The ideal in vitro recreation of the micro-tumor niche—although much needed for a better understanding of cancer etiology and development of better anticancer therapies—is highly challenging. Tumors are complex three-dimensional (3D) tissues that establish a dynamic cross-talk with the surrounding tissues through complex chemical signaling. An extensive body of experimental evidence has established that 3D culture systems more closely recapitulate the architecture and the physiology of human solid tumors when compared with traditional 2D systems. Moreover, conventional 3D culture systems fail to recreate the dynamics of the tumor niche. Tumor-on-chip systems, which are microfluidic devices that aim to recreate relevant features of the tumor physiology, have recently emerged as powerful tools in cancer research. In tumor-on-chip systems, the use of microfluidics adds another dimension of physiological mimicry by allowing a continuous feed of nutrients (and pharmaceutical compounds). Here, we discuss recently published literature related to the culture of solid tumor-like tissues in microfluidic systems (tumor-on-chip devices). Our aim is to provide the readers with an overview of the state of the art on this particular theme and to illustrate the toolbox available today for engineering tumor-like structures (and their environments) in microfluidic devices. The suitability of tumor-on-chip devices is increasing in many areas of cancer research, including the study of the physiology of solid tumors, the screening of novel anticancer pharmaceutical compounds before resourcing to animal models, and the development of personalized treatments. In the years to come, additive manufacturing (3D bioprinting and 3D printing), computational fluid dynamics, and medium- to high-throughput omics will become powerful enablers of a new wave of more sophisticated and effective tumor-on-chip devices.

Cancer continues to be a leading cause of mortality in modern societies; therefore, improved and more reliable in vitro cancer models are needed to expedite fundamental research and anti-cancer drug development. Here, we describe the use of a miniaturized continuous stirred tank reactor (mCSTR) to first fabricate and mature cancer spheroids (i.e, derived from MCF7 cells, DU145 cells, and a mix of MCF7 cells and fibroblasts), and then to conduct anti-cancer drug assays under continuous perfusion. This 3 mL mCSTR features an off-center agitation system that enables homogeneous chaotic laminar mixing at low speeds to support cell aggregation. We incubated cell suspensions for 3 days in ultra-low-adherence (ULA) plates to allow formation of discoid cell aggregates (~600 µm in diameter). These cell aggregates were then transferred into mCSTRs and continuously fed with culture medium. We characterized the spheroid morphology and the expression of relevant tumor biomarkers at different maturation times for up to 4 weeks. The spheroids progressively increased in size during the first 5 to 6 days of culture to reach a steady diameter between 600 and 800 µm. In proof-of-principle experiments, we demonstrated the use of this mCSTR in anti-cancer drug testing. Three drugs commonly used in breast cancer treatment (doxorubicin, docetaxel, and paclitaxel) were probed at different concentrations in MCF7 derived spheroids. In these experiments, we evaluated cell viability, glucose consumption, spheroid morphology, lactate dehydrogenase activity, and the expression of genes associated with drug resistance (ABCB1 and ABCC1) and anti-apoptosis (Bcl2). We envision the use of this agitated system as a tumor-on-a-chip platform to expedite efficacy and safety testing of novel anti-cancer drugs and possibly in personalized medicine applications.

Background: The endocardium is a crucial signaling center for cardiac valve development and maturation. Genetic analysis has identified several human endocardial genes whose inactivation leads to bicuspid aortic valve (BAV) formation and/or calcific aortic valve disease (CAVD), but knowledge is very limited about the role played in valve development and disease by non-coding endocardial regulatory regions and upstream factors. Methods: We manipulated the NOTCH signaling pathway in mouse embryonic endocardial cells, defining the transcriptional profile associated to each condition. The endocardial chromatin accessibility landscape for each condition was defined by high-throughput sequencing (ATAC-seq) determination of transposase-accessible chromatin. In vitro and in vivo models carrying deletions of different non-coding regulatory elements were generated by CRISPR-Cas9 gene editing. Results: We identified primary and secondary transcriptional responses to NOTCH ligands in the mouse embryonic endocardium. By integrating our gene expression data with data from developing valves of mice with NOTCH loss-of-function and from human valve calcification samples, we identified a NOTCH-dependent transcriptional signature in valve development and disease. Further, by defining the endocardial chromatin accessibility landscape after NOTCH pathway manipulation and integrating with in vivo data from developing mouse endocardium and adult human valves, we were able to identify a set of potential non-coding regulatory elements, validate representative candidates, propose co-factors interacting with them, and define the timeframe of their regulatory activity. Analysis of the transcriptional repression driven by NOTCH activation revealed cooperation between the NOTCH and HIPPO pathways in the endocardium during cardiac valve development. Conclusions: Transcriptional regulation in the embryonic endocardium after NOTCH pathway stimulation occurs in a sequential manner and requires the participation of several factors. NOTCH not only triggers the transcriptional activity of the non-coding elements recognized by these factors, but also represses those elements whose activity negatively affects the development and homeostasis of the cardiac valves.