SUPPLEMENTAL DATA MICROSCALE FUNCTIONAL CYTOMICS FOR STUDYING HEMATOLOGIC CANCERS Edmond W.K. Young,1,2 Chorom Pak,2,3 Brad S. Kahl,2,4,5 David T. Yang,5,6 Natalie S. Callander,4,5 Shigeki Miyamoto,2,3,5,7 and David J. Beebe1,2,5,8 1Department of Biomedical Engineering, 2 Wisconsin Institutes for Medical Research, 3 Program in Molecular & Cellular Pharmacology, 4 Department of Medicine, 5 University of Wisconsin Carbone Cancer Center, 6Department of Pathology and Laboratory Medicine, 7 Department of Oncology, University of Wisconsin-Madison, Madison, WI, USA 8 Correspondence should be addressed to D.J. Beebe (djbeebe@wisc.edu) Scientific section: LYMPHOID AND MYELOID NEOPLASIA Running Title: Microscale cytomics for hematologic cancers Keywords: Microfluidics, cytomics, cancer, nuclear translocation, NF-κB Corresponding author David J. Beebe

Wisconsin Institutes for Medical Research Department of Biomedical Engineering

University of Wisconsin-Madison 1111 Highland Avenue, Room 6009,

Madison, WI, USA, 53705, Tel: 1-608-262-2260

email: djbeebe@wisc.edu

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Methods

Microchamber device fabrication

Established soft lithography techniques1-2 were used to fabricate single-use devices consisting of

twelve independent microscale cell culture chambers (hereafter called microchambers) arranged in a 3 x

4 array (Figure S-1).

150-mm diameter silicon wafers were acquired from WRS Materials (San Jose, CA). Different

formulations of SU-8 negative photoresist were acquired from Microchem Corp. (Newton, MA).

Poly(dimethylsiloxane) (PDMS) was obtained from Dow Corning (Midland, MI). Corning #2947 glass

slides, and No. 1 thickness glass coverslips (for CLL experiments only) were obtained from Fisher

Scientific (Pittsburgh, PA). Acetone and isopropanol (IPA) were purchased from Fisher Scientific, and

propylene glycol monoethyl ether acetate (PG-MEA) was purchased from Sigma-Aldrich (St. Louis,

MO).

Two separate master molds were made, one for the channel layer consisting of the central well,

side chambers, diffusion ports, and inlet and outlet channels, and one for the port layer. For the port

layer, SU-8-100 was spincoated at 500 rpm for 60 s to yield a ~600 µm layer, soft-baked for 30 min at

65°C and 3 h at 95°C, exposed at 2700 mJ/cm2, post-exposure baked for 2 h at 95°C, cooled to room

temperature, and developed in PG-MEA on a rotator for a minimum of 3 h. After development, the

master mold was rinsed sequentially with fresh PG-MEA, acetone, and IPA, and blown dry with

compressed air.

For the channel layer, SU-8-10 was spincoated at 2800 rpm for 45 s to yield a 20 µm layer, soft-

baked for 2 min at 65°C and 7 min at 95°C, exposed at 250 mJ/cm2, post-exposure baked for 15 min,

cooled to room temperature and developed for 1 min. After development, the wafer was rinsed clean as

usual before applying the next layers. SU-8-100 was spincoated at 1500 rpm for 45 s to yield a 200 µm

layer of side chambers and inlet/outlet channels. The mold was then soft-baked for 30 min at 65°C and 2

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h at 95°C, exposed for 2000 mJ/cm2, and post-exposure baked for 90 min at 95°C. After cooling to room

temperature, the next layer of SU-8-100 was spincoated at 1200 rpm for 45 s to yield an additional 300

µm layer. The mold was soft-baked for 30 min at 65°C and 2.5 h at 95°C, exposed for 3000 mJ/cm2,

post-exposure baked for 2 h min at 95°C, cooled to room temperature, and developed in PG-MEA on a

rotator for a minimum of 3 h. After development, the master mold was rinsed sequentially with fresh

PG-MEA, acetone, and IPA, and blown dry with compressed air.

