運用非線性光學顯微術觀察腫瘤環境

Abstract

治療腫瘤的方法日新月異，傳統的化學治療不僅殺死腫瘤細胞且會傷害到正常組織，標靶治療藉著專一抗體來辨識腫瘤細胞上特有生物標記，進行選擇性的藥物輸送，可減少傳統化療的副作用。但因腫瘤具有基因表現的多樣性，即使發生在同一個器官上，這些標記也不見得一致。由於標靶治療仍有上述盲點，直接毒殺腫瘤細胞常會無法根除而引起基因突變，這也是產生抗藥性的主要原因。為了解決這個問題，近來科學家努力研究腫瘤成長所必需仰賴的微環境，希望能藉由阻斷腫瘤微環境有關的信息途徑，來抑制腫瘤的生長並防止腫瘤轉移。也因此，研究了解活體內腫瘤與周遭微環境的分子互動過程，是腫瘤信息途徑標靶治療一項重要的基礎研究工作。 本論文利用極低侵入性的鉻貴橄欖石飛秒脈衝雷射，建構一個小動物活體內長時間連續觀察腫瘤微環境的非線性光學顯微術平台，透過二倍頻、三倍頻、以及多光子螢光等生物體內的非線性光學對比，以活體虛擬切片的方式，觀察深埋皮下的腫瘤細胞與被改造的膠原蛋白、免疫細胞的活動、新生血管及其通透度變化等。本論文觀察腫瘤微環境主要的三種互動情境，即腫瘤-基質、腫瘤-血管、與腫瘤-免疫系統的動態演變。我們預料藉著本論文發展出的活體內影像平台，結合螢光分子探針，追蹤信息途徑的時間與空間分布，將是洞察腫瘤分子生物學上很有力的工具，不僅可以用來更進一步了解腫瘤細胞是如何逐步改變其周邊微環境並利用其被改變的周邊微環境來達到腫瘤細胞快速轉移的目的，同時也可利用這技術來驗證並發展出更新的腫瘤治療策略。

Strategies of treatments on cancers evolve with the understanding of their epidemiology and biology. Traditional chemotherapies not only kill cancers, but also do harms to normal tissues. To selectively treat on cancer, target therapies can deliver drugs to the biomarkers over expressed on cancer cells through a specific antibody binding. Because of the polymorphism of genetic phenotype, markers of tumors are quite different even though they grow in the same organ. With this blind spot of chemotherapy, treatments on cancers can further induce the genetic mutation. Therefore, treating cancers with markers just on themselves is not enough. Recently, people start to search markers necessary for the development of tumors. Candidate targets could be the critical ligands or secondary messengers on the transduction pathways among tumor cells, extracellular matrices, blood vessels, and immune cells. Through the blocking of tumor-associated signal transduction in the microenvironment, such pathway-targeting therapy can arrest the tumor growth and prevent tumor metastasis. Therefore, the understanding of the interaction between tumors with surrounding microenvironment in vivo is an important first step and basis for pathway-targeting cancer therapy. In this thesis, we develop a small animal nonlinear microscopic imaging platform for in vivo, high-resolution, long-term, consecutive observation on tumor microenvironments. It was achieved with the least invasive femtosecond Cr:forsterite laser and a laser scanning microscope. Through this unique platform, we can in vivo investigate three major contexts of tumor microenvironments with sub-micron spatial resolution, that is, the remodeling of collagens, the trafficking of immune cells, and the permeability of neovasculatures. We anticipate thus developed platform with ongoing developments in molecular probes can be a powerful tool to reveal spatial-temporal dynamics of molecular pathways in tumor microenvironments, enhance our understanding of tumor biology, and trigger new therapeutic approaches.