Stable tumor vessel normalization with pO₂ increase and endothelial PTEN activation by inositol trispyrophosphate brings novel tumor treatment

Abstract

Tumor hypoxia is a characteristic of cancer cell growth and invasion, promoting angiogenesis, which facilitates metastasis. Oxygen delivery remains impaired because tumor vessels are anarchic and leaky, contributing to tumor cell dissemination. Counteracting hypoxia by normalizing tumor vessels in order to improve drug and radio therapy efficacy and avoid cancer stem-like cell selection is a highly challenging issue. We show here that inositol trispyrophosphate (ITPP) treatment stably increases oxygen tension and blood flow in melanoma and breast cancer syngeneic models. It suppresses hypoxia-inducible factors (HIFs) and proangiogenic/glycolysis genes and proteins cascade. It selectively activates the tumor suppressor phosphatase and tensin homolog (PTEN) in vitro and in vivo at the endothelial cell (EC) level thus inhibiting PI3K and reducing tumor AKT phosphorylation. These mechanisms normalize tumor vessels by EC reorganization, maturation, pericytes attraction, and lowering progenitor cells recruitment in the tumor. It strongly reduces vascular leakage, tumor growth, drug resistance, and metastasis. ITPP treatment avoids cancer stem-like cell selection, multidrug resistance (MDR) activation and efficiently enhances chemotherapeutic drugs activity. These data show that counteracting tumor hypoxia by stably restoring healthy vasculature is achieved by ITPP treatment, which opens new therapeutic options overcoming hypoxia-related limitations of antiangiogenesis-restricted therapies. By achieving long-term vessels normalization, ITPP should provide the adjuvant treatment required in order to overcome the subtle definition of therapeutic windows for in vivo treatments aimed by the current strategies against angiogenesis-dependent tumors.

Figures

Fig. 1

ITPP reduces melanoma tumor growth and improves mice survival. a Effect of ITPP treatment on the kinetics of tumor growth measured by bioluminescence in treated and nontreated animals at days 18 and 24. Endpoint was fixed at 2 cm3 (n = 6 animals per group, one representative experiment out of N > 10, **p < 0.001). b Comparison of tumor size, 23 days after B16F10LucGFP cells injection, showing reduced tumor growth in treated mice. Representative groups of five animals among groups of n = 10 animals. One experiment out of N ≥ 5 separate experiments. Insets illustrate the extreme size ranges (minimal and maximal) that tumor reached in nontreated compared to treated mice. c Mean size of the tumors in treated and non treated animals at day 23 (n = 10 in each group; number of experiments N > 20, **p < 0.001). d Magnetic resonance imaging of B16 F10 induced tumor. Morphological pulse sequence (left). Strong volume variation of the tumor (untreated/ITPP = 1163 mm3/121 mm3) was observed by image analysis after volume reconstruction. One typical example out of n = 10/experimental group. MRA-TOF/saturation recovery pulse sequence (right): Necrotic areas appear darker. After ITPP treatment, their size decreased. One typical example out of n = 10/experimental group. e Analysis by flow cytometry of B16LucGFP cells in tumors. Luciferase was detected intracellularly by specific antibodies and labeled by PerCP-Cy7 antirabbit IgG confirming: the reduced growth of tumor cells in ITPP-treated mice (%) and counts by direct cytometry analysis. Cells were numbered on the basis of intracellular Luciferase detection (n = 8; *p < 0.05) from dot plots or inset from histogram analysis for quantification of B16F10LucGFP in the tumor

Fig. 2

Reduction of colonization and increased survival in cancer bearing mice treated by ITPP. a Reduced luciferase activity after ITPP treatment in lungs of mice bearing subcutaneously implanted melanoma, indicating reduced metastasis (n = 8 ; p < 0,001, one experiment out of 10). b Reduced lung colonization, after treatment, in artificial metastasis model where melanoma cells were injected intravenously. Representative samples out of 20 mice in each group. c Evolution of the animal body weights. Nontreated animals are losing weight compared to ITPP-treated animals (n = 10 in each group, the number of experiments is N > 10), inset shows the effect of daily ITPP injection compared to saline. d Survival curve showing the rescue of melanoma bearing mice treated by ITPP (n = 10 in each group, one typical experiment out 5)

