硼中子捕獲治療（Boron neutron capture therapy; BNCT）為一種透過硼10 同位素(10B )受到熱中子撞擊後裂解產生高能的特性，與具有腫瘤選擇性的含硼藥物結合的放射性療法。在現今BNCT 臨床應用中主要以borocaptate（BSH）與borophenylalanine （BPA）作為含硼藥物，並廣泛用於治療黑色素癌、頭頸癌與腦癌。然而過去的研究顯示，BSH 與BPA 侷限於其藥理特性，不適用於肝癌的硼中子捕獲治療。肝癌（Hepatocellular carcinoma, HCC）為世界常見的癌症類形，在亞洲地區肝癌致死率更高居第二名。目前治療肝癌的療法大多不適於多發性與末期肝癌患者。清華大學周鳳英教授團隊於近年發現以硼酸作為BNCT 藥物，在大鼠與兔肝癌模型上有顯著性的腫瘤專一性與治療效果，但硼酸在肝腫瘤中的傳遞與聚積相關機制尚未被完整研究。硼酸為最簡單的含硼化合物，被發現會與醣類結合。硼酸進入人體後在一般狀況下不會被代謝，其傳遞聚積與組織硼酸濃度相關，可透過簡單擴散的方式進入細胞當中。另一方面，許多文獻指出肝腫瘤的細胞特性及微環境與正常組織有所區別，包含醣類代謝、細胞膜組成及酸鹼值。因此，我們於本研究中，系統性的探討硼酸是如何被肝癌細胞所吸收。實驗結果顯示，硼酸主要透過擴散作用的進入癌細胞內，硼酸吸收量與醣類並無顯著關聯性，但可能會受到細胞膜通透性與酸鹼值影響。結合周鳳英教授團隊的動物實驗結果，我們推測肝腫瘤的異常血管床與其高通透性，可能為硼酸累積在肝腫瘤內的主要原因。本研究將有助於未來評估肝癌患者是否適合接受以硼酸為含硼藥物的BNCT 治療，亦有助於硼酸藥物的施放及臨床試驗規劃。

Boron neutron capture therapy (BNCT) is a radiation therapy which utilizes the high-energy particles produced from the nuclear reaction of boron isotope (10B) and thermal neutron to kill cancer cells with selective tumor targeting. However, borocaptate and borophenylalanine which are widely used in clinical studies, are not suitable for treating hepatocellular carcinoma (HCC). HCC is one of the most common cancer types worldwide, with the second highest mortality rate in Asia. The current treatments of HCC include surgical and chemical therapies. Nonetheless, these therapies are not suitable for patients with advance and multifocal HCC. In recent years, Chou, FI et al. found that boric acid showed significant HCC selectivity and efficacy in rat and rabbit liver tumor-bearing models after neutron radiation. However, the mechanism of transportation and accumulation of boric acid in the hepatocellular cells remains unclear. After entering the body, boric acid interacts with carbohydrates, but could not be metabolized in mammals. Previous reports indicated that the characteristics of liver tumor was different from normal liver tissue, including carbohydrates metabolism, cell membrane composition and pH value of tumor microenvironments. In this study, we systematically analyzed how boric acid was absorbed by hepatocellular carcinoma cells. Our results showed that boric acid was mainly transported into cancer cells by simple diffusion. The uptake level of boric acid in liver cancer cells was not associated with the interaction of boric acid and carbohydrates, but might be affected by cell membrane composition and pH value in the microenvironment. Combining with the findings by Chou, FI et al., we proposed that abnormal vasculature and enhanced vascular permeability of liver tumor might explain boric acid’s HCC targeting and selectivity effect. Our findings could help to design clinical trials for patient recruitment and treatment planning for boric acid-mediated BNCT of HCC in the future.

