Lab on a Chip PAPER Linking invasive motility to protein expression in † Cite this: Lab Chip,2018,18,371 single tumor cells Jung-Ming G. Lin, ab Chi-Chih Kang,b Yun Zhou,c Haiyan Huang,cde Amy E. Herr ab and Sanjay Kumar*abf The invasion of malignant cells into tissue is a critical step in the progression of cancer. While it is increas- ingly appreciated that cells within a tumor differ in their invasive potential, it remains nearly unknown how these differences relate to cell-to-cell variations in protein expression. Here, we introduce a microfluidic platform that integrates measurements of invasive motility and protein expression for single cells, which we use to scrutinize human glioblastoma tumor-initiating cells (TICs). Our live-cell imaging microdevice is comprised of polyacrylamide microchannels that exhibit tissue-like stiffness and present chemokine gradi- ents along each channel. Due to intrinsic differences in motility, cell subpopulations separate along the Received 19th September 2017, channel axis. The separated cells are then lysed in situ and each single-cell lysate is subjected to western Accepted 18th December 2017 blotting in the surrounding polyacrylamide matrix. We observe correlations between motility and Nestin and EphA2 expression. We identify protein–protein correlations within single TICs, which would be DOI: 10.1039/c7lc01008g obscured with population-based assays. The integration of motility traits with single-cell protein analysis – rsc.li/loc on the same cell – offers a new means to identify druggable targets of invasive capacity. – Solid tumors consist of cell subpopulations that differ widely assays.4,8 11 However, the vast majority of these approaches in gene and protein expression, tumor-forming ability, have focused entirely on the functional characterization of chemoresistance, and invasive capacity.1,2 Over the past de- single-cell motility, with few efforts to connect single-cell mi- cade, this notion has been formalized into the cancer stem gration with specific marker expression.4,10 In one such cell (a.k.a. tumor-initiating cell) paradigm, which argues that study, cell speed was measured within one microfluidic chan- tumor initiation, progression, and metastasis are driven by a nel device while expression of motility-relevant mRNAs for the rare-cell cohort whose properties are masked by population same cell was subsequently measured using molecular bea- measurements.3,4 Consequently, such population-based mea- cons in a separate device.4 However, mRNA levels are neither surements can obscure the understanding of cancer develop- fully predictive of protein expression nor capable of reporting ment, progression, and response to treatment. An important protein signaling events that occur after translation.12 hallmark of cancer progression is the invasion of tumor cells For single-cell protein detection, the de facto standard is into the surrounding tissue and, in some cases, metastasis to immunocytochemistry (ICC). While useful, ICC suffers from distant sites.5,6 However, little is definitively known about fixation artifacts (limiting specificity), background antibody how the invasive phenotype (or potential) relates to protein cross-reactivity (impacting multiplexing and increasing back- expression within a single cell.5,7 ground signal), and the challenge of accurately measuring to- The precision afforded by microfluidics tools allows the tal fluorescence intensity from a cell volume.13,14 On the scrutiny of tumor invasion potential with more nuanced and other hand, standard slab western blot detects much im- higher-dimensionality descriptors than population-based proved protein quantification but requires pooled populations of cells and is therefore unsuitable for single-cell analysis. To achieve the best of both approaches, the Herr a UC-Berkeley-UCSF Graduate Program in Bioengineering, University of California, Laboratory has recently developed the single-cell western blot Berkeley, CA 94720, USA. E-mail: [email protected] ∼ 3 b Department of Bioengineering, University of California, Berkeley, CA 94720, USA (scWB) assay, which can quantify protein levels in 10 sepa- c Division of Biostatistics, School of Public Health, University of California, rate cells within 4 h, with a lower limit of detection of ∼27 Berkeley, CA 94720, USA 000 copies of a protein.15 We have successfully used this as- d Department of Statistics, University of California, Berkeley, CA 94720, USA say to investigate heterogeneity in cell signaling, differentia- e Center for Computational Biology, University of California, Berkeley, CA 94720, USA f Department of Chemical and Biomolecular Engineering, University of California, tion and chemotherapeutic resistance in specific cell 15–18 Berkeley, CA 