Correlation of Cell Surface Biomarker Expression Levels with

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Correlation of Cell Surface Biomarker Expression Levels with Adhesion Contact Angle Measured by Lateral Microscopy Jenna A Walz, and Charles R. Mace Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b00268 • Publication Date (Web): 30 Apr 2018 Downloaded from http://pubs.acs.org on May 4, 2018

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Analytical Chemistry

Correlation of Cell Surface Biomarker Expression Levels with Adhesion Contact Angle Measured by Lateral Microscopy

Jenna A. Walz and Charles R. Mace*

Department of Chemistry, Tufts University, 62 Talbot Avenue, Medford, MA 02155 USA

*Corresponding author: [email protected]

ACS Paragon Plus Environment

Analytical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Abstract Immunophenotyping is typically achieved using flow cytometry, but any influence this biomarker may have on adhesion or surface recognition cannot be determined concurrently. In this manuscript, we demonstrate the utility of lateral microscopy for correlating cell surface biomarker expression levels with quantitative descriptions of cell morphology. With our imaging system, we observed single cells from two T cell lines and two B cell lines adhere to antibodycoated substrates and quantified this adhesion using contact angle measurements. We found that SUP-T1 and CEM CD4+ cells, both of which express similar levels of CD4, experienced average changes in contact angle that were not statistically different from one another on surfaces coated in anti-CD4. However, MAVER-1 and BJAB K20 cells, both of which express different levels of CD20, underwent average changes in contact angle that were significantly different from one another on surfaces coated in anti-CD20. Our results indicate that changes in cell contact angles on antibody-coated substrates reflect the expression levels of corresponding antigens on the surfaces of cells as determined by flow cytometry. Our lateral microscopy approach offers a more reproducible and quantitative alternative to evaluate adhesion compared to commonly used wash assays, and can be extended to many additional immunophenotyping applications to identify cells of interest within heterogeneous populations.


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Introduction Chemical and biochemical contributions to cell adhesion encompass the interactions that occur at the interface between a cell and its substrate. When exposed to ligands that interact favorably with proteins expressed on the surfaces of cells, molecular recognition will facilitate adhesion.1 For example, integrins are transmembrane proteins that are critical in establishing focal adhesions between cells and their extracellular environments.2 During an immune response, β2 integrin, specifically, is largely responsible for leukocyte arrest and subsequent penetration into wounded tissue.3,4 Conversely, cells that express high levels of sialic acid evade detection from the immune system by greatly reducing interactions with other cells, which has also been demonstrated using in vitro assays.5,6 For in vitro studies of cell adhesion, which are typically performed on planar transparent substrates, adhesion is characterized qualitatively by spreading where the morphology of a cell reorganizes substantially to assume a flattened appearance.7 Quantitatively, the extent of spreading is indicated by measurements of cell-substrate contact area.8,9,10 Spreading is not only dictated by the cell’s expression level of a specific surface marker, but it has also been demonstrated that increasing the density of a ligand on a substrate increases cell spreading and cell-substrate contact areas.11 Flow cytometry is the most commonly implemented technique for identifying and measuring biomarkers of interest expressed on the surfaces of cells.12 With this method, cells flow in a stream of liquid where they are interrogated by lasers to enable the detection of scattered light and fluorescence emitted from pre-labeled biomarkers.13 By way of design, cells must be in suspension to pass through the instrument and a relatively large number of cells (~0.5 × 106 cells/mL) is required for each analysis.14 Unless equipped with sorting, which can greatly increase the cost of flow cytometry instrumentation, cells cannot be recovered according to a



unique biomolecular feature for subsequent investigations. Additionally, only qualitative information on the physical appearance of cells (i.e., size and granularity based on forward and side scatter plots) is attainable.15 While imaging flow cytometers are now available commercially to view cell morphology during biomarker analysis,16 single cells cannot be analyzed over time, and cells must still be in suspension to perform experiments, which limits the correlation of surface marker expression levels and cell function.17 To determine if a specific interaction between a biomarker and ligand influences cell function by promoting adhesion (i.e., a favorable interaction), wash assays are often performed.18 These assays involve culturing cells on a desired substrate (e.g., a ligand-coated polystyrene dish) for a specified length of time (ca. hours), carefully removing the culture medium, and performing successive washes by gently adding and removing fresh buffer or medium.19 Each wash serves to eliminate nonadherent cells from the substrate, and the fraction of cells that remains is determined as a semi-quantitative measure of how well the cell population interacts with the substrate. While wash assays are simple to perform and enable relatively quick comparisons between different substrates and cell types with respect to their binding compatibility, the forces applied by manual washing can vary substantially from user to user—or even from substrate to substrate—making results difficult to reproduce.20 This lack of reproducibility also hinders the utility of this approach for isolating subsets of cells from a heterogeneous population. An ideal method to understand how surface marker expression influences cell function combines aspects of both flow cytometry and adhesion assays. Antibody (Ab) microarrays, in particular, have been used to analyze surface-bound cells.14,21,22,23 This solid-phase cytometry approach entails surface microfabrication to display antibodies to specific targets on cells of