List of figures ............................................................................................................................................. I
List of tables..............................................................................................................................................II
中文摘要................................................................................................................................................. III
Abstract ................................................................................................................................................... IV
1.Introduction............................................................................................................................................ 1
1.1 Hepatocellular carcinoma and its current treatments ............................................................ 1
1.2 Overview of boron neutron capture therapy ......................................................................... 2
1.3 Medical use of boric acid ...................................................................................................... 2
1.4 Mechanisms of boric acid transportation .............................................................................. 3
1.5 The characteristic and micro-environmental differences between normal tissue and tumor
tissue ............................................................................................................................................... 4
1.6 The objectives and applications of this study ....................................................................... 5
2.Materials and methods ........................................................................................................................... 6
2.1 Cell and cell culture .............................................................................................................. 6
2.2 Preparation of BA solution.................................................................................................... 6
2.3 Chemical treatment ............................................................................................................... 6
2.4 Cell harvesting ...................................................................................................................... 7
2.5 Boron quantitation................................................................................................................. 7
2.6 Total carbohydrates assay ..................................................................................................... 8
2.7 Statistical analysis ................................................................................................................. 8
3.Results.................................................................................................................................................... 9
3.1 Modification of cell harvesting method on BA treated cells ................................................ 9
3.2 Analysis of BA uptake in vitro............................................................................................ 10
3.3 Non-significant effect of glucose on uptake of BA in HepG2 cells.....................................11
3.4 Effect of total carbohydrates on uptake of BA in liver cancer cells and normal liver cells in
in vivo and in vitro study .................................................................................................................11
3.5 Evaluation of acid environment in uptake of BA in HepG2 cells....................................... 12
3.6 Effect of fluidity of cell membrane on uptake of BA in HepG2 cells................................. 13
3.7 Analysis of boron uptake in co-treatment of BA and BPA................................................. 13
4.Discussion ............................................................................................................................................ 15
4.1 Contribution of this study ................................................................................................... 15
4.1.1 Potential advantages of BA as the boron drug for BNCT.............................................. 15
4.1.2 Feasibility of developing cell membrane-based boron drug delivery system................ 16
4.1.3 Efficacy of BA-BNCT might correlate with tumor vascular properties in HCC........... 16
4.2 Limitation of BA-BNCT........................................................................................................... 17
4.2.1 Lack of imaging method for prognosis and management of BA-BNCT for HCC ........ 17
4.3 Tumor properties on BA uptake ................................................................................................ 17
4.3.1 The acidic microenvironment and tumor glucose metabolism ...................................... 17
4.3.2 Cell membrane fluidity related tumor properties on BA uptake in cancer cells ............ 18
4.4 Perspective ................................................................................................................................ 19
Reference ................................................................................................................................................ 20


List of figures

Figure 1. Differences of boron uptake after modification of cell-harvesting method .................................23
Figure 2. Boron uptake of HepG2 cells under boric acid treatment of various time .................................24
Figure 3. Boron uptake of HepG2 in different concentration and temperature treatment of BA .................................25
Figure 4. Boron uptake of HepG2 cells under different glucose condition .................................26
Figure 5. In vitro and in vivo study of total carbohydrates analysis .................................27
Figure 6. Boron uptake of HepG2 cells under different pH environment .................................28
Figure 7. Boron uptake of HepG2 cells under cholesterol treatment .................................29
Figure 8. Boron uptake of HepG2 cells under co-treatment of BA and BPA .................................30
Supplemental figure 1. Mechanism of boron neutron capture therapy .................................32
Supplemental figure 2. The pattern of simple diffusion and facilitated diffusion in rate of diffusion against concentration plot
.................................33
Supplemental figure 3A. Boric acid interactions with vicinal diol of sugar, which is adapted from .................................34
Supplemental figure 3B. Thin layer chromatography of boric acid and carbohydrates interaction (Original data provided by Prof. Chia-Min Yang’s laboratory).................................34
Supplemental figure 4. Time course study of BA from previous cell collecting method .................................35

List of tables


Table 2. Comparison between Cell-harvesting methods. .................................31

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