Multicellular aggregates of circulating tumor cells (CTC clusters) are potent initiators of distant organ metastasis. However, it is currently assumed that CTC clusters are too large to pass through narrow vessels to reach these organs. Here, we present evidence that challenges this assumption through the use of microfluidic devices designed to mimic human capillary constrictions and CTC clusters obtained from patient and cancer cell origins. Over 90% of clusters containing up to 20 cells successfully traversed 5-to 10-μm constrictions even in whole blood. Clusters rapidly and reversibly reorganized into single-file chain-like geometries that substantially reduced their hydrodynamic resistances. Xenotransplantation of human CTC clusters into zebrafish showed similar reorganization and transit through capillary-sized vessels in vivo. Preliminary experiments demonstrated that clusters could be disrupted during transit using drugs that affected cellular interaction energies. These findings suggest that CTC clusters may contribute a greater role to tumor dissemination than previously believed and may point to strategies for combating CTC cluster-initiated metastasis.microfluidics | cancer metastasis | CTC clusters | circulating tumor cell cluster microemboli | capillary microhemodynamics

We introduce the first lab-on-a-chip platform for complete mammalian cell culture. The new method is powered by digital microfluidics (DMF), a technique in which nanolitre-sized droplets are manipulated on an open surface of an array of electrodes. This is the first application of DMF to adherent cell culture and analysis, and more importantly, represents the first microfluidic platform capable of implementing all of the steps required for mammalian cell culture-cell seeding, growth, detachment, and re-seeding on a fresh surface. Three key innovations were required to implement complete cell culture on a microfluidic device: (1) a technique for growing cells on patterned islands (or "adhesion pads") positioned on an array of DMF actuation electrodes; (2) a method for rapidly and efficiently exchanging media and other reagents on cells grown on adhesion pads; and (3) a system capable of detachment and collection of cells from an (old) origin site and delivery to a (new) destination site for subculture. The new technique was applied to cells from several different lines which were seeded and repeatedly subcultured for weeks at a time in 150 nL droplets. Cells handled in this manner exhibited growth characteristics and morphology comparable to those cultured in standard tissue culture vessels. To illustrate an application for this system, a microfluidic method was developed to implement transient transfection-we propose that the combination of this technique with multigenerational culture allows for "on-demand" generation of transiently transfected cells. Broadly, we anticipate that the automated cell microculture technique presented here will be useful in myriad applications that would benefit from automated mammalian cell culture.

Circulating tumor cell clusters (CTC clusters) are potent initiators of metastasis and potentially useful clinical markers for patients with cancer. Although there are numerous devices developed to isolate individual circulating tumor cells from blood, these devices are ineffective at capturing CTC clusters, incapable of separating clusters from single cells and/or cause cluster damage or dissociation during processing. The only device currently able to specifically isolate CTC clusters from single CTCs and blood cells relies on the batch immobilization of clusters onto micropillars which necessitates long residence times and causes damage to clusters during release. Here, we present a two-stage continuous microfluidic chip that isolates and recovers viable CTC clusters from blood. This approach uses deterministic lateral displacement to sort clusters by capitalizing on two geometric properties: size and asymmetry. Cultured breast cancer CTC clusters containing between 2–100 + cells were recovered from whole blood using this integrated two-stage device with minimal cluster dissociation, 99% recovery of large clusters, cell viabilities over 87% and greater than five-log depletion of red blood cells. This continuous-flow cluster chip will enable further studies examining CTC clusters in research and clinical applications.