Louis Van der Meeren

Background Immunotherapy represents the future of clinical cancer treatment. The type of cancer cell death determines the antitumor immune response and thereby contributes to the efficacy of anticancer therapy and long-term survival of patients. Induction of immunogenic apoptosis or necroptosis in cancer cells does activate antitumor immunity, but resistance to these cell death modalities is common. Therefore, it is of great importance to find other ways to kill tumor cells. Recently, ferroptosis has been identified as a novel, iron-dependent form of regulated cell death but whether ferroptotic cancer cells are immunogenic is unknown. Methods Ferroptotic cell death in murine fibrosarcoma MCA205 or glioma GL261 cells was induced by RAS-selective lethal 3 and ferroptosis was analyzed by flow cytometry, atomic force and confocal microscopy. ATP and high-mobility group box 1 (HMGB1) release were detected by luminescence and ELISA assays, respectively. Immunogenicity in vitro was analyzed by coculturing of ferroptotic cancer cells with bone-marrow derived dendritic cells (BMDCs) and rate of phagocytosis and activation/maturation of BMDCs (CD11c + CD86 + , CD11c + CD40 + , CD11c + MHCII + , IL-6, RNAseq analysis). The tumor prophylactic vaccination model in immune-competent and immune compromised (Rag-2 −/− ) mice was used to analyze ferroptosis immunogenicity. Results Ferroptosis can be induced in cancer cells by inhibition of glutathione peroxidase 4, as evidenced by confocal and atomic force microscopy and inhibitors’ analysis. We demonstrate for the first time that ferroptosis is immunogenic in vitro and in vivo. Early, but not late, ferroptotic cells promote the phenotypic maturation of BMDCs and elicit a vaccination-like effect in immune-competent mice but not in Rag-2 −/− mice, suggesting that the mechanism of immunogenicity is very tightly regulated by the adaptive immune system and is time dependent. Also, ATP and HMGB1, the best-characterized damage-associated molecular patterns involved in immunogenic cell death, have proven to be passively released along the timeline of ferroptosis and act as immunogenic signal associated with the immunogenicity of early ferroptotic cancer cells. Conclusions These results pave the way for the development of new therapeutic strategies for cancers based on induction of ferroptosis, and thus broadens the current concept of immunogenic cell death and opens the door for the development of new strategies in cancer immunotherapy.

Regulated cell death (RCD) has a fundamental role in development, pathology, and tissue homeostasis. In order to understand the RCD mechanisms, it is essential to follow these processes in real time. Here, atomic force microscopy (AFM) is applied to morphologically and mechanically characterize four RCD modalities (intrinsic and extrinsic apoptosis, necroptosis, and ferroptosis) in murine tumor cell lines. The nano-topographical analysis revealed a distinct surface morphology in case of necroptosis, ∼ 200 nm membrane disruptions are observed. Using mechanical measurements, it is possible to detect the early onset of RCD. Combined elasticity and microrheology analysis allowed for a clear distinction between apoptotic and regulated necrotic cell death. Finally, immunofluorescence analysis of the cytoskeleton structure during the RCD processes confirm the measured mechanical changes. The results of this study not only demonstrate the possibility of early real-time cell death detection but also reveal important differences in the cytoskeletal dynamics between multiple RCD modalities.

Regulated cell death modalities such as apoptosis and necroptosis play an important role in regulating different cellular processes. Currently, regulated cell death is identified using the golden standard techniques such as fluorescence microscopy and flow cytometry. However, they require fluorescent labels, which are potentially phototoxic. Therefore, there is a need for the development of new label-free methods. In this work, we apply Digital Holographic Microscopy (DHM) coupled with a deep learning algorithm to distinguish between alive, apoptotic and necroptotic cells in murine cancer cells. This method is solely based on label-free quantitative phase images, where the phase delay of light by cells is quantified and is used to calculate their topography. We show that a combination of label-free DHM in a high-throughput set-up (~10,000 cells per condition) can discriminate between apoptosis, necroptosis and alive cells in the L929sAhFas cell line with a precision of over 85%. To the best of our knowledge, this is the first time deep learning in the form of convolutional neural networks is applied to distinguish-with a high accuracy-apoptosis and necroptosis and alive cancer cells from each other in a label-free manner. It is expected that the approach described here will have a profound impact on research in regulated cell death, biomedicine and the field of (cancer) cell biology in general.

Abstract Novel bone growth‐stimulating interfaces are designed via surface modification of titanium (Ti) surfaces using the bioceramic CaCO 3 in the vaterite phase, Ca‐crosslinked alginate hydrogel, or a blend of these two materials with an active enzyme, alkaline phosphatase (ALP), as an osteoinductive component. The surface morphology and chemistry of the engineered surfaces are investigated using scanning electron microscopy, atomic force microscopy, and Fourier transform infrared spectroscopy, while the vaterite crystal fraction within the inorganic phase of the different coating types is determined by X‐ray diffraction. The functionality of the osteoconductive assembled bioceramic–hydrogel interface on Ti surface in regard with an active ALP payload is verified by the surface ALP loading and its activity. The methods of loading of ALP onto a Ti surface, adsorption versus coprecipitation, have a significant influence on the activity of immobilized ALP amount. The osteoblasts cultivated on the engineered surfaces functionalized with ALP exhibit a higher viability. The proposed composite materials with an active surface and a high mineral content represent an attractive biointerface for tissue engineering.

Biomaterials engineered with specific cell binding sites, tunable mechanical properties, and complex architectures are essential to control cell adhesion and proliferation. The influence of the local properties, such as the local hardness and stability on the interaction with cells, has not been yet fully understood and exploited. This is particularly relevant for hydrogels, very promising materials with, unfortunately, poor cell adhesion properties, attributed mostly to their softness. Here, we propose a new approach for producing hybrid hydrogels by functionalizing them with particles and performing a thermal treatment. Exploring the interaction of cells with these materials we introduce a new concept, cells-grabbing-onto-particles, a facilitation of the cell adhesion through modulation of local properties. The approach is implemented on alginate hydrogels typically unsuitable for cell growth by turning them into a very effective cell culture growth platform. Specifically, alginate hydrogels are bio-mineralized with calcium carbonate (CaCO3) particles, where an additional thermal annealing (T-A) process has been applied. The local Young’s modulus of new T-A treated hybrid hydrogels has increased to over 3 MPa on areas of hydrogels containing particles and to around 1 MPa on areas without particles, which is drastically different from 130 to 180 kPa values for unmodified hydrogels. Intriguingly, our results show that enhancement of local mechanical properties alone is a necessary, but insufficient, condition; the particles must be stably fixed in gels for cell growth and proliferation. Extended for hydrogels functionalized with silica particles too, the cells-grab-on-particles concept is shown applicable to different materials and cells for cell biology and tissue engineering.