活體多光子顯微術於癌症診斷之研究

Abstract

在本研究第一部份，我們運用雙光子螢光和二倍頻顯微術於判別診斷正常、肺腺癌、以及鱗狀上皮細胞癌等肺臟組織。除了從型態上判別罹癌與否，我們也發展一項量化指標︰氧化還原比率。細胞氧化還原比率的值越低，代表細胞的代謝能力越強，這通常是癌細胞的特徵。研究結果顯示結合多光子造影和氧化還原比率指標能有效判別正常與罹癌的肺臟組織。 除了診斷活體外組織的型態特徵之外，第二部份的研究內容將此技術推廣到活體內癌症造影與分析。利用活體多光子顯微術與自體螢光對二倍頻的比例指標(MAFSI)，可讓我們觀察到癌化的上皮細胞和細胞外基質的互動。隨著造影深度增加，正常和癌化皮膚組織兩者MAFSI的消長亦有所不同。既然癌化皮膚最後演變成鱗狀上皮細胞癌，此結果代表活體多光子顯微術可用於追蹤生理狀態的改變。 最後我們針對癌細胞侵襲和外滲等生理現象直接進行活體造影。所關注的課題包括:腫瘤微環境的動態資訊、轉移性結腸直腸癌細胞對肝膽代謝的影響、癌細胞和免疫細胞如白血球及庫氏細胞的互動。藉由活體多光子顯微術以及各種量化指標，我們對於癌化過程中對皮膚和肝臟生理狀況的影響有了更深入的了解，並希望在未來有助於改善癌症患者的治療與照顧。

In the first part of this study, we performed two-photon excited autofluorescence and second harmonic generation microscopy for the distinction of normal, lung adenocarcinoma, and squamous cell carcinoma specimens. In addition to morphological distinction, we derived quantitative metrics of cellular redox ratios for cancer discrimination. The lower redox ratios in cancer specimens, indicating an increase in metabolic activity. These results show that the combination of morphological multiphoton imaging along with redox ratio indices can be used for the discrimination of normal and pulmonary cancer tissues. After performing the capability of tissue characterization under ex vivo condition, the second segment of our work is to image and analyze normal and carcinogen treated skin tissues of nude mice in vivo. Using intravital images and the quantitative pixel to pixel ratiometric processing of multiphoton autofluorescence to second harmonic generation index (MAFSI), we can visualize the interaction between epithelial cells and extracellular matrix. We found that as the imaging depth increases, MAFSI has different distribution in normal and treated cutaneous specimens. Since the treated skin eventually became squamous cell carcinoma, our results show that the physiological changes to mouse skin en route to become cancer can be effectively tracked by intravital multiphoton microscopy. In the final section, we have directly visualized the in vivo physiological process such as cancer invasion and extravsation on skin and liver of the mouse animal model. We focused on studying the dynamic information of the cancer microenvironment, the effects of the metastatic colon tumor cells on hepatobiliary metabolism, and the interaction of the tumor cells with immune-cells such as leukocyte and Kupffer cells. In this manner, we have gained a better understanding of the process of carcinogenesis and influence on normal physiology of the skin and liver. The improved understanding may lead to improved treatment and care strategies for cancer patients in the future.