Two-step transplantation with adipose tissue-derived stem cells increases follicle survival by enhancing vascularization in xenografted frozen-thawed human ovarian tissue.

STUDY QUESTION: Do adipose tissue-derived stem cells (ASCs) enhance vascularization and follicle survival in xenografted ovarian tissue using a two-step transplantation approach? SUMMARY ANSWER: Higher rates of oxygenation and vascularization of ovarian tissue, as well as increased follicle survival rates, were detected in the early post-grafting period. WHAT IS KNOWN ALREADY: ASCs have multilineage differentiation potential, proangiogenic properties and enhance vascularization in a peritoneal grafting site. Some studies suggest that using ASCs may improve ovarian tissue quality by enhancing graft angiogenesis. STUDY DESIGN, SIZE, DURATION: A total of 15 severe combined immunodeficient (SCID) mice were intraperitoneally grafted with frozen-thawed human ovarian tissue (OT) from five different patients. A peritoneal transplantation site had been previously prepared in a first step using either empty fibrin (Fi+OT group [n = 5]) or ASC-loaded fibrin (Fi/ASCs+OT group [n = 5]) for 14 days prior to grafting. Five mice underwent the standard one-step transplantation procedure and served as controls (OT group). Lithium phthalocyanine (LiPc) crystals were inserted into all grafted human ovarian tissue before transplantation. Levels of partial pressure of oxygen (pO2) in grafts were monitored in vivo by electron paramagnetic resonance (EPR) oximetry on Days 3 and 7. Samples for histology and immunohistochemistry (IHC) were collected after euthanizing the mice on Day 7 following EPR. One piece of ovarian tissue per patient was fixed for analysis to serve as non-grafted controls. PARTICIPANTS/MATERIALS, SETTING, METHODS: Prospective experimental study conducted at the Gynecology Research Unit, Université Catholique de Louvain. All materials were used to perform pO2 measurements (EPR oximetry), histological (haematoxylin and eosin staining), immunohistochemistry (anti-mouse and human double CD34 and anti-human Ki-67) and TUNEL analyses. MAIN RESULTS AND THE ROLE OF CHANCE: A significant increase in pO2 was observed in all groups between Days 3 and 7 (P < 0.001). A significantly higher pO2 level was observed in the Fi/ASCs+OT group compared to the OT group on Day 7 (P = 0.028). Total CD34-positive vessel area on Day 7 was greater in the Fi/ASCs+OT group than in any other group (vs non-grafted group: P = 0.0014; vs OT group: P = 0.013; vs Fi+OT group: P = 0.018). Primordial follicle survival rates after grafting were higher in the Fi/ASCs+OT group than in the OT (P = 0.0059) or Fi+OT groups (P = 0.0307). TUNEL-positive follicle percentages after grafting were significantly lower in the Fi/ASCs+OT group than in any other grafted tissue (vs OT group: P = 0.045; vs Fi+OT group: P = 0.0268). Percentages of Ki-67-positive primordial follicles were significantly higher in all grafted groups compared to non-grafted tissue controls (P < 0.01). LIMITATIONS REASONS FOR CAUTION: As demonstrated by our results, the proposed two-step ovarian tissue transplantation procedure using ASCs enhances vascularization in the early post-grafting period, leading to increased follicle survival rates and decreased apoptosis. However, mechanisms involved in the proangiogenic behavior of ASCs remain to be elucidated. WIDER IMPLICATIONS OF THE FINDINGS: Our results suggest that the proposed transplantation procedure with ASCs is a promising step towards potentially solving the problem of massive follicle loss after ovarian tissue grafting. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by grants from the Fonds National de la Recherche Scientifique de Belgique (FNRS-PDR Convention T.0077.14, grant Télévie No. 7.6515.16 F to DDM and grant 5/4/150/5 awarded to MMD and CAA is research associate, FRS-FNRS), Fonds Spéciaux de Recherche, Fondation St Luc, and Foundation Against Cancer, and donations from the Ferrero family.

Adipose tissue-derived stem cells in a fibrin implant enhance neovascularization in a peritoneal grafting site: a potential way to improve ovarian tissue transplantation.