94720, USA lines. In addition to acting as both a molecular sieve and † Electronic supplementary information (ESI) available. See DOI: 10.1039/c7lc01008g blotting membrane, the polyacrylamide (PA) gel used to This journal is © The Royal Society of Chemistry 2018 Lab Chip,2018,18,371–384 | 371 Paper Lab on a Chip create the scWB has the potential to be micropatterned with Materials and methods features that integrate important, complementary cellular Antibodies measurements, including single-cell resolution motility assays. Antibodies employed for the migratory scWB study include 19,20 Here, we describe an integrated microfluidic device that rabbit anti-EphA2 (1 : 10, 6997S, Cell Signaling, with anti- combines the scWB assay with live-cell imaging of invasive rabbit secondary antibody conjugated with Alexa-Fluor 647), 21,22 cell motility (Fig. 1), which we term SCAMPR (Single-Cell mouse anti-STAT3 (1 : 10, 9139S, Cell Signaling, with anti- Analysis of Motility and Proteotype). In this platform, cells mouse secondary antibody conjugated with Alexa-Fluor 488), 23,24 are induced to chemotactically migrate along microchannels mouse anti-Nestin (1 : 10, MAB5326, EMD-Millipore, with and imaged to capture instantaneous and time-averaged cel- anti-mouse secondary antibody conjugated with Alexa-Fluor β 17,25 lular migratory properties (i.e., speed, persistence and aspect 488), rabbit anti- -tubulin (1 : 10, ab6046, Abcam, with ratio) for later quantification. Each cell is then lysed in situ, anti-rabbit secondary antibody conjugated with Alexa-Fluor with the lysate electrophoresed through the wall of the chan- 647). nel and size-separated due to molecular sieving through the PA gel, photoimmobilized to the PA gel (via light-activated Cell culture benzophenone methacrylamide co-monomer),18 and then U373 Empty Vector and U373 DN Rac1 GBM cell lines26 were ° immunoprobed to quantify candidate proteins. Using the maintained at 37 C in a 5% CO2 humidified chamber and SCAMPR platform, we first separate cell populations with cultured in high glucose DMEM (Life Technologies) known differences in motility. We then investigate the corre- supplemented with 10% calf serum (JR Scientific), 100 U − − lation of protein levels with migratory behavior in primary mL 1 penicillin, 100 μgmL1 streptomycin, 1× MEM non- glioblastoma tumor-initiating cell (GBM TIC) cultures. essential amino acids, and 1 mM sodium pyruvate (Life Tech- − SCAMPR represents an important step towards the identifica- nologies). To induce gene expression, 25 ng mL 1 doxycycline tion of tumor-specific proteomic predictors of invasive motil- was added to the cell culture medium 48 h prior to any exper- ity, relevant to questions spanning basic discovery to preci- iments. As a technical note, we used U373 cells from the sion medicine. American Type Culture Collection (ATCC). U373 cells have Fig. 1 Microfluidic integration supports a Single-Cell Analysis of Motility and Proteotype (SCAMPR) assay. A heterogeneous population of primary cells is first dissociated into a single-cell suspension. The cells are then seeded into the SCAMPR device and tracked as each cell chemotactically migrates through the channels under a chemokine gradient, which reports motility and motility-related parameters, persistence and average as- pect ratio. Immediately following the live-cell tracking, cells are immobilized in an agarose layer and the scWB is run in order to measure protein expression on each tracked cells. Motility and proteotype information from single cells are then correlated to associate proteomic markers with in- vasive motility properties. 372 | Lab Chip,2018,18,371–384 This journal is © The Royal Society of Chemistry 2018 Lab on a Chip Paper recently been recognized to be a subclone of the human glio- PDMS layer fabrication blastoma line U251, with the two lines having subsequently Sylgard 184 base and curing agent was mixed in a 10 : 1 ratio 27 diverged to exhibit differential drug sensitivities. Cell lines and degassed. The resulting solution was degassed and were authenticated using short tandem repeat analysis and poured into a rectangular mold to a height of 1.5 mm. The tested negative for mycoplasma. PDMS was then cured at 80 °C for 2 h. After the PDMS was The primary GBM TIC line L0 was originally obtained cured, rectangular slabs (3 cm (L) × 7.5 cm (W)) were cut out from Dr. Brent Reynolds' laboratory at the University of Flor- of the mold. 3 mm diameter holes that match the inlet and 28 ida and has been described in an earlier study. L0 outlet wells on the PA gel were then punched out of the gliomaspheres were propagated in growth conditions with PDMS rectangular slab. serum-free medium (Neurocult NS-A Proliferation kit, Stem Cell Technologies) supplemented with epidermal