Immunogenic cell death induced by a new photodynamic therapy based on photosens and photodithazine

Background: Anti-cancer therapy is more successful when it can also induce an immunogenic form of cancer cell death (ICD). Therefore, when developing new treatment strategies, it is extremely important to choose methods that induce ICD and thereby activate anti-Tumor immune response leading to the most effective destruction of tumor cells. The aim of this work was to analyze whether the clinically widely used photosensitizers, photosens (PS) and photodithazine (PD), can induce ICD when used in photodynamic therapy (PDT). Methods: Cell death in murine glioma GL261 or fibrosarcoma MCA205 cells was induced by PS-or PD-PDT and cell death was analyzed by MTT or flow cytometry. Intracellular distribution of PS and PD was studied by using the laser scanning microscope. Calreticulin exposure and HMGB1 and ATP release were detected by flow cytometry, ELISA and luminescence assay, respectively. Immunogenicity in vitro was analyzed by co-culturing of dying cancer cells with bone-marrow derived dendritic cells (BMDCs) and rate of phagocytosis and maturation (CD11c+CD86+, CD11c+CD40+) of BMDCs and production of IL-6 in the supernatant were measured. In vivo immunogenicity was analyzed in mouse tumor prophylactic vaccination model. Results: We determined the optimal concentrations of the photosensitizers and found that at a light dose of 20 J/cm2 (\u3bbex 615-635 nm) both PS and PD efficiently induced cell death in glioma GL261 and fibrosarcoma MCA205 cells. We demonstrate that PS localized predominantly in the lysosomes and that the cell death induced by PS-PDT was inhibited by zVAD-fmk (apoptosis inhibitor) and by ferrostatin-1 and DFO (ferroptosis inhibitors), but not by the necroptosis inhibitor necrostatin-1 s. By contrast, PD accumulated in the endoplasmic reticulum and Golgi apparatus, and the cell death induced by PD-PDT was inhibited only by z-VAD-fmk. Dying cancer cells induced by PS-PDT or PD-PDT emit calreticulin, HMGB1 and ATP and they were efficiently engulfed by BMDCs, which then matured, became activated and produced IL-6. Using dying cancer cells induced by PS-PDT or PD-PDT, we demonstrate the efficient vaccination potential of ICD in vivo. Conclusions: Altogether, these results identify PS and PD as novel ICD inducers that could be effectively combined with PDT in cancer therapy

3D in vitro platform produced by two-photon polymerization for the analysis of neural network formation and function

Zr-Si organic-inorganic scaffolds fabricated by a two-photon polymerization technique were used for the primary culture of mouse embryonic neural cells. We observed that dissociated hippocampal cells adhere to the scaffolds, produce neurites, elongate and differentiate into adult neurons. Neuronal outgrowth and synaptogenesis were confirmed by immunohistochemical staining with antibodies against βIII-Tubulin and synaptophysin. The formation of a functional neural network was assessed by the measurement of spontaneous activity using Ca2+ imaging of dissociated hippocampal cultures grown on Zr-Si scaffolds. The results of this study suggest that two-photon-induced polymerization of organic-inorganic hybrid biomaterials provides a robust model for 3D neuronal tissue engineering studies

Long-term neurological and behavioral results of biodegradable scaffold implantation in mice brain

The aim of the study was to assess the integral outcomes of implantation biocompatible scaffold as the carrier of neural stem cells in the reconstructive surgery of open traumatic brain injury (TBI) by parameters of neurological and cognitive status of the animals in the experiment. Materials and Methods. Adult male C57BL/6 mice were injured with open-skull weight-drop method. 3D hydrogel scaffold based on modified chitosan in complex with hyaluronic acid was transplanted into the lesion cavity 1 week after TBI. Using a variety of behavioral and cognitive tests (modified neurological severity scores (mNSS), open field test as well as novel object recognition and passive avoidance tests) the short-and long-term neurological and memory functions sequelae induced by TBI were assessed. Magnetic resonance imaging was used to visualize the injury site. Results. Significant functional recovery was observed on both the mNSS and open field tests in the scaffold transplantation group compared to the control TBI group. In addition, enhanced improvement of short and long-term memory functions was found 5 months post injury. Magnetic resonance imaging data revealed that the scaffold transplantation result in decreasing of the volume of injury area compared to control TBI group and preventing the disruption of the neural networks of the brain. Conclusion. Taken together, our findings indicated that transplantation of 3D biodegradable scaffold into injury cavity contributed to the preservation of volume in the damaged region in the first months after TBI which in turn led to a better functional recovery in the remote period