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interest and has been used to sort and micropattern cells within a heterogeneous sample. For example, Ab microarrays have been used to capture pure leukocyte subsets from whole blood,14 as well as detect cytokines produced by captured leukocytes for multiparametric blood analysis.22 While Ab microarrays serve as a promising alternative to traditional immunophenotyping and adhesion techniques, they can be expensive and time-consuming to develop and perform: capture surfaces are either fabricated from polymeric materials to display certain antibodies or antibodies are printed in specific patterns on glass surfaces using a robotic microarrayer, and surfaces must be susceptible to flow in fluidic chambers to remove unbound cells. Flow rates must be determined experimentally to maximize the quantity and purity of captured cells. Moreover, in order to visualize captured cells, microarrays must be compatible with an upright or inverted microscope; thus, the materials with which they are fabricated must be optically transparent to enable the passage of light and of marginal thickness for the sample to be imaged within the working distance of the lens in use. A more generalizable method may be to use changes in cell morphology—specifically, the contact angle formed between a cell and a substrate—as a quantitative means to study adhesion. Such an approach has been applied successfully to studies of fundamental adhesion processes related to the differential adhesion hypothesis24 and the differential interfacial tension hypothesis,25 as well as to studies of adhesion interactions related to cell surface engineering and tissue engineering.26,27 In this paper, we demonstrate the utility of lateral microscopy28 to observe and quantify cell adhesion as it correlates with surface biomarker expression levels (Figure 1). We introduced different types of leukocytes to glass coverslips coated in surface antigen-specific antibodies (e.g., anti-CD4 specific for T cells expressing CD4). We then monitored changes in the contact angles of cells for 90 minutes from the time of initial cell-substrate contact. With the lateral



microscope, single cells can be imaged in the field of view orthogonal to that imaged with all standard upright or inverted microscopes such that cells appear like droplets wetting surfaces. Therefore, cells can be observed on any substrate, and the contact angles formed between a cell and substrate are easily measured over time, providing a more quantitative description of adhesion than what can be acquired using wash assays. Furthermore, we show that changes in the contact angles of cells correspond to the amount of antigen expressed or freely presented on their surfaces according to flow cytometry. Our approach combines descriptions of cell morphology and immunophenotyping to provide a unique and reproducible technique for cell capture and characterization.

Materials and Methods Cell Culture: The following reagent was obtained through the NIH AIDS Reagent Program, Division of AIDS, NIAID, NIH: SUP-T1 from Dr. Dharam Ablashi.29 The following reagent was obtained through the NIH AIDS Reagent Program, Division of AIDS, NIAID, NIH: CEM CD4+ Cells from Dr. J.P. Jacobs.30 SUP-T1, CEM CD4+, MAVER-1 (ATCC CRL-3008), and BJAB K20 (a gift from Dr. Michael Pawlita; DFKZ German Cancer Research Center) were cultured in suspension in RPMI (Corning) supplemented with 10% fetal bovine serum (FBS; Gibco) and 1% penicillin-streptomycin (Gibco).

Substrate Preparation: Glass coverslips (No. 1.5; VWR) were cut into 0.25 inch pieces using a deluxe diamond scribing pen (Ted Pella, Inc.). Each piece of glass was sterilized with 70% ethanol and dried with nitrogen gas to remove any surface contaminants. Using double-sided adhesive (DF051521, FLEXcon), each piece of glass was mounted onto a 0.25-inch piece of


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aluminum (McMaster-Carr) that had been previously cut using a band saw. This serves to ensure that the substrate remains immobilized at the bottom of the sample container in cell culture media for the duration of imaging experiments. To coat substrates with antibody, stock solutions of 500 µg/mL of mouse anti-human CD4 (BioLegend) and mouse anti-human CD20 (BioLegend) were diluted to 20 µg/mL, 10 µg/mL, and 5 µg/mL in 1X PBS (Fisher Scientific) to achieve a final volume of 200 µL. The entire volume was then pipetted on top of the glass substrate to enable antibody adsorption during a 45-minute incubation period at room temperature. Any remaining liquid was aspirated from the glass before adding the substrate to the sample container to proceed with imaging experiments.

Lateral Microscopy: The lateral microscope and sample containers to enable imaging in the lateral field of view were fabricated as previously described.28 Images of leukocytes adhering to antibody-coated substrates were acquired as follows: (i) The live-cell enclosure surrounding the lateral microscope was allowed to warm to 37 °C for approximately 30 minutes. (ii) An antibody-coated substrate was added to an ethanol-sterilized sample container and washed twice with RPMI. (iii) The sample container with the substrate and 4 mL of RPMI was positioned on the sample stage of the lateral microscope inside the 37 °C live cell enclosure. (iv) The front edge of the substrate inside the sample container was brought into focus in the field of view provided by the lateral microscope. (v) An aliquot of cells was pipetted directly onto the substrate in the sample container, and a custom-built lid that evenly distributes 5% CO2 in sample container (Figure S1) was immediately placed over the sample container to maintain physiological pH during imaging experiments. (vi) Upon settling onto the antibody-coated surface, cells of interest were imaged one at a time. The same cells were imaged every 15



minutes for the duration of 90 minutes. Using a 40× objective lens, we were able to image ~6–10 cells per imaging experiment by quickly adjusting the motorized sample stage to bring each cell into focus within ~2 seconds of one another.

