利用聲孔效應調控生物物理訊息—腫瘤微環境硬度與放射治療增敏

腫瘤微環境（TME）是影響腫瘤行為與治療反應的關鍵。放射治療利用游離輻射破壞癌細胞的DNA，是目前臨床上主要的癌症治療方法之一。輻射增敏劑，可透過增加DNA損傷，加強放射治療成效，但臨床上，這些輻射增敏藥物，多採用全身性給予，因此多有系統性的副作用。另一方面近年研究發現細胞內DNA的損傷可能對細胞外基質(ECM)的重塑有直接且重要的影響，但基質重塑導致腫瘤硬度的變化與放射治療反應之間的動態關係，仍有待深入研究。本研究目標在發展一治療策略，其同時考量癌細胞且針對腫瘤微環境硬度所造成的輻射敏感度影響，以追求放射治療成效的最大化。首先，我們利用奈米金桿作為輻射增敏劑，透過奈米金桿微氣泡與超音波穴蝕聲孔效應達成了輻射增敏。此一策略應用超音波聲孔效應在空間與時間上標定腫瘤釋放藥物的特性，增加了奈米金的在人類肝癌細胞內的濃度，提高了 DNA的損傷與細胞凋亡。在細胞培養實驗中，我們在10%的存活條件下，達成了1.56±0.45的dose modifying ratio。而在活體小鼠實驗中，採用 VEGFR2作為腫瘤血管系統的靶標，增強了奈米金微氣泡對腫瘤的靶向遞送，而 VEGFR2 已被許多研究證實其廣泛並大量表現在不同的實體腫瘤血管網內，因此將來也有潛力對不同組織腫瘤的輻射增敏策略。藉此，我們注入體內的奈米金含量低於文獻中的使用量，較低的全身性金劑量將更有可能為臨床轉譯研究提供機會。而本研究第二部分則是探討了腫瘤微環境的硬度對於放射敏感度的影響，透過剪力波彈性量測技術的運用，我們開發了單一超音波探頭的彈性影像測量(SWEI)技術，結合於毫米尺寸的三維細胞培養平台。該單一探頭產生剪力波並接收自邊界反射之剪力波，經接收時間與距離的計算，得出剪力波之波速，並另以雷射光斑剪切波影像加以驗證。而透過改變膠原蛋白的濃度，我們建立了三種不同基質硬度的細胞培養樣品。進一步對三維肝癌細胞培養照射16 Gy的放射治療，我們觀察其在96小時內的硬度變化。本研究在此首次建立了針對毫米尺寸之三維細胞培養養品，評估輻射敏感度的作業流程。利用流式細胞儀對γ-H2AX 和 PARP1 在癌細胞放射治療後的表現量進行檢測，DNA 損傷在基質硬度最低的膠體中最為明顯。值得提出討論的是，在不同起始硬度的培養膠體中，其放射照射後的硬度時間變化明顯不同。而在最軟的膠體切片中，細胞周圍的膠原蛋白螢光強度在放射治療後明顯增加，但在其他兩組硬度的組別中，則沒有觀察到此一現象。我們依此推測 DNA 損傷可能對胞外基質的重塑中有所影響。此SWEI平台具有非侵入性的，技術簡明的優勢，適合在體外細胞培養來研究胞外基質的重塑，特別是對於光學混濁的毫米尺度生物材料樣品，其後續的生物表現與整體硬度之間的關係，都可在這個平台量化描述。後續的研究包括探討 DNA 損傷引致胞外基質的訊號傳遞機轉，並開發結合腫瘤硬度靶向藥物的放射治療策略。

關鍵字

奈米金粒子；聲孔效應；放射治療；輻射增敏；剪力波彈性影像；腫瘤微環境硬度；三維細胞培養系統

並列摘要

The tumor microenvironment (TME) is a critical regulator of tumor behaviors shaping therapeutic responses. Radiotherapy (RT), posing DNA damage in cancer cells, is widely used to treat cancer patients. Radiosensitizers are used to produce DNA damage and thus increase therapeutic efficacy. However, most clinical radiosensitizers are administered systemically, so generalized adverse effects remain significant. On the other hand, emerging studies have suggested that DNA damages could directly introduce remodeling of the extracellular matrix (ECM). Meanwhile, the dynamic interaction between TME stiffness and the cancer response to RT has not been thoroughly examined. The research aims to develop a strategy that targets cancer cells and TME stiffness to maximize the clinical benefits of RT. First, we achieve radiosensitization through cavitation-induced sonoporation with gold-nanoparticle-encapsulated microbubbles (AuMBs). The spatially and temporally controlled release of gold nanoparticles (AuNPs) enhances the delivery of AuNPs and causes augmented DNA damage and apoptosis in human liver cancer cells. A dose modifying ratio of 1.56±0.45 for a 10% surviving fraction has been demonstrated in vitro. Moreover, VEGFR2, a common target on the vasculatures of various solid tumors, is adopted to enhance the tumor-specific delivery of AuMBs. The low systemic dose of gold in our animal study offers an opportunity for translation into clinics. The second part of the research is to characterize the influence of TME stiffness on radiosensitivity. We have developed a platform using a single-element transducer shear wave elasticity imaging (SWEI) setup on a millimeter-sized, ECM-based 3D culture. The shear wave speed inside the hydrogel is calculated using a time-of-flight algorithm on the reflected shear waves from the boundaries, which is then verified with a laser speckle contrast imaging system. By varying the concentrations of collagen, we create 3D cultures of three different stiffnesses. We irradiated the cultures of liver cancer with 16 Gy and measured the temporal dynamics of stiffness for 96 hours after RT. A workflow for assessing radiosensitivity in a millimeter-sized 3D culture is proposed for the first time. The expressions of γ-H2AX and PARP1 are examined via flow cytometry. We observe the most abundant DNA damage in the sample of the lowest matrix elasticity. The dynamics of shear modulus in cultures of varying stiffness after RT are considerably different. The peri-cellular collagen deposit increases after RT in the culture with the most compliant matrix. At the same time, there is no significant change in the peri-cellular collagen intensity of the other two groups. These findings imply that DNA damage has a role in subsequent ECM remodeling. The platform is noninvasive, technically straightforward, and appropriate to investigate how cells remodel their surrounding matrix, especially for optically-turbid millimeter-sized biomaterials. Future works include deciphering mechanistic pathways and exploring RT strategies with stiffness-targeted agents in the SWEI-incorporated 3D culture.

並列關鍵字

Gold nanoparticles ； Sonoporation ； Radiotherapy ； Radiosensitization ； Shear wave elasticity imaging ； Tumor microenvironment stiffness ； Three-dimensional cell culture

參考文獻

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