Inactive ERBB receptors cooperate with reactive oxygen species to suppress cancer progression.

The ERBB receptors are a family of heterodimerization partners capable of driving transformation and metastasis. While the therapeutic targeting of single receptors has proven efficacious, optimal targeting of this receptor family should target all oncogenic members simultaneously. The juxtamembrane domains of ERBB1, ERBB2, and ERBB3 are highly conserved and control various aspects of ERBB-dependent biology. In an effort to block those functions, we have targeted this domain with decoy peptides synthesized in tandem with a cell-penetrating peptide, termed EJ1. Treatment with EJ1 induces cell death, promotes the formation of inactive ERBB multimers, and results in simultaneous reduction of ERBB1, ERBB2, and ERBB3 activation. Treatment also results in the activation of myosin light chain-dependent cell blebbing while inactivating CaMKII signaling, coincident with the induction of cell death. EJ1 also directly translocates to mitochondria, correlating with a loss of mitochondrial membrane potential and production of reactive oxygen species. Finally, treatment of a mouse model of breast cancer with EJ1 results in the inhibition of tumor growth and metastasis without associated toxicities in normal cells. Overall, these data demonstrate that a portion of the ERBB jxm domain, when used as an intracellular decoy, can inhibit tumor growth and metastasis, representing a novel anticancer therapeutic.

MUC1 regulates nuclear localization and function of the epidermal growth factor receptor.

Alteration of protein trafficking and localization is associated with several diseases, including cystic fibrosis, breast cancer, colorectal cancer, leukemia and diabetes. Specifically, aberrant nuclear localization of the epidermal growth factor receptor (EGFR), a receptor tyrosine kinase, is a poor prognostic indicator in several epithelial carcinomas. It is now appreciated that in addition to signaling from the plasma membrane, EGFR also trafficks to the nucleus, and can directly bind the promoter regions of genes encoding cyclin D1 (CCND1) and B-Myb (MYBL2). We have previously established that loss of MUC1 in an EGFR-dependent transgenic mouse model of breast cancer correlates with the loss of cyclin D1 expression. Here, we provide evidence for a novel regulatory function of MUC1 in the trafficking and nuclear activity of EGFR. We found that MUC1 and EGFR interact in the nucleus of breast cancer cells, which promotes the accumulation of chromatin-bound EGFR. Additionally, the presence of MUC1 results in significant colocalization of EGFR and phosphorylated RNA polymerase II, indicating that MUC1 influences the association of EGFR with transcriptionally active promoter regions. Importantly, we found that the loss of MUC1 expression resulted in a decrease in the interaction between EGFR and the CCND1 promoter, which translated to a significant decrease in cyclin D1 protein expression. This data offers insights into a novel regulatory mechanism of EGFR nuclear function and could have important implications for evaluating nuclear localization in cancer.

Application of Microfluidic Systems for Breast Cancer Research.

Cancer is a disease in which cells in the body grow out of control; breast cancer is the most common cancer in women in the United States. Due to early screening and advancements in therapeutic interventions, deaths from breast cancer have declined over time, although breast cancer remains the second leading cause of cancer death among women. Most deaths are due to metastasis, as cancer cells from the primary tumor in the breast form secondary tumors in remote sites in distant organs. Over many years, the basic biological mechanisms of breast cancer initiation and progression, as well as the subsequent metastatic cascade, have been studied using cell cultures and animal models. These models, although extremely useful for delineating cellular mechanisms, are poor predictors of physiological responses, primarily due to lack of proper microenvironments. In the last decade, microfluidics has emerged as a technology that could lead to a paradigm shift in breast cancer research. With the introduction of the organ-on-a-chip concept, microfluidic-based systems have been developed to reconstitute the dominant functions of several organs. These systems enable the construction of 3D cellular co-cultures mimicking in vivo tissue-level microenvironments, including that of breast cancer. Several reviews have been presented focusing on breast cancer formation, growth and metastasis, including invasion, intravasation, and extravasation. In this review, realizing that breast cancer can recur decades following post-treatment disease-free survival, we expand the discussion to account for microfluidic applications in the important areas of breast cancer detection, dormancy, and therapeutic development. It appears that, in the future, the role of microfluidics will only increase in the effort to eradicate breast cancer.

