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SUPPLEMENTS
Antihypoxic Effects of Myo-Inositol Trispyrophosphate on Vasculature and
Therapy of Colorectal Liver Metastasis
Perparim Limani1, Michael Linecker1, Philipp Kron1, Christoph Tschuor1, Andrea Schlegel1,
Ekaterina Kachaylo1, Udo Ungethuem1, Jae Hwi Jang1, Stavroula Georgiopoulou2, Claude
Nicolau3, Jean-Marie Lehn4, Rolf Graf1, Bostjan Humar1*, Pierre-Alain Clavien1*
*shared senior authorship
- Supplementary Methods
- Supplementary Figure Legends
Supplementary Methods
Primary antibodies for immunohistochemistry
antigen
-catenin
Caspase 3 cleaved
Cd3
Cd31
Cd34
Cd45-B220
Cdh5
Cytochrome C
F4/80
Hif1a
Hif2a
Ki67
Notch1
Piminidazole
adduct
pHistone 3
Vimentin
company
Novocastra Laboratories Ltd, Newcastle, UK
Cell Signaling, Boston, MA
Diagnostic Biosystem, Pleasanton, CA
Abcam, Cambridge, MA
LifeSpan BioSciences, Seattle, WA
BD Biosystems, Franklin Lakes, NJ
Abcam, Cambridge, MA
Cell Signaling, Boston, MA
BMA Biomedicals AG, Augst, Switzerland
LifeSpan BioSciences, Seattle, WA
Abcam, Cambridge, MA
Cell Marque Lifescreen Ltd., Rocklin, CA
Cell Signaling, Boston, MA
Hypoxyprobe Plus Kit, Hypoxyprobe™, NPI Inc.,
Burlington, MA
Abcam, Cambridge, MA
Cell Signaling, Boston, MA
order number
NCL-B-CAT
9664
RMAB005
ab28364
LS-C180574
553084
ab33168
11940
T-2006
LS-B495
ab199
CMC27531021
36085
HP2-1000Kit
ab92628
5741
Enzyme-linked immunosorbent assays (ELISA)
ELISAs for Vegf (DY493), osteopontin (MOST00), Cxcl12 (MCX120), Scf (MCK00) and Plgf
(MP200) were from R&D Systems (Minneapolis, USA), and Hif1a/Hif2a ELISAs (E90798Mu/
E93466Mu) from NewLife BioChemEX (Bethesda, MD). Triplicate
measurements were
performed.
Western Blot
Antibodies used were against lamin B1 (sc-377001, Santa Cruz Biotechnology, Dallas, TX) and βtubulin (2128S, Cell Signaling).
Taqman gene expression assays
Gene
Arg1
Cd40
Cdh1
Cox4i1
Cpt1a
Drp1
Emr1
Fis1
Hadha
Hadhb
Hif1a
Hif2a
Il6
Ldha
Mfn2
Mgl1
Mrc1
Nos2a
Ocln
Opa1
Slc2a
Snai1
Src
Tnfa
Twist1
Vegfa
Vim
Taqman assay no.
Mm00475988_m1
Mm00441891_m1
Mm01247357_m1
Mm01250094_m1
Mm01231183_m1
Mm01342903_m1
Mm00802530_m1
Mm00481580_m1
Mm00805228_m1
Mm01217745_m1
Mm00468869_m1
Mm01236112_m1
Mm00446190_m1
Mm01612132_g1
Mm00500120_m1
Mm00546124_m1
Mm00443781_m1
Mm00440485_m1
Mm00500912_m1
Mm01349707_g1
Mm00441480_m1
Mm00441533_g1
Mm00436785_m1
Mm00443258_m1
Mm00442036_m1
Mm01281449_m1
Mm01333430_m1
All assays were from PE Applied Biosystems (Grand Island, NY). Ct values were normalized to
18SrRNA (TaqMan control reagents, PE Applied Biosystems) to represent relative fold induction
of mRNA expression ± 95% confidence intervals. Tumor values were not normalized to unaffected
liver, because tumor material originates from the intestine with a different basal expression pattern
than liver.
Supplementary Figure Legends
Supplementary Figure 1. ITPP increases O2 dissociation from hemoglobin. A, O2 dissociation
curve. ITPP shifts the dissociation curve to the right, reflecting the requirement of an increased
pO2 to saturate hemoglobin with oxygen. Comparing the percentage in right shift following
different administration routes (p.o, i.p., i.v.), intravenous injection was most effective. However, a
drop in the right shift on day 2 by about 50% indicated weakening of the ITPP effects over time.
Data are presented as means (n=3/group); error bars refer to ±95%CI. B, Immunoblot showing
separation of cytosolic and nuclear fractions from tumor lysates. Subcellular fractionation was
used to quantify by ELISA cytoplasmic and nuclear levels of Hifs.
Supplementary Figure 2. Impact of ITPP on tumor burden, hypoxia and survival in the CT26/BALB model. A, Tumor response towards ITPP. Analysis included tumor development
(representative MRI scans before and after treatment, ctrl=saline control), proliferation (Ki67, pH3,
magnification 10x), apoptosis (caspase 3), necrosis inflammation (serum Hmgb1), and tumor
volume/number (pathological and MRI assessment) immediately after treatment. B, Impact on
tumoral Hif expression and hypoxia at day 17. Nuclear and cytoplasmic Hif levels (ELISA) and
quantified pimonidazole staining for hypoxic areas immediately after treatment is shown. C,
Kaplan-Meier survival analysis. Day 15 represents treatment end (n=5/group).
