGT immune response; +, rare to 10% of GGT-positive tumor cells; ++, 10% to less than one-half of tumor cells were GGT-positive; +++, over one-half of the tumor cells were GGT-positive.

Efficacy of gGlu-HMRG for visualization of canine MGT

Fluorescence intensity (FI) after tissues dipping into gGlu-HMRG solution was evaluated. Unfixed fresh-frozen tissues were cut into approximately 30 mm3 piece and put on the eight-cell chamber slides (Ibidi, Munich, Bavaria, Germany) one by one. After they were brought back to room temperature, around 300 μl of gGlu-HMRG solution, final concentration 50 μM at 0.5% v/v DMSO/PBS, was dropped into the chamber to soak the tissue specimens completely. Fluorescence images were tracked and FI were measured before and 20 min after administration of gGlu-HMRG with Maestro in-vivo imaging system (Cambridge Research & Instrumentation, Inc., Hopkinton, MA, USA). Excitation was 420 nm and the observation filter with 515-nm long-pass emission was used. To evaluate FI, the regions of interests (ROIs) along the outline of each tissue were collected and the average of FI of each ROI was calculated with the Maestro software.

Statistical analysis

Statistical analysis was performed using XLSTAT Life Science 2019 (Addinsoft Inc., New York, NY, USA). Wilcoxon signed-rank test and Mann-Whitney U test were used to assess the mRNA expression levels and FI intensities of the tumor and control samples. Pearson’s correlation test was used for assessing correlation between mRNA and FI values. Kruskal Wallis and Steel-Dwass tests were performed to evaluate the relationship between mRNA, GGT immunoresponse levels and FI value. A P-value of less than 0.05 was considered to indicate a statistically significant difference.

RESULTS

Included animal profiles

In total, seventeen MGT tissues were included in this study. The median of the patient ages was 12 and a half years old (range; 8 years to 12 years 8 months old). Five of the patients were spayed female and the rest of them were intact female. No dogs had experience of pregnancy or had estrus in the perioperative period. The breeds of the patients were Miniature Dachshund (n=4), Welsh Corgi Pembroke (3), Papillon (2), American Cocker Spaniel (1), Chihuahua (1), Maltese (1), Miniature Schnauzer (1), and mixed (4). Their MGT tissues had been histopathologically diagnosed by the veterinary pathologists as follows; twelve malignant epithelial neoplasms which were complex (7) and simple (5) type carcinomas, and five mammary as benign epithelial neoplasms which were complex (3), and simple (2) type adenomas.

Expression activity of GGT in canine MGT

The gGlu-HMRG method might be established in the future using the evaluation criteria as the FI absolute values, or as the relative intensity compared to the peritumor tissues. Therefore, both of the statistical analyses for paired and unpaired samples were performed in this study (Figs. 1 and 2). To evaluate the transcriptional level of GGT in canine MGT tissues, qPCR was performed as shown in Fig. 1A. The expression level of GGT mRNA was highly observed in the tumor specimens compared with the peritumoral control samples (P<0.01 for both of Wilcoxon signed rank test and Mann-Whitney U test). GGT immunoresponse was also mostly observed on the cell membrane and cytosol of the alveolar and duct mammary epithelium of MGT tissues (Fig. 1B). The signals were strongly positive in the six cases while the signals existed mildly or weekly in the six cases and did not exist in the five cases. There were apparently no signals in the normal epithelial cells of all the peritumor control tissues or the connective tissues (Fig. 1B, 1C). The signal intensity scores of each subtype of MGT were shown as Table 2. There was no significant tendency correlated with malignancy, stages or grades.

Fig. 1.

Gamma-glutamyl transpeptidase (GGT) expression in canine mammary gland tumor tissues. GGT mRNA levels in the tumor tissues were compared with their peritumoral control tissues (A). Red and blue symbols mean the malignant (n=12) and benign (n=5) tumors, respectively. An asterisk indicates statistical significance (P<0.05). The representative immunohistochemistry images of the tumoral area (+++) and peritumoral control area (−) are shown as B and C. A scale bars mean 250 µm (white) and 20 µm (black).

