The development of different strategies and platforms to identify and characterize CSC have led novel approaches for cancer predictions and treatment. There is wide evidence that, this minority cell population is associated to local tumor recurrence, metastases and therapeutic resistance that impact in patient’s survival [8], [9], [28], [29].
New versatile, cost-effective, and efficient methods that can perform in vitro stem cells-based studies with clinical relevance help to improve the follow-up of cancer treatment. The rise of microfluidics platforms that allow a better use of samples to obtain information with statistical advantages, has gained relevance during the last decades [17], [30], [31]. In this study, we report a novel MDs that allows an in vitro CSC culture method. Work scale miniaturization has enabled to obtain many advantages as, get robust information with small samples, more homogeneous experimental conditions, and less reactive and time-consumption.
The device used in this article has 72 individual wells enabling the possibility of obtaining a higher number of replicates and the tracking of individual sphere growth. Furthermore, a better homogenous cell distribution was observed within this device than the one previously descripted [2]. Different methods have been described for cell seeding inside devices, some of them include cell injection with minimal flow using a syringe pump or pump-free approaches [20], [32]. Our work describes a pump-free method with robust information about CT in spheres that result useful for laboratories with limited technological capacity [19], [33].
Drug-screening studies in cell cultures are a good strategy to evaluate in vitro new antitumor compounds, providing valuable information to predict patient treatment response[34]–[36]. In this work, we could develop a MDs that can be a sphere culture platform, allowing to quantify the number of spheres, their size, the single-sphere growth and compare these results against CT with drugs that are used in clinics. These results are comparable with conventional culture plates, validating the use of this MDs.
The use of stem cell-enriched culture is guaranteed by evaluating the expression of some pluripotent genetic markers like Oct4, Sox2, Nanog and CD44 in chemotherapeutic resistant spheres [15], [37]. All of them are transcription factors of stemness expressed in adult stem-like cells but not in normal differentiated cells [38]. Therefore, the possibility of quantifying these genes will be useful to evaluate the biology of therapeutic resistant cells. In this sense, we demonstrate that the MDs has been useful for determining the expression of pluripotency genetic markers by qPCR and IF with the possibility of obtained high-quality RNA.
Interestingly, we observed in MDs and culture plates that despite the amount of spheres decreases after treatment, the expression of pluripotency genetic markers in the resistant spheres is higher than in untreated ones. There are different biological explanations for this event, such as cell heterogeneity, [2], [39], the selection of resistant CSC, or the activation of dedifferentiation processes by stemness-related transcription factors increased expression [40], [41]. However, these findings have also been reported in others cancer models like breast [41], [42], small-cells lung cancer [43] and glioblastoma [44].
We have shown that the MDs has an additional advantage allowing the monitoring of individual cells. The design of the device and wells facilitates the identification of individual spheres and makes it possible determine their growth rate. This fact will allow the identification and monitoring of different clones from tumor samples. It is already known that some CSC-targeted therapies can avoid the resistant clone selection after antitumoral treatment in cancer, hence the importance of developing MDs that allow the analysis and the efficacy of these therapeutic strategies [10], [11], [45], [46].
Finally, we develop a MDs that are suitable for CSC enrichment by sphere isolation directly from tumor samples. This strategy brings us closer to a more physiological condition, considering even elements of the tumor microenvironment. The study of CSC through tumor-derived spheres could be a first approach in the study of targeted therapies for this cell population. There are reported devices capable of generating spheroids from primary culture from different tumor samples, but the sphere isolation is enriched in stem-like cells and that have an additional benefit [16], [34], [35], [47]. In this work we were able to isolate spheres without functionalized surfaces for the capture of cells with stem potential using antibodies, which makes it an even simpler device [8], [48], [49]. We consider that we can bring a portable, economical, and versatile tool with great potential in personalized medicine.
The translational potential of MDs into clinics, specially applied to study of small patient samples, has been validated in different studies and it is still growing [27], [50], [51]. The limitations of different microfluidics platforms are associated with the complex manufacturing process that include the selection of material, the fabrication techniques standardization and drug-specific limitations before considering the miniaturization [31], [52].