STUDY QUESTION: Do two different concentrations of human adipose tissue-derived stem cells (ASCs) embedded inside a fibrin scaffold have the potential to differentiate into vessels and aid vascularization in a peritoneal grafting site intended for ovarian tissue transplantation? SUMMARY ANSWER: Human ASCs in low and high concentrations differentiated into vessels when transplanted to mouse peritoneum inside a fibrin matrix, but only high ASC concentrations significantly increased human vessel area 14 days after transplantation. WHAT IS KNOWN ALREADY: ASCs have multilineage differentiation potential, including proangiogenic properties and have been used in tissue engineering to enhance vascularization in transplanted tissues. Fibrin has been studied and used as an ASC-compatible biomaterial. STUDY DESIGN, SIZE, DURATION: In vivo experimental model using 22 severe combined immunodeficient mice. In total, 16 mice (eight per group) were intraperitoneally grafted with a fibrin scaffold loaded with two different human ASC concentrations (either 150 000 [L-ASC] or 1 500 000 [H-ASC] cells) and lithium phthalocyanine (LiPc) crystals as oxygen-sensitive probes. Six mice were grafted with an empty fibrin (EF) implant containing only LiPc and served as controls. Levels of partial pressure of oxygen (pO2) in implants were monitored in vivo by electron paramagnetic resonance oximetry (EPR). ASC identification, proliferation, and host and human vascularization were analyzed by immunohistochemistry (IHC). All analyses were performed on post-grafting Days 3, 7 and 14. PARTICIPANTS/MATERIALS, SETTING, METHODS: Prospective experimental study conducted at the Gynecology Research Unit, Université Catholique de Louvain. All materials were used to perform pO2 measurements (EPR oximetry), as well as histological (hematoxylin-eosin staining) and IHC (anti-human vimentin, anti-human Ki67, anti-mouse and human double CD34) analyses. MAIN RESULTS AND THE ROLE OF CHANCE: A significant increase in pO2 in implants was observed in all groups between Days 3 and 7 (P < 0.001). ASC-loaded implants displayed a tendency towards increased pO2 levels from Days 7 to 14, not observed in EF implants. ASC-loaded implants showed differentiation into human CD34-positive vessels. Total CD34-positive endothelial area was correlated to pO2 values obtained by EPR oximetry (r = 0.6506, P = 0.0019). In the H-ASC group, a greater human CD34-positive vascular surface area was found compared to the L-ASC group 14 days after transplantation (P < 0.0049). LARGE SCALE DATA: N/A. LIMITATIONS REASONS FOR CAUTION: As demonstrated by our results, ASCs transplanted inside a fibrin matrix can differentiate into CD34-positive human vessels. However, other possible mechanisms involved in ASC angiogenic behavior remain to be investigated. WIDER IMPLICATIONS OF THE FINDINGS: High concentrations of ASCs loaded inside a fibrin scaffold could serve as a substrate to prepare a peritoneal grafting site over 14 days, in order to enhance vascularization once human ovarian tissue is grafted. Our proposed preparation of the grafting site would not only benefit ovarian tissue transplantation, but also other experimental avascular grafting procedures. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by grants from the Fonds National de la Recherche Scientifique de Belgique (FNRS-PDR Convention T.0077.14, Télévie Grant no. 7.6515.16F awarded to DDM and Grant 5/4/150/5 awarded to M.M.D. [CAA is FRS-FNRS research associate]), Fonds Spéciaux de Recherche, and Fondation St Luc, Foundation Against Cancer, and donations from the Ferrero family. None of the authors have any competing interests to declare.

Tumour hypoxia determines the potential of combining mTOR and autophagy inhibitors to treat mammary tumours.

