Noh Choong-Kyun, Lim Chang Su, Lee Gil Ho, Cho Myung Ki, Lee Hyo Won, Roh Jin, Kim Young Bae, Lee Eunyoung, Park Bumhee, Kim Hwan Myung, Shin Sung Jae
Department of Gastroenterology, Ajou University School of Medicine, Suwon, South Korea.
Department of Energy Systems Research and Department of Chemistry, Ajou University, Suwon, South Korea.
Front Oncol. 2021 Aug 27;11:634219. doi: 10.3389/fonc.2021.634219. eCollection 2021.
Endoscopy is the most important tool for gastric cancer diagnosis. However, it relies on naked-eye evaluation by endoscopists, and the histopathologic confirmation is time-consuming. We aimed to visualize and measure the activity of various enzymes through two-photon microscopy (TPM) using fluorescent probes and assess its diagnostic potential in gastric cancer.
β-Galactosidase (β-gal), carboxylesterase (CES), and human NAD(P)H: quinone oxidoreductase (hNQO1) enzyme activities in the normal mucosa, ulcer, adenoma, and gastric cancer biopsy samples were measured using two-photon enzyme probes. The fluorescence emission ratio at long and short wavelengths (Ch2/Ch1) for each probe was comparatively analyzed. Approximately 8,000 - 9,000 sectional images in each group were obtained by measuring the Ch2/Ch1 ratio according to the tissue depth. Each probe was cross-validated by measuring enzymatic activity from a solution containing lysed tissue.
Total of 76 subjects were enrolled in this pilot study (normal 21, ulcer 18, adenoma 17, and cancer 20 patients, respectively). There were significant differences in the mean ratio values of β-gal (0.656 ± 0.142 . 1.127 ± 0.109, < 0.001) and CES (0.876 ± 0.049 . 0.579 ± 0.089, < 0.001) between the normal and cancer, respectively. The mean ratio value of cancer tissues was different compared to ulcer and adenoma ( < 0.001). The hNQO1 activity showed no significant difference between cancer and other conditions. Normal mucosa and cancer were visually and quantitatively distinguished through β-gal and CES analyses using TPM images, and enzymatic activity according to depth, was determined using sectional TPM ratiometric images. The results obtained from lysis buffer-treated tissue were consistent with TPM results.
TPM imaging using ratiometric fluorescent probes enabled the discrimination of gastric cancer from normal, ulcer, and adenoma. This novel method can help in a visual differentiation and provide quantitative depth profiling in gastric cancer diagnosis.
内镜检查是胃癌诊断的最重要工具。然而，它依赖于内镜医师的肉眼评估，且组织病理学确认耗时。我们旨在通过使用荧光探针的双光子显微镜（TPM）可视化并测量各种酶的活性，并评估其在胃癌诊断中的潜力。
使用双光子酶探针测量正常黏膜、溃疡、腺瘤和胃癌活检样本中的β-半乳糖苷酶（β-gal）、羧酸酯酶（CES）和人NAD(P)H：醌氧化还原酶（hNQO1）的酶活性。对每个探针在长波长和短波长处的荧光发射率（Ch2/Ch1）进行比较分析。通过根据组织深度测量Ch2/Ch1比值，在每组中获得约8000 - 9000张断层图像。通过测量来自含有裂解组织的溶液的酶活性对每个探针进行交叉验证。
本初步研究共纳入76名受试者（分别为正常21例、溃疡18例、腺瘤17例和癌症20例患者）。正常组与癌症组之间β-gal（0.656±0.142. 1.127±0.109，<0.001）和CES（0.876±0.049. 0.579±0.089，<0.001）的平均比值分别存在显著差异。癌组织的平均比值与溃疡和腺瘤相比有所不同（<0.001）。hNQO1活性在癌症与其他情况之间无显著差异。通过使用TPM图像的β-gal和CES分析在视觉上和定量上区分了正常黏膜和癌症，并使用断层TPM比率图像确定了酶活性随深度的变化。从裂解缓冲液处理的组织获得的结果与TPM结果一致。
使用比率荧光探针的TPM成像能够区分胃癌与正常、溃疡和腺瘤。这种新方法有助于在胃癌诊断中进行视觉区分并提供定量深度分析。