Fig. 3

Improved tumor oxygenation in cancer bearing mice treated by ITPP. a ITPP treatment increases oxygen pressure specifically inside a melanoma tumor within 30 min. No pO2 modification was observed in the healthy muscle. The probe was maintained in the same place, and pO2 was recorded in real time. Figure reports a representative example out of five animals treated the same day; identical data were acquired from n > 50 mice treated by ITPP. b Tumor oxygen pressure increase after double injection of ITPP is stable up to 72 h. Tumor oxygen pressure is enhanced 30 min after the first injection of ITPP. A second injection of ITPP applied after 24 h increases and stabilizes the pO2 increase for at least 72 h. Figure reports typical measurements randomly performed in treated animals (n = 10 per experiment). c ITPP treatment increases oxygen pressure inside a 4T1 breast tumor within 10 min. The increased pO2 (12 mmHg at day 14) is stable over 1 week (20 mmHg at day 22) and enhanced once more after a second ITPP injection. Picture shows data typically reporting several measurements randomly performed in treated animals (n = 8 per experiment). d Laser Doppler signals showing tumor blood flow improvement in ITPP-treated mice. Measurements were done randomly in each tumor treated as described in “Materials and methods,” day 22 (n = 8 out of 10 per group; p < 0.05)

Fig. 4

Reduction of hypoxia in cancer bearing mice treated by ITPP. a Images showing CD31+ blood vessels (green) and hypoxic sites detected by pimonidazole staining (red), in tumor sections. Nuclei are in blue. Pimonidazole was i.p. injected, 1 h before killing then staining by anti pimonidazole antibodies. After ITPP treatment, no staining for hypoxia was visible in tumors and weakly detected by image analysis estimating the fluorescence intensities and distribution in the green and red channels. One representative picture out N > 10 experiments (n = 10 animals per group). b Quantification by PET imaging of hypoxia by [18F]-FMISO fixation in melanoma bearing mice 14 days after subcutaneous implantation of the tumor cells (left, control) and upon serial ITPP treatments (right). The tumor radioactivity incorporation was quantified and expressed as: % of tumor activity = (total tumor activity/total whole body activity) × 100. After ITPP treatment, the tumor activity decreased from 14.58 ± 0.52 % (controls) to 7.6 ± 0.6 % (n = 8 animals in each group) showing the reversal of tumor hypoxia (N = 4). Representative PET imaging of control and treated animals, normalized with the same color scaling (0–21 on both images)

Fig. 5

Morphological and functional tumor vessel normalization induced by ITPP treatment. a ITPP-induced vessel normalization in ITPP-treated mice compared to untreated tumors imaged by: magnetic resonance angiography, 20 days after tumor induction showing chaotic vessel architecture in nontreated tumor (left panel, yellow arrows), vessel reorganization (normalization, yellow arrows, right panel) in ITPP-treated melanoma-bearing mice accompanied by tumor size reduction (see scale). Representative images from a typical example among 10 separate experiments. CD31 immunostaining of vascular endothelial cells in tumor sections showing disorganized aggregates in non treated tumors (n = 10) and vessel-like structures (green arrow) in treated mice (n = 10). Nuclei are stained with DAPI (blue); immunohistochemical staining for smooth muscle actin (SMA+) pericytes (white arrows) in frozen sections showing vessel organization in tumors from ITPP-treated mice (right panel, n = 10) compared to nontreated animals (left panel, n = 10). Tumor vessel architecture observed by confocal microscopy (Zeiss, LSM) imaging showing the close proximity of smooth muscle actin (SMA)-stained pericytes (green) with CD31+ endothelial cells (red) in the organized and normalized vessels, after ITPP treatment (n = 10) compared to nontreated tumor-bearing mice (n = 10). Scale bars represent 50 μm. b Reduction of tumor vessel leakiness measured by permeability to Evans Blue diffusion in tumor (n = 8; *p = 0,001). c Assessment of circulating VEGF by ELISA (n = 8; *, p = 0,001). d Angiogenesis-associated Endoglin (CD105) related to CD31 endothelial cell marker, quantified by flow cytometry among CD45-depleted tumor population, predicting endothelial cells lower activity and motility after treatment (n = 8; **p = 0.001). e Increased CD31+ and VEGF-Rs+ cells, in CD45 depleted tumor population after ITPP treatment (n = 10/group; *p = 0.05). f Flow cytometry analysis showing endothelial cells maturation markers: enhanced CD31 and Tie-2 (CD202) expressing cell numbers (n = 10/group; *p = 0.05)

