Interfacial geometry dictates cancer cell tumorigenicity

Supplementary Information (Junmin Lee1, 2014)

Interfacial geometry dictates cancer cell tumorigenicity

Junmin Lee1, Amr A. Abdeen1, Kathryn L. Wycislo2, Timothy M. Fan3, Kristopher A. Kilian1,*

1Department of Materials Science and Engineering, University of Illinois at Urbana-Champaign,

Urbana, IL 61801 2Department of Pathobiology, University of Illinois at Urbana-Champaign,
Urbana, IL 61801 3Department of Veterinary Clinical Medicine, University of Illinois at Urbana-
Champaign, Urbana, IL 61801 *Correspondence to: kakilian@illinois.edu
Figure S1. Tunable polyacrylamide hydrogel fabrication and conjugation. a, Proteins are patterned
on the surface of hydrazine activated polyacrylamide gels using PDMS stamps. b, Representative
immunofluorescence microscopy images of murine B16 cells cultured on polyacrylamide hydrogels
with or without protein conjugation. Staining for cell nuclei (blue). Scale bar: 100 m
Figure S2. Cancer stem cell marker expression of B16 cells is influenced by culture time and
geometry. a, Expression of cancer stem cell marker (ABCB5) depends on culture time for different
combinations of matrix stiffness and various geometries (1:1 and 1:8 aspect ratio shapes; 5,000
m2). (N=3, * P<0.05, Fishers exact test compared to glass). b, Representative immunofluorescence
microscopy images of ABCB5 expression for B16F10 cells on circular patterns (5,000 m2) or
nonpatterned surfaces with culture days. c, Quantitation of ABCB5 marker expression for B16F10
cells cultured for 5 days on different matrix elasticity and shapes (5,000 m2). (* P<0.05, Fishers
exact test compared to the glass control). (N=3, * P<0.05, Fishers exact test compared to glass).
Error bars represent standard deviation. Scale bar: 50 m.

Figure S3. Micropatterning tumor cells reveals an optimal size and curvature that guides expression
of cancer stem cell and pluripotency markers in B16F0 and B16F10 cells. a, Expression of cancer
stem cell (CD133) and pluripotency (Oct4 and Nanog) markers for B16F0 and B16F10 cells cultured
for 5 days on different matrix elasticity and pattern sizes (3,000-100,000 m2 and NP). (N=3, *
P<0.05, Fishers exact test compared to glass). b, Representative immunofluorescence images and
expression of the cancer stem cell marker ABCB5 for B16F10 cells cultured for 5 days on different
size circular patterns (3,000-100,000 m2) or non-patterned cells on different stiffness gels (1-100
kPa) and glass. (N=3, * P<0.05, Fishers exact test compared to glass). c, Representative
immunofluorescence microscopy images of B16F0 cells (5 days) on non-patterned surfaces (1-100
kPa) and glass. No significant difference was observed between the non-patterned and glass
conditions. (* P<0.05, Fishers exact test compared to the glass control). Error bars represent
standard deviation. Scale bars: 50 m.
Figure S4. Analysis of cell and nuclear shape, proliferation characteristics and integrin expression
levels show marked differences in perimeter cells consistent with enhanced invasiveness. a,
Immunofluorescence heatmaps of B16F10 cells cultured in a panel of 2D shapes for > 10 patterns
shows a semi-quantitative decrease in proliferation (BrdU) dependent on culture time; no geometric
effect on focal adhesion (Paxillin) expression and enhanced expression of 51 integrins on B16F0
cells at the perimeter of geometric features. Scale bar: 50 m. b, Nuclear shape index (NSI) and
alignment of B16F0 and B16F10 cells (N=421 each) cultured on glass or spiral patterned substrates.
We quantified nuclear elongation by calculating the NSI according to the formula, NSI = 4A/P2,
where A is projected nuclear area and P is nucleus perimeter. c, A flow chart to describe how NSI
data is filtered by nuclear area.