A Novel Approach to Inner Cancer Treatment through the Activation of Photosensitizers by Protons

Brain cancers like glioblastoma multiforme (GBM) are incurable due to their location and infiltrative nature. The current standard of care offers limited survival benefit with only 3% of patients surviving longer than five years after the initial diagnosis. In the work included in this thesis, we sought to combine the strengths of photodynamic (PDT) and proton therapies to simultaneously overcome their individual limitations. In our hypothesis, we envisaged a hybrid treatment modality, ‘protondynamic therapy’, where accelerated protons are used to excite photosensitizers (PSs, light activated drugs) by Coulomb interactions. This would elicit a ‘PDT-like’ effect to synergize with proton-radiotherapy and efficiently eradicate cancer cells, both in primary and disseminated GBM lesions. In the work included in this thesis, we showed for the first time that accelerated protons can indeed activate PSs to emit fluorescence and generate singlet oxygen in solutions and gels. We also provided proof of principle of the cytotoxic efficacy of ‘protondynamic therapy’ on two GBM cell lines, laying the foundations for a novel treatment, applicable to many difficult-to-cure cancer indications

Epigenetic regulation of neurogenic differentiation of adipose tissue stem cells

Mesenchymal stem cells (MSCs) derived from adipose tissue are multipotent stem cells able to give rise into multiple cell types, not only of the mesodermal lineage, but also of the neuroectodermal lineage. In this study, we show that there is an epigenetic basis related to the capacity of adipose tissue stem cells (ASCs) to differentiate towards the neurogenic pathway. Neurogenic differentiation was induced by a step of mitogenic stimulation (step 1), followed by neurogenic induction (step 2), and shown at the gene and protein expression levels. Nestin has been extensively used as a marker for neurogenesis. The DNA methylation status of several regions of the nestin (NES) gene, including the promoter, a muscle-specific enhancer in the first intron, and a neural enhancer in the second intron, was determined by bisulphite genomic sequencing prior to and after induction of proliferation (step 1) and neurogenic differentiation (step 2). There was a global demethylation of the second intron upon proliferation induction and this was followed by a strong upregulation of nestin expression at the mRNA and protein level. We observed re-methylation of these regions after induction of neurogenic differentiation in vitro, and that was accompanied by a steep decline in nestin expression at the mRNA and protein level. These data are consistent with nestin being a marker of early neurogenesis. Analysis of post-translational histone modifications by chromatin immunoprecipitation reveals dynamic changes at the NES locus and on the promoter of a housekeeping gene (GAPDH) associated with step 1 and step 2. Our data suggest an epigenetic ‘priming’ of ASCs (at least at the NES locus) towards neurogenic differentiation, which is primarily elicited by mitogenic stimulation

Photodynamic Efficacy of Cercosporin in 3D Tumor Cell Cultures

In the present work, we study the photodynamic action of cercosporin (cerco), a naturally occurring photosensitizer, on human cancer multicellular spheroids. U87 spheroids exhibit double the uptake of cerco than T47D and T98G spheroids as shown by flow cytometry on the single cell level. Moreover, cerco is efficiently internalized by cells throughout the spheroid as shown by confocal microscopy, for all three cell lines. Despite their higher cerco uptake, U87 spheroids show the least vulnerability to cerco‐PDT, in contrast to the other two cell lines (T47D and T98G). While 300 μm diameter spheroids consistently shrink and become necrotic after cerco PDT, bigger spheroids (>500 μm) start to regrow following blue‐light PDT and exhibit high viability. Cerco‐PDT was found to be effective on bigger spheroids reaching 1mm in diameter especially under longer exposure to yellow light (~590 nm). In terms of metabolism, T47D and T98G undergo a complete bioenergetic collapse (respiration and glycolysis) as a result of cerco‐PDT. U87 spheroids also experienced a respiratory collapse following cerco‐PDT, but retained half their glycolytic activity

Cellular reactions and compensatory tissue re-organization during spontaneous recovery after spinal cord injury in neonatal mice

International audienceFollowing incomplete spinal cord injuries, neonatal mammals display a remarkable degree of behavioral recovery. Previously, we have demonstrated in neonatal mice a wholesale re-establishment and reorganization of synaptic connections from some descending axon tracts (Boulland et al.: PLoS One 8 (2013)). To assess the potential cellular mechanisms contributing to this recovery, we have here characterized a variety of cellular sequelae following thoracic compression injuries, focusing particularly on cell loss and proliferation, inflammation and reactive gliosis, and the dynamics of specific types of synaptic terminals. Early during the period of recovery, regressive events dominated. Tissue loss near the injury was severe, with about 80% loss of neurons and a similar loss of axons that later make up the white matter. There was no sign of neurogenesis, no substantial astroglial or microglial proliferation, no change in the ratio of M1 and M2 microglia and no appreciable generation of the terminal complement peptide C5a. One day after injury the number of synaptic terminals on lumbar motoneurons had dropped by a factor of 2, but normalized by 6 days. The ratio of VGLUT1/2+ to VGAT+ terminals remained similar in injured and uninjured spinal cords during this period. By 24 days after injury, when functional recovery is nearly complete, the density of 5-HT+ fibers below the injury site had increased by a factor of 2.5. Altogether this study shows that cellular reactions are diverse and dynamic. Pronounced recovery of both excitatory and inhibitory terminals and an increase in serotonergic innervation below the injury, coupled with a general lack of inflammation and reactive gliosis, are likely to contribute to the recovery. © 2016 Wiley Periodicals, Inc. Develop Neurobiol 77: 928-946, 2017

Proton-dynamic therapy following photosensitiser activation by accelerated protons demonstrated through fluorescence and singlet oxygen production

We demonstrate excitation of photosensitisers (PSs) by accelerated protons to produce fluorescence and singlet oxygen. Their fluorescence follows a pattern similar to the proton energy loss in matter, while proton-derived fluorescence spectra match the photon-induced spectra. PSs excited in dry gelatin exhibit enhanced phosphorescence, suggesting an efficient PSs triplet state population. Singlet oxygen measurements, both optically at ~1270 nm and through the photoproduct of protoporphyrin IX (PpIX), demonstrate cytotoxic singlet oxygen generation by proton excitation. The singlet oxygen-specific scavenger 1,4-diazabicyclo[2.2.2]octane (DABCO) abrogates the photoproduct formation under proton excitation, but cannot countermand the overall loss of PpIX fluorescence. Furthermore, in two cell lines, M059K and T98G, we observe differential cell death upon the addition of the PS cercosporin, while in U87 cells we see no effect at any proton irradiation dose. Our results pave the way for a novel treatment combining proton therapy and “proton-dynamic therapy” for more efficient tumour eradication