Supplementary Figure 3. Impact of ITPP on metabolic and immune parameters in the CT26/BALB model at day 17. A, Impact on energy metabolism. Graphs show normalized gene
expression in control tumors (light gray bars) and ITPP-treated tumors (dark grey bars). The
upregulation of Cox4i1 to the expense of Slc2a and Ldha was less pronounced than in the MC-38/B6
model, but still is consistent with a switch from glycolysis towards oxidative phosphorylation
following ITPP treatment. In agreement, the genes controlling mitochondrial dynamics are shifted
from
mitochondrial
fission
(Fis1,
Dsp1)
towards
fusion
(Mfn2,
Opa
and -oxidation genes (Cpt1a, Hadha, Hadhb) are upregulated in ITPP tumors compared to
controls. Images (top right) show immunostaining for Cox4i1 protein in control (ctrl) and ITPPtreated tumors (magnification 40x). B, Impact on immunity. Graphs to the left show normalized
expression of inflammatory (upper graph; Tnfa, Il6, and Emr1 encoding the macrophage marker
F4/80) and macrophage polarization markers (lower graph; M1 markers: Cd40, Nos2a; M2 markers:
Arg1, Mrc1, Mgl1). Images to the right (magnification 20x) show increased tumor infiltration of
ITPP-treated tumors by macrophages (F4/80), B-cells (CD45B220), and NK cells (CD3).
Supplementary Figure 4. Impact of ITPP on malignant tumor phenotype in the CT-26/BALB
model at day 17. A, Immunohistochemistry for vimentin. Upper panels show control tumors with
invasive spread into adjacent tissue (magnification 5x, 10x, 40x). Lower panels (magnification
100x) show fibroblastoid and spheroid morphology of untreated and treated cancer cells,
respectively. B, Left: normalized gene expression of EMT inducers (Twist, Snai1, Src), a
mesenchymal marker (Vim) and epithelial markers (Ocln, Cdh1) in control and ITPP tumors. Right:
circulating levels of tumor-independent, systemic promoters of malignancy (Scf, osteopontin,
Cxcl12) in control and ITPP-treated tumors.
Supplementary Figure 5. Impact of oxygen levels and ITPP on cancer cells in vitro. One day
after seeding MC-38 and CT-26 cells at hypoxia, cells were exposed or not to normoxia (A-C), or
exposed to ITPP or saline (D-F) for 24h. A, Dependency of in vitro proliferation and
mitochondrial activity on oxygen levels. Cells seeded at different densities (A=0.5x106, B=1x106,
C=5x106) were exposed (5%O2) or not (1%O2) to normoxia. Cell numbers (growth) and
mitochondrial activity (MTT assay) were assessed. The right graph shows the same set of
experiments for CT-26 cells. B, Effect of normoxia on glucose consumption and lactate
production. Glucose and lactate levels (glycolytic activity) were determined in supernatants from
the experiments described under A (for the highest density only). C, Impact of normoxia on
invasiveness. Single cell suspensions from hypoxic cultures seeded at 70% (cell density)
and exposed or not to normoxia for 24h were assayed with Boyden chambers. D, Effect of ITPP
on proliferation and mitochondrial activity. The same set of experiments described under A was
performed for hypoxic MC-38 and CT-26 cells treated (+) or not (-) for 24h with ITPP (0.5mg/ml).
E, Effect of ITPP on glucose consumption and lactate production. Supernatants from the
experiments described under D were analyzed for glucose and lactate. All cell densities (A, B, C)
were considered for the comparison between saline (-) and ITPP (+) to reveal potential ITPP
effects. The patterned bars indicate glucose and lactate levels in fresh culture media. F, Effect of
ITPP on in vitro invasiveness. Following a 24h exposure to saline (-) or ITPP (+), cells seeded at
different densities were analyzed by Boyden chambers in analogy to C. Bars represent means and
error bars 95% CI from triplicate measurements. Two independent replicates were performed.
Supplementary Figure 6. Impact of ITPP on tumor vasculature in the MC-38/B6 model at day
17. Immunohistochemistry for Cd31, Cd34, Notch1 and VE-cadherin on liver samples
immediately after treatment is shown. To better visualize vascular expression patterns, colors
unrelated to the peroxidase-based staining were faded equally for all images using Adobe
Photoshop. For Notch1/VE-cadherin, note the positive staining (Notch1 20x, upper corner) and
(VE-cadherin, control, upper corner) in normal liver sinusoids, and the increases in both expression
and the density of positive vessels within ITPP tumors.
Supplementary Figure 7. Impact of ITPP on tumor vasculature in the CT-26/BALB model at day
17. Immunohistochemistry for Cd31, Cd34, Notch1 and VE-cadherin on liver samples immediately
after treatment is shown. Fading of background color was applied for better visualization of
staining patterns. For VE-cadherin/Notch1, note the increases in both expression and the density of
positive vessels within ITPP tumors.
Supplementary Figure 8. Long-term impact of ITPP on hypoxic response and tumor vasculature in
the
CT-26/BALB
model.
A,
Long-term
impact
on
the
hypoxic
response.
Tumoral
gene expression and serum quantifications are shown for control (-) and ITPP-treated mice (+)
immediately (day 17) and one month (day 45) after treatment (see bottom for labels). Note the
persistence of treatment effects for all parameters investigated. B, Long-term impact on tumor
vasculature. Immunohistochemistry for Cd31, Cd34, Notch1 and VE-cadherin in control and ITPPtreated tumors at day 45. Fading of background color was applied for better visualization of staining
patterns. Please compare with Supplementary Fig. 7B to note the similar expression patterns
immediately and one month after ITPP treatment.