Introduction



Characterization and separation of cancer cells with a wicking fiber deviceSuzanne M. Tabbaa1,2, Julia L. Sharp3, and Karen J.L. Burg1,2,41Department of Bioengineering; Clemson University, Clemson, SC 29634, United States 2Institute for Biological Interfaces of Engineering; Clemson University, Clemson, SC 29634, United States3Department of Mathematical Sciences; Clemson University, Clemson, SC 29634, United States4Department of Small Animal Medicine & Surgery; University of Georgia, Athens, GA 30602, United StatesAbstractCurrent cancer diagnostic methods lack the ability to quickly, simply, efficiently, and inexpensively screen cancer cells from a mixed population of cancer and normal cells. Methods based on biomarkers are unreliable due to complexity of cancer cells, plasticity of markers, and lack of common tumorigenic markers. Diagnostics are time intensive, require multiple tests, and provide limited information. In this study, we developed a novel wicking fiber device that separates cancer and normal cell types. To the best of our knowledge, no previous work has used vertical wicking of cells through fibers to identify and isolate cancer cells. The device separated mouse mammary tumor cells from a cellular mixture containing normal mouse mammary cells. Further investigation showed the device separated and isolated human cancer cells from a heterogeneous mixture of normal and cancerous human cells. We report a simple, inexpensive, and rapid technique that has potential to identify and isolate cancer cells from large volumes of liquid samples that can be translated to on-site clinic diagnosis. Key TermsBiomarkers, cancer, cell movement, circulating tumor cell, in vitro diagnostic IntroductionCurrent methods to diagnose and analyze the presence and progression of cancer involve histological and genetic analysis of a solid tumor to predict the stage of the cancer1. A major limitation of this type of diagnosis is the heterogeneity of the tumor as well as the fact that the analysis occurs after a palpable tumor has formed. Tumors are reported to have different subpopulations of cancer cells with varying phenotypes and degrees of tumor-initiating capabilities2. The subpopulations of cancer cells create tumorigenic regions and non-tumorigenic regions within the tumor3. This regionality limits the ability for a single biopsy to capture complete information about the tumor1. Common biopsy analysis involves detecting phenotypic and genotypic biomarkers; this type of analysis is unreliable and lacks specificity due to the heterogeneous phenotype and genotype of cancer cellsADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "DOI" : "10.1146/annurev-pathol-020712-163923", "abstract" : "Intratumor heterogeneity represents a major obstacle to effective cancer treatment and personalized medicine. However, investigators are now elucidating intratumor heterogeneity at the single-cell level due to improvements in technologies. Better understanding of the composition of tumors, and monitoring changes in cell populations during disease progression and treatment, will improve cancer diagnosis and therapeutic design. Measurements of intratumor heterogeneity may also be used as biomarkers to predict the risk of progression and therapeutic resistance. We summarize important considerations related to intratumor heterogeneity during tumor evolution. We also discuss experimental approaches that are commonly used to infer intratumor heterogeneity and describe how these methodologies can be translated into clinical practice.", "author" : [ { "dropping-particle" : "", "family" : "Almendro", "given" : "Vanessa", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Marusyk", "given" : "Andriy", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Polyak", "given" : "Kornelia", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Annu. Rev. Pathol.", "id" : "ITEM-1", "issued" : { "date-parts" : [ [ "2013", "1" ] ] }, "page" : "277-302", "title" : "Cellular heterogeneity and molecular evolution in cancer.", "type" : "article-journal", "volume" : "8" }, "uris" : [ "", "" ] }, { "id" : "ITEM-2", "itemData" : { "DOI" : "10.1016/j.gde.2008.01.017", "abstract" : "The theory of cancer stem cells states that a subset of cancer cells within a tumor has the ability to self-renew and differentiate. Only those cells within a tumor that have these two properties are called cancer stem cells. This concept was first demonstrated in the study of leukemia where only cells with specific surface antigen profiles were able to cause leukemia when engrafted into immunodeficient mice. In recent years solid tumors were studied utilizing similar techniques in mice. Human tumors where evidence of cancer stem cells has been published include tumors of the breast, brain, pancreas, head and neck, and colon. If this difference in tumorigenicity of cancer cells also occurs in patients, then the ability to enrich for cancer stem cells lays an important groundwork for future studies where mechanisms involved in cancer stem cells can now be investigated.", "author" : [ { "dropping-particle" : "", "family" : "Cho", "given" : "Robert W", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Clarke", "given" : "Michael F", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Curr. Opin. Genet. Dev.", "id" : "ITEM-2", "issue" : "1", "issued" : { "date-parts" : [ [ "2008", "2" ] ] }, "page" : "48-53", "title" : "Recent advances in cancer stem cells.", "type" : "article-journal", "volume" : "18" }, "uris" : [ "", "" ] }, { "id" : "ITEM-3", "itemData" : { "abstract" : "Tumors may be initiated and maintained by a cellular subcomponent that displays stem cell properties. We have used the expression of aldehyde dehydrogenase as assessed by the ALDEFLUOR assay to isolate and characterize cancer stem cell (CSC) populations in 33 cell lines derived from normal and malignant mammary tissue. Twenty-three of the 33 cell lines contained an ALDEFLUOR-positive population that displayed stem cell properties in vitro and in NOD/SCID xenografts. Gene expression profiling identified a 413-gene CSC profile that included genes known to play a role in stem cell function, as well as genes such as CXCR1/IL-8RA not previously known to play such a role. Recombinant interleukin-8 (IL-8) increased mammosphere formation and the ALDEFLUOR-positive population in breast cancer cell lines. Finally, we show that ALDEFLUOR-positive cells are responsible for mediating metastasis. These studies confirm the hierarchical organization of immortalized cell lines, establish techniques that can facilitate the characterization of regulatory pathways of CSCs, and identify potential stem cell markers and therapeutic targets.", "author" : [ { "dropping-particle" : "", "family" : "Charafe-Jauffret", "given" : "Emmanuelle", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Ginestier", "given" : "Christophe", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Iovino", "given" : "Flora", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Wicinski", "given" : "Julien", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Cervera", "given" : "Nathalie", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Finetti", "given" : "Pascal", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Hur", "given" : "Min-Hee", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Diebel", "given" : "Mark E", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Monville", "given" : "Florence", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Dutcher", "given" : "Julie", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Brown", "given" : "Marty", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Viens", "given" : "Patrice", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Xerri", "given" : "Luc", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Bertucci", "given" : "Fran\u00e7ois", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Stassi", "given" : "Giorgio", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Dontu", "given" : "Gabriela", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Birnbaum", "given" : "Daniel", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Wicha", "given" : "Max S", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Cancer Res.", "id" : "ITEM-3", "issue" : "4", "issued" : { "date-parts" : [ [ "2009", "2" ] ] }, "page" : "1302-1313", "title" : "Breast cancer cell lines contain functional cancer stem cells with metastatic capacity and a distinct molecular signature.", "type" : "article-journal", "volume" : "69" }, "uris" : [ "", "" ] }, { "id" : "ITEM-4", "itemData" : { "DOI" : "10.1016/r.2012.03.003", "abstract" : "The differentiation of tumorigenic cancer stem cells into nontumorigenic cancer cells confers heterogeneity to some cancers beyond that explained by clonal evolution or environmental differences. In such cancers, functional differences between tumorigenic and nontumorigenic cells influence response to therapy and prognosis. However, it remains uncertain whether the model applies to many, or few, cancers due to questions about the robustness of cancer stem cell markers and the extent to which existing assays underestimate the frequency of tumorigenic cells. In cancers with rapid genetic change, reversible changes in cell states, or biological variability among patients, the stem cell model may not be readily testable.", "author" : [ { "dropping-particle" : "", "family" : "Magee", "given" : "Jeffrey A", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Piskounova", "given" : "Elena", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Morrison", "given" : "Sean J", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Cancer cell", "id" : "ITEM-4", "issue" : "3", "issued" : { "date-parts" : [ [ "2012", "3" ] ] }, "page" : "283-296", "publisher" : "Elsevier Inc.", "title" : "Cancer stem cells: impact, heterogeneity, and uncertainty.", "type" : "article-journal", "volume" : "21" }, "uris" : [ "", "" ] } ], "mendeley" : { "formattedCitation" : "^1,5,6,15", "plainTextFormattedCitation" : "1,5,6,15", "previouslyFormattedCitation" : "(Cho and Clarke 2008; Charafe-Jauffret et al. 2009; Magee et al. 2012; Almendro et al. 2013)" }, "properties" : { "noteIndex" : 0 }, "schema" : "" }3,4,5,6. The standard techniques to analyze a tumor biopsy are time-consuming, expensive, provide limited information regarding the metastatic potential of subpopulations within the tumor, and require multiple forms of off-site analysis to confirm results. Most importantly, tumor interrogation occurs only after a mass has been identified, i.e. after the cancer process has evolved.Other diagnostic techniques are shifting from invasive tissue analysis toward liquid biopsy analysis, e.g. blood biopsy, which has the potential to detect the initial occurrence of cancer. A minimally invasive blood biopsy sample can be used for detecting circulating tumor cells (CTCs) and diagnosing the cancer stage, monitoring treatments, and providing insight into the metastatic process7. However, major limitations for detecting CTCs are the exceedingly low concentrations of CTCs in blood samples and the heterogeneous phenotypes within the CTC population8,9. As a result, the current CTC detection and isolation systems have low capture efficiencies and low specificities. Technologies for CTC detection and separation include macro-scale systems and microfluidics that use physical characteristics of the cells as well as cell surface labels. Most macro-scale systems use antibody-dependent techniques that detect epithelial markers to target CTCs. These approaches assume all CTCs are expressing epithelial markers, which limits these approaches to a specific subpopulation of CTCs. Aggressive CTC populations commonly exhibit mesenchymal phenotypes in the bloodstream and, because they lack the epithelial markers that current CTC technologies target, go undetected10,11,12,13,14. Other CTC detection methods include microfluidic devices and chips that use both antigen-dependent and antigen-independent techniques to detect and separate cancer cells. These approaches have better specificity and isolation efficiency but are restricted to small volumes of sample and low yield of isolated CTCsADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "DOI" : "10.1007/s10544-013-9739-y", "abstract" : "Ovarian cancer is the second most common of the gynecological cancers in Taiwan. It is challenging to diagnose at an early stage when proper treatment is the most effective. It is well recognized that the detection of tumor cells (TCs) is critical for determining cancer growth stages and may provide important information for accurate diagnosis and even prognosis. In this study, a new microfluidic platform integrated with a moving-wall micro-incubator, a micro flow cytometer and a molecular diagnosis module performed automated identification of ovarian cancer cells. By efficiently mixing the cells and immunomagnetic beads coated with specific antibodies, the target TCs were successfully isolated from the clinical samples. Then counting of the target cells was achieved by a combination of the micro flow cytometer and an optical detection module and showed a counting accuracy as high as 92.5 %. Finally, cancer-associated genes were amplified and detected by the downstream molecular diagnosis module. The fluorescence intensity of specific genes (CD24 and HE4) associated with ovarian cancer was amplified by the molecular diagnosis module and the results were comparable to traditional slab-gel electrophoresis analysis, with a limit of detection around 10 TCs. This integrated microfluidic platform realized the concept of a \"lab-on-a-chip\" and had advantages which included automation, disposability, lower cost and rapid diagnosis and, therefore, may provide a promising approach for the fast and accurate detection of cancer cells.", "author" : [ { "dropping-particle" : "", "family" : "Hung", "given" : "Lien Yu", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Chuang", "given" : "Ying Hsin", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Kuo", "given" : "Hsin Tzu", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Wang", "given" : "Chih Hung", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Hsu", "given" : "Keng Fu", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Chou", "given" : "ChengY ang", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "Bin", "family" : "Lee", "given" : "Gwo", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Biomed. Microdevices", "id" : "ITEM-1", "issue" : "2", "issued" : { "date-parts" : [ [ "2013", "4" ] ] }, "page" : "339-352", "title" : "An integrated microfluidic platform for rapid tumor cell isolation, counting and molecular diagnosis.", "type" : "article-journal", "volume" : "15" }, "uris" : [ "", "" ] }, { "id" : "ITEM-2", "itemData" : { "DOI" : "10.1038/nature06385.Isolation", "author" : [ { "dropping-particle" : "", "family" : "Nagrath", "given" : "Sunitha", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "V", "family" : "Sequist", "given" : "Lecia", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Maheswaran", "given" : "Shyamala", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Bell", "given" : "Daphne W", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Ryan", "given" : "Paula", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Balis", "given" : "Ulysses J", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Tompkins", "given" : "Ronald G", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Haber", "given" : "Daniel A", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Nature", "id" : "ITEM-2", "issue" : "7173", "issued" : { "date-parts" : [ [ "2007" ] ] }, "page" : "1235-1239", "title" : "Isolation of rare circulating tumour cells in cancer patients by microchip technology", "type" : "article-journal", "volume" : "450" }, "uris" : [ "", "" ] } ], "mendeley" : { "formattedCitation" : "^11,18", "plainTextFormattedCitation" : "11,18", "previouslyFormattedCitation" : "(Nagrath et al. 2007; Hung et al. 2013)" }, "properties" : { "noteIndex" : 0 }, "schema" : "" }8,15. We have developed a wicking fiber device that may lead to a simple and low cost approach to rapidly identify and capture cancer cells. A wicking fiber (Fig. 1) has a non-circular, grooved cross-section with channels that run the length of the fiber. The unique configuration facilitates and enhances wicking by capillary action along the channels. These fibers were originally used to improve wicking properties of textiles, but were later used at the stationary phase in chromatography applications such as proteomic separation and metal extraction processes16,17,18. Our research group established the use of wicking fibers to tissue engineering applications to improve vascularity and cell growth in scaffolds19. The aim of the current study was to evaluate the capability of wicking fibers to isolate cancer cells from a heterogeneous cellular liquid. We demonstrated the wicking fiber device separates (1) a mixture of normal mouse mammary epithelial cells (MMTV-neu) from mouse epithelial breast cancer cells (NMuMG) and (2) malignant human breast epithelial cells (MCF-7) and benign human breast epithelial cells (MCF-10A). Figure 1. Scanning electron microscope (SEM) image of a wicking fiber. Reprinted from Journal of Chromatography A, Vol. 986, R.K. Marcus et al., Capillary-channeled polymer fibers as stationary phases in liquid chromatography separations, pp. 17-31. 2003, with permission from Elsevier20. (a) Image of non-circular grooved cross-section and parallel channels of the wicking fiber which facilitate strong wicking action and greatly increase surface area20,21. (b) Image depicts the long axis of the wicking fiber, with parallel channels along the length of the fiber. Methods and MaterialsCell culture of mouse cellsCells from a normal mouse mammary epithelial cell line, NMuMG (ATCC), were stably transfected with Green Fluorescent Protein (NMuMG-GFP), as previously described22. Cells from a cancer mouse epithelial cell line (as previously described, cells isolated from a mammary tumor that spontaneously arose in a MMTV-neu transgenic female mouse23) were stably transfected with Red Fluorescent Protein (MMTV-neu-RFP)22. NMuMG-GFP cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, Invitrogen) supplemented with 10% fetal bovine serum (FBS, Gibco), and Mammary Epithelial Cell Growth Medium (MEGM) single quots (Lonza) while MMTV-neu-RFP cells were cultured in DMEM (Invitrogen) supplemented with 10% FBS (Gibco), 10,000 U penicillin, and 10 mg streptomycin/mL (Sigma-Aldrich). Cells were cultured in a T150 flask (Corning) and maintained in a humidified incubator at 37°C and 5% CO2. Once cells reached confluence, NMuMG-GFP at passage 4 and MMTV-neu-RFP at passage 5, they were detached using trypsin-EDTA solution (Sigma) and resuspended in growth medium. Cell culture of human cellsCells from a mammary epithelial cell line from benign breast tissue, MCF-10A (ATCC), were stably transfected with Green Fluorescent Protein (MCF-10A-GFP)22. Cells from a human breast cancer cell line, MCF-7, (ATCC) were stably transfected with Red Fluorescent Protein (MCF-7-RFP). To perform the lentiviral transfection of the MCF-7 cells, MCF-7 cells were seeded in a 96 well plate in normal growth medium and proliferated in an incubator at 37?C. The media was removed and Cignal Lentiviral particle-RFP (Qiagen; Venlo, Netherlands) with SureENTRY transduction reagent (Qiagen; Venlo, Netherlands) in growth media without antibiotics was added. The plate was incubated for 18-20 hrs, after which the media containing the lentiviral particles was removed and normal growth media containing antibiotics was added and the MCF-7 cells were allowed to grow to confluency. Once confluent growth media containing the selective agent, 2 ug/ml puromycin (Fisher; Fair Lawn, NJ) was added. The selection media was replaced every 3-4 days until puromycin resistant positive colonies appeared. The RFP-positive cells were expanded in selection media to obtain RFP positive cultures. MCF-10A-GFP cells, passage 5, were cultured in DMEM (Invitrogen) supplemented with 10% FBS (Gibco), 1% fungizone, and MEGM single quots (Lonza). MCF-7-RFP cells, passage 6, were cultured in DMEM (Invitrogen) supplemented with 10% FBS, 1% fungizone (Gibco), 10,000 U penicillin, and 10 mg streptomycin/mL (Sigma-Aldrich). Cells were cultured in a T-150 flask (Corning) and maintained in a humidified incubator at 37°C and 5% CO2. Once cells reached confluence, both cell types were removed with trypsin-EDTA solution (Sigma-Aldrich) and resuspended in culture medium to prepare for vertical testing.Preparation of fibersPoly-L-lactide (Natureworks) was extruded to obtain fibers with non-circular cross-sectional dimensions of 0.72 mm x 0.55 mm. Wicking fibers were sliced with a razor blade into individual single wicking fibers of 3.5 cm and 10 cm lengths. The 10-cm wicking fibers were used to form the wicking fiber bundles. To form bundles, three fibers were twisted, using a power drill, at 110 rotations per 10-cm fiber length. Each bundle was sliced into 3.5 cm lengths. Single and bundled wicking fibers were cleaned in three changes of ethanol, for 1 hour each, and placed under ultraviolet light for 6 hours. Samples were then soaked in a phosphate-buffered saline (PBS, Invitrogen) solution for 2 hours and air-dried overnight in a sterile hood. A total of eight single fibers and fifteen 3-fiber bundles were prepared.Testing of vertical wicking with individual fibersThe vertical wicking test of cells, shown in Fig. 2, includes a custom-made culture lid for a low-attachment 12-well plate (Corning). The lid contains fitted holes with columns to securely hold the 3.5 cm wicking fibers and wicking fiber bundles in a vertical position. A 50/50 mix of cancer (NMuMG-GFP) and normal (MMTV-neu-RFP) mouse cells were seeded in eight wells of a low attachment 12-well plate, at a density of one million cells per well (5x105 MMTV-neu-RFP and 5x105 NMuMG-GFP), along with 1 mL of growth medium per well. Eight single wicking fibers were vertically inserted through the columns, one per well, so only the bottom 3 mm of the fiber contacted the cell solution. The custom 12-well plate set up was placed on a flat top shaker (VWR) at 100 rpm in a humidified incubator at 37oC and 5% CO2. The vertical displacements of the cancerous and normal mouse mammary cells along the wicking fibers were determined at time points of 0.5 and 24 hours, with time point 0 being fiber placement into the cell solution. Four fibers were assessed