聚焦於黏附異質性:探討幾丁聚醣作為酸鹼應答平台應用於癌症醫學之研究

Abstract

癌細胞的轉移(Cancer metastasis)被視為是癌症的致命殺手鐧，目前文獻上主要藉分子生物標記(molecular biomarker)的方法來評估轉移發生機會。然而腫瘤異質性(tumor heterogeneity)的存在使得分子指標出現捉摸不定的現象，導致應用上缺乏專一性。鑒於高轉移力表型(metastatic phenotype)的癌細胞在腫瘤群體中展現相對較低的黏附能力，這項特殊性質反而能作為引導癌症治療方式發展的突破點，進而減少肇因於轉移發生的死亡。近年來，從生物物理角度出發的分析逐漸蔚為新勢力，它主要偵測癌症侵襲-轉移級聯中(invasion-metastasis cascade)，於特定階段時必然發生的細胞力學表型(mechanical phenotype)，故較為穩定可靠。先前實驗室的研究發現，幾丁聚醣(chitosan)能透過調整環境酸鹼值而成功從異質群體裡分選出黏附能力相異的細胞，使之脫附並收集。且過程中幾丁聚醣上細胞外基質蛋白的脫附與腫瘤發生轉移之初，周圍基質蛋白瓦解的情形極為相似。因此，在細胞脫附相關的生醫研究上，以幾丁聚醣作為免標記平台頗具前瞻性。本論文將利用幾丁聚醣上的細胞脫附分析來針對癌症轉移評估之相關主題進行探討。 第一部分，嘗試從人類非小細胞肺癌細胞株A549中分選出癌幹細胞(cancer stem-like cell)次族群。所涉及之培養基酸鹼值變化範圍皆控制在pH 6.99至pH 7.65間。研究首先利用西方墨點法確認於微酸性環境下在幾丁聚醣上進行之短時間培養，對細胞幹性表現及上皮間質轉化(epithelial-to-mesenchymal transition)並無明顯調控和改變。隨著酸鹼值的提升，15.7±1.9% 的細胞於一個小時內迅速脫附離開幾丁聚醣基材。經由一系列試驗後發現，脫附細胞群相較於基材上之未脫附細胞母群，呈現出更強勢的癌幹細胞特性，諸如細胞多能性、上皮間質轉化能力、細胞侵襲和轉移潛力、抗藥性、群落形成能力與裸鼠異種移植瘤生成能力。除此之外，細胞黏附分子家族中的integrin β4在脫附細胞群裡的大量基因表現相較於未脫附細胞母群具顯著差異。進一步在兩群體內加入抑制integrin β4功能之中和抗體後，於第二輪的細胞脫附分析中，脫附細胞群的脫附率由34.2±3.9% 顯著下滑至12.2±0.4%；反之，在未脫附細胞母群裡並未產生明顯的影響。此一現象同時也意味著反覆進行多次細胞脫附實驗能夠完成癌幹細胞池的富集。最後，經螢光染色疊圖的驗證，兩群體等比例混和並回種於幾丁聚醣基材上，較強integrin β4螢光表現的細胞於脫附實驗後便幾乎消失在顯微鏡視野下。此一研究證實了利用癌細胞的黏附異質性可於酸鹼應答幾丁聚醣上將A549之癌幹細胞篩選出來，且此過程是在integrin β4的驅動下完成。 第二部分，嘗試利用酸鹼應答幾丁聚醣上所得之細胞脫附率將癌細胞侵襲能力參數化。本研究選擇帶有異常ErbB受體訊號的非小細胞肺癌PC9及乳癌BT474細胞株，並採用已獲FDA核准之gefitinib作為標靶治療藥物。研究首先由細胞活性曲線圖找出gefitinib對各細胞株的最佳工作濃度，同時建立癌細胞抑制模型，再透過生物分析如ErbB相關訊號、細胞運動和侵襲能力以及異種移植瘤生成能力的衰退與減弱，驗證該模型的成功建立。接著從幾丁聚醣上細胞脫附分析結果可知，細胞脫附率會隨著抑制後癌化程度的衰退而同步消長。此外，反向利用人類轉化生長因子(transforming growth factor-β1)於頭頸癌、肺癌、肝癌、子宮頸癌、乳癌和大腸直腸癌等細胞株，分別誘導細胞進行上皮間質轉化，發現細胞脫附率亦隨癌化程度增加而提高。另一方面，在PC9及BT474細胞膜上受gefitinib抑制而顯著影響脫附能力之特定integrin，經蛋白陣列搭配抗體阻斷法(antibody blockade)得到驗證，而此結果最後也與免疫螢光染色具一致性。此一研究證實了於幾丁聚醣上所得之重要生醫參數-細胞脫附率，能作為量測癌細胞轉移潛力的工具並轉譯為個體反應以評估預後及藥物治療效果。 本論文使用幾丁聚醣作為免標記平台，以調控培養基酸鹼值的溫和手法成功揭露細胞脫附力與癌症侵襲之間的關聯性，同時這種採以生物物理為切入點的對策不僅拓展了生醫材料的應用性，也為癌症醫學領域向前邁出重要一步。