Cxcl10 Chemokine Induces Migration of ING4-Deficient Breast Cancer Cells via a Novel Cross Talk Mechanism between the Cxcr3 and Egfr Receptors.

The chemokine Cxcl10 has been associated with poor prognosis in breast cancer, but the mechanism is not well understood. Our previous study has shown that CXCL10 was repressed by the ING4 tumor suppressor, suggesting a potential inverse functional relationship. We thus investigated a role for Cxcl10 in the context of ING4 deficiencies in breast cancer. We first analyzed public gene expression data sets and found that patients with CXCL10-high/ING4-low expressing tumors had significantly reduced disease-free survival in breast cancer. In vitro, Cxcl10 induced migration of ING4-deleted breast cancer cells but not of ING4-intact cells. Using inhibitors, we found that Cxcl10-induced migration of ING4-deleted cells required Cxcr3, Egfr, and the Gßγ subunits downstream of Cxcr3 but not Gαi. Immunofluorescent imaging showed that Cxcl10 induced early transient colocalization between Cxcr3 and Egfr in both ING4-intact and ING4-deleted cells, which recurred only in ING4-deleted cells. A peptide agent that binds to the internal juxtamembrane domain of Egfr inhibited Cxcr3/Egfr colocalization and cell migration. Taken together, these results presented a novel mechanism of Cxcl10 that elicits migration of ING4-deleted cells, in part by inducing a physical or proximal association between Cxcr3 and Egfr and signaling downstream via Gßγ. These results further indicated that ING4 plays a critical role in the regulation of Cxcl10 signaling that enables breast cancer progression.

Heart-valve mesenchyme formation is dependent on hyaluronan-augmented activation of ErbB2-ErbB3 receptors.

Heart septation and valve malformations constitute the most common anatomical birth defects. These structures arise from the endocardial cushions within the atrioventricular canal (AVC) through dynamic interactions between cushion cells and the extracellular matrix (termed cardiac jelly). Transformation of endothelial cells to mesenchymal cells is essential for the proper development of the AVC and subsequent septation and valve formation. Atrioventricular septal defects can result from incomplete endocardial cushion morphogenesis. We show that hyaluronan-deficient AVC explants from Has2(-/-) embryos, which normally lack mesenchyme formation, are rescued by heregulin treatment, which restores phosphorylation of ErbB2 and ErbB3. These events were blocked using a soluble ErbB3 molecule, as well as with an inhibitor of ErbB2, herstatin. We show further that ErbB3 is activated during hyaluronan treatment of Has2(-/-) explants. These data provide a link between extracellular matrix-hyaluronan and ErbB receptor activation during development of early heart-valve and septal mesenchyme.

Pilot study on the effects of dietary conjugated linoleic acid on tumorigenesis and gene expression in PyMT transgenic mice.

Conjugated linoleic acid (CLA) is a class of commercially available fatty acids that have been associated with anticancer properties in rodent models of chemical carcinogenesis. We conducted a pilot study to examine the antitumor effect of dietary CLA in a polyoma virus-middle T antigen (PyMT) mouse model of invasive breast cancer. Virgin 4-week-old PyMT mice were administered a mixed-isomer CLA diet (1% wt/wt) or control AIN-93G diet for 4 weeks (N = 6 and 5, respectively) and tumor burden was assessed at 8 weeks of age. Thoracic mammary glands were prepared as whole mounts with other glands being formalin fixed and paraffin embedded for histology and immunohistochemistry (IHC). Total RNA was prepared for microarray and real-time reverse transcription-polymerase chain reaction analysis. Western blots were performed for protein expression analysis. Tumor incidence was significantly increased in CLA-treated animals compared with controls (P = 0.009) and occurred with extensive lobular-alveolar expansion and loss of mammary adipose tissue. More than 100 genes were downregulated > or = 2-fold in the CLA-treated group compared with controls, including adipose-specific markers, as wells as cytoskeletal and adhesion-related genes. This was supported by dramatic decreases in the epithelial adherens E-cadherin and beta-catenin as demonstrated by IHC. Taken together, these results suggest that dietary CLA affects the mammary stromal environment, leading to tumor progression and cellular expansion in the PyMT mouse model. Further studies of the potential for cancer promotion are needed, especially because mixed-isomer CLA formulations are sold commercially as a nutritional supplement.

