Supplemental Figure legends and tables
Supplemental Figure 1:
(A) Representative FACS analysis plots: 4T1-shNS and 4T1-shCAIX cells were cultured
as TSs for 1 week in normoxia or hypoxia. TSs were trypsinised to give a single cell
suspension and stained with Dapi, CD44, CD24 antibodies for FACS analysis. For each
experiment, staining controls were stained with Dapi, CD44 or CD24 antibodies alone
and analysis quadrants set accordingly. The % of CD44+CD24-/low cells in the upper left
quadrant was recorded. Representative analysis plots are shown.
(B) FACS cell collection: 4T1 cells were cultured as TSs for 1 week. TSs were
trypsinised to give a single cell suspension and stained with Dapi, CD44, CD24
antibodies. For each experiment, staining controls for Dapi alone, CD44 alone, CD24
alone were used to set collection gates, which were used for subsequent experimental
population gating. A more conservative gating strategy was used for FACS collection to
ensure sample purity. A representative image is shown.
(C) 4T1-shNS and 4T1-shCAIX(A) cells were seeded for sphere limited dilution
analysis (SLDA) and cultured in normoxia or hypoxia for 7 days. The presence or
absence of tumorspheres was scored at each cell dilution. The percentage of wells
without a TS was calculated and plotted as a function of the number of cells plated. 6
wells/cell dilution were analyzed in each condition. These data were used to derive the
number of cells required for TS formation reported in Figure 1. (D) 4T1-shNS and 4T1shCAIX(B) cells were seeded for sphere limited dilution analysis (SLDA) and cultured in
normoxia or hypoxia for 7 days. The number of cells required to initiate TS growth was
assessed. Mean and SEM of three independent experiments is shown. (E) 4T1-shNS
(repeated from Fig.2A) and 4T1-shCAIX(B) cells were cultured as TSs in normoxia (N)
or hypoxia (H) and lysates analyzed by Western blot. (F) Parental 4T1 cells were
cultured as TSs in hypoxia, with or without CAI017 for 7 days and cell lysates analyzed
by Western blot. (G) 4T1-shNS, 4T1-shCAIX(A) and 4T1-shCAIX(B) cells were cultured
as TSs in normoxia (N) or hypoxia (H) for 7 days and RNA extracted. mTOR, Raptor
and 4EBP1 mRNA levels were assessed by QPCR, relative to actin expression. (H)
4T1-shNS and 4T1-shCAIX(A) cells were cultured as TSs in normoxia (N) or hypoxia
(H) for 7 days, lysed directly in 3X sample buffer and lysates analyzed by Western blot.
Supplemental Figure 2:
(A) Parental 4T1 cells were cultured as TSs in hypoxia, and viable CD44+CD24-/low or
control cells collected by FACS, then cultured in hypoxia and immunostained for CAIX,
E-cadherin, smooth muscle actin or Snail. Scale bar = 50µm.
(B) 4T1-shNS and 4T1-shCAIX(B) cells were cultured as TSs in hypoxia for 7 days. TSs
were collected and subjected to CD44, CD24 FACS. Viable CD44+CD24-/low or CD44CD24+ cells collected by FACS (see supplemental Figure 1B for representative FACS
collection plot) and cultured in hypoxia, fixed and immunostained (scale bar = 50µm).
(C) Parental 4T1 cells were cultured under constant rotation as spinner cultures in
normoxia or hypoxia, frozen, sectioned and subjected to double immunostaining to
assess expression of CD133 and CAIX, E-cadherin and CAIX, smooth muscle actin and
CAIX, Snail and CAIX, 40x magnification.
(D) MDA-MB-231 LM2-4Luc+ cell cultures treated ± U104 in Figure 2D were fixed and
TUNEL-labeling was performed to assess cell death. Representative images of TUNELpositive cells (arrows) are shown. Scale bar = 100 μm.
Supplemental Figure 3:
IVIS images from untreated mice injected with 10 EpCAM+ or EpCAM- MDA-MB-231
LM2-4Luc+ primary tumor cells are shown.
Supplemental Table 1.
4T1-shNS and 4T1-shCAIX cells were cultured as TSs in hypoxia, disaggregated using
trypsin and cells stained for CD44, CD24 and collected by FACS (see Supplemental
Figure 1B for representative FACS collection plots). Viable CD44+CD24-/low or CD44CD24+ control cells were collected and seeded for sphere limited dilution analysis in
hypoxia for 7 days and TS-forming efficiency was recorded.
Minimum cell # required for positive TS growth
N=1 expt
N=2 expt
N=3 expt
N=4 expt
shNS CD44+CD24-/low
124
7
7
7
shCAIX CD44+CD24-
No TS
No TS
34
46
shNS CD44-CD24+
No TS
32
218
761
shCAIX CD44-CD24+
No TS
No TS
421
101
/low
Supplemental Table 2.
Table shows a selection of gene targets assessed by QPCR displaying no significant
changes across all cell populations.
Gene name
Rac1
Fold
change
mean
SEM
shNS CD44CD24+
1
0
shNS
CD44+CD24-/low
0.82
0.04
shCAIX
CD44+CD24-/low
1.15
0.16
Tmem132a
mean
SEM
1
0
1.04
0.02
1.08
0.46
Moesin
mean
SEM
1
0
1.01
0.08
1.02
0.04
Beta 1 Integrin
mean
SEM
1
0
1.03
0.20
1.00
0.10
Supplemental Table 3.
Tumor initiating capacity of limiting dilutions of EpCAM+ or EpCAM- cells from untreated
MDA-MB-231 LM2-4Luc+ orthotopic primary tumors.
Cell Type
EpCAM+
No. of cells injected
100 cells
10 cells
Total
EpCAM-
Tumors formed
4/12
1/12
3/14
0/14
7/26
1/26
Supplemental Table 4.
QPCR primer sequences used in Supplemental Figure 1F:
Raptor
F: CTGTTCACATCCTGCCTCAC
R: TCAAATCCAGTGTGACTCCAG
4E-BP1
F: GGGGACTACAGCACCACTC
R: CTC ATCGCTGGTAGGGCTA
mTOR
F: TTCTGGGGTGTTGGAATACG
R: GGACACAAACAAGGAAGACCC
Actin
F: AAGGCCAACCGTGAAAAGAT
R: GTGGTACGACCAGAGGCATAC