BACKGROUND: Hypoxia can activate autophagy, a self-digest adaptive process that maintains cell turnover. Mammalian target of rapamycin (mTOR) inhibitors are used to treat cancer but also stimulate autophagy. METHODS: Human mammary cancer cells and derived xenografts were used to examine whether hypoxia could exacerbate autophagy-mediated resistance to the mTOR inhibitor rapamycin. RESULTS: Rapamycin exerted potent antitumour effects in MCF-7 and MDA-MB-231 mammary tumours through a marked inhibition of angiogenesis, but the autophagy inhibitor chloroquine (CQ) failed to further sensitise tumours to mTOR inhibition. Rapamycin treatment actually led to tumour reoxygenation, thereby preventing the development of autophagy. Chloroquine alone, however, blocked the growth of MCF-7 tumours and in vitro blunted the hypoxia-induced component of autophagy in these cells. Finally, when initiating CQ treatment in large, hypoxic tumours, a robust antitumour effect could be observed, which also further increased the antiproliferative effects of rapamycin. CONCLUSION: The mTOR inhibitor rapamycin significantly contributes to tumour growth inhibition and normalisation of the tumour vasculature through potent antiangiogenic effects. The resulting reduction in hypoxia accounts for a lack of sensitisation by the autophagy inhibitor CQ, except if the tumours are already at an advanced stage, and thus largely hypoxic at the initiation of the combination of rapamycin and CQ treatment.

Electron paramagnetic resonance as a sensitive tool to assess the iron oxide content in cells for MRI cell labeling studies.

MRI cell tracking is a promising technique to track various cell types (stem cells, tumor cells, etc.) in living animals. Usually, cells are incubated with iron oxides (T(2) contrast agent) in order to take up the particles before being injected in vivo. Iron oxide quantification is important in such studies for validating the labeling protocols and assessing the dilution of the particles with cell proliferation. We here propose to implement electron paramagnetic resonance (EPR) as a very sensitive method to quantify iron oxide concentration in cells. Iron oxide particles exhibit a unique EPR spectrum, which directly reflects the number of particles in a sample. In order to compare EPR with existing methods (Perls's Prussian blue reaction, ICP-MS and fluorimetry), we labeled tumor cells (melanoma and renal adenocarcinoma cell lines) and fibroblasts with fluorescent iron oxide particles, and determined the limits of detection of the different techniques. We show that EPR is a very sensitive technique and is specific for iron oxide quantification as measurements are not affected by endogenous iron. As a consequence, EPR is well adapted to perform ex vivo analysis of tissues after cell tracking experiments in order to confirm MRI results.

Multimodal assessment of early tumor response to chemotherapy: comparison between diffusion-weighted MRI, 1H-MR spectroscopy of choline and USPIO particles targeted at cell death.

The aim of this study was to determine the value of different magnetic resonance (MR) protocols to assess early tumor response to chemotherapy. We used a murine tumor model (TLT) presenting different degrees of response to three different cytotoxic agents. As shown in survival curves, cyclophosphamide (CP) was the most efficient drug followed by 5-fluorouracil (5-FU), whereas the etoposide treatment had little impact on TLT tumors. Three different MR protocols were used at 9.4 Tesla 24 h post-treatment: diffusion-weighted (DW)-MRI, choline measurement by (1) H MRS, and contrast-enhanced MRI using ultrasmall iron oxide nanoparticles (USPIO) targeted at phosphatidylserine. Accumulation of contrast agent in apoptotic tumors was monitored by T(2) -weighted images and quantified by EPR spectroscopy. Necrosis and apoptosis were assessed by histology. Large variations were observed in the measurement of choline peak areas and could not be directly correlated to tumor response. Although the targeted USPIO particles were able to significantly differentiate between the efficiency of each cytotoxic agent and best correlated with survival endpoint, they present the main disadvantage of non-specific tumor accumulation, which could be problematic when transferring the method to the clinic. DW-MRI presents a better compromise by combining longitudinal studies with a high dynamic range; however, DW-MRI was unable to show any significant effect for 5-FU. This study illustrates the need for multimodal imaging in assessing tumor response to treatment to compensate for individual limitations.

Noninvasive mapping of spontaneous fluctuations in tumor oxygenation using 19F MRI.

