唾液腺腺样囊性癌源性exosomes诱导的转移前微环境微流控模型的构建及应用

Salivary gland adenoid cystic carcinoma (ACC) is a common malignancy in the head and neck region. It tends to metastasize to lung, bone, and liver. Recent evidence suggests that the primary tumor is able to influence and alter the distant organs by promoting the formation of supportive metastatic microenvironment, termed pre-metastatic niche, prior to tumor cell dissemination. Exosomes derived from tumor cells play an important role in the formation of pre-metastatic niche. Identifying the factors that promote pre-metastatic niche formation in ACC patients will offer promise for new therapeutic directions to prevent metastasis. Microfluidic technology has been proved to be an ideal platform to operate mammal cells with low consumption and high throughput. This project is to develop a biomimetic microfluidic model that reconstitutes artificial lung, bone, liver, and microvessels. Pre-metastatic niche formation induced by tumor-derived exosomes in these artifical organs will be reproduced on the microfluidic model. Vascular leakiness, remodeling of extracellular matrix, and recruiment of bone marrow derived cells will be regarded as biological indicators of pre-metastatic niche formation. Then the microfluidic model will be used to study the pre-metastatic niche formation in specific organs induced by ACC-derived exosomes. Results obtained from the microfludic model will be compared with that obtained from mouse models. At last, the microfluidic model will be applied to assess the capability of inducing pre-metastatic niche formation by ACC patient-derived exosomes in clinic. It will be assisstant to evaluate the metastatic potential of ACC patients and familiate individual therapy. The microfluidic model developed in this project can also be used to study pre-metastatic niche formation induced by exosomes derived from other cancers. We hope the achievements of this project can provide a new platform for pre-metastatic niche study.

唾液腺腺样囊性癌（ACC）是头颈部常见恶性肿瘤，易转移至肺、骨、肝等器官。肿瘤发生转移前，可在预转移器官形成适宜瘤细胞定植和生长的转移前微环境，exosomes（外泌体）对转移前微环境的形成具有重要调控作用。揭示影响ACC转移前微环境形成的因素，有助于采取措施预防转移。本项目拟以微流控芯片技术为主要手段构建包括肺、骨、肝和微血管的仿生模型，以血管渗透性增加、细胞外基质重建、骨髓源性细胞募集等为生物学指标，模拟肿瘤源性exosomes经循环到达仿生器官选择性诱导转移前微环境形成的过程，进而考察ACC源性exosomes在多器官诱导转移前微环境形成中的作用，并与小鼠模型对照；用对照确认后的微流控模型考察ACC患者源性exosomes诱导转移前微环境形成的能力，筛查高转移潜能患者，为个体化治疗提供依据。鉴于此仿生模型在很大程度上的普适性，本项目也将为其它肿瘤转移前微环境研究提供一种新技术平台。

唾液腺腺样囊性癌（SACC）是头颈部常见恶性肿瘤，易转移至肺、骨、肝等器官。肿瘤发生转移前，可在预转移器官形成适宜瘤细胞定植和生长的转移前微环境，exosomes（外泌体）对转移前微环境的形成具有重要调控作用。揭示诱导SACC转移前微环境形成的因素，有助于采取措施预防转移。. 在生物基础研究方面，我们发现人SACC细胞和SACC组织源性CAF细胞均可分泌EVs，这些EVs具有显著的肺趋向性和一定程度的肝趋向性，可在肺部诱导转移前微环境形成，进而促进SACC肺转移。CAF分泌的EVs在肺部通过Integrin α2β1识别肺成纤维细胞，并激活其TGF-β信号通路，使其高表达Fibronectin、LOX、Periostin等，诱导肺转移前微环境形成，易于肿瘤细胞的黏附和增殖。进一步研究发现Integrin α2β1的抑制剂在体外可以抑制肺成纤维细胞摄取CAF EV，在体内显著抑制CAF EV诱导的SACC肺转移和转移前微环境形成，裸鼠移植瘤模型显示血浆EV相关性 integrin β1可以预测SACC肺转移。为了深入研究CAF的作用，我们建立一个SACC源性CAF的细胞系。. 在微流控芯片平台研究方面，我们基于微流控技术构建了高通量单细胞EVs检测平台，利用该平台揭示了肿瘤单细胞在EVs分泌能力和分泌种类方面具有显著异质性。通过分析单细胞的EVs分泌，可以将高侵袭性OSCC细胞与低侵袭性的细胞区分开（，进而可区分淋巴结转移与非转移的OSCC临床样本，单细胞多维度分析还揭示群体细胞中EV分泌和蛋白分泌由不同细胞亚群负责。我们还构建了精密组织切片（PTS）体外培养技术微流控芯片。与传统Trasnwell比较，肝PTS在芯片模型上产生较高的趋化因子浓度梯度，微流控芯片PTS培养和动物实验结果均证实癌细胞EVs具有明显的肝趋向性，CXCL12/CXCR4生物轴在EVs的肝趋向性中发挥一定的作用。我们还基于微流控技术构建了一个采用免疫磁性纳米粒子分离检测EVs的技术平台，为开展以EVs为检测对象的肿瘤液体活检提供一个新的技术平台。. 本研究成果为筛查高转移潜能SACC患者提供了生物标志物，也为肿瘤EVs研究提供了一系列新技术平台。