Flow Cytometry: The expression levels of CD4 and CD20 on each of the four cells lines were determined by flow cytometry (EMD-Millipore Guava easyCyte 6HT-2L). 1 × 106 cells were incubated with 2 µg/mL of antibody in 1 mL of RPMI for 30 minutes at 37 °C and 5% CO2. Cells were then washed twice with 1 mL of 1X PBS, resuspended in 1 mL of RPMI, and incubated with 4 µg/mL of FITC-labeled anti-mouse IgG (BioLegend) for 30 minutes at 37 °C and 5% CO2. Cells were washed three times with 1 mL of 1X PBS, and 200 µL of the cell suspension were added to a round bottom 96 well plate to place inside the flow cytometer. Cell populations expressing each antibody were identified by gating for single cells that were positive for green fluorescence.

Blocking Surface Marker Presentation: To partially block the presentation of CD4 markers on the surface of CEM CD4+ cells, 1 × 106 cells were incubated with 4 µg/mL of mouse anti-human CD4 (BioLegend) in 1 mL of RPMI for 30 minutes at 37 °C and 5% CO2. Cells were then washed twice with 1 mL of 1X PBS and resuspended in 1 mL of RPMI. These cells, as well as a separate sample of 1 × 106 unblocked CEM CD4+ cells, were incubated with 2 µg/mL of FITClabeled mouse anti-human CD4 (BioLegend) for 30 minutes at 37 °C and 5% CO2. Cells were washed three times with 1 mL of 1X PBS, and 200 µL of the cell suspension were added to a round bottom 96 well plate to place inside the flow cytometer. Cell populations were identified by gating for single cells that were positive for green fluorescence.


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Wash Assays: Cells were stained with 10 µL/mL of DiI (ThermoFisher) or DiO (ThermoFisher), both of which are general membrane stains, in cell culture media for 30 minutes at 37 °C and 5% CO2. Cells were then washed twice with 1X PBS and split into two samples such that, after a third centrifugation step, one sample could be resuspended in RPMI and the second could be resuspended in 1X PBS. The sample in RPMI was used for wash assays. Two different cell types (stained with different dyes) were added in a 1:1 ratio of 50,000 cells each to a well coated in 10 µg/mL of antibody in a black, glass bottom 24 well plate (N = 3 for each cell mixture). The well plates were incubated at 37 °C and 5% CO2 for 90 minutes. Each well was then washed three times with 1X PBS by gently pipetting 500 µL down the side of the well, swirling, and then aspirating off the liquid. An additional 500 µL of 1X PBS was added to each well for plate reader analyses. The second sample of cells in 1X PBS was used to generate a calibration curve with the plate reader that correlates number of cells with fluorescence intensity. This calibration was achieved by adding cells in 10,000 cell increments (N = 3 for each increment) from 0 to 60,000 cells to separate wells in a black, glass-bottom 24 well plate. Each well was filled with 1X PBS to a final volume of 500 µL. Optimal parameters on a bottom-reading microplate reader (Spark 10M, Tecan) were determined for each cell line and stain using the calibration curve plates, and these parameters were then used when analyzing the wash assay plates. The linear regression analysis of each calibration curve was used to calculate numbers of cells remaining in each well after wash assays.

Inverted Microscopy: To reaffirm wash assay results obtained by the microplate reader, cells were imaged using fluorescence microscopy (Leica DMi8 with Andor Revolution DSD2 confocal imaging system).



Image and Statistical Analyses: The Contact Angle plug-in for ImageJ was used to measure the contract angles formed between cells and antibody-coated substrates as previously described.28 Left and right contact angle measurements were generated for each cell, and the average of the two was reported and used to calculate the change in contact angle from 0 to 90 minutes. Ten cells were imaged for each antibody coating condition, and any outlier cells—with changes in contact angle outside of the 95% confidence interval—were determined in Prism 7 (GraphPad) using the ROUT method (Q = 5%), which detects multiple outliers according to the false discovery rate. The average changes in the contact angles of 10 different cells (or less, if outliers were specified) were reported in Tables 1 and 2. A Mann-Whitney U-test, which is a nonparametric test that does not assume equal variances nor Gaussian distributions, was used in Prism 7 to determine if the average changes in contact angle between two different cell lines on each surface were significantly different (i.e., p < 0.05). However, when comparing the average number of cells remaining from two different cell lines after each wash assay performed, an unpaired t-test with Welch’s correction, which does not assume equal variances but does assume normality, was used to determine statistical differences.

