1,721,132 research outputs found
Molecular pathways of different types of cell death: many roads to death
No full textCell death is a fundamental cellular response that has a crucial role in
shaping our bodies during development and in regulating tissue homeostasis by
eliminating unwanted cells. Three major morphologies of cell death have been
described: apoptosis (type I), cell death associated with autophagy (type II) and
necrosis (type III). In mammalian cells, the apoptotic response is mediated by either
an intrinsic or an extrinsic pathway, depending on the origin of the death stimuli,
and is almost always caspase-dependent. For a long time necrosis has been considered
to be an accidental and uncontrolled form of cell death. However, evidence
is accumulating that necrotic cell death in some cases can be as well controlled
and programmed as caspase-dependent apoptosis. Autophagy is foremost a survival
mechanism that is activated in cells subjected to nutrient or obligate growth factor
deprivation. When cellular stress continues, cell death may continue by autophagy
alone, or else it often becomes associated with features of apoptotic or necrotic cell
death, depending on the stimulus and cell type. It is debatable whether autophagic
cell death is an alternative way of dying, different from apoptotic and necrotic cell
death, or whether failure of autophagy to rescue the cell can lead to cell death by
either pathway. The aim of this chapter is to provide a general overview of current
knowledge on signalling events that result in apoptosis, necrosis and cell death associated
with autophagy
Damage-associated molecular patterns : revealing the molecular communication between dying cancer cells and the immune system
No full textCell surface-exposed calreticulin (ecto-CRT) and secreted ATP are crucial damage-associated molecular patterns (DAMPs) for immunogenic apoptosis. Here, immunogenic apoptosis is a type of cancer cell death subroutine which is apoptotic in nature but accompanied by enhanced immunogenicity as opposed to immunosuppression or tolerogenicity which accompanies the normal physiological apoptosis. DAMPs, which play an important role in mediating this enhanced immunogenicity, are molecules that are normally hidden within live cells (where they perform pre-dominantly non-immunological functions) however they tend to acquire immunomodulatory functions once secreted/surface exposed by dying/stressed/damaged cells. Previously described inducers of immunogenic apoptosis (certain chemotherapeutics) relied on an endoplasmic reticulum (ER)-based (reactive oxygen species (ROS)-regulated) pathway for DAMP exposure/secretion. However, these inducers caused immunogenic apoptosis through an off target effect thereby making the resultant immunogenicity secondary in character and prone to tumour/cancer microevolution-based resistance.During my PhD research, we found that after hypericin-based photodynamic therapy (Hyp-PDT), which generates on target ROS-mediated ER stress, dying cancer cells undergo bona fide immunogenic apoptosis characterized by phenotypic maturation and functional stimulation of (human) dendritic cells (DCs) as well as induction of a protective anti-tumour immune response, in vivo. Intriguingly, early after Hyp-PDT the cancer cells displayed ecto-CRT and secreted ATP through overlapping PERK-orchestrated pathways that required a functional secretory pathway and phosphoinositide 3-kinase (PI3K) p110alfa-mediated plasma membrane/extracellular trafficking. Interestingly, eIF2alfa phosphorylation and caspase-8 signalling which were important for chemotherapeutics-induced ecto-CRT were dispensable for Hyp-PDT induced ecto-CRT. Moreover, we found that Hyp-PDT induced ecto-CRT (in contrast to chemotherapy-induced ecto-CRT) was ERp57-independent. We also identified LRP1 as the surface docking site for ecto-CRT and found that depletion of PERK, PI3K p110alfa and LRP1 but not caspase-8 reduced the immunogenicity of the cancer cells. These results unravelled a novel PERK-dependent subroutine for the early and simultaneous emission of two critical DAMPs during Hyp-PDT induced on target immunogenic apoptosis. Thus, research done during my PhD has shown that there is a great need to increase the awareness (amongst patients and oncologists) that it is possible for the immunogenicity of a dying cancer/tumour cell to be accentuated to cause instigation of potent anti-tumour immunity if a proper therapy like Hyp-PDT is used; a message that has important socio-economic relevance. In terms of a practical implementation, the research done during my PhD can help in efficient Hyp-PDT based production of autologous anti-cancer DC vaccines against cancers like glioblastoma and melanoma. Moreover, for the preparation of autologous anti-cancer DC vaccines, Hyp-PDT has a higher probability of overcoming various immunogenicity resistance mechanisms (e.g. caspase-8 ablation based) employed by tumours than currently characterized chemotherapeutic inducers of immunogenicity. Hence, Hyp-PDT induced immunogenicity (if implemented clinically) promises to have better health economic implications.status: Publishe
