Abstract
Metastatic spread is mainly sustained by cancer stem cells (CSC), a subpopulation of cancer cells that displays stemness features. CSC are thought to be derived from cancer cells that undergo epithelial to mesenchymal transition (EMT), thus acquiring resistance to anoikis and anti-cancer drugs. After detachment from the primary tumor mass, CSC reach the blood and lymphatic flow, and disseminate to the target tissue. This process is by nature dynamic and in vitro models are quite far from the in vivo situation. In this study, we have tried to reproduce the adhesion process of CSC to a target tissue by using a 3D dynamic cell culture system. We isolated two populations of 3D tumor spheroids displaying CSC-like features from breast carcinoma (MCF-7) and lung carcinoma (A549) cell lines. Human fibroblasts were layered on a polystyrene scaffold placed in a dynamically perfused millifluidic system and then the adhesion of tumor cell derived from spheroids to fibroblasts was investigated under continuous perfusion. After 24 h of perfusion, we found that spheroid cells tightly adhered to fibroblasts layered on the scaffold, as assessed by a scanning electron microscope (SEM). To further investigate mechanisms involved in spheroid cell adhesion to fibroblasts, we tested the effect of three RGD integrin antagonists with different molecular structures on cell adhesion; when injected into the circuit, only cilengitide was able to inhibit cell adhesion to fibroblasts. Although our model needs further refinements and improvements, we do believe this study could represent a promising approach in improving current models to study metastatic infiltration in vitro and a new tool to screen new potential anti-metastatic molecules.
Highlights
Over 90% of cancer-associated deaths are caused by metastatic disease, rather than primary tumors [1]
Fibroblasts were grown on polystyrene scaffolds in LB1 chambers and at day 10 before the methods); briefly, the alamar blue reagent (Tox8, resazurin) was added to the culture media at experiments were shifted to dynamic culture conditions with a medium flux of 100 μL/min for other increasing times and fluorescence was read at an excitation wavelength of 590 nm
This scenario makes the study of metastasis a daunting task; in the past, in this field, there is an urgent need to implement reliable in vitro models that could mimic as close several drug candidates failed in translation from in vitro experimental models to in vivo tests
Summary
Over 90% of cancer-associated deaths are caused by metastatic disease, rather than primary tumors [1]. We have tried to set up an in vitro 3D dynamic model to resemble as close as possible the first steps of metastatic spread This model is clearly an approximation of in vivo processes, it could be useful to investigate molecular mechanisms underlying pre-metastatic niche formation and metastatic cell adhesion. We tested the ability of three integrin antagonists with different chemical structures, FR-72, 1a-RGD, and cilengitide [9,11,12], to inhibit cancer cell adhesion to 3D fibroblasts, and found that only the well-known integrin antagonist cilengitide markedly inhibits CSC adhesion after 24 h We suggest that this dynamic model could be a reliable experimental model to study metastasis-related processes, suitable for the screening of new molecules with anti-metastatic activity
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