Intracellular MUC1 peptides inhibit cancer progression.

PURPOSE: During cancer progression, the oncoprotein MUC1 binds beta-catenin while simultaneously inhibiting the degradation of the epidermal growth factor receptor (EGFR), resulting in enhanced transformation and metastasis. The purpose of this study was to design a peptide-based therapy that would block these intracellular protein-protein interactions as a treatment for metastatic breast cancer. EXPERIMENTAL DESIGN: The amino acid residues responsible for these interactions lie in tandem in the cytoplasmic domain of MUC1, and we have targeted this sequence to produce a MUC1 peptide that blocks the protumorigenic functions of MUC1. We designed the MUC1 inhibitory peptide (MIP) to block the intracellular interactions between MUC1/beta-catenin and MUC1/EGFR. To allow for cellular uptake we synthesized MIP adjacent to the protein transduction domain, PTD4 (PMIP). RESULTS: We have found that PMIP acts in a dominant-negative fashion, blocking both MUC1/beta-catenin and MUC1/EGFR interactions. In addition, PMIP induces ligand-dependent reduction of EGFR levels. These effects correspond to a significant reduction in proliferation, migration, and invasion of metastatic breast cancer cells in vitro, and inhibition of tumor growth and recurrence in an established MDA-MB-231 immunocompromised (SCID) mouse model. Importantly, PMIP also inhibits genetically driven breast cancer progression, as injection of tumor-bearing MMTV-pyV mT transgenic mice with PMIP results in tumor regression and a significant inhibition of tumor growth rate. CONCLUSIONS: These data show that intracellular MUC1 peptides possess significant antitumor activity and have important clinical applications in the treatment of cancer.

Detachment of captured cancer cells under flow acceleration in a bio-functionalized microchannel.

Attachment, deformation and detachment of N-cadherin expressing prostate and breast cancer cell lines in a functionalized microchannel under hydrodynamic loading have been studied. N-cadherin antibodies are immobilized on the microchannel surface to capture the target cancer cells, PC3N and MDA-MB-231-N, from a homogeneous cell suspension. Although difficult, a significant fraction of moving cells can be captured under a low flow rate. More than 90% of the target cells are captured after a certain incubation time under no flow condition. The mechanical response of a captured cancer cell to hydrodynamic flow field is investigated and, in particular, the effect of flow acceleration is examined. The observed cell deformation is dramatic under low acceleration, but is negligible under high acceleration. Consequently, the detachment of captured cells depends on both flow rate and flow acceleration. The flow rate required for cell detachment is a random variable that can be described by a log-normal distribution. Two flow acceleration limits have been identified for proper scaling of the flow rate required to detach captured cells. A time constant for the mechanical response of a captured cell, on the order of 1 min, has been identified for scaling the flow acceleration. Based on these acceleration limits and time constant, an exponential-like empirical model is proposed to predict the flow rate required for cell detachment as a function of flow acceleration.

Transforming growth factor alpha dependent cancer progression is modulated by Muc1.