PDMS at a 10:1 ratio of elastomer base and curing agent was mixed vigorously with a plastic

fork and allowed to degas in a vacuum dessicator for ~1 h. After degassing, PDMS was poured over

each of the two master molds (10 g of mixed PDMS each), which were each placed on separate hot

plates. Transparency film (AF4300, 3M, St. Paul, MN) was cut to size, and placed on top of the PDMS

with sufficient care to not trap any bubbles. Silicone foam, a polycarbonate block, and 30 lbs of weight

were then placed on top of the master mold, and the hot plate was set to 80°C for 4 h.

Once cured, the PDMS slabs were removed from the SU-8 master molds, and placed in a Soxhlet

extractor to extract uncrosslinked oligomers using 100% ethanol.3 All PDMS slabs were bonded in the

orientation and sequence as shown in Figure S-1. Glass slides were rinsed in acetone and IPA, and

blown dry with compressed air. Top side of channel layer (red arrow) and glass were plasma-treated,

then bonded in step 5 (which requires that the channel PDMS layer be flipped). The top of the device

from step 5 and the bottom of the port layer (side without red arrow) were plasma-treated, and bonded in

step 6. The entire device was finally placed on a hot plate for 15 min at 120°C to increase bond strength.

COMSOL Numerical Simulation

Numerical simulations were performed using the COMSOL multiphysics package (COMSOL,

Inc., Los Angeles, CA) to determine wall shear stress at the bottom of the well due to typical passive

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pumping flows (Figure S-2). While flow generated by passive pumping is unsteady,4 the typical flow

rates used with the current geometry corresponded to low Reynolds number flow (i.e., Re ~ O(1)), and

thus a simplified quasi-steady solution was found to be a suitable approximation. We used the Fluid

Dynamics module within the Multiphysics package (version 3.4), and used an incompressible Navier-

Stokes equation (steady state analysis) to model the problem. Steady flow was simulated at an inlet flow

rate of 1 µL/s (velocity of 1 cm/s with 500 µm wide and 200 µm tall inlet channel), with zero outlet

pressure at the outlet end and no slip boundary conditions on all other surfaces. Fluid density was set at

1000 kg/m3, and fluid viscosity was set at 0.72 x 10-3 kg/m s2, to model an aqueous solution at 37°C.

Lagrangian-P2P1 elements were used in the mesh, which contained 16,683 elements in total. Results

estimated that the velocity decreased abruptly from a maximum of 1.8 cm/s in the inlet/outlet channels

to < 0.2 cm/s near the center of the well (Figure S-2). Wall shear stress along the channel centerline

ranged from 8 x 10-3 Pa at the center of the well to a maximum of 23 x 10-3 Pa at the two “hot spots”

(Figure S-2B, red regions).

Cell Retention Results

In all microchambers tested, the number of counted cells increased after the first VR, followed

by the expected trends associated with culturing time. The increase in counted cells was due to

displacement of the cells in the inlet port and inlet channel leading into the well (Figure S-3G, red cells),

which entered the well after the initial VR. Since these cells experienced a shear stress approximately

0.6 Pa, 25x larger than at the maximum shear stress at the bottom of the well, they were easily dislodged

and allowed to pass into the central well. During the depletion tests, the cells from the inlet remained

suspended in the well, above the bottom surface, and were not given sufficient time to settle and interact

with the surface. Hence, each successive VR propelled the suspended cells toward the outlet, until they

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reached the outlet channel after ~20-25 VRs (red cells in Figure S-3G-I).

Overall, the cell depletion results indicated that settling time was an important consideration for

developing an immunostaining procedure for non-adherent cells. We concluded that cell depletion was

negligible as long as cells were cultured for > 12 h prior to the application of the first VR. Furthermore,

while ~ 1% of cells may be lost even after 16-h culturing time, the use of population characteristics over

thousands of cells attenuated this issue.