IRONic cell death : unveiling the immunogenicity and efferocytosis of ferroptosis in cancer
No full textEnhancing and educating our body's immune system is undoubtedly one of the paramount objectives in the design of efficient cancer treatments. Traditional cancer therapies like chemotherapy and γ-radiation primarily aim to kill cancer cells directly. However, these treatments often come with significant side effects and may not fully engage the immune system in tackling cancer. Cancer immunotherapy stands as a revolutionary approach in combating cancer, leveraging the body's immune system to target and eliminate malignant cells. Central to this strategy is the concept of immunogenic cell death (ICD), where cancer cells succumb to treatment in a manner that triggers an immune response against the cancer. ICD operates through a dual mechanism: firstly, cancer cells are directly targeted and eliminated by various therapeutic agents or therapeutic modalities, and secondly, the dying cancer cells release immunostimulatory signals such as damage-associated molecular patterns (DAMPs) and, in combination with tumor association antigens (TAAs), induce a strong anti-cancer immunity leading to eradication of the cancer and generation of long-lasting immunological memory preventing the cancer relapse. Various therapeutic modalities have been shown to induce ICD in both murine and human cancer cell lines in vitro. Moreover, their immunogenic potential has been demonstrated in tumor therapeutic and vaccination models in mice in vivo. Most importantly, ICD-inducing agents and therapies have been already admitted into the clinics and are used for the treatment of cancer in patients.
There is a persistent challenge in cancer therapy being the development of drug resistance and immune evasion mechanisms, allowing cancer cells to withstand various forms of anti-cancer therapy and targeted therapies. The way cancer cells die influences the body's anti-cancer immunity, which plays a role in the effectiveness of cancer treatments and the prolonged survival of patients. While many agents triggering immunogenic apoptosis or necroptosis in cancer cells can stimulate anti-cancer immune responses, yet resistance to these forms of cell death is frequently encountered. Consequently, identifying alternative methods for eliminating cancer cells is critically important. As a result, there is a pressing need for alternative therapeutic modalities capable of overcoming the cell death resistance displayed by cancer cells.
Among the emerging types of ICD, ferroptosis offers notable advantages over apoptosis and necroptosis. Ferroptosis, distinct in both morphology and biochemistry from apoptosis and necroptosis, offers an alternative strategy for targeting resistant cancers. Ferroptosis leads to lipid peroxidation via abnormal iron metabolism, which can overcome previously mentioned cell death resistance and more effectively induce anti-cancer immunity leading to more potent cancer eradication. At the beginning of the PhD thesis, it was not known whether ferroptotic cancer cells are immunogenic.
In the initial part of this study, our objective was to assess the immunogenic properties of cancer cells undergoing ferroptotic cell death. To achieve this, we utilized a murine MCA205 fibrosarcoma cell line commonly employed in ICD studies. Our findings reveal that only early (1h and 3h cell death induction) and not late (24h cell death induction) ferroptotic MCA205 cells release DAMPs (ATP and HMGB1) and activate bone marrow derived dendritic cells (CD11c+CD86+, CD11c+CD80+, CD11c+MHCII+, IL-6) in vitro. Upon immunization of mice with early ferroptotic cancer cells, we observed efficient anti-tumor protection against the challenge with viable cancer cells in immune-competent mice but not in Rag-2-/- mice suggesting that the
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mechanism of immunogenicity is tightly regulated by the adaptive immune system and is time dependent. This contrasts with cancer cells undergoing freeze-thaw-induced accidental necrosis, which fail to elicit an efficient anti-cancer immune response. This work demonstrated that cancer cells in the early time-points of ferroptosis are immunogenic in vitro and in vivo.