Fig. 6

Phenotypic effect of ITPP treatment on tumor metabolism. a RT-PCR analysis revealing: downregulation of hypoxia/oxygen sensing genes and prometastasis genes; upregulation of genes implicated in endothelial cells maturation. Results are percent of non treated samples level (n = 8 animals, five separate experiments; **p = 0.001; ***p = 0.0001). b Reduction of the number of CXCR4+ CD34+ CD45− precursor cells among tumor cells upon ITPP treatment. Quantification by flow cytometry from separate tumor samples (n = 10/group p < 0.001). c Flow cytometry quantitative analysis showing a dramatic decrease in the number of cells expressing hypoxia-, stress-, and metastasis- related markers in primary tumors (day 22) after ITPP treatments as described in “Materials and methods” (n = 8/group; 5 separate experiments, **p = 0.001; ***p = 0.0001). d Flow cytometry quantification analysis showing a dramatic decrease in the number of cells expressing markers of high energetic metabolism in primary tumors (day 22) after ITPP treatments as described in “Materials and methods” (n = 8/group; five separate experiments; ***p = 0.0001)

Fig. 7

Effect of ITPP treatment on activation of endothelial PTEN and loss of tumor AKT phosphorylation. a PTEN, P-AKT (Ser473) and CD31 immunostainings. PTEN was expressed (red arrows) and colocalized with CD31+ endothelial cells (green arrows and green/red channels analysis of the label distribution, by image analysis) in nontreated tumor-bearing animals (left panel, n = 10/group). Markers separately localized after ITPP treatment (right panel, n = 10/group). The red/green channels display separate distribution by image analysis. b P-AKT distribution over the tumor (red arrows and red curve of the image analysis) observed in tumor stroma and endothelial cells, colocalized with CD31 staining (green arrows and green curve) in nontreated tumors. Expression of P-AKT was strongly reduced to punctual sites upon ITPP treatment. Image analysis point the separate localization with CD31 (right four panels, n = 10/group)

Fig. 8

Effect of ITPP on activation of endothelial PTEN in vitro upon hypoxia reoxygenation. In vitro activation of endothelial PTEN by ITPP upon hypoxia reoxygenation experiments. Murine lungs endothelial cells, MLuMEC cell line immortalized from FVB mice, were submitted to hypoxia (25 h) (b) then reoxygenated (22 h) (c) in the presence or not of ITPP (25 mM). PTEN activation was evidenced by its relocalization from the cytoplasm in hypoxia towards the inner face of the plasma membrane upon ITPP treatment. ITPP induced localization effect of PTEN was as enhanced by reoxygenation

Fig. 9

Effect of ITPP treatment on tumor hypoxia-induced resistance, stem cell selection, and enhancement of chemotherapeutic efficacy. a The P-glycoprotein immunostaining showing a reduced number of multidrug resistance positive tumor cells after ITPP treatment. Frozen sections of primary tumors from experiments described in Fig. 6 were histochemically labeled (day 22, ITPP treatments as described in “Materials and methods” (n = 8/group; five separate experiments). Scale bars = 50 μm. b Quantification by flow cytometry showing the reduction of cells positive for precursor and stem cell-associated markers (CD133, Oct3-4, ABCG-2) after ITPP treatment. CD133+ immunostaining corroborated the reduction visible on frozen section staining of primary tumors as in a. Scale bars = 50 μm. c Lung metastasis is suppressed by chemotherapeutic drugs (Paclitaxel and Cisplatin), when treatment is preceded by ITPP injection. Tumor cells are detected by their Lucifease activity in the lungs of animals from control, ITPP, CisPt plus Paclitaxel and combined treatments ITPP+ drugs as described in “Materials and methods.” Data are reported for day 22 (n = 10/group; 5 experiments; ***; p = 0.001). d CD31 staining of endothelial cells (green) and eosin/hematoxylin staining obtained in primary tumor frozen sections from experiment described in c. Efficient tissue necrosis was obtained when chemotherapeutic treatment is preceded by ITPP injection as described in “Materials and methods.” Scale bars = 50 μm

Fig. 10

Schematic outline of the proposed action of ITPP on HIFs regulation. 1 ITPP regulates angiogenesis by activating PTEN that inhibits PI3K action, AkT phosphorylation and mTOR actitivy towards HIF at the endothelial cell level. This regulates the vessels and increases the blood flow, while 2 hypoxia mediated O2 delivery by ITPP, allosteric effector of hemoglobin [–18], increases the intra tumor pO2, which also acts to destabilize HIF1α and down regulates VEGFs and VEGFs-related gene cascade. This directly reduces the endothelial cell mobilization, activation and growth, thus regulating the tumor VEGF-mediated pathological angiogenesis