Cell Composition Analysis of Matched Salivary Organoid and Adherent Cultures: Choose Your Sjögren Disease Research Tool Carefully.

Sjögren disease (SjD) is widely recognized by the presence of B cell-dominated lymphocytic infiltration in the salivary glands (SGs). Contrary to what was originally presumed, however, SG hypofunction in SjD is not strongly correlated with the degree of SG lymphocytic infiltration in the SGs.

Organoid-based personalized medicine: from tumor outcome prediction to autologous transplantation.

Inter-individual variation largely influences disease susceptibility, as well as response to therapy. In a clinical context, the optimal treatment of a disease should consider inter-individual variation and formulate tailored decisions at an individual level. In recent years, emerging organoid technologies promise to capture part of an individual's phenotypic variability and prove helpful in providing clinically relevant molecular insights. Organoids are stem cell-derived 3-dimensional models that contain multiple cell types that can self-organize and give rise to complex structures mimicking the organization and functionality of the tissue of origin. Organoids therefore represent a more faithful recapitulation of the dynamics of the tissues of interest, compared to conventional monolayer cultures, thus supporting their use in evaluating disease prognosis, or as a tool to predict treatment outcomes. Additionally, the individualized nature of patient-derived organoids enables the use of autologous organoids as a source of transplantable material not limited by histocompatibility. An increasing amount of preclinical evidence has paved the way for clinical trials exploring the applications of organoid-based technologies, some of which are in phase I/II. This review focuses on the recent progress concerning the use of patient-derived organoids in personalized medicine, including (1) diagnostics and disease prognosis, (2) treatment outcome prediction to guide therapeutic advice, and (3) organoid transplantation or cell-based therapies. We discuss examples of these potential applications and the challenges associated with their future implementation.

In-vivo screening implicates endoribonuclease Regnase-1 in modulating senescence-associated lysosomal changes.

Accumulation of senescent cells accelerates aging and age-related diseases, whereas preventing this accumulation extends the lifespan in mice. A characteristic of senescent cells is increased staining with ß-galactosidase (ß-gal) ex vivo. Here, we describe a progressive accumulation of ß-gal staining in the model organism C. elegans during aging. We show that distinct pharmacological and genetic interventions targeting the mitochondria and the mTORC1 to the nuclear core complex axis, the non-canonical apoptotic, and lysosomal-autophagy pathways slow the age-dependent accumulation of ß-gal. We identify a novel gene, rege-1/Regnase-1/ZC3H12A/MCPIP1, modulating ß-gal staining via the transcription factor ets-4/SPDEF. We demonstrate that knocking down Regnase-1 in human cell culture prevents senescence-associated ß-gal accumulation. Our data provide a screening pipeline to identify genes and drugs modulating senescence-associated lysosomal phenotypes.

Mitophagy induction improves salivary gland stem/progenitor cell function by reducing senescence after irradiation.

BACKGROUND AND PURPOSE: Patients undergoing radiotherapy for head and neck cancer often experience a decline in their quality of life due to the co-irradiation of salivary glands. Radiation-induced cellular senescence is a key factor contributing to salivary gland dysfunction. Interestingly, mitochondrial dysfunction and cellular senescence have been reported to be strongly interconnected and thus implicated in several aging-related diseases. This study aims to investigate the role of mitochondrial dysfunction in senescence induction in salivary gland stem/progenitor cells after irradiation. MATERIALS AND METHODS: A dose of 7 Gy photons was used to irradiate mouse salivary gland organoids. Senescent markers and mitochondrial function were assessed using rt-qPCR, western blot analysis, SA-ß-Gal staining and flow cytometry analysis. Mitochondrial dynamics-related proteins were detected by western blot analysis while Mdivi-1 and MFI8 were used to modulate the mitochondrial fission process. To induce mitophagy, organoids were treated with Urolithin A and PMI and subsequently stem/progenitor cell self-renewal capacity was assessed as organoid forming efficiency. RESULTS: Irradiation led to increased senescence and accumulation of dysfunctional mitochondria. This was accompanied by a strong downregulation of mitochondrial fission-related proteins and mitophagy-related genes. After irradiation, treatment with the mitophagy inducer Urolithin A attenuated the senescent phenotype and improved organoid growth and stem/progenitor cell self-renewal capacity. CONCLUSION: This study shows the important interplay between senescence and mitochondrial dysfunction after irradiation. Importantly, activation of mitophagy improved salivary gland stem/progenitor cell function thereby providing a novel therapeutic strategy to restore the regenerative capacity of salivary glands following irradiation.

Studying Hepatic Stellate Cell Senescence.

Hepatic stellate cells (HSCs) are the key effector cells in liver fibrosis. They are the main producers of excessive amounts of extracellular matrix components during fibrogenesis and therefore a potential target for the treatment of liver fibrosis. Induction of senescence in HSCs may be a promising strategy to slow down, stop, or even reverse fibrogenesis. Senescence is a complex and heterogeneous process linked to fibrosis and cancer, but the exact mechanism and relevant markers can be cell-type dependent. Therefore, many markers of senescence have been proposed, and many methods to detect senescence have been developed. In this chapter, we review relevant methods and biomarkers to detect cellular senescence in hepatic stellate cells.

