In vivo quantification of the structural changes of collagens and tumor angiogenesis permeability in the melanoma microenvironment with nonlinear optical microscopy

惡性腫瘤連續 30 年蟬聯國人十大死因首位。目前癌症的主要治療方式為手術、化學治療與放射治療，但這些療法不只是治療腫瘤細胞亦會傷害許多正常細胞。為了能較專一性地治療腫瘤細胞，近年來發展出了標靶治療。近期許多學者把腫瘤微環境做為發展新的抗癌標靶治療的可能標的，因此本研究的目標為探討黑色素細胞癌微環境中膠原蛋白重組、新生血管、血管滲漏及腫瘤幹細胞的交互關係。腫瘤微環境中的膠原蛋白纖維，在正常組織下扮演調節細胞間溝通的角色，也被發現與腫瘤轉移有關，因此也能成為判斷腫瘤預後的指標之一。為了在活體內能依時序追蹤黑色素細胞癌長大過程中微環境之膠原蛋白變化，我們採用二倍頻及三倍頻顯微術來做觀察。並且在沒有染色情況下，發展方向指數、灰階共生矩陣、及二三倍頻訊號比等指標來量化膠原蛋白變化。我們發現黑色素細胞癌附近之膠原蛋白在早期的方向指數及二倍頻訊號較高;而在晚期則較呈多方向性且分布不均勻。此外，為了能在活體中觀察血管滲漏與黑色素細胞癌幹細胞之間的關係，我們將表現mCherry紅色螢光及以ALDH1A1啟動子調控表現綠色螢光蛋白的細胞株種入C57BL/6-c2J小鼠耳朵且透過非線性光學系統來進行觀察。我們透過小鼠尾靜脈打入150 kD (8.9 nm)葡聚醣，以葡聚醣所散發的螢光訊號來觀察及分析腫瘤新生血管的滲漏情形，結果顯示，腫瘤組織中黑色素細胞癌腫瘤幹細胞集中區域具有較高的血管滲透率。此外，我們也發現這些高滲漏之血管大多為動脈血管，且新生血管密度會隨著腫瘤生長而增加。Malignant neoplasm is the leading cause of death for 30 years in a row in Taiwan. The major approaches for cancer treatment are surgery, chemotherapy, and radiation therapy, but these approaches not only can hurt all kinds of cancers but also do harm to the normal cells. In order to kill cancer cells specifically, targeted therapy is developed. Many researchers focused on the tumor microenvironment as a target for cancer treatment recently and they found it would be an effective and potential target. The goal of this thesis is to investigate the relationship between collagen remodeling, angiogenesis, vessel permeability and cancer stem cells (CSC) in the microenvironment of melanoma. In tumor microenvironment, the collagen fibers in normal tissues play a role in the regulation of cell communication. Recent studies found collagen fibers surrounding cancer cells may regulate cancer metastasis, indicating collagen fibers might be used as indicators for cancer prognosis. We tracked the collagen remodeling in melanoma microenvironment by using in vivo second harmonic generation (SHG) and third harmonic generation (THG) microscopy. The corresponding structural and morphological changes of collagen were quantitatively analyzed without labeling using an orientation index (OI), the gray level co-occurrence matrix (GLCM) method, and the intensity ratio of THG to SHG (RTHG/SHG). We found that collagen fibers surrounding melanoma cells had higher OI values and SHG intensities initially after implantation of melanoma cells, and these collagen networks had lower OI values and less homogeneity following the growth of tumors. To investigate the roles of melanoma CSC and the vessel permeability, we implanted the mCherry and ALDH1A1 regulatory element–driven EGFP melanoma cells into C57BL/6-c2J mice ears. The 150-kDa (the radius is about 8.9 nm) dextran labeled with fluorescein were injected via tail veins of mice with different tumor sizes. We found that the tumor region enriched with melanoma CSC correlates with higher vessel permeability. Besides, we observed that vessels with increased permeability were mainly arterioles and the density of neoangiogenesis was related to tumor growth