Results and Discussion Using lateral microscopy, we imaged single cells from four different leukocyte lines— two T cell lines and two B cells lines—on antibody-coated surfaces to compare surface marker expression levels with changes in cell morphology (Figure 2). SUP-T1 cells are human T cell lymphoblastic lymphoma T lymphoblasts that express high levels of CD4 but do not express CD20.29,31 CEM CD4+ cells are human T acute lymphoblastic leukemia T lymphoblasts that also express high levels of CD4 and do not express CD20.30,32 MAVER-1 cells are human mantle cell


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lymphoma B lymphoblasts that do not express the CD4 marker but express high levels of the CD20 marker.33 BJAB K20 cells are human Burkitt lymphoma B lymphocytes that do not express CD4, but are distinguishable from MAVER-1 cells because they express only moderate levels of CD20.34 All of these cell lines grow in suspension using recommended cell culture procedures. We introduced each of the four cell lines to glass coverslips coated in CD4 and CD20 antibodies and observed changes in cell morphology spanning the first 90 minutes of contact. To quantify these changes in morphology, we measured contact angles formed between single cells and the surface in 15 minute increments. We also varied the coating conditions of each antibody to determine if changes in cell morphology are exaggerated on surfaces coated with larger concentrations of applied antibody. For these experiments, we relied only on passive adsorption to coat glass surfaces with antibody, and did not apply methods of site-directed immobilization to control coating density or presentation of binding sites. Finally, we partially blocked the presentation of a particular antigen on the surfaces of cells before their introduction to the antibody-coated substrate to further demonstrate our ability to measure differences cell morphology that reflect surface biomarker expression levels.

Adhesion of Cells with Similar Expression Levels of a Common Surface Marker On anti-CD4-coated glass coverslips, we observed changes in the morphologies of SUPT1 cells that correspond with adhesion, a process that these cells do not typically experience using traditional cell culture methods. We imaged SUP-T1 cells spreading to increase contact area with the anti-CD4-coated surface, which was indicated by decreasing cell contact angles over the course of 90 minutes (Figure 2). Thus, by coating glass surfaces in an antibody for a



highly expressed target antigen on the cell surface—CD4—we were able to immobilize SUP-T1 cells on glass. We compared contact angle measurements of SUP-T1 cells on surfaces coated in 20, 10, and 5 µg/mL of anti-CD4 with the hypothesis that cells would spread more on surfaces coated with larger amounts of the antibody. SUP-T1 cells experienced average changes in contact angle of 84.6 ± 10.0, 81.6 ± 20.5, 66.5 ± 25.4 degrees on surfaces coated in 20, 10, and 5 µg/mL of anti-CD4, respectively (Table 1). While this trend seems to support our hypothesis, only the average changes in contact angle of SUP-T1 cells on 5 and 20 µg/mL of anti-CD4 were statistically different from one another (p = 0.009). CEM CD4+ cells express similar levels of CD4 as SUP-T1 cells, as determined by flow cytometry, and these results were reflected in our lateral microscopy experiments (Figure 2). On surfaces coated in 20, 10, and 5 µg/mL of anti-CD4, CEM CD4+ cells experienced average changes in contact angle of 91.0 ± 8.8, 75.7 ± 23.2, 70.2 ± 20.6 degrees, respectively (Table 1). The average changes in contact angle of CEM CD4+ cells on 5 and 20 µg/mL of anti-CD4 were statistically different from one another (p = 0.02). Furthermore, we compared the average changes in contact angle of SUP-T1 cells and CEM CD4+ cells on each surface coating and found that none of the measurements were statistically different from one another (p = 0.28, 0.85, and 0.68 for 20, 10, and 5 µg/mL, respectively). On anti-CD20-coated glass coverslips, SUP-T1 and CEM CD4+ cells experienced small average changes in contact angle of 6.0 ± 9.0 and 2.0 ± 4.9 degrees, respectively, which reflects their lack of expression of the CD20 biomarker (Figures S2 and S3). Further, on uncoated glass, SUP-T1 and CEM CD4+ cells underwent average changes in contact angle of 3.7 ± 5.9 and 7.9 ± 12.4 degrees, respectively (Table 1). These values were significantly different from those measured on anti-CD4-coated glass (p < 0.05), which confirms that the large changes in contact angle that occurred on coated


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glass were caused by antigen-antibody interactions and not the physical properties of the glass substrate. Therefore, our quantitative comparison of two T cell lines that express similar levels of CD4 indicates that changes in cell morphology determined by lateral microscopy are specific and correlate well with surface marker expression levels determined using by flow cytometry.

Adhesion of Cells with Different Expression Levels of a Common Surface Marker We additionally desired to demonstrate that our approach to correlating surface marker expression by flow cytometry with contact angles measured by lateral microscopy could distinguish between cell lines that are positive for a surface marker but differ in overall amount. For this reason, we chose to study two CD20-positive B cell lines, MAVER-1 and BJAB K20, on anti-CD20-coated glass coverslips (Figure 2). On surfaces coated in 20, 10, and 5 µg/mL of anti-CD20, MAVER-1 cells experienced average changes in contact angle of 78.7 ± 17.4, 77.2 ± 12.2, 4.4 ± 4.9 degrees, respectively (Table 2). The average change in contact angle on 5 µg/mL was statistically different from those on 10 µg/mL and 20 µg/mL (p < 0.0001 for both cases), which suggests that for the MAVER-1 cell line, a surface coated with concentrations of antibody greater than 5 µg/mL is needed to cause changes in morphology that are more representative of adhesion. Like MAVER-1 cells, BJAB K20 cells also express CD20 but at lower levels. This difference in CD20 expression was reflected by the results obtained using the lateral microscope: BJAB K20 cells experienced smaller average changes in contact angle after 90 minutes of contact with anti-CD20-coated glass than MAVER-1 cells. On surfaces coated in 20, 10, and 5 µg/mL of anti-CD20, BJAB K20 cells experienced average changes in contact angle of 52.1 ± 22.4, 47.6 ± 12.8, 35.6 ± 8.9 degrees, respectively (Table 2). The average change in contact