Metastasis is what proves fatal in cancer, not the primary tumor. Current approaches associated with metastatic assessment in the literature such as the cancer stem-like cell (CSLC) targeting and the prognostic judgment are primary by the employment of molecular biomarkers. However, the changeable molecular signature beneath the tumor heterogeneity results in an unsatisfactory specificity in practice. Given the low adhesive profile observed in the metastatic phenotype among heterogeneous cancer populations, this distinctive hierarchy holds a crucial breakthrough to shepherd the evolution of more refined treatment solutions against the cancer mortality that majorly caused by the metastasis. In the past decades, biophysical assays that have become a rising trend are focus on mechanical phenotyping at specific steps of the invasion-metastasis cascade. While molecular signaling network is diversified, capturing the mechanical features underlying metastasis is rather stable, compared to tracing the curious molecular biomarkers. Beneficial from its polycationic nature, the biopolymer chitosan enables cell adhesion selection from a miscellaneous population simply via pH adjustment and thereupon has the potential to serve as a label-free platform. Of most importance, the extra-cellular matrix (ECM) protein withdrawal on the de-protonated chitosan resembles the collapse of matrix components at the beginning of tumor invasion. Hence, chitosan is a promising candidate for solution realization of the biomedical demands associated with cell detachment. The purpose of the current dissertation is to apply cell detachment assays on chitosan for addressing the unsolved issues in cancer research. In chapter 2, the pH-dependent cell detachment assay (CDA) on chitosan was utilized as an alternative to isolating the stem-like subpopulation from non-small cell lung cancer (NSCLC) cell line A549. Manipulation of the medium pH was in the range between 6.99 and 7.65. The assay began with a short-term cell incubation on chitosan at pH 6.99 for 24 h. Immuno-blotting found no obvious regulation of cell pluripotency and epithelial-to-mesenchymal transition (EMT) at this period. As the culture medium pH elevated from 6.99 to 7.65, 15.7±1.9% of A549 rapidly detached from the chitosan substrate within an hour. Compared with their remaining high-adhesive counterpart, this subpopulation of cells with low adhesiveness exhibited superior CSLC hallmarks, including cell pluripotency, EMT potential, invasive and metastatic ability, therapeutic resistance, colony formation in vitro as well as nude mice xenograft in vivo for tumorigenicity. Subsequently, the integrin family of adhesion receptors was probed in the detached population and their adherent counterpart. From the genetic analyses, integrin β4 (ITGb4) in the detached population outstood among a subset of integrin subunits, in contrast to that in the adherent counterpart. These two populations were then subjected to the second round of CDA with and without the addition of ITGb4 neutralizing antibody. The acquired cell detachment ratio (CDR) indicated a drastic drop from 34.2±3.9% to 12.2±0.4% (p<.001) in the detached population, while there was no significant difference in the adherent population after experiencing functional inhibition of ITGb4. This phenomenon also implied that the enrichment of a CSLC pool may be achieved by multiple rounds of CDA. For further validation, those highly ITGb4-expressing cells were mostly absent in the field of vision following the detachment process as visualized by the fluorescence. To conclude, pH-responsive chitosan is capable of discriminating CSLC from A549 by adhesion heterogeneity and this process is driven by the ITGb4 on cell surfaces. In chapter 3, the CDR derived from pH-responsive chitosan was introduced for the parameterization of the cancer aggressiveness in cells harboring aberrant ErbB signals (NSCLC cell line PC9 and breast cancer cell line BT474). The FDA-approved drug gefitinib was administered as a selective epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitor of the targeted therapy. Prior to applying the detachment assay on chitosan, gefitinib dosage was optimized in respective cell lines along with the identification of the drug-induced tumor regression by the attenuated ErbB-related signaling, the reduced migratory and invasive ability in vitro as well as the repressed tumor engraftment in nude mice. From the gathered results, the corresponding CDR acquired from the pH-dependent detachment process goes synchronously with the extent of tumor regression in response to the medication. Inversely, the elevated CDR to an advanced malignancy also validated by the employment of EMT inducer transforming growth factor-beta 1 (TGF-β1) in several tumor cell lines, including hypopharyngeal (FaDu), hepatocellular (HepG2), lung (PC9), cervical (HeLa), breast (BT474), and colorectal carcinoma (HCT116). Moreover, the integrin protein array in company with the antibody blockade disclosed the definite integrin subunits in PC9 and BT474 respectively that mediate the detachment step in the presence of gefitinib. In the visualization of immuno-fluorescence, the differential expression of the dictated integrins in the drug-treated groups and the vehicle controls of both cell lines were collectively characterized. To conclude, the biometrical identifier CDR is capable of measuring metastatic potential and reasonably translate into the patient response for further prognostic judgment and drug efficacy assessment. The intimate correspondence between the cell detachment ability and cancer aggressiveness was successfully unveiled through the mild approach on pH-responsive chitosan as demonstrated in this dissertation. This biophysical-associated strategy not only expands the horizons of biomaterial-based applications but also represents a step forward in cancer medicine.