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Anaplastic lymphoma kinase (ALK) gene rearrangement in non-small cell lung cancer (NSCLC) is routinely evaluated by fluorescent in-situ hybridization (FISH) testing on biopsy tissues. Testing can be challenging however, when suitable tissue samples are unavailable. We examined the relevance of circulating tumor cells (CTC) as a surrogate for biopsy-based FISH testing. We assessed paired tumor and CTC samples from patients with ALK rearranged lung cancer (n = 14), ALK-negative lung cancer (n = 12), and healthy controls (n = 5) to derive discriminant CTC counts, and to compare ALK rearrangement patterns. Blood samples were enriched for CTCs to be used for ALK FISH testing. ALK-positive CTCs counts were higher in ALK-positive NSCLC patients (3-15 cells/1.88 mL of blood) compared with ALK-negative NSCLC patients and healthy donors (0-2 cells/1.88 mL of blood). The latter range was validated as the 'false positive' cutoff for ALK FISH testing of CTCs. ALK FISH signal patterns observed on tumor biopsies were recapitulated in CTCs in all cases. Sequential CTC counts in an index case of lung cancer with no evaluable tumor tissue treated with crizotinib showed six, three and eleven ALK-positive CTCs per 1.88 mL blood at baseline, partial response and post-progression time points, respectively. Furthermore, ALK FISH rearrangement suggestive of gene copy number increase was observed in CTCs following progression. Recapitulation of ALK rearrangement patterns in the tumor on CTCs, suggested that CTCs might be used to complement tissue-based ALK testing in NSCLC to guide ALK-targeted therapy when suitable tissue biopsy samples are unavailable for testing.

There has been an increased use of silicone gel for applications such as cell traction force measurements as well as lab- and organ-on-chip systems. However, silicone gel is a viscoelastic material which tends to undergo non-elastic deformation and displays time-dependent and strain rate-dependent responses. Here, we evaluated the mechanical responses of two types of commonly used silicone gels, Sylgard-184 and CY52-276, when subjected to nanoNewton force and micrometer displacement length scales. Using different mechanical characterization tools and theoretical models, we characterized and quantified the viscoelastic parameters of these substrates. Our experimental results showed that silicone substrates with high stiffness and elasticity and negligible strain rate-dependency and creep responses will be most suited for use at the nanoNewton force and micrometer displacement length scales such as that encountered in cell traction force assays.

ObjectivesWe aimed to study the prevalence of CTCs in breast cancer (BC) patients undergoing neoadjuvant or palliative therapy with a label-free microfluidic platform (ClearCell FX), and its prognostic relevance in metastatic BC (mBC).Materials and methodsPeripheral blood samples were collected from 108 BC patients before starting a new line of treatment ("baseline"), majority of whom had mBC (76/108; 70.4%). CTCs were retrieved by dean flow fractionation that enriched for larger cells, and enumerated using immunofluorescence-based staining. Progression-free survival (PFS) in mBC patients was analysed using Kaplan-Meier method; cox proportional hazard models were used for univariable and multivariable analyses.ResultsThe detection rate of CTCs before starting a new line of treatment was 75.9% (n = 108; median: 8 CTCs/7.5 ml blood) at a cut off of ≥2 CTCs. PFS was inferior for mBC patients with baseline CTC count ≥5 CTCs/7.5 ml blood vs. those with < 5 CTCs/7.5 ml blood (median PFS: 4.3 vs. 7.0 months; p-value: 0.037). The prognostic relevance of CTCs was most significant in patients with HER2- mBC (median PFS: 4.1 vs. 8.3 months; p-value: 0.032), luminal (HR+HER2-) subtype (median PFS: 4.2 vs. 8.3 months; p-value: 0.048), and patients who had one or more prior treatments (median PFS: 4.2 vs. 7.0 months; p-value: 0.02). On multivariable analysis, baseline CTC level (hazard ratio (HR): 1.84, p-value: 0.02) and pre-treatment status (HR: 1.87, p-value: 0.05) were independent predictors of PFS.ConclusionsThis work demonstrates the prognostic significance of CTCs in mBC detected using a label-free size-based enrichment platform.

Cell mechanics is a highly interdisciplinary research area which has made significant progress over the last decade, particularly in the study of human diseases. In diseases such as malaria and cancer, diseased cells undergo changes in both composition and organization of its cellular structures, which may eventually manifest as changes in the cell mechanical properties such as size and shape, deformability and cell adhesion. Despite the development of state-of-the-art experimental tools to manipulate and probe the cellular mechanical properties, microfluidics has emerged as an enabling technology for study of cell and molecular mechanics due to its numerous inherent advantages including small length scale, reduced sample and reagent volumes and low device cost. This paper presents an overview of the recent efforts in the study of cellular biomechanics using microfluidic approaches. Different areas of studies such as probing of cellular mechanical properties, cell separation using physical biomarkers (size, deformability, density) and effect of shear stress on cellular behavior and responses will be highlighted. Finally, we will discuss on the limitations and challenges associated with current microfluidics-based technologies and offer perspectives for future studies relating to cell biomechanics.