angle of BJAB K20 cells on 5 µg/mL was statistically different from those on 20 µg/mL (p = 0.046). More importantly, we compared the average changes in contact angle of MAVER-1 cells and BJAB K20 cells and found that, with each concentration of antibody used to coat surfaces, the contact angle results were significantly different between the two cell lines (p = 0.02, p < 0.0001, and p < 0.0001 for 20, 10, and 5 µg/mL, respectively). On anti-CD4-coated glass coverslips, MAVER-1 and BJAB K20 cells underwent small average changes in contact angle of 4.7 ± 9.0 and 8.2 ± 9.3 degrees, respectively, which is consistent with their lack of expression of the CD4 biomarker (Figures S4 and S5). Additionally, on uncoated glass, MAVER-1 and BJAB K20 cells experienced average changes in contact angle of 6.2 ± 8.0 and 1.7 ± 6.8 degrees, respectively (Table 2). With the exception of MAVER-1 cells on 5 µg/mL of anti-CD20, these values are significantly different from those measured on anti-CD20-coated glass (p < 0.05), which further suggests that the physical properties of the glass substrate did not influence cell adhesion. Taken together, these results indicate that our technique can discern between the adhesion of cells with different expression levels of a common surface marker.

Adhesion of Cells with Blocked Surface Marker Presentation To further demonstrate our ability to quantitatively study surface biomarker expression levels using lateral microscopy, we altered the apparent expression of CD4 on CEM CD4+ cells by partially blocking the surface presentation of this marker with anti-CD4, which we confirmed by flow cytometry (Figure 3). We then introduced the CD4-blocked CEM CD4+ cells to glass coated in 10 µg/mL of anti-CD4 and compared the average change in contact angle of these cells to CEM CD4+ cells with normal CD4 expression on the same substrate. CD4-blocked CEM CD4+ cells experienced an average change in contact angle of 52.0 ± 22.5 degrees, which was


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significantly different from the unblocked CEM CD4+ cells (p = 0.03) (Figure 3). Therefore, our approach can also be implemented to understand the expression levels of surface biomarkers on cells under non-native, perturbed conditions.

Comparison with Wash Assays To demonstrate that our approach provides a more quantitative and reproducible option for studying cell adhesion than other commonly implemented assays, we performed wash assays using identical conditions to those used for our lateral microscopy experiments. Wash assay results support those from lateral microscopy experiments in that leukocytes expressing greater levels of a specific antigen adhere better to surfaces coated in the corresponding antibody than leukocytes that express lower levels (or none) of the same antigen (Figure 4, Figure S6). However, we observed nonspecific binding even after three subsequent washes, which may have been due to weak adhesion interactions that were not disrupted by the gentle shear forces introduced by pipetting. These observations contrast the minor changes in contact angle that were observed with SUP-T1 and CEM CD4+ cells on anti-CD20-coated surfaces and MAVER-1 and BJAB K20 cells on anti-CD4-coated surfaces using lateral microscopy. In addition to nonspecific binding, wash assay results were difficult to reproduce: the coefficient of variation (CV) for the number of SUP-T1 cells remaining on anti-CD4-coated glass after three different wash assays (Figure 4a, c, d) was 27.5%, and the CV for MAVER-1 cells remaining on antiCD20-coated glass after two different wash assays (Figure 4b, c) was 81.9%. These large CV values were likely caused by differences in the forces applied with each manual wash. Another limitation of this approach was the use of fluorescent stains to enable the detection and specific identification of cells by a microplate reader. We found that each cell line incorporated the



general membrane stains we used (DiI and DiO) differently, and therefore, we had to create separate calibration curves for each cell line using cells from the same batch stained for wash assays (Figure S7). To bypass this time-consuming step, adherent cells can be counted manually or with cell counting software using brightfield microscopy images, but it would be equally (if not more) time-consuming to image numerous fields of view to acquire a large population of cells. Furthermore, measurements of adherent cell morphologies are limited to cell-substrate contact areas, but not all cells that remained adhered after washing have an increased contact area as expected (Figure S6). Our approach to quantify cell morphology using changes in contact angle with the lateral microscope enables a more rapid, reproducible, and quantitative method to study adhesion.