after 0.5 hours, the remainder after 24 hours. The vertical displacements of the cancerous (MCF-7-RFP) and benign (MCF-10A-GFP) human epithelial cells along the bundles were determined at time points of 0.25 hours, 2 hours, 12 hours and 24 hours, with time point 0 being fiber placement into the cell solution. A similar set-up to the single fiber experiment was conducted, with fifteen 3-fiber bundles, each bundle populating a well of a 12-well plate. Twelve of the bundles were designated for image analysis of cell displacment and the remaining three were designated for quantitative analysis. To assess the cell displacement of both cell types, three fiber bundles were transferred to a 6-well plate, one bundle per well, at each timepoint. Qualitative assessments were made and photographs were taken to document the progression of cells along the length of the bundle at each timepoint. The bundles were rinsed twice with phosphate-buffered saline solution, and fixed for 15 min with 4% paraformaldehyde. After the cells on the wicking fiber constructs were fixed, the fibers were transferred to microscope slides and the vertical displacement was evaluated using fluorescent microscopy and imaging software. The entire length of each bundle was imaged, using fluorescent microscopy, beginning with the seeded end of the wicking fiber construct, using 25x total magnification. A fluorescein isothiocyanate (FITC) filter was used to view the vertical movement of normal cells transfected with Green Fluorescent Protein, and a tetramethyl rhodamine iso-thiocyanate (TRITC) filter was used to view the vertical movement of cancer cells transfected with Red Fluorescent Protein. Imaging software (ImageJ, National Institutes of Health) was used to determine the vertical displacement (μm) of the cells along the fiber. Images were aligned to qualitatively show the total displacement of both normal and cancer cells, with total vertical displacement quantified by the maximum summation of the displacements in the individual images. Figure 2. Schematic of vertical wicking test apparatus. The lid of the low-attachment 12-well plate was modified with holes and fitted columns to support the wicking fiber in the vertical position. Cancer and normal cells, tracked with specific fluorescent proteins, were added to each individual well at equivalent cell densities. The wicking fiber or wicking fiber bundle was vertically placed through the column and into a mixed cellular solution so 3 mm of the bottom fiber region was submerged. The fiber was removed at various time points to analyze the vertical cell movement. Isolation of cancer cells from wicking fiber bundleAfter 24 hours, the remaining three vertical wicking fiber bundles were removed from the customized 12-well plate and the fibers were sectioned, with a razor blade, to separate the top and bottom fiber regions. The fiber sections were placed in separate wells of a 24-well plate. The samples were rinsed with PBS twice and untwisted using forceps. The cells were removed by adding 500 μL of trysin-ethylenediaminetetracetic acid (EDTA) solution to the well with the fiber and placing the plate on a flat-top (VWR) shaker at 200 rpm in a 37oC incubator. After 15 min, the cells were resuspended in 500 μL of growth medium and the number of MCF-10A and MCF-7, in both regions of the fibers, was evaluated using a Guava easyCyteTM flow cytometer (Guava Technologies). The number of MCF-10A-GFP and MCF-7-RFP was determined for each region, following the manufacturer’s instructions for InCyte software (Guava Technologies). Positive and negative controls with known cell densities were used to calibrate the machine before measurements of the treatment groups were made. Determination of cell size The average cell size of MCF-7-RFP and MCF-10A-GFP cells was measured in cell solution. MCF-7-RFP cells were removed from culture flasks with trypsin-EDTA solution. After incubation with solution for 15 min, 5x105 cells were resuspended in 1 mL of growth medium in a 15 mL centrifuge tube. The centrifuge tube was vortexed to maintain the cells in solution. A volume of 100 μL of cell solution was added to each of three microscope slides. Similarly, three slides of MCF-10A-GFP cells were prepared. The cellular slides were imaged with the fluorescence microscope and measured with imaging software (ImageJ). The measurement function of the software was used to determine the average diameter of each cell line. Imaging of wicking bundle cross-sectionsWicking fiber bundles, each containing three individual fibers of non-circular cross-sectional dimensions of 0.72 mm x 0.55 mm, were sectioned into 3-cm lengths. Samples were placed vertically in embedding molds for the microtome and infiltrated and embedded with Embed-itTM Low Viscosity Epoxy Resin (Polysciences, Inc.). The wicking fiber samples were embedded and sectioned, following the manufacturer’s protocol. Sections, 5 μm in thickness, were cut with a microtome and transferred to microscope slides. The sections were imaged with a light microscope, and ImageJ imaging software was used to measure and characterize the inter- and intra- fiber spaces. Statistical analysisMatched pairs analysis was conducted using JMP statistical software to compare (p<0.05) the vertical displacement of MMTV-neu-RFP (mouse cancer cells) and NMuMG-GFP (normal mouse cells) at 0.5 and 24 hours. A matched pairs analysis was also used to compare (p<0.05) the fraction of MCF-7-RFP cells in top and bottom regions of the fiber bundle. The results showed there were significantly more MCF-7-RFP cells in the top region of the fiber bundles than in the bottom region. ResultsMouse cancer cell separation using wicking fiber system We developed a vertical test system (Fig. 2) to analyze the vertical movement of different cell mixtures along individual fibers. The vertical displacements of mouse mammary normal cells expressing green fluorescent protein (NMuMG-GFP) and mouse mammary cancer cells expressing red fluorescent protein (MMTV-neu-RFP) were determined at time points of 0.5 and 24 hours using fluorescence microscopy. Fig. 3 is a composite of fluorescent images, showing the cancer and normal cells along the length of the fiber, aligned to qualitatively show the total displacement of both cell types after 24 hours of contacting cell solution. Fig. 3A demonstrates that MMTV-neu-RFP cells, labeled red, have vertically displaced 16.2 mm along the wicking fiber after 24 hours. Fig. 3B shows NMuMG-GFP cells, labeled green, vertically moved 3.0 mm, significantly less than the MMTV-neu-RFP cells, on the same wicking fiber. Images also depict higher cell densities of MMTV-neu-RFP cells along the fiber. Images qualitatively show a vertical separation of MMTV-neu-RFP and NMuMG-GFP cells, indicating different cell types move differently along the wicking fibers. Imaging software was used to quantify the vertical displacement (μm) of the cells along the length of the fiber. The total vertical displacement was found by assessing each individual image. The vertical displacement of the cancer cells, MMTV-neu-RFP, along the wicking fiber was significantly (p<0.05) greater than that of the normal cells (NMuMG-GFP) at the 24-hour time point (Fig. 3C). No significant difference was shown after 0.5-hour time point. 