肺腺癌细胞在基质胶-Ⅰ型胶原共混水凝胶支架类器官体系中的生物学研究

Biological study of lung adenocarcinoma cells in matrigel-collagen type Ⅰ hybrid scaffolds-based organoid systems

目的探讨在类器官培养体系下使用不同三维支架培养肺癌细胞A549所出现的生物学行为,观察并表征在模拟肺癌早期基质的基质胶和模拟肺癌进展期基质的基质胶-Ⅰ型胶原共混水凝胶中分别培养A549类器官出现的增殖、侵袭和药物敏感性方面的差异。方法通过扫描电镜(SEM)观察基质胶与Ⅰ型胶原共混后水凝胶的三维支架结构变化。A549细胞在基质胶中和基质胶-Ⅰ型胶原共混水凝胶中进行体外类器官培养,在光镜下观测类器官平均直径变化和第9天成球率评估类器官的生长趋势;使用HE染色和免疫组织化学染色评估类器官微组织特征和肿瘤增殖标志物Ki-67表达量;使用鬼笔环肽染色检测类器官中F-actin表达位置与空间排列;通过免疫印迹法检测类器官中EMT相关蛋白(E-cadherin、N-cadherin和Vimentin)变化;通过3D glo法检测类器官在吉非替尼处理后的活力以分析类器官药物敏感性的变化。结果A549类器官在基质胶中形成表面光滑的实心球体,在共混水凝胶中出现丝状伪足、片状伪足和单细胞散布到基质的现象;在两种支架中培养的A549类器官生长趋势无明显差异,但共混水凝胶组Ki-67阳性表达率明显高于基质胶组;A549类器官细胞骨架在基质胶中呈疏松立体球状结构,在共混水凝胶中出现分支结构且单细胞分支尖端细胞发生细胞骨架重塑;共混水凝胶组类器官相较于基质胶组其上皮标志物表达降低、间充质标志物表达升高;共混水凝胶组肺癌类器官相较于基质胶组对吉非替尼更耐药。结论在基质胶-I型胶原共混水凝胶中培养的肺癌类器官形态结构改变、增殖能力增强、侵袭能力增强以及对吉非替尼的药物敏感性降低。

Objective Explore the biological behavior of lung cancer cells A549 in organoid culture systems with different three-dimensional culture scaffolds,observing and characterizing the differences in proliferation,invasion,and drug sensitivity of A549 in Matrigel,which simulates the early matrix of lung cancer,and in Matrigel-collagen typeⅠhybrid scaffolds,representing the advanced stage matrix.Methods Observing the 3D scaffold structure of the hybrid scaffolds of matrigel and typeⅠcollagen by scanning electron microscopy(SEM).A549 cells were cultured in vitro in organoid systems using matrigel and matrigel-collagen typeⅠmixed scaffolds.The organoid formation process and morphological characteristics were observed.The average diameters of A549 lung cancer organoids on day0,4,7,10 and the percentage of organoids formed on day 9 were recorded to assess the growth of organoids.H&E staining and immunohistochemical staining were used to evaluate the microtissue features and the expression of the proliferation marker Ki-67.Phalloidin staining was employed to detect the expression location and spatial arrangement of F-actin.Western blot was used to detect changes in EMT-related proteins(E-cadherin,N-cadherin,and Vimentin).3D glo method was utilized to assess the vitality of organoids treated with gefitinib for analyzing drug sensitivity.Results A549 organoids formed smooth,solid spheroids in Matrigel,while in the hybrid scaffolds,filamentous pseudopodia,lamellar pseudopodia,and single-cell dispersion into the matrix were observed;there was no significant difference in growth trends between the two groups,but the hybrid scaffold group showed a significantly higher Ki-67 positive expression rate than the Matrigel group.The cytoskeleton exhibited a loose,three-dimensional spherical structure in Matrigel,while in the hybrid scaffolds,it displayed branching structures and actin cytoskeleton remodeling at the tips of the branches.The expression of Epithelial marker was decreased and mesenchymal marker was increased in the hybrid scaffold group compared to the Matrigel group;lung cancer organoids cultured in mixed scaffolds exhibited increased resistance to gefitinib.Conclusion Under the organoid system of Matrigel-collagen typeⅠhybrid scaffolds,lung cancer organoids exhibit changes in morphological structure,enhanced proliferative and invasive capabilities,and increased resistance to gefitinib.