Conclusions We have developed a new approach using lateral microscopy to correlate changes in cell morphology with surface marker expression levels. With our method, changes in cell morphology that are representative of adhesion are obvious and quantifiable. Our results on the changes in contact angles of cells on antibody-coated substrates reflect the expression levels of corresponding antigens on the surfaces of cells as determined by flow cytometry. Moreover, when compared to wash assays that are commonly used to assess adhesion, our technique is more rapid and reproducible. Importantly, with the lateral microscope, the morphologies of single cells can be quantified over time on any substrate without prior fluorescence staining. Although our current experimental approach requires the user to locate, track, and analyze images of cells manually, there is an obvious opportunity to develop methods to automate these processes as additional applications of lateral microscopy are developed. For example, while we


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have demonstrated the utility of this approach to classify T and B cells, which are often isolated for studies pertaining to the immune response, our assay can be extended to the study of a number of other cellular systems that rely on immunophenotyping to generate phenotypic or functional information. With cancer, in particular, only one invasive cell is required to initiate metastasis, but with the high throughput techniques that are used routinely to characterize cells, any invasive cells may be ignored as statistical outliers.35,36 Recently, a number of analytical techniques have been developed for the specific purpose of enabling experimentation (e.g., genomics, proteomics, and immunophenotyping) with the resolution of single cells.37,38,39 We believe that lateral microscopy offers another enabling platform for the study of single cells. With our lateral microscopy adhesion assay, desired cells within heterogeneous populations may be identified rapidly by exploring different surface coatings and conditions. Ultimately, we have demonstrated that the combination cell morphology and immunophenotyping provides a unique and reproducible technique for characterizing cells during adhesion processes.

Acknowledgments This work was supported by Tufts University. We thank Dr. Jessica C. Brooks for designing and fabricating the custom 3D printed gas distributor for the lateral microscope live cell enclosure. We thank Prof. Rebecca Scheck for providing training and access to a plate reader to perform wash assays. The BJAB K20 cell line was a generous gift provided by Prof. Michael Pawlita (DFKZ German Cancer Research Center). The following reagent was obtained through the NIH AIDS Reagent Program, Division of AIDS, NIAID, NIH: SUP-T1 from Dr. Dharam Ablashi. The following reagent was obtained through the NIH AIDS Reagent Program, Division of AIDS, NIAID, NIH: CEM CD4+ Cells from Dr. J.P. Jacobs.



Figure 1. Schematic representation of using lateral microscopy to quantitatively determine if a specific interaction between a surface biomarker and ligand promotes adhesion. Large changes in contact angle (θc) indicate an interaction between surfaces with a high coating of antibody and a cell with a high expression of a surface marker (e.g., surface coated with anti-A and cell expressing high levels [++] of antigen A). Moderate changes in θc indicate that the interaction occurs with a sub-optimal coating of antibody or lower expression of a surface marker (e.g., high surface coating of anti-A and a cell expressing low levels [+] of antigen A). Finally, small to no changes in θc reflect a mismatched interaction (e.g., surface coated with anti-A and a cell expressing antigen B) or an uncoated surface.


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Figure 2. Using lateral microscopy to compare changes in the morphologies of SUP-T1, CEM CD4+, MAVER-1, and BJAB K20 cells after 90 minutes of contact with glass coverslips coated in 10 µg/mL of antibody. SUP-T1 and CEM CD4+ cells express similar levels of CD4, while MAVER-1 and BJAB K20 cells express different levels of CD20 as determined by flow cytometry.



Table 1. Average changes in contact angle of SUP-T1 and CEM CD4+ cells on anti-CD4-coated glass and uncoated glass (N = 10 cells per condition).

∆θc (deg) SUP-T1

CEM CD4+

20 µg/mL

85.1 ± 10.4

91.0 ± 8.8

10 µg/mL

81.6 ± 20.5

75.7 ± 23.2

5 µg/mL

66.5 ± 25.4

70.2 ± 20.6

uncoated glass

3.7 ± 5.9

7.9 ± 12.4


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Table 2. Average changes in contact angle of MAVER-1 and BJAB K20 cells on anti-CD20coated glass and uncoated glass (N = 10 cells per condition).

∆θc (deg) MAVER-1

BJAB K20

20 µg/mL

78.7 ± 17.4

52.1 ± 22.4

10 µg/mL

77.2 ± 12.2

47.6 ± 12.8

5 µg/mL

4.4 ± 4.9 a

35.6 ± 8.9 b

uncoated glass

6.2 ± 8.0

1.7 ± 6.8

a. one statistical outlier b. two statistical outliers



Figure 3. Using lateral microscopy to compare changes in the morphologies of CEM CD4+ cells with normal and partially-blocked surface presentation of CD4. On glass coverslips coated in 10 µg/mL of anti-CD4, the average change in contact angle of normal CEM CD4+ cells was 75.7 ± 23.2 degrees, whereas the average change in contact angle of CD4-blocked CEM CD4+ cells was 52.0 ± 22.5 degrees (N = 10 cells per condition).


