Supplemental material:
Stable tumor vessel normalization with pO2 increase and endothelial
PTEN activation by inositol tris pyrophosphate brings novel tumor
treatment
Claudine Kieda et al.
Journal of Molecular Medicine 2013
Supplemental materials and methods
Vectors
were produced using pBMN-Luc-I-GFP plasmid (kindly gifted by Dr. Magnus Essand, Uppsala,
Sweden) and PT67 packaging cell line (Clontech) stably expressing the gag, pol, and env genes.
Additionally, the pM13 plasmid providing gag and pol gene (kindly gifted by Dr. Christine Brostjan,
Vienna, Austria) was used to increase the production efficiency. The packaging cells were cultured in
DMEM HG medium (PAA Laboratories) supplemented with 10% FCS, penicillin (100 U/mL) and
streptomycin (100 μg/mL), and co-transfected with pBMN-Luc-I-EGFP and pM13 plasmids using
SuperFect reagent (Qiagen) according to manufacturer instruction. After transfection the cells were
cultured at 32 ºC for 48 h. Then media containing the retroviral vectors were collected, mixed with
complete RPMI medium in the v/v ratio of 1:1 and used for transduction of B16F10 cells.
Transduction efficiency, estimated three days later, by fluorescence microscopy, for EGFP was about
5%. Passaged EGFP positive colonies, three times sorted using MoFlo Flow Cytometer (Dako
Cytomation) provided the B16F10LucGFP cell line of more than 99% purity. Cell transduction was
found stable for Luciferase expression as opposed to EGFP.
Experimental metastasis assay
After intravenous injection of B16LucGFP (105 cells) in the tail vein, mice were treated by 1,5 g/kg
ITPP injected intraperitoneally every 5 days. Treatment was initiated at day 5 after tumor cells
inoculation. 27 days later, mice were euthanized and lungs collected. Macroscopic lung foci were
counted and luciferase was determined by chemoluminescence assay (Promega) in order to quantify
the presence of melanoma cells in tissues.
Magnetic resonance imaging
MR experiments on mice were performed on 9.4 T horizontal magnet dedicated to small animal
(94/21 USR Bruker Biospec, Wissembourg, France), equipped with a 950mT/m gradient set. The
anesthetized mice were placed in a linear homogeneous coil (inner diameter: 35 mm). The body
temperature (36°C) was maintained constant by a warm water circulation heating bed.
Morphological pulse sequence: A first preparatory anatomical series of coronal images (21 slices) was
performed to localize the tumor. The imaging sequence was a Multi Slices Multi Echoes (MSME) on
the leg with the tumor (FOV=1.5 x 1.5 cm, matrix size = 128 x 128, slice thickness = 1 mm, TE = 14 ms,
TR = 4 s) leading to an in plane spatial resolution of 195 x 195 µm.
MRA-TOF sequence: Measurement of the tumor vascularization was performed by Magnetic
Resonance Angiography (MRA) Time of Flight (TOF) pulse sequence both in the axial and coronal
plane with the following parameters : (TR/TE = 30ms/5ms), matrix size = 256*256, slice thickness :
1mm leading to 21 images with an in plane resolution of 98*98 micrometers.
Supplemental figures:
Figure S1: Luciferase activity is not modified in B16F10LucGFP cells in hypoxia (1% O2) compared to
normoxia (21% O2). No significant difference was observed indicating that reporter gene expression
is not regulated by oxygen pressure.
Figure
S2:
Oxygen supply by ITPP inhibits lung metastasis and reverses hypoxia-induced gene cascade in
experimental melanoma model.
The subcutaneous tumor model data were confirmed in metastatic lung nodules induced by
intravenous injection of melanoma cells. Protein and mRNA levels were assessed in lung lysates of
healthy, melanoma bearing and ITPP treated mice. Compared to the melanoma bearing lungs, ITPP
reverses the expression of the all studied markers toward the basal expression in healthy mice
(A) Quantification of lung metastasis by Luciferase assay showing drastically reduced metastases.
(B) HIF-1α protein expression;
(C) HIF-dependent VEGF protein expression;
(D) Tie-2 protein expression significantly reduced in hypoxic lungs, was re-induced by ITPP- treatment
(E) HO-1 protein level.
(F) LOX mRNA content, indicating the reduction of the invasive process by ITPP.