Immunocytochemical Staining Procedure

Rabbit polyclonal IgG antibodies for RelA (an NF-κB subunit) were obtained from Santa Cruz

Biotechnology (Santa Cruz, CA; sc-372) and used at 1:200 dilution. Rabbit polyclonal IgG antibodies

for prostate specific antigen (PSA), as negative control for RelA non-specific binding, were obtained

from Abcam (Cambridge, MA; ab9537) and used at 1:100 dilution. Mouse monoclonal IgG antibodies

for STAT3 were obtained from Cell Signaling Technologies (Danvers, MA; 124H6) and used at 1:400

dilution. Mouse monoclonal IgG antibodies for cadherin-5 (VE-cadherin), as negative control for

STAT3 non-specific binding, were obtained from BD Transduction Laboratories (Lexington, KY; 51-

9001928) and used at 1:400 dilution. Alexa Fluor 488 goat anti-rabbit (A11034; 1:400) and Alexa Fluor

488 goat anti-mouse (A11029, 1:500) secondary antibodies, Hoechst 33342 nuclear dye (H1399; 10

ng/mL), and Texas Red-X phalloidin (T7471; 1:50) were obtained from Molecular Probes (Invitrogen,

Carlsbad, CA), and used at the dilutions and concentrations indicated.

Cells cultured and treated in microchambers were immunostained following a procedure that

involved sequential volume replacements (VRs) of reagents by passive pumping. Different numbers of

VRs were used for different reagents, depending on the reagent and the step in the procedure. Cells were

fixed in 4% paraformaldehyde (PFA) for 15 min (3 VRs), and then rinsed with PBS (5 VRs). At this

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stage, it was permissible to store the samples at 4°C for up to a week before staining. Samples were

permeabilized with 0.1% Triton X-100 for 30 min (3 VRs), and then rinsed with PBS (5 VRs). Cells

were blocked with 3% bovine serum albumin (BSA) containing 0.1% Tween-20 in PBS for 1 h (3 VRs).

After blocking, primary antibodies were added and incubated for 2.5 h at 37°C (3 VRs). Primary

antibodies were subsequently rinsed with PBS (5 VRs), and 10% goat serum in PBS was applied as

blocking buffer for 1 h at room temperature (3 VRs). Secondary antibodies were added and incubated

for 1 h at room temperature (3 VRs) while protected from light, and then rinsed with PBS (3 VRs).

Hoechst 33342 nuclear dye was added and incubated at room temperature for 10 min (3 VRs). Hoechst

was finally rinsed with PBS (5 VRs) prior to imaging. In coculture experiments, one representative

microchamber was selected for phalloidin staining. Texas Red-X phalloidin was added after Hoechst

staining and incubated for 30 min prior to final rinsing. Two immunostaining controls were included for

each experiment, one with no primary antibody, which controlled for background from the secondary

antibody, and one with an irrelevant primary antibody of the same species and type as the one tested,

which controlled for specificity. In all experiments both controls were performed and found to be

negative with negligible signal, and were omitted in further quantitative analyses (Figure S-5 and S-6).

Fluorescence Microscopy and Image Analysis

All fluorescent images for the µSCeNT assay were taken using a Nikon Eclipse Ti inverted

fluorescent microscope coupled to a Nikon DS-Qi1Mc CCD camera (Nikon Instruments Inc., Melville,

NY, USA) at a total magnification of 150x (10x objective, 1.5x intermediate magnification, 10x

eyepiece). Two images, one for RelA and one for Hoechst nuclear stain, were taken at five non-

overlapping locations per well of the microchamber to cover the center and four cardinal directions,

resulting in 34% coverage of the well area (denser grids were possible for higher or complete cell

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counts). In the CLL experiments only, a 20x objective was used, and 16 non-overlapping locations were

imaged for 62% coverage of the well area. For image processing and analysis, a custom ImageJ plugin

was developed and used in conjunction with an in-house database management system (Je’Xperiment or

JeX: http://jexperiment.wikidot.com/, screenshot in Figure S-7) to manage 2,280 raw images acquired

during this study. The plugin accepted the raw stained images as inputs and used them to generate a

binary cytoplasmic mask that isolated each cell in the viewfield, and separated the RelA stain into

nuclear and cytoplasmic regions based on the location of the nucleus. Mean nuclear and cytoplasmic

intensities were calculated by dividing the total fluorescence intensities in each region by their

respective areas. The ratio of the means, termed the intensity ratio IR, was then determined for each cell

in the population by dividing the means:

!