Transforming growth factor alpha (TGFalpha) is a potent inducer of cellular transformation, through its binding and activation of the epidermal growth factor receptor (EGFR). Previous studies in our laboratory showed that EGFR could also be affected by the glycoprotein MUC1, which inhibits ligand-stimulated degradation of EGFR in breast epithelial cell lines. To determine the effect of Muc1 expression on TGFalpha/EGFR-dependent breast transformation, we crossed the WAP-TGFalpha transgenic mouse model of breast cancer onto a Muc1-null background. We found that the loss of Muc1 expression dramatically affects mammary gland transformation and progression. Although 100% of WAP-TGFalpha/Muc1(+/+) mice form mammary gland tumors by 1 year, only 37% of WAP-TGFalpha/Muc1(-/-) form tumors by this time. This difference is also associated with a delay in onset, with a doubling of onset time observed in the WAP-TGFalpha/Muc1(-/-) compared with the WAP-TGFalpha/Muc1(+/+) mice. Analysis of signal transduction pathways revealed that activation of cyclin D1 expression is significantly suppressed in tumors derived from WAP-TGFalpha/Muc1(-/-) animals compared with those expressing Muc1. The loss of Muc1 expression also results in a significant inhibition in the formation of hyperplastic lesions during tumor progression. On the C57Bl/6 inbred background, pulmonary lesions were observed in 28 of 29 WAP-TGFalpha/Muc1(+/+) animals (including one metastatic pulmonary adenocarcinoma and multiple perivascular lymphomas), although none were detected in the WAP-TGFalpha/Muc1(-/-) animals. Together, these data indicate that Muc1 is an important modulator of TGFalpha-dependent tumor progression.

miR-9a mediates the role of Lethal giant larvae as an epithelial growth inhibitor in Drosophila.

Drosophila lethal giant larvae (lgl) encodes a conserved tumor suppressor with established roles in cell polarity, asymmetric division, and proliferation control. Lgl's human orthologs, HUGL1 and HUGL2, are altered in human cancers, however, its mechanistic role as a tumor suppressor remains poorly understood. Based on a previously established connection between Lgl and Fragile X protein (FMRP), a miRNA-associated translational regulator, we hypothesized that Lgl may exert its role as a tumor suppressor by interacting with the miRNA pathway. Consistent with this model, we found that lgl is a dominant modifier of Argonaute1 overexpression in the eye neuroepithelium. Using microarray profiling we identified a core set of ten miRNAs that are altered throughout tumorigenesis in Drosophila lgl mutants. Among these are several miRNAs previously linked to human cancers including miR-9a, which we found to be downregulated in lgl neuroepithelial tissues. To determine whether miR-9a can act as an effector of Lgl in vivo, we overexpressed it in the context of lgl knock-down by RNAi and found it able to reduce the overgrowth phenotype caused by Lgl loss in epithelia. Furthermore, cross-comparisons between miRNA and mRNA profiling in lgl mutant tissues and human breast cancer cells identified thrombospondin (tsp) as a common factor altered in both fly and human breast cancer tumorigenesis models. Our work provides the first evidence of a functional connection between Lgl and the miRNA pathway, demonstrates that miR-9a mediates Lgl's role in restricting epithelial proliferation, and provides novel insights into pathways controlled by Lgl during tumor progression.

CD44 attenuates metastatic invasion during breast cancer progression.

Metastatic invasion is the primary cause of breast cancer mortality, and adhesion receptors, such as CD44, are believed to be critical in this process. Historically, primary breast tumor epithelium has been investigated in isolation from other tissue components, leading to the common interpretation that CD44 and its primary ligand, hyaluronan, promote invasion. Here, we provide in vivo evidence showing CD44 antagonism to breast cancer metastasis. In a mouse model of spontaneously metastasizing breast cancer (MMTV-PyV mT), we found that loss of CD44 promotes metastasis to the lung. Localization studies, in combination with a novel hyaluronan synthase-GFP transgenic mouse, show a restricted pattern of expression for CD44 and hyaluronan. Whereas CD44 is expressed in tumor epithelium, hyaluronan synthase expression is restricted to stromal-associated cells. This distinct CD44 and hyaluronan pattern of distribution suggests a role for epithelial-stromal interaction in CD44 function. To define the relevance of this spatial regulation, we developed an in vitro invasion assay to emulate invasion into the extracellular matrix. Invasion of CD44-positive tumor cells was inhibited in hyaluronan-containing matrices, whereas blocking CD44-hyaluronan association increased invasion. Collectively, these data show that during breast cancer progression, hyaluronan-CD44 dynamics occurring through epithelial-stromal interactions are protective against metastasis.

