基于三维共培养体系的人乳腺癌靶向肺侵袭与转移模型的构建及机制研究

Invasive breast cancer is the second leading course of women death in the world. Unfortunately, much is yet to uncover to reach a full understanding of its complexity and related mechanisms due to the limitation of current cultivation system in recapitulating the process of cancer invasion or metastasis in tumor microenvironment. To address this issue, development of an ideal pathologically relevant, surrogate culture model for breast cancer invasion or metastasis is essential. It has been demonstrated that tumor microenvironment plays an important role in determining the fate of breast cancer cells, especially their invasion or metastasis behavior. Thus, to better understand the mechanism of cancer metastasis, especially its targeted metastasis to the target tissue, how to replicate the tissue microenvironment in vitro is the key issue. In this proposal, the PI is proposing to reconstruct an improved 3D co-culture model of human breast cancer by using silk scaffold-based tissue engineering techniques. It's hypothesized that the mciroenvironment of breast cancer could be reconstructed through co-culturing human breast cancer cells and multiple types of stromal cells seeded within silk scaffolds with appropriate extracellular matrix incorporation as well as the bioreactor culture system. It is expected that this bioengineered 3D coculture compartment could mimic the tissue microenvironment more vividly and replicate the behavior of in vivo breast cancer cells as close as possible. Based on this engineered 3D co-culture model of human breast cancer, a systemic study on the tumor microenvironment effect on breast cancer metastasis would be carried out and its related molecular mechanism will be explored. In addition, a tissue engineered human lung tissue model would be developed in the current study with an aim to further explore the mechanism of breast cancer metastasis to lung in vitro or in vivo. This is important because of its high frequency and mortality in the breast cancer patients. This model system constructed through integrating research approaches from the disciplines of tissue engineering and cancer biology would offer an improved approach to characterize the metastatic microenvironment of lung in breast cancer, allowing better understanding and manipulation of the microenviornment effect on cancer invasion or metastasis of human breast cancer.

侵袭性乳腺癌是威胁世界女性生命健康的重要疾病。但有关乳腺癌靶向侵袭与转移的具体机制却远未被揭示。制约该研究工作的主要"瓶颈"是目前尚缺乏可真实模拟在体乳腺癌侵袭与转移过程的理想模型。因此，建立与在体乳腺癌靶向侵袭/转移这一病理过程高度相关的疾病模型是侵袭性乳腺癌研究中的关键。研究证明：乳腺癌细胞的生物学行为尤其是其侵袭与转移特性与原位肿瘤或靶组织的微环境密切相关。由此，本项目拟采用组织工程技术体外模拟肿瘤组织微环境，在构建人乳腺癌组织三维共培养模型的基础上，深入考察微环境因素对乳腺癌细胞侵袭与转移的调控作用及相关机制；同时以组织工程化人源肺组织为靶组织，进一步在体外组织化水平探讨共培养体系内乳腺癌细胞靶向肺侵袭/转移的潜能及分子基础，并通过体内实验进行验证，以期建立一种基于3D共培养体系的乳腺癌靶向肺转移模型，从而为乳腺癌靶向侵袭/转移的研究提供有力的研究手段并奠定相应的理论和实验基础。

侵袭性乳腺癌是威胁世界女性生命健康的重要疾病。但受限于现有研究模型所存在的缺陷，有关乳腺癌靶向侵袭与转移的具体机制却远未被揭示。因此，建立与在体乳腺癌靶向侵袭/转移这一病理过程高度相关的疾病模型是侵袭性乳腺癌研究中的关键。研究证明：乳腺癌细胞的生物学行为尤其是其侵袭与转移特性与原位肿瘤或靶组织的微环境密切相关。基于此，本课题研究中首先采用组织工程技术体外模拟乳腺组织微环境，即以胶原和丝素蛋白为培养支架，接种人乳腺癌细胞MDA-MB-231-成纤维细胞成功构建乳腺癌3D共培养模型。通过对该模型进行细胞生物学表征，我们发现：对照2D传统培养模型，该共培养体系内乳腺癌细胞的增殖、耐药以及侵袭转移能力均显著提高；明确了微环境中培养维度和间质细胞因素对乳腺癌细胞EMT的调控作用及integrin信号相关分子机制；接下来我们建立并优化了体外灌流法和浸泡法以制备去细胞化肺，在对其进行DNA含量、组分表征评价的基础上，通过接种人肺上皮细胞-成纤维细胞-血管内皮细胞体外构建了具有复杂细胞组分的组织工程化人源肺组织，该体系内细胞不但保持良好的活性，且具有理想的表型:上皮细胞的表型和功能均对比2D培养体系显著提升；血管内皮细胞也可形成vascular-like结构。采用旋转生物反应器可大大改善培养体系内的传质，从而显著提高细胞在去细胞化肺支架内的活性和分布；同时，我们以去细胞化肺为靶组织，接种乳腺癌细胞和间质细胞建立共培养模型，对照平面培养和单独培养组，分别在分子、细胞以及组织水平对该模型体系进行评价，进一步明确了细胞外基质、间质细胞微环境因素可显著提高乳腺癌细胞的侵袭及转移能力，促进其高表达EMT信号通路中相关分子，降低E-cad蛋白/基因的表达，从而明确了乳腺癌细胞在肺基质内侵袭的分子基础，成功构建了一种基于3D共培养体系的乳腺癌靶向肺侵袭/转移模型。该研究结果将乳腺癌靶向肺侵袭/转移的研究提供有力的研究手段并奠定的理论和实验基础，并可能为侵袭肿瘤的药物筛选提供理想的模型.