Fig. 2.

Fluorescent intensity of gamma-glutamyl hydroxymethyl rhodamine green (gGlu-HMRG) with canine mammary gland tumor tissue pieces. The photos were representative images of time-lapse fluorescence observation (A). The leftmost photos were taken under the visible light. The others were taken through the 515-nm long-pass emission filter. They were obtained 0 (before), 5, 10, and 20 min after treatment of gGlu-HMRG solution. FI intensities in the 17 tumor tissues were compared with their own peritumoral control tissues (B). Red and blue symbols mean the malignant (n=12) and benign (n=5) tumors, respectively. An asterisk indicates statistical significance (P<0.05). The receiver operating characteristic curve is shown as C.

Table 2.

The immunoresponse intensity of gamma-glutamyl transpeptidase

Classification		Total	Immunoresponsive score
			-	+	++	+++
Adenoma	Simple	2	-	-	-	2
	Complex	3	1	1	-	1
Carcinoma	Simple	4	3	-	-	1
	Complex	8	1	2	3	2
		Total	5	3	3	6

To evaluate the efficacy of gGlu-HMRG for visualization of canine MGT tissues, the probe reagent was dripped into each well of chamber slides with the fresh tissue pieces. As shown in Fig. 2, FI values in MGT tissues was significantly increased comparing with each control tissues within 20 min (P<0.01 for both of Wilcoxon signed rank test and Mann-Whitney U test). When the receiver operating characteristic (ROC) curve was calculated based on the individual values of FI, the area under the curve (AUC) was 0.834 to distinguish the tumor tissues with the control tissues. Based on Youden index method said that the maximum sensitivity and specificity was 82.4% and 82.4% (cut off FI=0.30).

Correlation between GGT mRNA levels, protein expression, and Glu-HMRG FI values

To confirm whether gGlu-HMRG fluorescent intensity reflect GGT expression levels, the correlation between FI values and GGT mRNA levels or immunoresponse intensitiy was analyzed. All the measurement values of the tumor and peritumor tissues were used for the unpaired independent samples. As shown Fig. 3, logarithmic values of GGT mRNA levels were mildly correlated with FI values (R=0.686, P>0.01). GGT immune-signal intensity was also positively correlated with mRNA levels (P>0.05 for (−) vs. (+++)) and FI values (P>0.01 for (−) vs. (+++)).

Fig. 3.

Correlation between gamma-glutamyl hydroxymethyl rhodamine green (gGlu-HMRG) fluorescence intensity and gamma-glutamyl transpeptidase (GGT) expression. The scattered plots of fluorescence intensity with mRNA logarithmic values and subjective scores of GGT immunoresponsive intensity were shown (A, B, and C). Red, blue and black symbols mean the malignant (n=12), benign (n=5) mammary gland tumor tumors and peritumoral control tissue pieces (n=17), respectively. A dotted circle was 95% confidence interval and a solid line was regression line (A) (R=0.686). Horizontal lines were the median (B and C). Asterisks indicate statistical significance (P<0.05).

DISCUSSION

The novel GGT-enzymatically activatable fluorescent probe, named as gGlu-HMRG, has being developed in the human clinical oncology field [13, 18, 23, 24]. Although it has not been established yet, clinical application of gGlu-HMRG for visualization of human breast cancer is the on-going project to decide the margin area on the surgery. Since there has not been any evidence of GGT expression in canine MGT, GGT activity in canine MGT was observed in this study. GGT mRNA and protein were highly expressed in a part of MGT tissues, and they were not expressed in any peritumor normal tissues. To our knowledge, this is a first report indicating GGT expression in canine MGT. In addition, gGlu-HMRG fluorescent intensity was positively correlated with GGT mRNA and immunohistochemistry levels. It is suggested that gGlu-HMRG fluorescence could be dependent to GGT expression and useful to visualize at least a part of MGT tissues in dogs.