MUC1 alters beta-catenin-dependent tumor formation and promotes cellular invasion.

MUC1 is aberrantly expressed in greater than 90% of all breast carcinomas, yet its function as a tumor antigen is not fully understood. Recently, studies have shown that MUC1 interacts with beta-catenin, erbB receptors, src, GSK-3beta and protein kinase Cdelta, possibly in a complex that promotes the disassembly of adherens junctions and the invasion of cells. Here we show that the deletion of Muc1 expression from MMTV-Wnt-1 transgenic mice results in a significant increase in the time to mammary gland tumor onset. Analysis of MMTV-Wnt-1 tumors on a wild-type Muc1 background shows a tumor-specific complex formation between Muc1 and beta-catenin that can be observed in both the membrane and the cytoplasm of transformed epithelium. Analysis of primary human adenocarcinomas revealed that this MUC1/beta-catenin interaction occurs in both primary and metastatic tumors, but is dramatically increased in metastatic lesions. Addition of MUC1-cytoplasmic domain peptides to the invasive MDA-MB-468 and MDA-MB-231 cell lines increases their invasive capability, and these peptides colocalize with both beta-catenin and the focal adhesion protein vinculin, primarily at sites of membrane invasion into a collagen matrix. These data indicate a potential mechanism for MUC1 promotion of invasive tumorigenesis in the breast through the modulation of beta-catenin localization and subsequent cytoskeletal dynamics.

MUC1 overexpression results in mammary gland tumorigenesis and prolonged alveolar differentiation.

MUC1 is a transmembrane mucin that was initially cloned from malignant mammary epithelial cells as a tumor antigen. More than 90% of human breast carcinomas overexpress MUC1. Numerous studies have demonstrated an interaction between MUC1 and other oncogenic proteins such as beta-catenin, erbB receptors and c-Src, but a functional role for MUC1 in transformation has not been identified. We previously reported the development of transgenic mice that overexpress human MUC1 in the mouse mammary gland (MMTV-MUC1). Analysis of these transgenic mice at an early age demonstrated the ability of MUC1 to potentiate EGF-dependent activation of MAP kinase signaling pathways in the lactating mammary gland. We now report that multiparous MMTV-MUC1 transgenic mice stochastically develop unifocal mammary gland carcinomas late in life. Molecular analysis of these tumors shows a tumor-specific coimmunoprecipitation between MUC1 and beta-catenin. Examination of the contralateral glands in MMTV-MUC1 transgenics demonstrates that the development of frank carcinomas is accompanied by a failure of multiparous glands to undergo postlactational involution. Furthermore, uniparous MMTV-MUC1 transgenic mice display decreased postlactational apoptosis, elevated whey acidic protein expression and aberrant pErk2 activation. These findings are the first to determine that MUC1 overexpression promotes in vivo transformation of the mammary gland.

Form and function of developing heart valves: coordination by extracellular matrix and growth factor signaling.

It is becoming clear that converging pathways coordinate early heart valve development and remodeling into functional valve leaflets. The integration of these pathways begins with macro and molecular interactions outside the cell in the extracellular matrix separating the myocardial and endocardial tissue components of the rudimentary heart. Such interactions regulate events at the cell surface through receptors, proteases, and other membrane molecules which in turn transduce signals into the cell. These signals trigger intracellular cascades that transduce cellular responses through both transcription factor and cofactor activation mediating gene induction or suppression. Chamber septation and valve formation occur from these coordinated molecular events within the endocardial cushions to sustain unidirectional blood flow and embryo viability. This review discusses the emerging connection between extracellular matrix and growth factor receptor signaling during endocardial cushion morphogenesis by highlighting the extracellular component, hyaluronan, and erbB receptor functions during early valve development.

