During lung cancer metastasis,tumor cells undergo epithelial-to-mesenchymal transition(EMT),enabling them to intravasate through the vascular barrier and enter the circulation before colonizing secondary sites.Here,a ...During lung cancer metastasis,tumor cells undergo epithelial-to-mesenchymal transition(EMT),enabling them to intravasate through the vascular barrier and enter the circulation before colonizing secondary sites.Here,a human in vitro microphysiological model of EMT-driven lung cancer intravasation-on-a-chip was developed and coupled with machine learning(ML)-assisted automatic identification and quantification of intravasation events.A robust EMT-inducing cocktail(EMT-IC)was formulated by augmenting macrophage-conditioned medium with transforming growth factor-β1.When introduced into microvascular networks(MVNs)in microfluidic devices,EMT-IC did not affect MVN stability and physiologically relevant barrier functions.To model lung cancer intravasation on-a-chip,EMT-IC was supplemented into co-cultures of lung tumor micromasses and MVNs.Wihin 24 h of exposure,EMT-IC facilitated the insertion of membrane protrusions of migratory A549 cells into microvascular structures,followed by successful intravasation.EMT-IC reduced key basement membrane and vascular junction proteins-laminin and VE-Cadherin-rendering vessel walls more permissive to intravasating cells.ML-assisted vessel segmentation combined with co-localization analysis to detect intravasation events confirmed that EMT induction significantly increased the number of intravasation events.Introducing metastatic(NCI-H1975)and non-metastatic(BEAS-2B)cell lines demonstrated that both,baseline intravasation potential and responsiveness to EMT-IC,are reflected in the metastatic predisposition of lung cancer cell lines,highlighting the model’s universal applicability and cell-specific sensitivity.The reproducible detection of intravasation events in the established model provides a physiologically relevant platform to study processes of cancer metastasis with high spatio-temporal resolution and short timeframe.This approach holds promise for improved drug development and informed personalized patient treatment plans.展开更多
Tissue (re)vascularization strategies face various challenges, as therapeutic cells do not survive long enough in situ, while the administration of pro-angiogenic factors is hampered by fast clearance and insufficient...Tissue (re)vascularization strategies face various challenges, as therapeutic cells do not survive long enough in situ, while the administration of pro-angiogenic factors is hampered by fast clearance and insufficient ability to emulate complex spatiotemporal signaling. Here, we propose to address these limitations by engineering a functional biomaterial capable of capturing and concentrating the pro-angiogenic activities of mesenchymal stem cells (MSCs). In particular, dextran sulfate, a high molecular weight sulfated glucose polymer, supplemented to MSC cul-tures, interacts with MSC-derived extracellular matrix (ECM) components and facilitates their co-assembly and accumulation in the pericellular space. Upon decellularization, the resulting dextran sulfate-ECM hybrid material can be processed into MIcroparticles of SOlidified Secretome (MIPSOS). The insoluble format of MIPSOS protects protein components from degradation, while facilitating their sustained release. Proteomic analysis demonstrates that MIPSOS are highly enriched in pro-angiogenic factors, resulting in an enhanced pro-angiogenic bioactivity when compared to naïve MSC-derived ECM (cECM). Consequently, intravital microscopy of full-thickness skin wounds treated with MIPSOS demonstrates accelerated revascularization and healing, far superior to the ther-apeutic potential of cECM. Hence, the microparticle-based solidified stem cell secretome provides a promising platform to address major limitations of current therapeutic angiogenesis approaches.展开更多
基金supported by the research fund to the Center for Neuromusculoskeletal Restorative Medicine from Health@InnoHK program launched by Innovation and Technology Commission,the Government of the Hong Kong Special Administrative Region of the People’s Republic of China(AB),by the Health and Medical Research Fund(08191066,AB)a direct grant(4054732,AB)from the Faculty of Medicine,CUHK+1 种基金supported by the Lee Quo Wei and Lee Yick Hoi Lun Professorship in Tissue Engineering and Regenerative Medicine.A.J.receives a Walter Benjamin postdoctoral fellowship from the Deutsche Forschungsgemeinschaft(DFG,German Research Foundation,Germany521343357).
