RNA Sequencing of Pancreatic Circulating Tumour Cells Implicates WNT Signaling in Metastasis

Circulating tumour cells (CTCs) shed into blood from primary cancers include putative precursors that initiate distal metastases. While these cells are extraordinarily rare, they may identify cellular pathways contributing to the blood-borne dissemination of cancer. Here, we adapted a microfluidic device for efficient capture of CTCs from an endogenous mouse pancreatic cancer model and subjected CTCs to single molecule RNA sequencing, identifying Wnt2 as a candidate gene enriched in CTCs. Expression of Wnt2 in pancreatic cancer cells suppresses anoikis, enhances anchorage-independent sphere formation, and increases metastatic propensity in vivo. This effect is correlated with fibronectin upregulation and suppressed by inhibition of Map3k7 (Tak1) kinase. In humans, formation of non-adherent tumour spheres by pancreatic cancer cells is associated with upregulation of multiple Wnt genes, and pancreatic CTCs revealed enrichment for Wnt signaling in 5 of 11 cases. Thus, molecular analysis of CTCs may identify candidate therapeutic targets to prevent the distal spread of cancer

A microengineered vascularized bleeding model that integrates the principal components of hemostasis

Hemostasis encompasses an ensemble of interactions among platelets, coagulation factors, blood cells, endothelium, and hemodynamic forces, but current assays assess only isolated aspects of this complex process. Accordingly, here we develop a comprehensive in vitro mechanical injury bleeding model comprising an “endothelialized” microfluidic system coupled with a microengineered pneumatic valve that induces a vascular “injury”. With perfusion of whole blood, hemostatic plug formation is visualized and “in vitro bleeding time” is measured. We investigate the interaction of different components of hemostasis, gaining insight into several unresolved hematologic issues. Specifically, we visualize and quantitatively demonstrate: the effect of anti-platelet agent on clot contraction and hemostatic plug formation, that von Willebrand factor is essential for hemostasis at high shear, that hemophilia A blood confers unstable hemostatic plug formation and altered fibrin architecture, and the importance of endothelial phosphatidylserine in hemostasis. These results establish the versatility and clinical utility of our microfluidic bleeding model

Developing microfluidic systems to resolve longstanding hematological questions

Recent research has revealed that cells dynamically sense and respond to their physical microenvironments. In hematology, it has been shown that shear mediated red blood cell (RBC) deformation results in release, and that platelets attenuate contraction force based on substrate stiffness. Blood cells exist in a dynamic fluidic microenvironment, and they also interact with various matrices such as fibrin clots and the extracellular matrix proteins. The objective of this dissertation is thus to create microfluidic systems in which the biophysical and biochemical aspects of hematological processes are independently investigated toward the aim of discovering new solution spaces for diagnostics and therapeutics. To that end, this work presents novel microfluidic systems: 1) an “endothelial”-ized, T-junction fluidic to elucidate the biophysical processes that define the mechanism of action of the ferric chloride thrombosis model and 2) microfluidic devices with single-micron features (pillars and canals) to examine the effects of physical interactions between blood cell and geometrically relevant, non-biological matrices. Using the T-junction fluidic I resolved the mechanism of action of the most commonly used thrombosis injury model – the ferric chloride- thrombosis model. I show that the mechanism of action of ferric chloride is non-biological in nature. Rather, the chemical induces charge-based aggregation of blood cells that then triggers the conventional clotting cascade. This finding both reconciles discrepancies in the findings in the literature, and cautions researchers against using this injury model, especially for the study of clot initiation. My work also highlights the importance of considering mass transfer properties in biological processes. The second suite of devices recapitulates the physical dimensions of vascular matrices that blood cells interact with. By combining electron beam lithography, photolithography, and soft lithography, I considered hematological questions that were previously technologically infeasible. I found that the physical presence of a micropillar array creates a shear microgradient that leads to the localized adherence and aggregation of platelets that propagates to stem blood flow in the absence of exogenous agonists. Furthermore, I showed RBC fragmentation (schistocyte formation) real-time in an in vitro system by creating microcanals that forced RBCs to deform through a small cross-sectional area over various lengths of time. This further shows that RBC fragmentation is a time dependent process, contrary to what is historically cited in medical literature. Finally, by perfusing neutrophils through the microcanals, I show that mechanical forces can cause neutrophils to fragment into membranous debris that has neutrophil extracellular trap (NET)-like properties. This system provides insights into the synergy between hemolytic anemias, sepsis, and thrombosis. The mechanistic understandings gained by creating systems that successfully decouple the biophysical and biological aspects of blood cells, as is done in this work, can result in enhanced understanding of the etiology of pathologies, improved diagnostic assays for blood cell activity, and new targets for therapeutics.Ph.D

Single-Cell RNA Sequencing Identifies Extracellular Matrix Gene Expression by Pancreatic Circulating Tumor Cells

Circulating tumor cells (CTCs) are shed from primary tumors into the bloodstream, mediating the hematogenous spread of cancer to distant organs. To define their composition, we compared genome-wide expression profiles of CTCs with matched primary tumors in a mouse model of pancreatic cancer, isolating individual CTCs using epitope-independent microfluidic capture, followed by single-cell RNA sequencing. CTCs clustered separately from primary tumors and tumor-derived cell lines, showing low-proliferative signatures, enrichment for the stem-cell-associated gene Aldh1a2, biphenotypic expression of epithelial and mesenchymal markers, and expression of Igfbp5, a gene transcript enriched at the epithelial-stromal interface. Mouse as well as human pancreatic CTCs exhibit a very high expression of stromal-derived extracellular matrix (ECM) proteins, including SPARC, whose knockdown in cancer cells suppresses cell migration and invasiveness. The aberrant expression by CTCs of stromal ECM genes points to their contribution of microenvironmental signals for the spread of cancer to distant organs