Biological functions of therapy-induced senescence in cancer.

Therapy-induced cellular senescence is a state of stable growth arrest induced by common cancer treatments such as chemotherapy and radiation. In an oncogenic context, therapy-induced senescence can have different consequences. By blocking cellular proliferation and by facilitating immune cell infiltration, it functions as tumor suppressive mechanism. By fueling the proliferation of bystander cells and facilitating metastasis, it acts as a tumor promoting factor. This dual role is mainly attributed to the differential expression and secretion of a set of pro-inflammatory cytokines and tissue remodeling factors, collectively known as the Senescence-Associated Secretory Phenotype (SASP). Here, we describe cell-autonomous and non-cell-autonomous mechanisms that senescent cells activate in response to chemotherapy and radiation leading to tumor suppression and tumor promotion. We present the current state of knowledge on the stimuli that affect the activation of these opposing mechanisms and the effect of senescent cells on their micro-environment eg. by regulating the functions of immune cells in tumor clearance as well as strategies to eliminate senescent tumor cells before exerting their deleterious side-effects.

Enhanced extrinsic apoptosis of therapy-induced senescent cancer cells using a death receptor 5 (DR5) selective agonist.

Genotoxic agents are widely used anti-cancer therapies because of their ability to interfere with highly proliferative cells. An important outcome of these interventions is the induction of a state of permanent arrest also known as cellular senescence. However, senescent cancer cells are characterized by genomic instability and are at risk of escaping the growth arrest to eventually facilitate cancer relapse. The tumor necrosis factor related apoptosis inducing ligand (TRAIL) signals extrinsic apoptosis via Death Receptors (DR) 4 and 5, while Decoy Receptors (DcR) 1 and 2, and Osteoprotegerin (OPG) are homologous to death receptors but incapable of transducing an apoptotic signal. The use of recombinant TRAIL as an anti-cancer strategy in combination with chemotherapy is currently in development, and a major question remains whether senescent cancer cells respond to TRAIL. Here, we show variable sensitivity of cancer cells to TRAIL after senescence induction, and upregulation of both pro-apoptotic and anti-apoptotic receptors in therapy-induced senescent cancer cells. A DR5-selective TRAIL variant (DHER), unable to bind to DcR1 or OPG, was more effective in inducing apoptosis of senescent cancer cells compared to wild-type TRAIL. Importantly, no apoptosis induction was observed in non-cancerous cells, even at the highest concentrations tested. Our results suggest that targeting DR5 can serve as a novel therapeutic strategy for the elimination of therapy-induced senescent cancer cells.

Dihydroartemisinin-Transferrin Adducts Enhance TRAIL-Induced Apoptosis in Triple-Negative Breast Cancer in a P53-Independent and ROS-Dependent Manner.

Triple-negative breast cancer (TNBC) is a highly aggressive breast cancer subtype independent of estrogen receptor, progesterone receptor, or human epidermal growth factor receptor 2. It has a poor prognosis and high recurrence. Due to its limited treatment options in the clinic, novel therapies are urgently needed. Single treatment with the death receptor ligand TRAIL was shown to be poorly effective. Recently, we have shown that artemisinin derivatives enhance TRAIL-induced apoptosis in colon cancer cells. Here, we utilized transferrin (TF) to enhance the effectiveness of dihydroartemisinin (DHA) in inducing cell death in TNBC cell lines (MDA-MB-231, MDA-MB-436, MDA-MB-468 and BT549). We found that the combination of DHA-TF and the death receptor 5-specific TRAIL variant DHER leads to an increase in DR5 expression in all four TNBC cell lines, while higher cytotoxicity was observed in MDA-MB-231, and MDA-MB-436. All the data point to the finding that DHA-TF stimulates cell death in TNBC cells, while the combination of DHA-TF with TRAIL variants will trigger more cell death in TRAIL-sensitive cells. Overall, DHA-TF in combination with TRAIL variants represents a potential novel combination therapy for triple-negative breast cancer.

Artemisinin Derivatives Stimulate DR5-Specific TRAIL-Induced Apoptosis by Regulating Wildtype P53.

Artemisinin derivatives, widely known as commercial anti-malaria drugs, may also have huge potential in treating cancer cells. It has been reported that artemisinin derivatives can overcome resistance to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis in liver and cervical cancer cells. In our study, we demonstrated that artesunate (ATS) and dihydroartemisinin (DHA) are more efficient in killing colon cancer cells compared to artemisinin (ART). ATS/DHA induces the expression of DR5 in a P53 dependent manner in HCT116 and DLD-1 cells. Both ATS and DHA overcome the resistance to DHER-induced apoptosis in HCT116, mainly through upregulating death receptor 5 (DR5). We also demonstrate that DHA sensitizes HCT116 cells to DHER-induced apoptosis via P53 regulated DR5 expression in P53 knockdown assays. Nevertheless, a lower effect was observed in DLD-1 cells, which has a single Ser241Phe mutation in the P53 DNA binding domain. Thus, the status of P53 could be one of the determinants of TRAIL resistance in some cancer cells. Finally, the combination treatment of DHA and the TRAIL variant DHER increases cell death in 3D colon cancer spheroid models, which shows its potential as a novel therapy.