MUC1 and metastatic cancer: expression, function and therapeutic targeting.

MUC1 is a transmembrane mucin that is often overexpressed in metastatic cancers and often used as a diagnostic marker for metastatic progression. The extracellular domain of MUC1 can serve as a ligand for stromal and endothelial cell adhesion receptors, and the cytoplasmic domain engages in several interactions that can result in increased migration and invasion, as well as survival. In this review, we address the role of MUC1 in metastatic progression by assessing clinical studies reporting MUC1 levels at various disease stages, reviewing mouse models utilized to study the role of MUC1 in metastatic progression, discuss mechanisms of MUC1 upregulation, and detail MUC1 protein interactions and signaling events. We review interactions between MUC1 and the extracellular environment, with proteins colocalized in the plasma membrane and/or cytoplasmic proteins, and summarize the role of MUC1 in the nucleus as a transcriptional cofactor. Finally, we review recent publications describing current therapies targeting MUC1 in patients with advanced disease and the stage of these therapies in preclinical development or clinical trials.

Hugl1 and Hugl2 in mammary epithelial cells: polarity, proliferation, and differentiation.

Loss of epithelial polarity is described as a hallmark of epithelial cancer. To determine the role of Hugl1 and Hugl2 expression in the breast, we investigated their localization in human mammary duct tissue and the effects of expression modulation in normal and cancer cell lines on polarity, proliferation and differentiation. Expression of Hugl1 and Hugl2 was silenced in both MCF10A cells and Human Mammary Epithelial Cells and cell lines were grown in 2-D on plastic and in 3-D in Matrigel to form acini. Cells in monolayer were compared for proliferative and phenotypic changes while acini were examined for differences in size, ability to form a hollow lumen, nuclear size and shape, and localization of key domain-specific proteins as a measure of polarity. We detected overlapping but distinct localization of Hugl1 and Hugl2 in the human mammary gland, with Hugl1 expressed in both luminal and myoepithelium and Hugl2 largely restricted to myoepithelium. On a plastic surface, loss of Hugl1 or Hugl2 in normal epithelium induced a mesenchymal phenotype, and these cells formed large cellular masses when grown in Matrigel. In addition, loss of Hugl1 or Hugl2 expression in MCF10A cells resulted in increased proliferation on Matrigel, while gain of Hugl1 expression in tumor cells suppressed proliferation. Loss of polarity was also observed with knockdown of either Hugl1 or Hugl2, with cells growing in Matrigel appearing as a multilayered epithelium, with randomly oriented Golgi and multiple enlarged nuclei. Furthermore, Hugl1 knock down resulted in a loss of membrane identity and the development of cellular asymmetries in Human Mammary Epithelial Cells. Overall, these data demonstrate an essential role for both Hugl1 and Hugl2 in the maintenance of breast epithelial polarity and differentiated cell morphology, as well as growth control.

MUC1 drives c-Met-dependent migration and scattering.

The transmembrane mucin MUC1 is overexpressed in most ductal carcinomas, and its overexpression is frequently associated with metastatic progression. MUC1 can drive tumor initiation and progression via interactions with many oncogenic partners, including ß-catenin, the EGF receptor (EGFR) and Src. The decoy peptide protein transduction domain MUC1 inhibitory peptide (PMIP) has been shown to inhibit the tumor promoting activities of MUC1 in breast and lung cancer, including cell growth and invasion, and its usage suppresses metastatic progression in mouse models of breast cancer. To further characterize the reduced metastasis observed upon PMIP treatment, we conducted motility assays and observed that PMIP inhibits cell motility of breast cancer cells. To determine the mechanism by which PMIP inhibits motility, we evaluated changes in global gene transcription upon PMIP treatment, and identified a number of genes with altered expression in response to PMIP. Among these genes is the metastatic mediator, c-Met, a transmembrane tyrosine kinase that can promote cell scattering, migration, and invasion. To further investigate the role of c-Met in MUC1-dependent metastatic events, we evaluated the effects of MUC1 expression and EGFR activation on breast cancer cell scattering, branching, and migration. We found that MUC1 strongly promoted all of these events and this effect was further amplified by EGF treatment. Importantly, the effect of MUC1 and EGF on these phenotypes was dependent upon c-Met activity. Overall, these results indicate that PMIP can block the expression of a key metastatic mediator, further advancing its potential use as a clinical therapeutic.