IR =I nI c

=In /An

Ic /Ac

(1)

where In is the total nuclear intensity, An is the nucleus area, Ic is the total cytoplasmic intensity, Ac is the

cytoplasm area,

!

I n is the mean nuclear intensity, and

!

I c is the mean cytoplasmic intensity. Because of

the definition of IR in Eq. 1, only cells with distinct nuclear and cytoplasmic regions were counted, i.e.,

cells were not counted if either An or Ic were zero and IR was not defined.

The typical workflow for image analysis is shown in Figure S-7. Once image acquisition is

completed, the JeX program is started, and a new experiment file is opened. Experimental setup and

conditions are recorded into an experimental layout (3 min), and images are uploaded and distributed

according to the layout (2 min, screenshot of interface in Figure S-7). The desired custom ImageJ plugin

(“Live/Dead – Find Maxima” for the viability assay or “Nuclear Intensity Analysis – p65” for µSCeNT

assay) is then selected and the calculation carried out automatically (10 min). The raw data is

automatically streamed into a .csv file, read into MS Excel, and processed manually to generate

distributions (15 min) that are later graphed as histograms in Plot for Mac (10 min).

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Statistical Analysis

Population means of single-cell IR values were obtained for all cell culture samples by combining IR

values for all five images within one microchamber, and averaging over the whole population. For TNF-

α dose response experiments, each TNF-α concentration was tested in triplicate (i.e., three separate

microchambers in the same device), and for bortezomib dose response experiments, each concentration

was tested in duplicate. For coculture experiments, coculture and monoculture conditions were tested in

quadruplicate. For CLL experiments, conditions were tested in triplicate. In each case, replicate

averages were calculated within individual experiments, and these replicate averages were statistically

analyzed for all experiments (minimum n = 3 independent experiments per condition). ANOVA was

performed for each set of experiments, followed by Tukey’s procedure for multiple comparisons, except

for CLL experiments where a Wilcoxon rank sum test was used. Hartigan’s Dip Test was used to

determine bimodality of distributions.5 Data was considered statistically significant if p < 0.05.

Micropipette Error Analysis

We used Eppendorf Research® adjustable-volume 0.5-10 µL micropipette for all microdevice

pipetting operations presented in this manuscript. We acquired systematic (Es) and random (Er) error

estimates from the Eppendorf technical manual for 1, 5, and 10 µL volumes (Table S-1, light grey), and

linearly interpolated error estimates for 2 and 8 µL volumes (Table S-1, dark grey and bold). We

calculated the total error ET for n = 25 dispenses (used in cell retention assay) as simply 25 times the

error for one dispense, for the worst-case scenario.

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Table S-1. Error analysis for Eppendorf Research 0.5-10 µL micropipette

Dispensed Volume

Systematic Error, Es

(Inaccuracy)

Random Error, Er

(Imprecision)

Total Error per Dispense, ET

Total Error, ET(n) (n = 25 Dispenses)

Volume Percent Volume Percent Volume Percent Volume Percent 1 µL 25 nL 2.5% 18 nL ≤ 1.8% 30.8 nL 3.1% 770 nL 3.1% 2 µL 37.5 nL 1.875% 23.5 nL ≤ 1.2% 44.3 nL 2.2% 1108 nL 2.2% 5 µL 75 nL 1.5% 40 nL ≤ 0.8% 85.0 nL 1.7% 2125 nL 1.7% 8 µL 90 nL 1.125% 40 nL ≤ 0.5% 98.5 nL 1.2% 2463 nL 1.2% 10 µL 100 nL 1.0% 40 nL ≤ 0.4% 107.7 nL 1.1% 2693 nL 1.1%

References 1. Duffy D, McDonald J, Schueller O, Whitesides G. Rapid prototyping of microfluidic systems in

poly(dimethylsiloxane). Analytical Chemistry. 1998;70(23):4974-4984.