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Figure 4. Wash assay results. Number of cells remaining after adding a 1:1 mixture of (a) SUPT1 and CEM CD4+ cells to anti-CD4-coated glass, (b) MAVER-1 and BJAB K20 cells to antiCD20-coated glass, (c) SUP-T1 and MAVER-1 cells to anti-CD4- and anti-CD20-coated glass, and (d) SUP-T1 and BJAB K20 cells to anti-CD4- and anti-CD20-coated glass. Error bars represent the standard error of the mean of three replicates (*p < 0.05, **p < 0.01).




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For TOC Only



References

1.

Sampson, N. S.; Mrksich, M.; Bertozzi, C. R. Surface molecular recognition. Proc. Natl. Acad. Sci. U. S. A. 2001, 98, 12870−12871.

2.

Geiger, B.; Spatz, J. P.; Bershadsky, A. D. Environmental sensing through focal adhesions. Nat. Rev. Mol. Cell Biol. 2009, 10, 21–33.

3.

Evans, R.; Patzak, I.; Svensson, L.; De Filippo, K.; Jones, K.; McDowall, A.; Hogg, N. Integrins in immunity. J. Cell Sci. 2009, 122, 215–225.

4.

Zhang, Y.; Wang, H. Integrin signaling and function in immune cells. Immunology 2012, 135, 268–275.

5.

Varki, N.M.; Varki, A. Diversity in cell surface sialic acid presentations: implications for biology and disease. Lab. Investig. 2007, 87, 851–857.

6.

Dube, D.H.; Bertozzi, C.R. Glycans in cancer and inflammation—potential for therapeutics and diagnostics. Nat. Rev. Drug Discov. 2005, 4, 477–488.

7.

McGrath, J. L. Cell spreading: the power to simplify. Curr. Biol. 2007, 17, R357–R358.

8.

Brugmans, M.; Cassiman, J.-J., Vanderheydt, L.; Oosterlinck, A. J. J.; Vlietinck, R.; Van den Berghe, H. Quantification of the degree of cell spreading of human fibroblasts by semiautomated analysis of the cell perimeter. Cytometry 1982, 3, 262–268.

9.

Cuvelier, D.; Thery, M.; Chu, Y.-S.; Dufour, S.; Thiery, J.-P.; Bornens, M.; Nassoy, P.; Mahadevan, L. The universal dynamics of cell spreading. Curr. Biol. 2007, 17, 694−699.

10. Manning, M. L.; Foty, R. A.; Steinberg, M. S.; Schoetz, E.-M. Coaction of intercellular adhesion and cortical tension specifies tissue surface tension. Proc. Natl. Acad. Sci. U. S. A. 2010, 107, 12517−12522.


Page 26 of 30

Page 27 of 30 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


11. Reinhart-King, C. A.; Dembo, M.; Hammer, D. A. Endothelial cell traction forces on RGDderivatized polyacrylamide substrata. Langmuir 2002, 19, 1573–1579. 12. Picot, J.; Guerin, C. L.; Le Van Kim, C.; Boulanger, C. M. Flow cytometry: retrospective, fundamentals and recent instrumentation. Cytotechnology 2012, 64, 109–130. 13. Brown, M.; Wittwer, C. Flow cytometry: principles and clinical applications in hematology. Clin. Chem. 2000, 46, 1221–1229. 14. Zhu, H.; Macal, M.; Jones, C. N.; George, M. D.; Dandekar, S.; Revzin, A. A miniature cytometry platform for capture and characterization of T-lymphocytes from human blood. Anal. Chim. Acta 2008, 608, 186–196. 15. Zamai, L.; Falcieri, E.; Zauli, G.; Cataldi, A.; Vitale, M. Optimal detection of apoptosis by flow cytometry depends on cell morphology. Cytometry 1993, 14, 891–897. 16. Pelletier, M. G. H.; Szymczak, K.; Barbeau, A. M.; Prata, G. N.; O’Fallon, K. S.; Gaines, P. Characterization of neutrophils and macrophages from ex vivo-cultured murine bone marrow for morphologic maturation and functional responses by imaging flow cytometry. Methods 2017, 112, 124–146. 17. Barteneva, N. S.; Fasler-Kan, E.; Vorobjev, I. A. Imaging flow cytometry: coping with heterogeneity in biological systems. J. Histochem. Cytochem. 2012, 60, 723– 733. 18. Khalili, A. A.; Ahmad, M. R. A review of cell adhesion studies for biomedical and biological applications. Int. J. Mol. Sci. 2015, 16, 18149–18184. 19. Humphries, M. Cell adhesion assays. J. Mol. Biotechnol. 2001, 18, 57–61. 20. Zhou, D. W.; Garcia, A. J. Measurement systems for cell adhesive forces J. Biomech. Eng. 2015, 137, 0209081–0209088.



