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Supplementary Figure Legends
Supplementary Figure S1. Characterization of cancer spheroids. A, Bright field
images of H1650 cancer cell spheroids (1,000 cells/well) were monitored at the
indicated time-points. B, Growth curve of H1650 cancer cells in 2D and 3D culture
system. On indicated days, 2D cells and 3D spheroids were dissociated to single cells
by the cell detachment buffer and cell number was counted. C, Correlation between
cell number seeded and spheroid area. The spheroid area was calculated using Nikon
microscopy program. Correlation coefficient (r) was 0.98. D, Expression levels of the
indicated proteins were compared between 2D and 3D culture system by Western blot
analysis at the indicated time points. E, qRT-PCR analysis of hypoxia-induced gene
expression. Relative gene expression level, including HIF-1 and VEGF, was
normalized by that of 2D monolayer cells. F, Hypoxia status in cancer spheroids.
Spheroids, cultured for 5 days in 3D plate, were incubated with LOX-1, the 3D
permeable hypoxia probe, for 24 h (left). LOX-1 staining was also conducted on 2D
monolayer cells as a control (right). Red fluorescence was monitored with EMCCD.
G, Luminal cancer cell survival in spheroids. Luminal cell survival was analyzed by
DAPI staining in cancer spheroid. The upper and lower panels are non-sectioned and
sectioned images, respectively (10 m).
Supplementary Figure S2. Characteristics of spheroids based on morphologic
classification. A, Cell growth comparison of spheroid-type representative cancer cells
in 2D and 3D culture. 2D and 3D cultured cells were disrupted by cell detachment
buffer and cell number was counted at time interval. B and C, Hypoxia status in 3D
(B) and 2D (C) culture system was observed with LOX-1 staining. Hypoxia status in
3D and 2D culture system was monitored on day 6 and 2, respectively, after cell
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seeding. Fluorescence signal was monitored by fluorescence microscopy. D, Bright
field images of colon cancer cell line spheroids. Morphological type-representative
cells (LoVo: R type, SW480: A type) were seeded on the 3D plate and then bright
field images were monitored by microscopy at 6 days. E and F, Hypoxia status in
colon cancer spheroids. LoVo and SW480 cells were cultured in 3D culture plate for
5 days. qRT-PCR analysis of hypoxia-induced gene expression, including HIF1
) and VEGF (F) relative to GAPDH level. Relative gene expression of spheroid
was normalized to that of 2D cultured cells.
Supplementary Figure S3. Validation of spheroid morphology-dependent
characteristics. Nine different cancer cell lines (lung cancer: H441, H23, Calu-6;
colon cancer: LoVo, SW480, Colo205; GBM: LN18, U251MG, U251E4) were used
to validate spheroid morphology-dependent characteristics. A and B, Cell growth was
determined by cell counting in 2D (A) and 3D (B) culture system. C, Using 2D and
3D culture system, hypoxia status and drug sensitivity of 5-FU were determined by
the LOX-1 staining and LDH cell viability assay, respectively. To quantify hypoxia,
mean fluorescence intensity was measured by fluorescence microscopy. P values of
hypoxia and drug sensitivity were determined by one-way and two-way (spheroid
type x culture system) ANOVA, respectively. *P<0.01 (one-way ANOVA),
**P<0.01 (two-way ANOVA)
Supplementary Figure S4. Validation of PLS-DA model. A, Selection of
discriminatory genes. Among 22,285 genes with CNV, mutation, and mRNA
expression data in 70 cell lines (1st Venn diagram), 2,202 discriminatory genes were
selected by MPLS-DA (P<0.05; 2nd Venn diagram). Of them, 560 showing subtype-
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specific molecular patterns were further identified by individual PLS-DAs (3rd Venn
diagram). B-D, mRNA expression, CNV and mutation profiles. Columns include 70
tested cancer cells (R, M, A, N for round, mass, aggregate and none spheroid types,
respectively, across the top) and rows include tested genes for each data types (926,
65, and 1,333 genes for mRNA (B), CNV (C) and mutation (D) profiles, respectively,
down the side). Colors indicate increase (red) and decrease (green) in the levels of
molecular signatures relative to the median levels. Dendrograms were generated using
2-D hierarchical clustering (complete linkage and Euclidean distance as a similarity
measure). E and F, Accuracy (E) and misclassification error rate (F) during leaveone-out-cross-validations (LOOCVs) of the PLS-DA model. The model was
constructed with 560 subtype-specific discriminatory genes in the prediction of
spheroid subtypes. G, Block contribution of MPLS-DA.
Supplementary Figure S5. Network models describing cellular processes
associated with spheroid subtypes. A, Network associated with round type-specific
genes. B and C, Network models denoting cellular processes overrepresented by
mass type-specific genes (B) and aggregate type-specific genes (C) are also provided.