In the second part of the PhD thesis, we focused on potential strategies to modulate the interaction of late ferroptotic cancer cells with the cells of the immune system, more specifically, macrophages. An approach applying nanomaterials was selected which is based on the coating of late ferroptotic cancer cells with biopolymers by the Layer-by-Layer (LbL) method. As a primary step of the analysis, changes in mechanobiological properties of late ferroptotic cancer cells after the coating were measured by atomic force microscopy (AFM) and, in the next step, utilized to gain further understanding of the interaction between ferroptotic cancer cells and bone marrow derived macrophages. This work revealed that enhancing the elasticity of late ferroptotic cancer cells through LbL coatings led to increased uptake by macrophages (i.e., efferocytosis). The findings presented in this study highlight the significance of mechanobiological characteristics of dying cancer cells in the process of efferocytosis. Furthermore, these results offer promising prospects for nanomaterial-based therapeutic applications to increase the efferocytosis of dying cancer cells and thereby improve their adjuvanticity and anti-cancer immunity.
Overall, our work opens new avenues for creating cancer treatment modalities that focus on triggering ferroptosis. Moreover, the work presented in the second part of the PhD thesis highlights the importance of the mechanobiology of ferroptotic cancer cells in controlling how macrophages engulf them, a process known as efferocytosis. This insight not only expands our understanding of immunogenic cell death but can be leveraged to create new therapeutic approaches for conditions where altering efferocytosis could be advantageous, as well as in the development of drug delivery mechanisms for cancer treatment
Apoptotic, necrotic and autophagic cell death types in pathophysiological conditions: morphological and histological aspects
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Mechanobiology dynamics in regulated cell death : cells under pressure
No full textEvery day, over 50 billion cells die in our body. Luckily, one does not have to worry about this since regulated cell death (RCD) is an essential process to maintain homeostasis in our tissues and bodies. Apoptosis was for a long time seen as the only form of RCD. Since the beginning of 21st century an increasing number of different cellular pathways that lead to RCD have been uncovered, which are referred to as different RCD modalities (e.g., necroptosis, ferroptosis, pyroptosis, NETosis, …). With increasingly broad research performed in the field of RCD, increasing understanding emerged on how these cell death modalities and more importantly malfunctions in these processes are involved in ubiquitous pathologies among which are neurodegenerative diseases, systemic inflammation, and cancer.
The omnipresence of RCD processes throughout life underlines the importance of having thorough understanding of what is occurring during these RCD modalities and what differentiates them from each other. Previously research in this field has been focusing on the morphological and biochemical changes during the different RCD modalities. In this work the goal is to explore how mechanobiology is involved in different RCD modalities and how this knowledge can be used for increased insight in the occurring immune reactions towards RCD which will be essential in developing new treatment strategies for diseases such as Alzheimer’s disease and cancer.
First, by using a single cell Atomic Force Microscopy (AFM) analysis, it is possible to detect distinct changes in the (visco)elastic properties of cells during four different RCD modalities (intrinsic apoptosis, extrinsic apoptosis, necroptosis and ferroptosis) (1). While AFM provides a high sensitivity single cell data analysis, the low throughput hinders its use as a real diagnostic tool. Therefore, to address this, a high throughput mechano-cytometry method is developed and applied to investigate their correlation with data obtained by AFM. Results from this analysis indicated a clear clustering between viable and dead cancer cells based on differences in mechanical properties (2). Finally, knowledge gained on these changes of the mechanical properties of dying/dead cancer cells was used to gain further understanding in interaction between dead cancer cells and immune cells. From this analysis, it could be concluded that increasing the Young’s modulus of dead cancer cells (using Layer-by-Layer coatings) leads to an increase of their uptake (i.e., efferocytosis) by macrophages (3). The results presented in this work shed a light on the importance of mechanobiological characteristics in essential cellular processes such as cancer cell death. Furthermore, these results provide important future outlook for label-free diagnostic purposes to discriminate different types of RCD and development of new treatment strategies for such diseases as cancer.