Collective behavior refers to the emergence of complex migration patterns over scales larger than those of the individual elements constituting a system. It plays a pivotal role in biological systems in regulating various processes such as gastrulation, morphogenesis and tissue organization. Here, by combining experimental approaches and numerical modeling, we explore the role of cell density (‘crowding’), strength of intercellular adhesion (‘cohesion’) and boundary conditions imposed by extracellular matrix (ECM) proteins (‘constraints’) in regulating the emergence of collective behavior within epithelial cell sheets. Our results show that the geometrical confinement of cells into well-defined circles induces a persistent, coordinated and synchronized rotation of cells that depends on cell density. The speed of such rotating large-scale movements slows down as the density increases. Furthermore, such collective rotation behavior depends on the size of the micropatterned circles: we observe a rotating motion of the overall cell population in the same direction for sizes of up to 200 μm. The rotating cells move as a solid body, with a uniform angular velocity. Interestingly, this upper limit leads to length scales that are similar to the natural correlation length observed for unconfined epithelial cell sheets. This behavior is strongly altered in cells that present a downregulation of adherens junctions and in cancerous cell types. We anticipate that our system provides a simple and easy approach to investigate collective cell behavior in a well-controlled and systematic manner.

Migrating cells constantly interact with their immediate microenvironment and neighbors. Although studies on single cell migration offer us insights into the molecular and biochemical signaling pathways, they cannot predict the influence of cell crowding and geometrical cues. Using microfabrication techniques, we examine the influence of cell density and geometrical constraints on migrating fibroblasts. Fibroblasts were allowed to migrate on fibronectin strips of different widths. Under such conditions, cells experience various physical guidance cues including boundary effect, confinement and contact inhibition from neighboring cells. Fibroblasts migrating along the edge of the fibronectin pattern exhibit spindle-like morphology, reminiscent of migrating cells within confined space and high cell density are associated with increased alignment and higher speed in migrating fibroblasts.

Background: HER2-positive tumors are often associated with poor prognosis, chemo-resistances and some patients eventually develop refractory disease during HER2 targeted therapy. While different mechanisms of trastuzumab resistance are being identified in recent years, contribution of confounding factors such as inherent genomic heterogeneity, equivocal HER2 amplifications and presence of chromosome 17 polysomy has been less understood thus far. In this pilot study, we aim to examine HER2 heterogeneity in CTCs obtained from breast cancer patients in the Asian population setting. Methods: CTCs were enriched from blood samples using a label-free spiral microfluidics-based ClearCell® FX system. A total of 26 samples were collected from patients diagnosed with HER2 positive (17/26) and HER2 negative (9/26) breast cancer. The enriched CTCs were analyzed using conventional diagnostic modalities (fluorescence in-situ hybridisation (FISH) and immunocytochemistry) to examine HER2 status. Concordance rate between CTCs and matched primary tumor was evaluated. Results: HER2-positive CTCs were successfully identified in 14 out of 17 HER2+ patients (82.4%); HER2 gene amplification and chromosome 17 polysomy were observed in 10(58.8%) and 13(76.5%) patients respectively. HER2+ CTC counts ranged from 2 to 30 cells from 7.5ml blood (median: 4 HER2+ CTCs/7.5ml). HER2 amplification was not observed in any of the 9 patients with HER2-negative tumors, though 5 out of 9 patients (55.6%) were identified with CTCs harbouring gain in chromosome 17 (median: 2 HER2+ CTCs/7.5ml). A “false positive” cut-off of more than 2 cells/7.5ml blood were established using receiver operating characteristic (ROC) curve analysis and a concordance rate of approximately 70% between paired tumor tissue and CTC among the 26 patients. Immunofluorescence labelling of CTCs with cytokeratin, CD45 and HER2 antibodies further revealed heterogeneity in HER2 expression on CTCs. Conclusion: CTCs capture the heterogeneity of breast cancer, and could potentially overcome limitations of tissue biopsy which are site specific. HER2- patients, as confirmed by tissue biopsy, with HER2+ CTCs pose interesting questions while determining treatment regime. Citation Format: Man Chun Leong, Tse Hui Lim, Chye Ling Tan, Yong Wei Chua, Elaine Hsuen Lim, Kiley Wei Jen Loh, Guek Eng Lee, Rebecca Dent, Raymond Chee Hui Ng, Andrew Wu, Wan-Teck Lim, Alvin Soon-Tiong Lim, Yoon-Sim Yap. Elucidating HER2 molecular heterogeneity of circulating tumor cells among breast cancer patients. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5008.

e23122Background: Tumor heterogeneity represents a key hallmark of cancer which complicates anti-cancer treatments. Presence of sub-clonal tumor population may be undetected through bulk tumor sequ...

e23086Background: Fluorescence in-situ hybridization (FISH) is the reference assay for ROS1 gene rearrangement. Molecular detection of ROS1 rearrangement in lung cancer has therapeutic relevance in...

The role of geometrical confinement on collective cell migration has been recognized but has not been elucidated yet. Here, we show that the geometrical properties of the environment regulate the formation of collective cell migration patterns through cell–cell interactions. Using microfabrication techniques to allow epithelial cell sheets to migrate into strips whose width was varied from one up to several cell diameters, we identified the modes of collective migration in response to geometrical constraints. We observed that a decrease in the width of the strips is accompanied by an overall increase in the speed of the migrating cell sheet. Moreover, large-scale vortices over tens of cell lengths appeared in the wide strips whereas a contraction-elongation type of motion is observed in the narrow strips. Velocity fields and traction force signatures within the cellular population revealed migration modes with alternative pulling and/or pushing mechanisms that depend on extrinsic constraints. Force transmission through intercellular contacts plays a key role in this process because the disruption of cell–cell junctions abolishes directed collective migration and passive cell–cell adhesions tend to move the cells uniformly together independent of the geometry. Altogether, these findings not only demonstrate the existence of patterns of collective cell migration depending on external constraints but also provide a mechanical explanation for how large-scale interactions through cell–cell junctions can feed back to regulate the organization of migrating tissues.

e22023 Background: Detection and characterization of rare circulating tumor cells (CTCs) are pivotal to early cancer detection and can provide insights into tumor biology leading to better cancer m...