PURPOSE: Acute hypoxia (transient cycles of hypoxia-reoxygenation) is known to occur in solid tumors and may be a poorly appreciated therapeutic problem as it can be associated with resistance to radiation therapy, impaired delivery of chemotherapeutic agents, or metastasis development. The objective of the present study was to use MR 19F relaxometry maps to analyze the spontaneous fluctuations of partial pressure of oxygen (pO2) over time in experimental tumors. METHODS: The pO2 maps were generated after direct intratumoral administration of a fluorine compound (hexafluorobenzene) whose relaxation rate (1/T1) is proportional to the % O2. The authors used a SNAP inversion-recovery sequence at 4.7 T to acquire parametric images of the T1 relaxation time with a high spatial and temporal resolution. Homemade routines were developed to perform regions of interest analysis, as well as pixel by pixel analysis of pO2 over time. RESULTS: The authors were able to quantify and probe the heterogeneity of spontaneous fluctuations in tumor pO2: (i) Spontaneous fluctuations in pO2 occurred regardless of the basal oxygenation state (i.e., both in oxygenated and in hypoxic regions) and (ii) spontaneous fluctuations occurred at a rate of 1 cycle/12-47 min. For validation, the analysis was performed in dead mice for which acute changes did not occur. The authors thereby demonstrated that 19F MRI technique is sensitive to acute change in pO2 in tumors. CONCLUSIONS: This is the first approach that allows quantitative minimally invasive measurement of the spontaneous fluctuations of tumor oxygenation using a look-locker approach (e.g., SNAP IR). This approach could be an important tool to characterize the phenomenon of tumor acute hypoxia, to understand its physiopathology, and to improve therapies.

Iron oxide particles covered with hexapeptides targeted at phosphatidylserine as MR biomarkers of tumor cell death.

The aim of the study was to evaluate the ability of a new MR contrast agent to detect cell death as a biomarker of the efficacy of anti-cancer treatment. The phosphatidylserine-targeted hexapeptide (E3) was coupled to pegylated ultrasmall iron oxide nanoparticles (USPIO) that can be detected by magnetic resonance imaging (MRI) and by electron paramagnetic resonance (EPR). USPIO binding to staurosporine-treated TLT (transplantable liver tumor) cells, evaluated by X-Band EPR, indicated twice as much binding of USPIO grafted with the E3 peptide, compared with USPIO grafted with a scrambled peptide or ungrafted USPIO. In vivo experiments were carried out using TLT cells implanted intramuscularly into NMRI mice, and tumor cell death was induced by irradiation. After intravenous injection of the different types of USPIO, the accumulation of contrast agent was evaluated ex vivo by X-band EPR, in vivo by L-band EPR and by T(2)-weighted MRI. In irradiated tumors there was greater accumulation of the targeted USPIO particles compared with control particles or compared with the targeted particles in untreated tissues. In conclusion, phosphatidylserine-targeting of USPIO particles can detect dying tissues. This molecular targeted system should be evaluated further as a potential biomarker of tumor response to treatment.

Removal of local field gradient artefacts in BOLD contrast imaging of head and neck tumours.

Monitoring of oxygenation in tumours is an important issue in predicting the success of anti-cancer treatments such as radiotherapy. Gradient echo (GE) imaging sequences can be used for monitoring changes in tumour blood flow and oxygenation. However, the application of this method in head and neck tumours is hampered by significant artefacts and losses of the MR signal near air-tissue interfaces. We investigated the usefulness of a gradient-echo slice excitation profile (GESEPI) sequence that should keep the oxygen contrast while recovering the signal loss caused by susceptibility artefacts. A tumour model was implanted in the neck and in the leg of mice. MR imaging was performed at 4.7 T. GE and GESEPI sequences were used for monitoring the blood oxygen level dependent (BOLD) contrast after carbogen breathing. The pO2 was also monitored in tumours using an OxyLite probe (Oxford Optronics). Using the tumours implanted in the leg, we found that the variations of signal intensity after carbogen breathing were similarin both sequences. In the tumour implanted in the neck, it was possible, using GESEPI sequences, to recover the signal loss caused by susceptibility artefacts and to monitor the effect of carbogen-induced changes in the tumour.

Changes in tumor oxygenation/perfusion induced by the no donor, isosorbide dinitrate, in comparison with carbogen: monitoring by EPR and MRI.