Although GGT expression intensity in canine MGT tissues was statistically high compared with the normal mammary gland, there was variability among the individual samples. Several cases did not have substantial GGT expression intensities. Although it was not precisely checked using the double immunochemical method, GGT was expressed in most regions of the FI positive tumor cells, but not all the areas. In addition, the relationship between GGT expression intensity and the malignancy or subtypes of canine MGT classification was not found. Indeed, these characteristics are partially consistent with the previous reports of human breast cancer. GGT expression are quite variable in complex and simple breast carcinoma in human [4]. GGT is also expressed in several regions of human breast cancer cells, but not all tumor areas [22]. In addition, the measurement values of FI using the gGlu-HMRG method could not distinguish malignant and benign breast cancers [19, 22]. However, FI values were substantially strong to distinguish almost all human breast cancers and normal tissues [19, 22], which is different from our present canine study. While similarity of the human and dog mammary tumor has been discussed, there are several differences of the tumor pathophysiology between species [1], which could be one of the reasons. Although it cannot be concluded because of the small sample size, it is suggested that there could not be fundamental difference between human and dogs, and canine MGTs could express GGT more variably than human cases.

Sensitivity and specificity based on the FI values are important for clinical application of gGlu-HMRG. In the previous human reports, the sensitivity and specificity were so high as 92% and 94% when the ROC curves were constructed using the data at 20 min after gGlu-HMRG dropping to the tissue pieces [19, 22]. In this study, the most effective cut-off values calculated by Youden index method was 0.30 and the sensitivity and specificity were 82.4% and 82.4%, which was lower than the human report [19, 22]. These characteristics could be the biggest weak point when the surgical margin is rapidly diagnosed using this method in the future. It is mandatory to reduce the false-negative areas using some appropriate method for establishment of this method as a clinical diagnostic test such as evaluating the relative ratio, treating to avoid autofluorescence, improving or replacing the fluorescent measurement system, gathering other examination results.

Over expression of GGT has been observed in human other tumor tissues such as hepatocellular carcinoma and intrahepatic cholangiocarcinoma [14]. Then, in human various cancers, the efficacy of gGlu-HMRG on cancer imaging has been demonstrated. In human head and neck cancer [15], lung cancer [8], primary colorectal cancer [17], pancreatic cancer [10] and oral cancer [18] has been evaluated. In these studies, fluorescence imaging using gGlu-HMRG is reported to be able to identify these tumors with the high sensitivity and specificity. We don’t have substantial data for other tumor tissues in dogs because of the small sample sizes. However, gGlu-HMRG dripping tended to enhance the fluorescence of tumor tissues besides canine MGT such as pulmonary adenocarcinoma, squamous carcinoma and GIST in dogs (data not shown). The gGlu-HMRG reagent might be also useful in some other specific tumor types than MGT in dogs although future studies are needed.

There are several limitations to this study. The sample size for this study was quite small to consider the relationship of FI values to the grade or classification of the mammary gland tumor or conclude the clinical feasibility of this method. Only one gene, GAPDH was used as the internal control for qPCR. The tissue samples in this study were fresh-frozen tissues, not completely fresh. In addition, they were just only approximately 30 mm3, not full size of the excised lesions. It is needed to improve the sensitivity and specificity of this method for the clinical approach.

In conclusion, this study indicates the efficacy of the specific hydrolytic enzyme-activated fluorescent probe for canine MGTs. Successful oncologic procedures rely on the rapid and accurate localization of cancerous tissues followed by their complete resection or ablation. The achievement of this study leads to establishment of the new method for a useful intraoperative navigation tool.

CONFLICT OF INTEREST

The authors declare that there is no conflict of interest.