2. Xia Y, Whitesides G. Soft lithography. Annual Review of Materials Science. 1998;28:153-184. 3. Regehr KJ, Domenech M, Koepsel JT, et al. Biological implications of polydimethylsiloxane-based

microfluidic cell culture. Lab on a Chip. 2009;9(15):2132-2139. 4. Berthier E, Beebe DJ. Flow rate analysis of a surface tension driven passive micropump. Lab on a

Chip. 2007;7:1475-1478. 5. Hartigan J, Hartigan P. The Dip Test Of Unimodality. Annals of Statistics. 1985;13:70-84.

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Supplemental Figures

Figure S-1. Device fabrication bonding sequence. PDMS layers were removed from their respective SU-8 masters. Top side of channel layer (red arrow) and glass were first plasma-treated, then bonded in step 5. Then the top of the device from step 5 and the bottom of the port layer (side without red arrow) were plasma-treated, and bonded in step 6.

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Figure S-2. Numerical simulation results for (A) velocity magnitude through the channel midplanes (isometric, top, and side views shown), and (B) wall shear stress at the well bottom. The Fluid Dynamics module within COMSOL Multiphysics package (version 3.4) was used, and steady state analysis of the incompressible Navier-Stokes equation was performed. Model parameters included inlet flow rate of 1 µL/s (velocity of 1 cm/s), zero outlet pressure at the outlet, no slip boundary conditions on all other surfaces, fluid density of 1000 kg/m3, and fluid viscosity of 0.72 x 10-3 kg/m s2. Lagrangian-P2P1

elements were used in the mesh, with 16,683 elements in total.

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Figure S-3. Cell retention characterization. (A-I) Reconstructed phase contrast images of microchambers after various volume replacements (VRs), for 2-h and 16-h culturing times. (A-C) For culturing time of 2 h, cell population was noticeably depleted after 10 VRs. (D-F) For culturing time of 16 h, cell population was not disrupted even after 20 VRs (25 VRs tested). Scalebar = 1 mm. (G-I) Cells from inlet (red) were carried by flow through the central well to the outlet during VRs. Cells already in the well (blue) were not disrupted, but inlet cells were counted during the test, resulting in > 100% cell fraction counted after 1 VR. (J) Fraction of retained cells as a function of number of VRs. Error bars represent SD for n = 5 for all culturing times.

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Figure S-4. RPMI8226 cell population distributions for 10 ng/mL TNF-α timecourse of 20 min, 1 h, 2 h, and 4 h (red histogram = no treatment control; blue histogram = TNF-α treatment; n = 1).

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Figure S-5. Representative images of antibody controls for RelA experiments. Both RelA and PSA antibodies were polyclonal rabbit IgG antibodies. AlexaFluor 488 was of goat anti-rabbit. Fluorescence signal from PSA was negligible. Scalebar = 100 µm.

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Figure S-6. Representative images of antibody controls for STAT3 experiments. Both STAT3 and cadherin-5 antibodies were monoclonal mouse IgG antibodies. AlexaFluor 488 was of goat anti-mouse. Fluorescence signal from cadherin-5 was negligible. Scalebar = 100 µm.

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Figure S-7. Workflow for image analysis. Raw data analysis for a single 3 x 4 array (12 microchambers) used for a typical µSCeNT assay, with 120 total images acquired, takes roughly 5 minutes to set up and 10 minutes of computation time. Post-processing of raw data into histograms takes an additional 25 minutes manually using MS Excel and Plot for Mac. Screenshot shows JeX interface and a typical experimental layout for a 3 x 4 array with treatment conditions labeled for archiving.