The role of MIF in chronic lung diseases: looking beyond inflammation.

Macrophage migration inhibitory factor (MIF) is a pleiotropic cytokine that has been associated with many diseases. Most studies found in literature describe MIF as a proinflammatory cytokine involved in chronic inflammatory conditions, but evidence from last years suggests that many of its key effects are not directly related to inflammation. In fact, MIF is constitutively expressed in most human tissues and in some cases in high levels, which does not reflect the pattern of expression of a classic proinflammatory cytokine. Moreover, MIF is highly expressed during embryonic development and decreases during adulthood, which point toward a more likely role as growth factor. Accordingly, MIF knockout mice develop age-related spontaneous emphysema, suggesting that MIF presence (e.g., in younger individuals and wild-type animals) is part of a healthy lung. In view of this new line of evidence, we aimed to review data on the role of MIF in the pathogenesis of chronic lung diseases.

A Bispecific Inhibitor of the EGFR/ADAM17 Axis Decreases Cell Proliferation and Migration of EGFR-Dependent Cancer Cells.

Dysregulated epidermal growth factor receptor (EGFR) is an oncogenic driver of many human cancers, promoting aberrant cell proliferation, migration, and survival. Pharmacological targeting of EGFR is often challenged by acquired mechanisms of resistance. Ligand-dependent mechanisms in EGFR wild-type cells rely on ligand or receptor overexpression, allowing cells to outcompete inhibitors and perpetuate signaling in an autocrine manner. Importantly, EGFR ligands are synthesized as membrane-bound precursors that must be solubilized to enable receptor-ligand interactions. The A disintegrin and metalloproteinase 17 (ADAM17) is considered the main sheddase of several EGFR ligands, and a potential pharmacological target. However, its broad substrate range and ubiquitous expression complicate its therapeutic targeting. Here, we present a novel bispecific fusion protein construct consisting of the inhibitory prodomain of ADAM17 (TPD), fused to an EGFR-targeting designed ankyrin repeat protein (DARPin). TPD is a natural inhibitor of ADAM17, maintaining the protease in a zymogen-like form. Meanwhile, the high affinity anti-EGFR DARPin E01 binds to EGFR and inhibits ligand binding. The resulting fusion protein E01-GS-TPD retained binding ability to both molecular targets EGFR and ADAM17. The large difference in affinity for each target resulted in enrichment of the fusion protein in EGFR-positive cells compared to EGFR-negative cells, suggesting a possible application in autocrine signaling inhibition. Accordingly, E01-GS-TPD decreased migration and proliferation of EGFR-dependent cell lines with no significant increase in apoptotic cell death. Finally, inhibition of proliferation was observed through EGFR ligand-dependent mechanisms as growth inhibition was not observed in EGFR mutant or KRAS mutant cell lines. The use of bispecific proteins targeting the EGFR/ADAM17 axis could be an innovative strategy for the treatment of EGFR-dependent cancers.

Regulation of Survival Networks in Senescent Cells: From Mechanisms to Interventions.

Cellular senescence is a state of stable cell cycle arrest arising in response to DNA and mitochondrial damages. Senescent cells undergo morphological, structural and functional changes that are influenced by a number of variables, including time, stress, tissue, and cell type. The heterogeneity of the senescent phenotype is exemplified by the many biological properties that senescent cells can cover. The advent of innovative model organisms has demonstrated a functional role of senescent cells during embryogenesis, tissue remodeling, tumorigenesis and aging. Importantly, prolonged and aberrant persistence of senescent cells is often associated with tissue dysfunction and pathology, and is partially the consequence of mechanisms that enhance survival and resistance to cell death. Here, we describe the main molecular players involved in promoting survival of senescent cells, with particular emphasis on the regulation of senescence-associated anti-apoptotic pathways. We discuss the consequences these pathways have in providing resistance to intrinsic and extrinsic pro-apoptotic signals. Finally, we highlight the importance of these pathways in the development of targets for senolytic interventions.

Therapeutic interventions for aging: the case of cellular senescence.

Organismal aging is a multifactorial process characterized by the onset of degenerative conditions and cancer. One of the key drivers of aging is cellular senescence, a state of irreversible growth arrest induced by many pro-tumorigenic stresses. Senescent cells accumulate late in life and at sites of age-related pathologies, where they contribute to disease onset and progression through complex cell and non-cell autonomous effects. Here, we summarize the mechanisms by which cellular senescence can promote aging, and we offer an extensive description of current potential pharmacological interventions for senescent cells, highlighting limitations and suggesting alternatives.