B. Schnoor, R.K. Kottana, A.L. Papa
George Washington University,
Keywords: tumor cell clusters, light transmission aggregometry, tumor cell dissociation
Summary:During cancer metastasis, circulating tumor cells (CTCs) travel either as single cells or as a multi-cell clusters. These CTC clusters have a significantly increased metastatic potential and an enhanced survival compared to single CTCs. As such, there is a critical need to study the formation and dissociation of CTC clusters. However, current approaches for examining cluster formation are focused on monitoring CTC clusters in patients or are constrained by user-dependent, less directly quantifiable imaging methods. Furthermore, current procedures to create tumor-cells clusters do not account for platelets interacting with CTCs. To address these challenges, we have developed a method utilizing light transmission aggregometry (LTA) to quantitatively characterize the association and dissociation of tumor cell clusters and can be adapted to produce consistent platelet-tumor cell clusters in vitro. Our method enables the precise quantification of tumor cell cluster dissociation with the fibrinolytic agents, such as clinical plasminogen activator Alteplase (TPA) and Tenecteplase (TNK). This dissociation was observed using both breast cancer (MDA-MB-231) and lung cancer (A549) cell lines. The fibrinolytic data was analyzed for lag time for tumor cell dissociation, time to 25% dissociation, and dissociation over time (Fig 1). The results successfully show the dissociation curve for the tumor cell clusters with the predicted trends of decreased lag time and time to 25% dissociation for higher concentrations of fibrinolytic treatments (Fig. 1). As such, our method can be used to identify key differences in the dissociation curves. Subsequently, we validated our method using a cell count and microscopy method. The quantitative cell counts and the qualitative analysis of imaged cell clusters both indicated the same trends as the data obtained using our LTA method (Fig 2). This corroborating data demonstrates that our LTA method is able to accurately measure trends in the dissociation of clustered tumor cells. Furthermore, our method has also been adapted to produce platelet-tumor cell clusters that can be utilized in downstream applications. The interaction of CTC clusters with platelets in the bloodstream promotes cancer cell survival and metastasis. In order to characterize the effect of these platelet-tumor cell clusters and develop treatments to target these interactions, it is valuable to produce clusters that can be monitored and modulated for different size parameters. Our method allows for the modulation of cluster size by altering the cell concentration, while the clustering is monitored using LTA (Fig. 3). The formation and monitoring of these platelet-tumor cell clusters was validated via cell counting and microscopy (Fig. 3). Utilizing an LTA approach, our method is able to successfully quantify the dissociation of tumor cell clusters in vitro, allowing for the characterization of the dissociation curve and the evaluation of agents, such as fibrinolytics used to disperse clusters. Our method also allowed for the consistent, monitored production of platelet cancer cell clusters. Therefore, our method can provide a new effective approach to study tumor cell clustering and explore treatments to disperse or inhibit these highly metastatic clusters.