Understanding the dual nature of CD44 in breast cancer progression.

CD44 has been the subject of extensive research for more than 3 decades because of its role in breast cancer, in addition to many physiological processes, but interestingly, conflicting data implicate CD44 in both tumor suppression and tumor promotion. CD44 has been shown to promote protumorigenic signaling and advance the metastatic cascade. On the other hand, CD44 has been shown to suppress growth and metastasis. Histopathological studies of human breast cancer have correlated CD44 expression with both favorable and unfavorable clinical outcomes. In recent years, CD44 has garnered significant attention because of its utility as a stem cell marker and has surfaced as a potential therapeutic target, necessitating a greater understanding of CD44 in breast cancer. In this review, we attempt to unify the literature implicating CD44 in both tumor promotion and suppression, and explain its dualistic nature.

A high-performance microsystem for isolating circulating tumor cells.

A unique flow field pattern in a bio-functional microchannel is utilized to significantly enhance the performance of a microsystem developed for selectively isolating circulating tumor cells from cell suspensions. For high performance of such systems, disposal of maximum non-target species is just as important as retention of maximum target species; unfortunately, most studies ignore or fail to report this aspect. Therefore, sensitivity and specificity are introduced as quantitative criteria to evaluate the system performance enabling a direct comparison among systems employing different techniques. The newly proposed fluidic scheme combines a slow flow field, for maximum target-cell attachment, followed by a faster flow field, for maximum detachment of non-target cells. Suspensions of homogeneous or binary mixtures of circulating breast tumor cells, with varying relative concentrations, were driven through antibody-functionalized microchannels. Either EpCAM or cadherin-11 transmembrane receptors were targeted to selectively capture target cells from the suspensions. Cadherin-11-expressing MDA-MB-231 cancer cells were used as target cells, while BT-20 cells were used as non-target cells as they do not express cadherin-11. The attachment and detachment of these two cell lines are characterized, and a two-step attachment/detachment flow field pattern is implemented to enhance the system performance in capturing target cells from binary mixtures. While the system sensitivity remains high, above 0.95, the specificity increases from about 0.85 to 0.95 solely due to the second detachment step even for a 1 : 1000 relative concentration of the target cells.

Cell receptor and surface ligand density effects on dynamic states of adhering circulating tumor cells.

Dynamic states of cancer cells moving under shear flow in an antibody-functionalized microchannel are investigated experimentally and theoretically. The cell motion is analyzed with the aid of a simplified physical model featuring a receptor-coated rigid sphere moving above a solid surface with immobilized ligands. The motion of the sphere is described by the Langevin equation accounting for the hydrodynamic loadings, gravitational force, receptor-ligand bindings, and thermal fluctuations; the receptor-ligand bonds are modeled as linear springs. Depending on the applied shear flow rate, three dynamic states of cell motion have been identified: (i) free motion, (ii) rolling adhesion, and (iii) firm adhesion. Of particular interest is the fraction of captured circulating tumor cells, defined as the capture ratio, via specific receptor-ligand bonds. The cell capture ratio decreases with increasing shear flow rate with a characteristic rate. Based on both experimental and theoretical results, the characteristic flow rate increases monotonically with increasing either cell-receptor or surface-ligand density within certain ranges. Utilizing it as a scaling parameter, flow-rate dependent capture ratios for various cell-surface combinations collapse onto a single curve described by an exponential formula.