To obtain the results shown in this work, cells were put “under pressure”, both literally and figuratively
Multiple label-free microscopy techniques and machine learning to investigate regulated cancer cell death modalities
No full textGereguleerde cel dood (RCD) is een essentieel proces in de normale homeostase in menselijk weefsel. Indien teveel of te weinig celdood plaatsvindt kan dit leiden tot verschillende ziektebeelden zoals kankers en neurodegeneratieve ziektes. Het bestuderen van deze balans door RCD te (de-)activeren kan een mogelijke bron zijn voor de ontwikkeling van nieuwe medicijnen. De term RCD is een overkoepelende term van (>12) verschillende celdood processen die beschikken over een ander genetische machinerie en zijn biochemisch verschillend zijn van elkaar. In deze thesis werden drie verschillende RCD modi onderzocht, nl. apoptose, ferroptose en necroptose. Momenteel zijn methoden om naar RCD te bestuderen vaak gebaseerd op fluorescentie microscopie, waarbij fluorescente merkers worden gebruikt om een specifiek moleculair doel te visualiseren. Het is bewezen dat deze merkers celdood kunnen verstoren. Daarnaast hebben huidige methoden een gebrek aan specificiteit om accuraat te kunnen differentiëren tussen de verschillende RCD modi. Hierdoor wordt de bepaling van de correcte RCD modus sterk bemoeilijkt wanneer er zich verschillende RCD modi tegelijk voordoen. In deze thesis, wordt er onderzoek gedaan naar het potentieel van microscopie om zonder deze merkers (genaamd label-free microscopie) verschillen te observeren tussen deze RCD modi. Zoals de naam al aanduidt, gebruikt label-free microscopie geen merkers en zal dus niet interfereren met het RCD proces. Twee verschillende label-free microscopietechnieken werden in deze thesis gebruikt: digitale holografische microscopie (DHM) en Raman microscopie. De eerste kwantificeert de vertraging van licht door een transparant object, in dit geval een cel. Dit zodat we de morfologie (hoe de cel eruit ziet) veranderingen na RCD te kunnen bestuderen. De tweede methode kijkt naar de biochemische samenstelling van een cel. Deze biochemische samenstelling gebruiken we als “vingerafdruk” van de cel. Veranderingen in de data kunnen gelinkt worden aan de verschillende RCD modi onderzocht in deze studie. Deze label-free microscopietechnieken geven een enorme hoeveelheid informatie om te analyseren. Om deze data te kunnen verwerken wordt er gebruik gemaakt van kunstmatige intelligentie en specifieker machine learning (ML). Deze ML methoden proberen een model op te stellen dat past bij de gegeven data. Dit model kan vervolgens gebruikt worden om andere soortgelijke data te kunnen classificeren. Specifiek wordt van stalen voorspeld welke RCD plaatsvindt. Beide microscopietechnieken (DHM en Raman microscopie) worden gedetailleerd besproken in, respectievelijk, hoofdstuk drie en vier. Deze thesis laat zien dat het gebruik van label-free microscopie verschillende RCD modi correct kan identificeren met het gebruik van ML modellen. Deze modellen kunnen gebruikt worden in het veld van RCD onderzoek, maar deze aanpak kan ook gebruikt worden in het bredere veld van cel biologie. Potentieel kan dit onderzoek bijdragen aan het ontdekken van nieuwe medicijnen of gepersonaliseerde behandelingen door te kijken welk medicijn het beste aan zou slaan bij de patiënt
Role and contribution of mitochondrial connexin43, complex I and calcium sensitive potassium channels in cardiac ischemia-reperfusion injury
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