PURPOSE: In an effort to improve radiotherapy treatments, methods aimed at increasing the quantity of oxygen delivered to tumors were investigated. The aim of this study was to evaluate the effect of one nitric oxide (NO) donor (isosorbide dinitrate) on pO(2) and blood flow in a murine tumor model. The effect was compared to carbogen, used as a reference treatment. METHODS AND MATERIALS: Thirty-six liver tumors implanted in mouse thighs were imaged using magnetic resonance imaging (MRI) at 4.7 Tesla with dynamic Gd-DTPA and blood oxygen level-dependent (BOLD) contrast-enhanced imaging after administration of isosorbide dinitrate or carbogen. The effect on the pO(2) was also tested by EPR oximetry (1.1 GHz) on 52 mice. RESULTS: A significant increase in MRI intensity was observed for both treatments in comparison with the control group. EPR oximetry showed a dose-dependant increase in tumor pO(2) for isosorbide dinitrate (by 5.9 mmHg at 0.2 mg/kg) and a substantially greater change for carbogen breathing (by 23 mmHg). CONCLUSION: Both tumor blood flow and pO(2) were increased by isosorbide dinitrate and carbogen. Carbogen is more efficient than isosorbide dinitrate in increasing the BOLD image intensity, as well as the tumor pO(2), but as efficient as isosorbide dinitrate in the Gd-DTPA contrast-enhanced imaging. We conclude that the effects of carbogen on improving tumor pO(2) involve both improved blood flow and improved hemoglobin oxygenation, whereas the effects of isosorbide dinitrate are predominantly mediated by improved blood flow alone.

Pharmacological modifications of the partial pressure of oxygen in murine tumors: evaluation using in vivo EPR oximetry.

EPR oximetry using an implantable paramagnetic probe was used to quantify the partial pressure of oxygen (pO(2)) in tissues in a transplantable mouse tumor model (TLT) after administration of 34 different vasodilators belonging to one of the following classes: angiotensin-converting enzyme inhibitors, calcium antagonists, alpha antagonists, potassium channel openers, beta-blockers, NO donors, and peripheral vasoactive agents. Twenty-four compounds were efficient in significantly increasing the local pO(2) in a majority of tumors. The increase of local pO(2) using pharmacological treatments was lower than that achieved by using oxygen or carbogen breathing. This technique offers an unprecedented tool for rapidly and accurately measuring treatment-induced modifications of pO(2) in tumors. Magn Reson Med 42:627-630, 1999.

Microencapsulation of paramagnetic particles by pyrroxylin to preserve their responsiveness to oxygen when used as sensors for in vivo EPR oximetry.

Using the broadening of the electron paramagentic resonance (EPR) linewidth of paramagnetic particles by oxygen, it is possible to make measurements of the partial pressure of oxygen in vivo. While the results obtained so far with EPR oximetry are very encouraging, several paramagnetic materials may lose their responsiveness to oxygen in tissues. This aim of this study was to provide evidence that an appropriate coating can preserve the oxygen sensitivity of paramagnetic materials in vivo. Two charcoals that have the oxygen-sensing properties required for EPR oximetry (combined with a tendency to lose responsiveness to oxygen when placed in tissues) were coated using pyroxylin. Sensitivity to variations in pO2 was checked by inducing hypoxia in the muscles of mice injected with charcoal. While the uncoated material lost responsiveness to oxygen within few days, the particles coated with 20-30% of pyroxylin did not lose their responsiveness for more than 2 months.

Carbon-centered radicals as oxygen sensors for in vivo electron paramagnetic resonance: screening for an optimal probe among commercially available charcoals.

It is known that some charcoals possess paramagnetic centers with an electron paramagnetic resonance (EPR) linewidth which can be broadened by oxygen. In order to identify potential candidates as sensors for in vivo EPR oximetry, we carried out a systematic study among commercially available charcoals. A total of 34 charcoals were tested. The steps used for the screening were: (1) to check the presence of paramagnetic centers in the material; (2) to measure the EPR linewidth in nitrogen and in air on the dry material and on a aqueous suspension of particles; (3) to calibrate the oxygen sensitive materials (EPR linewidth vs. pO2); (4) to test the sensitivity and stability of the response to changes of pO2 in a simple model of hypoxia induced in mice. Seventeen charcoals contained paramagnetic centers detectable by low-frequency EPR (1.1 GHz). The EPR spectrum consist of one single line which is typical of carbon-centered radicals (g-factor approximately 2). Eight charcoals presented sufficient interesting EPR properties (linewidth in nitrogen < 0.1 mT, linewidth in air for an aqueous suspension of particles > 0.15 mT) to be further characterized in vivo. Only three charcoals presented a stable, reproducible, and sensitive response to pO2 for more than 2 months. These three coals should be considered as good candidates to be used as oxygen sensor using in vivo EPR spectroscopy.