43180002216150*00*40005001452245*00*Figure 3. Visualization and quantification of the vertical displacement of cancer and normal cells. The 3.0 mm arrow indicates the portion of fiber in solution. Left image A depicts compiled fluorescent images of the vertical displacement of MMTV-neu-RFP, cancer cells, along the length of the fiber after 24 hours. Image B shows NMuMG-GFP, normal cells, progressed significantly less than the cancer cells. Graph shows quantitative analysis of the vertical displacement of cancer cells (2757 ± 928.9 (SD), n=4) and mouse normal cells (2219 ± 940.6 (SD), n=4) after various time points. After 24 hours there was significantly greater vertical displacement of MMTV-neu-RFP (14380 ± 1192 (SD), n=4) than displacement of NMuMG-GFP (6220 ± 994.2 (SD), n=4). p<0.05, indicated by (*) in graph. Human cancer cell separation and isolation using wicking fiber systemTo enhance the effects of individual wicking fibers on cancer cell separation, we developed a wicking fiber bundle that demonstrated improved wicking properties, i.e. the overall wicking rate and volume of liquid transported were enhanced over that of single fibers. The wicking fiber bundle was used to separate and isolate malignant human breast epithelial cells expressing red fluorescent protein (MCF-7-RFP) from benign human breast epithelial cells expressing green fluorescent protein (MCF-10A-GFP). Fluorescent images of the bundles were used to evaluate the vertical displacement and separation of MCF-7-RFP and MCF-10A-GFP after 0.25, 2, 12, and 24 hours. Images show that, after 0.25 hours and 2 hours, MCF-7-RFP cells progressed along the entire fiber but in low numbers. After 12 and 24-hour time points, much higher numbers of MCF-7-RFP traveled to the top region of the fiber. Images indicate after 0.25 hours, few benign epithelial cells progressed along the fiber bundles. To quantify the number of cancerous and benign cells along the fiber bundles after 24 hours, cells were removed from top and bottom regions of the bundles (Fig. 4). The number of MCF-7-RFP and MCF-10A-GFP cells was determined using flow cytometry (Guava Technologies) with InCyte software (Guava Technologies). Fig. 4 illustrates the percentage of each cell type in top and bottom regions; there is a significant difference (p<0.05) between the percentage of MCF-7-RFP and the percentage of MCF-10A-GFP in the top region of the fiber bundles. These results suggest that the wicking fiber bundle separates cancerous cells from a mixture; as the graph indicates, 82% of cells isolated from the top fiber bundle regions are cancerous. 05773420Figure 5. Fluorescent images of MCF-7 and MCF-10A cells in solution. Imaging software was used to measure diameters (indicated in red) of both cell types in solution, see images above, each bar is the average of three 100?L samples. Left image shows smaller diameter MCF-7 cells and right image reveals larger diameter MCF-10A cells. The graph shows the average diameters of the cancerous and benign cell types. The cancerous cells, MCF-7s, are significantly smaller (p < 0.05) than the benign cells, MCF-10As.Figure 5. Fluorescent images of MCF-7 and MCF-10A cells in solution. Imaging software was used to measure diameters (indicated in red) of both cell types in solution, see images above, each bar is the average of three 100?L samples. Left image shows smaller diameter MCF-7 cells and right image reveals larger diameter MCF-10A cells. The graph shows the average diameters of the cancerous and benign cell types. The cancerous cells, MCF-7s, are significantly smaller (p < 0.05) than the benign cells, MCF-10As.03617843-6352329180Figure 4. Schematic of the fiber regions and data depicting percentage of each cell type in fiber regions. Left image illustrates axial slicing of fiber bundle to extract top and bottom region cells. Graph exhibits quantitative analysis of top and bottom regions of the fiber bundles after 24 hours, demonstrating significantly higher concentration of MCF-7-RFP cells in the top region of the fiber bundle (82.4 ± 4.3 (SD), n=3 bundles) than the bottom region of the fiber bundle (61.6 ± 3.04 (SD), n=3 bundles) The graph indicates a significantly lower concentration of MCF-10A-GFP cells in top region (17.6 ± 4.3 (SD), n=3 bundles) than bottom region (38.4 ±3.04 (SD), n=3 bundles) (* indicates significant difference, p<0.05).0Figure 4. Schematic of the fiber regions and data depicting percentage of each cell type in fiber regions. Left image illustrates axial slicing of fiber bundle to extract top and bottom region cells. Graph exhibits quantitative analysis of top and bottom regions of the fiber bundles after 24 hours, demonstrating significantly higher concentration of MCF-7-RFP cells in the top region of the fiber bundle (82.4 ± 4.3 (SD), n=3 bundles) than the bottom region of the fiber bundle (61.6 ± 3.04 (SD), n=3 bundles) The graph indicates a significantly lower concentration of MCF-10A-GFP cells in top region (17.6 ± 4.3 (SD), n=3 bundles) than bottom region (38.4 ±3.04 (SD), n=3 bundles) (* indicates significant difference, p<0.05).3310Determination of cell size and wicking bundle cross-sectionsFluorescent imaging and imaging software was used to determine the average cell size of cancer and normal cells in solution; MCF-7-RFP cells are significantly smaller, by approximately 5μm, than MCF-10A-GFP (Fig. 5). To understand the interaction that cell size may have on migration, cross-sectional images of the wicking fiber bundle were taken. These images depict channel and interfiber space sizes (Fig. 6). Figure 6. Axial cross-section of a representative wicking fiber bundle illustrates channels and inter-fiber spaces up to 95μm. The inter- and intra- fiber spaces affect capillary pressure and ability for fluid and cells to penetrate the fiber and vertically move through the fiber. Cell size and deformability will influence the ability of the cells to penetrate and travel through the intra- or inter- fiber spaces. DiscussionOur wicking fiber device has potential to separate, isolate, and identify cancerous breast epithelial cells from a mixture containing benign breast epithelial cells. The separation is due to the physical and functional properties of the cells, wicking characteristics of the fiber bundle, and the cell-fiber interaction. Physical properties that may play a role include cell size, deformability, surface friction or charge, and expression of cell adhesion molecules. The size and shape of the cells may play a role in the ability of cells to penetrate the smaller channels created from the bundling of the fibers. Fiber bundle architecture will affect the wicking rate and vertical displacement of different cell types. The size of the individual fibers, the tension of the bundle, the hydrophobicity, and the inter-fiber space will play a role in the wicking of liquid as well as cell-interaction. Further studies are necessary to reveal which characteristics are most influential on wicking and the relevant underlying mechanistic principles.Other cellular properties influencing vertical movement include cell membrane impedance, expression of adhesion molecules, and cell stiffness. Membrane impedance, cell dielectric properties or cell electric properties vary between cell typesADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "abstract" : "Electrorotation measurements were used to demonstrate that the dielectric properties of the metastatic human breast cancer cell line MDA231 were significantly different from those of erythrocytes and T lymphocytes. These dielectric differences were exploited to separate the cancer cells from normal blood cells by appropriately balancing the hydrodynamic and dielectrophoretic forces acting on the cells within a dielectric affinity column