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Sickle cell vasoocclusion and rescue in a microfluidic device.

ABSTRACT: The pathophysiology of sickle cell disease is complicated by the multiscale processes that link the molecular genotype to the organismal phenotype: hemoglobin polymerization occurring in milliseconds, microscopic cellular sickling in a few seconds or less [Eaton WA, Hofrichter J (1990) Adv Protein Chem 40:63-279], and macroscopic vessel occlusion over a time scale of minutes, the last of which is necessary for a crisis [Bunn HF (1997) N Engl J Med 337:762-769]. Using a minimal but robust artificial microfluidic environment, we show that it is possible to evoke, control, and inhibit the collective vasoocclusive or jamming event in sickle cell disease. We use a combination of geometric, physical, chemical, and biological means to quantify the phase space for the onset of a jamming event, as well as its dissolution, and find that oxygen-dependent sickle hemoglobin polymerization and melting alone are sufficient to recreate jamming and rescue. We further show that a key source of the heterogeneity in occlusion arises from the slow collective jamming of a confined, flowing suspension of soft cells that change their morphology and rheology relatively quickly. Finally, we quantify and investigate the effects of small-molecule inhibitors of polymerization and therapeutic red blood cell exchange on this dynamical process. Our experimental study integrates the dynamics of collective processes associated with occlusion at the molecular, polymer, cellular, and tissue level; lays the foundation for a quantitative understanding of the rate-limiting processes; and provides a potential tool for optimizing and individualizing treatment, and identifying new therapies.

SUBMITTER: Higgins JM

PROVIDER: S-EPMC2154459 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Cancer cells metastasize through the bloodstream either as single migratory circulating tumor cells (CTCs) or as multicellular groupings (CTC-clusters). Existing technologies for CTC enrichment are designed primarily to isolate single CTCs, and while CTC-clusters are detectable in some cases, their true prevalence and significance remain to be determined. Here, we developed a microchip technology (Cluster-Chip) specifically designed to capture CTC-clusters independent of tumor-specific markers from unprocessed blood. CTC-clusters are isolated through specialized bifurcating traps under low shear-stress conditions that preserve their integrity and even two-cell clusters are captured efficiently. Highly parallel architecture of the chip allows deterministic screening of clinically relevant volumes of blood samples at slow, and hence, non-damaging flow rates. Using the Cluster-Chip, we identify CTC-clusters in 30-40% of patients with metastatic cancers of the breast, prostate and melanoma. RNA sequencing of CTC-clusters confirms their tumor origin and identifies leukocytes within the clusters as being tissue-derived macrophages. Together, the development of a device for efficient capture of CTC-clusters will enable detailed characterization of their biological properties and role in cancer metastasis. We used the Cluster-Chip to capture CTC-clusters from the blood of a breast cancer patient with high CTC counts, released CTC-clusters in solution, stained them with TexasRed-conjugated antibodies against the leukocyte cell surface markers CD45, CD14 and CD16, and then isolated intact CTC-clusters individually using a micromanipulator. From a single time point, we retrieved 15 CTC-clusters, and each of those clusters was individually subjected to RNA-sequencing analysis using a next generation platform (SOLiD 5500). In addition, two leukocytes were isolated from the blood of a healthy donor were individually subjected to RNA-sequencing analysis using the same platform.

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Sickle cell vasoocclusion and rescue in a microfluidic device.

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