In the networks, the nodes with the same GOBPs or KEGG pathways were grouped
together. The asterisk symbols were attached to the labels of the GOBPs or KEGG
pathways overrepresented by round (186), mass (158) and aggregate (152) typespecific genes. Node colors represent VIPs of mRNA expression (center) and CNVs
(boundary). Node labels represent the presence of mutations (bold and purple).
Underline text represents cancer drug targets reported in NCI chemical index. Blue
edges represent protein-protein interactions selected from HotNet and obtained from
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the databases with experimental evidence (HitPredict, STRING, and GeneGO). Purple
lines denote signaling pathways obtained from KEGG pathway database.
Supplementary Figure S6. Drug penetration test in spheroids using bimolecular
fluorescence complementation (BiFC). A, Significance of the JAK-STAT signaling
in the round type spheroids. Bright field images of the round type spheroids of H226
and H441 cells are shown in the absence (con) or presence of AG490 (JAK2 inhibitor,
150 M) for 3 days. B, 293T cells expressing either or both of VN-FKBP12 and VCFRB were culture for 3 days to form spheroids. The proteins were then extracted from
the spheroids and the expression levels of VN- FKBP12 and VC-FRB were
determined by immunoblotting. C, 293T cells expressing each BiFC vector for
FKBP12 and FRB in 2D system were seeded on 3D plate (5,000 cells/well). After 3
days incubation, the spheroids were incubated with or without rapamycin (1 M) and
the BiFC signal was monitored with fluorescence microscopy. D, 293T cells were
transfected with BiFC vectors. Using these cells, spheroids were formed and then
treated with or without AG490. After 2 days incubation, spheroids were treated with
rapamycin. S6K phosphorylation was monitored by immunoblotting.
Supplementary Figure S7. Molecular signature of GBM patient-derived round
type spheroid. A, Selection of the discriminatory genes specific to the round type
spheroids. For the 186 round type-specific genes, the arrays include 167 genes (164
and 150 genes on mRNA expression and CNV arrays, respectively; 1st Venn diagram).
Among them, 36 (each 20 identified by mRNA expression and CNV) were selected as
the round type-specific discriminatory genes in GBM patients-derived cells (2nd Venn
diagram). B, mRNA expression (left panel) and CNV (right panel) profiles. Columns
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represent GBM patient-derived spheroids, and rows represent differential levels of
molecular signatures. Colors indicate increase (red) and decrease (green) in the levels
of molecular signatures relative to the median levels. C, The genes overlapped
between the JAK-STAT signature from cell lines and the 36 genes from patientderived R type spheroids (a) and their interactors (b). The significance (P value from
the hypergeometric test) of the overlaps is indicated. D, Integration of the 36 genes
identified from patient-derived R type spheroids into JAK-STAT pathway in the R
type-specific network (Fig. 2C). Round and diamond nodes are the genes in the R
type-specific network and the 36 genes, respectively. Node colors represent the
increase (red) and decrease (green) in mRNA or CNV levels, as well as the decrease
in CNV levels, but the increase in mRNA levels (blue). Gray edges represent proteinprotein interactions, and purple lines denote activations (arrows) and inhibitions
(inhibition symbols) obtained from KEGG pathway database.
Supplementary Figure S8. Xenograft model to validate JAK-STAT pathway for
drug sensitivity. A, H1650 xenografted mouse weights were measured every 2 days.
B, After sacrificing mice, two representative tumors isolated from each group were
shown.
Supplementary Table S1. Summary of cancer cell lines and their spheroid
morphology. Spheroid type, tissue origin, and genomic data of 100 different cancer
cell lines were represented.
Supplementary Table S2. Percent variance explained by PLS model of 560 type
specific discriminatory genes. We determined the number of latent variables (LVs)
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to 6 so that the PLS model captures more than 80% of variance of Y-block; LV1-10,
1st to 10th PLS LVs we tested; X1-, X2- and X3-blocks, mRNA expression data,
mutation data and CNV data, respectively; Y-block, data matrix containing the
subtypes of the 70 cancer cells. ‘This LV’ includes a percent variance captured by an
LV in X1-, X2-, X3- or Y-blocks which reflects the amount of the information in X1-,
X2-, and X3-block used to explain the amount of the separation explained by the LV
among the binary values in Y-block. ‘Total’ includes cumulative sum of percent
variance captured by LVs from LV1 to the specific LVs we tested.
Supplementary Movie S1. Spheroid formation of cancer cells. A-C, H1650 (A),
H460 (B) and A549 cells (C) (1,000 cells/well) were seeded on the 3D culture plate.
The cells were imaged in 2 h after seeding on live cell imager equipped with x10
objective lens. Images were taken every 10 min for 36 h, and the movie is played 15
frames per second.
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