containing a microelectrode array. The operational criteria for successful particle separation in such a column are analyzed and our findings indicate that the dielectric affinity technique may prove useful in a wide variety of cell separation and characterization applications.", "author" : [ { "dropping-particle" : "", "family" : "Becker", "given" : "F F", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Wang", "given" : "X B", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Huang", "given" : "Y", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Pethig", "given" : "R", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Vykoukal", "given" : "J", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Gascoyne", "given" : "P R", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "PNAS", "id" : "ITEM-1", "issue" : "3", "issued" : { "date-parts" : [ [ "1995", "1" ] ] }, "page" : "860-864", "title" : "Separation of human breast cancer cells from blood by differential dielectric affinity.", "type" : "article-journal", "volume" : "92" }, "uris" : [ "", "" ] }, { "id" : "ITEM-2", "itemData" : { "DOI" : "10.1016/S1350-4533(02)00194-7", "author" : [ { "dropping-particle" : "", "family" : "Zou", "given" : "Y", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Guo", "given" : "Z", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Med. Eng. Phys.", "id" : "ITEM-2", "issue" : "2", "issued" : { "date-parts" : [ [ "2003", "3" ] ] }, "page" : "79-90", "title" : "A review of electrical impedance techniques for breast cancer detection", "type" : "article-journal", "volume" : "25" }, "uris" : [ "", "" ] } ], "mendeley" : { "formattedCitation" : "^2,31", "plainTextFormattedCitation" : "2,31", "previouslyFormattedCitation" : "(Becker et al. 1995; Zou and Guo 2003)" }, "properties" : { "noteIndex" : 0 }, "schema" : "" }24,25. Electrical properties of the cell will influence the cell-interaction with the fiber and the resultant vertical movement. Cells with greater impedance or surface charge may have more interactions with the fiber, thus hindering the vertical cell movement. Abdolahab and coworkers evaluated the cellular impedance for cancer cells of varying aggressiveness and demonstrated that a more aggressive cancer cell type (MDA-MD231) has significantly lower impedance than a less aggressive cancer cell (MCF-7). Additionally they found a correlation between metastatic progression and membrane impedance reduction. Cell-fiber interaction and overall cell displacement will be affected by different membrane impedances. Expression of cell adhesion molecules can play a role in the cell-fiber interaction and ability of a cell to move through a confined space. Metastatic cells are known to lose endothelial adhesion molecules and transition into more motile mesenchymal phenotypesADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "DOI" : "10.1038/nrc3080.The", "author" : [ { "dropping-particle" : "", "family" : "Wirtz", "given" : "Denis", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Konstantopoulos", "given" : "Konstantinos", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Searson", "given" : "Peter", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Nat. Rev. Cancer", "id" : "ITEM-1", "issue" : "7", "issued" : { "date-parts" : [ [ "2012" ] ] }, "page" : "512-522", "title" : "The physics of cancer: the role of physical interactions and mechanical forces in metastasis", "type" : "article-journal", "volume" : "11" }, "uris" : [ "", "" ] } ], "mendeley" : { "formattedCitation" : "²⁶", "plainTextFormattedCitation" : "26", "previouslyFormattedCitation" : "(Wirtz et al. 2012)" }, "properties" : { "noteIndex" : 0 }, "schema" : "" }12. Cancerous cells with fewer adhesion molecules may have less surface friction and altered shapes that have less interaction with the fibers, allowing the cells to wick greater distances. Cell deformability will influence the ability for cells to deform, penetrate, and wick vertically through the twisted channels. Researchers have generally shown that cancer cells have a significantly lower Young’s modulus compared to that of normal cellsADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "DOI" : "10.1016/j.abb.2011.12.013", "abstract" : "Currently, cancer diagnosis relies mostly on morphological examination of exfoliated, aspirated cells or surgically removed tissue. As long as standard diagnosis is concerned, this classical approach seems to be satisfactory. In the recent years, cancer progression has been shown to be accompanied by alterations in mechanical properties of cells. This offers the detection of otherwise unnoticed cancer cell disregarded by histological analysis due to insignificant manifestations. One of techniques, sensitive to changes in mechanical properties, is the atomic force microscopy, which detects cancer cells through their elastic properties. Such measurements were applied to tissue sections collected from patients suffering from various cancers. Despite of heterogeneity and complexity of cancer cell sections, the use of the Young's modulus as an indicator of cell elasticity allow for detection of cancer cells in tissue slices.", "author" : [ { "dropping-particle" : "", "family" : "Lekka", "given" : "Ma\u0142gorzata", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Gil", "given" : "Dorota", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Pogoda", "given" : "Katarzyna", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Duli\u0144ska-Litewka", "given" : "Joanna", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Jach", "given" : "Robert", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Gostek", "given" : "Justyna", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Klymenko", "given" : "Olesya", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Prauzner-Bechcicki", "given" : "Szymon", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Stachura", "given" : "Zbigniew", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Wiltowska-Zuber", "given" : "Joanna", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Oko\u0144", "given" : "Krzysztof", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Laidler", "given" : "Piotr", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Arch Biochem Biophys", "id" : "ITEM-1", "issue" : "2", "issued" : { "date-parts" : [ [ "2012", "2" ] ] }, "page" : "151-156", "title" : "Cancer cell detection in tissue sections using AFM.", "type" : "article-journal", "volume" : "518" }, "uris" : [ "", "" ] }, { "id" : "ITEM-2", "itemData" : { "DOI" : "10.1073/pnas.1209893109", "abstract" : "Here we report a microfluidics method to enrich physically deformable cells by mechanical manipulation through artificial microbarriers. Driven by hydrodynamic forces, flexible cells or cells with high metastatic propensity change shape to pass through the microbarriers and exit the separation device, whereas stiff cells remain trapped. We demonstrate the separation of (i) a mixture of two breast cancer cell types (MDA-MB-436 and MCF-7) with distinct deformabilities and metastatic potentials, and (ii) a heterogeneous breast cancer cell line (SUM149), into enriched flexible and stiff subpopulations. We show that the flexible phenotype is associated with overexpression of multiple genes involved in cancer cell motility and metastasis, and greater mammosphere formation efficiency. Our observations support the relationship between tumor-initiating capacity and cell deformability, and demonstrate that tumor-initiating cells are less differentiated in terms of cell