21. Zhu, H.; Stybayeva, G.; Silangcruz, J.; Yan, J.; Ramanculov, E.; Dandekar, S.; George, M. D.; Revzin, A. Detecting Cytokine Release from Single Human T-cells. Anal. Chem. 2009, 81, 8150–8156. 22. Stybayeva, G.; Mudanyali, O.; Seo, S.; Silangcruz, J.; Macal, M.; Ramanculov, E.; Dandekar, S.; Erlinger, A.; Ozcan, A.; Revzin, A. Lensfree holographic imaging of antibody microarrays for high-throughput detection of leukocyte numbers and function. Anal. Chem. 2010, 82, 3736–3744. 23. Chen, A.; Vu, T.; Stybayeva, G.; Pan, T.; Revzin, A. Reconfigurable microfluidics combined with antibody microarrays for enhanced detection of T-cell secreted cytokines. Biomicrofluidics 2013, 7, 24105. 24. Foty, R. A.; Steinberg, M. S. The differential adhesion hypothesis: a direct evaluation. Dev. Biol. 2005, 278, 255–263. 25. Brodland, G. W. The Differential Interfacial Tension Hypothesis (DITH): a comprehensive theory for the self-rearrangement of embryonic cells and tissues. J. Biomech. Eng. 2002, 124, 188−197. 26. Diz-Munoz, A.; Fletcher, D. A.; Weiner, O. D. Use the force: membrane tension as an organizer of cell shape and motility. Trends Cell Biol. 2013, 23, 47–53. 27. Todhunter, M. E.; Jee, N. Y.; Hughes, A. J.; Coyle, M. C.; Cerchiari, A.; Farlow, J.; Garbe, J. C.; LaBarge, M. A.; Desai, T. A.; Gartner, Z. J. Programmed synthesis of threedimensional tissues. Nature Methods 2015, 12, 975–981. 28. Walz, J. A.; Lui, I.; Wilson, D. J.; Mace, C. R. Lateral microscope enables the direct observation of cellular interfaces and quantification of changes in cell morphology during adhesion. ACS Biomater. Sci. Eng. 2016, 2, 1367–1375.


Page 28 of 30

Page 29 of 30 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


29. Ablashi, D. V.; Berneman, Z. N.; Kramarsky, B.; Whitman, J. Jr.; Asano, Y.; Pearson, G. R. Human herpesvirus-7 (HHV-7): current status. Clin. Diagn. Virol. 1995, 4, 1–13. 30. Foley, G.E.; Lazarus, H.; Farber, S.; Uzman, B. G.; Boone, B. A.; McCarthy, R. E. Continuous culture of human lymphoblasts from peripheral blood of a child with acute leukemia. Cancer 1965, 18, 522–529. 31. Smith, S. D.; Shatsky, M.; Cohen, P. S.; Warnke, R.; Link, M. P.; Glader, B. E. Monoclonal antibody and enzymatic profiles of human malignant T lymphoid cells and derived cell lines. Cancer Res. 1984, 44, 5657–5660. 32. Suzuki, H.; Zelphati, O.; Hildebrand, G.; Leserman, L. CD4 and CD7 molecules as targets for drug delivery from antibody bearing liposomes. Exp. Cell Res. 1991, 193, 112–119. 33. Zamo, A.; Ott, G.; Katzenberger, T.; Adam, P.; Parolini, C.; Scarpa, A.; Lestani, M.; Menestrina, F.; Chilosi, M. Establishment of the MAVER-1 cell line, a model for leukemic and aggressive mantle cell lymphoma. Haematologica 2006, 91, 40–47. 34. Keppler, O. T.; Hinderlich, S.; Langner, J.; Schwartz-Albiez, R.; Reutter, W.; Pawlita, M. UDP-GlcNAc 2-epimerase: a regulator of cell surface sialylation. Science 1999, 284, 1372– 1376. 35. Marusyk, A.; Polyak, K. Tumor heterogeneity: causes and consequences. Biochim. Biophys. Acta 2010, 1805, 105–117. 36. Meacham, C. E.; Morrison, S. J. Tumour heterogeneity and cancer cell plasticity. Nature 2013, 501, 328–337. 37. Sinkala, E.; Sollier-Christen, E.; Renier, C.; Rosàs-Canyelles, E.; Che, J.; Heirich, K.; Duncombe, T. A.; Vlassakis, J.; Yamauchi, K. A.; Huang, H.; Jeffrey, S. S.; Herr, A. E.



Profiling protein expression in circulating tumour cells using microfluidic western blotting. Nat. Commun. 2017, 8, 14622. 38. Poudineh, M.; Aldridge, P. M.; Ahmed, S.; Green, B. J.; Kermanshah, L.; Nguyen, V.; Tu, C.; Mohamadi, R.M.; Nam, R. K.; Hansen, A.; Sridhar, S. S.; Finelli, A.; Fleshner, N. E.; Joshua, A. M.; Sargent, E. H.; Kelley, S. O. Tracking the dynamics of circulating tumour cell phenotypes using nanoparticle-mediated magnetic ranking. Nat. Nanotechnol. 2017, 12, 274–281. 39. Welch, J. D.; Williams, L. A.; DiSalvo, M.; Brandt, A. T.; Marayati. R.; Sims, C. E.; Allbritton, N. L.; Prins, J. F.; Yeh, J. J.; Jones, C. D. Selective single cell isolation for genomics using microraft arrays. Nucleic Acids Res. 2016, 44, 8292–8301.


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Correlation of Cell Surface Biomarker Expression Levels with

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