文摘During lung cancer metastasis,tumor cells undergo epithelial-to-mesenchymal transition(EMT),enabling them to intravasate through the vascular barrier and enter the circulation before colonizing secondary sites.Here,a human in vitro microphysiological model of EMT-driven lung cancer intravasation-on-a-chip was developed and coupled with machine learning(ML)-assisted automatic identification and quantification of intravasation events.A robust EMT-inducing cocktail(EMT-IC)was formulated by augmenting macrophage-conditioned medium with transforming growth factor-β1.When introduced into microvascular networks(MVNs)in microfluidic devices,EMT-IC did not affect MVN stability and physiologically relevant barrier functions.To model lung cancer intravasation on-a-chip,EMT-IC was supplemented into co-cultures of lung tumor micromasses and MVNs.Wihin 24 h of exposure,EMT-IC facilitated the insertion of membrane protrusions of migratory A549 cells into microvascular structures,followed by successful intravasation.EMT-IC reduced key basement membrane and vascular junction proteins-laminin and VE-Cadherin-rendering vessel walls more permissive to intravasating cells.ML-assisted vessel segmentation combined with co-localization analysis to detect intravasation events confirmed that EMT induction significantly increased the number of intravasation events.Introducing metastatic(NCI-H1975)and non-metastatic(BEAS-2B)cell lines demonstrated that both,baseline intravasation potential and responsiveness to EMT-IC,are reflected in the metastatic predisposition of lung cancer cell lines,highlighting the model’s universal applicability and cell-specific sensitivity.The reproducible detection of intravasation events in the established model provides a physiologically relevant platform to study processes of cancer metastasis with high spatio-temporal resolution and short timeframe.This approach holds promise for improved drug development and informed personalized patient treatment plans.
基金Funding support for material synthesis and in vitro work includes a laboratory start-up grant(8508266)from CUHK(AB),a direct grant(2019.016)from the Faculty of Medicine,CUHK(AB)and a grant from the Shun Hing Institute of Advanced Engineering(SHIAE,BME-p5-20,AB)Hong Kong SAR China.R.S.T.would like to acknowledge the Lee Quo Wei and Lee Yick Hoi Lun Professorship in Tissue Engineering and Regenerative Medicine(RST).J.G.and G.G.acknowledge financial support from the National Natural Science Foundation of China(J.G.,No.22178233)+1 种基金the National Global Talents Recruitment Program,the Talents Program of Sichuan Province,State Key Laboratory of Polymer Materials Engineering(Grant No.sklpme 2020-3-01)Key Laboratory of Leather Chemistry and En-gineering,and the National Engineering Research Center of Clean Technology in Leather Industry.The experimental data analyzed by Orbitrap Fusion mass spectrometer were acquired at the Academia Sinica Common Mass Spectrometry Facilities for Proteomics and Protein Modification Analysis located at the Institute of Biological Chemistry,Academia Sinica,supported by Academia Sinica Core Facility and Innovative Instrument Project Grant(AS-CFII-108-107).
文摘Tissue (re)vascularization strategies face various challenges, as therapeutic cells do not survive long enough in situ, while the administration of pro-angiogenic factors is hampered by fast clearance and insufficient ability to emulate complex spatiotemporal signaling. Here, we propose to address these limitations by engineering a functional biomaterial capable of capturing and concentrating the pro-angiogenic activities of mesenchymal stem cells (MSCs). In particular, dextran sulfate, a high molecular weight sulfated glucose polymer, supplemented to MSC cul-tures, interacts with MSC-derived extracellular matrix (ECM) components and facilitates their co-assembly and accumulation in the pericellular space. Upon decellularization, the resulting dextran sulfate-ECM hybrid material can be processed into MIcroparticles of SOlidified Secretome (MIPSOS). The insoluble format of MIPSOS protects protein components from degradation, while facilitating their sustained release. Proteomic analysis demonstrates that MIPSOS are highly enriched in pro-angiogenic factors, resulting in an enhanced pro-angiogenic bioactivity when compared to naïve MSC-derived ECM (cECM). Consequently, intravital microscopy of full-thickness skin wounds treated with MIPSOS demonstrates accelerated revascularization and healing, far superior to the ther-apeutic potential of cECM. Hence, the microparticle-based solidified stem cell secretome provides a promising platform to address major limitations of current therapeutic angiogenesis approaches.