biomechanics.", "author" : [ { "dropping-particle" : "", "family" : "Zhang", "given" : "Weijia", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Kai", "given" : "Kazuharu", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Choi", "given" : "Dong Soon", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Iwamoto", "given" : "Takayuki", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Nguyen", "given" : "Yen H", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Wong", "given" : "Helen", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Landis", "given" : "Melissa D", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Ueno", "given" : "Naoto T", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Chang", "given" : "Jenny", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Qin", "given" : "Lidong", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "PNAS", "id" : "ITEM-2", "issue" : "46", "issued" : { "date-parts" : [ [ "2012", "11" ] ] }, "page" : "18707-18712", "title" : "Microfluidics separation reveals the stem-cell-like deformability of tumor-initiating cells.", "type" : "article-journal", "volume" : "109" }, "uris" : [ "", "" ] }, { "id" : "ITEM-3", "itemData" : { "DOI" : "10.1016/j.actbio.2007.04.002.Biomechanics", "author" : [ { "dropping-particle" : "", "family" : "Suresh", "given" : "Subra", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Acta Biomater.", "id" : "ITEM-3", "issue" : "4", "issued" : { "date-parts" : [ [ "2010" ] ] }, "page" : "413-438", "title" : "Biomechanics and biophysics of cancer cells", "type" : "article-journal", "volume" : "3" }, "uris" : [ "", "" ] } ], "mendeley" : { "formattedCitation" : "^12,24,30", "plainTextFormattedCitation" : "12,24,30", "previouslyFormattedCitation" : "(Suresh 2010; Lekka et al. 2012; Zhang et al. 2012)" }, "properties" : { "noteIndex" : 0 }, "schema" : "" }26,27,28. Researchers have found that cancer cells of increased metastatic potential have higher deformabilityADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "DOI" : "10.1371/journal.pone.0046609", "abstract" : "The metastatic potential of cells is an important parameter in the design of optimal strategies for the personalized treatment of cancer. Using atomic force microscopy (AFM), we show, consistent with previous studies conducted in other types of epithelial cancer, that ovarian cancer cells are generally softer and display lower intrinsic variability in cell stiffness than non-malignant ovarian epithelial cells. A detailed examination of highly invasive ovarian cancer cells (HEY A8) relative to their less invasive parental cells (HEY), demonstrates that deformability is also an accurate biomarker of metastatic potential. Comparative gene expression analyses indicate that the reduced stiffness of highly metastatic HEY A8 cells is associated with actin cytoskeleton remodeling and microscopic examination of actin fiber structure in these cell lines is consistent with this prediction. 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This point suggests that MCF-7 cells are much softer and deform more easily than normal cells, allowing the cancer cells to migrate more readily. Microfluidic devices have been employed to distinguish cancer and normal cells, based on cellular deformability, by analyzing the ability of cells to squeeze and pass though confined constrictionsADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "DOI" : "10.1073/pnas.1218806110/-/DCSupplemental.cgi/doi/10.1073/pnas.1218806110", "author" : [ { "dropping-particle" : "", "family" : "Byun", "given" : "Sangwon", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Son", "given" : "Sungmin", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Amodei", "given" : "Dario", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Cermak", "given" : "Nathan", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Shaw", "given" : "Josephine", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Ho", "given" : "Joon", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Hecht", "given" : "Vivian C", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "PNAS", "id" : "ITEM-1", "issue" : "19", "issued" : { "date-parts" : [ [ "2013" ] ] }, "page" : "7580-7585", "title" : "Characterizing deformability and surface friction of cancer cells", "type" : "article-journal", "volume" : "110" }, "uris" : [ "", "" ] }, { "id" : "ITEM-2", "itemData" : { "DOI" : "10.1115/1.4002180", "abstract" : "In this paper, frequency response (dynamic compression and recovery) is suggested as a new physical marker to differentiate between breast cancer cells (MCF7) and normal cells (MCF10A). A single cell is placed on the laminated piezoelectric actuator and a piezoresistive microcantilever is placed on the upper surface of the cell at a specified preload displacement (or an equivalent force). The piezoelectric actuator excites the single cell in a sinusoidal fashion and its dynamic deformation is then evaluated from the displacement converted by measuring the voltage output through a piezoresistor in the microcantilever. The microcantilever has a flat contact surface with no sharp tip, making it possible to measure the overall properties of the cell rather than the local properties. These results indicate that the MCF7 cells are more deformable in quasi-static conditions compared with MCF10A cells, consistent with known characteristics. Under conditions of high frequency of over 50 Hz at a 1 \u03bcm preload displacement, 1 Hz at a 2 \u03bcm preload displacement, and all frequency ranges tested at a 3 \u03bcm preload displacement, MCF7 cells showed smaller deformation than MCF10A cells. MCF7 cells have higher absorption than MCF10A cells such that MCF7 cells appear to have higher deformability according to increasing frequency. Moreover, larger preload and higher frequencies are shown to enhance the differences in cell deformability between the MCF7 cells and MCF10A cells, which can be used as a physical marker for differentiating between MCF10A cells and MCF7 cells, even for high-speed screening devices.", "author" : [ { "dropping-particle" : "", "family" : "Shim", "given" : "Sangjo", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Kim", "given" : "Man Geun", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Jo", "given" : "Kyoungwoo", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Kang", "given" : "Yong Seok", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Lee", "given" : "Boreum", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Yang", "given" : "Sung", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Shin", "given" : "Sang-Mo", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Lee", "given" : "Jong-Hyun", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "J Biomech. Eng.", "id" : "ITEM-2", "issue" : "10", "issued" : { "date-parts" : [ [ "2010", "10" ] ] }, "page" : "104501", "title" : "Dynamic characterization of human breast cancer cells using a piezoresistive microcantilever.", "type" : "article-journal", "volume" : "132" }, "uris" : [ "", "" ] }, { "id" : "ITEM-3", "itemData" : { "DOI" : "10.1073/pnas.1209893109", "abstract" : "Here we report a microfluidics method to enrich physically deformable cells by mechanical manipulation through artificial microbarriers. Driven by hydrodynamic forces, flexible cells or cells with high metastatic propensity change shape to pass through the microbarriers and exit the separation device, whereas stiff cells remain trapped. We demonstrate the separation of (i) a mixture of two breast cancer cell types (MDA-MB-436 and MCF-7) with distinct deformabilities and metastatic potentials, and (ii) a heterogeneous breast cancer cell line (SUM149), into enriched flexible and stiff subpopulations. We show that the flexible phenotype is associated with overexpression of multiple genes involved in cancer cell motility and metastasis, and greater mammosphere formation efficiency. Our observations support the relationship between tumor-initiating capacity and cell deformability, and demonstrate that tumor-initiating cells are less differentiated in terms of cell biomechanics.", "author" : [ { "dropping-particle" : "", "family" : "Zhang", "given" : "Weijia", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Kai", "given" : "Kazuharu", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Choi", "given" : "Dong Soon", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Iwamoto", "given" : "Takayuki", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Nguyen", "given" : "Yen H", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Wong", "given" : "Helen", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Landis", "given" : "Melissa D", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Ueno", "given" : "Naoto T", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Chang", "given" : "Jenny", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Qin", "given" : "Lidong", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "PNAS", "id" : "ITEM-3", "issue" : "46", "issued" : { "date-parts" : [ [ "2012", "11" ] ] }, "page" : "18707-18712", "title" : "Microfluidics separation reveals the stem-cell-like deformability of tumor-initiating cells.", "type" : "article-journal", "volume" : "109" }, "uris" : [ "", "" ] } ], "mendeley" : { "formattedCitation" : "^4,21,30", "plainTextFormattedCitation" : "4,21,30", "previouslyFormattedCitation" : "(Shim et al. 2010; Zhang et al. 2012; Byun et al. 2013)" }, "properties" : { "noteIndex" : 0 }, "schema" : "" }28,31,32. Indeed, Li and coworkers found the apparent Young’s modulus of MCF-10A, non-malignant breast epithelial cells, to be significantly higher than that of MCF-7ADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "DOI" : "10.1016/j.bbrc.2008.07.078", "abstract" : "Mechanical properties of individual living cells are known to be closely related to the health and function of the human body. Here, atomic force microscopy (AFM) indentation using a micro-sized spherical probe was carried out to characterize the elasticity of benign (MCF-10A) and cancerous (MCF-7) human breast epithelial cells. AFM imaging and confocal fluorescence imaging were also used to investigate their corresponding sub-membrane cytoskeletal structures. Malignant (MCF-7) breast cells were found to have an apparent Young's modulus significantly lower (1.4-1.8 times) than that of their non-malignant (MCF-10A) counterparts at physiological temperature (37 degrees C), and their apparent Young's modulus increase with loading rate. Both confocal and AFM images showed a significant difference in the organization of their sub-membrane actin structures which directly contribute to their difference in cell elasticity. This change may have facilitated easy migration and invasion of malignant cells during metastasis.", "author" : [ { "dropping-particle" : "", "family" : "Li", "given" : "Q S", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Lee", "given" : "G Y H", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Ong", "given" : "C N", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Lim", "given" : "C T", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Biochem. Biophys. Res. Commun.", "id" : "ITEM-1", "issue" : "4", "issued" : { "date-parts" : [ [ "2008", "10" ] ] }, "page" : "609-613", "title" : "AFM indentation study of breast cancer cells.", "type" : "article-journal", "volume" : "374" }, "uris" : [ "", "" ] } ], "mendeley" : { "formattedCitation" : "¹⁴", "plainTextFormattedCitation" : "14", "previouslyFormattedCitation" : "(Li et al. 2008)" }, "properties" : { "noteIndex" : 0 }, "schema" : "" }33. The softer cytoskeleton and deformability of the MCF-7 cells may play a role in their ability to squeeze through channels and wick upward through the confined spaces. We demonstrated the wicking fiber approach can separate normal mouse mammary epithelial cells (MMTV-neu) from mouse breast cancer cells and (NMuMG). We showed the wicking fiber bundle rapidly separates human cancerous breast cells from human benign breast cells. Fluorescent images indicate separation of cells after 15 min of fiber bundle contact with cellular liquid. After 24 hours, large quantities of cells penetrated the fibers, and cancerous breast cells were efficiently isolated from the top region of the fiber. Properties of the fiber and cells will influence the cell-fiber interaction and resultant separation of the cells. The architecture, material properties, and surface properties of the fiber bundle will influence the separation of cells and their wicking profiles. Relevant cell properties, including cell surface charge, cell stiffness, cell size, and cell surface adhesion molecules, will vary between cancerous cells of different metastatic potential and play a role in cell-fiber interaction and the ability of cells to wick through the channels. We have developed a simple, inexpensive approach to separate cancer cells from benign cells; the approach could be applied to separating cancerous cells of varying metastatic potential. Current diagnostic methods are time-consuming, expensive, and lack information. The current methods require external laboratories with complex systems and trained research personnel to perform multiple tests to confirm results. Common reliance on biomarkers may only capture a small cellular subpopulation of a heterogeneous tumor and does not provide information about the metastatic potential of the cancer cells. The rigid design of microfluidic devices that separate cancer cells requires the use of an external pump and antigen labels, which limits the populations of cancer cells targeted. These technologies are only capable of analyzing small amounts of sample and provide limited information about the metastatic potential of heterogeneous populations within the tumor. Our simple and rapid approach allows the analysis of large volumes of liquid biopsy and requires no external pump. Our results demonstrate this device can separate cancerous breast epithelial cells from benign breast epithelial cells based on their wicking capabilities. The flexible design of the fiber bundle allows identification and separation of subpopulations of varying metastatic potential within a tumor. The wicking fiber diagnostic may provide a rapid approach to identify cancer cells from a liquid biopsy that can easily be translated into a method for on-site clinic testing. AcknowledgmentsFunding for the work was provided, in part, by the Avon Foundation for Women Grant 02-2013-076 and the Clemson University Hunter Endowment.References1.Crowley, E., F. Di Nicolantonio, F. Loupakis, and A. Bardelli. Liquid biopsy: monitoring cancer-genetics in the blood. Nat. Rev. Clin. Oncol. 10:472–484, 2013.2.Hanahan, D., and R. Weinberg. Hallmarks of cancer: the next generation. Cell 144:646–674, 2011.3.Magee, J. A., E. Piskounova, and S. J. Morrison. Cancer stem cells: impact, heterogeneity, and uncertainty. Cancer Cell 21:283–296, 2012.niversity. ADDIN Mendeley Bibliography CSL_BIBLIOGRAPHY 4.Almendro, V., A. Marusyk, and K. Polyak. 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