包封多細胞腫瘤球體之可光交聯透明質酸-明膠基質作為3D生物列印體外工程化胰腺癌組織研究

Abstract

三維 (3D) 生物列印技術可用於製造類癌症組織，以模擬腫瘤微環境中的複雜交互作用。在人類胰腺導管腺癌 (PDAC) 中，其主要涉及了細胞外基質 (ECM)、癌細胞和胰腺星狀細胞之間的交互作用。透明質酸 (HA) 是ECM的主要成分，有利於促進PDAC的腫瘤進展和化療耐藥性。在目前的研究中，通過將多細胞胰腺腫瘤樣球體包封於新型HA-明膠光交聯水凝膠 (即GHP水凝膠) 中，並利用3D生物列印技術在體外創建出類PDAC組織平台。這種最佳化的GHP水凝膠 (含7 wt%明膠和0.2 wt%酚官能基化HA) 達到了與臨床人類PDAC樣本相似的模量 (~5.46 kPa)，其更加緻密且均勻的結構優於純明膠水凝膠 (即GN水凝膠)。於3D生物列印之GHP水凝膠 (GHP 3DP構建體) 內的腫瘤樣球體呈現出顯著的侵襲性和轉移性，伴隨典型上皮-間質轉化 (EMT) 標誌基因的表達上調。此外，其餘基因表達分析顯示CD44基因的表達增加約290倍，而S100A9 (一種用於早期診斷的新型胰腺癌生物標誌物) 則增加了7.3倍的表達。在GHP 3DP構建體的腫瘤樣球體中還觀察到顯著的化療耐藥性，在接受臨床聯合藥物 (吉西他濱和白蛋白結合-紫杉醇) 處理48小時後，其腫瘤樣球體的細胞存活率仍高達98.5%。這與未含水凝膠之單獨培養的腫瘤樣球體和雙細胞類型的共同培養組別所觀察到的較低耐藥性形成鮮明對比 (細胞存活率分别為32.5%和28.9%)。深入研究3D生物列印之類PDAC組織構建體內的HA分布，可發現其分布模式與真實胰腺癌樣本中的結果相似，此展現了於體外再現腫瘤生物學中惡性程度及癌細胞重編程的可能性。綜上所述，該3D生物列印之類PDAC模型具有推動胰腺癌研究和臨床前藥物篩選的巨大潛力。

3D bioprinting can be utilized to fabricate cancer-like tissue that models complex interactions within the cancer microenvironment. In human pancreatic ductal adenocarcinoma (PDAC), these interactions involve the extracellular matrix (ECM), cancer cells, and pancreatic stellate cells. Hyaluronan (HA) is a major component of ECM supporting tumor progression and chemoresistance in PDAC. In the current study, an in vitro PDAC-like tissue platform was developed by embedding multicellular pancreatic tumor-like spheroids within a novel 3D bioprinting HA-gelatin photocrosslinked hydrogel (GHP). This optimized GHP bioink (7 wt% gelatin and 0.2 wt% phenolic HA) achieved a modulus (~5.46 kPa) closely resembling that of clinical PDAC tissue, with a dense and uniform structure superior to gelatin-only hydrogel (GN). The bioprinted 3D tumor-like spheroids within GHP exhibited distinct invasive and metastatic behavior, along with up-regulated expression of epithelial-mesenchymal transition (EMT) markers. Furthermore, gene expression analysis also revealed a ~290-fold increase in CD44 gene and a 7.3-fold rise in S100A9 (a novel pancreatic cancer biomarker for early diagnosis). These tumor-like spheroids within 3D-bioprinted GHP constructs further demonstrated substantial chemoresistance, maintaining remarkable 98.5% viability after 48 h of exposure to a Gemcitabine and Abraxane combination, in contrast to significantly lower resistance observed in spheroids alone or co-cultured monolayers. An in-depth investigation of HA distribution within the 3D-bioprinted PDAC-like construct revealed a pattern consistent with clinical PDAC, indicating enhanced malignancy and potential tumor reprogramming. This 3D-bioprinted PDAC model holds significant potential for advancing pancreatic cancer research and preclinical drug testing.